The Gram-negative bacterium *L. pneumophila* was first identified as the causative agent of an epidemic of pneumonia at an American Legion convention in Philadelphia, PA, USA in 1976 (97). This disease is characterized by the inhalation of aerosols containing high numbers of *L. pneumophila* (98). Although usually found in freshwater protozoa and amebae, *L. pneumophila* can accidentally replicate in alveolar macrophages in human lung, especially in immune-compromised patients (99, 100). In order to replicate within its eukaryotic host, *L. pneumophila* employs strategies that involve blocking the fusion of phagosomes with lysosomes after phagocytic ingestion of the bacteria and the generation of endoplasmic reticulum (ER)-like compartment that affords its replication (7, 101). The *L. pneumophila*-containing vacuoles (LCVs) present features that are shared by autophagosomes, including its close association with ER membrane (100, 102). This led to the speculation that the formation of biogenesis could involve the autophagy machinery (103, 104). Initial studies that focused on the characterization of the LCV reported that this compartment did not fuse with acidic vesicles since proteins that localize to endolysosomes, such as Lamp-1 and Rab7 were absent in LCV membrane and that the ability of *L. pneumophila* to evade phagosomal maturation was dependent on its viability (98, 105). Further studies using avirulent strains of *L. pneumophila* identified the intracellular multiplication (icm) and defect in organelle trafficking (dot) loci as the genetic loci determinants required for intracellular multiplication and evasion phagosome–lysosome fusion (106, 107). The emergence of autophagy as an antimicrobial effector led to the examination of the role

apparatus in order to deconjugate LC3 from ER-derived vesicles and block autophagy in order to escape from autophagic degradation.

of this process in the pathogenesis of *L. pneumophila* infection. Since LC3 is a major marker for autophagosome membranes, several cell biology approaches aimed to analyze the recruitment of LC3<sup>+</sup> compartments to LCVs (100). Interestingly, following infection of macrophages with *L. pneumophila,* the formation of autophagosomes was blunted. In line with the role of Dot/Icm in the virulence of this bacterium, infection of macrophages with an isogenic Dot/Icm-deficient dotA mutant was unable to induce defects in autophagy induction (108). To identify the bacterial factors involved in autophagy inhibition, Choy et al. conducted a genetic screen that mapped the defect in autophagy to a chromosomal region encoding for 10 effectors. Analysis of the effects of the individual effectors revealed the protein RavZ as necessary and sufficient for blocking autophagy (108). *In vitro* analysis demonstrated that RavZ, which displays cysteine-protease activity, acts to deconjugate LC3 from autophagosomes and block its reconjugation (108). Furthermore, recent reports demonstrate that RavZ might participate not only in the deconjugation of LC3 but also in other steps that interfere with xenophagy. Kubori et al. have found in co-infection experiments with *L. pneumophila* and *S. typhimurium* that the recruitment of ubiquitin, p62, and NDP52 to the surface of *S. typhimurium* was dampened, suggesting a deubiquitinase-like enzymatic activity for RavZ (109). The resolution of the crystal structure of RavZ yielded new clues to its mechanisms. According to this study, by targeting autophagosomes through PIP3- and curvature-sensing motifs, RavZ limits its activity only to LC3 that is bound to autophagosomes (110) (**Figure 6**). Other RavZ-independent mechanisms for *L. pneumophila* evasion of autophagy have been described as well. Phylogenetic analyses suggested a high degree of similarity between one *L. pneumophila* and the eukaryotic sphingosine-1 phosphate lyase (SPL) (111). The *L. pneumophila* SPL homolog (LpSlp) has similar enzymatic activities to the eukaryotic SPL, which finely regulates intracellular levels of sphingosine-1-phosphate (S1P) (112), which have been shown to stimulate autophagy (111, 113). Infection of macrophages with wild-type *L. pneumophila* but not the LpSpl-deficient mutant leads to a depletion in S1P levels and inhibition of autophagy, indicating that *L. pneumophila* uses molecular mimicry to block autophagy and replicate within macrophages (111).

## The Role AMP-Dependent Protein Kinase (AMPK) Activation and Bacterial-Induced Amino Acid Starvation in Bacterial Xenophagy

Living organisms obtain energy from the catabolism of nutrients whose molecular blocks are then converted into ATP and NADPH. The fact that cells are continually synthesizing ATP keeps its level close to maximal, with only small variations (114). However, under nutrient stress, when ATP levels drop, adenylate kinase shifts to an ATP synthesis mode to restore its levels. In turn, AMP levels increase significantly and, physiologically, changes in AMP concentrations are much higher than those observed to ATP (115), which makes the AMP/ATP ratio the most reliable marker of the cellular energetic status (114). Under such conditions, AMPK detects tiny changes in AMP levels and represents the principal cellular metabolism regulator (114). One of the main direct consequences of AMPK engagement is the activation of ULK1, suppressing mTORC1 inhibitory activity to allow the formation of autophagosomes (116).

In addition to its crucial role as a metabolic sensor, AMPK has also been widely reported to be involved in the activation of autophagy by bacteria. In a bacterial peritonitis-induced sepsis model, the use of the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR) increased bacterial killing, suggesting the implication of AMPK in the enhancement of the activity of phagocytic cells. Indeed, the use of these activators led to increased chemotaxis, phagocytosis, and bacterial killing of neutrophils infected with *Escherichia coli* (117). Evidence from the literature demonstrates that activation of AMPK by treatment with AICAR can also increase targeting of *M. tuberculosis* to LC3-positive compartments. Of note, when key autophagic proteins such as ATG7 were silenced, this effect was not observed, suggesting AICAR promotes the targeting of *M. tuberculosis* to autophagosomes. Moreover, AICAR-induced xenophagy was shown to contribute to bacterial killing *in vitro*, in a mechanism involving mTOR inhibition and increased mitochondrial biogenesis and ATP generation, likely as a result of energy drop during *M. tuberculosis* infection. Since it has been previously demonstrated that peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) is important for the regulation of mitochondrial gene expression and glucose metabolism, it was speculated that PPARGC1A was involved in AICAR-induced xenophagy of *M. tuberculosis*. When PPARGC1A expression was silenced in macrophages infected with *M. tuberculosis*, the robust increase in mitochondrial biogenesis, ATP generation, and decreased *M. tuberculosis* replication induced by AICAR treatment were not observed (118, 119). The findings of this study support those from Gutierrez et al. (3) demonstrating that induction of autophagy through rapamycin enhances antimicrobial defenses against *M. tuberculosis*. Although AMPK activation was found to be involved in the efficient xenophagy-dependent control of *M. tuberculosis*, this pathogen developed sophisticated mechanisms to manipulate AMPK activity in order to favor its replication. In another recent evidence, miRNAs emerged as important "fine-tuners" of gene expression in response to pathophysiological stimuli. These RNAs bind to the 3'-untranslated region of specific mRNAs to reduce protein expression by blocking mRNA translation or inducing its degradation (120). Accumulating evidence shows that many miRNAs regulate the complex interplay between mycobacterial survival strategies and host innate immune and metabolic pathways (121). One of these miRNAs, miR33 has been shown to the regulation of fatty acid metabolism and insulin signaling (122). *M. tuberculosis* seems to use the expression of miRNAs to subvert autophagy to create a favorable replicative niche. *M. tuberculosis* infection of macrophages induces the expression of miR-33 and its passenger strand miR-33\* to dampen mitochondrial fatty acid oxidation and lipophagy (autophagy of lipid droplets) to increase cellular lipid content, which is essential for the bacilli as a nutrient source during infection (34). According to this study, autophagy inhibition was achieved by inhibition of AMPK, which controls transcription factor EB and Forkhead box transcription factor class O (FOXO3), transcriptional regulators of autophagy and lysosomal biogenesis gene programs, respectively (123, 124). Altogether, these studies demonstrate that AMPK is activated during infection with intracellular bacteria.

The mechanisms by which intracellular bacteria initiate xenophagy are not completely elucidated, but compelling evidence from the literature suggests that these pathogens trigger energy imbalance and cellular nutritional stress that result in the activation of cellular responses culminating in the upregulation of autophagic activity (93). It has been reported that the infection of epithelial cells with *S. flexneri* infection induces a general and persistent loss of amino acids, leading to amino acid starvation-induced stress (94). In contrast, *S. typhimurium* induces only a rapid and transient depletion of amino acid pools. Of note, during *S. flexneri*- and *S. typhimurium*-induced amino acid depletion, robust relocalization of mTORC1 is observed. While in *S. flexneri*-infected cells, S6K1 and 4EBP1, two major targets of mTOR, are downregulated, and mTORC1 dispersed in the cytosol throughout infection, during the infection with *S. typhimurium*, this is observed only in early timepoints, suggesting that this bacterial pathogen developed means to manipulate mTOR signaling to favor its intracellular survival (94) (**Figure 7**). Indeed, in a recent study, Ganesan et al. demonstrated that despite sustained low levels of ATP in macrophages infected by *S. typhimurium*, AMPK was only transiently activated at early timepoints and then returned to basal levels (125). AMPK activation is known to be regulated by a cytosolic complex consisting of liver kinase B1 (LKB1) and Sirtuin-1 (Sirt1), where Sirt1 is necessary for deacetylation and activation of LKB1 (126). Interestingly, the study by Ganesan et al. reports that *S. typhimurium* induces the lysosomal degradation of AMPK, LKB1, and Sirt1 to reactivate mTORC1 activity in order to inhibit autophagosome formation of autophagosomes.

and escape from xenophagy. Notably, this reactivation was shown to be dependent on SsrB, a regulator of pathogenicity island 2 (SPI2) encoded virulence factors (127), and SsaV a component of the SPI2 type III secretion apparatus (128). *S. typhimurium* mutants lacking SsrB and SsaV failed to induce lysosomal degradation of the AMPK/ LKB1/Sirt1 circuit and are efficiently targeted to autophagosomes (125). Together, the studies from Tattoli et al. and Ganesan et al. demonstrate that nutritional cellular stress induced by bacterial infection triggers xenophagy to control bacterial replication (in the case of *S. flexneri* infection) and that *S. typhimurium* developed mechanisms to escape from autophagy by reactivating mTORC1 activity. Together, the studies from Tattoli et al. and Ganesan et al. demonstrate that nutritional cellular stress induced by bacterial infection triggers xenophagy to control bacterial replication (in the case of *S. flexneri* infection) and that *S. typhimurium* developed mechanisms to escape from autophagy by reactivating mTORC1 activity.

*Listeria monocytogenes* has also been reported to induce amino acid starvation-induced cellular stress and activation of the GCN2-eIF2α pathway upstream mTOR. Upon de detection of a decrease in the amino acid pool, mTOR activity is reduced leading to autophagy activation in order to normalize this condition. Unlike what is observed during the infection of epithelial cells with *S. flexneri*, in *L. monocytogenes* cells, autophagy is kept repressed, suggesting that *L. monocytogenes* possesses other virulence weapons to block autophagy (93–95) (**Figure 7**). Finally, AMPK has also been implicated in the enhancement of xenophagy during the infection with *E. coli*. According to this study, *E. coli* infection leads to an increase in intracellular calcium levels, which activates Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) to promote AMPK activation. AMPK was undoubtfully implicated in CaMKKβ-mediated xenophagy when macrophages were silenced for AMPK and control of *E. coli* replication was dampened (129).

# CONCLUDING REMARKS

Xenophagy has been widely reported to target bacteria for autophagic degradation, with clear impact on intracellular bacterial handling. Even with major advances in our understanding of the mechanisms involved in cargo selection, many questions remain unanswered. For example, why so many different mechanisms to target bacterial pathogens exposed to the cytosol? Still, why different autophagic adaptors and ubiquitin-ligases with apparent redundant functions? Although no evidence in this direction has been reported, we cannot exclude that different types of autophagosomes exist. Thus, p62, NDP52, NBR1, and Optineurin would function as sorters for different autophagosomes. It is possible that the different ubiquitin-ligases work in this direction as well by adding different ubiquitin linkages to the bacterial surface. Regarding bacteria-induced nutritional stress and autophagy induction, it is still to be elucidated whether amino acid starvation is induced upon infection with bacterial pathogens other than *Shigella*, *Salmonella*, and *Listeria.* Also, why bacteria induce amino acid starvation that leads to autophagy to subsequently inhibit it?

In *in vitro* studies, it is clear that only a fraction of the intracellular bacterial population is targeted to autophagosomes, with modest impact in bacterial replication control following autophagy ablation. This is in sharp contrast to *in vivo* studies, which demonstrate much more pronounced differences in bacterial replication in the absence of autophagy. How would these differences be explained?

Future goals in the field must address these open questions to provide a full understanding of the role of autophagy in bacterial infections.

# AUTHOR CONTRIBUTIONS

LT, MS, and RM wrote the paper. LT also revised the final document.

# REFERENCES

## FUNDING

Research in Leonardo Travassos' Lab was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). LT is research fellow from Faperj (Cientista do Nosso Estado) and CNPq (1D). MS was supported by a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). RM was supported by a fellowship from CNPQ.

autophagy of intracellular *Salmonella typhimurium*. *Cell Host Microbe* (2012) 12:778–90. doi:10.1016/j.chom.2012.10.019

to impair autophagy. *PLoS Pathog* (2017) 13:e1006227. doi:10.1371/journal. ppat.1006227

**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 Siqueira, Ribeiro and Travassos. 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 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.*

*Larissa O. C. P. Rodrigues, Rodrigo S. F. Graça and Leticia A. M. Carneiro\**

*Laboratório de Inflamação e Imunidade, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil*

Activation of an appropriate innate immune response to bacterial infection is critical to limit microbial spread and generate cytokines and chemokines to instruct appropriate adaptive immune responses. Recognition of bacteria or bacterial products by pattern recognition molecules is crucial to initiate this response. However, it is increasingly clear that the context in which this recognition occurs can dictate the quality of the response and determine the outcome of an infection. The cross talk established between host and pathogen results in profound alterations on cellular homeostasis triggering specific cellular stress responses. In particular, the highly conserved integrated stress response (ISR) has been shown to shape the host response to bacterial pathogens by sensing cellular insults resulting from infection and modulating transcription of key genes, translation of new proteins and cell autonomous antimicrobial mechanisms such as autophagy. Here, we review the growing body of evidence demonstrating a role for the ISR as an integral part of the innate immune response to bacterial pathogens.

#### *Edited by:*

*Francesca Granucci, Università degli studi di Milano Bicocca, Italy*

### *Reviewed by:*

*Michaela Semeraro, INSERM U1151 Institut Necker Enfants Malades Centre de médecine moléculaire (INEM), France Barbara M. Bröker, University of Greifswald, Germany*

> *\*Correspondence: Leticia A. M. Carneiro<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 20 February 2018 Accepted: 25 May 2018 Published: 07 June 2018*

### *Citation:*

*Rodrigues LOCP, Graça RSF and Carneiro LAM (2018) Integrated Stress Responses to Bacterial Pathogenesis Patterns. Front. Immunol. 9:1306. doi: 10.3389/fimmu.2018.01306*

Keywords: eukaryotic translation initiation factor 2 alpha, cellular stress, bacterial pathogens, heme-regulated eIF2**α** kinase, general control non-derepressible 2, PKR-like ER kinase, PKR

# INTRODUCTION

Microbial sensing by pattern recognition molecules (PRMs) triggers a robust innate immune response with the production of cytokines, chemokines, and antimicrobial factors (1–4). In the last decade, the concept that, in addition to microbial-associated molecular patterns recognition by PRMs, the host response can be tuned by the recognition of alterations in homeostasis induced by pathogens during progression of disease has been established (5–7). Such alterations on cell homeostasis allow the host to differentiate pathogenic organisms from those that do not represent a threat and, thus, adequate the immune responses to deal with the attack being mounted accordingly. It is interesting that despite the multitude of virulence mechanisms among bacterial species, most of them converge to few common "patterns of pathogenesis" that include membrane damage, access to the cytosol, disruption of cytoskeleton, and protein aggregation among others (5–10). In a sense, these patterns of pathogenesis would align with the concept of danger-associated molecular patterns (DAMPs), which are host molecules whose presence indicate that there has been tissue damage such as, for example, extracellular ATP or the chromatin-associated protein high-mobility group box 1 (11–13). But they are not exactly the same as DAMPs as these are molecules that are released as a result of cellular death and that are recognized by receptors on other cells whereas patterns of pathogenesis induce alterations of cell homeostasis during infection and provides the infected cell with information to mount a more refined response and to adapt and, in many cases recover from the insult.

In this context, the cellular mechanisms to sense and respond to stress can be regarded as an integral part of the innate immune response. The integrated stress response (ISR), a common adaptive pathway that eukaryotic cells activate in response to diverse stress stimuli is one such mechanism. The core event in this pathway is the phosphorylation of eukaryotic translation

**82**

Figure 1 | Integrated stress response (ISR) activation by bacterial pathogenesis patterns. This figure summarizes how cellular damage induced by different bacterial species is sensed by one or more eukaryotic translation initiation factor 2 alpha (eIF2α) kinases to activate defense mechanisms and homeostatic programs. We intentionally included a simplified representation of pattern recognition molecules (PRMs) recognition of microbes, PAMPs, and danger-associated molecular patterns (DAMPs) in all cartoons to strengthen the notion that these system act together to refine the cell response to the infection. (A) Pathogenesis pattern: bacterial growth; (B) pathogenesis pattern: membrane damage; (C) pathogenesis pattern: access to cytosol; (D) pathogenesis pattern: cytoskeleton disruption and protein aggregation.

initiation factor 2 alpha (eIF2α) by one or more of four members of the eIF2α kinase family (6). The phosphorylation of eIF2α results in a marked decrease in global protein synthesis accompanied by the induction of selected genes, including the transcription factor ATF4, both of which are important to promote cellular recovery (6, 7, 14). This type of response to stress mediated by the eIF2α kinases, parallels those mediated by the mTOR pathway or by autophagy in the sense that are highly conserved signaling modules that regulate essential metabolic circuits, both in homeostatic and stress conditions, from yeast to mammals (6, 8, 15). In the context of an infection, the power of this type of "sensing system" relies on the fact that it does not recognizes pathogens *per se* but rather utilizes an ancient system that detects cellular stress/damage to sense insults that are caused by pathogenic bacteria regardless of its specific virulence factors.

In the present review, we focus on the emerging role of the ISR on host response to bacterial pathogens, which only recently began to be appreciated, in contrast to its well-established role in response to viruses. As obligate intracellular pathogens that highjack the host cell machinery to produce its own proteins, the link between viruses and the ISR is more obvious and more generally accepted. The impact of the ISR on viral infections has been extensively reviewed elsewhere (16–21). Here, we discuss recent data that implicate the ISR as an important component of cell autonomous anti-bacterial responses. As an emerging topic, there are still many gaps in our understanding of the mechanisms underlying this process but we believe that our current knowledge already provides a conceptual framework to work with. As much as we tried to bring together evidence of a role for ISR in different bacterial infections, this is by no means an exhaustive review.

# eIF2**α** AND eIF2**α**-KINASES

Regulation of translation may be useful to coordinate several innate immune functions such as microbial sensing, microbial replication control, and induction of inflammatory cytokines. Translation shut down can help cells to cope with stress conditions and prevent further damage until the insult is gone. However, this happens in a context where cells still need to communicate that they are under attack in order to prevent infection spread and initiate adequate immune responses. Among metabolic sensors, eIF2α kinases have major roles in adjusting the protein synthesis machinery to enhance translation of mRNAs that are relevant to deal with the source of stress, including those induced by PRM activation, while shutting down the translation of unrelated proteins (6, 7, 21). This ability to screen and modulate host protein synthesis can affect the quality of the innate immune responses both at the transcriptional and translational levels. In addition, the gene expression program induced during ISR adjusts the stress response according to cellular context, nature, and intensity of stress stimuli (6, 7). Finally, although ISR is primarily a homeostatic-preserving program by which cells adapt to survive, severe and/or long-lasting stress can tip the balance toward cell death signaling by regulating the cell autonomous processes of autophagy and apoptosis (6, 7, 15).

The eIF2α kinases act as early responders to alterations in cellular homeostasis which is mainly due to the fact that these proteins are at the same time the sensors of stress and the kinases that phosphorylate eIF2α (6, 10, 20, 22). Each kinase dimerizes and autophosphorilates for full activation in response to distinct environmental and physiological types of stress. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is activated mainly by dsRNA during viral infection but also by oxidative and ER stress, growth factor deprivation, cytokines, bacterial infections, and ribotoxic stress (23–27). Interestingly, caspase activity in the early stages of apoptosis was also shown to activate PKR, indicating a role for protein synthesis inhibition in apoptosis (28). PKR-like ER kinase (PERK) is activated by accumulation of unfolded proteins in the ER or perturbations in calcium homeostasis, cellular energy, or redox status (29–31). It has also been reported to respond to ATP depletion and subsequent sarcoplasmic/ER Ca2<sup>+</sup>-ATPase pump inhibition in the context of glucose deprivation in neuronal cells and in pancreatic β cells (32, 33). Heme-regulated eIF2α kinase (HRI) is a sensor for low levels of intracellular heme as well as arsenite-induced oxidative stress, heat shock, nitric oxide, 26S proteasome inhibition, and osmotic stress (34–37). This array of types of stress activate HRI independently of heme but require the presence of heat shock proteins HSP90 and HSP70 (37). General control non-derepressible 2 (GCN2) is highly conserved from yeasts to humans and is activated in response to amino acid deprivation when it binds to deacylated transfer RNAs (tRNAs) *via* histidyl-tRNA synthetase-related domain (38, 39). As one can appreciate, some types of stress can potentially activate more than one of these four kinases. Most likely, the eIF2α kinases act cooperatively to specifically tune cellular responses stress. Of note, all of these kinases have been reported to have roles independent of eIF2α phosphorylation but here we will focus on the ISR, which signals through eIF2α phosphorylation.

The common signaling hub for all the stress stimuli that activate ISR is phosphorylation of the subunit α of eIF2 on serine 51 (6, 10, 20, 22). eIF2 is constituted by three subunits (α, β, and γ). When bound to GTP and Met-tRNAi Met (initiator methionyl-tRNA), eIF2 form a ternary complex that delivers the initiator tRNA to the 40S ribosomal subunit. eIF2 is released from the ribosome bound to a GDP and to be ready for another round of translation initiation, the eIF2 complex must be recycled back to its active GTP-bound form. The guanine nucleotide exchange factor eIF2B exchanges GDP for GTP on the γ subunit and maintains the levels of the ternary complex available for new rounds of translation. Under a variety of stress conditions, however, phosphorylation of the α subunit of eIF2 at Ser51 blocks general translation initiation, as it converts eIF2 to a competitive inhibitor of eIF2B by blocking the GDP–GTP exchange reaction and reducing the dissociation rate of eIF2 from eIF2B (6, 40, 41). Phosphorylation of eIF2α leads to a global arrest in translation but it does not affect all mRNA transcripts alike. A subset of mRNAs that contain upstream open reading frames and often encode proteins that are important for stress recovery and re-establishment of homeostasis have selective increased translation (6).

One of the genes that are upregulated following eIF2α phosphorylation is the transcription factor ATF4. Studies using ATF4-deficient mice have shown it has critical roles in the regulation of normal metabolic as well as redox processes such as regulation of obesity, glucose homeostasis, energy expenditure, and neural plasticity (42–44). Under stress conditions, increased ATF4 expression represents a signature of the ISR and is mainly due to translational control, as *Atf4* is one of those mRNAs that have its translation augmented upon eIF2α phosphorylation in contrast with the general translational arrest observed for most transcripts (6, 45). As a transcription factor, ATF4 can activate several transcriptional programs that will ultimately determine the cell fate—from cell death to re-establishment of homeostasis. The ability of ATF4 to interact with multiple other transcription factors allows it to generate distinct tailored responses to different types of cellular stress. Thus, despite ATF4 being a master common regulator of ISR, its target genes will be highly dependent on stress intensity and cellular context (45–47). For example, when acting in combination with ATF3, ATF4 is a part of a program that aims to re-establish cellular homeostasis and promote survival (48). Conversely, when interacting with CHOP, ATF4 promotes cell death following ER stress (49). In addition to the interacting partners that cooperate with ATF4 to promote transcription of target genes, another set of interacting partners prevent ATF4 transcriptional activity as is the case for PHD3 during hypoxia and TRIB3 during amino acid starvation and ER (50–52).

# ISR AND BACTERIAL INFECTIONS

Eukaryotes have evolved in a context of constant interactions with prokaryotes and it is clear that the latter have contributed to shape those organisms throughout evolution. A human being harbors more than 1,000 bacterial species as part of their microbiota and interacts with another incalculable number of bacterial species during its lifetime (53). The vast majority of these interactions does not result in disease and, in many cases, they are actually beneficial to the hosts. However, despite representing less than 1% of the total number of estimated bacterial species in our planet, pathogenic bacteria still cause millions of deaths every year.

In general, those bacteria that are considered as pathogenic are the ones endowed with certain attributes that allow them to (1) colonize the host; (2) find a nutritionally compatible niche in the host body; (3) avoid, subvert, or circumvent the host innate and adaptive immune responses; (4) replicate, using host resources; and (5) exit and spread to a new host (54). However, even though some bacteria display very well-defined virulence attributes, the pathogenic potential of a given bacterium can only really be observed upon interaction with its host. The final outcome of an infection is never the result of bacterial virulence alone but rather a cross talk between the host and the pathogen. This complicates the definition of "true pathogen" as the same bacterial pathogen can have different impact in different individuals. Thus, for the host, it is critical to be able to assess the potential threat that a given pathogen represents in order to establish an appropriate response.

During a bacterial infection, a multitude of signals exchanged by the two organisms establishes a cross talk that will ultimately determine the outcome of the infectious process. Many known bacterial virulence factors are only synthesized when bacteria go through major changes in metabolism in order to adapt to the dynamic conditions of the host environment (55). While doing that, bacterial pathogens may have profound effects on host cell homeostasis that, in turn, trigger cellular stress responses. Bellow, we will discuss how the ISR can be triggered by cellular alterations caused by bacterial infections and the impact of this response on host–pathogen interactions. The data discussed in the next sections are summarized in **Figure 1**.

# BACTERIAL GROWTH

The ability to survive and grow inside the host upon infection is one of the most common pathogenesis patterns as it represents the ability of a given pathogen to scape the host response and establish a replicative niche. For the host, being able to differentiate growing and dying bacteria, especially in the context of an acute infection, is key to mount a proper response. Molecules whose presence could indicate bacterial growth include peptidoglycan fragments released during bacterial cell division, quorumsensing inducers that are produced once the bacterial population reaches a certain density and bacterial pyrophosphates such as HMB-PP (5, 56–58). Alternatively, instead of direct detection of a molecule, bacterial growth sensing could be achieved by sensing of altered local levels of cellular nutrients such amino acids or oxygen (59–61).

Recently, the definition of PAMPs has been updated to allow the classification of those produced specifically by living microorganisms, the so-called *Vita*-PAMPs, and those that represent the degradation products of dead microorganisms, named PAPMs-*postmortem* (PAMPs-PM), as two different categories that have different biological activities (10, 62). Moretti et al. (10) has recently identified cyclic-di-adenosine monophosphate (c-di-AMP), a second messenger that is produced by live Gram-positive bacteria, as a *Vita*-PAMP. The authors show that phagocytes are able to discriminate live and dead *Listeria innocua* by sensing this *Vita*-PAMP through the innate immune sensor stimulator of interferon genes resulting in ER stress, PERK and eIF2α phosphorylation. Subsequently, an autophagic response ensued to sequester stressed ER membranes and prevent stress-induced cell death while also inducing an IFN-dependent response. Importantly, this response was blunted in phagocytes lacking PERK. Finally, following *L. monocytogenes* infection, mice engineered to have PERK-deficient macrophages presented lower systemic levels of IFN-I and higher bacterial burden on both liver and spleen when compared with WT controls. In this model, at a cellular level, there was no difference between the response induced by live *L. innocua*, a non-pathogenic bacteria, and live *L. monocytogenes*, but there was differences when these were compared to dead bacteria. The response to any infection is multilayered and dependent on the interaction of multiple sensing systems—each one of these systems provides the cells with different information that when combined determine the cells response and, ultimately, its fate. In this case, the ISR provided the cells with the ability to distinguish live from dead bacteria, which is crucial to mount appropriate response even though it was not able to differentiate between a pathogenic from a non-pathogenic species. Of note, when the authors tested their hypothesis *in vivo,* they used only *L monocytogenes*, most likely because *L. innocua* would have been readily cleared given its lack of virulence and would have not generated any of the responses observed against *L. monocytogenes*. This, once again, illustrates how important the context is: in the natural course of a real infection, *L. innocua* would probably have never caused the systemic infection that *L. monocytogenes* does and would have not reached circulating phagocytic cells or the liver or the spleen. However, when given directly to these cells *in vitro*, it induced the same response that the bacterial species that would have encountered these cells during infection. It would also be interesting to investigate if this response is restricted to phagocytic cells or can occur in other cell types.

A contrasting study showed that PERK activation and IFN-I production by myeloid cells during infection with *L. monocytogens* or treatment with the pore-forming toxin LLO is actually detrimental to the host. In this model, the PERK pathway is amplified by IFN-I resulting in the activation of another eIF2αkinase, PKR. This, in turn, served as an amplification loop for PERK-signaling leading to excessive ER stress and cell death. Consistent with this, mice deficient on CHOP, a pro-apoptotic factor that is downstream of PERK, are more resistant to *L. monocytogenes* infection than WT controls (63). This model could provide a partial explanation for why mice lacking IFN-I receptor have been consistently reported to be more resistant to *L. monocytogenes* than WT mice (64–66).

The opportunistic Gram-negative *Pseudomonas aeruginosa* causes both acute and chronic infections, especially in the respiratory tract (31, 67). Its ability to scape or subvert the host immune response constitutes its main virulence attribute. *P. aeruginosa* is able to form biofilms, a complex biological system that protects the bacteria from host immune defense mechanisms and promotes persistent infection. This bacterium coordinates the production of biofilms and other virulence factors using quorum sensing, a cell-to-cell communication system that allow bacteria to perceive their population density by producing and sensing diffusible signal molecules. One of the quorum-sensing auto inducers produced by *P. aeruginosa* to regulate gene expression and communicate is *N*-(30oxododecanoyl)-homoserin lactone (HSL-C12) (31, 67). HSL-C12 is a lipid-like diffusible molecule that has multiple effects on mammalian cells including apoptosis and release of Ca2<sup>+</sup> from the ER stores. By perturbing ER homeostasis, HSL-C12 induces the activation of PERK and eIF2α phosphorylation resulting in protein synthesis inhibition (31). If in the one hand this inhibition results in increased NF-κB activation and transcription of pro-inflammatory genes because IκB re-synthesis is blocked, on the other hand it prevents the translation into proteins of the transcribed genes resulting in an overall downregulation of the host response and, thus, can be considered a pathogen scape mechanism. This would be one instance where the pathogen evolved to manipulate and take advantage of a cell host sensing system.

As mentioned above, nutrient availability is a critical limitation for invading microorganisms. Iron is a nutrient indispensable for growth of almost living organisms and is unlikely to be readily available for invading microorganisms resulting in fierce competition between host and pathogens (59). Like many other bacteria, *P. aeruginosa* has developed several mechanisms to acquire iron during infection. In a recent study, it was demonstrated that the iron-chelating siderophore pyoverdine produced by *P. aeruginosa* limits the concentration of iron in the cell medium resulting in the activation of HRI, eIF2α phosphorylation, and induction of *Gadd34* transcription in human bronchial epithelial cells. This response had cytoprotective effect and was turned off when the medium was supplemented with iron (61).

These few examples demonstrate that host cells can detect growth of bacteria by sensing molecules that accumulate as the number of bacteria increase including those that bacteria use to communicate with each other, such as quorum-sensing auto inducers and second messengers as well as molecules that bacteria use to acquire nutrients.

# MEMBRANE INTEGRITY

The detection of this type of stress is highly conserved. Damage of the plasma membrane is an archaic threat that needs to be faced with efficient cell autonomous defense mechanisms (5). Recently, a pivotal role for GCN2 in the response to membrane damage has been uncovered in different models. For example, it has been demonstrated that membrane permeabilization by the detergent digitonin induces a robust response characterized by GCN2 phosphorilation and ATF3 expression (68). In *Drosophila*, the damage caused by *Pseudomonas entomophila* in gut cells induces a starvation-like state, resulting in GCN2 and eIF2α phosphorylation and concomitant inhibition of the mTOR pathway by the AMP-activated kinase (AMPK). In this model, these two stress response pathways together shut down translation of new proteins and trigger innate immune responses (69).

In mammalian cells, disturbance of membrane integrity caused by bacterial pathogens can also trigger stress responses (8, 15, 68). Pore-forming toxins represent an important class of bacterial exoproducts that can induce membrane damage leading to stress responses (70). In human epithelial cells, the α-toxin produced by *Staphylococcus aureus* induces the formation of pores on cellular membranes resulting in potassium efflux, failure of nutrient transport and loss of ATP which, in turn, activates both GCN2 and the energy sensor AMPK, with subsequent eIF2α phosphorylation and mTORC1 deactivation (similar to what was reported in *Drosophila*) (15, 71). Low intracellular concentrations of potassium is known to trigger several responses in infected or stressed cells including the activation of inflammasomes and caspases (15, 71), as well as activation of multiple kinases such as p38 and CREB, in addition to the aforementioned AMPK and GCN2 (71–73). Activation of GCN2 induced by potassium efflux caused by membrane perforation indicates that cells may exploit the dependence of nutrient transport across the plasma membrane on physiological ion gradients to indirectly sense perturbations on ion concentration. Both removal of the pore from the plasma membrane by dynamindependent endocytosis and the metabolic reprogramming activated by the ISR are essential for cellular recovery as cells that are not able to activate this program are more susceptible to α-toxin (74, 75).

Invasive bacteria such as *Salmonella* Tiphymurium, *Shigella flexneri*, and *Listeria monocytogenes* also cause membrane damage during their internalization process. Similar to what was described above, all three bacteria trigger an acute intracellular amino acid starvation program that induces stress responses dependent on GCN2 and eIF2α phosphorylation at the same time as it disarms mTOR signaling unleashing an autophagic response (8, 68). However, the response that ensues is different for these three bacteria. (i) During infection with *S. flexneri*, a Gram-negative bacterium that escapes to and replicates in the host cell cytoplasm, amino acid starvation persists up to 4 h after infection allowing not only the induction of autophagy but also GCN2- and eIF2α-dependent formation of stress granules in the cytosol as well as reprogramming of the transcriptional response orchestrated by ATF3 (8, 76). (ii) *L. monocytogenes*, a Gram-positive bacterium that similar to *S. flexneri* escapes to and replicates in the cytosol, also triggers a state of amino acid starvation characterized by activation of GCN2, eIF2α phosphorylation, and transcriptional upregulation of ATF3. In this case, however, this response is very transient peaking at 1 h and is completely normalized after 4 h post-infection. The kinetics of this response parallels the kinetics of the poreforming toxin LLO-dependent scape from the internalization vacuole and coincides with the maximal targeting of *L. monocytogenes* to autophagosomes (68). (iii) *Salmonella*, in contrast to the bacteria described above, remains in vesicles known as *Salmonella*-containing vacuoles (SCV) after its internalization. The damages to the SCV membranes trigger the same GCN2 dependent early amino acid starvation program described above. However, following *Salmonella* infection membrane integrity and cytosolic amino acid concentration are readily normalized allowing mTOR to be reactivated at the surface of the SCV and promoting bacterial scape from autophagy (8). Thus, these three model invasive bacteria all induce GCN2-dependent ISR during their entry processes but each one of them deal with it in different ways once again highlighting that bacteria have also evolved to counteract ISR-mediated responses.

Adherent-invasive *Escherichia coli* (AIEC), which is abnormally abundant in the intestinal mucosa of Crohn's disease patients, also induces phosphorylation of GCN2 with subsequent eIF2α phosphorylation and increased ATF4 levels. Upon activation of this pathway, ATF4 binds to promoters of multiple autophagy-related genes including *MAP1LC3B*, *Becn1*, *SQSTM1*, *ATG3*, and *ATG7*. This is necessary to initiate autophagy and restrict bacterial growth as depleting cells from GCN2 resulted in impaired autophagy, increased bacterial replication, and elevated pro-inflammatory cytokine production both *in vitro* and *in vivo*. The authors go on to show that the GCN2–eIF2α–ATF4 pathway is activated in ileal biopsies from patients with noninflamed Crohn's disease but not on those with inflamed Crohn's disease, indicating that failure to activate this stress response could be one of the mechanisms contributing to active disease (77).

Thus, it appears that a nutrient sensor, GCN2, may also function as a sentinel of membrane integrity and that the responses it triggers are essential to prevent abyssal ATP loss and irreversible damage. In addition, in the case of invasive pathogens, this response might affect their ability to replicate within the host cell due to increase in autophagic activity as a consequence of amino acid starvation as well as production of inflammatory factors induced by the stress transcription factors ATF3 and ATF4.

# ACCESS TO CYTOSOL

Many pathogens are able to deliver molecules directly into the cytosol of host cell. This may be achieved by AB-toxins when the B subunit binds to specific receptors on the surface of the cells and translocates the active subunit A into the cell (78), by pore-forming toxins such as listeriolysin O (mentioned above) and streptolysin O (70), or secretion systems such as the type III secretion systems of *Yersinia* and *Salmonella* (79), the type IV secretion system of *Legionella*, *Coxiella*, and *Brucella* (80), and the type VI secretion system of *Pseudomonas* and *Vibrio* (81, 82).

Shiga-toxigenic *E. coli* produces Shiga toxin (Stx) 1 and 2 that cause hemorrhagic colitis and hemolytic uremic syndrome. A newly described toxin, namely subtilase cytotoxin (SubAB), was shown to bind to and be internalized by target cells through clathrin-, lipid rafts-, and actin-dependent pathways. Once it reaches the ER, SubAB cleaves the chaperone Bip/Grp78 initiating an ER-stress induced ISR resulting in cytotoxicity. This response also included the formation of stress granules induced not only by PERK but also as a result of PKR activation (14).

Yang et al. (83) show that *P. aeruginosa* infection induces a strong activation of the GCN2–eIF2α–ATF4 pathway that is largely dependent on production of pyocianin during initial infection and that ultimately results in bacterial clearance through autophagy. Pyocianin is a cell permeable toxin considered to be a major virulence factor for *P aeruginosa*. *In vivo*, in rats, infection with a mutant bacterial strain that does not produce pyocianin and, thus, does not activate of the GCN2–eIF2α–ATF4 pathway results in higher number of colony-forming units in the lungs, more extensive alveolar wall thickening and higher mortality when compared to infection with the WT strain. Although indirect, these data suggest a role for the ISR in preventing prolonged infection and immunopathology. Interestingly, reduction of pyocianin production by *P. aeruginosa* in chronic airways infections has been associated with better host adaptation and worse outcomes in cystic fibrosis patients (84).

# ACTIN CYTOSKELETON DISRUPTION

Another common feature employed by various highly divergent pathogenic bacterial species is the disruption of the host cell cytoskeleton. Invasive bacteria such as *S. flexneri*, *L. monocytogenes*, *Mycobacterium marinum*, and Rickettsial species exploit the actin-based motility to move inside the cell and from one cell to the other without never being exposed to immune defenses outside the cells (5, 85). Other bacterial pathogens, such as *E. coli* and *Citrobacter freundii,* produce hallmark attaching and effacing lesions that are characterized by localized destruction of the brush border villi of enterocytes, intimate attachment of bacteria to the residual apical membrane and formation of a dense plaque of actin cytoskeletal filaments beneath adherent bacteria that is essential for their pathogenesis (86, 87). Finally, some pathogens manipulate host actin cytoskeleton to either induce their own uptake or to avoid phagocytosis (88–90).

Polysomes, mRNAs, elongations factors, and aminoacyltRNA synthetases are found associated with actin filaments indicating that the cytoskeleton might actually act as a platform to facilitate the assembly of components involved translation (91–93). GCN2 has been recently implicated as a sensor of F-actin depolymerization. Disruption of the actin cytoskeleton by drugs such as latruculin-B and cytochalasin-D induces GCN2 activation followed by eIF2α phosphorylation, attenuation of global translation, and augmented ATF4 and CHOP expression (94). In nutrient-replete cells, GCN2 is kept in a latent state by the interaction with other proteins such as the eukaryotic elongation factor 1A (eEF1A) that delivers aminoacyl-tRNAs to ribosomes during the elongation step of protein synthesis (95, 96). During starvation periods, however, uncharged tRNA displaces eEF1A from GCN2 allowing its autophosphorilation and eIF2α phosphorylation (97, 98). Another binding partner of eEF1A is F-actin. In yeasts, the same mutations that affect binding of eEF1A to aminoacyl-tRNAs also result in actin binding and buding defects that lead to GCN2-dependent eIF2α phosphorylation (99, 100). Thus, it has been proposed that upon F-actin disruption eEF1A is displaced from GCN2 and bound to F-actin leaving GCN2 free to initiate the ISR (94). In addition, F-actin disruption also leads to deacylated tRNA accumulation, which in turn might also contribute to the activation of GCN2 resulting in global protein synthesis arrest and reduction of amino acylated tRNA levels (94).

As mentioned above, two invasive pathogens—*L. monocytogenes* and *S. flexneri*—that exploit the actin cytoskeleton of the cell to move around the cell and infect neighboring cells were shown to induce a GCN2-dependent starvation program as a consequence of membrane damage. It is possible that disruption of F-actin could impede the proper function of amino acid transporters on the plasma membrane triggering this response. In summary, infection with *L. monocytogenes* and *S. flexneri* could potentially activate GCN2 in multiple ways: when bacteria escape from the vacuole into the cytosol causing membrane damage (as it has been experimentally demonstrated) or by disrupting the actin cytoskeleton.

# PROTEIN AGGREGATION

Heme-regulated eIF2α kinase is able to sense and respond to a variety of types of cellular stress including heme deprivation, oxidative stress, heat shock, and proteasome inhibition, all of which are known to result in accumulation of misfolded protein aggregates in the cytosol (35, 36, 101, 102). As it has been previously shown that infection with bacterial pathogens trigger the formation of large PRM oligomeric complexes in the cytosol, one may speculate that this is the common feature among all these types of stresses that is actually sensed by HRI (103–106). This could serve as a sensing system to monitor misfolding of large protein complexes and formation of toxic aggregates in the cytosol and trigger damage control mechanisms such as ISR and autophagy.

# CONCLUDING REMARKS

Even at a single cell level, the response to an infection is multilayered and involves sensing, effector, and homeostatic mechanisms. Each one of these elements has, in itself, multiple layers of complexity and, together, they generate a full-blown response. Sensing of microbes or their products by PRMs is pivotal and activates robust inflammatory responses. Since the discovery of PRMs, there has been much debate on how the cells can tailor the response to specific pathogens using a limited number of receptors that recognize structures that are present in many different microorganisms, including non-pathogenic. This can be achieved by different means including the combinatorial effect of several PRMs (107), the compartmentalization of PRMs that only allows recognition of certain PAMPs when presented in specific compartments of the cell (108) and the sensing of *vita*-PAMPs versus PAMPs-PM (62). The recognition of pathogenesis patterns by the ISR represents another layer in the host response. Sensing alterations on homeostasis and cell damaged caused by infection can instruct the host to generate a more refined and specific response while triggering protective gene expression programs that enable cells to recover from the initial stress and re-establish homeostasis. Given its origins early on evolution, stress responses may actually represent an ancient innate defense mechanism against invading pathogens.

In this review, we discussed evidence showing that the ISR can have an important role in shaping the autonomous cell response to bacteria with varying levels of virulence. In this context, the ISR acts in concert with other sensing systems to adequate the response to the threat. Thus, the ISR during bacterial infection cannot be analyzed isolated from the context. This generates a complexity that represents a challenge for dissecting the precise role and the relevance of each component in the final response. While there are still many gaps to be filled before we have a more comprehensive overview, the picture that emerges is that the ISR can influence the quality of the response initiated by innate immune recognition.

For the most part, the studies discussed in this review show that several bacteria are able to activate or manipulate the ISR during infection, through different eIF2α kinases and signaling pathways, resulting in specific transcriptional programs. However, in many cases, it is yet to be defined how this affects the outcome of the infection. In some cases, it is clear that it can affect the ensuing immune response. For example, ISR activation in phagocytic cells infected by *Listeria* was shown to be critical for IFN-I production and bacterial clearance. On the other hand, eIF2α phosphorylation induced by the HSL-C12 from *P. aeruginosa* results in downregulation of translation of proinflammatory cytokines such as IL-6 and KC. We believe that understanding how the ISR can affect qualitatively the response to a given pathogen is a major avenue for future work. In this sense, as we progress, it would be important to determine how the ISR impact on how cells communicate infection to the neighboring cells as well as to immune cells and how this can qualitatively affect the immune response, including in subsequent exposures to the same pathogen. Finally, it would be interesting to see if the homeostatic adaptations during infection can lead to persistent alterations in the infected cell rendering it more resistant to following infections.

The interplay between ISR and autophagy is also a common theme in most of the studies mentioned here. Interestingly, two studies discussed above showed that in the absence of the GCN2–eIF2α–ATF4-autophagy pathway, opportunistic bacteria such as AIEC and *P. aeruginosa* establish persistent infection that perpetuate inflammation contributing to worsen the pathology in Crohn's disease and cystic fibrosis, respectively. It will interesting to investigate how ISR could affect the development of chronic complex diseases that are known to have a microbial component to it such as the two mentioned above.

As we mentioned above, co-evolution of pathogens and their hosts have shaped (and continue to do so) their interactions. In this constant arms race, both sides try to adapt in order to survive. Thus, it should come as no surprise that some bacteria might be able to scape or even take advantage of the ISR to manipulate the host cell response. Indeed, being able to subvert host responses is part of the very definition of what a pathogen is. In this case, failure to activate the ISR properly could lead the host to underestimate the infectious threat. As it is well documented for several viruses, we expect that as our knowledge increases, we will uncover many bacterial strategies to tamper with the ISR.

Because of the significant overlap between the eIF2α kinases in addition to the complexity of many host–pathogen interactions, at this point, it is difficult to clear define the role of each of the ISR sensors in response to bacterial pathogens and most likely a combination of them are responsible for an appropriate response. Future work will help us understand how these pathways are activated and manipulated by bacterial pathogens and how can we use this knowledge to develop new treatments to prevent or cure infection. For example, if we are able to safely increase the signals generated by cells of the innate immunity by manipulating the ISR, we might be able to improve the adaptive immunity generated by vaccines.

# AUTHOR CONTRIBUTIONS

LR, RG, and LC wrote the manuscript. LC edited and revised the final version of the manuscript.

# REFERENCES

# FUNDING

Work in the lab of LAMC is supported by Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). LR and RG were supported by studentships from PIBIC/ UFRJ and CNPq, respectively.

membrane at the site of bacterial entry. *Nat Immunol* (2010) 11:55–62. doi:10.1038/ni.1823

initiation factor 2. *Mol Cell Biol* (2009) 29:1592–607. doi:10.1128/MCB. 01446-08

**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 Rodrigues, Graça and Carneiro. 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 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.*

# Pattern Recognition Receptors and the Host Cell Death Molecular Machinery

Gustavo P. Amarante-Mendes 1,2 \*, Sandy Adjemian3,4, Laura Migliari Branco5,6 , Larissa C. Zanetti <sup>7</sup> , Ricardo Weinlich<sup>7</sup> \* and Karina R. Bortoluci 5,6 \*

<sup>1</sup> Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil, <sup>2</sup> Instituto de Investigação em Imunologia, Instituto Nacional de Ciência e Tecnologia (INCT), São Paulo, Brazil, <sup>3</sup> Molecular Signaling and Cell Death Unit, Inflammation Research Center, VIB, Ghent, Belgium, <sup>4</sup> Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, <sup>5</sup> Departamento de Ciências Biológicas, Universidade Federal de São Paulo, Diadema, Brazil, <sup>6</sup> Centro de Terapia Celular e Molecular (CTC-Mol), Universidade Federal de São Paulo, São Paulo, Brazil, <sup>7</sup> Instituto Israelita de Ensino e Pesquisa, Hospital Israelita Albert Einstein, São Paulo, Brazil

### Edited by:

Leticia A. Carneiro, Universidade Federal do Rio de Janeiro, Brazil

#### Reviewed by:

Fabianno Ferreira Dutra, Universidade Federal do Estado do Rio de Janeiro, Brazil Marco Di Gioia, Boston Children's Hospital, Harvard University, United States

#### \*Correspondence:

Gustavo P. Amarante-Mendes<EMAIL_ADDRESS>Ricardo Weinlich<EMAIL_ADDRESS>Karina R. Bortoluci<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 02 August 2018 Accepted: 25 September 2018 Published: 16 October 2018

#### Citation:

Amarante-Mendes GP, Adjemian S, Branco LM, Zanetti LC, Weinlich R and Bortoluci KR (2018) Pattern Recognition Receptors and the Host Cell Death Molecular Machinery. Front. Immunol. 9:2379. doi: 10.3389/fimmu.2018.02379 Pattern Recognition Receptors (PRRs) are proteins capable of recognizing molecules frequently found in pathogens (the so-called Pathogen-Associated Molecular Patterns—PAMPs), or molecules released by damaged cells (the Damage-Associated Molecular Patterns—DAMPs). They emerged phylogenetically prior to the appearance of the adaptive immunity and, therefore, are considered part of the innate immune system. Signals derived from the engagement of PRRs on the immune cells activate microbicidal and pro-inflammatory responses required to eliminate or, at least, to contain infectious agents. Molecularly controlled forms of cell death are also part of a very ancestral mechanism involved in key aspects of the physiology of multicellular organism, including the elimination of unwanted, damaged or infected cells. Interestingly, each form of cell death has its particular effect on inflammation and on the development of innate and adaptive immune responses. In this review article, we discuss some aspects of the molecular interplay between the cell death machinery and signals initiated by the activation of PRRs by PAMPs and DAMPs.

Keywords: PRR, pathogen recognition receptor, apoptosis, necroptosis, pyroptosis, inflammation

# INTRODUCTION

In 1989, Charles Janeway Jr. proposed the existence of a collection of receptors expressed by innate immune cells responsible for detecting conserved products of microbial origin (1). After 25 years of intense research, fierce debates, and a Nobel Prize granted on this subject, it is unquestionable that Janeway's ingenious idea has revolutionized our understanding of the immune system. Indeed, his seminal article is considered as one of the pillars of immunology (2).

The so-called Pattern Recognition Receptors (PRRs) are proteins capable of recognizing molecules frequently associated with pathogens (aka Pathogen-Associated Molecular Patterns—PAMPs). A more comprehensive description of PRRs and their signaling transduction pathways can be found elsewhere (3). Briefly, PRRs can be found associated to subcellular compartments, such as the cellular and endosomal membranes, the cytosol, as well as extracellularly, in secreted forms present in the bloodstream and interstitial fluids (3). There are four major sub-families of PRRs—the Toll-like receptors (TLRs), the nucleotide-binding oligomerization domain (NOD)- Leucin Rich Repeats (LRR)-containing receptors (NLR), the retinoic acid-inducible gene 1 (RIG-1) -like receptors (RLR; aka RIG-1-like helicases—RLH), and the C-type lectin receptors (CLRs) (4). As predicted by Janeway, the engagement of PRRs on the innate immune cells induces co-stimulatory signals for the adaptive immune cells (particularly T lymphocytes) (5). In addition, they activate microbicidal and pro-inflammatory responses required to eliminate (or at least to contain) infectious agents, including the induction of infected cell death (6), as discussed below.

Another ingenious idea came from Polly Matzinger (7), who proposed that the immune system is less concerned with the origin of the antigens (self vs. non-self) than with the context of their encounter with our body (tissue damage vs. tissue homeostasis). In her "Danger Theory," Matzinger suggested that during tissue stress or damage, endogenous molecules are released or activated and initiate or propagate the inflammatory response, which, among other things, empower antigenpresenting cells to activate the adaptive immune response. Today, these molecules are collectively known as DAMPs (Damage-Associated Molecular Patterns). Importantly, soon enough it became clear that similarly to PAMPs, DAMPs could also engage PRRs.

These two theories together put forward the idea that our body is equipped to distinguish "healthy," homeostatic tissue turnover or encounters with foreign "friendly" microorganisms, from potential "danger" that may come from pathogens and/or tissue damage.

# CELL DEATH PROGRAMS

Molecularly controlled forms of cell death are part of a very ancestral mechanism involved in key aspects of the physiology of multicellular organism, including the elimination of unwanted, damaged or infected cells. Importantly to our discussion, cell death can have a direct or an indirect impact upon the course of infection, as the elimination of infected cells may eradicate or at least restrain the growth of a given pathogen. Moreover, the recognition of dying cells or their by-products modulates both inflammatory and immune responses. In the following sections, we will briefly describe the mechanisms that govern the three major types of molecularly controlled forms of cell death, namely apoptosis, necroptosis and pyroptosis, that participate in host defense through elimination of infected cells, and how they are regulated by signals derived from PRRs. For information regarding other cell death modes please refer to the work published by the Nomenclature on Cell Death Committee 2018 (8)

# Apoptosis

Apoptosis was the first type of programmed cell death to be described, initially based on morphological features that distinguished it from necrosis, an uncontrolled, accidental form of cell death observed upon extreme physicochemical insults (9). In this regard, apoptosis is characterized by chromatin condensation, nuclear fragmentation, cell shrinkage with formation of cellular membrane blebs, and, finally, cellular disintegration into fragments known as apoptotic bodies (10). Importantly, during apoptosis, the plasma membrane integrity is preserved, avoiding the release of intracellular contents to the extracellular milieu. This feature contributes to the concept that apoptosis is an (relatively) inflammatory-silent form of cell death. Indeed, recognition and elimination of apoptotic cells during physiological circumstances, such as tissue/organ sculpture during development and tissue homeostasis, occurs without the cardinal signs of inflammation. In addition, it is well established that recognition of apoptotic cells by macrophages, in particular, results in the production of anti-inflammatory molecules, such as TGF-β and PGE<sup>2</sup> (11). On the other hand, it is also known that apoptotic cells release a series of so-called "find-me" signals, such as extracellular ATP and lysophosphatidylcholine (LPC), capable of recruiting phagocytes to the site of apoptotic corpses, characterizing, therefore, at least one aspect of an inflammatory reaction (12, 13). Besides, more recently, it was shown that apoptosis initiated via the FAS/CD95 death receptor is associated with the release of chemokines and other immunologically active proteins that coordinates the migration of phagocytes and proper removal of apoptotic cells (14). Taken together, it is reasonable to say that although not completely "silent," apoptosis is a form of cell death that does not trigger an overt inflammatory response.

From the molecular point of view, much of our knowledge about the regulation of apoptosis came from works with the nematode Caenorhabditis elegans. In a series of elegant studies, Bob Horvitz and colleagues identified four crucial genes (Ced-3, Ced-4, Ced-9, and Egl-1) responsible for the control of developmental cell death in C. elegans (15), which granted him the Nobel Prize in Physiology or Medicine in 2002, together with John Sulston and Sidney Brenner, "for their discoveries concerning genetic regulation of organ development and programmed cell death." Soon after Horvitz discoveries, it became clear that cell death in C. elegans and apoptosis in mammals shared a very similar, phylogenetically conserved mechanism. Apoptosis is executed by certain members of a family of cysteine aspartatespecific proteases called caspases (16–18). Importantly, not all caspases induces apoptosis. Caspases-1, -4, -5, -11, -12, -13, and-14 are inflammatory caspases not related to the initiation or execution of the apoptotic program. Caspases are produced as an inactive pro-form (zymogen) that can be activated either through proteolytic processing by upstream caspases (in the case of caspases-3, -6, and-7) or via dimerization in the context of multimolecular platforms, such as the apoptosome (caspase-9), the DISC (death-inducing signaling complex) (caspases-8 and-10), the PIDDosome (caspase-2), and the inflammasome (caspase-1 and-11) (16). Executioner or effector caspases, such as caspase-3, -6, and-7 (and CED-3 in C. elegans), are responsible for the induction of the morphological as well as the biochemical features associated with apoptosis, including oligonucleosomal DNA fragmentation and externalization of phosphatidylserine (PS) residues from the inner to the outer leaflet of the plasma membrane (19). Interestingly, in mammals, although the inhibition of effector caspases prevents apoptosis, it does not preclude cell death, which proceeds with different morphological and biochemical characteristics (20). Because of this, it has been proposed that apoptosis in mammals may not be actually a cell death mechanism, but perhaps a termination step of a cell-death

program aimed to properly dispose damaged or unwanted cells without initiating inflammatory responses (18).

There are two signaling pathways of apoptosis (**Figure 1**). The intrinsic pathway deals with signals derived from intracellular stress, such as DNA damage, oxidative stress, dysregulation of Ca2<sup>+</sup> homeostasis, interference with the cytoskeleton structure, endoplasmic reticulum stress, etc. Its first layer of regulation comprises the differential expression/activation of BCL-2 family members, responsible for controlling the mitochondria outer membrane permeabilization (MOMP) (21). When the proapoptotic stress is too strong for a given cell, MOMP allows the selective release of certain mitochondrial proteins, such as SMAC (second mitochondria-derived activator of caspases)/Diablo (direct IAP binding protein with low pI), HtrA2 (high temperature requirement protein A2)/Omi, and cytochrome c to the cytosol. Cytochrome c associates with APAF-1 (apoptosis-activating factor-1), the mammalian CED-4 homolog, and pro-caspase-9, thereby assembling the apoptosome and enabling caspase-9 to activate the downstream effector caspases. SMAC/Diablo and HtrA2/Omi facilitate apoptosis by preventing the inhibitory action of the inhibitors of apoptosis proteins (IAPs) on the effector caspases. The extrinsic pathway, in comparison, is initiated by the interaction of trimeric, extracellular ligands (TNF-α, CD95L, and TRAIL) to their cognate receptors (TNFR1, CD95 and TRAILRI, or TRAILRII, respectively) present on the plasma membrane (10, 22, 23). The stimulation of these so-called death receptors (DRs) leads to the recruitment of adaptor molecules, such as TRADD (Tumor necrosis factor receptor type 1-associated death domain protein) and/or FADD (Fas-associated protein with death domain), and the pro-caspase-8, giving rise to the conventional DISC. Next, caspase-8 directly activates the effector caspases or amplifies the cell death signal by engaging BID (BH3 interacting-domain death agonist), a pro-apoptotic member of the BCL-2 (B-cell lymphoma 2) family, leading to MOMP, cytochrome c release and assembly of the apoptosome (**Figure 1**). It is important to mention that the activation of caspase-8 in the context of DISC can be regulated by c-FLIP (cellular FLICE-like inhibitory protein), a catalytically-dead caspase-8 homolog (24).

In some instances, apoptosis can also be triggered by TLR stimulation, as a defense mechanism against infection. TLR2 was the first PRR to be associated with induction of apoptosis, by virtue of its ability to recruit FADD via MyD88 (Myeloid differentiation primary response 88), and the consequent activation of caspase-8 (25). Likewise, bacterial lipoproteins were reported to trigger apoptosis through this TLR2 pathway (26, 27) and Mycobacterium tuberculosis was also shown to induce TLR-2/caspase-8-dependent apoptosis in macrophages (28). Interestingly, TLR3-induced apoptosis is mediated via TRIF (TIR-domain-containing adapter-inducing interferon-β), which interacts with RIPK1 (Receptor Interacting Serine/Threonine Kinase 1) through its RHIM (RIP homotypic interaction motif) domain (please refer to necroptosis section for further information on these protein-protein interactions). FADD is then recruited, and activates caspase-8 leading to apoptosis (25, 29). In human keratinocytes, poly I:C-induced apoptosis required the stimulation of TLR3 and its adaptor TRIF, thus inducing caspase-8 activation (30); the same molecules were shown to induce apoptosis in human breast cancer cells (31). Not surprisingly, TLR4 can induce apoptosis either via MyD88 or TRIF, and depending on the cell type or conditions engage the extrinsic or intrinsic pathways. For instance, Yersinia was shown to induce TLR4-mediated apoptosis of macrophages through TRIF (32, 33). TRIF-mediated apoptosis seems to be executed through the extrinsic pathway, with no evidence of the involvement of the mitochondrial pathway (34). Interestingly, UV irradiation was shown to induce apoptosis in murine macrophages through TLR4 and MyD88 (35). Despite these observations and a number of other examples that we have not presented here, it is important to emphasize that PRR-induced apoptosis is a relatively minor event compared to all other triggers of apoptosis and that PRR activation leads preferentially to other forms of regulated cell death, as we will discuss below.

## Necroptosis

Evidence of a molecularly controlled necrotic cell death was first provided by studies showing that Tumor Necrosis Factor Receptor 1 (TNFR1) and CD95 ligation were capable of inducing necrosis, particularly when caspase activity was inhibited (36, 37). This idea was further supported by a study that demonstrated that the cowpox virus could induce necrosis in porcine kidney cells when it harbored the caspase inhibitor CrmA (cytokine response modifier A) (38). This cell death mode was named "Necroptosis," as it reflects the existence of a molecular pathway (like apoptosis) but with a necrotic phenotype.

The first molecule to be identified in the necroptotic pathway was RIPK1 as its kinase activity inhibitor, necrostatin-1 (Nec-1), was shown to suppress cell death triggered by caspase inhibition during TNFR1/Fas stimulation (39). RIPK1 has been previously involved in apoptotic and survival pathways, functioning as a scaffold protein to the assembly of the respective signaling platforms (40). Contrastingly, the RIPK1 kinase activity is indispensible for death receptor-triggered necroptosis, as its auto-phosphorylation induces a conformational change that allows RIPK1 to recruit, via their respective RHIM domains, the next member of this pathway, namely RIPK3 (41–43). Once recruited, RIPK3 gets activated by auto-phosphorylation and forms an amyloid-like structure, which promotes the recruitment and activation of Mixed Lineage Kinase Domain-Like (MLKL) (42, 44–47). RIPK3-phosphorylated MLKL oligomerizes and translocates to the plasma membrane, where it interacts with phosphatidylinositides and induces plasma membrane disruption [(48–51); **Figure 2**]. Distinct effector mechanisms were raised to account for the MLKL-driven permeabilization of the plasma membrane, either directly by pore or cation channel formation, or indirectly, by activation of TRPM or other ion channels (48–52). It is still unclear, however, which of these mechanisms are physiologically relevant. Nonetheless, in all cases, MLKL induces a loss of osmolality control, which causes cell swelling and membrane rupture. Recently, ESCRT-III machinery was suggested to counter these effects by shedding out the MLKL-damaged plasma membrane regions (53).

Necroptosis can be initiated by a variety of signals. The first to be described and most thoroughly studied was TNFR1

pathway of apoptosis.

ligation (36). Upon its ligation, TNFR1 typically assembles a multimolecular complex (Complex I) composed by TRADD, RIPK1, TRAF2, TRAF5, cIAP1, cIAP2, and LUBAC (linear ubiquitin chain assembly complex), which is involved in NF-κB activation, pro-inflammatory cytokines synthesis and cell survival (54). Sustained TNFRI ligation leads to CYLDmediated deubiquitination of this complex, which disassembles, allowing the formation of a secondary complex (Complex II) in the cytosol, constituted by TRADD, FADD, RIPK1, caspase-8, and occasionally c-FLIP (54). As pointed out above, when c-FLIP levels are low, caspase-8 forms active homodimers and triggers downstream events that culminate in apoptosis. However, in the absence of FADD, c-FLIP or a functional caspase-8, TNFRI signaling results in the recruitment of TRADD and RIPK1, forming a platform called complex IIb or necrosome, wherein RIPK3 and MLKL are activated to execute necroptosis (55). Although slightly differing on how RIPK1 is brought to the complex, this molecule has also a central role in Fas and TRAILR-induced necroptosis, as RIPK1 is, in all these cases, mandatory to

recruit RIPK3 via their RHIM homotypic domain interactions (54).

Necroptosis can also be triggered by PRRs, such as TLR3 and TLR4, intracellular sensing proteins, such as DAI, RIG-I and MDA-5 as well as interferon signaling [(56–59); **Figure 2**]. Intriguingly, however, RIPK1 is dispensable for or even inhibitory of the necrosome formation during TLR3-, TLR4- , DAI-, and interferon-mediated necroptosis (57, 60). In these cases, RIPK3 is directly recruited to the signaling platforms, and the presence of RIPK1 slows down or halts the RIPK3 mediated activation of MLKL (60, 61). The ability of RIPK1 to recruit FADD, and consequently, caspase-8 and FLIP accounts, at least in part, for its inhibitory property. Therefore, from the molecular point of view, necroptosis ought to be defined as a RIPK3-dependent form of cell death.

Many other stimuli have been described as capable to induce necroptosis, ranging from UV irradiation, chemotherapeutic drugs (such as cisplatin, etoposide, and staurosporine), natural compounds (such as shikonin and its analogs), to DNA damage, hypoxia, ischemia/reperfusion and oxidative stress (62). The signaling pathways that lead to necroptosis in each of these cases are still to be fully elucidated. Further studies are required to evaluate whether they are dependent on RIPK1 and also whether they directly signal to a RIPK3-activating platform or indirectly, via up regulation of a classic necroptotic inducer, such as TNF or FasL. For example, UV irradiation was reported to induce necroptosis via TNF upregulation but also via spontaneous aggregation of RIPK1 and RIPK3, independently of any death receptor ligation (29, 63). Particularly puzzling is the fact that shikonin, a naphthoquinone compound obtained from a plant extract, can induce necroptosis even in the absence of FADD/caspase-8/FLIP inhibition, which is thought to be mandatory for this type of cell death (64). Thus, either this compound can itself somehow block their activity, or it shall be instrumental to decipher alternative ways in which MLKL is activated and necroptosis is executed.

Nonetheless, despite the different mechanisms that initiate necroptosis, in all cases cells undergo rapid MLKL-mediated plasma membrane permeabilization with consequent release of intracellular contents, including many DAMPs, such as lysosomal proteases, DNA, mtDNA, ATP, and HMGB1 [(55); **Table 1**]. Therefore, similarly to pyroptosis (see below), necroptosis is considered a pro-inflammatory form of cell death. Even so, it is still to be determined whether the pro-inflammatory properties of necroptotic cells are the result of the intracellular content leakage or, rather, they can actively produce and/or modify specific DAMPs. Evidence for the latter comes from ESCRT-III-deficient cells that undergo necroptosis much faster, which limits the amount of inflammatory cytokines and chemokines produced and hinders antigen cross-presentation (53). Moreover, both RIPK3 and MLKL have been associated with inflammasome and NF-κB activation, supporting the notion that the proinflammatory potential of necroptotic cells goes beyond the passive release of their intracellular content (110–112).

Necroptotic cells not only induce a potent inflammatory response but they are also highly immunogenic, which may be instrumental against infection and during anti-tumoral responses. For example, mice injected with necroptotic cells present a higher CD8<sup>+</sup> T cell cross-priming and increased tumor immunity when compared with animals injected with apoptotic cells (113, 114). Likewise, RIPK3 deficiency in mice inhibits immune cell infiltration and attenuates organ injury during sepsis

### TABLE 1 | DAMPs released by cell death and its role in the immune system.

NCD stands for Necrotic Cell Death, which means the referred papers only characterized the cell death by its necrotic morphology. AIM2, absent in melanoma 2; ASC, Apoptosisassociated speck-like protein containing a CARD; CCR, CC chemokine receptor; CD, cluster of differentiation; CXCR, CXC chemokine receptor; DC, dendritic cells; FEEL-1, fasciclin EGF-like laminin-type EGF-like and link domain-containing scavenger receptor-1; FPR-1, formyl peptides receptor-1; G2A, G2 accumulation; GP130, Glycoprotein 130; HMGB1, highmobility group box 1 protein; HMGN1, high-mobility group nucleosome-binding domain 1 protein; IL, interleukin; LRR and PYD domains-containing protein 3; Mincle, Macrophage inducible Ca2+-dependent lectin receptor; Mϕ, macrophage; NLRP3, NACHT LRR and PYD domains-containing protein 3; P2XR, P2X receptor; P2YR, P2Y receptor; PMNs, polymorphonuclear leukocytes; RAGE, receptor for advanced glycation end-products; SAP130, Sin3A Associated Protein 130; SREC-I, Scavenger receptor expressed by endothelial cells; ST2, Interleukin 1 receptor-like 1; TLR, Toll-like receptor.

(115). Therefore, given that necroptosis is highly immunogenic, disruption in the necroptotic pathway would be expected in some pathophysiological conditions. Indeed, it was reported that most of the in vitro transformed cells as well as human tumor samples have low or no expression of RIPK3 (116), and a cohort of chronic lymphocytic leukemia patients present down regulation of CYLD (117). Furthermore, patients with lower expression of RIPK3 or MLKL have worse prognosis for breast cancer or ovarian cancer, respectively (116, 118), suggesting that resistance to necroptosis is positively selected during tumor growth and/or development. This may be associated with an increased ability to evade immune attack, either by prolonging the lifespan of the transformed cells, by decreasing the availability of DAMPs, or by avoiding the activation of antigen-presenting cells during the immune responses. Therefore, induction of necroptosis in tumors may change its immunogenicity and promote a better immune response against it. This is particularly exciting, as we are currently witnessing novel and promising approaches in tumor treatment that are based on stimulation of the immune system. On the other hand, it is possible that the inflammation generated by necroptosis may promote tumor development by stimulating angiogenesis and metastasis (119). Therefore, thorough investigation of the benefits and pitfalls of inducing inflammatory cell death for each cancer type will be required in order to determine whether inducing necroptosis is indeed a good option in the specific cancer treatment.

Besides its impact on tumorigenesis and tumor progression, deficient necroptotic signaling can be detrimental during viral infection. Mice lacking RIPK3 are highly sensitive to vaccinia virus due to widespread infection (120). Likewise, RIPK3 deficient mice are more susceptible to Influenza A virus (IAV) than the wild-type animals (121). Remarkably, seasonal IAV, but not the 1918 and 2009 pandemic IAV strains, induces RIPK3 mediated immunogenic death of dendritic cells (122). The pandemic strains' ability to suppress necroptosis was mapped to the hemagglutinin (HA) genomic segment (122), indicating that either the pandemic strains' HA do not induce necroptosis or it may directly interfere with the necroptotic signaling pathway.

Keeping with the notion that suppressing necroptosis is advantageous to the infectious agent, there is accumulating evidence that viruses can encode molecules that are able to directly interfere with the necroptotic signaling. vIRA, a molecule expressed by MCMV that contains a RHIM-like domain blocks RIPK3 recruitment to RIPK1 and to DAI (57). MCMV expressing vIRA mutated in its RHIM domain produces an attenuated viremia in wild-type mice, which is reverted in RIPK3-deficient animals (57). Likewise, HSV-1 and HSV-2 express ICP-6 and ICP-10, respectively, which are able to suppress necroptosis in human cells through a similar RHIM-dependent mechanism (123, 124). Curiously, in mice, ICP-6 was shown to promote necroptosis through direct aggregation with RIPK3, restricting virus propagation (124, 125). A different mode of action was reported for the IE1-regulated gene product expressed by HCMV, which suppresses necroptosis downstream of RIPK3 activation and MLKL recruitment (126).

Bacteria can also induce necroptosis, at least in vitro. It is less clear, though, whether necroptosis plays a central role in bacterial infections in vivo. Loss of RIPK3 in combination with deletion or inhibition of caspase-8 or FADD renders mice susceptible to a number of pathogens, including Yersinia and Citrobacter (127, 128). However, the relative contribution of necroptosis and caspase-8-mediated apoptosis in these models were not yet tested, as caspase-8- or FADD–deficient animals are not viable (129–131).

Necroptosis, though, may not always be protective against infection. Macrophage death by necroptosis correlates with increased susceptibility to Salmonella infection (132). Also, HIVspecific CD8<sup>+</sup> T cell response, which is a key indicator of infection control, is impaired due to increased necroptosis levels in this cell population (133). Taken together, necroptosis seems to be detrimental when it eliminates the population that is central for the immune control of the infection. In the other cases, necroptosis limits infection, mostly likely by destroying the pathogen's replicative niche through a cell death mode that generates a pro-inflammatory and immunogenic environment. However, it is important to note that, as mentioned above, RIPK3 and MLKL were shown to participate of additional signaling platforms, including inflammasome activation, NF-κB signaling and even apoptosis induction (134). Therefore, in the light of these novel RIPK3 and MLKL roles, it is essential to reevaluate the relative contribution of necroptosis to the phenotypes observed. A good illustration comes from the fact that while RIPK3 deficient mice are more susceptible to IAV, MLKL-deficient animals are not, indicating that necroptosis is not the sole RIPK3 mediated mechanism important in IAV control (121). In fact, it was shown that IAV also triggers RIPK3-mediated apoptosis, via recruitment of RIPK1, FADD and caspase-8. This was further supported by the fact that MLKL-caspase-8 double deficient mice present similar levels of susceptibility to IAV infection observed with the RIPK3-deficient animals (121). Another example is that RIPK3-deficient mice are less susceptible to Staphylococcus aureus lung damage and present reduced bacterial loads and inflammation, while MLKL-deficient animals present an opposite outcome, suggesting that these molecules have independent, nonnecroptotic roles (135).

# Pyroptosis

Pyroptosis is a necrotic form of regulated cell death distinct from necroptosis, mainly due to the requirement of inflammatory caspase-1 and/or caspase-11 (murine caspase-11 corresponds to caspases-4 and -5 in humans) [(136); **Figure 3**]. It is the result of pore formation in the plasma membrane that increases osmotic pressure ensuing in osmotic lysis and, consequently, the release of the intracellular content, including pro-inflammatory cytokines and DAMPs (137). Although distinct from the typical oligonucleosomal fragmentation observed during apoptosis, DNA fragmentation is also a hallmark of pyroptosis, which seems to occur independently of the caspase-activated DNase (CAD) (138).

Pyroptosis is a form of cell death initiated in response to the engagement of certain members of the PRRs, which are capable of assembling complex structures called inflammasomes. These platforms are composed by a sensor protein, either from the NLR or the pyrin and HIN domain-containing protein

(PYHIN) families of cytosolic PRRs, in addition to the adaptor molecule apoptosis-associated speck-like protein containing a caspase activating and recruitment domain (ASC) and procaspase-1. There are five major types of the so-called canonical inflammasomes—NLR pyrin domain-containing 3 (NLRP3), NLRP1, neuronal apoptosis inhibitory protein (NAIP)/NLR CARD-containing 4 (NLRC4), absent in melanoma 2 (AIM2), and PYRIN inflammasomes (139). Activation of one of these cytosolic sensors in response to PAMPs, DAMPs or cytosolic disturbances such as ionic imbalance leads to the recruitment and activation of caspase-1 either directly or through the ASC adaptor molecule [(140); **Figure 3**]. Besides the induction of pyroptosis, caspase-1 also leads to the processing and release of the pro-inflammatory cytokines IL-1β and IL-18 (139).

In contrast to the canonical inflammasomes, which require multicomplex structures, the non-canonical inflammasome seems to be composed solely by pro-caspase-11, which plays the role of the sensor as well as the executor (141, 142). During intracellular gram-negative bacteria infections, Lipid A, a component of LPS, can directly bind to the CARD domain of pro-caspase-11 (143), which gets activated and induces pyroptosis. Interestingly, the non-canonical caspase-11 inflammasome acts independently of LPS recognition by TLR4 and does not directly induce IL1-β and IL-18 maturation [(141, 142); **Figure 3**]. In monocytes, however, non-canonical inflammasome stimulation may result in minor production of IL-1β and IL-18 through the bystander induction of NLRP3 activation (144). Interestingly, LPS-induced lethal shock is driven by the activation of the non-canonical inflammasome. Since IL-1β and IL-18 release are not a major outcome of caspase-11 activation, the exacerbated inflammatory response observed in sepsis seems to be mainly driven by pyroptosis, probably due to the efflux of DAMPs, such as High Mobility Group Box 1 (HMGB1) and IL-1α [**Table 1**; (145)].

Such non-canonical inflammasome-mediated responses have drawn the attention of different research groups that became interested in unraveling the relevant as well as the pathogenic caspase-11 downstream targets. In 2015, two concurrent studies reported that Gasdermin D (GSDMD), a member of the GSDM family, was the effector component of the non-canonical inflammasome pathway (146, 147), which was later confirmed by a third study (148). Kayagaki and colleagues performed N-ethyl-N-nitrosourea mutagenesis screening for mutations that compromised LPS-induced IL-1β release and pyroptosis (146) while Shi and colleagues employed the clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 genomeediting screens in TLR4 deficient mouse bone marrow-derived macrophages for guide RNAs that protected from LPS-induced cell death (147). Both studies hit GSDMD as a substrate for caspase-11 and the effector of pyroptosis. GSDMD is composed by a C-terminal and a N-terminal domain linked by a long loop. Caspase-11 cleaves an aspartate residue within the linking loop, releasing the N-terminal fragment from the inhibitory C-terminus (146, 147). The N-terminal domain, also called Pore-Forming Domain (PFD) (149) oligomerizes and associates with lipids in the inner plasma membrane to form 10–33 nm pores leading to cell swelling and eventually to cell lysis (150– 153). Importantly, it was also demonstrated that caspase-1 cleaves GSDMD at the same site as caspase-11, establishing that GSDMD is also required for the canonical inflammasome-driven pyroptosis (147).

Until the discovery of GSDMD as the pyroptosis executioner, the physiological function of GSDM proteins was largely unknown. However, recent studies described that the PFD is highly conserved among several members the GSMD family. Indeed, expression of PFD from GSDMA, GSDMA3, GSDMB, GSDMC, GSDME, or GSDMA3 in HEK293 was able to induce pore formation and a cell death phenotype similar to pyroptosis (147, 151). Moreover, GSDMA3 cleavage by caspase-3 in HEK293 and macrophages results in a secondary necrotic cell death after apoptosis (154). This necrotic cell death might contribute to hearing loss in GSDMA3 spontaneous mutations that are associated with deafness (155). Thus, given the cytotoxic activity of different GSDM PFD, some authors have proposed a redefinition of pyroptosis as a GSDM-mediated cell death (146). However, it is controversial how other GSDM members are activated and whether these proteins participate in cell death pathways. Also, GSDMD seems not to be required for pyroptosis during prolonged inflammasome activation in response to the classical agonists, ATP, and flagellin (146). Moreover, in the absence of caspase-1 protease activity, caspase-8 accounts for GSDMD-independent cell death in response to inflammasome agonists (156–158). Since some of these processes share features of pyroptosis, it is hard to define pyroptosis solely as being a process of cell death regulated by inflammatory caspases or mediated by GSDM proteins, since we can find exceptions to the rules that govern both concepts.

From the biological point-of-view, cell death by pyroptosis results in a fast removal of infected cell leading to the elimination of the replication niche. Conversely to the previous idea of liberation of bacteria to the extracellular milieu by pyroptotic cells (159), the current knowledge predicts that, instead, the damaged bacteria remain trapped within the pyroptotic corpses. This structure is called pore-induced trap (PIT) and it prevents bacterial dissemination (160, 161). Despite that PIT does not directly kill intracellular bacteria, pyroptosis renders them more susceptible to H202, to the antimicrobial peptide polymyxin B and to the antibiotic ciprofloxacin (157). As a consequence, the recovered bacteria from PIT are less capable to infect neighbor cells.

The inflammatory milieu created by the release of the intracellular content from pyroptotic cells recruits circulating phagocytes to the infectious site.  Extracellular bacteria can also be controlled by the action of antimicrobial peptides (160, 161) and potentially by the GSDMD Nterminal domain released during cell lysis due to its affinity to cardiolipin and phosphatidylserine expressed in some bacterial cell membranes, such as Escherichia coli and Listeria monocytogenes (152, 162). Interestingly, canonical and noncanonical inflammasomes are required for intestinal epithelial cells (IECs) responses to infections (163, 164). The activation of NLRC4 inflammasomes in IECs results in a lytic cell death prior to a non-conventional process of cell expulsion that contributes to control bacterial replication. Although caspase-1 and Gasdermin-D were required for IEC pyroptosis, both molecules were dispensable for cell expulsion, demonstrating that coordinated inflammasome responses in IECs are important to prevent bacterial translocation to deeper tissues (163, 164).

Interestingly, neutrophils seems to be more resistant to pyroptosis than macrophages in response to Salmonella and are able to maintain a sustained IL-1β production and secretion, which could be important to control the infection (165). However, Kambara et al. (166) recently described that a specific neutrophil elastase (ELANE) is able to cleave GSDMD independently of caspases activity, promoting a lytic cell death in these cells. Interestingly, these authors demonstrated that GSDMD-dependent neutrophil death impairs the control of extracellular bacteria E. coli, thus suggesting that GSDMD could exert an anti-inflammatory role depending on the infection context.

In addition to its role in the elimination of replicative niche, the pyroptosis machinery is involved in IL-1β and IL-18 release. As these cytokines lack the signal peptide, their release is considered to occur by non-conventional pathways (167). Among the different pathways that have been proposed to explain their secretion, mechanisms involving cell death are particularly subject to intense debate in the literature. Growing evidences suggest that IL-1β can be released by viable monocytes (168), dendritic cells (DCs) (169), and macrophages (170). GSDMD pore is large enough to allow IL-1β release concomitant with the influx of cationic ions (148). Notably, in viable cells, GSDMD seems to be required for IL-1β translocation to the extracellular space in response to stimuli that hyperactivate phagocytes, such as oxidized phospholipids (oxPAPC) in DCs or LPS in human monocytes (170–172). Nonetheless, it is difficult to establish whether the cells were actually viable, since cell death can precede cell lysis, thus suggesting that pyroptosis and cell lysis can be uncoupled events (173). Moreover, the assessment of cell death by the detection of lactate dehydrogenase release (LDH), used in several studies as the only viability assay, might be insufficient to discriminate viable cells from dying cells since both viable and unviable cells can release LDH to the cell culture (170, 173, 174).

Although many studies have demonstrated the requirement of canonical and non-canonical inflammasomes to host defense against pathogens, the precise contribution of pyroptosis and other inflammasome-related mechanisms are poorly understood and arose mainly from in vitro assays or bacterial infection models in mice deficient for molecules that compose these platforms (159, 175). In L. monocytogenes, S. typhimurium, and B. thailandensis in vivo infections, the lack of caspase-1/11 was more deleterious to the host than IL-1β/IL-18 deficiency (176– 179), while in mice infected with F. novicida, the treatment with recombinant IL-1β/IL-18 only partially recovered the resistance to infection, suggesting that cytokine secretion was not sufficient to protect the host (180). The susceptibility of GSDMD-deficient mice seems to correlate with that demonstrated by caspase-1/11 deficient mice, although the deletion of GSDMD also culminated in reduced IL-1β/IL-18 release (146–148). Even though (a) IL-1β/IL-18 secretion might occur independently of pyroptosis and (b) caspase-1 or caspase-11 deletion is more severe than IL-1β/IL-18 ablation in some bacterial infections, usually cytokine release and cell death are overlapping events necessary for optimal host defense (159). In any case, the susceptibility of GSDMD and other GSDM deficient mouse strains to infectious agents and its comparison to mice lacking caspase-1, caspase-11, IL-1β, and/or IL-18 remains to be established, especially in non-bacterial infection contexts. For example, despite clear evidences of the involvement of inflammatory caspases in the host control of some fungal infections such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis (172), the requirement of GSDMD to cell death and the consequences to the host resistance against these infections is still to be elucidated.

Notwithstanding, the highly pro inflammatory outcome of pyroptosis as well as the cell loss can be prejudicial to the host during the response to pathogens. In HIV patients, the quiescent CD4 T cells depletion seems to be mainly mediated by pyroptosis (181, 182). During HIV abortive infection, the engagement of the interferon-gamma-inducible-protein 16 (IFI16) in response to cytosolic viral DNA leads to inflammasome assembly and caspase-1 mediated CD4 T cells pyroptosis in lymphoid tissues (181, 182). Interestingly, co-cultivation of lymphoid-derived cells sensitizes blood-derived CD4 T cells to HIV-induced pyroptosis (183). Moreover, pyroptotic peripheral blood CD14+CD16<sup>−</sup> monocytes from HIV-infected patients release ASC specks, a hallmark of inflammasome activation. Therefore, besides the depletion of CD4 T cells, pyroptosis of CD4 T cells and monocytes contributes to the chronic inflammation that characterizes the disease (184).

The identification of the non-canonical inflammasome and the discovery of GSDMD as the executioner of pyroptosis have expanded our understanding of the mechanisms driving this type of cell death. However, further studies are necessary to elucidate the precise role of inflammatory and noninflammatory caspases and the participation of members from GSDM family and/or other effector proteases in the molecular regulation of pyroptosis. In addition, the understanding of its role during infection or inflammatory processes in vivo will contribute to better understand the biological relevance of this regulated cell death induced in response to the PRRs activation.

# PRR SENSING OF CELL DEATH AND CELL DEATH PRODUCTS

The notion that cells undergoing cell death release or expose several intracellular molecules regardless of the accidental nature or the different regulated death programs (apoptosis, necrosis or pyroptosis) is widely recognized. Although mainly non-inflammatory in the intracellular space, molecules released/exposed from damaged cells can participate in the activation of inflammation and immune responses. Indeed, a broad range of receptors, including PRR, sense these DAMPs and alert the immune system by inducing immune cell migration, increasing phagocytosis by macrophages and DCs, stimulating the production of pro-inflammatory cytokines or even contributing to the maturation of DCs, among other key functions (72, 80, 185). A number of studies have been dedicated to the characterization of putative DAMPs, and it became apparent that the type of cell death, as well as the nature of cell death stimuli, influence the quality and quantity of DAMPs release (**Table 1**).

Importantly, the stress or damage before the cellular demise itself is determinant to set in motion a sequence of events leading to an immunogenic cell death (ICD). The sensing of this stress regulates the cell death process thus initiating signaling pathways that will actively—or not—generate danger signals (186). Other DAMPs will be passively released as a result of membrane rupture during necroptosis or pyroptosis. These DAMPs define in part the immunogenicity of cell death, but are not sufficient to elicit a specific anti-tumor immune response, for instance. Indeed, they are released or exposed by the dying cells and act as adjuvant providing that antigens are exhibited conjointly (187). In contrast, a non-immunogenic cell death does not provide the required levels of DAMPs and antigens to evoke an adaptive immune response (187).

Together, these concepts redefined the widely accepted paradigm stating that apoptosis is always a silent cell death modality as opposed to necrosis, which is inflammatory and immunogenic. Therefore, a non-immunogenic apoptosis is characterized by the absence of plasma membrane leakage and the rapid phagocytosis of apoptotic bodies prevents the release of DAMPs and the consequent inflammatory reaction. Indeed the apoptotic process reduce cell immunogenicity by diverse ways including, (1) preservation of intracellular structures and plasma membrane, thereby blocking the release of DAMPS; (2) reduction of cellular volume, by condensation of the nucleus and shedding of small vesicles, which favors its rapid elimination by the surrounding tissue; (3) expression of "find-me" and "eat-me" signals, which increases the speed of cell clearance (12, 188); (4) inhibition of the production of interferons and pro-inflammatory cytokines, as the DNA is being chopped and condensed (189); (5) induction of an antigenic tolerance in the engulfing APCs (190); and (6) post-translational modifications on DAMPS and alarmins that decrease their pro-inflammatory potential (191, 192).

Interestingly, depending on the trigger, apoptosis can be immunogenic. Indeed, some chemotherapeutic agents, such as anthracyclines, as well as radiation and hypericin-based photodynamic therapy, were found to strongly prime immune

TABLE 2 | PRR agonists and consequent cell death program.

AIM2, absent in melanoma 2; DAI (DLM-1/ZBP), DNA-dependent activator of IFNregulatory factors; dsDNA, double stranded DNA; dsRNA, double stranded RNA; HMGB1, high-mobility group box 1 protein; LPS, Lipopolysaccharides; NAIP/NLRC4, NLR family CARD domain-containing protein 4; NALP1, NACHT, LRR and PYD domainscontaining protein 1; P2X7, P2X purinoceptor 7; PAM3CSK4/Pam3CysK, TLR2 receptor agonist; Poly(I:C), Polyinosinic:polycytidylic acid; RIG-I, retinoic acid-inducible gene-I-like receptors; shRNA, short hairpin RNA; ssRNA, single stranded RNA; T3SS/T4SS, Type III/IV secretion system; TLR, Toll-like receptor.

responses through the induction of ICD (65, 185). Among these, immunogenic chemotherapies are well characterized and involve the emission of a number of danger signals. The pre-apoptotic release or exposure on the plasma membrane of ER-chaperones, such as calreticulin and Heat Shock Proteins (HSPs), constitutes an early event of ICD, which relies on the induction of an ERstress. Calreticulin promotes the uptake of dying cells by DCs (72) and the inhibition of its exposure during anthracyclineinduced apoptosis of murine tumor cell lines abolished their immunogenic potential (72). The early apoptotic secretion of ATP, which binds to P2X7 or P2Y2 purinergic receptor on DCs, stimulates the formation of the NLRP3 inflammasome, thereby inducing the release of IL-1β (66). Moreover, ATP

released by dying cells undergoing ICD is responsible for the recruitment and differentiation of myeloid precursors into inflammatory DCs, mediating a specific antitumor immune response (193). Passive release of the nuclear protein HMGB1 occurs during secondary necrosis (i.e., late-stage apoptotic cells), which interacts with TLR4 on DCs, and through Myd88 signaling, enables efficient tumor antigen processing and crosspresentation (80). Additionally, anthracyclines have been shown to induce the release of RNA, thereby stimulating TLR3 as a mimic of viral infection. Activation of TLR3 is then responsible for type I IFNs production that acts in an autocrine and paracrine manner to promote the secretion of CXCL10 (194). Release of Annexin A1 has also been described after anthracyclines treatment, stimulating the Formyl Peptide Receptor 1 (FPR1), thus directing the final trajectory of DCs to dying tumor cells (195).

Besides chemotherapeutic agents, bacterial and viral infection can also trigger an immunogenic apoptosis. In this case, PAMPs, such as LPS or double-stranded RNA, expressed by the pathogen can stimulate TLR signaling and induce an immune response. Indeed, phagocytosis of infected apoptotic cells could trigger TLR activation and lead to IL-6 and TGF-β production that induce the development of infection-specific as well as self-reactive TH17 cells, linking infection to autoimmunity (196, 197). Finally, defects in mechanisms of apoptotic cell clearance are linked to autoimmunity disorders, including lupus and rheumatoid arthritis, likely due to the increased risk of loss of cell integrity with the consequent release of DAMPs and increased availability of circulating self-antigens (198, 199).

Accidental or programmed forms of necrosis are responsible for the release of an usually larger panel of DAMPs compared to apoptotic cells, mainly due to plasma membrane permeabilization. Also, RIPK1-mediated activation of the NF-κB pathway, through upregulation of pro-inflammatory cytokines and increased antigen presentation, was reported to play a role in the immunogenicity of necroptotic cell death mode (186). Recently, it was show that necroptosis is accompanied by the release of the classical and potent DAMPs—HSPs, ATP, and HMGB1 (200, 201). While HSPs were shown to stimulate TLR2 and 4, inducing DCs maturation, HMGB1 was reported to interact with TLR3, 4 and 9, as well as RAGE, to activate DCs and macrophages (202–204). Necrotic cells can potentially release intact mitochondria, a major source of ATP, which may activate the NLRP3 inflammasome resulting in IL-1β secretion and neutrophil recruitment (67, 96, 205). Moreover, mitochondrial DAMPs, such as formyl peptides and mitochondrial DNA, can potentially act on FPR1 and TLR9, respectively, inducing neutrophils recruitment and degranulation (97, 115). Additionally, Mincle, the C-type lectin receptor 4E was reported to interact with the necrotic DAMP SAP130 (spliceosome-associated protein 130), normally involved in spliceosomes assembly. The stimulation of this PRR was also reported to induce recruitment of neutrophils (106). Uric acid has been described as a product of accidental necrosis (108). Once released, this DAMP can precipitate and form monosodium urate (MSU) crystals able to induce the expression of costimulatory molecules by DCs, as well as activating the NLRP3 inflammasome to trigger the production of IL-1β and IL-18 (206, 207). RNA and double-stranded DNA (dsDNA) are also released during necrotic cell death, and while RNA stimulates TLR3, dsDNA stimulates TLR9 and two members of the RLR family, RIG-I and MDA5, responsible for the release of IFN-β and CXCL10 through IRF3 and NF-κb pathways. The AIM2 inflammasome can sense dsDNA released by necrotic cells, thereby inducing IL-1β secretion (204). Finally, it is important to mention that some proteins considered DAMPs also stimulate receptors that are not PRRs. For instance, IL-1α acts on IL-1R1 to trigger an inflammatory response mediated by the Myd88 pathway (208) and full-length IL-33 is another DAMP released during necrosis, with immunological property due to the absence of caspase processing (191).

# REFERENCES

1. Janeway CA Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol. (1989) 54(Pt 1):1–13. doi: 10.1101/SQB.19<IP_ADDRESS>

# CONCLUSIONS

The protective response of our body against pathogens and tumor cells depends on proper activation of both innate and adaptive immunity. Particularly, macrophages and DCs reside on the center of these two arms of immunity. They are powerful antigen-presenting cells that may elicit effector T cell responses (protection) or induce T cells to become regulatory (tolerance), depending on their activation status. They express PRRs, which are very ancient proteins that help us identify and react to pathogens and danger signals. Upon engagement, through the interaction with conserved molecular patterns frequently associated with pathogens (PAMPs), PRRs trigger a series of biochemical signaling cascades that activates proinflammatory programs on DCs that enable the differentiation of antigen-specific T cells into protective effector TH1, TH2, and TH17 cells. PRR engagement also triggers programs of cell death, particularly necroptosis and pyroptosis, the necrotic forms of cell death associated with a pro-inflammatory outcome (**Table 2**). These forms of cell death release larger amounts of DAMPs, which in turn, stimulate surrounding cells via PRRs, thus constituting a positive feedback loop capable of amplifying host defense mechanisms (**Figure 4**). Apoptosis, on the other hand, is a cell death program mostly related to non-inflammatory outcomes and likely to take major role in the maintenance of homeostasis by "silently" eliminating unwanted or damaged cells. However, apoptosis may also participate in elimination of infectious agents or tumor cells. Therefore, recognition of pathogen- and damage/danger-associated molecules by the same set of immune receptors (PRRs) is a powerful strategy that bridges intrinsic cell death programs and complex immune cell interactions to preserve homeostasis and at the same time protects the organism against infection and cellular transformation (**Figure 4**).

# AUTHOR CONTRIBUTIONS

LM designed the figures. LZ and SA prepared Table. GA-M, RW, and KB designed the review organization. All authors contributed to the writing, reviewed, and approved the manuscript.

# ACKNOWLEDGMENTS

LM and LZ are recipients of Ph.D. fellowships from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). SA is recipient of a postdoctoral fellowship from FWO (Fonds voor Wetenschappelijk Onderzoek—Vlaanderen, Belgium). GA-M, KB, and RW are supported by grants from FAPESP and from the Brazilian Research Council (CNPq).

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

The reviewer FD and handling Editor declared their shared affiliation at the time of review.

Copyright © 2018 Amarante-Mendes, Adjemian, Branco, Zanetti, Weinlich and Bortoluci. 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.

# Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis

*Áislan de Carvalho Vivarini1 , Teresa Cristina Calegari-Silva1 , Alessandra Mattos Saliba2 , Viviane Sampaio Boaventura3,4, Jaqueline França-Costa3,4, Ricardo Khouri3,4, Tim Dierckx5 , Karina Luiza Dias-Teixeira1 , Nicolas Fasel6 , Aldina Maria Prado Barral3,4, Valéria Matos Borges3,4, Johan Van Weyenbergh5 \* and Ulisses Gazos Lopes1 \**

*<sup>1</sup> Laboratory of Molecular Parasitology, Carlos Chagas Filho Biophysics Institute, Center of Health Science, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 2Department of Microbiology, Immunology and Parasitology – FCM/ UERJ, State University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 3Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil, 4 Federal University of Bahia, Salvador, Bahia, Brazil, 5Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium, 6 Faculty of Biology and Medicine, Department of Biochemistry, University of Lausanne, Lausanne, Switzerland*

## *Edited by:*

*Leticia A. Carneiro, Federal University of Rio de Janeiro, Brazil*

#### *Reviewed by:*

*Min Zhang, Indiana University School of Medicine, United States Peter Epeh Kima, University of Florida, United States*

### *\*Correspondence:*

*Johan Van Weyenbergh<EMAIL_ADDRESS>Ulisses Gazos Lopes<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 29 May 2017 Accepted: 28 August 2017 Published: 15 September 2017*

### *Citation:*

*Vivarini ÁC, Calegari-Silva TC, Saliba AM, Boaventura VS, França-Costa J, Khouri R, Dierckx T, Dias-Teixeira KL, Fasel N, Barral AMP, Borges VM, Van Weyenbergh J and Lopes UG (2017) Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis. Front. Immunol. 8:1127. doi: 10.3389/fimmu.2017.01127*

*Leishmania* parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive *in vitro* and *ex vivo* mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of *in situ* (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in *Leishmania amazonensis* infection *in vitro*. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of *Sod1* Nrf2-dependent gene and reduced parasite load. *L. amazonensis* counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 *via* PKR. This Nrf2/Keap1 observation was confirmed *in situ* in skin biopsies from *Leishmania*-infected patients. Next, we explored the *ex vivo* transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. *In silico* enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our *in vitro* findings. Our integrated *in vitro, ex vivo*, and *in silico* approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.

### Keywords: *Leishmania*, macrophage, nuclear factor erythroid 2-related factor 2, PKR, Sod1

# INTRODUCTION

Human cutaneous leishmaniasis (CL) is spread worldwide, and the incidence is estimated to be from 0.7 to 1.2 million cases each year (1). Different clinical manifestations occur in humans due to the immune response and the infection by distinct *Leishmania* species (2). *Leishmania* parasites exhibit a plethora of adaptive mechanisms that interfere with several macrophage functions through the manipulation of host signaling pathways (3).

The imbalance between oxidative stress and cytoprotective systems of detoxification dictates the outcome of intracellular parasitic infections (4). The transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] is a master regulator of phase II defense gene expression that may protect cells from oxidative stress. The DNA promoter sequences of phase II defense genes share the canonical antioxidant response element (ARE), which is recognized by Nrf2 (5). Nrf2-dependent expression leads to profound effects on the suppression of the inflammatory response and immune activation through *Toll*like receptors (6, 7).

The control of Nrf2 activation is dictated by different posttranslational modifications. Multiple sites on the Nrf2 protein are phosphorylated by kinases, such as PERK, members of the MAPK family, PKCζ, and GSK3β, increasing the nuclear translocation and binding of the protein to ARE elements on the promoters of target genes (8–10).

The PI3K/Akt pathway modulates Nrf2 signaling (11) and, importantly, recent reports have demonstrated the modulation of the PI3K/Akt pathway upon *Leishmania amazonensis* infection (12). The enzyme GSK3, a target of Akt1, phosphorylates the Nh6 domain of Nrf2 and facilitates the action of ubiquitin ligase, leading to proteasomal degradation. The inhibition of GSK3 by phosphorylation allows the nuclear translocation of Nrf2 (13).

Kelch-like ECH-associated protein 1 (Keap1) is a major inhibitor of Nrf2 that constitutively induces the ubiquitination of the Nh2 domain, directing Nrf2 to proteasomal degradation. Keap1 is uncoupled from Nrf2 because of post-translational modifications due to oxidative stress, releasing Nrf2 for nuclear translocation (14).

Autophagy may modulate Nrf2 activation *via* Keap1 degradation (15). The components of the autophagy pathway are sensors of oxidative stress (16), and the increase in the expression of *p62* (*Sqstm1*), an Nrf2 target autophagy gene (17), favors the cellular capacity to process proteins destined for the autophagosome, decreasing oxidative stress (18).

The phosphorylation of p62 allows its binding to several cargo proteins, including Keap1, leading to autophagy as well as the Nrf2 stability and activation (19). The activation of Nrf2 through the p62/autophagy non-canonical pathway has also been demonstrated in macrophages treated with LPS, PolyI:C and peptidoglycan (PGN) upon the engagement of TLR4, TLR3, and TLR2, respectively (20).

Double-stranded RNA-dependent protein kinase [protein kinase R (PKR)] has in the Nh2-terminal domain two doublestranded RNA-binding motifs, and its kinase catalytic domain is located in the carboxyl-terminal (21, 22). On binding dsRNA, PKR dimerizes and undergoes autophosphorylation at multiple sites (23). Expression of catalytically defective mutant PKR (K296R) in cells inhibited the autophosphorylation and subsequent the activation of its major substrate eIF2-α (24, 25). PKR-mediated signaling may promote autophagy through eIF2-α phosphorylation (26). In several viral infections, PKR plays an essential role in the autophagy trigger (27). In STAT3<sup>−</sup>/<sup>−</sup> cells, PKR is able to induce autophagy through LC3-I to LC3-II conversion and the formation of vacuole compartments (28). In cells treated with type I interferon (IFN-I), both LC3 activation and p62 expression are increased (29).

In this work, we unveil the mechanisms that regulate Nrf2 gene expression in a PKR-dependent fashion. We describe for the first time the signaling pathway that coordinates Nrf2 activation during *Leishmania* infection. Finally, the induction of cytoprotective genes through the novel PKR/Nrf2 pathway may represent a prominent therapeutic mechanism for treatment and guide the development of novel targets in both infectious and inflammatory diseases.

# MATERIALS AND METHODS

# Reagents

Chloroquine diphosphate salt, dl-sulforaphane (SFN), phorbol-12 myristate-13 acetate (PMA), *N*-acetyl-l-cysteine (NAC), Wortmannin, and LY294002 hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). AKTi (AKT inhibitor VIII, Akt1/2) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Poly (cytidylic-inosic) acid potassium salt (PolyI:C) and the PKR inhibitor CAS 608512-97-6 were purchase from Calbiochem-Millipore (Darmstadt, Germany). Human recombinant interferon-alpha 2b was obtained from Blausiegel (Cotia, SP, Brazil).

# Cell Lines and Culture

The mouse macrophage leukemia cell line RAW 264.7 (TIB-71; American Type Culture Collection (ATCC), Manassas, VA, USA), the human monocytic leukemia cell line THP-1 (ATCC:- TIB202TM) and the human embryonic kidney cell line HEK-293T (ATCC:CRL-11268) were maintained in DMEM medium with high glucose (Vitrocell Embriolife, Campinas, SP, Brazil) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). THP-1 cells were differentiated to macrophages with 40 ng/mL of PMA for 3 days. Afterward, the cells were washed three times with PBS and incubated with fresh medium for an additional 3 days. RAW 264.7 cells expressing either empty vector (RAW-Bla cells) or a dominantnegative PKR K296R (RAW-DN-PKR cells) were donated by Dr. Aristóbolo Silva, Federal University of Minas Gerais, Brazil.

# Peritoneal Macrophages

Ten-week-old male 129/SvEv PKR−/− (PKR-ko) and their respective wild-type littermates (WT) were used for experiments. Briefly, 4 days before peritoneal lavage, 2 mL of 3% thioglycolate were intra-peritoneally injected in each mouse. Thioglycolate-elicited peritoneal macrophages from wild-type or PKR-knockout 129Sv/ Ev were obtained by injecting 8 mL of serum-free DMEM into the peritoneal cavity. After 1 h, the cells were washed once in PBS and then plated in in DMEM medium supplemented with 10% FBS on glass coverslips at 2 × 105 /well in 6-well or 24-well polystyrene plates for subsequent *Leishmania* infection assays.

# Cell Treatment

To induce the activation of Nrf2, 10 mM SFN were used as positive controls. For the inhibition of PKR activity, we pretreated the cells for 1 h with 300 nM of the PKR inhibitor (PKRi). To induce PKR activation, poly(inosinic-cytidylic-) acid potassium salt (PolyI:C) at a final concentration of 25 µg/mL or recombinant IFNα-2b at 1,000 U/mL were used. PI3K/Akt inhibition was accomplished by cell treatment with 10 µM LY294002, 10 mM Wortmannin or 5 mM AKTI (AKT inhibitor VIII Akt1/2). To inhibit autophagy, we used 40 µM chloroquine. *N*-acetylcysteine (NAC) was used at a concentration of 10 mM.

# Parasites, Culture Conditions, and Infection

*Leishmania (Leishmania) amazonensis* (WHOM/BR/75/Josefa) and *Leishmania (Viannia) braziliensis* (BA788) were used in this study. The *L. (L.) amazonensis* strains obtained from biopsies of patients with diffuse cutaneous leishmaniasis (DCL) (Ba276, Ba336, and Ba760) or localized cutaneous leishmaniasis (Ba69, Ba73, and Ba125) were also used *in vitro* assays. The promastigote forms were grown at 26°C in Schneider's Insect Medium (Sigma-Aldrich) with 10% fetal bovine serum, and metacyclic promastigotes were collected from stationary cultures and used for cell infections. Macrophages were infected with *Leishmania* promastigotes at a parasite:cell ratio of 10:1 at 37°C. Infected macrophages were counted in a Neubauer Chamber by light microscopy to assess the infection index, which was calculated by multiplying the percentage of infected macrophages by the average number of parasites per macrophage in Giemsastained slides.

# Immunoblotting