In conclusion, the above-reported data concur to reach the following conclusions: (1) differences in the genetic background among different geographic populations account for significant variances observed in psoriasis populations, both in terms of frequency and of severity (localized palmoplantar versus generalized forms) of pustular psoriasis (107, 108), (2) despite the dramatic *in vitro* effects of some CARD14 variants on the keratinocytes, there is a wide range of phenotypes, even among individuals who carry the same substitution, suggesting that in many instances, the variable phenotypes are likely to multiple other factors besides the genetic background.

## NLRP12-AID

NLRP12 is an NLR encoded by *NLRP12* (also known as *NALP12*, Monarch-1, or PYPAF7) and functions as a negative regulator of NF-κB activation (109). NLRP12 interacts via its pyrin domain with the pyrin domain of apoptosis-associated speck-like (ASC) protein (110) leading to the formation of an intracellular aggregate called speck to active IL-1B (111). Sequencing of *NLRP12* revealed a heterozygous nonsense mutation c.850C>T (p.Arg284X) in identical twin brothers presenting with symptoms overlapping FCAS and MWS. A second NLRP12 mutation (c.2072+3insT) was identified in a patient presenting with a periodic fever syndrome, including clinical manifestation of FCAS. This mutation, affecting the donor splice site of intron 3, activates a cryptic splice site located upstream in exon 3 and results in a frameshift, followed by a premature stop codon (112). These two mutations were demonstrated to be functionally associated with high levels of NF-κB activity, thus accounting for the autoinflammatory phenotype. Jèru et al. identified a missense mutation c.1054C\_T (p.Arg352Cys) within the NBS domain of the protein in two unrelated patients. This missense mutation is associated with a gain of function of caspase 1 processing (110). The c.882C>G (p.Asp294Glu) mutation was found to mostly segregate with a particular sensitivity to cold exposure (especially arthralgias and myalgia), even in the absence of urticarial rash, fever, or elevation in the levels of acute-phase reactants. In any case, the clinical manifestations presented by the carriers were generally mild, although quality of life was affected, especially during the winter season (112–114). Several reports identified the NLRP12 variant F402L (c.1206 C>G) (115–117).

# Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome

Pyogenic sterile arthritis, pyoderma gangrenosum, and acne syndrome is an autosomal dominant AID caused by mutations in the *PSTPIP1* gene, which is located in chromosomal position 15q24–q25.1. Pyrin protein is a cytosolic receptor for PSTPIP1. Ligation between pyrin and PSTPIP1 induces pyrin to interact with ASC protein, inducing the creation of an active ASC pyroptosome. A possible explanation of PAPA syndrome is a constitutive ligation and consequent activation of pyrin with the mutated PSTPIP1 proteins (112). The disease is extremely rare, with less than 10 families described worldwide. The first case of PAPA syndrome was reported in 1975 (118) and in 1997, in the same family, PAPA syndrome was described as a heritable disease (119).

In 2000, 93 genomic loci were investigated in patients with a pleiotropic inflammatory syndrome characterized by pyoderma gangrenosum, cystic acne, and erosive arthritis, demonstrating PAPA syndrome maps in chromosomal position 15q (120).

There are two hot-spots mutations, c.688G>A (p.A230T) and c.748G>C (p.E250Q), which occur in exons 10 and 11 and have been found in many familial (121–126) and sporadic cases (127, 128). Mutations are thought to disrupt the binding of PSTPIP-1 with protein tyrosine phosphatase–PEST, a regulatory phosphatase, increasing its avidity for pyrin in the cytosol, thereby causing dysregulation of IL-1β production (129).

Until 2016, a total of 27 genetic variants were reported for *PSTPIP1* gene (23 substitutions, 1 insertion, 2 deletions, and 1 duplication), 17 of which are PAPA phenotype associated (16).

# Deficiency of Adenosine Deaminase 2

Searching for mutations in systemic inflammation and vasculopathy and/or necrotizing vasculitis polyarteritis nodosa patients, *CECR1* (cat eye syndrome chromosome region, candidate 1) gene mutations were discovered by two independent groups. Pattern of inheritance was autosomal recessive. Subsequent studies described another case with a fatal vasculopathy (130). Common clinical signs are early onset recurrent stroke, neurologic manifestations, and fever. Being the *CECR1* gene highly polymorphic, as other AID causative genes, the correlation between clinical signs and familial ancestry is important. The *CECR1* gene encodes the adenosine deaminase 2 (ADA2) protein, which has partial homology with ADA1 protein. Both ADA1 and ADA2 act as intracellular enzymes that regulate the purinergic signaling pathway. Mutations in ADA1 are known to cause severe combined immunodeficiency disease, characterized by a defect in T- and B-lymphocytes. ADA2 mutations, conversely, cause only mild hypogammaglobulinemia due to a defect in terminal differentiation of B-cells (131).

## NLRC4-Associated Inflammatory Diseases (SCAN4, NLRC4-MAS, and NLRC4-FCAS)

Patients with macrophage activation syndrome or a milder phenotype like FCAS may carry gain of function mutations in the *NLRC4* (IPAF; CARD12) gene (132–134). Two novel causal mutations, p.T337S and p.V341A, have been diagnosed by whole exome sequencing (WES) in two sporadic patients (trios); the reported clinical symptoms were early onset fever, failure to thrive, rash, joint pain, and elevated inflammatory markers, including hyperferritinemia (133). The two mutations map in a highly conserved HD1 region of the NLRC4 nucleotide-binding domain and may decrease the function of NLRC4 to maintain itself in an auto-inhibited state. A third mutation, p.H443P, was identified in a Japanese family with milder symptoms including cold induced rash, fever, and arthralgia, even if the patient has a different phenotype from those other two previously patients reported. Mutations are supposed to regulate the mutant proteins into a constitutively active state, with the influence of environmental factors such as cold and stress which act as trigger, causing inflammasome activation. Further studies are necessary to explore full spectrum of monogenic inflammasome-related diseases. Interestingly, the *NLRC4* gene (IPAF, CARD12) is supposed to initiate inflammation in response to bacterial ligands, such as flagellin (135, 136).

# Stimulator of Interferon Genes (STING)- Associated Vasculopathy with Onset in Infancy

In a trio with a patient affected by early onset symptoms of systemic inflammation, cutaneous rash, and pulmonary manifestations, and his unaffected parents, WES was performed, identifying a *de novo* mutation, p.N154S, in the TMEM173 gene; this gene encodes for a STING (138). Extending the analysis to other five sporadic cases of different ancestries but with similar phenotype led to the identification of missense mutations in the TMEM173 gene. Functional studies showed that a particular missense mutation, p.V155M, has been previously associated with a phenotypically different disease like systemic lupus erythematosus (139). Most of the mutations are located in the exon 5 of TMEM173 gene, which encodes for a domain important for the STING dimerization site. The second study highlights that in families with dominantly inherited traits, the possibility of reduced penetrance should not be ignored. Sting knockout mice are prone to viral infections, because of the lacking of the ability to upregulate IFN-beta (137). As a result, all these data support the evidence that TMEM173 associated mutations are gain of function (138, 139).

# TNFRSF11A-Associated Hereditary Fever Disease

Patients with this disease have a phenotype similar to those affected by TRAPS. In a single patient with complex phenotype including neonatal onset of systemic inflammation and congenital abnormalities, a *de novo* genomic duplication containing the TNFRSF11A gene was identified (140). The TNFRSF11A gene is one of the 30 genes in the 10-Mb genomic duplication. Another study, using a different approach based on candidate gene screening, identified two other patients (mother and daughter) with a novel heterozygous 1-bp deletion (p.Met416Cysfs\*110) in exon 9 of *TNFRSF11A* gene. The resulting protein lacks the C-terminal intracellular domain. As TNFRSF1A, also TNFRSF11A gene encodes for a protein member of the TNF-receptor superfamily. *TNFRSF11A* (RANK, PDB, ODFR) gene encodes a signaling receptor that functions in osteoclast differentiation and bone remodeling (141, 142). RANK-ligand (RANKL) mediates essential signal for osteclastogenesis (143).

Rank-deficient mice present osteopetrosis caused from a block in osteoclast differentiation and the lack of peripheral lymph nodes (144). The pathogenesis of this disorder is unclear; in fact, the *TNFRSF11A* gene duplication suggests a gain of function mutation, while the heterozygous deletion is more consistent with a haploinsufficiency or a dominant negative effect.

## TRNT1 Deficiency

Patients presenting with a variable phenotype of congenital sideroblastic anemia, B cell immunodeficiency, and developmental delay have been termed SIFD (145). SIFD is characterized by an early onset, with frequently associated neurological symptoms, and metabolic abnormalities. SIFD has been associated to AID because it is characterized with a pediatric onset with periodic fevers and gastrointestinal involvement with sideroblastic anemia. Genetic cause of SIFD has been found in the *TRNT1* gene in an autosomal recessive inheritance (146, 147). The *TRNT1* gene encodes the ubiquitously expressed CCA-adding enzyme, essential for template-independent maturation of nuclear and mitochondrial transfer RNAs (148). Functional studies in yeast showed deficiency of TRNT1 homolog causes partial loss of function of TRNT1 affecting variable degrees of enzyme activity (146). Knock-out yeast for *TRNT1* was fully restored with human TRNT1 and partially rescued by human mutant proteins.

## Monogenic Form of Systemic Juvenile Idiopathic Arthritis

Systemic-onset juvenile idiopathic arthritis is a polygenic inflammatory disease characterized by fever, rash, and symmetrical polyarthritis, with persistent systemic inflammation that seems to be linked to altered innate immune system (149, 150). Mendelian inheritance is autosomal recessive. Studying five consanguineous families with 13 affected patients from the Saudi Arabia with several genomic approaches, such as linkage analysis, homozygosity mapping, and WES, a homozygous missense mutation, p.C284R, located in exon 4 of the laccase domain containing 1 (LACC1) gene was identified (151). This private mutation is highly conserved during evolutionary scale and was not described in more than 2,000 Arab controls, suggesting its role in the pathogenesis.

Laccase domain containing 1 gene belongs to a family of Laccases, multi-copper oxidoreductases able to catalyze the oxidation of a variety of phenolic and non-phenolic compounds. Protein function is largely unknown, even if may regulate the innate immune responses. In a recent study, the gene product of LACC1 gene has been named FAMIN (fatty acid metabolism– immunity nexus), important for the synthesis of endogenous fatty acids and their mitochondrial oxidation, controlling the glycolytic activity and ATP regeneration (145).

Genetic variants in the LACC1 gene have been previously associated with susceptibility to leprosy (152–154). LACC1 gene belongs to a family of Laccases, multi-copper oxidoreductases able to catalyze the oxidation of a variety of phenolic and nonphenolic compounds. Protein function is largely unknown, even if may regulate the innate immune responses.

# GENETIC DIAGNOSIS WITH NEW TECHNOLOGIES

In recent years, with the increased use of instruments for NGS, it has become feasible to analyze several genes in a single experiment. This method is of great interest for AID, because of the increasing number of genes associated with the different forms of AID. In some cases, it is difficult to differentiate the different diseases, in particular, in those patients with intermediate phenotypes or a difficult clinical diagnosis. The analysis of a panel of several candidate genes is feasible with targeted sequencing investigating causative mutations, rare variants, or regions associated with the disease.

With the large number of genetic variants found with NGS, it is extremely important to find information about their possible role in the development of a specific disease. Several softwares have been developed in recent years and most of them have been based on the assumption that protein sequences derived from living organisms have survived a natural selection. The goal is to find if a genetic variant is a causative mutation or a common polymorphism.

There is also the possibility to analyze all the 18,000 genes of the human genome. WES is a recent strategy designed to sequence only the coding regions of the genome (which represents 1% of the human genome, about 30 megabases); this is an effective method alternative to whole genome sequencing, cheaper and less complicated in the bioinformatical/statistical analysis. Exons are generally short, functional sequences of DNA which represent the portion of the genome translated in protein. The WES has the potential to identify the coding variants responsible for both Mendelian and common diseases.

In recent years, with the use of WES, several undiagnosed Mendelian genetic conditions have been investigated in order to search the involved gene (155, 156). WES has been applied to the study of trios (unaffected parents and the sporadic case) and unrelated patients with phenotypic similarities. Identification of causal gene in a single sporadic patient can then be confirmed in other patients with similar phenotypes. The potential of such strategies is high in polygenic human disorders such as AID. The emerging genetic technologies complemented by the development of public databases of human variation can allow discovery of disease causal genes in sporadic and unrelated patients.

Whole genome sequencing allows to sequencing the entire genomic DNA, both chromosomal and mitochondrial. Unlike WES, this method allows the sequencing of both exons and introns, for a total amount of 3 Gigabases.

In a single center study (157), more than 2,000 diagnostic patients have been analyzed with Sanger sequencing for the NLRP3, MVK and TNFRSF1A genes, and other AID gene portions, failing to find mutations in 86% of samples. Possible explanations of this high failure are the restricted number of tested genes, clinical misdiagnosis, genetic heterogeneity, and/or a complex mode of inheritance. In order to improve the sensitivity of the genetic tests, 50 patients were re-analyzed with a gene panel of 10 genes for NGS procedure. The 10 genes were *MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2,* and *PSMB8* (157). In order to better understand the possible role of the detected variants, allele frequencies have been compared with those of 1,000 Genomes Project, and searching for a possible genotype–phenotype correlation. Some genes, such as *NOD2, LPIN2*, and *NLRP12*, showed a high frequency of genetic variants, which in theory may alter the clinical phenotype with mild or atypical symptoms. In the next future, NGS data combined with clinical information may help diagnosis for those patients with intermediate phenotype (156); in fact, the interaction between geneticists and physicians will allow to improve the diagnosis of AID patients (157).

#### REFERENCES

# CONCLUSION

In AID, genetic and environmental factors act modulating the clinical presentation of a specific disorder. The knowledge of the biological pathways at the basis of different AIDs is very important; the elucidation of these novel factors may have clinical relevance, because it may be included in genetic-risk modeling approaches. The genetic variants previously identified as playing a role in the same pathway represent new potential therapeutic targets. The new age of the *-omics* has allowed the improvement of the knowledge of AID. By means of genetic fine mapping, targeted sequencing, transcriptomics, proteomics, and metabolomics, physicians may improve treatment and therapy tailored on the single patient.

# AUTHOR CONTRIBUTIONS

DM contributed in conception or design of the work, drafting the article, and final approval of the version to be published. FB and PM contributed in drafting the article and final approval of the version to be published. AV contributed in drafting the article, critical revision of the article, and final approval of the version to be published. AP contributed in conception or design of the work, drafting the article, critical revision of the article, and final approval of the version to be published.

# FUNDING

Supported in part by the grant "A tailored approach to the immune-monitoring and clinical management of viral and autoimmune diseases" funded by the Regione Emilia-Romagna within the Programma di Ricerca Regione Università 2010–2012, in part by "Tailoring Rituximab treatment in ANCA-associated vasculitis; a genetic and an immunological approach" funded by the Regione Emilia-Romagna within the Programma di ricerca Regione-Università Giovani Ricercatori 'Alessandro Liberati' 2013 and by the "Fondazione Emma ed Ernesto Rulfo per la Genetica Medica" (Italy).

(Tunisia) in the first half of the 20th century. *Clin Exp Rheumatol* (2003) 21(30):S53–4.

of 103 patients with hyperimmunoglobulinemia D syndrome. *Medicine (Baltimore)* (2008) 87:301–10. doi:10.1097/MD.0b013e318190cfb7

fever. *J Allergy Clin Immunol* (2001) 108(4):615–20. doi:10.1067/mai.2001. 118790

incidence and prevalence. *J Invest Dermatol* (2013) 133:377–85. doi:10.1038/ jid.2012.339

gangrenosum and acne) syndrome with the recombinant human interleukin-1 receptor antagonist anakinra. *Br J Dermatol* (2009) 161(5):1199–201. doi:10.1111/j.1365-2133.2009.09404.x

specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1. *Hum Mol Genet* (2015) 24(10):2841–7. doi:10.1093/ hmg/ddv044

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Martorana, Bonatti, Mozzoni, Vaglio and Percesepe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Dysregulated iL-1**β** secretion in Autoinflammatory Diseases: A Matter of stress?

#### *Sonia Carta1†, Claudia Semino2,3†, Roberto Sitia2,3 and Anna Rubartelli1 \**

*1Cell Biology Unit, IRCCS AOU San Martino-IST, Genova, Italy, 2Unit of Protein Transport and Secretion, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy, 3Vita-Salute San Raffaele University, Milan, Italy*

Infectious and sterile inflammation is induced by activation of innate immune cells. Triggering of toll-like receptors by pathogen-associated molecular pattern or damage-associated molecular pattern (PAMP or DAMP) molecules generates reactive oxygen species that in turn induce production and activation of pro-inflammatory cytokines such as IL-1β. Recent evidence indicates that cell stress due to common events, like starvation, enhanced metabolic demand, cold or heat, not only potentiates inflammation but may also directly trigger it in the absence of PAMPs or DAMPs. Stress-mediated inflammation is also a common feature of many hereditary disorders, due to the proteotoxic effects of mutant proteins. We propose that harmful mutant proteins can induce dysregulated IL-1β production and inflammation through different pathways depending on the cell type involved. When expressed in professional inflammatory cells, stress induced by the mutant protein activates in a cell-autonomous way the onset of inflammation and mediates its aberrant development, resulting in the explosive responses that hallmark autoinflammatory diseases. When expressed in non-immune cells, the mutant protein may cause the release of transcellular stress signals that trigger and propagate inflammation.

Keywords: autoinflammatory syndromes, endoplasmic reticulum stress, IL-1**β**, inflammation, monocytes, NLRP3 inflammasome, oxidative stress, toll-like receptor

# INTRODUCTION

The term "autoinflammation" (1) groups syndromes with different etiologies characterized by systemic inflammation in the absence of detectable infections and/or autoimmunity. Autoinflammatory diseases are disorders of the innate immune system, sharing recurrent episodes of fever, rash, joint pain, neutrophilia, and increased inflammatory markers. Most of them are monogenic, and the causative gene relates to the innate immune system. Examples are MEFV/pyrin in familial Mediterranean fever (FMF), TNFRSF1A/TNF receptor type 1 in TNF receptor-associated periodic syndrome (TRAPS), and nucleotide-binding domain, leucine-rich-containing family, pyrin domaincontaining 3 (NLRP3) in cryopyrin-associated periodic syndromes (CAPS) (2).

#### *Edited by:*

*Alessandra Soriano, Università Campus Bio-Medico, Italy*

#### *Reviewed by:*

*Anja Schwenzer, Oxford University, UK Nicolas Riteau, National Institutes of Health, USA*

#### *\*Correspondence:*

*Anna Rubartelli<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 22 December 2016 Accepted: 10 March 2017 Published: 04 April 2017*

#### *Citation:*

*Carta S, Semino C, Sitia R and Rubartelli A (2017) Dysregulated IL-1β Secretion in Autoinflammatory Diseases: A Matter of Stress? Front. Immunol. 8:345. doi: 10.3389/fimmu.2017.00345*

**81**

**Abbreviations:** CAPS, cryopyrin-associated periodic syndromes; DAMPs, damage-associated molecular patterns; ER, endoplasmic reticulum; IL, interleukin; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domaincontaining 3; NOX, NADPH oxidases; PAMPs, pathogen-associated molecular patterns; ROS, reactive oxygen species; TLRs, toll-like receptors; TRAPS, TNFRSF1A/TNF receptor type 1 in TNF receptor-associated periodic syndrome; UPR, unfolded protein response.

The reversal of clinical symptoms in CAPS patients upon treatment with recombinant IL-1 receptor antagonist (*Anakinra*) or with IL-1β blocking agents (e.g., *Canakinumab*, a neutralizing antibody) provided compelling *ex adjuvantibus* evidence for the key role of IL-1β (3). The efficacy of anti-IL-1 drugs suggested that "gain-of-function" mutations in NLRP3, a central component of the inflammasome, cause uncontrolled IL-1β production, in turn responsible for the severe inflammatory symptoms (4, 5). Less expectedly, the same drugs displayed strong therapeutic effects also in autoinflammatory diseases, where the causative gene is not directly involved in IL-1β production and regulation (2, 3). A representative case is TRAPS, a disease characterized by recurrent episodes of long-lasting fever, pain, and fasciitis. Despite TRAPS is caused by mutations in p55 TNF receptor type I, patients showed no or modest response to TNFα inhibition (6), whereas IL-1β-blocking agents have high efficacy (7). These observations suggest that the presence of a mutated protein in inflammatory cells, independently from its function, activates mechanisms converging on dysregulated IL-1β secretion.

In this perspective article, we propose a pro-inflammatory role for cell stress and the responses it elicits in some hereditary diseases, and suggest that stress is a central player in the pathophysiology of autoinflammatory disorders, due to its presence in innate immune cells.

#### STRESS AND INFLAMMATION

Inflammation is traditionally defined as a reaction to infectious or sterile injuries, aimed at recruiting molecules and cells of the immune system to the tissue where the damage is taking place and restoring homeostasis. Inflammation is initiated by activation of pattern recognition receptors on inflammatory cells, by two subclasses of ligands responsible for infectious and sterile inflammation, respectively (8, 9): pathogen-associated molecular patterns and damage-associated molecular patterns (PAMPs and DAMPs). The former are part of pathogens, while the latter are components of cells or extracellular matrix released or degraded upon cell and tissue damage. Additional factors concur in determining the onset, duration, and intensity of inflammatory responses. Among these, particularly important is cell stress due to starvation, enhanced metabolic demand, cold or heat, altered proteostasis. The most common and well studied cell stresses are endoplasmic reticulum (ER) stress and oxidative stress that are counteracted by highly conserved responses. These responses share common traits, for example, eIF2α phosphorylation, with transient translational inhibition and transcriptional activation of chaperones and antioxidants (10). This integrated stress response prevents the toxicity caused by misfolded proteins [named "proteotoxicity" (10)] and limits reactive oxygen species (ROS)-based vicious circles. If excessive or prolonged, however, virtually all stress responses become maladaptive and induce inflammation due to activation of chemokine genes or, in case of cell damage, release of DAMPs that recruit inflammatory cells (11).

Oxidative stress is due to excessive production and/or deficient detoxification of ROS. These can be abundantly generated by mitochondria during oxidative phosphorylation (12) and by flavoenzymes like NADPH oxidases (NOX) (13). In cells of the innate immune system, phagocytosis and toll-like receptor (TLR) triggering activate NOX to produce abundant H2O2 (14). H2O2 is released into phagosomes to clear microorganisms and induces pro-inflammatory cytokines and inflammation: however, it may generate oxidative stress (13, 14). ROS are also produced in the ER as a by-product of oxidative protein folding, particularly in conditions of ER stress, which elicit the unfolded protein response (UPR) (15, 16). ER stress occurs when misfolded proteins accumulate in the secretory pathway, and also during infections, lipid unbalance, and other metabolic defects (15). UPR, a complex set of intracellular signaling pathways, has evolved to respond to protein misfolding and restore ER homeostasis. In addition, UPR signaling has a recognized role in immunity and inflammation (16). Oxidative and ER stresses are intimately linked: the former can induce misfolding of secretory proteins impacting disulfide bond formation. On the other hand, ER stress leads to ROS production (17). In concert with ROS, a prolonged UPR can induce NF-κB-mediated chemokine production and recruit inflammatory cells. In turn, PAMP or DAMP can potentiate the UPR (16).

These vicious circuits are evident in many chronic disorders such as type 2 diabetes (18), obesity (19), lung respiratory disease (20), inflammatory bowel disease (21), non-alcoholic fatty liver disease (22), and cancer (23).

Also in many hereditary diseases, the mutant protein may alter proteostasis: if this occurs, stress and inflammation are induced. For example, in cystic fibrosis, different mechanisms contribute to the inflammatory lung disease that is the major cause of morbidity and mortality in patients affected by this disease. Firstly, the mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein cannot fold properly into the ER lumen, causing accumulation of misfolded CFTR aggregates, ER stress, and UPR. In turn, UPR activates NF-κB inducing production of chemokines, such as IL-8, that recruit polymorphonuclear leukocytes (PMN). PMN increase the oxidative burden in the lung, with generation of ROS that amplify the production of IL-8 thus locally increasing PMNs (24). Moreover, upregulation of ROS inhibits autophagy with consequent accumulation of protein aggregates and lung inflammation (25). Finally, the mutant CFTR transporter is unable to channel antioxidants into the airways: oxidative stress is worsened and concurs to the hyperinflammatory phenotype (24).

In Duchenne muscle dystrophy, due to the defect of dystrophin, oxidative stress and UPR-activated NF-κB interactively promote fiber necrosis. Recruited macrophages generate inflammatory cytokines and ROS, thereby triggering vicious inflammatory waves (26, 27).

Differently from autoinflammatory disorders, in these cases, the mutant protein, being synthesized by epithelia or muscle, determines the release of stress signals that recruit leukocytes ultimately causing inflammation. These signals include small molecules like ROS and antioxidants, and proteins such as thioredoxin (28) and chemokines (as described above for cystic fibrosis, 24), which induce inflammation transcellularly, i.e., by recruiting and activating other cells (**Figure 1A**). When instead it is a professional inflammatory cell that produces a proteotoxic mutant, inflammation is generated in a cell-autonomous way and

the onset, development, and outcome of it will be much worse for the host (**Figure 1B**).

# STRESS IN CAPS MONOCYTES

This hypothesis is supported by the observation that monocytes from CAPS patients, which express mutated NLRP3 molecules, display redox distress even before PAMP stimulation. Why mutant NLRP3 causes stress is unclear. A possible explanation is that it changes the affinity for the other components of the inflammasome complex (4), causing a disruption of the cytosolic homeostasis with induction of stress and integrated stress responses (10). Whatever the reason of NLRP3-induced stress, CAPS monocytes have higher basal ROS levels than monocytes from healthy donors but also higher expression of antioxidant systems (29–31) that allow them to maintain the redox homeostasis despite their stressed state. This equilibrium is, however, precarious, and CAPS monocytes can easily be induced to overreact, through pathways that largely depend on extracellular ATP, the most common inflammasome-activating signal (32). ATP is released by injured tissues, activated platelets, and other cells through pathways that are still ill defined (32). Unlike other pro-inflammatory cells, however, human monocytes do not need ATP from external sources. The accumulation of ROS upon TLR triggering (33) induces them to secrete ATP (34) that autocrinally or paracrinally stimulates cognate purinergic receptors (P2X7R) at the cell surface (32, 34). The ensuing lower intracellular [K<sup>+</sup>] induces inflammasome assembly and IL-1β secretion (35). The higher ROS levels in CAPS monocytes following TLR triggering facilitate ATP release that increases and accelerates IL-1β secretion (31) (**Figure 1B**).

Cell stress also decreases the threshold for IL-1β processing and secretion: minute amounts of TLR agonists, that in healthy monocytes are sufficient to trigger pro-IL-1β synthesis but not its processing and secretion, drive large amounts of IL-1β release in CAPS monocytes (31). Probably owing to their "pre-activated state," small doses of TLR agonists increase ROS, inducing abundant ATP release, and IL-1β processing and secretion (31). This circuit explains why small traumas or infections that go undetected in healthy subjects can cause severe inflammatory manifestations in CAPS patients.

The effects described above occur soon after TLR stimulation. In later phases, the precarious redox equilibrium of CAPS monocytes is broken as antioxidant responses collapse. CAPS monocytes display damaged mitochondria (30), a further indication of the presence of oxidative stress [(12), **Figure 2**].

Interestingly, mitochondria are normal in CAPS lymphocytes, which do not express NLRP3, and in monocytes from healthy donors, which express wild-type NLRP3 (30), suggesting that mutant NLRP3 is indeed the causative agent of the oxidative stress. In this crucial phase, stress impacts also the production of IL-1Ra, normally secreted by activated monocytes a few hours after IL-1β to limit inflammation [(30), **Figure 1**]. Thus, deficient IL-1Ra production likely concurs in increasing the severity of the disease. Highlighting the dangerous stress-inflammation liaisons, insufficient IL-1Ra production may depend on eIF2α phosphorylation. Indeed, TLR-activated monocytes from CAPS patients, but not from healthy donors, display attenuated protein translation (30). Thus, IL-1ra mRNA is transcribed but stress prevents translation. Once more, oxidative and ER stress appear to be linked because IL-1Ra secretion is restored by antioxidants.

# HYPER-STIMULATED HEALTHY MONOCYTES RECAPITULATE THE BEHAVIOR OF CAPS MONOCYTES

The above observations suggest that the increased IL-1β/IL-1Ra ratio in CAPS depends on the synergistic effects of NLRP3 mutations and stress. Combinations of PAMPs that stimulate surface and intracellular TLRs (LPS, R848, zymosan) were then used to induce a CAPS-like stress state in healthy monocytes (36). When given alone, each TLR agonist triggered the secretion of IL-1β and IL-1Ra by healthy monocytes. When provided simultaneously, however, they induced a superstimulation resulting in enhanced secretion of IL-1β but impaired release of IL-1Ra (36). The underlying molecular mechanisms are similar to those described in CAPS monocytes (29–31): super-stimulation induces ROS accumulation, responsible of the massive ATP release and IL-1β secretion, and of the consequent oxidative stress leading to inhibition of IL-1Ra production, despite normal IL-1Ra mRNA levels. Antioxidants restore IL-1Ra release by super-stimulated healthy monocytes, confirming the role of oxidative stress and recapitulating the phenotype of CAPS monocytes (36). However, the latter are constitutively stressed by the mutation (37) so that stimulation with low doses of a single TLR agonist strongly increases stress that drives prompt and abundant IL-1β secretion and, in a second phase, lowers IL-1Ra (30, 31). In healthy monocytes with balanced basal redox state (29, 36), instead, multiple TLR co-stimulation is needed to cause cell stress and derange the normal cytokine network (36). These observations may suggest that, in CAPS, mutations in NLRP3 are more important indirectly, triggering and enduring stress, than directly activating inflammasome.

# DIFFERENT MECHANISMS FOR IL-1**β** SECRETION: DOES STRESS DETERMINE THE PATHWAYS OF SECRETION?

Since IL-1β is a potent and potentially dangerous mediator of inflammation, its production is tightly controlled virtually at all levels, including post-translationally (38, 39). IL-1β is synthesized as an inactive precursor, pro-IL-1β, and processed mainly by caspase-1, which in turn must be activated by the inflammasome. Only mature, 17 kDa IL-1β is then secreted. The underlying mechanisms are still poorly understood. Indeed, IL-1β secretion has been a problem for cell biologists, since it was shown that the cytokine lacks a secretory signal sequence (40). Initially, a popular view was that the cytokine was released by dying cells. However, further studies demonstrated that secretion of mature IL-1β is an active process, requires living cells, avoids the ER-Golgi route, and involves secretory lysosomes (41–44). In addition to this pathway, recent studies revealed another route for IL-1β release, involving pyroptosis. This is a highly inflammatory form of programmed cell death, which has been proposed to mediate IL-1β secretion under condition of strong stimulation such as infection with intracellular pathogens (45–48). According to this model of secretion, stressful stimuli (e.g., intracellular LPS) activate caspase-11 (the mouse homologous of human caspase-4). In turn, caspase-11 cleaves gasdermin D, generating toxin-like peptides that form pores on the plasma membrane, which allow secretion of mature IL-1β, but not of the 33 kDa precursor (48, 49). It remains to be determined how the pores guarantee transport selectivity.

The two pathways are not mutually exclusive, and the choice of lysosomal or pyroptotic secretion may depend on the strength of pro-inflammatory signals (**Figure 2**). Mild stimuli, such as low amounts of PAMPs triggering surface bound TLRs, would induce the less efficient but more regulated lysosomal pathway. Accordingly, low doses of LPS induce pro-IL-1β synthesis, but not ATP secretion (31): in the absence of a second trigger, therefore, pro-IL-1β is degraded by lysosomal proteases (31, 42) preventing unnecessary inflammation. Stronger stimuli, such as intracellular infections with gram-negative bacteria (45) could instead induce pyroptosis, causing massive release of IL-1β and possibly DAMPs, and dysregulated cytokine production (36).

Support to this hypothesis comes from our preliminary observations that human monocytes display more IL-1β-containing lysosomes when stimulated with LPS alone than with three agonists simultaneously triggering extra- and intracellular TLRs (unpublished results). Moreover, only in monocytes stimulated with extracellular LPS alone, do drugs interfering with lysosomal function modulate IL-1β secretion. Conversely, caspase-4 inhibitors block IL-1β release only in super-stimulated monocytes (unpublished results).

It is possible that the secretory lysosome-mediated mechanism is more active in low pathogen load or small trauma, as a way to restore the homeostasis. Differently, the pyroptosismediated secretion would intervene in severe inflammatory responses, characterized by strong or multiple stimuli such as it may occur in sepsis (50), diabetes (51) or cancer (52).

The ongoing stress could also determine the route of IL-1β secretion. Owing to the high ROS levels that favor ATP release, we predict that CAPS monocytes utilize preferentially the pyroptotic pathway. Accordingly, caspase-4 inhibition blocks IL-1β secretion by CAPS monocytes stimulated with a single TLR agonist, a condition that neither involves caspase-4 nor induces pyroptosis in healthy monocytes (unpublished) (**Figure 2**).

# CELL-AUTONOMOUS PROTEOTOXIC STRESS IN MONOCYTES INCREASES IL-1**β** SECRETION IN AUTOINFLAMMATORY DISEASES

Increased IL-1β secretion has been reported *in vitro* by monocytes from other autoinflammatory diseases, including FMF (53), TRAPS (54) hyperimmunoglobulinemia D syndrome (55), pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA) (56), and also in the milder NLRP-12-associated periodic syndrome (57). As introduced above, anti-IL-1β therapies are the standard of care in these syndromes (58), suggesting that IL-1β is a key culprit. Nonetheless, the links between the mutated gene and IL-1β secretion are elusive. Remarkably, in these diseases, the mutant genes are expressed by monocytes that are under stress (53, 57, 59–61). It is tempting to speculate that stress and the ensuing responses converge to induce excessive IL-1β secretion, possibly switching from lysosomal to pyroptotic secretion (**Figure 2**)*.* The consequences on disease severity are many, since pyroptosis-mediated secretion would alter the networks of proand anti-inflammatory cytokine production.

Stress-induced hyperinflammatory response may occur in other inherited diseases that are not (yet) classified as autoinflammatory diseases. This is the case of chronic granulomatous disease (CGD), a disorder linked to mutations in NOX2. Because of these mutations, phagocytes of CGD patients fail to produce ROS with consequent deficiency in bactericidal activity and increased susceptibility to infections (62). In addition, and consistent with the evidence that CGD is associated with increased inflammasome activation (63–65), patients often develop hyperinflammatory traits. Moreover, *Anakinra* induced significant clinical improvement in two cases with colitis (66). Thus, CGD was defined as a potentially lethal combination of immunodeficiency and excess inflammation (67), most likely due to cell-autonomous stress responses. Likewise, evidence is accumulating for a role of stress and inflammation in the pathogenesis of Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase (68). Monocyte/macrophages from these patients display increased secretion of IL-1β that depends on increased inflammasome activation, in turn due to the impaired autophagy secondary to the lysosomal enzyme deficiency (68). A further example is mucopolysaccharidosis type I, where, in innate immune cells, stress induced by lysosomal storage defects can upregulate immunity-related genes. In turn, these may be responsible for the severe inflammation-dependent pathologies observed in patients (69).

# CONCLUSION AND PERSPECTIVES

In essence, we propose that stress hallmarks monocytes from patients affected by autoinflammatory syndromes (and possibly other inherited diseases) that express mutant proteins not necessarily directly involved in IL-1β production. Stress induces inflammation and is, therefore, a key pathogenetic factor in these diseases. The stress levels contribute to determine the severity of the disease, and so do individual differences in resistance to oxidative stress, as proposed for chronic inflammation-mediated diseases such as diabetes (70). Accordingly, we showed clear correlations among basal stress, ongoing antioxidant responses, and disease severity in two CAPS patients sharing the same NLRP3 mutation (31). Extending these concepts, we suggest that a similar stress-related mechanism may be operative in other genetic diseases, where the mutant protein is present in monocytes and inflammation participates to disease progression. Considering that individual tolerance plays a major role (71), improving the responses to stress represents a promising therapeutic opportunity for these serious diseases.

# AUTHOR CONTRIBUTIONS

SC, CS, RS, and AR designed, wrote, and approved the final manuscript.

## REFERENCES

#### ACKNOWLEDGMENTS

The authors are grateful to Prof. M. E. Bianchi from Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy, for discussion and support and Dr. M. Gattorno from Pediatrics II Unit, G. Gaslini Institute, Genoa, Italy, for advice.

#### FUNDING

This study was supported in part by the Italian Ministry of Health ("Cinque per mille" and Ricerca Corrente to AR; PE-2011-02352286 to RS), by Telethon, Italy (grants no. GGP14144 to AR and GGP15059 to RS), by Associazione Italiana per la Ricerca sul Cancro (grants no. IG15434 to AR and IG18824 to RS), and Fondazione Cariplo (2015-0591 to RS).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Carta, Semino, Sitia and Rubartelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# NF-**κ**B Pathway in Autoinflammatory Diseases: Dysregulation of Protein Modifications by Ubiquitin Defines a New Category of Autoinflammatory Diseases

#### *Ivona Aksentijevich\* and Qing Zhou*

*Inflammatory Disease Section, National Human Genome Research Institute, Bethesda, MD, USA*

Autoinflammatory diseases are caused by defects in genes that regulate the innate immunity. Recently, the scope of autoinflammation has been broadened to include diseases that result from dysregulations in protein modifications by the highly conserved ubiquitin (Ub) peptides. Thus far these diseases consist of linear ubiquitin chain assembly complex (LUBAC) and OTULIN deficiencies, and haploinsufficiency of A20. The LUBAC is critical for linear ubiquitination of key signaling molecules in immune response pathways, while deubiquitinase enzymes, OTULIN and TNFAIP3/A20, reverse the effects of ubiquitination by hydrolyzing linear (Met1) and Lys63 (K63) Ub moieties, respectively, from conjugated proteins. Consequently, OTULIN or A20-deficient cells have an excess of Met1 or K63 Ub chains on NEMO, RIPK1, and other target substrates, which lead to constitutive activation of the NF-kB pathway. Mutant cells produce elevated levels of many proinflammatory cytokines and respond to therapy with cytokine inhibitors. Patients with an impairment in LUBAC stability have compromised NF-kB responses in non-immune cells such as fibroblasts, while their monocytes are hyperresponsive to IL-1β. Discoveries of germline mutations in enzymes that regulate protein modifications by Ub define a new category of autoinflammatory diseases caused by upregulations in the NF-kB signaling. The primary aim of this review is to summarize the latest developments in our understanding of the etiology of autoinflammation.

Keywords: TNFAIP3/A20, linear ubiquitin chain assembly complex, OTULIN, haploinsufficiency of A20, LUBAC deficiency, otulipenia/otulin-related autoinflammatory syndrome

## INTRODUCTION

Autoinflammatory diseases are a diverse group of inherited conditions characterized by early-onset systemic inflammation and are accompanied by a range of organ-specific manifestations. The genetic etiology involves abnormalities in molecules such as inflammasomes, cytokine inhibitors, cytokine receptors, enzymes, and proteasome complex. The excessive secretion of proinflammatory cytokines can lead to chronic morbidity and may be life-threatening. Therapies with cytokine inhibitors are efficacious in most patients; however, considering the high cost of biologics there is a need to develop more affordable treatment options for these lifelong conditions. The discovery of germline mutations

#### *Edited by:*

*José Hernández-Rodríguez, Hospital Clinic of Barcelona, Spain*

#### *Reviewed by:*

*Jordi Yagüe, Hospital Clinic of Barcelona, Spain Kyeong Kyu Kim, Sungkyunkwan University, South Korea*

> *\*Correspondence: Ivona Aksentijevich<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 22 December 2016 Accepted: 21 March 2017 Published: 19 April 2017*

#### *Citation:*

*Aksentijevich I and Zhou Q (2017) NF-κB Pathway in Autoinflammatory Diseases: Dysregulation of Protein Modifications by Ubiquitin Defines a New Category of Autoinflammatory Diseases. Front. Immunol. 8:399. doi: 10.3389/fimmu.2017.00399*

linked to autoinflammation offer an opportunity to search for new therapeutic targets.

# UBIQUITIN (Ub) PATHWAY

Posttranslational protein modification by ubiquitination (also known as ubiquitylation) is critical for the regulation of many biological processes including DNA repair, endocytosis, transcription, protein degradation, and preservation of cellular homeostasis. Ubiquitination involves the attachment of evolutionarily conserved 76-aa Ub molecules to target proteins in the form of a monomer or polymers (Ub chains). The type of conjugation determines the fate of the modified protein by directing protein localization and regulating protein interactions, activity, and degradation (1).

The ubiquitination process is initiated by the attachment of a single Ub molecule to a target protein through a three-step enzymatic pathway that includes Ub-activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub-ligating enzymes (E3) (2). Ub chains are generated by the sequential addition of Ub monomers through one of seven lysine residues that serve as a linker, thus there are seven types of Ub Lys-linkages. Ub can be also conjugated through an N-terminal methionine residue (Met1 linkage; linear linkage). Lys11-, Lys48-, Lys63-, and Met1 linked chains are the best known and most studied. Significance of four other Ub chains Lys6-, Lys27-, Lys29-, and Lys33- is poorly understood. In addition, there is increasing evidence that more than one linkage type can exist on modified proteins either in the form of mixed (hybrid) Ub chains or branched Ub chains, and this may provide a new layer of complexity to the Ub-mediated modifications (2, 3). Ub proteins are also subject to modifications by acetylation, phosphorylation, and ubiquitin-like (UBL) molecules (SUMO or NEDD8) (4). Ub-conjugated proteins are recognized by Ub sensors, Ub-binding proteins (Ub receptors) that can translate each linkage type of Ub modifications into specific functional outcomes. For example, proteins conjugated with Lys (K11) or Lys48 (K48) Ub chains are targeted for proteosomal degradation *via* the ubiquitin–proteasome system (UPS) (5). In addition, Lys11 Ub chains have a role in cell cycle control and may have other functions in the context of mixed Ub chains (6). Lys63 (K63) Ub chains are involved in cell signaling and are essential for DNA damage response (7). Linear (Met1) Ub chains regulate a wide range of immune signaling pathways (8–10). Ubiquitination is a highly dynamic and reversible process whereby Ub chains are removed from modified substrates by a class of enzymes known as deubiqutylases or deubiquitinases (DUBs) (11). There are close to 100 proteases that have DUB activity with different degrees of specificity for Ub linkages. Several DUBs, including A20, OTULIN, CYLD, and Cezanne, function as negative regulators of NF-kB signaling (12).

Alterations in various components of the Ub–proteasome machinery have been linked to many human conditions including immune diseases. Recently, deregulations in the UPS were reported in patients with autoinflammatory disorders including chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (13), linear ubiquitin chain assembly complex (LUBAC) deficiency (14, 15), haploinsufficiency of A20 (HA20) (16), and otulipenia/otulin-related autoinflammatory syndrome (ORAS) (17, 18). This review will primarily focus on two diseases caused by malfunction in DUB enzymes, TNFAIP3/ A20 and OTULIN, which are known to hydrolyze Lys63- and Met1-linked Ub chains, respectively. In both conditions, HA20 and otulipenia/ORAS, a defect in DUB activity results in excessive ubiquitination and increased activity of key signaling molecules in the canonical NF-kB pathway. LUBAC-associated diseases will be briefly discussed in the context of LUBAC–OTULIN interactions.

# LINEAR Ub CHAINS IN IMMUNE SIGNALING

Linear ubiquitin chain assembly complex (LUBAC)-mediated Met1 ubiquitination has emerged pivotal for regulation of innate and adaptive immune responses and regulation of cell death (9, 19). The E3 ligase complex, LUBAC, has been shown to maintain the stability of the TNF receptor 1 (TNFR1), TLRs, IL-1R, CD40, RLR, and inflammasome receptor signaling complexes (RSCs). Upon stimulation with proinflammatory signals, LUBAC is recruited to attach linear Ub chains to target substrates such as IKK (NEMO), RIPK1, RIPK2, IRAKs, MyD88, and ASC (8, 20, 21). Attachment of linear Ub chains is critical for the assembly of RSCs. LUBAC depletion leads to attenuation of NF-kB and the mitogen-activated protein kinases (MAPK)-mediated signaling and increases cell death.

Linear ubiquitin chain assembly complex consists of HOIP (HOIL-1 interacting protein; *RNF31*) and two accessory proteins: HOIL-1 (heme-oxidized IRP2 ubiquitin ligase 1; *RBCK1*) and SHARPIN (SHANK-interacting protein like 1; *SIPL1*) (**Figure 1**). The catalytic subunit HOIP is auto-inhibited until the complex is fully assembled. Depletion of any subunits greatly reduces the stability of LUBAC in cells (14, 15). As the immune responses must be tightly regulated to avoid chronic inflammation, LUBAC activity is counter-regulated by the specific DUB OTULIN (also known as *gumby*; *FAM105B*).

OTULIN is a highly conserved protease with a specific activity to hydrolize linear (Met1)-linked Ub chains (22) (**Figure 1**). OTULIN interacts with the N-terminal PUB domain of HOIP *via* an evolutionarily conserved PUB-interacting motif (23). Loss of HOIP–OTULIN interaction reduces OTULIN capacity to restrict LUBAC-induced NF-kB activation (24). A recent study showed that the activity of LUBAC is also negatively regulated by its interaction with tumor necrosis factor receptor-associated factor 1 (TRAF1). TRAF1 directly interacts with LUBAC to interfere with the activation of IKK/NEMO. Reduced expression of TRAF1 could explain the association of susceptibility alleles in *TRAF1* with rheumatoid arthritis (RA) and other autoimmune diseases (25).

The importance of the linear ubiquitination in regulation of inflammatory pathways has been demonstrated in murine models. Genetic ablation of the catalytic HOIP subunit results in embryonic lethality at day 10.5 due to TNF-mediated endothelial cell death and vascular abnormalities (26). Mice deficient for non-catalytic subunits have variable degrees of inflammation manifesting with chronic proliferative dermatitis in the case

TNF receptor 1; TRADD, TNFR1-associated death domain protein; RIPK1, the death domain-containing protein kinase receptor-interacting protein1; IKKγ/NEMO, inhibitor of nuclear factor kappa B kinase subunit gamma.

of SHARPIN-deficient mice (*cpdm*) (27, 28) or with no overt inflammation in mice lacking HOIL-1 (21). Skin inflammation in Sharpin-KO mice is largely dependent on the TNFR1-induced apoptosis (19, 29, 30). LUBAC activity is also important for proper B and T cell development, activation, and maintenance of adaptive immune responses (31, 32). Genetic loss of *Otulin* (*gumby*/*gumby*) causes embryonic lethality (E12.5–E14) due to compromised angiogenesis and defects in neuronal development (33). Tamoxifen-induced *Otulin* deficiency in immune cells results in an acute severe multiorgan inflammatory phenotype (18). Targeted ablation of *Otulin* in myeloid cells leads to chronic inflammation with features of autoimmunity, while *Otulin* deficiency in adaptive immune cells does not produce overt phenotype (18). Together, these data show critical and cell-specific function of OTULIN in the maintenance of immune homeostasis.

## LUBAC DEFICIENCIES

Patients with defects in the LUBAC components develop immunodeficiency, autoinflammation, muscular amylopectinosis, and die in early childhood (**Tables 1** and **2**). HOIL-1 and HOIP deficiencies are recessively inherited diseases caused by mutations

#### TABLE 1 | Comparison of genetics and mechanisms of disease in otulipenia, haploinsufficiency of A20 (HA20), and linear ubiquitin chain assembly complex (LUBAC) deficiencies.

that either create truncating proteins or affect a highly conserved PUB domain of HOIP (14, 15) (**Figure 2**). Pathogenic mutations in one subunit destabilize the expression of the entire LUBAC complex. As LUBAC is important for activation of immune signaling, stimulated patient fibroblasts and B cells fail to upregulate NF-kB activity, which manifests in recurrent bacterial infections. In contrast to immunodeficient fibroblasts, peripheral blood mononuclear cells (PBMCs) of HOIP and HOIL-1-deficient patients are highly responsive to IL-1 stimulation and produce high levels of proinflammatory cytokines IL-6 and MIP-1α. One patient was noted to have severe T cell lymphopenia and impaired antibody production. Muscular amylopectinosis/myopathy appear to be unrelated to a defect of linear ubiquitination in immune cells and its mechanism remains to be investigated. Abnormalities in the lymphatic system have been observed in the HOIP-deficient patient and HOIP-deficient mice, which suggests TABLE 2 | Clinical manifestations in patients with otulipenia, haploinsufficiency of A20 (HA20), and linear ubiquitin chain assembly complex deficiencies.

Aksentijevich and Zhou

(*Continued*)

that HOIP, independently of other LUBAC components, may have a function in the regulation of angiogenesis. LUBAC appears to be important for toll-like receptor 3 (TLR3)-mediated innate immune responses to influenza A virus infection, by enabling TLR3-mediated activation of NF-kB, MAPK, and IRF3 (34). In summary, the identification of patients with LUBAC deficiencies showed the critical role of LUBAC in regulation of

# OTULIPENIA/ORAS

immune responses.

Recessively inherited loss-of-function mutations in the linear (Met1)-specific DUB OTULIN have been linked to the earlyonset severe inflammatory disease, named otulipenia/ORAS (**Table 1**) (17, 18). Patients present with prolonged recurrent fevers, joint swelling, GI inflammation/diarrhea, and failure to thrive (**Table 2**; **Figure 3**). The cutaneous manifestations include painful erythematous rash with skin nodules, lipodystrophy, and episodes of pustular rash in one patient (**Figure 3B**). Skin biopsy showed evidence for neutrophilic dermatitis, mixed type panniculitis, and vasculitis of small and medium-sized blood vessels (**Figure 3C**) (17). In contrast to patients with the LUBAC deficiency, OTULIN-deficient patients have no obvious immunodeficiency, although some of them suffered from iatrogenic infections induced by immunosuppressive therapies. Initial analyses showed adequate specific antibody responses to vaccines, adequate T and B cell proliferative responses, normal levels of immunoglobulins, and normal T, B, and NK cell counts.

The four patients identified carry novel homozygous mutations in the *FAM105B* gene that encodes OTULIN. Heterozygote carriers are asymptomatic, which suggests that reduced protein expression of OTULIN may not be critical for maintenance of immune homeostasis. OTULIN is a 352-residue protein that consists of N-terminal LUBAC-binding domain and C-terminal ovarian tumor (OTU) domain. Disease-associated mutations affect the OTU domain and binding of OTULIN to linear Ub chains (**Figure 2**).

OTULIN functions as a negative regulator of the canonical NF-kB pathway and as such is essential for resolving inflammation (**Table 1**; **Figure 1**). Mutant OTULIN proteins cannot restrict the accumulation of Met1 Ub chains on target substrates IKK/ NEMO, RIPK1, and ASC. Patients' mononuclear leukocytes and fibroblasts have a strong inflammatory signature as evidenced by increased degradation of IkBα and increased phosphorylation of IKKα/IKKβ and IkBα, the hallmarks of the activated NF-kB pathway. Mutant cells overproduce many proinflammatory cytokines, including cytokines associated with activation of adaptive immune cells, and therapy with TNF inhibitors is very effective in controlling disease activity. Tamoxifen-induced *Otulin* ablation in murine immune cells (CreERT2-*OtulinLacZ*/flox chimeras) resembles the phenotype described in patients with otulipenia/ORAS including responsiveness to therapy with TNF inhibitors (18).

In summary, combined data from human and murine studies suggest that deficiency of the linear DUB, OTULIN, leads to amplification of the Met1 Ub regulated signaling in the canonical NF-kB pathway, most notably in myeloid cells.

repeat sequence (reported as c.1245\_1248del) should be assigned as the most 3′ position in the repeat, i.e., c.1249\_1252del; therefore, the proposed nomenclature for the mutation is p.Lys417Serfs\*4.

Frontiers in Immunology | www.frontiersin.org

mediated by the OTU domain, and the ZnF domains mediate A20 ubiquitin (Ub) E3 ligase activity, binding to Lys63-linked Ub chains and dimerization. Mutations reported by Zhou at al. are shown on the top of the diagram, while three mutations reported in Japanese patients are shown bellow the diagram. To stay consistent with the Human Genome Variation Society nomenclature the reported p.Gln415fs (35) should be described as p.Lys417Serfs\*4. The four nucleotide deletion in the

(A) Prominent fat loss (lipodystrophy) and (B) erythematous skin lesions and subcutaneous nodules in a patient with otulipenia. (C) Skin biopsy showed dense inflammatory infiltrate throughout the subcutaneous lobules (right upper corner of the image) and subcutaneous lobular atrophy or lipodystrophy (similar findings have been reported in chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature patients) on the left lower corner of the image. The middle part of the image showed vasculitis affecting a medium-sized artery, characterized by dense intramural and perivascular inflammation with endothelial proliferation and vascular occlusion. Adjacent the affected artery is a medium-sized vein (left lower) showing mild inflammation of the vessel wall. (D) Dermal abscesses in a patient with HA20. (E) Recurrent aphthous (oral) ulcers in a patient with HA20.

#### Lys63-LINKED Ub CHAINS IN IMMUNE SIGNALING

Lys(K)63-Ub modification was first described as a mechanism for the activation of the canonical NF-κB pathway (38). K63 linked Ub chains are generated following cell stimulation with inflammatory cytokines, and they function as a scaffold for the formation of receptor signaling complexes. Molecules such as NEMO/IKK, TRAF6, and RIPK1 are ubiquitinated both by linear and K63 Ub chains, which suggest a substantial regulatory redundancy in immune signaling (39–42). TNFAIP3/A20 restricts inflammatory responses *via* its dual yet synergistic functions: its deubiquitinase activity by hydrolyzing K63 linkages and its E3 ligase activity by conjugating substrates with K48 Ub chains to target them for proteasomal degradation (43, 44) (**Figure 1**). A20 is also subject to regulations as it undergoes posttranslational phosphorylation (45) and is cleaved by mucosa-associated lymphoid tissue lymphoma translocation protein 1 (46). In tumor cells, A20 acts as a tumor-suppressor gene and is frequently inactivated by somatic mutations and deletions in diffuse large B-cell and Hodgkin lymphomas (47, 48).

The *TNFAIP3* gene is highly conserved and intolerant to genetic variations, in particular to loss-of-function mutations. Common low-penetrance mostly non-coding variants in *TNFAIP3* have been associated with many autoimmune diseases including systemic lupus erythematosus (SLE) (49–51), RA (52), psoriasis (53), type 1 diabetes (54), celiac disease (55), coronary artery disease (56), inflammatory bowel disease (57), and more recently with protection to allergy and asthma (58, 59). Given the potent anti-inflammatory function of A20, these susceptibility alleles are predicted to decrease A20 expression and function, although that has been experimentally demonstrated only for a single non-coding variant associated with SLE. The dinucleotide functional variant downstream of *TNFAIP3*, TT > A, likely alters the binding of transcription factors in response to proinflammatory signals (51, 60).

Genetic ablation of A20 leads to spontaneous inflammation in mice with a range of cell-specific phenotypes. A20-deficient (A20<sup>−</sup>/<sup>−</sup>) mice exhibit multiorgan inflammation, cachexia, and early lethality (61). Although A20 was initially described as required for termination of TNF-induced signals, the excessive inflammation observed in double-deficient mice, A20-TNF or A20-TNFR1, suggested that A20 might be critical for the regulation of TNF-independent signals including the termination of TLR-induced activity of NF-kB (62). Cell-specific deletions of A20 resemble human autoimmune diseases, from a mild autoimmune phenotype in *Tnfaip3 Cd19* (B-cell) KO mice to severe spontaneous inflammation in mice with A20-deficient dendritic cells. Loss of A20 in macrophages mimics human RA, although the phenotype appears to be TNF-independent. Deficiency of A20 in keratinocytes leads to hyperkeratosis, while loss of A20 in intestinal epithelial cells causes DSS-induced TNF-dependent colitis (63). Aging heterozygous mice (A20+/−) develop spontaneous autoantibodies. In summary, multiple murine models with cell-specific ablation of A20 demonstrate closer approximation of human diseases than the complete knock-out mice.

This past year, Zhou et al. reported 11 patients from 6 families with a new dominantly inherited autoinflammatory disease, termed haploinsufficiency of A20, characterized by childhood-onset episodic fevers, arthralgia/arthritis, oral and/or genital ulcers, skin pathergy, GI, and ocular inflammation (16) (**Table 2**; **Figures 3D,E**). These symptoms resemble Behcet's disease (BD). One patient was initially diagnosed with SLE and presented with CNS vasculitis and idiopathic thrombocytopenic purpura. Subsequently, two families of Japanese ancestry diagnosed with entero-BD and one Japanese patient diagnosed with autoimmune lymphoproliferative syndrome (ALPS) were reported to carry novel LOF mutations in the gene (**Figure 2**) (35–37). A patient with ALPS presented with fevers, bilateral cervical lymphadenopathy, extensive skin rash, and massive hepatosplenomegaly. The patient's immunophenotyping revealed an increased percentage of DNT cells and a decreased number of IgM memory B cells, which is characteristic of ALPS. However, unlike in ALPS the patient's central memory (TCM), naïve, TEMRA, and effector memory (TEM) subpopulations of CD3+CD8<sup>+</sup> cells were normal (35).

TNFAIP3/A20 is a 790-residue protein that consists of an amino-terminal OTU followed by seven zinc finger (ZnF) domains. HA20-associated mutations create truncated mutant proteins, and most of them are located in the OTU domain (**Figure 2**). In addition to its OTU domain-mediated DUB activity, A20 can downregulate IKK activation by blocking IKK phosphorylation (64). Two pathogenic mutations have been identified in the ZnF domains 1 and 4. The ZnF4 domain is essential for A20 E3 ligase activity and dimerization (65).

Similar to patients with otulipenia/ORAS, mutant A20 cells have enhanced NF-kB activity as demonstrated by increased phosphorylation of IKKα/β and increased degradation of IkBα. Stimulated patient PBMCs and fibroblasts failed to hydrolyze K63 Ub chains from NEMO/IKK, RIPK1, and TNFR1 (16). Accumulation of K63 Ub proteins on these molecules triggers activity of the NF-kB and the MAPK pathways (**Table 1**; **Figure 1**). In murine models, A20/Tnfaip3 was shown to downregulate the activity of NLRP3 inflammasome (40, 66). Zhou et al. demonstrated constitutive NLRP3 activity in PBMCs of HA20 patients. Stimulated PBMCs and serum samples of HA20 patients have highly elevated levels of many proinflammatory cytokines produced by myeloid cells (IL-1, TNF, IL-6, IL-18, and IP-10) and T cells (IL-9, IL-17, and IFNγ). Therapies with cytokine inhibitors, anti-TNF, or anti-IL-1, have been very efficient in suppressing systemic inflammation.

Taken together, human genetic studies and murine models of A20 deficiency provide strong evidence that the reduced expression of A20 is associated with a range of inflammatory phenotypes.

# CONCLUSION

Maintenance of immune homeostasis is a highly balanced act that requires coordinated action of many proteins to allow optimal and efficient immune responses. Discovery of otulipenia/ORAS, HA20, and LUBAC-associated diseases has reiterated the importance of ubiquitination in regulation of immune signaling and revealed cell-specific functions of these proteins.

Despite similar function of OTULIN and A20 in restricting immune responses, the DUB activity of A20 appears to be less critical than the one of OTULIN (22). This may explain a milder inflammatory phenotype in HA20 than in patients with otulipenia. In addition, patients with otulipenia have more profound protein deficiency (less than 50%) than the patients with HA20 who retain one functional allele of the gene (50% protein deficiency).

Given the importance of the ubiquitination in cellular physiology, the UPS system has elicited a significant interest for drug development. The list of human diseases related to abnormalities in UPS has been steadily increasing and includes neurodegenerative diseases, cancer, and immune diseases. Ub-mediated protein degradation is critical for homeostasis in aging neuronal cells (67). Deficiency of A20 has been linked to lymphomas, and its most aggressive subtype is associated with constitutive activation of NF-kB. Reconstitution of A20 in mutant cell lines induced apoptosis and suppressed tumor growth (47). In immune diseases, A20 and OTULIN might be new therapeutic targets for development of immunomodulatory drugs that can potentially increase or stabilize their expression.

A key challenge for finding effective drugs will be in developing cell-based therapies. The ubiquitination process is regulated at multiple levels: generation, recognition, and removal. Targeting more components of the Ub–proteasome pathway may provide new opportunities for therapeutic exploitation and drug discovery (68, 69).

# AUTHOR CONTRIBUTIONS

IA reviewed the literature and wrote the manuscript. QZ reviewed the literature to make figures and tables and helped with writing the manuscript.

# ACKNOWLEDGMENTS

We would like to thank Dr. C. Richard Lee from National Cancer Institute for his help with interpretation of the dermal histology slide and Ms. Julia Fekecs from NHGRI for her help with the figures.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer, JY, and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2017 Aksentijevich and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# CANDLe Syndrome As a Paradigm of Proteasome-Related Autoinflammation

#### *Antonio Torrelo\**

*Department of Dermatology, Hospital Infantil del Niño Jesús, Madrid, Spain*

CANDLE syndrome (*C*hronic *A*typical *N*eutrophilic *D*ermatosis with *L*ipodystrophy and *E*levated temperature) is a rare, genetic autoinflammatory disease due to abnormal functioning of the multicatalytic system proteasome–immunoproteasome. Several recessive mutations in different protein subunits of this system, located in one single subunit (monogenic, homozygous, or compound heterozygous) or in two different ones (digenic and compound heterozygous), cause variable defects in catalytic activity of the proteasome–immunoproteasome. The final result is a sustained production of type 1 interferons (IFNs) that can be very much increased by banal triggers such as cold, stress, or viral infections. Patients start very early in infancy with recurrent or even daily fevers, characteristic skin lesions, wasting, and a typical fat loss, all conferring the patients a unique and unmistakable phenotype. So far, no treatment has been effective for the treatment of CANDLE syndrome; the JAK inhibitor baricitinib seems to be partially helpful. In this article, a review in depth all the pathophysiological, clinical, and laboratory features of CANDLE syndrome is provided.

#### *Edited by:*

*José Hernández-Rodríguez, Hospital Clinic of Barcelona, Spain*

#### *Reviewed by:*

*Maryam Piram, CHU de Bicêtre, France Karoline Krause, Charité Universitätsmedizin Berlin, Germany*

#### *\*Correspondence:*

*Antonio Torrelo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 05 February 2017 Accepted: 20 July 2017 Published: 09 August 2017*

#### *Citation:*

*Torrelo A (2017) CANDLE Syndrome As a Paradigm of Proteasome-Related Autoinflammation. Front. Immunol. 8:927. doi: 10.3389/fimmu.2017.00927*

Keywords: CANDLE, neutrophilic dermatosis, proteasome, immunoproteasome, interferonopathy, autoinflammation

# DEFINITION

CANDLE is an acronym standing for *C*hronic *A*typical *N*eutrophilic *D*ermatosis with *L*ipodystrophy and *E*levated temperature (1–3). CANDLE syndrome is an autoinflammatory disease (AID) characterized by the appearance of recurrent fever in the first months of life, along with characteristic skin lesions, lipodystrophy, and manifestations of multisystem inflammation. Mutations in different genes encoding protein subunits of the proteasome–immunoproteasome system are the cause of CANDLE syndrome.

# HISTORY

CANDLE syndrome was described in 2010 by Torrelo et al. (1). They reported on four children collected in two centers in Madrid and Chicago, two of whom were siblings, who showed striking skin lesions which on histopathology displayed an infiltration of immature, myeloid, mononuclear cells, resembling leukemia cutis. Because in many parts of the skin biopsies there was some maturation into polymorphonuclears and karyorrhexis, a type of yet undescribed "neutrophilic dermatosis" was suspected. The skin lesions had appeared very early in infancy, in attacks that occurred after common triggers (especially cold and viral infections), but some degree of skin involvement was present all the time. Furthermore, the patients had recurrent, almost daily, fevers or temperature elevations below 38.3°C, also since very early in life. The disease seemed to cause some general growth delay, and after more than 10 years of follow-up most patients looked wasted, with a striking loss of fat. With all these features, the chronic eruption with skin neutrophilic and mononuclear immature infiltration, the fevers and the lipodystrophy, an acronym was coined. The dermatological aspects were emphasized in this description, because they had been the most constant features, but it was recorded that the patients had suffered unexplained inflammatory attacks in many different organs of the body, such as the central nervous system (CNS), cartilage, joints, testes, and many others. One of the patients died of an attack of "carditis," but autopsy was not performed.

Also, in 2010, Garg et al. (4) reported three adult patients with a disease they named JMP (*J*oint Contractures, *M*uscle atrophy, microcytic anemia, and *P*anniculitis-induced lipodystrophy syndrome). The authors emphasized the lipodystrophic features that were related to panniculitis, and the joint features, but did not mention on the skin manifestations of the disease. However, they anticipated that their patients would suffer a disease of the innate immune system.

A similar constellation of signs had been reported in the Japanese literature. It had been reported by Nakajo in 1939 and Nishimura in 1950, under the names "secondary hypertrophic osteoperiostosis with pernio," a syndrome with nodular erythema, elongated and thickened fingers, and emaciation, and "hereditary lipomuscular atrophy with joint contracture, skin eruptions and hyper-γ-globulinemia" (5). An eponym for the syndrome was proposed by the Japanese authors as "Nakajo–Nishimura syndrome." Overall, patients described started in early infancy with a pernio-like rash, periodic high fever, nodular erythemalike eruptions, and myositis. Lipoatrophy and joint contractures occurred progressively in life, mainly on the upper part of the body, leading to a very characteristic facial appearance.

After the description of CANDLE and JMP, and the appearance of new cases from Japan, a number of cases from different parts of the world were published. It was later disclosed that most patients described under these descriptions had homozygous or compound heterozygous mutations in the gene *PSMB8*, encoding the subunit β5i of the immunoproteasome (2, 6–8). However, some patients with CANDLE syndrome did not bear mutations in *PSMB8* (2). Further genetic studies disclosed that some patients have homozygous and compound heterozygous mutations in other subunits of the proteasome–immunoproteasome, as well as digenic heterozygous mutations in two different genes encoding subunits (9). Finally, mutations in the proteasome maturation protein (*POMP*) gene have also been reported in a patient of Lebanese origin that had been reported by Mégarbané et al. (9, 10) in 2002 under the name "unknown autoinflammatory syndrome associated with short stature and dysmorphic features."

Several terms have been proposed to encompass the three denominations, such as PRAAS (proteasome-associated autoinflammatory syndrome) (11) or ALDD (autoinflammation, lipodystrophy, and dermatitis) (12). However, all of them represent the same entity, and it seems that the most accepted nomenclature is CANDLE syndrome. The general appearance of consumption in the patients, like a burntout candle, emphasizes the name of the syndrome.

# PATHOPHYSIOLOGY

Overall, proteasome–immunoproteasome dysfuntion causes a continuous state of inflammation with exacerbations in CANDLE syndrome. Proteasome–immunoproteasome dysfunction leads to constitutional hypersecretion of type 1 Interferons (IFNs), which by several mechanisms will lead to accumulation of waste proteins within the cells (13). Thus, further proteasome– immunoproteasome activity is required, which cannot be achieved. Accumulation of waste proteins within the cell causes cellular stress, which in turn stimulates type 1 IFN, and finally closes the circle of inflammation. Common triggers, such as cold, physical or psychical stress, banal infections or others cause stimulation of type 1 IFN secretion, and thus provoke severe inflammatory attacks that can occur in any organ of the body.

## The Proteasome–Immunoproteasome System

The proteasome is a multiprotein structure present both in the nucleus and the cytoplasm of all eukaryotic cells (**Figure 1**) (14). It is constitutively expressed, and has multicatalytic activity. It has a cylindrical shape, and there are several types. All of them contain at least two different complexes: the 20S or core complex, which contains the proteolytic activity, and the 19S or regulatory complex, responsible for recognizing ubiquitinated proteins and transporting them into the 20S complex. The 20S complex is composed of two α-rings flanking two β-rings; each ring is composed of seven different protein subunits, named α-1 to α-7 and β-1 to β-7.

The immunoproteasome is very similar to the constitutive proteasome (**Figure 1**). The 19S complex of the proteasome is replaced with the PA289 (or 9S) complex, and the β1, β2, and β5 subunits in the beta rings are substituted by specialized units, named β1i, β2i, and β5i (i = inducible) (14). The β1 subunits have caspase-like activity, β2 subunits have trypsin-like activity, and β5 subunits have chemotrypsin-like activity (9). Immunoproteasome formation is mainly induced by IFNs, to account for an increased demand of catalytic activity within the cell.

The assembly and maturation of proteasome and immunoproteasomes are governed by facilitating proteins. POMP is essential for proteasome formation and is strongly involved in the incorporation of the β5i subunit into the immunoproteasome. Proteasomal β subunits but β3 and β4, are synthesized as preforms that require autocatalytic cleavage during assembly to liberate the active-site threonines (9, 15, 16).

The proteasome is mostly involved in the removal of waste intracellular proteins. The immunoproteasome is also responsible for the degradation of foreign proteins. Whereas the proteasome is constitutively expressed in every cell, only proinflammatory cytokines and mostly type 1 IFNs induce the immunoproteasome formation. However, immunoproteasomes are constitutively expressed in hematopoietic cells (17, 18). The catalytic activity of the proteasome and the immunoproteasome cleaves protein substrates to generate smaller peptides that can be easily removed

from the cell or may act as antigens that can be presented through the MHC type I molecules to the adaptive immune system. In this way, more specifically the immunoproteasome acts as a link between innate and adaptive immunity.

(core) immunoproteasome.

# Normal Functioning of the Proteasome– Immunoproteasome System

Cellular proteins destined for degradation or cleavage should be first marked with ubiquitin. Ubiquitinization allows for recognition by the 19S complex. The ubiquitinized protein is entered into the 20S particle, which degrades the protein through enzymatic proteolysis (**Figure 2**). The small peptides resulting from degradation can thus be easily removed or enter the endoplasmic reticulum to be eventually presented to T lymphocytes by means of the MCH type I on the cell surface (19, 20).

Viral infections or other triggers such as cellular stress or cold can induce secretion of type 1 (α or β) IFNs. When IFNs are recognized by their cell surface receptor, transphosphorylation of JAK1 and TYK2 occurs (21), as the first step of JAK/STAT signaling pathway activation. As a result of activated JAKs, the STAT proteins STAT1 and STAT2 dimerize and enter the nucleus, leading to transcription of type 1 IFN genes (**Figure 3A**). On the other hand, danger signals such as an irritant or infection provoke inflammation, which is associated with the production by immune cells of microbiocidal reactive oxygen and reactive nitrogen species during the innate immune response (22). Such increased oxidative stress conditions have profound consequences for the functional integrity of proteins.

During JAK/STAT pathway activation and increased oxidative stress, many waste proteins are generated, as well as many irreversibly oxidant-damaged and potentially toxic proteins. All these require increased proteolytic degradation machinery for their degradation to preserve cell viability and basic cellular functions (22, 23). The constitutive proteasome may not be sufficient to accomplish this extra effort, and hence the immunoproteasomes are recruited and assembled, mainly induced by type 1 IFNs themselves.

# Proteasome Dysfunction and CANDLE Syndrome

In CANDLE syndrome (**Figure 3B**), proteasome system dysfunction leads to an inability of the cell to get rid of waste proteins. This

cells that will cause tissue damage.

situation may lead to a weak or moderate state of proinflammation in the absence of triggers, but under situations of stress, such as cold, viral infections, or physical stress, higher requirements of removing waste proteins are not met.

When a cell is infected by a virus, viral genetic material is sensed in the cytoplasm and activation of the central protein STING (stimulator of interferon genes) ensues. As a result, type 1 IFN genes are transcripted, and IFNs are released (24, 25). As stated above, when the IFN receptor is activated by IFNs, many waste proteins are produced, which must be removed by the proteasome and the IFN-induced immunoproteasome. On the other hand, cells infected by viruses produce viral proteins that are also a substrate for the proteasome system for their removal. Other triggers causing cellular stress also produce type 1 IFN release. If the proteasome system does not work properly, waste proteins accumulate in the cell and are also further marked with more ubiquitin (poly-ubiquitinization) (7, 8). The accumulation of poly-ubiquitinized proteins causes further cellular stress and more type 1 IFN production, thus feeding a vicious cycle of inflammation (2, 7–9).

Type 1 IFNs are virtually produced by every cell in the body, but the plasmacytoid dendritic cells are the most potent producers of type 1 IFNs and are involved in the pathophysiology of CANDLE (26). Type 1 IFNs exert many different actions (27). They increase release of proinflammatory substances and recruit inflammatory cells, including neutrophils and myeloid cells (28). Due to the enhanced and continuous type 1 IFN release, myeloid cells are rapidly mobilized from the bone marrow, and thus "atypical" or immature cells reach the target organs, contributing to the "atypical" skin infiltrate (26).

The IFN signature is very strong in CANDLE syndrome. Microarray analyses have shown an intense expression of IFN signature genes, and the serum of patients with CANDLE contains high levels of proteins of the IFN pathway such as IFN-derived protein 10 (2).

## GENETIC BACKGROUND

The first gene mutations detected in patients with CANDLE syndrome were located in the gene *PSMB8* (proteasome subunit, beta-type, 8) in chromosome 6p21.32, encoding for the β5i (i = inducible) subunit of the immunoproteasome (2, 6–8). Mutations in PSMB8 were responsible for CANDLE, JMP, and Nakajo–Nishimura syndromes. However, mutations in other genes, encoding other proteasome–immunoproteasome subunits or the regulatory protein *POMP*, were later discovered in patients with CANDLE syndrome, thus expanding the CANDLE genotype (9). All mutations were located in highly conserved sites in vertebrates and were thus predicted to be pathogenetic. It was proven that CANDLE syndrome is a disease of proteasome–immunoproteasome dysfunction, which could be inherited as a recessive homozygous, compound heterozygous or digenic trait, or less commonly in a dominant fashion (9). The following mutations have been so far identified in patients with CANDLE (9):

• *PSMB4 mutations*. The gene *PSMB4* (proteasome subunit, beta-type, 4), located in chromosome 1q21, encodes for the β7 subunit of the proteasome. It seems to be important for proteasome assembly and stabilization.

The c.-9G>A mutation originates a β7 subunit protein with lower expression than that of wild type, which is therefore less incorporated into proteasome complexes than the wild-type counterpart. It is also possible that this mutant β7 subunit also impairs propeptide cleavage of the β5i subunit. A deletion of three aminoacids in β7, p.D212-V214, affects the N terminus of an α-helix forming an intramolecular hydrogen-bonding network that stabilizes its C-terminal extension. The C-terminal extension is essential for proteasome assembly (29). Two other mutations affect the C-terminal extension: (1) the c.44insG insertion, which causes a frameshift mutation (p.P16Sfs\*45) and leads to non-expression of the mutant allele; and (2) the missense p.Y222X, which causes the loss of the C-terminal extension of the β7 subunit; although β7 subunit is expressed, it fails to incorporate into the 20S or 26S proteasome complexes.

Finally, the deletion causing the p.D212\_V214del mutant of β7 leads to a poor maturation of the β7 subunit. Although the mutant protein is detected in proteasome assembly intermediates, it is poorly incorporated into 20S or 26S proteasomes.

• *PSMA3 mutations*. The gene *PSMA3* (proteasome subunit, alpha-type, 3), located in chromosome 14q23.1, encodes for the α7 subunit of the proteasome.

Two mutations in *PSMA3* have been reported in patients with CANDLE syndrome. A p.R233del deletion (c. 696\_698delAAG) most likely affects the subunit folding and prevents incorporation of the subunit to the mature proteasomes. Overall reduced proteasome content is thus resulting (9). On the other hand, a c.404+2T>C mutation affects a splice site and causes an unstable transcript due to exon 5 skipping.

• *PSMB8 mutations*. The gene *PSMB8* (proteasome subunit, betatype, 8), located in chromosome 6p21.32, encodes for the β5i subunit of the immunoproteasome. Incorporation of β5i subunit to the maturing immunoproteasome requires proteolytic removal of a prosequence by proteolytically active subunits. The β5i subunit has chemotrypsin-like activity, crucial for the immunoproteasome function.

Mutations in PSMB8 in CANDLE syndrome may affect the chemotrypsin activity or impair immunoproteasome assembly or maturation. The most common mutation in CANDLE syndrome is T75M; when found in homozygosis, it causes selective impairment in chemotryptic-like activity. The A92T mutation produces a similar effect, as well as the mutations K105Q and M117V.

The K105Q mutation is also associated with defects in incorporation and/or maturation of proteasome subunits and with inability to completely trim the β5i propeptide (9). The common T75M and the G201V mutations also cause decreased proteasome assembly (7, 8). Finally, the C135X mutation leads to truncation and non-expression of the protein (2, 9); when found in homozygosis, the subunit β5i is absent in all immunoproteasomes and most likely impairs immunoproteasome assembly, thus showing reduction in all three proteasome activities (trypsin-like, caspase-like, and chemotrypsin-like) (9).

• *PSMB9 mutations*. The gene *PSMB9* (proteasome subunit, beta-type, 3), located in chromosome 6p21.32, encodes for the β1i subunit of the immunoproteasome. The β1i subunit has a caspase-like proteolytic activity.

The only β1i variant described so far is a missense mutation, p.G165D, located in a loop interconnecting 2 α-helices that define the position of a β1i/caspase-like activity conferred by threonine (9).

• *POMP mutations*. The gene *POMP*, located in chromosome 13q12.3, encodes for the POMP, which is key for the maturation and assembly of the proteasome subunits. POMP associates specifically with proteasome precursor intermediates and facilitates the sequential assembly of β subunits onto the preformed α subunit rings (15).

A single patient with CANDLE has been found to bear no mutations in proteasome subunit genes, but a heterozygous, dominant, insertion in POMP causing a frameshift, p.E115Dfs\*20 (c.344\_345insTTTGA) and a truncated protein, which is likely unstable. POMP insufficiency causes proteasome precursor accumulation, reduced mature proteasome formation, and reduced overall proteasome activity (9).

Patients with CANDLE have shown variable combinations of these mutations (9). Most frequently, patients are homozygous or compound heterozygous for *PSMB8* mutations, but others are compound heterozygous for *PSMB4*, or are heterozygous for combinations such as *PSMA3*/*PSMB8*, *PSMB9*/*PSMB4*, or *PSMB8*/*PSMB4*. In the latter situation, a digenic inheritance is suggested causing additive proteasome defects. Patients with digenic inheritance have variable proteolytic defects. For example, a combination of *PSMB8*/*PSMA3* causes impairment in all three proteolytic activities, somewhat similar to patients' compound heterozygous for *PSMB4* in whom proteasome assembly is severely impaired. Patients with combined *PSMB9*/*PSMB4* mutations have reduced caspase-like activity, which is conferred by subunit β1i. Finally, patients with double *PSMB8* mutations experience a severe decrease in chemotrypsin-like activity.

# CLINICAL FEATURES

#### Onset and Course

CANDLE syndrome usually starts in the first months of life (1, 30). The most common presenting sign is fever or temperature elevations below 38.3°C. These appear daily or almost daily, but the general state is minimally affected or even normal. Sometimes, cold exposure can trigger temperature elevation and skin lesions. Skin lesions are the first clinical sign to appear in CANDLE, and usually are present all along the disease course, although they may be less conspicuous after puberty. Lipodystrophy usually starts in early childhood and is usually well established before puberty. Finally, disabling joint manifestations usually occur in the long term. During patients' life, different acute attacks of disease may ensue, spontaneously or after common triggers, which may affect virtually every organ in the body.

# Skin Manifestations of CANDLE

The skin lesions of CANDLE syndrome are very characteristic and should raise the diagnosis. The combination of fever, typical skin lesions, and classic histopathologic features should allow for a rapid diagnosis of CANDLE (**Figures 4**–**6**). The skin lesions in CANDLE are of three types (1):

Figure 4 | Periorbital erythema and edema and flat nose in a patient with CANDLE.

macule. New, active lesions coexist all over time with residual, purpuric macules, which confers a very typical appearance to the patients. These lesions are very conspicuous during childhood, but in adult life they may be less visible and may be absent in long-standing disease.

3. Perioral and periocular edema. Patients with CANDLE develop in infancy or childhood a persistent erythematous to violaceous edema affecting the periorbital and less commonly the perioral area. It may be less visible after puberty and in long-standing disease.

The histological features of the skin lesions in CANDLE are very characteristic and may permit a diagnosis in early stages of the disease (1, 27). The papillary and reticular dermis contains a perivascular and interstitial infiltrate of varying intensity, extending to the subcutaneous fat as lobar panniculitis. The infiltrate is predominantly composed of mononuclear cells, many of which have large, irregularly shaped nuclei; the atypical appearance of the infiltrate may lead to a diagnosis of skin malignancy.

Figure 5 | Annular, purpuric plaques of CANDLE syndrome.

Figure 6 | CANDLE hands: skin lesions and swollen joints.

The infiltrate also contains mature neutrophils, some eosinophils, and a few mature lymphocytes. Leukocytoclasia is often seen, without fibrinoid necrosis of the vessels (1, 27). Immunohistochemistry shows that the infiltrate is mixed, with an important presence of myeloid cells (positive for myeloperoxidase) and a prominent cell population of macrophages (positive for CD163 and CD68/PMG1) (27). CD123-positive plasmacytoid dendritic cells are seen in clusters (27).

#### Lipodystrophy

Fat loss is a key manifestation of CANDLE (1, 3, 30–32). It can be seen in most patients before the age of two, but it is progressive and may take some years to become fully developed. The loss of subcutaneous fat usually starts on the face and progresses to the trunk and upper limbs. The lower limbs are usually less affected. The cause of lipodystrophy is not well known, but it can be related to chronic inflammation involving the fatty tissue (33, 34). Alternatively, increased expression of proinflammatory cytokines in adipose tissue and reduced secretion of adiponectin and leptin may be involved (35, 36). An intense type 1 IFN signature is believed to be associated with fat loss in children with lipoatrophic panniculitis (37), which reinforces the role of IFN in CANDLE syndrome. Type 1 IFNs may be toxic to adipocytes, as is suggested by the development of lobar panniculitis with lipophagia and lipoatrophy in patients treated with intramuscular injections of IFN-β (38, 39).

Lipodystrophy and the typical skin lesions confer to CANDLE patients a unique phenotype. On the face, the loss of fat on the cheeks and the periorbital and periocular edemas are pathognomonic (1). In adulthood, the eyelids and the lips are retracted, causing a false proptosis and exposure of the teeth; coupled with the severe fat loss, these features cause an unmistakable appearance (4). On the limbs, a progressive fat loss, coupled with muscle wasting (see later), is seen. A prominent abdomen may be related to increased visceral fat, which remains the only fat storage capability of the patient. An increased distance between nipples is also typical in CANDLE (1). Metabolic disturbance due to absence of body fat can also impair hydrocarbon metabolism and lead to acanthosis nigricans and hirsutism.

#### General Examination

Patients with CANDLE have a mild to moderate growth delay and show low weight and height (1, 30–32). Chronic inflammation may explain this physical delay, but muscle wasting and lipoatrophy are also major contributors. However, most patents with CANDLE do not show mental retardation.

CANDLE patients show variable degrees of hepatomegaly, which can be related to a secondary metabolic disturbance due to extensive lipoatrophy. Splenomegaly and generalized lymphadenopathy are common findings, reflecting the persistent autoinflammatory activity.

#### Musculoskeletal Signs

Arthralgias are very common in children with CANDLE, but patients do not show radiologic features of arthritis. Some joint swelling can appear in the interphalangeal joints, but the absence of overt arthritis distinguishes CANDLE from rheumatoid arthritis or juvenile idiopathic arthritis (1, 30–32). With time, hyperextensibility of interphalangeal joints can occur, and during adulthood, most patients will develop variable degrees of joint contractures on the hands and feet, which are often severely disabling (4).

Cartilage inflammation has been reported in CANDLE patients (1). Recurrent and also chronic chondritis of the ears and nose causes partial loss of auricles and a flat, saddle-like nose. Because both ears and nose are usually exposed, a role of triggering by cold has been suspected.

Muscle involvement is also a feature of CANDLE (4). Acute attacks of inflammatory myositis have been reported that can be demonstrated by MRI. Possibly, there is also a role for chronic muscle inflammation in the development of severe muscular wasting.

#### Nervous System

As stated above, mental delay is not a common feature in CANDLE (1). However, patients may suffer attacks of aseptic meningitis, meningoencephalitis, and possibly some degree of chronic inflammation in the CNS. Basal ganglia calcifications have been reported (1), most likely as a result of encephalitis; these are similar to those present in Aicardi–Goutières syndrome, which also features a high type 1 IFN production.

#### Other Organ Involvement

Episodes of inflammation may occur in any organ, as well as some degree of persistent generalized inflammation. Attacks of acute sterile epididymitis, conjunctivitis and nodular episcleritis, parotitis, pneumonitis, nephritis, carditis, and otitis have been reported. The clinical manifestations are related to the organs involved. Some of these complications have been reported to be fatal (1).

## LABORATORY INVESTIGATIONS

As is the case with other AIDs, laboratory findings are not very striking (1, 3, 32). The most common features are elevation of acute phase reactants (ESR, CRP, and thrombocytosis) and a chronic, hypochromic anemia. Liver enzymes are usually moderately elevated, but this may be caused by lipodystrophy itself; also, increased triglyceride levels can occur in relation to metabolic disturbance by lipodystrophy. Less frequently, elevated muscle enzymes (CPK and aldolase) reveal chronic muscle involvement (1). Studies for autoimmunity and autoantibodies are usually negative, but some patients show increased levels of antinuclear antibodies. Serum levels of immunoglobulins are regularly normal. Bone marrow and lymph node biopsies have been unconspicuous, revealing only reactive changes.

Other laboratory and imaging anomalies are seen during acute inflammatory attacks; these are dependent on the organs affected by inflammation.

## DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

The diagnosis of CANDLE is suspected by the early onset of fevers, skin lesions, and lipodystrophy. A skin biopsy with immunohistochemistry studies can be characteristic enough to permit an accurate diagnosis. The genetic study of the genes involved establishes a confirmation diagnosis.

Other AIDs may share some features with CANDLE syndrome, including NOMID syndrome, TRAPS, or hyper-IgD syndrome. Lipodystrophy in CANDLE syndrome is a very characteristic feature of the disease, but other causes of loss of fat must be considered, including generalized congenital lipodystrophy, partial familial lipodystrophy, leprechaunism, or acquired partial lipodystrophy of Barraquer–Simons. Aicardi–Goutières syndrome and other type 1 interferonopathies (such as SAVI syndrome, familial chilblain lupus, or C1q deficiency) may show features similar to CANDLE syndrome (28, 40, 41). Sweet syndrome in infants may present with violaceous ring lesions reminiscent of CANDLE syndrome, and histology may be misleading in some cases. Fat loss and skin lesions are clinical manifestations of a recently described autoinflammatory syndrome named otulipenia, due to loss-of-function mutations in OTULIN, encoding a deubiquitinase that cleaves Met1-linked chains (42).

#### PROGNOSIS AND FOLLOW-UP

CANDLE patients have a variable outcome. Some patients have had a lethal course due to acute attacks of inflammation in important organs of the body. In other patients, a long survival is possible, with variable degrees of disability (1, 3, 4).

Regular clinical follow-up is mandatory. A protocol has not been established, but attention must be paid to identify inflammatory attacks as early as possible. Regular skin, eye, and joint exams are recommended. Endocrinologist consultation is mandatory for diet and metabolic control because of lipodystrophy. Basic laboratory follow-up must include CBC (with special attention to anemia, leukocytosis with increased neutrophil count, and thrombocytosis), ESR, CRP, serum liver enzymes, muscle enzymes, and metabolic profile (including glucose, triglyceride, and cholesterol levels). Ultrasound may be helpful in detecting enlarged liver, spleen, or lymph nodes. Specific tests for organ involvement must be considered in patients with abnormal clinical examination.

#### REFERENCES

#### TREATMENT

So far, no individual treatment has been consistently effective in CANDLE syndrome. Oral corticosteroids and methotrexate can provide some improvement. Methotrexate can be considered the first line therapy. NSAIDs may provide partial control of fevers. Dapsone or colchicine has been ineffective. Cyclosporine, azathioprine, or intravenous immunoglobulins have achieved minimal improvements, if any. Anti-TNF drugs such as etanercept have not been helpful and have even been the cause of disease exacerbations (1). Acute attacks may need systemic corticosteroids as well as organ-specific therapy.

A compassionate use treatment protocol has been started for CANDLE syndrome with the selective JAK1/2 kinase inhibitor baricitinib. Oral baricitinib was used in patients who failed to achieve control or required high doses of corticosteroids. Eight patients treated with this drug showed clinical and analytical improvement (43), but these results still await confirmation.

Finally, physical therapy to prevent joint contractures and specific organ therapy must be provided.

#### CONCLUDING REMARKS

CANDLE syndrome is an AID due to gene mutations leading to protesome–immunoproteasome dysfunction. CANDLE syndrome can be diagnosed very early in life because the skin signs and their histopathology are very characteristic. Genetic confirmation is necessary. Early therapy to prevent disabling manifestations is desirable, but still no agent has been truly effective. Prevention and treatment of acute inflammatory attacks will permit longer life expectancy in these patients.

#### AUTHOR CONTRIBUTIONS

There is one single author for this article. The manuscript has been completely written by AT.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Torrelo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Diagnostic criteria for adult-Onset Periodic Fever, aphthous stomatitis, Pharyngitis, and cervical adenitis (PFaPa) syndrome

*Luca Cantarini1 \*, Antonio Vitale1 , Ludovico Luca Sicignano2 , Giacomo Emmi3 , Elena Verrecchia2 , Isabella Patisso2 , Lucia Cerrito2 , Claudia Fabiani <sup>4</sup> , Gabriele Cevenini <sup>5</sup> , Bruno Frediani <sup>1</sup> , Mauro Galeazzi1 , Donato Rigante6 and Raffaele Manna2*

*1Research Center of Systemic Autoinflammatory Diseases, Behçet's Disease and Rheumatology-Ophthalmology Collaborative Uveitis Center, Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy, 2Periodic Fevers Research Center, Institute of Internal Medicine, Fondazione Policlinico Universitario A. Gemelli, Università Cattolica Sacro Cuore, Rome, Italy, 3Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy, 4 Department of Ophthalmology, Humanitas Research Center, Rozzano, Italy, 5Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy, 6 Institute of Pediatrics, Fondazione Policlinico Universitario A. Gemelli, Università Cattolica Sacro Cuore, Rome, Italy*

#### *Edited by:*

*José Hernández-Rodríguez, Hospital Clinic of Barcelona, Spain*

#### *Reviewed by:*

*Sinisa Savic, University of Leeds, United Kingdom Dong Li, Jilin University, China*

*\*Correspondence: Luca Cantarini<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 21 June 2017 Accepted: 08 August 2017 Published: 24 August 2017*

#### *Citation:*

*Cantarini L, Vitale A, Sicignano LL, Emmi G, Verrecchia E, Patisso I, Cerrito L, Fabiani C, Cevenini G, Frediani B, Galeazzi M, Rigante D and Manna R (2017) Diagnostic Criteria for Adult-Onset Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis (PFAPA) Syndrome. Front. Immunol. 8:1018. doi: 10.3389/fimmu.2017.01018*

Objective: To identify a set of variables that could discriminate patients with adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome from subjects with fever of unknown origin (FUO).

Methods: We enrolled 74 adults diagnosed with PFAPA syndrome according to the currently used pediatric diagnostic criteria and 62 additional patients with FUO. After having collected clinical and laboratory data from both groups, univariate and multivariate analyses were performed to identify the variables associated with PFAPA diagnosis. Odds ratio (OR) values, their statistical significance, and corresponding 95% confidence interval (CI) were evaluated for each diagnostic factor both at the univariate and multivariate analyses. Diagnostic accuracy was evaluated by the area under receiver operating characteristic (ROC) curve, while the leave-one-out cross-validation procedure was used to ensure that the model maintains the same diagnostic power when applied to new data.

results: According to the multivariate analysis, the clinical variables that discriminated PFAPA patients were: fever episodes associated with cervical lymphadenitis (OR = 92; *p* < 0.0001), fever attacks associated with erythematous pharyngitis (OR = 231; *p* < 0.0001), increased inflammatory markers during fever attacks (OR = 588; *p* = 0.001), and the lack of clinical and laboratory signs of inflammation between flares (OR = 1202; *p* < 0.0001). These variables were considered for a diagnostic model which accounted for their OR values. The diagnostic accuracy of the proposed set of criteria corresponded to an area under ROC curve of 0.978 (95% CI 0.958–0.998), with a model sensitivity and specificity equal to 93.4% (95% CI 87.5–96.5%) and 91.7% (95% CI 82.8–96.7%), respectively.

Keywords: PFAPA syndrome, autoinflammatory disease, differential diagnosis, diagnostic criteria, adults, fever of unknown origin

#### INTRODUCTION

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome belongs to the spectrum of multifactorial autoinflammatory diseases (AIDs) and is characterized by spontaneous flares of systemic inflammation characterized by fever and other clinical manifestations, especially cardinal signs described by the PFAPA acronym (1).

To date, the pathogenesis of this syndrome remains still obscure, but studies aimed at assessing immunological mechanisms, also supported by therapeutic evidences (2, 3), have highlighted an abnormal interleukin-1 release in response to many environmental triggers, which associates PFAPA syndrome to other hereditary periodic fever disorders (4, 5). However, unlike other AIDs characterized by recurrent fever attacks, no genetic mutations have been clearly associated with PFAPA syndrome (6, 7).

In addition to fever (often achieving and overcoming 40°C), aphthous stomatitis, pharyngitis, and cervical adenitis, many other clinical manifestations may enrich the clinical framework of PFAPA patients, including abdominal pain, headache, nausea, skin manifestations, and arthralgia (1, 8–10). Inflammatory flares arise every 3–8 weeks with no premonitory symptoms and generally last 3–6 days. Patients are typically healthy between febrile episodes and the overall growth of children affected by this syndrome is not stunted (11, 12). Although during the last decades diagnosis of PFAPA syndrome has been relegated to children aged under 5 years, increasing evidence has recently shown that the disease can also arise in older children as well as during adulthood (2, 3, 9, 10, 13–18). The treatment of PFAPA patients is based on intermittent corticosteroid administration, as patients are generally responsive to a single dose of a corticosteroid given at the onset of febrile flares (1, 12, 14, 19, 20).

No laboratory or instrumental tools are available to support the diagnosis of PFAPA syndrome, which is currently based on the fulfillment of clinical diagnostic criteria. In particular, to date, clinical criteria proposed by Marshall et al. in 1986 (21) and later modified by Thomas et al. in 1999 (11) represent the most used set of criteria in the clinical practice. However, these criteria are tailored on pediatric patients and their application on adults is categorically excluded by the first item that requires the presence of recurrent fever in patients under 5 years of age. In addition, the fifth item imposes the lack of normal growth and development for patients affected, which is not applicable to adult-onset PFAPA patients. In this context, Padeh et al. employed a further set of inclusion criteria valid for both children and adult patients (8). This set included the presence of monthly fever attacks, exudative tonsillitis, possibly oral ulcers, cervical lymph node enlargement, negative throat cultures, and failure of antibiotic treatment during the acute episodes or as prophylactic treatment, while normal growth/development and a rapid response to a single corticosteroid administration were later added as further items (8, 22). However, to the best of our knowledge, no statistical procedures were employed to identify variables useful in discriminating PFAPA patients among subjects presenting with recurrent fever attacks. In addition, recent evidences have proved that erythematous pharyngitis is more typical than sterile exudative pharyngitis in adult-onset PFAPA patients (10). Therefore, the need for a new set of diagnostic criteria for patients experiencing PFAPA syndrome during adulthood has prompted our group to evaluate a set of variables on both clinical and statistical basis that could discriminate such patients from subjects with fever of unknown origin (FUO).

#### MATERIALS AND METHODS

#### Patients

Seventy-four consecutive adult patients who had been referred to our Units from September 2007 to December 2016 because of recurrent fever attacks and other clinical manifestations consistent with PFAPA syndrome were classified as suffering from adult-onset PFAPA syndrome (PFAPA group) according to the Marshall criteria modified by Thomas et al. (11, 21), which are the most frequently used diagnostic tool in the clinical practice. As this set of criteria is tapered on pediatric patients, the item requiring a disease onset before the age of 5 was neglected, while the item requiring a normal growth and development was retrospectively applied, as previously made in other studies (9, 10, 13). Two patients out of 74 were included in the PFAPA group despite the lack of symptom-free intervals. In both cases, the patients showed the resolution of fever and of cardinal symptoms as well as the normalization of acute phase reactants. Conversely, the sole arthralgia and myalgia persisted in both cases and were attributed to the presence of concomitant degenerative, not inflammatory musculoskeletal diseases.

Sixty-two additional adult subjects admitted in our Units between September 2016 and March 2017 for recurrent FUO were consecutively enrolled in the study as control group (control group). FUO diagnosis was based on the currently available diagnostic criteria (23). As for patients with adult-onset PFAPA syndrome, any specific disease related to fever or inflammatory manifestations had been ruled out at the time of enrollment in this study. The control group was included into a follow-up protocol aimed at early identify any sign or symptom potentially useful for a prompt specific diagnosis; patients were treated with non-steroidal anti-inflammatory drugs or low-to-high dosage corticosteroids.

#### Assessment Parameters

Each patient's medical record was reviewed for demographic and clinical data. In particular, we looked for the age at disease onset, characteristics of the febrile pattern (peak temperature, duration of flares, frequency of fever episodes per year), clinical manifestations accompanying fever (oral and/or genital aphthosis, exudative and/or erythematous pharyngitis, cervical and/or widespread lymphadenitis, abdominal pain, vomiting, diarrhea, thoracic pain, arthralgia, arthritis, myalgia, urticarial-like rash, maculopapular rash, erysipelas-like rash, erythematous rash, periorbital edema, conjunctivitis, asthenia, and headache), any increase of inflammatory markers (erythrocyte sedimentation rate and/or C-reactive protein and/or serum amyloid A) during attacks and the presence or absence of clinical manifestations and positive laboratory inflammatory markers during fever-free intervals.

None of the patients with adult-onset PFAPA syndrome showed upper respiratory infections, while the throat swab was negative in all cases presenting with pharyngitis or cervical lymphadenitis. Both PFAPA group and control group patients underwent detailed laboratory and instrumental screening tests to rule out potential underlying diseases, such as infections, autoimmune diseases, and malignancies. In all patients enrolled, previous antibiotic therapies administered during flares did not change the progression of clinical manifestations. Monogenic periodic fever syndromes were ruled out by performing molecular analysis of *MEFV*, *MVK*, *TNFRSF1A*, and *NLRP3* genes, respectively responsible for familial Mediterranean fever (FMF), mevalonate kinase deficiency, tumor necrosis factor receptor-associated periodic syndrome, and cryopyrin-associated periodic syndrome (CAPS). Moreover, neither PFAPA patients nor subjects included in the control group fulfilled clinical diagnostic criteria for FMF or CAPS as well as for Still's disease, Schnitzler's syndrome, and Behçet's disease (24–33).

The study was approved by the local Ethics Committee of Azienda Ospedaliera Universitaria Senese, Siena (Italy) and each patient provided a written consent for both genetic testing and clinical data processing, in accordance with the Declaration of Helsinki.

#### Statistical Analysis

Descriptive statistics are expressed as mean and SD for quantitative variables as well as frequency counts and percentages for quantitative binary variables.

Multivariate stepwise logistic regression analysis was performed to identify, among all possible diagnostic factors (predictive variables), a statistically significant minimum subset of factors with the highest possible accuracy to establish a diagnosis of PFAFA syndrome. In the stepwise process, one independent variable was added to or removed from the discriminant model at each step, on the basis of maximum likelihood-ratio statistics. The process stops when no statistical significant variables can be more entered or removed. We used the leave-one-out (LOO) cross-validation procedure to ensure that the model maintains the same diagnostic power when applied to new data. LOO uses all available data to train and test model: it executes a number of training sessions equal to the sample size (*N*) and in each of them it classifies each patient (LOO testing case) in turn by using all other patients as training set.

Diagnostic accuracy was evaluated by the area under receiver operating characteristic (ROC) curve (AUC) along with its 95% confidence interval (95% CI). Model sensitivity and specificity together with their 95% CIs were also estimated by selecting a probability threshold giving comparable sensitivity and specificity values, along with their 95% CIs. The Hosmer–Lemeshow goodness-of-fit test was used to evaluate model calibration, that is its prognostic ability.

Finally, the odds ratio (OR), its statistical significance, and corresponding 95% CI were evaluated for each diagnostic factor, taken singularly (univariate analysis), and for the model selected factors, taken together (multivariate analysis). The SPSS software, version 10, was used for all statistical computations, always considering a significance level of 95% (*p* value < 0.05).

#### RESULTS

Both patients with adult-onset PFAPA syndrome and subjects belonging to the control group experienced a disease onset over the age of 16. Specifically, the mean age at disease onset was 26.55 ± 10.03 years for PFAPA patients and 27.94 ± 17.67 for those with FUO. **Table 1** summarizes demographic and clinical features of patients enrolled.

Univariate analysis performed on patients with PFAPA syndrome and subjects with FUO recognized clinical variables positively or negatively associated with PFAPA syndrome by an OR significantly different from 1, i.e., with a 95% CI not including 1.0. The results of univariate analysis are summarized in **Table 2**.

As reported in **Table 3**, according to multivariate analysis performed on the two groups of patients, clinical variables that showed a statistical significant (*p* < 0.05) discriminant power to identify PFAPA patients were: recurrent fever accompanied by cervical lymphadenitis (OR = 92), recurrent fever with concomitant erythematous pharyngitis (OR = 231), increased inflammatory markers during attacks (OR = 588), and symptom-free intervals corresponding to the lack of clinical manifestations and laboratory abnormalities between flares (OR = 1,202). These variables were then considered for a diagnostic model that accounts for their OR values. In particular, the occurrence of symptomfree intervals and the increase of inflammatory markers during attacks, which have higher OR values, represent mandatory items in the proposed diagnostic model. Conversely, on the basis of their lower OR values, only one between fever associated with erythematous pharyngitis and fever with cervical lymphadenitis is required for the diagnosis of PFAPA syndrome. **Table 4** shows the resulting set of criteria proposed in this study.

The diagnostic accuracy of the proposed diagnostic criteria corresponded to an AUC of 0.978 (95% CI 0.958–0.998), with sensitivity and specificity equal to 93.4% (95% CI 87.5–96.5%) and 91.7% (95% CI 82.8–96.7%), respectively. **Figure 1** represents the ROC curve assessing the performance of the criteria for our PFAPA patients and the control group with FUO.

*Quantitative data are referred as mean* ± *SD values; qualitative data are reported as frequency counts and percentages.*

# DISCUSSION

Despite the increasing evidence on the possible delayed onset of PFAPA syndrome during adulthood, current diagnostic criteria are tailored on pediatric patients (34) and their application on adults requires specific adjustments not yet validated. On this basis, we looked for clinical variables that can identify patients with adult-onset PFAPA syndrome among patients presenting with FUO. Therefore, we analyzed the occurrence of inflammatory features in patients with a clinical picture consistent with adult-onset PFAPA syndrome, being excluded all the known causes of recurrent fever, as well as in patients consecutively visited in our Units because of FUO during a 6-month period. Multivariate analysis allowed to identify a set of clinical variables capable of discriminating adult-onset PFAPA patients. These variables were then rearranged into a diagnostic model in which items with a higher OR were considered mandatory for the diagnosis of PFAPA, while just one out of the two variables with a lower OR value had to be fulfilled.

Noteworthy, these proposed diagnostic criteria should be applied after having ruled out the known causes of fever in terms of infective, autoimmune, and neoplastic diseases. Monogenic AIDs should be also excluded on the basis of clinical presentation, as required by the clinical classification criteria recently proposed by Federici et al. to drive genetic analysis for patients with periodic fevers (35). According to Federici et al., we have also reported that the diagnosis of monogenic AIDs in adulthood is not unworkable when patients' symptoms are carefully classified (36, 37). Therefore, a correct evaluation of the patients' clinical picture integrated by familiar and laboratory data may allow the identification of adult-onset monogenic AIDs by specifically performing genetic testing. In addition to this, specific clinical diagnostic and classification criteria, when available, should also be applied to preventively recognize both monogenic (i.e., FMF and CAPS) and multifactorial AIDs (i.e., Behçet's disease, Still's disease, and Schnitzler's disease) (24–33). Moreover, our present criteria should not be applied in patients with positive throat swab during fever episodes and in patients responsive to antibiotics, as for previous diagnostic and classification criteria (11, 22).

Among the cardinal signs of PFAPA syndrome, the occurrence of recurrent fever with erythematous pharyngitis represented the variable most strongly associated with diagnosis of PFAPA syndrome in adulthood, while exudative pharyngitis and oral aphthosis during attacks were not included in the model. Accordingly, we had previously found that the exudative form of pharyngitis is almost rare in patients with a delayed onset of PFAPA syndrome (9, 10), while univariate analysis performed in this study even highlights a protective role of exudative pharyngitis against the diagnosis of PFAPA syndrome, further remarking a less important role of this clinical manifestation in adults. In relation to lymph node involvement, the specific observation of cervical lymphadenitis was strongly correlated to PFAPA syndrome both when considered individually and at the overall multivariate assessment. Conversely, at the univariate analysis, generalized lymphadenitis represented a variable tending to exclude the diagnosis of PFAPA syndrome in adults.

Regarding oral aphthosis, although significantly discriminant when considered singularly, it was not included into the multivariate model as its diagnostic information resulted to be absorbed by "recurrent fever accompanied by erythematous pharyngitis" and "symptom-free intervals." Therefore, most patients with oral aphthosis also presented at least one out of these two variables included in the model, thus minimizing the diagnostic value of oral aphthosis as an additional item. Interestingly, according with our results, Padeh had already suggested oral ulcers just as "possible" among the classification items proposed in 2005 (22). Furthermore, other authors have also highlighted that oral aphthosis is less frequently encountered in adult-onset PFAPA patients than among children (9, 11, 14). These observations seem to corroborate that aphthous stomatitis, although important for clinical evaluation, does not necessarily have to be included for diagnostic purposes in adults.

In relation to non-cardinal symptoms, beyond generalized lymphadenitis, also thoracic pain, conjunctivitis, maculopapular, and urticarial-like skin rash appeared to be protective factors against PFAPA diagnosis when considered individually. Consequently, the TABLE 2 | Results of univariate logistic regression analysis performed on adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) patients and subjects with fever of unknown origin by evaluating clinical manifestations described in both groups.

*NE, not evaluable; CI, confidence interval; OR, odds ratio.*

TABLE 3 | Estimations derived from multivariate logistic regression analysis performed on adult periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) patients and patients with fever of unknown origin, representing the control group.

Clinical variable *p*-Value OR 95% CI Erythematous pharyngitis <0.0001 231 14.463–3,715.288 Cervical lymphadenitis <0.0001 92 8.865–953.279 Increased inflammatory markers during attacks 0.001 588 3,534.3–40,879.463 Symptom-free intervals <0.0001 1202 12.631–27,937.885

*CI, confidence interval; OR, odds ratio.*

observation of these manifestations in patients with a suspected PFAPA syndrome should call for caution before assigning the diagnosis. In addition, univariate analysis shows that PFAPA syndrome is mostly connected with a fever duration ranging between 2 and 5 days, while fever attacks lasting less than 48 h and longer than 10 days should point to other diagnoses than PFAPA syndrome.

Although results obtained by univariate analysis are clinically interesting and potentially useful to identify or exclude adultonset PFAPA syndrome, we aimed at creating a set of diagnostic criteria easy to be applied in the clinical practice and reproducible for further studies. Therefore, we deliberately avoided a longer list of diagnostic items as well as concomitant exclusion criteria, TABLE 4 | Clinical diagnostic criteria resulting from the multivariate analysis.

Diagnostic criteria for adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome

*were established as mandatory in the final diagnostic model; conversely, on the basis of a lower OR value, only one item between erythematous pharyngitis during fever and cervical lymphadenitis during fever is required for the diagnosis of PFAPA syndrome. These diagnostic criteria should be applied on patients aged at least 16 years and after having excluded infective, autoimmune, and neoplastic diseases as well as monogenic autoinflammatory diseases (AIDs) and febrile polygenic AIDs. In addition, throat swab performed during fever have to be negative and antibiotic therapy ineffective.*