In all, autoimmunity disorders are one of the main mechanisms of PD pathogenesis and development and have gained increasing attention in recent years. Based on the latest research advances, including our laboratory data, conclusions can be drawn that both innate and adaptive immunity become pathogenic when

# REFERENCES

self-antigen tolerance is lost. From all evidence, when facing an autoimmunity attack, the CNS cannot escape. To decipher the mechanisms of autoimmunity disorders involved in PD, the deletion or mutation of PD-related genes and the dysfunction of their encoded proteins should be studied. The involvement of α-syn provides strong evidence that this protein quite possibly acts as the first target of autoimmune attack, followed by the sustained activation of DCs and microglia, inflammation, and immune cell recruitment. Our review suggests that α-syn will no longer merely be the PD pathological hallmark but will also become one of the main targets of autoimmunity attack. In addition, NM, as another novel autoantigen released from dead DNs, is phagocytized by DCs and then induces the activation of microglia, contributing to the autoimmune aggravation of PD. Through developing neuroimmunoregulatory therapies, many new therapeutic options will become available to PD patients. These achievements will benefit both diagnostics and treatments. This review sheds light on autoimmunity associated with the etiology and pathogenesis of PD from a new perspective and further proposes some possible therapeutic targets and methods for PD (**Table 1**).

# AUTHOR CONTRIBUTIONS

TJ and GL wrote the paper. JX supervised the figures. SG and XC supervised and wrote the paper.

# ACKNOWLEDGMENTS

This work is supported by the Guangzhou Setzer biological Polytron Technologies Inc.

regulatory immune responses in the CNS. J Neurosci. (2009) 29:140–52. doi: 10.1523/jneurosci.2199-08.2009

proteins and sporadic Parkinson's disease? Antioxid Redox Signal. (2014) 21:2143–8. doi: 10.1089/ars.2014.6072

**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 © 2018 Jiang, Li, Xu, Gao and Chen. 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) and the copyright owner(s) 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.

# Tertiary Lymphoid Structures: Autoimmunity Goes Local

Elena Pipi 1,2†, Saba Nayar 1†, David H. Gardner 1 , Serena Colafrancesco3 , Charlotte Smith1 and Francesca Barone1 \*

1 Rheumatology Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom, 2 Experimental Medicine Unit, Immuno-Inflammation Therapeutic Area, GSK Medicines Research Centre, Stevenage, United Kingdom, 3 Reumatologia, University of Rome, Sapienza, Italy

Tertiary lymphoid structures (TLS) are frequently observed in target organs of autoimmune diseases. TLS present features of secondary lymphoid organs such as segregated T and B cell zones, presence of follicular dendritic cell networks, high endothelial venules and specialized lymphoid fibroblasts and display the mechanisms to support local adaptive immune responses toward locally displayed antigens. TLS detection in the tissue is often associated with poor prognosis of disease, auto-antibody production and malignancy development. This review focuses on the contribution of TLS toward the persistence of the inflammatory drive, the survival of autoreactive lymphocyte clones and post-translational modifications, responsible for the pathogenicity of locally formed autoantibodies, during autoimmune disease development.

#### Edited by:

Ralf J. Ludwig, Universität zu Lübeck, Germany

#### Reviewed by:

Andreas Habenicht, Ludwig-Maximilians-Universität München, Germany Karen Willard-Gallo, Free University of Brussels, Belgium

> \*Correspondence: Francesca Barone<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: 11 February 2018 Accepted: 07 August 2018 Published: 12 September 2018

#### Citation:

Pipi E, Nayar S, Gardner DH, Colafrancesco S, Smith C and Barone F (2018) Tertiary Lymphoid Structures: Autoimmunity Goes Local. Front. Immunol. 9:1952. doi: 10.3389/fimmu.2018.01952 Keywords: tertiary lymphoid structures (TLS), autoantibodies, germinal center response, glycosylation, B-cells

# INTRODUCTION

The polyclonal expansion of autoreactive B cells is a cardinal feature of autoimmune conditions. Whether directed against a single antigen or playing part in a poly-specific response, autoreactive B cells support the persistence of the autoimmune process and, in several cases are directly pathogenic.

The development of an autoreactive B cell repertoire during the natural history of the autoimmune condition is regulated by the process of affinity maturation against single or multiple autoantigens that occurs within the inner part of the B cell follicles, classically within secondary lymphoid organs (SLOs) (1). Formation of B cell follicles and germinal centers (GC) has been also described in ectopic or tertiary lymphoid structures (TLS) in a process defined "ectopic lymphoneogenesis." TLS form at target organs of chronic inflammatory/autoimmune process, localized infections and in the areas surrounding solid tumors (2–11). The prognostic value of these structures is debated. TLS formation in target organs autoimmune disease is classically associated with disease persistence and worst clinical manifestations. In solid tumors TLS have instead been associated with the generation of an anti-tumor response, however in some cases the ability of tumor cells to induce T regulatory cells (Treg) and suppress the host immune response has been described **Table 1**.

Often indicated as "tertiary lymphoid organs," TLS fail to adhere to the proper definition of organs as they lack a stable structural organization, including a capsule, and are better classified as "tertiary lymphoid structures" or TLS (97). TLS are not present in embryonic life and form in adult life to support local aggregation of lymphocytes at the target organ of disease. Other terms

**44**

#### TABLE 1 | TLS in different conditions.

GPA/WG, Granulomatosis polyangiitis/Wegener's granulomatosis; COPD, Chronic Obstructive Pulmonary Disease; IBD, Inflammatory Bowel Disease; PSC, Primary Sclerosing Cholangitis AID, Autoimmune disease; CID, Chronic inflammatory disease; PDC, Pancreatic duct carcinoma; HCV, Hepatitis C virus; Can, Cancer; Tran, Transplantation; Inf, Infection. (Note: Studies on mice are presented only if there is evidence from human studies for the presence of TLSs in these different conditions).

including "ectopic lymphoid structures" (ELS) or "ectopic germinal centers". The latter, however, should only be used when GC formation is determined histologically within the ectopic lymphoid tissue (97–101). The cross-over between TLS and SLO is the subject of debate and has been reviewed by ourselves and others in recent publications (9, 98).

The term "tertiary lymphoid" tissue in the literature dates back to 1992 and was introduced by Louis Picker and Eugene Butcher (102) to describe the formation of extra-lymphoid sites, where memory lymphocytes and/or precursors can be re-stimulated by antigen to induce further clonal expansion or terminal effector responses. By definition, TLS arise in tissues whose main function is other than the generation of immune cells or the initiation of an adaptive immune response. This excludes the bone marrow and thymus, (as primary lymphoid organs) and spleen, lymph nodes and Peyer's patches (which are defined as SLOs). The kidneys, heart, pancreas, synovium and salivary glands are not embryologically predisposed to host the presence of lymphoid tissue therefore lymphocyte assembly at these sites should be considered TLS. The liver provides a hematopoietic function during embryonic development (103) however, this function is lost postnatally, thus including this organ among those that host TLS in adult life (97).

TLS form in response to a series of pro-inflammatory cytokines and TNF receptor family members following the local cross-talk between inflammatory immune cells and resident stromal cells. Fibroblasts, perivascular myo-fibroblasts and resident mesenchymal cells have been differently implicated in TLS development (39, 75, 104–112). Their role has been recently reviewed elsewhere (97, 113, 114). Probably evolved before SLO, TLS might have developed in ectopic tissues to fulfill the survival need of aggregated leucocytes, prior to placentation and development of SLOs. As such, the ability of TLS to be initiated independently from Lymphotoxin (LT) upon the expression of inflammatory cytokines and in absence of lymphoid tissue inducer cells (LTi) might have remained as heritage of their developmental ancestry (97).

Physiologically, the generation of a humoral response requires the physical interaction of naïve B cells with antigen experienced T cells within the confined space of a microenvironment rich in survival and chemotactic factors (115). Lymphocytes are recruited from the bloodstream to the SLO in response to a chemotactic gradient that regulates cell positioning and interactions (116, 117). CXCL13 and CCL19/CCL21, ligands for the chemokine receptors CXCR5 and CCR7, respectively, regulate the recirculation of naïve B cells between the inner part of the B cell follicle to the outer area of the T/B cell boundary (118), thus enabling the contact of B cells with antigenexperienced, activated T cells (119). Within the follicles, antigenexperienced B cells migrate toward the dark zone of the GC, a highly hypoxic CXCL12-rich area. Within this area they become highly proliferating centroblasts and upregulate the enzyme activation-induced cytidine deaminase (AID) (120, 121), that regulates the introduction of single base-pair substitutions of antibody gene segments in the immunoglobulin (Ig) variableregion genes, in a process defined as somatic hypermutation (SHM) (122).

Following SHM, B cells stop proliferating and undergo the process of affinity maturation (123). Differentiated, non-dividing B cells (centrocytes) upregulate CXCR5 and migrate along the CXCL13 gradient toward the GC light zone (120), herby establishing connections with the network of follicular dendritic cells (FDC) that provide survival factors (124, 125) and antigen presentation via the CR2 receptor (125, 126). Within the light zone, centrocytes also encounter mature T follicular helper cells (Tfh), known to provide signals for selection and terminal differentiation into long-lived plasma cells or memory B cells (127–130). Once exited from the GC, affinity matured B cells undergo the process of class switch recombination (CSR), that regulates isotype switching and ultimate effector function of the immunoglobulins (Igs). This latter process is also regulated by AID (130–142) (**Figure 1A**).

This organizational program in SLOs is maintained by the anatomical differentiation of specialized, resident stromal cells that regulate migration and functional activation of the immune cells in the different part of the follicle (138, 143– 149). The development of this stromal network and the signals required for his homeostasis have been reviewed elsewhere (150). TLS display a similar anatomical structure to support naïve B and T cell recirculation, including the expression of homeostatic lymphoid chemokines CXCL13, CCL21 and CCL19 and the molecular complex peripheral node addressin (PNAd) (97, 98, 151, 152). However, the complex anatomical compartmentalization displayed in SLO is rarely acquired in TLS. While the majority of reports on TLS describe a certain degree of T/B cell segregation, vascular/stromal cell specialization and expression of lymphoid chemokines, the presence of a more complex organization of the TLS and the formation of functional GC is highly variable within and amongst diseases (4, 153– 155). In TLS that form during chronic autoimmune processes, the establishment of such disorganized microenvironment, rich in survival factors and pro-inflammatory cytokines, but likely missing key checkpoints for autoreactive cells screening, is likely responsible for the local generation of pathogenic autoantibody specificities and oncogenic mutations, ultimately favoring disease progression (1, 9, 97, 98).

# TLS IN AUTOIMMUNE CONDITIONS: A LESSON FROM RHEUMATOID ARTHRITIS AND SJÖGREN'S SYNDROME

In 1996, Nancy Ruddle described the presence of a "structural chronic inflammatory process" caused by ectopic production of lymphotoxin, in the context of chronic inflammation of the pancreas (156). Since then, TLS formation has been associated with a localized process of inflammation at sites of infection, autoimmunity, cancer, and allograft rejection. The ultimate pathogenic role of TLS is still debated (98, 151) and most likely depends on the context, organ and type of disease. For the scope of this review we will focus on the role of TLS in supporting the autoimmune process in chronic autoimmune conditions and we will discuss the role of TLS in Rheumatoid Arthritis (RA) and primary Sjögren's Syndrome (pSS) (33, 36, 135, 151, 157–162).

RA is the most common rheumatic autoimmune condition, affecting 0.5–1% of the global adult population. The pathological features of the disease include severe inflammation of the synovial membrane that, in some cases, leads to tissue destruction and subchondral bone erosions (163–166). Histologically, the disease can be classified in 3 main histopathological subtypes: a lymphoid type, mainly characterized by T and B cell aggregates that form TLS; a myeloid type, characterized by diffuse infiltration of prevalent monocyte and macrophages; and the fibroid type, defined by scarce or no immune cell infiltration and prevalent synovial fibroblast hyperplasia (151).

The presence of a ". . .marked infiltration of chronic inflammatory cells (lymphocytes or plasma cells predominating) with tendency to form "lymphoid nodules" was recognized already in the 1957 RA classification criteria (167). In 1972, Munthe and Natvig suggested that the RA synovial membrane is similar to an active lymphoid organ, containing many lymphoid follicles with GC that undergo local division and differentiation into plasma cells with restricted Ig production (168). Later, Steere and colleagues described "elements found in normal organized lymphoid tissue" in synovial lesions from both RA and Lyme disease patients (169); suggesting that the formation of GC-like structures in the synovium is not specific for RA and can be driven by the local antigenic stimulation. It took, however, more than 40 years after these first descriptions to introduce the concept that B-cell affinity maturation could arise within the inflamed synovium (170). It is now accepted that TLS are present in less than half of RA patients who display so called "lymphoid" synovitis (151) and that, in those patients, the presence of TLS is associated with differential prognosis and disease manifestations (151). TLS formation in the synovia have been also identified in patients with psoriatic arthritis (171) and ankylosing spondylitis (172, 173).

A similar phenomenon of leucocyte aggregation in lymphoid like structures occurs in the salivary glands of patients affected

by pSS, a disease characterized by chronic inflammation of the exocrine glands, with progressive loss of function (sicca syndrome) and systemic activation of the humoral response (174). Excessive B cell hyperactivity and extra-glandular manifestation are observed in ∼30% of pSS patients and an increased risk for lymphoma development has been described in this condition. In 1974, Chused et al. first described the presence of lymphoid-like structures in the salivary glands of patients with pSS (175). This was followed by the report of local antigenic stimulation within GC-like structures in the salivary glands (176) and, 10 years later, by the description of FDC network formation within the aggregates (177). In 1998, Stott and colleagues provided the first experimental evidence of an antigen-driven GC response, defined by clonal B cell proliferation and clone hypermutation within the salivary gland inflammatory foci (37), and, since then, several features associated with

predominant infiltration of MB and marginal zone B cells (MB). Aberrant production of survival and chemoattractant signals is observed at these sites.

lymphoneogenesis have been reported within pSS aggregates (157, 178).

It is now recognized that during pSS, TLS form in the minor salivary and/or parotid gland in around 30–40% of patients (151) and those structures host a phenomenon of oligoclonal B cell expansion and SHM of the Ig variable genes (37). The formation of TLS in pSS salivary glands correlates with increased B cell hyperactivity, the presence of anti-SSA and anti-SSB autoantibodies, hypergammaglobulinemia and cryoglobulinemia, supporting the hypothesis that TLS persistence contributes to disease progression in pSS (179). Our group has contributed to these reports, describing both the expression of lymphoid chemokines and of AID within highly organized aggregates that harbor in the salivary gland of patients with pSS and MALT lymphoma (135, 180). The relationship between TLS formation and disease progression in pSS is still debated. TLS detection has been associated with high antibody titer, systemic manifestations and lymphoma development. However, the direct correlation between GC formation in the salivary glands and lymphoma formation has not been demonstrated, suggesting that the development of GC+ TLS within the salivary glands represent one of the stages in the process of lymphomagenesis but is not per se sufficient to induce lymphoma (135, 154, 161, 180–182).

In order to better understand the pathogenic effect that TLS play in disease it is important to dissect the elements, present within these structures that contribute to their function and persistence in the tissue.

# STRUCTURAL ELEMENTS OF TLS

# Antigen

There is enough evidence to support the hypothesis that TLS form to provide an immune response against locally displayed antigens. There are suggestions that TLS formation is an antigen (Ag)-driven process. In the mucosal associated lymphoid tissue that forms during Helicobacter Pilori gastritis antigen clearance following antibiotic treatment impacts on TLS maintenance and progression to lymphoma (183), similarly inducible bronchial associated lymphoid tissue can dissolve upon antigen clearance (184). Maffia and colleagues explored the properties of Ag presentation within TLS (58, 185) demonstrating that Ag presentation is regulated by a random process of diffusion, rather than selective Ag uptake by DCs. Those data are reinforced by the anatomical structure of TLS where conduits, able to support Ag movement and APC migration have been described (186). In this context, the absence of a capsule could favor not only the initial Ag delivery in the tissue, but the progressive accumulation of new antigen specificities during the course of the immune response, favoring the persistence of these structures in the tissue.

During a classical immune response, the antigens are collected by antigen presenting cells in the periphery and moved, via a complex network of lymphatic vessels, to draining lymph nodes (LNs) (187–189). LN space is pre-formed during the embryonic development and anatomically set before the generation of the immune response to accommodate optimal interaction between APC, Ag and immune cells. Differently by SLOs, TLS organization is not anatomically predisposed to organize such a response and Ag presentation is often provided by non-immune cells, such as stromal cells and epithelial cells (190–193).

Lack of Ag drainage could mechanistically explain TLS formation. TLS form spontaneously in the lungs of mice deficient for CCR7, a chemokine receptor required for the migration of antigen-charged dendritic cells (DCs) to draining lymph nodes (194). However, the reconstitution of these animals with CCR7 sufficient cells is enough to re-establish the physiological delivery of the antigen to the lymph node and to induce TLS resolution in the tissue. This evidence appears to suggest that an intrinsic defect in DCs is sufficient to trigger TLS establishment. However, it is not clear whether this phenomenon could be also supported by a defect of lymphatic drainage from the inflamed tissue.

The expansion of a functional network of lymphatic vessels is required for appropriate antigen delivery to the SLOs. There are several reports describing the dramatic remodeling of the lymphatic vessels during inflammation, whereby the activation of NF-κB pathway supported by the expression of LT, IL-1 and TNFα, stimulates the expression of Prox1 and increases the transcripts for the VEGF-R3, both of which are factors involved in lymphoangiogenesis (195–201). TLS lack the presence of an organized lymphatic system such as the one described in SLOs (152). However, the expansion of the lymphatic vascular system has been observed in these structures, in response to the same cytokine milieu that regulates the maturation of the non-vascular stroma at these sites (97, 105). It is not clear whether these newly formed vascular structures are, however, able to establish viable connections with pre-existing lymphatics. The failure to do so would prevent efficient drainage of the antigen to the SLOs and support the excessive antigenic stimulation in the peripheral tissue (89, 202–206).

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. Defects in lymphangiogenesis in RA present with a reduction in lymphatic flow, absence of lymphatic pulse and collapse of draining LNs is observed during disease and is associated with flare onsets as has been shown in vivo and ex vivo studies performed by Schwarz and colleagues (207). Accordingly, effective therapeutic approaches in RA, including anti-TNF and B cell have been associated with the expansion of the lymphatic bed (208) and increase in cell drainage from the synovium (209).

In a model of pSS our group demonstrated that during TLS assembly an expansion of the lymphatic vascular network takes place and this is regulated by the sequential engagement of IL-7 and LTβR signaling; suggesting the presence of a natural proresolving mechanism for lymphocyte exit from the tissues during TLS establishment (105).

The open questions related to the mobilization of Ag loaded APC to the draining SLOs could be addressed in the future by inducing TLS formation and tracking the movement of labeled antigen-loaded DCs across vessels. The possibility to interfere pharmacologically or genetically with the process of lymphoangiogenesis and with the molecules responsible for cell migration across these structures, is likely to elucidate this complex phenomenon and to provide evidences on the role of aberrant antigen presentation and vascular disturbances in TLS establishment and persistence.

Both RA and pSS are characterized by antibody production against a discrete set of autoantigens and a large body of research in this area has been focused around the identification of antigen specificities within the TLS in the context of these diseases. The presence of citrullinated proteins has been reported within the synovia of RA patients by Baeten (210) and others, and associated with the local expression of the enzyme peptidyl arginine deiminase (PAD) in patients characterized by high systemic and local levels of anti-citrullinated antibodies (APCA) (211). This report fails to demonstrate the presence of the citrullinated proteins within the synovial TLS and is in disagreement with other studies reporting the detection of citrullinated proteins in non-RA synovium lacking classical TLS (212); casting doubts on the exclusive association between citrullinated protein expression and TLS development in RA. Additional evidence that associate the presence of TLS with the generation of auto-antibody specificity against citrullinated peptides (but not necessarily local display of the defined antigen) will be discussed in a different section of this review.

Stronger evidences supporting the link between TLS and local auto-antigen presentation have been provided in pSS. Ro/SSA 52 kDa, Ro/SSA 60 kDa and La/SSB belong to a intracellular complex of RNA binding proteins that is physiologically involved in the intrinsic response to viruses (213). The aberrant expression of Ro and La has been reported in pSS patients upon cellular apoptosis or extracellular transport in vesicles (214–216). Moreover, the presence of anti-Ro52/TRIM21 specific plasma cells has been demonstrated, at the boundaries of well-organized TLS in pSS salivary glands, establishing a clear connection between local antigen presentation and TLS formation in this disease (158). The presence of extractable nuclear antigen (ENA) antibodies against these two ribonucleoproteins is pathognomonic for Sjogren's and associated with more severe systemic manifestation and worst prognosis (214, 216, 217).

# Lymphocytic Components of TLS

We have recently reviewed the role of non-haematopoietic cells in TLS establishment and organization (97, 98) and for the scope of this issue focused on autoantibodies, we will limit the discussion in this manuscript to the lymphocytic compartment.

Whilst mainly constituted of B cells and associated with aberrant humoral responses and GC formation, TLS establishment and maintenance strongly relies on T cells. In humans, the presence of a shared TCR specificity among different follicles in the RA synovium, has been described, suggesting the presence of a common antigen for different TLS that form within the synovial tissue (218). In line with this finding, depletion of CD8+ T cells in human synovium-SCID mouse chimeras hinders the formation on TLS (218).

Recently, efforts have been made to identify the cells and signals required for TLS establishment and a series of reports have highlighted the important role of IL-17+ T cells. Th17 cells are required for iBALT formation (219) and for TLS establishment in a model of experimental autoimmune encephalomyelitis (EAE); the latter, dependent on the production of LT-αβ, IL-17 and IL-22 (22, 23, 220, 221). In human renal allograft rejection, Th17 cells have been shown to promote ectopic GC formation in an IL-21 dependent manner (222). Aberrant differentiation of Th17 cells in the absence of IL-27 has been also associated with aberrant TLS formation in an experimental model of arthritis and in a model of pSS (223, 224).

Our group has recently demonstrated the requirement of IL-22 producing T cells in the early phases of TLS establishment in murine salivary glands (39). In this model, IL-22 production, similarly to the IL-17 production in the lungs and brain, appears to regulate, independently but also in synergy with lymphotoxin and TNF, the ectopic production of lymphoid chemokines that defines TLS formation (97). These studies demonstrate that T cells, and in particular Th17/Th22 cells, play an important role in shaping the constituents of TLS in a manner that can support subsequent B cell recruitment and germinal center formation.

Whether TLS provide a site of aggregation for naïve T cell is not clear and whilst naïve T cell recruitment and priming has been reported within TLS that form in pancreatic tissue in NOD mice (225), it is more likely that effector T cells and central memory recirculate in these structures, in particular in the earliest phases of TLS assembly. On the contrary it is now well accepted that TLS function as a site for functional T cell polarization. TLS maintenance appears to hinge on the functional relationship between T-follicular helper cells and regulatory T cell populations. T cells displaying a Tfh phenotype have been described in TLS, where they are expected to regulate the GC reaction and the activation of resident proliferating B cells (1). In the TLS that form outside the arterial wall and control the atherosclerotic plaque development, the presence of Tfh correlates with the organization and maintenance of the ectopic B cell clusters (226). Functional interference of the Tfh by ICOS-L blockade results in decreased TLS formation and aberrant atherosclerotic plaque formation. The opposite effect is obtained by depletion of T regulatory cells, previously demonstrated to play a critical role in the homeostatic control of the TLS and in the atherosclerotic process (58).

The developmental program of Tfh in TLS is debated. There are suggestions that this population in TLS derives from a population of peripheral CXCR5+ T cells that migrate to the peripheral tissue following the newly established CXCL13 gradient. These circulating CXCR5+ cells do not bear classical signs of activation and would, by definition, preferentially migrate to SLOs; however, the local differentiation of HEVs and the expression of PNAd (the ligand for L-selectin) supports their homing to the TLS (227). Others suggest that, within TLS, Tfh locally differentiate from other T cell subpopulations, including Th17. In support of this hypothesis, in EAE, Th17 cells appear to acquire some characteristics of Tfh including the expression of CXCR5, ICOS and Bcl6 (23). Similarly, within the inflamed joints of RA patients, a population of PD1hiCXCR5−CD4+ T cells termed "peripheral helpers" has been described that appear to fulfill the function of Tfh within the periphery (228). In pSS the expansion of Tfh cells has been reported and correlates with the increasing frequency of memory B and plasma cells in the tissue and blood (229, 230).

Genetic manipulation in conditional knockouts is currently in use to induce TLS formation in mice deficient for specific T cell populations and will allow better definition of cellular requirements for TLS formation.

Classically, fully established TLS are mainly characterized by B cell infiltration and the inversion of the B/T cell ratio within TLS has been used as an index of disease severity (231, 232). In SLO, naïve B cells are known to receive antigen education and co-stimulation; however, whether a similar phenomenon would regularly occur in TLS is debated. Patients with pSS display altered peripheral blood B cell frequencies with a predominance of CD27− naïve B cells, diminished frequencies/absolute numbers of CD27+ memory B cells in the periphery, and an enrichment of mature B cells in the salivary glands (233, 234). The presence of CD20+CD27+ B cells and plasmablasts is a consistent finding in pSS salivary glands biopsies (235). Whilst we have reported the presence of IgD+ naïve B cells, in particular in large TLS (180), memory B cells remain the predominant component of the infiltrates (180, 236). This casts doubts over the possibility that naïve B cells are primed within the TLS (235). In support of this hypothesis, bona fide GC B cells (CD10+CD21+/−CD24+/−CD27−CD38+IgD− that express AID) are rarely found within the B cell aggregates of TLS, that are mainly inhabited by CD10−CD21+CD24+CD27±CD38−IgD+ marginal zone-like type II transitional B cells (159) (**Figure 1B**).

The connection between the marginal zone (MZ) and TLS establishment is also not clear. There are several evidences supporting the involvement of MZ B cells in autoimmunity, including reports of preferential SHM and B cell proliferation in MRL Fas/lpr mice spleen (237) and the presence of RF+ cells in the splenic marginal sinus bridging channels (238). The low threshold of BCR activation, the numerous effector functions of MZ B cells and the link between autoimmunity, TLS and MZ lymphoma development in pSS suggests a direct involvement of this population in TLS pathology (239, 240). However, the origin of the MZ-like B cells and the relationship between those and the ectopic GC has not been proven. In humans, MZ B cells are allegedly able to recirculate and carry a highly mutated B cell receptor (241–243), thus suggesting a post-GC origin of this population. This is not the case in mice, where MZ B cells are stable and permanently located in the spleen (242–244). Interestingly, however, MZ-B cells in humans share some phenotypic features of transitional B cells, a highly autoreactive B cell population that emerge from the BM and mature inside the spleen before entering the follicle (245–248), suggesting the possibility that transitional immature autoreactive cells are inhabiting the ectopic follicles. The recirculation pattern and screening of transitional B cells has been described from Spencer and co-authors in an elegant work that describes the migration and BCR editing of this population in the gutassociated lymphoid tissue (GALT) (245). This process, aimed at modifying the specificity of autoreactive clones, is altered in systemic lupus erythematosus (SLE), resulting in the expansion of the autoreactive B cell repertoire (245). In diseases characterized by TLS formation, such as pSS and RA, this recirculation pathway could be also altered, favoring the migration of autoreactive clones from the lymphoid organs to the TLS. Hereby, the aberrant expression of survival factors and chemokines would support clonal expansion in the absence of BCR editing and support persistence of autoimmunity.

The use of mass cytometry on digested tissue and sections are needed to better characterize in humans the phenotype and functional features of the B cells inhabiting the TLS. The use of transgenic mice engineered to track cells in vivo (249) will be useful in inducible models of TLS to perform migration studies in vivo.

# TLS AS ABERRANT MICROENVIRONMENTS FOR AUTOREACTIVE B CELL SURVIVAL AND DIFFERENTIATION

More than simply acting as a hub for lymphocyte migration, TLS have also been shown to provide critical survival signals for incoming lymphocytes and differentiated long-lived plasma cells such as BAFF, IL-7, and CXCL12 (98, 250). The persistence of TLS in the tissue, despite peripheral B cell depletion of post Rituximab, has been reported in RA (251), SS and lymphoma (252) and, more recently in peri-bronchial TLS described in two patients with cystic fibrosis and chronic Pseudomonas aeruginosa infection treated with B cell depletion therapy before transplantation. The reason for this persistence most probably resides on the excess survival factors, such as B cell activating factor (BAFF) or IL-7 present within the TLS that protects tissue infiltrating cells.

BAFF is a potent B-cell survival factor produced within SLO GCs and in the periphery by fibroblasts and epithelial cells (159, 248–253) Excess BAFF is known to rescue selfreactive B-cells from peripheral deletion and allows their entry into forbidden follicular and marginal zone niches (253). The connection between BAFF, MZ B cells, loss of tolerance and TLS emerged from studies in mice transgenic for BAFF (BAFF-Tg), that develop a lupus-like syndrome followed by infiltration of MZ-like B cells within salivary glands TLS (254). Interestingly, BAFF-Tg asplenic mice that lack MZ-B and B1a cells, but retain normal B1b cell numbers, develop lupus nephritis but lack TLS in the salivary glands, suggesting that both BAFF and MZ-B cells are required for TLS establishment in this model (255).

Other lymphoid survival cytokines including IL-7 have been described in association with TLS establishment in chronic diseases (162, 256–258). Gene expression levels of IL-7, IL-7 receptor (both IL-7Rα and IL-2Rγ subunits) and its downstream signaling gene JAK3 are significantly elevated in RA patient biopsies displaying TLS (259). Similarly, engagement of the IL-7/IL-7R axis has been linked to formation of TLS in salivary glands and associated with pSS pathology (22, 23, 33, 36, 37, 39, 58, 75, 89, 102–112, 112–262). Among other critical homeostatic functions, IL-7 can abrogate the suppressive ability of Treg, altering the balance between pro-inflammatory effector cells vs. suppressive T cells (162, 256, 258). Consistent with these observations, in vivo studies demonstrated the ability of IL-7 to induce TLS formation (263–265). The reciprocal expression of T and B cell survival factors in TLS is somehow strictly regulated by the critical balance between infiltrating T and B cells, probably in response to gradients of lymphotoxin and TNF family members. The mechanism regulating this production and the resulting segregation of lymphocytes in T or B cell rich areas is still under investigation (266).

We and others have provided evidence that a functional GC response takes place within these structures. This supports the concept that even if TLS do not initiate disease they are involved in its progression. In particular, we have demonstrated that AID is expressed in pSS salivary gland TLS in association with networks of follicular dendritic cells (135) and that its expression is retained in the large GCs found in parotid pSS-MALT lymphomas. On the contrary, neoplastic B cells are found to be consistently negative for AID expression (135). AID expression in GC B cells controls susceptibility to apoptosis, ultimately regulating the magnitude of the GC response (267). In SLO, low levels of AID expression have been associated with defective somatic hypermutation and decreased peripheral B cell tolerance (268). AID expression in TLS is consistently low (as compared to SLOs), thus potentially explaining the aberrant survival and lack of selection of autoreactive B-cell clones in ectopic GCs.

Other data have been generated supporting the functional role of TLS in sustaining the generation of novel antibody specificities. Transplantation of TLS from pSS salivary glands infected with Epstein-Barr virus (EBV) into SCID mice have been shown to support the production of anti-Ro 52/anti-La 48 and anti-EBV antibodies and the survival of autoreactive B cell clones (158). Similar data have been produced for RA. The presence of CD138+ plasma cells, characterized by immune reactivity against citrullinated fibrinogen, has been described within AID+/CD21+ follicular structures (33). Moreover, the survival of these clones in a transfer model of human biopsies in SCID mice, alongside the detection of gamma-Cmu circular transcripts in synovial grafts, has been reported. These observations provide evidence that synovial TLS represents an independent compartment for B cell maturation (33).

# AUTOANTIBODY PRODUCTION GOES LOCAL

The contribution of the immune response that arises within TLS toward disease severity, including the production of autoantibodies, remains controversial (151). Nonetheless there are substantial evidences in support of local antibody production within the inflamed synovium and convincing documentation that the synovial microenvironment could independently favor the production of RA specific antibodies (33)

Mellors et al. firstly described the presence of "plasma B cells" that are able to react with FITC-labeled human IgG, interpreting this result as evidence of synovial production of rheumatoid factors (RF) by tissue-resident plasma cells (269). The first solid indication of local IgG production in RA is dated to 1968 with the report of Ig synthesis in rheumatoid synovium in vitro (270). Further studies supported this observation suggesting that gene selection, usage of kappa/lambda chains and class switching follows a non-stochastic process in the RA synovium (168). Similarly, the enrichment in RF+ B cells producing monoreactive, affinity matured, class switched antibodies in the RA synovium is highly suggestive of a local process of affinity maturation (271–273). On the contrary, clones producing monoreactive RF have not been obtained from the synovial tissue of patients with osteoarthritis, where TLS do not form, supporting the link between chronic autoimmune diseases and TLS (271– 275).

The production of anti-citrullinated protein antibodies (ACPA) has been firmly associated with RA development (276) and there are convincing evidences that these specificities can be locally produced in the RA synovium within the TLS (277, 278). Both anti-cyclic citrullinated peptide (CCP) antibodies (279) and anti-CCP producing B cells (280) have been detected in the synovial fluid of RA patients and antibodies against different citrullinated RA candidate antigens (vimentin, type II collagen, fibrinogen and α-enolase) appear to be enriched in the joint compared to paired serum (281). Notably, the presence of anti-CCP antibodies in the synovium has been also reported in RA seronegative patients (279, 282), thus highlighting the dissociation between the systemic and local autoimmune response. In support of this notion, the presence of FcRL4+ ACPA producing IgA-B has been reported in the synovium, but not in the blood of RA patients (283). This observation provides an indication that inflammatory joints provide a specific microenvironment able to shape and influence B cell immune phenotype and output.

The ability of TLS to sustain the whole autoimmune process in the absence of SLO is debated. However, cloning of the local B cell repertoire isolated from inflamed organs bearing TLS is highly suggestive of the presence of a functional and SLO-independent process of affinity maturation. Terminally differentiated CD20−CD38+ cells, rheumatoid factor (RF) producing B cells have been detected in the inflamed joints of RA patients (284). Moreover, clonal analysis has provided evidence of an antigen-dependent process of SHM, selection and isotype switching in TLS positive RA synovium, indicating that a dominant antigen-specific local immune response shapes the synovial plasma cell repertoire (170, 285–290). Similarly, in pSS, the multiclonal expansion of B cells within the salivary glands has been described. Expansion of B cell clones bearing Humkv325, a conserved V kappa gene usually associated with lymphomas, was described previously in 1989 (291). Additional studies further supported the notion that an antigen-driven germinal center-type B cell response and somatic hypermutation occurs within the salivary glands (37, 292, 293). The presence of clones that expand and mature in the TLS does not prove that the autoimmune process is initiated within the TLS, or that the presence of TLS is causative of disease. However, a certain degree of antigenexperience and affinity maturation of the B cell repertoire undoubtedly occurs within TLS (33, 135, 153, 160, 294–296) and, whilst the causal role of these structures in disease initiation cannot be proved, TLS certainly display the ability to host and perpetuate the autoimmune process. Production of Ig and RF has been shown in other tissues, in addition to the synovium, including rheumatoid pericardium (297), pleura (298), muscles (299), and in the inducible bronchus-associated lymphoid tissue

(iBALT) in patients with pulmonary complications of RA (219). The presence of sputum autoantibodies in the absence of systemic seropositivity, and the increased autoantibody:total Ig ratio in the sputum (300) suggest that lymphoid tissue present in the bronchi of RA lungs can also act as sites of antibody development.

Independent IgG and IgM synthesis has been also described in pSS salivary glands (301) with later studies confirming the presence of RF+ clones in ∼43% of patients with pSS (302) and with the ability of salivary gland infiltrating B cells to secrete antibodies specific for the Ro52/TRIM21, Ro60 and La autoantigens (36, 179, 217, 303). In vitro expression of recombinant antibodies derived from either newly emigrant/transitional mature naïve B cells from pSS patients and healthy individuals confirmed the presence of high frequencies of autoreactive antibodies in both populations. This suggests a general defective peripheral B cell tolerance in this condition (304).

Analysis of Ig levels in different compartments (blood, saliva) has further contributed to our understanding of the ability of TLS to independently produce antibodies. Increased levels of IgA, but not IgG- and IgM-RF, has been detected in the saliva of patients with pSS (305). A study on isotype distribution of anti-Ro/SS-A and anti-La/SS-B antibodies in the plasma and saliva of patients with pSS demonstrated a correlation between the focus score (the measured degree of salivary gland inflammation) and autoantibody titers in saliva or blood. This report establishes a pathogenic link between locally displayed autoantigens, presence of antigen specific B cells in the inflamed tissue and autoreactive Ig levels (306).

# IMMUNOGLOBULINS AND GLYCOSYLATION: THE SWEETER THE BETTER?

It is becoming increasingly clear that antibody post-translational modifications, in particular glycosylation, can influence their function and pathogenicity. However, a relationship between the pathogenic microenvironment established in the TLS and the progressive acquisition of pathogenic post-translational modifications has not been demonstrated.

Glycosylation is the process by which glycans are attached to proteins, lipids and other molecules, thereby altering their structure and influencing their biological activity. Whilst IgG presents a single conserved N glycosylation site within the Fc region, other subclasses are more heavily glycosylated (307). IgG Fc glycosylation determines the binding of the globulins to their receptors, FcRs type I (FcgammaRs) and II (SIGN-R1, DC-SIGN, DCIR, CD22, and CD23), thereby influencing Ig downstream pro-inflammatory, anti-inflammatory or immunomodulatory effects (308, 309). In addition to conserved IgG Fc glycans, ∼15–25% of serum IgG contain glycans within the Fab domain. Intriguingly, the attachment sites for N-glycans to the Fab portion is determined by the process of somatic hypermutation and, accordingly, Fab glycosylation could influence antibody binding, activity, half-life, formation of immunocomplexes and strength of BCR signaling [extensively reviewed in (310)].

The presence of altered glycosylation in RA was suggested in the 1970s, but it wasn't until 1985 when two studies from Oxford and Japan demonstrated different galactosylation profiles between normal individuals and patients with RA or OA (311). Later, Axford and colleagues reported the presence of reduced circulating B cell galactosyltransferase activity in RA (312), which was later confirmed in other studies (313–315). Other posttranslational modifications have been described in RA. Several studies have demonstrated the presence of an altered overall glycosylation status within specific Ig subclasses (316) that can be detected before disease onset (317). This correlates with measures of disease activity (318, 319) and decreased sialylation of RF-IgG, but not in non-RF-IgG (318, 320, 321).

More recently, the degree of IgG glycosylation has been used to monitor treatment effectiveness (321) and, whilst no differences have been observed in the Fc glycosylation pattern between ACPA-IgG1 and total IgG1 in arthralgia patients, a decrease in galactose residues have been observed in patients with preclinical synovitis before the onset of RA; a change probably supported by the increasingly inflammatory microenvironment (322). The increased presence of agalactosylated IgG in the synovial fluid as compared to serum samples of RA has also been reported (323). Finally, Scherer et al. recently demonstrated the presence of autoreactive IgG in synovial fluid with decreased number of galactosylation and sialylation sites as compared to serum. This latter difference appeared to be specific for autoreactive specificities as no difference was observed in total IgG glycosylation (324).

Elevated levels of asialylated IgG have been detected in 60% of pSS patients and those appear to correlate with clinical manifestations, such as Raynaud's phenomenon and arthritis. A strong correlation with rheumatoid factor or IgAcontaining immune complexes was reported (325). Based on IgG galactosylation, the pSS patients can be classified into two groups: one with comparable galactosylation status as in RA patients with the presence of RF, and the other similar to healthy individuals, and RF seronegative (326).

More recently, studies on Fab glycosylation and disease have been performed. Corsiero and colleagues reported the relationship between increased molecular weight of anti-NET antibodies and the presence of N-glycans onto the Fab domain of autoreactive clones in RA, suggesting that the process of SHM occurring in the synovium is responsible for the acquisition of-N glycosylation sites (286). Acquisition of N-glycosylation sites and subsequent enrichment in Fab-glycans in the variable domain of ACPA-IgG has been further confirmed (327, 328). On a similar note, it has been reported that there is a selective increase in Fab-N glycosylation sites in ACPA specific clones. However, the presence of those glycans didn't appear to significantly alter the antigen binding of the APCA. Accordingly, in silico analysis suggested that the added glycans were not located on the antibody binding sites (329). Moreover, an increased frequency of N-glycans in the Fab ACPA domain, but in association with altered antibody affinity, has been also reported (330). Interestingly, this enrichment was more prominent on ACPA isolated from synovial fluid compared with peripheral blood (264), providing evidence that the local microenvironment influences the immunoglobulin glycosylation pattern. Hamza et al. recently reported the high prevalence of acquired IgG Nglycosylation sites in pSS suggesting a hypothesis that in pSS, the selection pressures that shape the antibody repertoire in the parotid glands results from an antigen-independent mechanism and is driven by interactions between glycosylated B cell receptors and lectins within the microenvironment (328). In summary, the glycan composition can have different associations with the disease, depending on the site of glycosylation. Decreased and increased glycosylation for the Fc and Fab portion, respectively, have been associated with RA and SS.

A relationship between post-translational modifications and antibody pathogenicity has been proposed. Leader et al. reported the presence of agalactosylated IgG in synovial immunocomplexes, suggesting a pathogenic role for agalactosylated IgG (331). However, the relationship between glycosylation and RF activity is debated (318, 332). The presence of N-linked glycosylation sites within the Fc portion of target IgG has been also shown to markedly reduce RF binding in vitro (333) whilst the ability of rheumatoid factors to selectively bind hypogalactosylated IgG has been suggested (334). In mice, desialylated but not sialylated immune complexes appear to enhance osteoclastogenesis in vitro (335). Accordingly, artificial sialylation of anti-type II collagen antibodies, including ACPAs, but not other IgG can supress the development of collagen-induced arthritis (CIA) (320, 336).

A potential pathogenic role of IgG glycosylation in pSS pathogenesis, to our knowledge, has not been addressed yet. A recent study pointed out that the Fc-mucin binding is enhanced when antibodies are agalactosylated, offering a mechanistic concept for increased binding on mucosal surfaces of the inflammatory agalocysylated antibodies and potential antibody pathogenicity (337).

Agalactosylated IgG levels were not found to be correlated in twin pairs indicating a low influence from genetic factors for IgG glycosylation (338). However, four loci contained genes for glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3) have been highlighted in genome-wide association studies for loci associated with IgG N-glycosylation (339). There is evidence to support the notion that the microenvironment can influence Fc IgG glycosylation. A recent study illustrates the ability of CpG, IFN-gamma and IL-21 to increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels (340). Stimulation of a mouse B cell lymphoma line with IL-4 and IL-5, but not LPS, has been shown to significantly decrease the terminal glycosylation of secreted IgA (341) and IgM (342), but substantially increase the terminal glycosylation of MHC Class-I (342), suggesting that the glycosylation machinery works in a protein-specific manner.

A mechanistic link between inflammation and posttranslational modification has been recently established by G. Schett's group in a manuscript illustrating the ability of IL-21 and IL-22 to regulate the expression of α2,6-sialyltransferase-1 in newly differentiating plasma cells, thus controlling the glycosylation profile of secreted IgG (320). Interestingly, both T cell-independent B cell activation (343) and tolerance induction with T cell-dependent protein antigens (344) results in the production of sialylated IgG. However, T cell independent vaccination seems to result in a stronger induction of sialylated antigen specific antibodies (345).

IgG glycosylation can also be controlled at an extracellular level. IgG sialylation has been reported in the bloodstream, through secreted ST6Gal1 (326). S-glycosyltransferases have also been shown to alter the IgG molecule at sites of inflammation with local platelets serving as nucleotide-sugar donors (346). Other reports link the process of altered glycosylation to a post-secretory degradative process involving oxygen free radicals (347). All together these reports suggest the possibility that the site of antibody synthesis can profoundly affect the posttranslational profile of the immunoglobulins.

Due to technological limitations, the extent of the disease-related glycan alterations and the role of these modifications in disease pathophysiology has not been thoroughly addressed. A novel microfluidic-based method to identify trace sulphated IgG N-glycans as biomarkers for rheumatoid arthritis has been recently described (348) and high-throughput methods for analysing IgG glycosylation have also been introduced (349). These tools have been only used in selected populations and their application on a larger scale could, in the future, unveil differences and patterns not yet captured.

To our knowledge, there has been no attempt to use these stratification tools in the context of TLS associated pathologies. The differential profile of glycosylation observed in Ig isolated, respectively from serum and synovial compartments suggest the fascinating hypothesis that SLO and TLS differentially regulate these post-translational modifications (323, 324, 350). However, the possibility that Ig derived from SLO and TLS present substantially different "sugary fingerprints" and that those patterns correlate with a certain degree of tissue involvement and disease progression has still to be proven.

# TLS AND LYMPHOMAGENESIS

If the concept of an association between progressive posttranslational modifications of the Ig repertoire and continuous antigen exposure within a highly inflammatory environment is true, we should be able to detect progressive accumulation of Ig in patients undergoing malignant transformation through the course of autoimmunity. The occurrence of non-Hodgkin's lymphoma (NHL) is pathogenically linked to TLS and represents the most serious complication of pSS, but not RA (351).

B cell VH and VL gene analysis for pSS patients with lymphoma revealed several point mutations in the germline genes and intra-clonal sequence heterogeneity, in line with an ongoing somatic hypermutation process sustaining lymphoma growth (352, 353). It is believed that the emergence of monoclonal RF B cells within the polyclonal infiltrate of the salivary gland TLS represents a key developmental step in lymphomagenesis. Chromosomal abnormalities and mutation eventually converge in these B clones that present a proliferative advantage, ultimately converting them into malignant clones. Indeed, there is a strong bias for RF specific B cells in the salivary gland MALT-type lymphoma (354–356), whilst alternative analysis of the B cell repertoire in micro dissected labial salivary glands could not convincingly demonstrate predominance of RF reactivity in the infiltrating clones (357). The relationship between GC formation and lymphomagenesis has been recently challenged and further studies will be required to clarify the pathogenic link between TLS persistence and emergency of malignant clones (161, 182, 358, 359).

The high incidence of acquired N-glycosylation sites found in follicular lymphoma (360) would be suggestive of a similar phenomenon in pSS associated MALT lymphoma; however, contrary to these expectations, patients with MALT lymphoma present low frequency of N-glycosylation sites (161, 182, 358, 359, 361). Longitudinal analysis of the glycosylation and sialylation profile in patients with TLS undergoing lymphoma transformations are needed to address these questions.

# FUTURE PROSPECTIVE AND CONCLUSIONS

In conclusion, TLS formation can be easily considered as a hallmark of tissue autoimmunity. In the past few years a large body of work has been generated aimed at dissecting key aspects of TLS biology, however, many areas have to be further addressed. The inter-dependency between SLOs and TLS has to be better dissected in order to understand whether these immune hubs are functional, both in the early phases as tolerance is broken and, later, during disease progression. The signals regulating migration pathways and differentiation of immune cells within the TLS should also be investigated in vivo with

# REFERENCES

the prospective to target these pathways therapeutically. A better knowledge around the signals involved in TLS establishment and maturation, but in particular, the mechanisms regulating GC formation and regulation should be acquired. Moreover, a specific effort should be made to dissect the functional role of TLS GCs in the development of lymphoma. Finally, key questions should be answered around the cross-talk between the TLS and their surrounding environment, dissecting the permissive factors for TLS formation and persistence in the tissue.

The acquired knowledge on the role of non-haematopoietic stromal cells in TLS biology has been critically important in explaining why these structures are resistant to classical immune cell therapy. In the future, potential combined therapy could be utilized to interfere with the microenvironment alongside targeting immune cells in TLS associated disease that is nonresponsive to classical immunosuppression.

# AUTHOR CONTRIBUTIONS

EP and FB defined the content of the manuscript, contributed to literature search and manuscript writing. SN, DG and SC contributed to literature search and manuscript writing. CS contributed to manuscript writing. EP and FB created graphical illustrations. All authors approved the final version of the manuscript.

# FUNDING

FB is supported by ARUK.

expression in chemokine-containing active intrathyroidal germinal centers. Am J Pathol. (2001) 159:861–73. doi: 10.1016/S0002-9440(10)61762-2

low-grade malignant lymphoma. Am J Surg Pathol. (1999) 23:302–8. doi: 10.1097/00000478-199903000-00009

hastens clinical chronic rejection. J Immunol. (2010) 184:5344–51. doi: 10.4049/jimmunol.0902999

of the interleukin-7 signaling pathway in tissues with lymphoid neogenesis. Arthritis Rheum. (2007) 56:2492–502. doi: 10.1002/art.22748

develop from rheumatoid factor B cells. Arthritis Rheum. (2000) 43:908–16. doi: 10.1002/1529-0131(200004)43:4<908::AID-ANR24>3.0.CO;2-K

**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 © 2018 Pipi, Nayar, Gardner, Colafrancesco, Smith and Barone. 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) and the copyright owner(s) 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.

# Immunoadsorption of Desmoglein-3-Specific IgG Abolishes the Blister-Inducing Capacity of Pemphigus Vulgaris IgG in Neonatal Mice

Maxi Hofrichter 1†, Jenny Dworschak 2†, Shirin Emtenani 1 , Jana Langenhan2 , Fanny Weiß1 , Lars Komorowski 2 , Detlef Zillikens 3 , Winfried Stöcker 2 , Christian Probst 2 , Enno Schmidt 1,3 \* ‡ and Stephanie Goletz 1‡

1 Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany, 2 Institute of Experimental Immunology, Euroimmun AG, Lübeck, Germany, 3 Department of Dermatology, University of Lübeck, Lübeck, Germany

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease which is associated with autoantibodies directed against two desmosomal proteins, desmoglein (Dsg) 3 and 1. Treatment of PV is rather challenging and relies on the long-term use of systemic corticosteroids and additional immunosuppressants. More recently, autoantibody-depleting therapies such as rituximab, high-dose intravenous immunoglobulins, and immunoadsorption were shown to be valuable treatment options in PV. Specific removal of pathogenic autoantibodies would further increase efficacy and usability of immunoadsorption. Here, we tested the capacity of our recently developed prototypic Dsg1- and Dsg3-specific adsorbers to remove circulating pathogenic autoantibodies from three different PV patients. The pathogenic potential of the Dsg3/1-depleted IgG fractions and the anti-Dsg3-specific IgG was explored in two different in vitro assays based on cultured human keratinocytes, the desmosome degradation assay and the dispase-based dissociation assay. In addition, the neonatal mouse model of PV was used. In both in vitro assays, no difference between the pathogenic effect of total PV IgG and anti-Dsg3-specific IgG was seen, while Dsg3/1-depleted and control IgG were not pathogenic. For the samples of all 3 PV patients, depletion of anti-Dsg3/1 IgG resulted in a complete loss of pathogenicity when injected into neonatal mice. In contrast, injection of anti-Dsg3-specific IgG, eluted from the column, induced gross blistering in the mice. Our data clearly show that anti-Dsg3-specific IgG alone is pathogenic in vitro and in vivo, whereas Dsg3/1-depletion results in a complete loss of pathogenicity. Furthermore, our data suggest that Dsg-specific adsorption may be a suitable therapeutic modality to efficiently reduce pathogenic autoantibodies in patients with severe PV.

Keywords: acantholysis, autoantibody, desmoglein, desmosome, immunoadsorption, pemphigus, skin, treatment

#### Edited by:

David William Scott, Uniformed Services University of the Health Sciences, United States

#### Reviewed by:

Cornelia Deeg, Ludwig-Maximilians-Universität München, Germany Robert Gniadecki, University of Alberta, Canada David Andrew Fulcher, Australian National University, Australia

\*Correspondence:

Enno Schmidt<EMAIL_ADDRESS>
†These authors have contributed equally to this work as first authors

‡These authors have contributed equally to this work as last authors

#### Specialty section:

This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology

Received: 12 December 2017 Accepted: 06 August 2018 Published: 03 September 2018

#### Citation:

Hofrichter M, Dworschak J, Emtenani S, Langenhan J, Weiß F, Komorowski L, Zillikens D, Stöcker W, Probst C, Schmidt E and Goletz S (2018) Immunoadsorption of Desmoglein-3-Specific IgG Abolishes the Blister-Inducing Capacity of Pemphigus Vulgaris IgG in Neonatal Mice. Front. Immunol. 9:1935. doi: 10.3389/fimmu.2018.01935

# INTRODUCTION

Pemphigus vulgaris (PV) is a potentially life-threatening intraepidermal blistering autoimmune disease (1–4). Desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1) have been identified as autoantigens in PV (5–8). Dsg1 and Dsg3 are desmosomal transmembrane cadherins that mediate intercellular adhesion of keratinocytes in the skin and surface-close epithelia (3, 6, 9). In PV patients with exclusive mucosal involvement (mPV), autoantibodies are restricted to Dsg3, whereas autoantibodies against both Dsg3 and Dsg1 are associated with skin and mucosal lesions (mucocutaneous type of PV, mcPV) (10–12). In pemphigus foliaceus (PF), autoantibody reactivity is limited to Dsg1 and patients only develop skin lesions. In addition to Dsg1 and Dsg3, a variety of other target antigens have been described in PV including muscarinic and nicotinic acethylcholine receptors, annexins, thyroid peroxidase, desmocollins, and mitochondrial proteins (13–15). While good, albeit not undisputed, evidence for the pathogenic effect of anti-Dsg1/3 antibodies has been provided, less data were reported about the pathogenicity of non-Dsg antibodies (13, 15–20).

Treatment of PV is challenging and has required the long-term use of prednisolone and other immunosuppressants such as azathioprine and mycophenoles (4, 21, 22). Very recently, first-line rituximab, an anti-CD20 antibody that depletes B cells from the circulation for 3–9 months, in conjunction with the short term use of prednisolone has been shown to be significantly more effective and safe compared to the long term use of prednisolone alone (23). Highdose intravenous immunoglobulin and immunoadsorption are two other treatment modalities that reduce serum anti-Dsg autoantibodies and are recommended in refractory and/or severely affected PV patients (21, 22, 24). The reduction of serum autoantibodies in PV appears to be a particularly attractive therapeutic approach since the direct pathogenic importance of pemphigus autoantibodies has been shown in various experimental settings in vitro and in vivo (13, 25).

Whereas plasmapheresis requires substitution with freshfrozen plasma or human albumin, immunoadsorption specifically removes antibodies from the circulation (24). Unfortunately, the use of immunoadsorption is limited by the increased risk of infections due to the parallel reduction of protective immunoglobulins. Thus, removal of Dsg-specific antibodies appeared to be advantageous leading to the recent development of prototypic anti-Dsg1 and anti-Dsg3 adsorbers. The Dsg1/3-specific adsorbers are based on the recombinant Dsg ectodomains coupled to sepharose and allowed the effective removal of anti-Dsg reactivity from PV and PF serum samples in vitro (26). The aim of the present study was to show that by the use of the Dsg1/3-specific adsorbers removal of anti-Dsg antibodies from PV sera is sufficient to abolish the pathogenic effect of pemphigus IgG not only in vitro but also in vivo in neonatal mice. We now also show that anti-Dsg3-specific IgG is sufficient for acantholysis in cultured keratinocytes and blister formation in neonatal mice.

# MATERIAL AND METHODS

# Patients

IgG from 3 PV patients (PV1, PV2, PV3) that were treated with conventional protein A immunoadsorption at the Department of Dermatology, Lübeck, was used (27, 28). The clinical phenotype, age, sex, indirect immunofluorescence (IF) serum titers on monkey esophagus, and anti-Dsg1/3 IgG serum levels by ELISA (Euroimmun, Lübeck, Germany) are shown in **Table 1**. IgG bound to protein A was eluted by glycine buffer (pH 2.8) and immediately neutralized with 1M Tris pH 9.0 followed by precipitation with ammonium sulfate and dialysis against PBS. As no immunoadsorption material from healthy donors is available, we use affinity-purified IgG from sera of healthy volunteers as control. The study was performed following the Declaration of Helsinki. Pathogenicity experiments were positively reviewed by the ethics committees of the University of Lübeck, Germany (file reference, 09-090).

# Affinity Purification of Dsg-Specific PV IgG Using the Entire Ectodomain of Dsg3 and Dsg1

For antigen-specific immunoaffinity purification of anti-Dsg3 and anti-Dsg1 IgG, the entire ectodomains of Dsg3 and Dsg1, respectively, were immobilized on Nhydroxysuccinimide-activated Sepharose 4 Fast Flow (GE Healthcare, Buckinghamshire, UK) as previously described (26). Immunoaffinity purifications were performed as follows. The immobilized protein matrix was transferred into microcentrifuge spin columns (Thermo Fisher Scientific, Darmstadt, Germany) and washed three times with tris-buffered saline supplemented with 5 mM CaCl2 (Ca2+-TBS). The concentrated IgG of the PV patients was diluted 1:1 with Ca2+-TBS and incubated with the immobilized protein for 30 min at room temperature. The flow-through fraction was collected by centrifugation at 500x g for 30 s. After several washing steps with Ca2+-TBS (until OD280 < 0.05) the anti-Dsg3 and anti-Dsg1 IgG fractions were eluted from the matrix with IgG elution buffer (Thermo Fisher Scientific) until the OD280 was below 0.05, and immediately neutralized with 1M Tris pH 9.0. All eluted fractions were pooled and buffer was exchanged to PBS using Vivaspin 500 centrifugal filter units (Sartorius AG, Göttingen, Germany). Finally, Dsg3/1-depleted PV IgG (flow-through fractions) and anti-Dsg3 PV IgG (eluted fractions) were analyzed for anti-Dsg3 and anti-Dsg1 autoantibody reactivity by ELISA (Euroimmun).

## Immunoblotting With HaCaT Extract

HaCaT cells were grown in low calcium Keratinocyte Growth Medium 2, KGM2 (Promocell, Heidelberg, Germany) containing 0.06 mM CaCl2 to confluence and lysed in Laemmli sample buffer. Lysates were fractionated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted as reported (29). After blocking, nitrocellulose membranes were incubated

**Abbreviations:** Dsg, desmoglein; IF, immunofluorescence; NH IgG, normal human immunoglobulin G; PF, pemphigus foliaceus; PV, pemphigus vulgaris; mPV, mucosal pemphigus vulgaris; mcPV, mucocutaneous pemphigus vulgaris.

PV2 M 44 mcPV 1:1,280 103 9,748 PV3 F 72 mPV 1:320 – 6,001

TABLE 1 | Pemphigus vulgaris (PV) patients' characteristics.

mcPV, mucocutaneous PV; mPV, mucosal PV.

with anti-Dsg3 specific IgG (2µg/ml), a monoclonal anti-Dsg3 antibody (1:100, Bio-Rad, Munich, Germany), control IgG (2µg/ml) from a healthy donor and IVIg (2µg/ml, Biotest, Dreieich, Germany) diluted in TBST containing 5% skimmed milk powder plus 1% BSA. As secondary antibodies a horseradish peroxidase (HRP)-conjugated polyclonal goat anti-human IgG antibody (1:1,000, DAKO, Hamburg, Germany,) and a polyclonal rabbit anti-mouse IgG antibody (1:1,000; DAKO) were used. The proteins were visualized using Super Signal West Femto (Thermo Fisher Scientific).

# Desmosome Degradation Assay

The desmosome degradation assay was performed as described previously (26, 30, 31). In brief, HaCaT cells were grown in 8-well chamber slides (BDBiosciences, Heidelberg, Germany) to confluent monolayers. Low calcium Keratinocyte Growth Medium 2, KGM2 (Promocell) containing 0.06 mM CaCl2 was changed to high calcium medium by adding sterile 0.15 M CaCl2 to a final concentration of 1.5 mM calcium. Monolayers were treated with PV IgG, control IgG and IgG fractions collected from Dsg3 and Dsg1 immunoaffinity purification as indicated in the results part. After 24 h incubation at 37◦C in a humidified atmosphere, culture medium was removed and monolayers were washed with DPBS (Thermo Fisher Scientific) and subsequently fixed with 4% paraformaldehyde. After washing, monolayers were treated with 0.1% Triton X-100 (Sigma Aldrich, Steinheim, Germany), incubated with 10% normal goat serum (DAKO) plus 1% BSA (Carl Roth, Karlsruhe, Germany) and then with mouse anti-human Dsg3 IgG1 (1:100 in PBS, clone 5G11; Acris, Herford, Germany) for 30 min at 37◦C and after three times washing with Cy3-labeled goat anti-mouse IgG (1:100 in PBS; Dianova, Hamburg, Germany). Slides were mounted with ProLong R Gold antifade reagent (Life Technologies, Carlsbad, USA) and examined microscopically (BZ-9000, Keyence, Neu-Isenburg, Germany).

# Dispase-Based Dissociation Assay

The assay was performed as reported previously (26, 30–32) with minor modifications. In brief, HaCaT cells were cultivated in 12-well-plates (Greiner Bio-One, Solingen, Germany) with KGM2 (Promocell) containing 0.06 mM CaCl2 in a humidified atmosphere (5 CO2) at 37◦C. At confluence, fresh KGM2 containing 1.5 mM CaCl2 was added. PV IgG (2 mg/mL), control IgG (2 mg/mL), anti-Dsg3-specific IgG fractions collected from Dsg3- and Dsg1-specific affinity purification (20µg/mL) and anti-Dsg3/1-depleted IgG (2 mg/mL), respectively were added and incubated for 24 h. Subsequently, the cells were treated with dispase solution (2.5 U/ml for 30 min; Stemcell Techn., Vancouver, Canada) and subjected to mechanical stress by pipetting 10 times (5 times moderate, 5 times strong) with a 1 ml pipette. Cell fragments were fixed and stained with crystal violet (Sigma Aldrich). Photos were taken from each well and cell fragments were counted manually. Every experiment was performed at least in triplicate.

# Passive Transfer Neonatal Mouse Model

Neonatals from C57BL/6 mice (Charles River Laboratories, Sulzfeld, Germany) were injected subcutaneously <24 h after birth with the respective PV IgG fraction at doses of 3–7 mg/g of total and anti-Dsg3/1-depleted IgG and 300µg/g anti-Dsg3-specific IgG (each IgG batch was applied in 3 mice) with or without exfoliative toxin A (ETA; Toxin Technology Inc., Sarasota, USA) as described in parts previously (33, 34). ETA is a serine protease produced from Staphylococcus aureus which specifically degrades Dsg1 (35). Due to the different expression patterns of Dsg1 and Dsg3 in mucous membranes and the skin, anti-Dsg3 IgG is only pathogenic in the skin when Dsg1 is graded concomitantly (either by anti-Dsg1 IgG or ETA). In contrast, in mucous membranes, anti-Dsg3 antibodies alone are sufficient to induce intraepithelial splitting (12). Here, half of the minimum ETA dose was applied that in preliminary experiments had induced clinical blistering (usually 0.1µg/g bodyweight). After 16–24 h, the mice were clinically evaluated before and after application of mechanical stress at the back and sides of mice (Nikolsky phenomenon). Blood was obtained as well as biopsies from the back for histopathology (H&E staining) and direct IF microscopy. All animal experiments were approved by the Animal Rights Commission of the Ministry of Agriculture and Environment, Schleswig Holstein (98-8/14).

# Immunofluorescence Microscopy

For direct IF microscopy, a polyclonal rabbit anti-human IgG-FITC antibody (Bio-Rad, Hercules, USA) at a dilution of 1:50 in PBS was used for 1 h at room temperature. For indirect IF microscopy, 6 µm sections of monkey esophagus were incubated with human IgG and mouse sera in a dilution range of 1:20– 1:5120 in PBS for 1 h at room temperature. For detection, a FITClabeled polyclonal anti-human IgG (DAKO) at 1:50 in PBS was employed for 30 min at room temperature.

# Statistics

Graphpad Prism 6 was used for the statistical analysis. The dispase assay data across different groups within each patient was compared for its statistical significance using the Kruskal-Wallis test. For all three patients, correction for multiple comparisons was done by post-hoc Dunn's tests to identify significant pairwise differences between the groups.

# RESULTS

# Dsg1- and Dsg3-Specific Adsorption of PV Patient IgG

Anti-Dsg3-specific IgG was immunoadsorbed from total PV IgG in all three PV patients as previously described in Langenhan et al. (26). In addition, in PV1 and PV2, the present Dsg1-reactivity was removed by Dsg1-specific immunoadsorption. To study the effect of Dsg-specific IgG we focused on anti-Dsg3-specific IgG since only two of the three PV patients revealed Dsg1 specifc IgG. Characteristics of patient IgG after Dsg3/1-specific adsorption are summarized in **Tables 2**, **3**. Western blot analysis of cellular extracts of cultured human keratinocytes confirmed the specific purification of anti-Dsg3 PV IgG (**Figure 1**). Indirect IF microscopy on monkey esophagus revealed that total IgG from PV patients as well as Dsg3-specific IgG, but not Dsg3/1-depleted IgG and control IgG from a healthy blood donor, showed the PV-typical intercellular staining of the stratified squamous epithelium (**Figure 2**, **Table 2**).

# Dsg3/1-Specific Depletion of PV IgG Abolishes Pathogenicity in vitro

To evaluate the pathogenicity of the Dsg3-specific PV IgG and anti-Dsg3/1 IgG-depleted fractions in vitro, the desmosome degradation assay and the dispase-based dissociation assay were performed. The PV IgG-induced loss of Dsg3 expression on the keratinocyte cell surface due to internalization of Dsg3 into endosomes and degradation was determined microscopically. Incubation of HaCaT cell monolayers with either total PV patient IgG or anti-Dsg3-specific IgG resulted in an equivalent discontinuous Dsg3 staining at the keratinocyte cell borders (**Figure 3**). In contrast, when cells were treated with anti-Dsg3/1 IgG-depleted fractions and normal human IgG, respectively, Dsg3 staining was uniformly localized to the cell membrane of keratinocytes (**Figure 3**).

In the dispase-based dissociation assay, treatment with PV1, PV2, and PV3 IgG, respectively, as expected resulted in significantly more keratinocyte fragments compared to incubation with normal human IgG (PV1: p = 0.0011; PV2: p = 0.0045; PV3: p = 0.003; **Figure 4**). Incubation of monolayers with purified Dsg3-specific PV IgG from the three patients generated a significantly higher fragmentation level compared to incubation with anti-Dsg3/1-depleted IgG (PV1: p = 0.046; PV2:

extract of cultured keratinocytes. Western blotting of a monoclonal anti-Dsg3 antibody (lane 1; Bio-Rad, Munich, Germany), Dsg3-specific IgG (2 µg/ml) from pemphigus vulgaris (PV) patient PV1 (lane 2), PV2 (lane 3), and PV3 (lane 4) with extract of cultured HaCaT keratinocytes. IgG affinity purified from a healthy volunteer (2 µg/ml) and IVIg (2 µg/ml, Biotest, Dreieich, Germany) are shown in lanes 5 and 6. Molecular weight markers are shown to the left (kDa). The arrow indicates the migration position of desmoglein 3.

aBefore subjection to Dsg-specific adsorption.

bBy ELISA (Euroimmun; lower cut-off 20 U/ml).

cBy indirect immunofluorescence (IIF) microscopy on monkey esophagus.

Dsg, desmoglein; neg., negative.

TABLE 3 | Characteristics of pemphigus vulgaris (PV) IgG fractions (dispase-based dissociation assay).

aBefore subjection to Dsg-specific adsorption.

bBy ELISA (Euroimmun; lower cut-off 20 U/ml).

Dsg, desmoglein; neg., negative.

p = 0.011; PV3: p = 0.02; **Figure 4**). No difference was observed between treatment with anti-Dsg3/1-depleted IgG and normal human IgG (**Figure 4**).

# Anti-Dsg3/1 IgG-Depleted PV IgG Prevents Pathogenicity While Anti-Dsg3-Specific IgG Results in Blister Formation in Neonatal Mice

When injected into neonatal mice, only total PV1 IgG contained enough anti-Dsg1 antibodies for the induction of skin blisters without co-injection of a subclinical dose of ETA (**Figure 5A,** lane 1). For mice injected with PV2 or PV3 IgG, gentle mechanical friction was required to obtain macroscopic blistering (**Figure 5A,** lanes 4 and 7). The injection of anti-Dsg3 specific IgG fractions from all three PV patients (combined with subclinical ETA doses) induced gross skin blistering (**Figure 5A,** lanes 2, 5, and 8). In contrast, anti-Dsg3/1 IgG-depleted IgG from all three PV patients (combined with subclinical ETA doses) failed to induce blistering in neonatal mice (**Figure 5A,** lanes 3, 6, and 9). Lesional skin biopsies revealed suprabasal acantholysis, the characteristic histological finding of PV, after injection of PV IgG and anti-Dsg3-specific IgG, but not after injection of Dsg3/1-depleted PV IgG fractions or ETA alone (**Figure 5D**). By direct IF microscopy, intercellular IgG depositions were found in the epidermis in all PV IgG and anti-Dsg3-specific IgG-injected mice, but not in mice injected with anti-Dsg3/1 IgG-depleted PV IgG or ETA alone (**Figure 5B**). By indirect IF microscopy on monkey esophagus, the characteristic intercellular staining was seen with sera of mice injected with PV IgG and anti-Dsg3 specific PV IgG, but not after injection of anti-Dsg1/3-depleted IgG or ETA alone (**Figure 5C**).

degradation was detected after incubation with total PV IgG (A,D,G) and Dsg3-specific IgG (B,E,H) but not with the PV IgG fractions depleted of anti-Dsg1/3 IgG (C,F,I) and normal human IgG (50 µg/ml; NH IgG; J).

# DISCUSSION

Adjuvant immunoadsorption is a well-established treatment option in a variety of autoantibody-mediated diseases including PV. So far, more than 100 pemphigus patients were reported to have been subjected to immunoadsorption which has been recommended in the guideline of the German Dermatological Society for the treatment of refractory or severe PV (21, 36).

FIGURE 5 | In vivo pathogenicity of pemphigus vulgaris (PV) IgG fractions. Injection of neonatal mice (n = 3/ group) with PV IgG and anti-Dsg3-specific IgG purified from three different PV patients PV1, PV2, PV3) induced flaccid macroscopic blisters (A; lanes 1, 2, 4, 5, 7, and 8; white arrows) and suprabasal splitting as seen by lesional histopathology (D; lanes 1, 2, 4, 5, 7, and 8). No macroscopic and microscopic blistering was induced by PV IgG depleted of anti-Dsg3/1 IgG from the three PV patients (A,D; lanes 3, 6, and 9) or ETA alone (A,D; lane 10). By direct immunofluorescence (IF) microscopy of back skin, an intercellular epidermal staining was observed in mice injected with PV IgG (B; lanes 1, 4, and 7) or anti-Dsg3-specific IgG (B; lanes 2, 5, and 8) but not after injection of anti-Dsg3/1 IgG-depleted IgG (B; lanes 3, 6, and 9) or ETA alone (B, lane 10). By indirect IF microscopy on monkey esophagus, the characteristic intercellular staining (1:80 dilutions are shown) was seen with sera of mice injected with PV IgG (C; lanes 1, 4, and 7) and anti-Dsg3-specific PV IgG (C; lanes 2, 5, and 8), but not after injection of PV IgG depleted of Dsg3-specific IgG (C; lanes 3, 6, and 9) or ETA alone (C, lane 10). Nuclei were counterstained with DAPI.

Furthermore, the results of a randomized control trial comparing the efficacy and safety of immunoadsorption plus best medical treatment with best medical treatment alone in pemphigus are currently evaluated. In PV, it may be of particular value to rapidly reduce the amount of circulating autoantibodies at the beginning of treatment at a stage when other therapies, i.e., corticosteroids, azathioprine, and rituximab, are not yet effective. This assumption is supported by the clear evidence of a direct pathogenic effect of pemphigus autoantibodies as demonstrated by the occurrence of transient pemphigus in neonates of mothers with PV, the correlation of disease activity with serum levels of anti-Dsg1/3 IgG and various experimental models in vitro and in vivo (2, 15, 20, 33). Conventional immunoadsorption is, however, limited due to the risk of hypogammaglobulinaemia and the subsequent risk of infections. This disadvantage would not be applicable for the use of autoantibody-specific adsorbers. Therefore, we have recently developed Dsg1- and Dsg3-specific adsorbers based on the recombinant Dsg ectodomains. We could show that the prototypic adsorbers effectively removed anti-Dsg1/3 IgG from PV and PF sera and eliminated the pathogenic effect of PV and PF IgG in vitro (26).

In the present study, the prototypic adsorbers were employed to investigate whether Dsg1/3-specific adsorption can also abolish the pathogenic effect of PV IgG in vivo. Extending our previous studies (26), we asked the question whether anti-Dsg3 specific IgG alone, i.e., without the addition of non-desmoglein antibodies, is sufficient to induce pathogenic effects in vitro and intraepidermal blistering in mice. Our experiments are of particular importance since the concept that in the great majority of PV patients, the pathogenic effects of autoantibodies are mediated by anti-Dsg antibodies is challenged (13, 16). Furthermore, although Amagai et al. previously showed that affinity-purified anti-Dsg3 IgG prevented pathogenicity in vivo (37), there are still doubts about the specificity of the affinity purification as the recombinant Dsg3 fragment used for this process contained the constant region of human IgG1 that might have also bound to non-Dsg PV autoantibodies (13, 38). In our Dsg1/3-specific adsorbers, only the ectodomains of Dsg1 and 3 were used (26).

Here, we initially demonstrated the high efficiency of the Dsg3/1-specific adsorbers since no anti-Dsg3 or anti-Dsg1 IgG antibodies could be detected in the Dsg3/1-depleted IgG fraction by ELISA. This result was corroborated by Western blotting of anti-Dsg1/3-specific IgG and anti-Dsg1/3 IgG-depleted PV IgG fractions with extract of human keratinocytes. In line, by indirect IF microscopy on monkey esophagus both, total PV IgG and anti-Dsg3-specific IgG but not anti-Dsg3/1 IgG-depleted PV IgG and normal human IgG stained the epithelium.

Next, we demonstrated in two different in vitro assays that PV IgG, depleted from anti-Dsg1/3 reactivity by the use of the Dsg1/3-specific adsorbers, lost their pathogenic effect. No difference between anti-Dsg1/3 IgG-depleted IgG and normal human IgG was observed in both, the desmosome degradation assay and the dispase-based dissociation assay. In contrast, PV IgG and anti-Dsg3-specific IgG obtained after elution from our Dsg1/3-specific adsorbers led to increased desmosome degradation and keratinocyte dissociation, respectively. It has already previously been shown that human keratinocytes loose Dsg3 expression on their cell surface after incubation with PV IgG (39, 40). Nevertheless, we observed that total PV IgG and anti-Dsg3 specific IgG from PV3 resulted in less Dsg3 degradation (**Figures 3G,H**) compared to the IgG fractions of PV1 and PV2. We hypothesize that the weaker desmosome-degrading capacity of both PV3 IgG and PV3 anti-Dsg3-specific IgG may be explained by the lower anti-Dsg3 IgG titers in this patient (**Table 2**).

Furthermore, the different PV IgG fractions were also assayed in the neonatal mouse model of PV. Initially, Anhalt and coworkers reported that the injection of PV serum in neonatal mice recapitulated major clinical and immunopathological characteristics of the human diseases, i.e., flaccid blisters that easily erode when mechanical friction is applied, intraepidermal split formation as detected by histopathology, and the intercellular binding of PV antibodies in the epidermis as seen by direct IF microscopy (33). In the present study, the injection of PV IgG and Dsg1/3-specific IgG led to macroscopic and microscopic blisters indicating that anti-Dsg1/3 IgG alone is pathogenic and does not require the presence of non-Dsg PV autoantibodies. These data are supported by the previous observations that injection of the monoclonal anti-Dsg3 antibody AK23 resulted in blister formation in neonatal as well as in adult mice (41, 42). More important for the future use of the Dsg1/3-specific adsorbers in the treatment of PV patients is our observation that PV IgG fractions depleted from anti-Dsg1/3 reactivity did not induce skin lesions when injected into neonatal mice. These results unequivocally show that non-Dsg antibodies that had been previously described in PV sera directed e.g., against muscarinic and nicotinic acethylcholine receptors, annexins, thyroid peroxidase, and mitochondrial proteins are not a prerequisite for blister formation in PV. In line, these non-Dsg antibodies have not yet been described to be pathogenic in vivo while co-pathogenic effects have been reported in vitro (13, 15, 17, 18, 20, 43, 44). One may speculate that the previously proposed pathogenic effect of non-Dsg antibodies in PV is not a key element for the initiation of blister formation.

In contrast, anti-desmocollin autoantibodies that have been described in pemphigus sera caused desmosome degradation in the desmosome degradation assay, cell fragmentation in the dispase-based dissociation assay, and suprabasal splitting in an ex vivo skin model (45–47). In line, desmocollin 3-deficient mice present with skin erosions and suprabasal intraepidermal blistering (48). However, evidence is accumulating that anti-desmocollin autoantibodies may be more relevant in paraneoplastic and atypical pemphigus than in PV and PF (49–51). In fact, in a large prospective study with more than 330 pemphigus patients, only 4% of all pemphigus sera and 2.7% of PV and PF sera exhibited anti-desmocollin reactivity, while 98% of sera contained anti-Dsg3 and/or anti-Dsg1 IgG (52). These data indicate that only in a small number of PV and PF patients, Dsg1/3-specific immunoadsorption may not be clinically effective although anti-Dsg1/3 antibodies have effectively been decreased. Future studies now aim at applying the Dsg3/1-specific adsorbers in a clinical trial with PV patients.

# AUTHOR CONTRIBUTIONS

MH contributed to the performance of the experiments and the writing of the manuscript. JD contributed to the planning of the project and the performance of the experiments. SE and JL contributed to the performance of the experiments and to the revision of the manuscript. FW contributed to the performance of the experiments and to the revision of the manuscript. LK contributed to the planning of the project. DZ contributed to the

# REFERENCES

planning of the project and to the revision of the manuscript. WS contributed to the planning of the project and to the revision of the manuscript. CP contributed to the planning of the project and to the revision of the manuscript. ES contributed to the planning of the project and to the writing of the manuscript. SG contributed to the planning of the project and to the writing of the manuscript.

# FUNDING

This work was supported by the European Regional Development Fund (project no. 122-09-017) and the Excellence Cluster Inflammation@Interfaces (DFG EXC306/2).

signaling in pemphigus vulgaris. J Invest Dermatol. (2012) 132:346–55. doi: 10.1038/jid.2011.299

**Conflict of Interest Statement:** JD, JL, LK, and CP are employees of Euroimmun AG. Winfried Stöcker is board members of Euroimmun AG. DZ and ES have a research cooperation with Euroimmun.

The remaining 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 © 2018 Hofrichter, Dworschak, Emtenani, Langenhan, Weiß, Komorowski, Zillikens, Stöcker, Probst, Schmidt and Goletz. This is an openaccess 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) and the copyright owner(s) 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.

# Flightless i alters the inflammatory response and autoantibody Profile in an OVa-induced atopic Dermatitis skin-like Disease

*Zlatko Kopecki\*† , Natalie E. Stevens, Heng T. Chong, Gink N. Yang and Allison J. Cowin†*

*Regenerative Medicine, Future Industries Institute, University of South Australia, Adelaide, SA, Australia*

#### *Edited by:*

*Ralf J. Ludwig, Universität zu Lübeck, Germany*

#### *Reviewed by:*

*Luciana D'Apice, Consiglio Nazionale Delle Ricerche (CNR), Italy Kentaro Izumi, Hokkaido University, Japan*

*\*Correspondence: Zlatko Kopecki*

<EMAIL_ADDRESS>These authors have contributed*

*†*

#### *Specialty section:*

*equally to this work.*

*This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology*

*Received: 21 December 2017 Accepted: 25 July 2018 Published: 10 August 2018*

#### *Citation:*

*Kopecki Z, Stevens NE, Chong HT, Yang GN and Cowin AJ (2018) Flightless I Alters the Inflammatory Response and Autoantibody Profile in an OVA-Induced Atopic Dermatitis Skin-Like Disease. Front. Immunol. 9:1833. doi: 10.3389/fimmu.2018.01833*

Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease characterized by excessive inflammation and disrupted skin barrier function. Although the etiology of AD is not completely understood, clinical and basic studies suggest increasing involvement of autoantibodies against intracellular proteins. An actin remodeling protein, Flightless I (Flii), has been shown to promote development of inflammatory mediated skin conditions and impairment of skin barrier development and function. Here, we sought to determine the effect of altering *Flii* expression on the development of AD and its contribution to autoimmune aspects of inflammatory skin conditions. Ovalbumin (OVA)-induced AD skin-like disease was induced in *Flii* heterozygous (*Flii+/−*), wild-type (*Flii+/+*), and *Flii* transgenic (*FliiTg/Tg*) mice by epicutaneous exposure to OVA for 3 weeks; each week was separated by 2-week resting period. Reduced *Flii* expression resulted in decreased disease severity and tissue inflammation as determined by histology, lymphocytic, and mast cell infiltrate and increased anti-inflammatory IL-10 cytokine levels and a marked IFN-γ Th1 response. In contrast, *Flii* over-expression lead to a Th2 skewed response characterized by increased pro-inflammatory TNF-α cytokine production, Th2 chemokine levels, and Th2 cell numbers. Sera from OVA-induced AD skin-like disease *Flii+/−* mice showed a decreased level of autoreactivity while sera from *Flii*Tg/Tg mice counterparts showed an altered autoantibody profile with strong nuclear localization favoring development of a more severe disease. These findings demonstrate autoimmune responses in this model of OVA-induced AD-like skin disease and suggest that Flii is a novel target, whose manipulation could be a potential approach for the treatment of patients with AD.

Keywords: atopic dermatitis, flightless I, autoantibody, inflammation, skin barrier

# INTRODUCTION

Atopic dermatitis (AD) is one of the most common heterogeneous inflammatory skin diseases affecting 20% of children and 1–3% of adults worldwide (1). The disease is associated with impairments in the skin barrier and variable clinical indicators including occurrence of eczematous lesions, pruritus, and cheilitis (1). The etiology of AD is complex and is often characterized by abnormal immunological pathways that manifest in an imbalance of T-helper (Th)1 and Th2 responses (2). Typically, AD is described as having a biphasic course consisting of an acute inflammatory Th2 dominated phase associated with IgE production and a chronic phase distinguished by reappearance of Th1 responses, tissue remodeling, and dermal thickening (3). Histopathologically, AD is characterized by an inflammatory infiltrate consisting of CD4+ memory T cells, mast cells, and eosinophils and a controlled temporal-spatial expression of proinflammatory cytokines and chemokines driving atopic inflammation of the skin (4).

Research within the last decade has found an association between AD and autoantibody development, suggesting the contribution of autoantibodies to the pathogenesis of AD (3, 5–8). It is proposed that tissue damage induced by AD allows exposure of intracellular antigens that are normally inaccessible by antibodies to the extracellular space, where they can interact with B cells and antibodies (9). A broad spectrum of IgE targeting self-antigens have been identified in above 90% of severe AD patients and high-avidity IgG autoantibodies have been proposed as potential diagnostic markers for severe AD (5, 10–12). The fact that these autoantibodies are associated with disease severity implicates their role in both humoral and cellular immunity in AD pathogenesis (5, 12). Antinuclear antibodies have been shown to have both biological and clinical significance acting as sensors of cellular stress and inflammation associated with environmental factors (13). While the presence of autoantibodies can have a protective role as natural autoantibodies (14), the presence of nuclear autoantibodies in AD has been suggested to lead to the continual provocation of the immune system hence contributing to the severity and chronicity of disease.

Flightless I (Flii) is a highly conserved and unique member of the gelsolin family of actin remodeling proteins and a nuclear receptor activator affecting the transcriptional activity of many modulators of tissue remodeling and inflammation (15, 16). Flii has been demonstrated to regulate cytokine secretion and cellular inflammatory responses *via* its intracellular and extracellular effects on toll-like receptors (17–20). Flii expression increases in skin during development and in response to inflammation, injury, skin cancer development, wound healing, and skin blistering (21–23). Over-expression of *Flii* delays the development of an intact skin barrier in the embryonic skin of mice and impairs the recovery of the epidermal barrier post injury *via* its effects on tight-junction formation (22). A recent study has shown that reducing Flii levels either genetically or using Flii neutralizing antibodies decreases erythema, inflammatory cell infiltrate, and pro-inflammatory cytokine secretion in a mouse model of psoriasiform dermatitis (24). Taken together, these findings suggest a possible role for Flii in inflammatory responses mediating AD. Using an OVA-induced AD-like skin mouse model (25), this study aimed to investigate the effect of differential *Flii* gene expression on development of AD *via* characterization of inflammatory and autoimmune responses responsible for AD pathogenesis.

# MATERIALS AND METHODS

# Animal Studies

Mice were maintained according to the Australian Code for the Care and Use of Animals for Scientific Purposes under protocols approved by the Child Youth and Women's Health Service Animal Ethics Committee (AEC916/06/2015). Mice with the BALB/c background and wild-type controls were obtained from inbred litters. *Flii*-deficient heterozygous null mice (*Flii+/−*) and mice carrying the complete human *Flii* gene on a cosmid transgene were maintained as described previously (26, 27). Heterozygous transgenic mice *FliiTg/+* were made by crossing *Flii+/+* with cosmid transgene *Flii+/−*. These transgenic mice were intercrossed to obtain animals homozygous for the transgene *FliiTg/Tg* which carry two copies of the mouse *Flii* gene and two copies of the human *Flii* transgene.

Atopic dermatitis was induced *via* epicutaneous exposure of mice to OVA as previously described (25). Briefly, 10- to 12-weekold female wild-type (*Flii+/+*), *Flii* heterozygous (*Flii+/−*), and *Flii* transgenic (*FliiTg/Tg*) mice (*n* = 8/genotype) were anesthetized using isoflurane inhalation, the skin on the back of mice was shaved and then tape stripped four times by a transparent adhesive tape (Tegaderm) to introduce a standardized skin injury. A gauze patch (1 × 1 cm2 ) soaked with 100 µl of 0.1% OVA (OVA group) in saline or 0.9% saline (control group) was placed on the back skin and secured with Tegaderm dressing. The experiment comprised three 1-week exposures with a 2-week interval between each exposure week (**Figure 1A**). Clinical images of affected skin, transepidermal water loss (TEWL), and erythema measurements were recorded at the end of the third sensitization week. The level of skin erythema was measured daily using a handheld DermaLab Unit (Cortex Technology) following manufacturer's instructions. This instrument uses skin reflectance spectroscopy to determine the redness of inflamed skin. The instrument was blanked prior to placing a probe directly onto the inflamed AD-like lesions on dorsal skin and a reading obtained as previously described (24). Measurements of TEWL were obtained with a calibrated Vapometer evaporimeter (Delfin Technologies, Finland). The probe was allowed to equilibrate for approximately 30 s before a brief measurement period (8 s) as indicated in manufacturer's guidelines. The Vapometer was placed directly onto the inflamed AD-like lesions on dorsal skin (using an 11-mm adaptor) and held there securely for the duration of the measurement. For both erythema and TEWL analysis, three separate measurements were taken per mouse to ensure the entire region of AD-like inflamed back skin was assessed as previously described (24). Mice were then euthanized and skin biopsies collected for histology analysis, immunohistochemistry, and mRNA extraction. Blood was collected for autoimmunity experiments.

# Histology and Immunohistochemistry

Paraffin-embedded fixed tissue samples were stained with hematoxylin and eosin or toluidine blue following established protocols (28). Level of skin inflammation was assessed using skin thickness measurements, and analysis of inflammatory and mast cell numbers in lesional skin of OVA-induced AD-like skin lesions of *Flii+/−*, wild-type, and *FliiTg/Tg* mice using high magnification images and light microscopy using optimized protocols using Image Pro-Plus 5.1 program (MediaCybernetics Inc.) as previously described (24). Skin thickness included epidermal and dermal measurements of OVA-induced AD-like lesions; inflammatory cell number was assessed by counting total inflammatory cells in 10 different HPF of view in OVA-induced AD-like lesions, and mast cell analysis included counting toludine blue positive

and scaling (black arrowhead) in WT and *FliiTg/Tg* mice exposed to OVA. (C,D) OVA exposure leads to development of AD-like lesions demonstrated by increased erythema and transepidermal water loss (TEWL). Decreasing *Flii* expression reduces development of AD-like lesions with significantly decreased erythema and TEWL measurements compared to OVA exposed skin of WT and *FliiTg/Tg* animals. *n* = 8. Mean ± SEM. \*<0.05.

mast cells in entire sections of OVA-induced AD-like skin lesions per mm2 . Immunohistochemistry was also performed on paraffinembedded fixed AD-like skin lesions following antigen retrieval according to the manufacturer's protocols (DAKO Corporation, Glostrup, Denmark). Following blocking in 3% normal goat serum, primary antibodies against CD4 [rat monoclonal, #14- 9766-82 (Thermo Scientific, Australia) (1:200)], T-bet [rabbit polyclonal, #PA5-40573 (Thermo Scientific, Australia) (1:100)], GATA-3 [rabbit polyclonal, #ab106625 (Abcam Australia) (1:100)], and ROR-γ [rabbit monoclonal, #ab207082 (Abcam Australia) (1:3,000)] were applied and slides were incubated at 4°C overnight in a humidified chamber before application of species-specific, Alexa Flour-488 or Alexa Flour-594 secondary antibodies (Invitrogen, Australia) for 1 h at room temperature. Finally, slides were washed and mounted in Fluorescence Mounting Medium (Dako, Australia). Images were captured on Olympus microscope and CellSense Live Science Imaging Software program (Olympus, Germany) used for counting the positive cells in the AD-like lesions of *Flii+/−*, wild-type, and *FliiTg/Tg* mice. Negative controls included replacing primary antibodies with normal rabbit IgG, or normal mouse IgG. For verification of staining, non-specific binding was determined by omitting primary or secondary antibodies. All control sections had negligible immunofluorescence.

# RTq-PCR

Harvested tissue was snap-frozen in liquid nitrogen and total RNA was isolated using Ultraclean Tissue and Cell RNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. Total cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer's protocol. Quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in triplicate reactions. The plates were placed in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Reactions underwent 30 s at 95°C, then 40 cycles of 5 s at 95°C, 20 s at 60°C, and 10 s at 95°C before determination of melt curve between 65 and 95°C. GAPDH and CyPA were used as reference genes and inter-reaction calculator method was applied for all plates. For relative comparison, the cycle threshold value (Ct) was analyzed using the ΔΔCt method and data reported as Ct normalized to reference genes. Sequences for PCR primers are listed in Table S1 in Supplementary Material.

# Autoantibody Immunofluorescence

In order to assess the degree of autoimmunity in OVA-induced AD-like skin of *Flii+/−*, wild-type, and *FliiTg/Tg* mice, sub-confluent primary wild-type mouse keratinocytes were stained with mouse sera of AD-induced mice following established protocols (13, 29). Briefly, primary keratinocytes were isolated from murine epidermis as previously described (30), grown on glass coverslips and washed in 1× phosphate-buffered saline before paraformaldehyde (4%) fixation (10 min at room temperature). Fixed cells were subsequently permeabilized using 0.2% Triton-X-100 and 0.5% BSA in 1× phosphate-buffered saline (5 min at room temperature) before incubation with pooled murine serum (*n* = 3) diluted (1 in 10) in 0.5% BSA in 1× phosphate-buffered saline for 1 h. Bound murine IgG was detected with Alexa Flour 633 goat anti-mouse IgG (1 in 1,000; #A21050; Invitrogen, Mulgrave, VIC, Australia) in 1× phosphate-buffered saline for 1 h at room temperature. Following repeated 2 min washes with 1× phosphatebuffered saline, cells were stained with DAPI nucleic acid stain (Sigma-Aldrich) for 5 min at room temperature before washing and mounting for imaging. Negative controls included omitting the incubation with mouse serum. All control sections had negligible immunofluorescence. Staining pattern of autoantibodies was assessed using the Olympus microscope and CellSense Live Science Imaging Software program (Olympus, Germany).

# Immunoblot Analysis of AD Serum Autoreactive Antibodies

Protein was extracted from *WT* murine keratinocytes using standard protein extraction protocols (31). Samples of extracted protein (20 µg) were run on 10% SDS-PAGE gels (30 min; 200V) and transferred to nitrocellulose by wet transfer (1 h; 100V). Membranes were cut into strips, blocked in 3% bovine serum albumin (Sigma) for 30 min, and probed with murine serum diluted (1 in 10) in tris-buffered saline containing 3% BSA and 0.1% Tween overnight. After washing, horseradish peroxidaseconjugated goat anti-murine immunoglobulin secondary antibody was added for a further 1 h at room temperature. Stringent washes were performed before detection of horseradish peroxidase and exposure using GeneSnap analysis program (SynGene, Frederick, MD, USA).

# Statistical Analysis

Statistical differences were determined using the Student's *t*-test or one-way ANOVA. For data not following a normal distribution, the Mann–Whitney *U* test was performed. A *P* value of less than 0.05 was considered significant.

# RESULTS

# Flii-Deficient Mice Exhibit Reduced OVA-Induced AD-Like Skin Disease

The repeated epicutaneous exposure of OVA (**Figure 1A**) on mouse skin results in an AD-like skin disease with histological and phenotypic features of human AD, including erythema, skin thickening, and a localized immune response (25). *Flii* homozygous (*Flii−/−)* mice are embryonic lethal (26); therefore, using the OVA-induced AD-like skin mouse model, the severity of OVAinduced AD-like lesions was determined in response to different *Flii* gene levels in *Flii* heterozygous (*Flii+/−*), normal (*Flii+/+*), and *Flii* transgenic (*FliiTg/Tg*) mice. All three mice genotypes developed localized inflammation which increased following each week of OVA exposure. *Flii*-deficient mice showed reduced levels of inflammation and scaling (**Figure 1B**). Spectrophotometric measurement of the redness of the OVA exposed skin showed that *Flii*deficient mice had significantly less erythema (**Figure 1B**) than wild-type and *FliiTg/Tg* counterparts at day 50 of the experiment. Similarly, the degree of TEWL was also significantly reduced in *Flii+/−* mice and very similar to that observed in control animals (**Figure 1C**). Control mice administered saline only showed no evidence of AD-like inflammation macroscopically or any development of erythema or TEWL (**Figures 1B–D**).

A hallmark of AD is skin thickening with a marked influx of leukocyte and mast cell inflammatory infiltrate. All OVA-induced AD-like skin sections showed evidence of AD compared to control saline exposed skin including a degree of epidermal hyperplasia and edema coupled with inflammatory lymphocytic and mast cell dermal infiltrate (**Figures 2A–C**). Examining the skin thickness in OVA-induced AD-like skin lesions revealed that deficient *Flii* mice had significantly thinner skin than OVA-induced AD-like skin lesions of wild-type and *FliiTg/Tg* mice (**Figure 2A**). Assessment of lymphocytic and mast cell dermal infiltrate showed that *Flii* deficiency consistently resulted in significantly decreased inflammatory cell numbers (**Figures 2B,C**). In contrast, both normal and increased *Flii* gene expression resulted in thickened epidermis, increased numbers of inflammatory cells (**Figure 2B**) and mast cells (**Figure 2C**). Control mice skin exposed to saline only gauze patch showed no evidence of AD-like dermatitis features microscopically and had low levels of inflammatory infiltrate in the dermis (**Figures 2A–C**).

# Anti-Inflammatory Cytokine mRNA Profiles Are Increased in OVA-Induced AD-Like Skin of *Flii*-Deficient Mice

OVA-induced AD-like skin lesions of *Flii+/−*, wild-type, and *FliiTg/Tg* mice were assessed for *Flii* and levels of cytokines and chemokines mediating the Th1 and Th2 inflammatory responses

during AD pathogenesis. *Flii-*deficient mice showed approximately 25% decrease in *Flii* levels, while *Flii* over-expressing mice had a twofold increase in *Flii* levels compared to wild-type counterparts (**Figure 3**). *Flii* gene levels were found to affect number of key cytokines and chemokines responsible for development of AD-like lesions. Notably, main *Flii* deficiency favored a Th1 immune response and decreased inflammation with significant threefold increase in IFN-γ mRNA levels compared to *FliiTg/Tg* mice, decreased pro-inflammatory IL-4, and increased antiinflammatory IL-10 mRNA levels compared to both wild-type and *FliiTg/Tg* mice (**Figure 3**). Interestingly, *Flii+/−* OVA-induced AD-like skin lesions also showed increased IL-5 and IL-6 mRNA levels when compared to *FliiTg/Tg* mice but not wild-type counterparts (**Figure 3**). In contrast, over-expression of *Flii* resulted in similar cytokine levels to wild-type counterparts, except significantly increased TNF-α mRNA expression and significantly reduced IFN-γ mRNA expression when compared to both *Flii*deficient and wild-type mice, which would favor more severe AD manifestation. Cytokine mRNA levels of IL-13, IL-23, and IL-17A did not differ between OVA-induced AD-like skin lesions of three

inflammation in *Flii+/−* mice skin. Magnification 4× scale bar 200 µm. *n* = 8. Mean ± SEM. \*<0.05.

genotypes (**Figure 3**). Expression of CCL22 mRNA was significantly increased in OVA-induced AD-like skin lesions of *FliiTg/Tg* mice compared to wild-type mice, while OVA-induced AD-like skin lesions of *Flii+/−* mice showed elevated CCL17 chemokine mRNA expression compared to *FliiTg/Tg* mice (**Figure 4**). CXCL9 chemokine mRNA levels were found to be similar in all three genotypes (**Figure 4**). The unbalanced skewed ratio of Th1/Th2 response in *FliiTg/Tg* mice was confirmed by immunohistochemical co-staining of T cell subset markers, namely Th1 (CD4 and T-bet), Th2 (CD4 and GATA-3), and Th17 (CD4 and ROR-γ) showing increased Th2 cell numbers in dermal area of *FliiTg/Tg* mice (**Figures 5A–D**).

# *FliiTg/Tg* OVA-Induced AD-Like Disease Mice Have Altered Autoreactivity Profiles Compared With WT and *Flii−/−* Mice

To compare autoantibody formation in the OVA-induced AD-like disease model between *Flii*-deficient, wild-type, and *Flii*-overexpressing mice, serum samples collected at day 50

Figure 3 | OVA-induced atopic dermatitis (AD) skin-like lesions of *Flii*-deficient mice exhibit increased anti-inflammatory cytokine levels. mRNA levels of Flii, pro-inflammatory, and anti-inflammatory cytokines responsible for manifestation of AD were analyzed in the OVA-induced AD skin-like lesions of *Flii+/−*, wild-type, and *FliiTg/Tg* animals. OVA-induced AD skin-like lesions of *Flii*-deficient mice have significantly higher IFN-γ, reduced IL-4 signaling, and significantly higher levels of anti-inflammatory IL-10 while the *Flii* transgenic counterparts show significantly increased TNF-α and significantly reduced IFN-γ compared to wild-type animals. *n* = 6. Mean ± SEM. \*<0.05.

were assessed by immunofluorescence and immunoblot analysis. Primary keratinocytes from wild-type mice were fixed and probed with pooled sera from experimental mice and staining patterns analyzed using fluorescence microscopy. Cytoplasmic and perinuclear staining within keratinocytes was apparent after incubation with sera from all three genotypes, however, only sera from OVA-induced AD-like disease *FliiTg/Tg* mice produced strong nuclear staining patterns (**Figure 6A**). To further elucidate autoreactivity in these samples, keratinocyte whole-cell lysates were separated *via* SDS-PAGE, transferred to nitrocellulose membrane, and probed with pooled sera (**Figure 6B**). Additionally, autoreactivity patterns of individual mouse sera are shown in Figure S1 in Supplementary Material. Regions of differential autoreactivity were detected at 145, 70, 60, and 43 kDa band sizes (black arrows). Immunoblot analysis of sera (**Figure 6C**) from individual mice found that autoreactivity was highest in wild-type mice (50%) and *FliiTg/Tg* mice (43%), while Flii-deficient (*Flii+/−)* mice showed the lowest degree of autoreactivity (25%). Positive autoreactive bands at 70 kDa were also different in *FliiTg/Tg* mice compared to the two other genotypes, which may be responsible for the nuclear staining pattern observed in immunofluorescence analysis. Immunoblot analysis of sera from normal non-dermatitis mice from each genotype were also analyzed; no autoreactivity was detected (Figure S2 in Supplementary Material).

# DISCUSSION

Flii is a cytoskeletal protein with important roles in skin development, tight junction function, skin barrier establishment, and recovery post injury which is upregulated in response to tissue

inflammation and wounding (22, 31). The secreted form of Flii has been shown to affect innate immune signaling pathways and modulate cell activity and cytokine secretion from fibroblasts and macrophages *in vitro* (17, 20). Recent studies have described the contribution of *Flii* over-expression in exacerbation of inflammatory conditions, including psoriasiform dermatitis (24) and an autoimmune inflammation-mediated epidermolysis bullosa acquisita (23, 32).

mice. Magnification 4× scale bar 200 µm. *n* = 6. Mean ± SEM. \*<0.05.

To determine if Flii affects AD development and severity, mice with low (*Flii+/−*)*,* normal (*Flii+/+*), and high (*FliiTg/Tg*) expression of the *Flii* gene were exposed to repeated epicutaneous exposure to OVA and induced to form AD-like skin lesions. In contrast to our original hypothesis, *Flii* over-expression did not result in a more exacerbated development of erythema and AD-like skin lesions. However, reduced Flii levels in *Flii*-deficient mice led to significantly decreased development of AD-like skin lesions as marked by decreased erythema, TEWL, epidermal hyperplasia, and lymphocytic and mast cell tissue infiltrate compared to both wild-type and *FliiTg/Tg* mice. Despite increased IFN-γ levels, the AD lesions of *Flii+/−* mice had reduced IL-4, which may contribute significantly to the reduced skin thickening in this model of AD (33). These findings are in agreement with studies which demonstrated that reducing Flii expression, either genetically or using Flii neutralizing antibodies, decreased tissue inflammation, and disease severity in mouse models of psoriasiform dermatitis and epidermolysis bullosa acquisita (23, 24, 32).

Genetically modified mice engineered to over-express Th2 cytokines develop skin barrier defects and AD spontaneously (34). The formation of AD lesions is known to be triggered by production of Th2 cytokines by mast cells and CD4+ T cells which also promote IgE production by B cells, while Th1 cells secrete IFN-γ to suppress proliferation of Th2 cells and IgE synthesis (35, 36). The dominance of Th2 cytokines in AD cause decreased expression of fillagrin and other barrier promoting molecules found in the skin (3). Examining the levels of cytokines and chemokines produced in AD-like lesional skin of *Flii+/−*, wild-type, and *FliiTg/Tg* mice revealed decreased IFN-γ levels in *FliiTg/Tg* mice suggestive of Th2 immune responses, while *Flii+/−* had increased IFN-γ levels and decreased clinical AD severity suggestive of potential Flii effect on IgE synthesis, however, this is yet to be investigated. Analysis of T-helper subsets within lesional skin showed a significantly Th2 skewed response in OVA-induced AD-like lesions of *FliiTg/Tg* mice with higher numbers of CD4+GATA3+ cells compared to AD-like lesions of *Flii+/−* or wild-type counterparts who showed significantly higher Th1:Th2 ratio. Additionally, AD-like lesions of *FliiTg/Tg* mice showed increased pro-inflammatory TNF-α cytokine levels and while TNF-α has been demonstrated to promote the AD development (37) we did not observe increased

and perinuclear (green arrow) staining patterns, while antibodies from OVA-induced AD skin-like lesions of *FliiTg/Tg* mice produce a strong cytoplasmic, perinuclear (green arrow), and nuclear (black arrow) staining patterns. (B) Western blot analysis of murine keratinocyte proteins probed with pooled sera from OVA-induced AD-like mice (2–3 mice per lane); black arrows represent regions of autoreactivity. (C) Autoreactivity summary of Western blot results from murine keratinocyte lysate probed with sera from individual mice.

AD severity in these mice in this model of OVA-induced AD. As mast cell numbers were not significantly increased in *FliiTg/Tg* mice, and T-helper cells were heavily skewed toward a Th2 phenotype within *FliiTg/Tg* lesions, excess TNF-α was likely produced by other cell types such as macrophages, which is further supported by increased CCL22 chemokine levels observed in *FliiTg/Tg* mice. Interestingly, previous studies have shown that secreted Flii reduces macrophage secretion of TNF-α *in vitro* (20) while *in vivo* studies using a mouse model of psoriasiform dermatitis showed reduced TNF-α levels in response to reduced Flii (24) which was not observed in this model of OVA-induced AD. These findings may reflect inherent differences between *in vivo* and *in vitro* studies, for example differences in complex *in vivo*, multicellular environments, as well as differences in different models of inflammatory skin diseases.

In agreement with previous studies demonstrating decreased tissue inflammation and inflammatory cytokine secretion in *Flii*-deficient mice (24, 32), OVA-induced AD-like skin lesions of *Flii+/−* mice showed a reduced inflammatory response marked by significantly increased anti-inflammatory IL-10 secretion as well as significantly increased IFN-γ and significantly reduced IL-4 levels. Indeed, IL-4 has been shown to be essential for eosinophil recruitment, Th2 cell differentiation, and IgE production (38). The increased CCL17 expression observed in AD lesions of *Flii+/−* mice compared to *FliiTg/Tg* mice may be a potential mechanism to restore Th1/Th2 balance, as CCL17 has previously been shown to induce a Th2-dominated inflammatory reaction (39). Reduction of *Flii* levels did not alter IL-23 and IL-17A cytokine levels as previously observed in psoriasiform dermatitis (24) suggesting that Th17 responses are not involved in this model of AD. Despite significant differences in IFN-γ levels between three genotypes, CXCL9 chemokine levels were not altered between genotypes in this model of AD. IL-13 levels were also not significantly different between three genotypes, however, there was a trend to higher levels in *FliiTg/Tg* mice OVA-induced AD-like skin lesions and IL-13 cytokine has previously been linked to autoantibody production in early rheumatoid arthritis (40).

Autoimmunity has been increasingly recognized to play part in exacerbating the severity of AD (6, 9, 12) as a consequence of both humoral and cellular and immunity (6). It is postulated that IFN-γ signaling during AD pathogenesis may promote the development of autoimmunity as IFN-γ overexpressing mice spontaneously develop autoantibodies (41, 42) and deletion of the IFN-γ receptor inhibits autoantibody production in lupusprone mice (43). On the basis of our findings showing altered AD severity and altered Th1/Th2 responses including significantly altered IFN-γ expression with different *Flii* genotypes, we examined the effect of Flii levels on autoimmunity in OVA-induced AD-like skin lesions. The decreased severity of AD observed in *Flii+/−* mice also correlated with the reduced degree of autoreactivity (50% reduction vs WT), and further studies are required to investigate whether the autoantibodies from OVA induced AD-like skin mice contribute to or are a product of the observed AD-like symptoms. Immunoblot analysis of sera from normal non-dermatitis mice showed no autoreactivity suggesting that autoantibody development was disease-specific. Interestingly, both *Flii+/−* and wild-type mice showed similar autoantibody staining patterns and regions of differential autoreactivity. *FliiTg/Tg* mice showed similar levels of autoreactivity compared to wildtype mice, however, in addition to the cytoplasmic and perinuclear staining pattern observed in all genotypes, the sera of these mice had a strong nuclear staining pattern. This pattern may indicate the presence of disease-mediating antinuclear antibodies which have been clinically associated with AD and other inflammatory skin disorders (44–46). Flii has previously been shown to affect TLR signaling pathways, both intracellularly and extracellularly, hence modulating innate inflammatory responses and directly impacting immune signaling, however, the potential role of Flii in autoimmunity has not been explored to date. Further studies are required to determine if autoreactivity in *Flii* genetic mice is a direct result of the impact of *Flii* on immune signaling or if it is secondary to increased pathology observed in these animals. In addition, determining the subtype of autoreactive immunoglobulins developed in the OVA-challenged murine model of AD would allow more comparison to be drawn to findings of previous clinical cohorts (10, 47).

While major differences between human AD and murine models have been demonstrated (47, 48), models of AD promote our understanding of the complex pathogenesis of human AD, and identify potential novel targets for design of targeted biologics (49). Here, we have demonstrated that Flii is a novel target in AD and that reducing its levels decreased the severity of AD in

# REFERENCES

the ovalbumin-challenged murine model of AD. Additionally, we have examined the role of autoimmunity in this model of AD and while the exact mechanisms are yet to be identified, our results suggest that the effects of Flii upon Th1/Th2 balance and autoimmunity are important during AD pathogenesis.

# ETHICS STATEMENT

Mice were maintained according to the Australian Code for the Care and Use of Animals for Scientific Purposes under protocols approved by the Child Youth and Women's Health Service Animal Ethics Committee (AEC916/06/2015).

# AUTHOR CONTRIBUTIONS

AC and ZK conceived all the experiments with assistance from NS. ZK and NS carried out experiments and analysis with the assistance of HC and GY. ZK, NS, and AC wrote the manuscript and all authors contributed to the manuscript preparation and approved the final submitted and published versions.

# ACKNOWLEDGMENTS

AC is supported by NHMRC Senior Research Fellowship (GNT#1102617) and ZK is supported by a Future Industries Institute Foundation Fellowship.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01833/ full#supplementary-material.

Table S1 | Primer sequences used in real-time qPCR.

**Conflict of Interest Statement:** IP associated with this project has been filed by AbRegen Pty Ltd., of which AC is a shareholder and both AC and ZK are named inventors on associated patents.

The handling Editor declared a past co-authorship with one of the authors AC.

*Copyright © 2018 Kopecki, Stevens, Chong, Yang and Cowin. 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) and the copyright owner(s) 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.*

# The p.Arg435His Variation of IgG3 With High Affinity to FcRn Is Associated With Susceptibility for Pemphigus Vulgaris—Analysis of Four Different Ethnic Cohorts