In the context of acute infection, *KIR2DS3* and the IFN-λ3 SNP rs12979860 unfavorable T allele synergized to increase the risk of chronic infection (58). This study also suggested a direct link between IFN-λ and NK cells, showing reduced IFN-γ production by NK cells upon IFN-λ treatment (58). However, these data were difficult to reproduce in other cohorts. The IFN-λ3 rs12979860 CC genotype was associated with decreased levels of the inhibitory receptor NKG2A after infection resolution (31). Individuals bearing the CC genotype also displayed increased NK cell function measured by IFN-γ production after stimulation irrespective of infectious outcome suggesting that IFN-λ genotype influenced NK cell function but that this was not sufficient to achieve spontaneous HCV clearance (31). Although *IFNLR1* mRNA expression could be detected in NK cells (59, 60), they express very low levels of the specific type III IFN receptor (IFN-λR1) on cell surface, even after IFN-α stimulation (31, 61, 62). Treatment of purified NK cells with IFN-λ had no effect on neither NK cytotoxicity nor cytokine production (31, 60, 62, 63). On the other hand, it was reported that the level of expression of IFN-λR1 could be upregulated by IFN-λ treatment (59) and studies in IFN-λR1−/− mice have demonstrated that this receptor is required for optimal antitumoral *in vivo* activity of NK cells (64), suggesting that in some activation context, NK cells could become sensitive to type III IFNs.

Given the lack of activation of NK cells by IFN-λ (31, 60, 62, 63), indirect mechanism were investigated. IFN-λ1 affected NK cells indirectly *via* the activation of monocyte derived macrophages. Macrophages activated by IFN-λ1 produced cytokines of the IL-12 family (IL-12p40) that could then activate NK cells leading to increased IFN-γ production. This activation was determined by polymorphisms in the IFN-λ3 gene, and the presence of monocytes was essential (53). This suggests that HCV-infected individuals bearing the unfavorable IFN-λ3 allele have an impaired monocyte function. Monocytes can activate NK cells through the production of IL-12 or IL-18. Stimulated monocytes from CC genotype background produced significantly more IL-12p40 and IL-12p70 compared to monocytes of the CT or TT genotype. Blocking IL-12 and not IL-18 abolished the IFN-λ association with the level of NK cell activation by monocytes, suggesting that IL-12 is a major player in the interplay between monocyte and NK cells that is associated with IFN-λ3 genotype in HCV-infected subjects (53).

Analysis of NK cell phenotype and function in chronic HCV infection demonstrated that CD56bright NK cell subsets are significantly more cytotoxic than in healthy donors based on TRAIL and CD107a expression (65). This effect was independent of the IFN-λ3 rs12979860 genotype, but subjects carrying the TT genotype exhibited the highest levels of TRAIL+ and CD107a+IFNγ+NK cells. In the same study, CD56dim NK cells of TT genotype individuals produced more TNF-α. Accordingly, individuals with the TT genotype also had a higher proportion of polyfunctional NK cells (65).

In conclusion, whether by a direct or indirect mechanism, it appears that type III IFNs can modulate NK cells activation and functions but further investigation is required to identify the exact mechanism.

#### Dendritic Cells

Dendritic cells are important antigen-presenting cells and have a central role in mediating the link between the innate and adaptive immune response. DCs also are a major source of type III IFNs (8, 60, 66). Stimulation of DCs (*in vitro* and *ex vivo*) with HCV RNA induced the production of both type I and type III IFNs and the levels were associated with IFN-λ3 rs12979860 genotype, with the favorable CC allele leading to the highest IFN type III production (66). Type I and type III IFNs produced by DCs could control HCV replication *in vitro*, suggesting again an important role for type III IFN in HCV infection (66). During chronic HCV infection, serum levels of IFN-λ1 were lower compared to HCV resolvers and healthy controls (29). In acute HCV infection, IFN-λ1 serum levels were variable as described earlier (29, 31). Interestingly, HCV proteins E2 and NS3 inhibited IFN-λ1 production by stimulated DCs, suggesting that IFN-λ1 is an important immune mediator in HCV infection (29). On the other hand, treatment of DCs with IFN-λ altered their function toward a dysfunctional help to T cells (7). IFN-λ-treated DCs exhibited decreased T cell stimulation capacity by upregulating PDL1 expression. In addition, IFN-λ-treated DCs promoted the expansion of regulatory T cells (Tregs), further impeding with the immune response. Further research will be needed to clarify the role of IFN-λ and DCs during a viral infection, such as HCV.

#### CD4 and CD8 T Cells

The link between IFN-λ and T cells is less studied compared to the link with cells of the innate immune response. A study by Bes et al. showed that CD4 T cell responses to HCV, assessed by IFNγ enzyme-linked immunospot (ELISpot) assay, were of a higher frequency in the unfavorable allele (non CC) group (67). This was unexpected since a stronger immune response is normally associated with HCV spontaneous clearance. However, they tested a limited number of patient (n = 69; with 38 samples with positive ELISpot response), and there was a lot of variability between samples. In a more recent study, Scheurich et al. tested the breadth and frequency of CD4 responses to HCV and stratified their results according to the IFN-λ3 rs12979860 genotype (68). However, they did not find any difference between the various IFN-λ groups. Genetics studies showed that polymorphisms of MHC class I and MHC class II are associated with spontaneous clearance of HCV infection, independently of the IFN-λ3 polymorphism (69, 70). Protective alleles were shown to have additive effect, suggesting that innate and adaptive immunity contribute independently to the prediction of a favorable outcome following acute HCV infection. However, these independent associations do not rule out the possibility that type III IFN could modulate CD4 T cells responses. Indeed, data from other models suggest that IFN-λ can indeed impact CD4 T cells (71–73). It was reported that naive and memory CD4 T cells express IFN-λR1 mRNA and that T cell stimulation in presence of IFN-λ inhibited IL-4, IL-5, and IL-13 production, thus impeding the development of Th2 helper response without affecting cell proliferation (72, 73). IFN-λ also inhibited the upregulation of IL-4Rα on the surface of stimulated naive CD4 T cells, thus limiting the Th2 polarizing effect of IL-4 on these cells. IFN-λ-treated cells expressed significantly less GATA3, the transcription factor that is master regulator of Th2 differentiation, further supporting the hypothesis that IFN-λ has inhibitory effect on the development of a Th2 response (73). Whether such mechanisms are implicated during HCV infection remains to be seen. It is also tempting to speculate that the effect of IFN-λ polymorphism on production of IL-12 by monocytes may indirectly influence priming of the HCV-specific CD4 and CD8 T cells and the generation of antiviral Th1 responses.

#### B Cells

The role of the B cells and neutralizing antibody responses during acute HCV infection remains unclear. Some studies showed that early anti-HCV antibody response is associated with higher rate of spontaneous clearance of the virus during primary infection and reinfection (74–78), but other studies showed no association (79–82). To our knowledge, no data are currently available on the role of IFN-λ polymorphisms in the B cell response against HCV but it can be inferred from other models. Generally, the role of IFN-λ on B cells was only modestly studied. One recent article by de Groen et al. demonstrated that both naive and memory B cells express IFNLR1 mRNA and that IFN-λ1 can activate B cells (83). Treatment of B cells with IFN-λ1 led to increased expression of ISGs (Mx1 and OAS1) as well as increased expression of TLR7. IFN-λ1 also enhanced IgM and IgG production from TLR7/8 stimulated B cells. Finally, IFN-λ1 stimulation increased the proliferation of B cells stimulated with TLR7/8 agonist. However, after TLR2 or TLR9 stimulation, IFN-λ1 had no effect on the antibody production or proliferation (83). Globally, this study suggests that IFN-λ1 can enhance B cell response, but only under certain stimulation conditions.

In another context, Egli et al. showed that the IFN-λ3 rs8099917 GG genotype was associated with a higher rate of seroconversion after influenza vaccination in a cohort of immune-suppressed transplant patients (84). In addition, GG genotype carriers showed lower Th1 responses after PBMC stimulation with influenza antigens, suggesting that the IFN-λ3 rs8099917 genotype can affect the Th1/Th2 balance. Accordingly, adding IFN-λ3 in the stimulation medium increased the Th1 cytokine production and reduced Th2 cytokine production (84). These observations were confirmed in a cohort of healthy volunteers where IFN-λ3 treatment enhanced Th1 cytokine profile and reduced production of Th2 cytokines after influenza stimulation (84). Also, IFN-λ3 decreased B cell proliferation and antibody production. Interestingly, it was also demonstrated that adding peptides that block the effect of IFN-λ3 during the stimulation led to an increased antibody production, suggesting that blocking IFN-λ3 during influenza vaccination could improve the seroconversion rate and thus have a better protective effect (84).

These two aforementioned studies used different type III IFNs to stimulate B cells (IFN-λ1 or IFN-λ3) as well as different antigenic stimulation. Hence, there is no clear conclusion on whether type III IFNs exert a beneficial or detrimental effect on B cell function and antibody production, and additional studies with standardized stimuli are warranted.

# SUMMARY OF THE ROLE OF TYPE III IFNs ON HEMATOPOIETIC CELLS

Within the hematopoietic compartment, DCs are the main producers of IFN-λ, and they can travel between the liver and peripheral blood. The production of IFN-λ by DCs can inhibit HCV RNA replication in hepatocytes. However, HCV proteins E2 and NS3 can also inhibit IFN-λ production by DCs. Hematopoietic cells express variable levels and splice variants of IFN-λR1, and conflicting results were obtained about the effect of IFN-λ treatment on these cells. HCV exposed DCs or DCs treated with IFN-λ display reduced stimulation of T cells by upregulating expression of PDL1 and enhanced proliferation of Tregs. Monocytes are responsive to IFN-λ treatment resulting in IL-12 and IL-18 production. In turn, these cytokines can influence NK cell functions, and thus, IFN-λ is an important component of the innate immune response to HCV. The role of IFN-λ on CD4 and CD8 T cells as well as on B cells in the context of HCV remains understudied, but studies suggest that IFN-λ could modulate the CD4 Th1/Th2 balance and can also have a positive or negative impact on IgG production by B cells (**Figure 2**).

## TYPE III IFNs AND HCV-SPECIFIC IMMUNITY DURING PREGNANCY

In HCV-infected women, a sharp decrease in HCV viral load is sometimes observed after childbirth, suggestive of a boost in the immune response following delivery (85, 86). It was recently demonstrated that beside a stronger T cell response, the presence of the favorable CC IFN-λ3 rs12979860 genotype was significantly associated with this high decrease in viral load (87). Considering the high linkage disequilibrium between IFNλ3 rs12979860 genotype and IFN-λ4 rs368234815 genotype, the IFN-λ4 was also associated with the decrease in viral load postpartum (87). It is well known that women's immune system is altered through pregnancy to avoid a reaction against the fetus (88). It is postulated that the innate immune system will play a significant role against pathogens, while the adaptive immune responses are dampened by increased Tregs activity (89, 90). Another recent study showed that the expression of innate immunity genes is enriched in postpartum women compared to control (91). Interestingly, ISGs level in women of the CT or TT IFN-λ3 rs12979860 genotype remained elevated as late as 24 weeks after childbirth, while women with the CC IFN-λ3 genotype were comparable to non-pregnant controls (91). This reflects what was observed in the context of acute HCV where individuals with non-favorable CT or TT IFN-λ3 genotype had a higher baseline ISG expression in the liver.

#### IFN-**λ** DURING TREATMENT WITH DIRECT-ACTING ANTIVIRALS (DAA) AND HCV-RELATED LIVER DISEASE

With the development of novel DAAs that are highly effective (~100%) against most genotypes, treatment has switched to IFNfree regimens. Limited studies have indicated that polymorphism

in the IFN-λ region may influence the response to DAAs, especially if IFN is still used in the combination (92, 93). However, given the high rate of response to DAAs and the availability of multiple products on the market, IFN-λ has lost its predictive value, and testing for it before treatment is no longer recommended except in specific situations where a DAA and IFN combination may still be warranted (94).

Chronic HCV infection is associated with an increased risk of liver-related illness, such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The link between IFN-λ polymorphisms and HCV-related liver disease is not completely clear and was reviewed in detail elsewhere (95). One study showed that the favorable CC IFN-λ3 rs12979860 allele, associated with better chance of HCV clearance, is also associated with higher blood ALT levels, indicative of increased liver inflammation. However, IFN-λ rs12979860 polymorphism was not associated with fibrosis progression in the same cohort (96). Another study did not find any association of the IFN-λ polymorphism with any of the observed associated liver illness (decompensated cirrhosis, HCC, liver-related death, and all-cause mortality) (97). On the other hand, Bochud et al. demonstrated that particularly in non-genotype 1 HCV-infected individuals, the favorable IFN-λ alleles were associated with increased inflammation and higher fibrosis scores (98). In agreement with this, Eslam et al. observed a significant association between IFN-λ3 rs12979860 polymorphism and liver necroinflammatory activity, serum level of AST and ALT, as well as fibrosis score and progression (99). Once again the association was stronger in individuals infected with HCV genotype 3 than those infected with HCV genotype 1. Also, in patients carrying the IFN-λ4 rs368234815 unfavorable allele (ΔG), there was a correlation between the frequency of CD107a expressing cells and the serum ALT levels, suggestive of increased liver damage (49). In the context of HCC, two studies associated IFN-λ3 rs12979860 unfavorable CT or TT alleles with liver cirrhosis and the development of HCC in patients chronically infected with HCV (100, 101). However, this was not confirmed in two other independent studies in Japanese (102) or Italian cohorts (97). Finally, HCV-related liver disease is a multifactorial problem, and the independent association of genetic factors may not be a clear cut. Fortunately, with the development of highly effective DAAs, it is expected that these complications will be less frequent as SVR will be achievable in most patients.

#### CONCLUDING REMARKS

Type III IFNs exhibit strong antiviral activity, and yet, the expression of a functional IFN-λ4 protein was strongly associated with failure to clear HCV infection either spontaneously or after IFN-based treatment. A recent study suggested that humans suppress IFN-λ4 expression through various mechanisms and hence immune functions may be dependent on other type III IFNs (103). Data accumulated so far suggest that a higher baseline ISG expression level is associated with induction of a refractory state, where further IFN treatment has no beneficial effect. With the new era of anti-HCV IFN-free DAA therapies, the role of type III IFNs during therapy has become somewhat irrelevant but its role in mediating spontaneous clearance during acute HCV infection and modulating the cross-talk between innate and adaptive immunity remains highly pertinent. This is applicable not only for HCV infection but also for other viral infections and response to vaccines. Furthermore, the recently described role of type III IFN polymorphisms in driving immunity postpartum is

#### REFERENCES

just the tip of the iceberg as it will become increasingly relevant to mother–infant health and vertical transmission of various pathogens.

#### AUTHOR CONTRIBUTIONS

Both the authors reviewed the literature and wrote this manuscript.

#### ACKNOWLEDGMENTS

The authors would like to thank Thomas Fabre for preparation of the figures. Their research is funded by the Canadian Institutes of Health Research (CIHR) (MOP-133680), Alberta Innovates Health Solutions and the Canadian Liver Foundation. MB is supported by postdoctoral fellowships from the American Liver Foundation, the Fonds de Recherche du Québec–Santé (FRQS), and the Canadian Network on Hepatitis C (CanHepC). NS is supported by a Chercheur Boursier salary award from the FRQS.

persistence, and disease. *Proc Natl Acad Sci U S A* (2002) 99:15661–8. doi:10.1073/pnas.202608299

non-viral chronic liver disease. *Nat Commun* (2015) 6:6422. doi:10.1038/ ncomms7422

**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 © 2016 Boisvert and Shoukry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Type i interferons in Bacterial infections: A Balancing Act

*Pavel Kovarik\*, Virginia Castiglia, Masa Ivin and Florian Ebner*

*Max F. Perutz Laboratories, University of Vienna, Vienna, Austria*

Defense against bacterial infections requires activation of the immune response as well as timely reestablishment of tissue and immune homeostasis. Instauration of homeostasis is critical for tissue regeneration, wound healing, and host recovery. Recent studies revealed that severe infectious diseases frequently result from failures in homeostatic processes rather than from inefficient pathogen eradication. Type I interferons (IFN) appear to play a key role in such processes. Remarkably, the involvement of type I IFNs in the regulation of immune and tissue homeostasis upon bacterial insult may have beneficial or detrimental consequences for the host. The reasons for such ambivalent function of type I IFNs are not understood. The disparate effects of type I IFNs on bacterial infections are in marked contrast to their well-established protective roles in most viral infections. In this review, we will focus on type I IFN effector mechanisms which balance processes involved in immune and tissue homeostasis during specific bacterial infections and highlight the most important missing links in our understanding of type I IFN functions.

#### *Edited by:*

*Claudia U. Duerr, McGill University, Canada*

#### *Reviewed by:*

*Didier Soulat, Universitätsklinikum Erlangen, Germany Stefanie Scheu, University of Düsseldorf, Germany*

#### *\*Correspondence:*

*Pavel Kovarik<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 01 November 2016 Accepted: 14 December 2016 Published: 26 December 2016*

#### *Citation:*

*Kovarik P, Castiglia V, Ivin M and Ebner F (2016) Type I Interferons in Bacterial Infections: A Balancing Act. Front. Immunol. 7:652. doi: 10.3389/fimmu.2016.00652*

Keywords: type I interferon, bacterial infection, cytokines, chemokines, innate immunity, resilience to infections, immunomodulation, immunosuppression

#### INTRODUCTION

Successful defense against pathogens requires both, the eradication of the infectious agent by the immune system as well as tissue protection against the damaging effects of the immune response. Increasing evidence indicates that many if not most infectious diseases result from insufficient resilience, i.e., from a failure of the infected host to repair and regenerate destroyed tissues, rather than from inefficient pathogen clearance (1–4). Mechanisms which preserve the integrity of host tissues during the intensive inflammatory response against the pathogen remain incompletely understood. Recent studies established that successful tissue protection during infection requires systems which balance the immune response as well as mechanisms which restore tissue homeostasis. These mechanisms are often interdependent and result from messengers like growth factors, cytokines, or lipids produced by immune cells (1–4). Examples include the anti-inflammatory cytokine IL-10, the tissue regeneration promoting IL-22 and amphiregulin, the tissue remodeler TGF-β, or the pro-resolving lipid lipoxin (5–8). Remarkably, several recent studies demonstrated that type I interferons (IFNs) can also act as critical resilience-promoting cytokines during infections with several streptococcal species (9–11). Such protective functions are in marked contrast to detrimental effects of type I IFN during infections with many other bacterial species (12, 13). The reasons for the ambivalent roles of type I IFNs in bacterial infections remain poorly understood. However, it appears that the ability of type I IFNs to both suppress and stimulate immune responses is of critical importance (**Table 1**; **Figure 1**). This review focuses on the role of type I IFNs in balancing pro- and anti-inflammatory processes as well as cell survival and cell death programs during antibacterial defense and discusses how these effects determine the outcome of an infection.

# TYPE I IFN INDUCTION BY BACTERIA

Type I IFNs were described more than a half century ago as products which are secreted by virus-infected cells and interfere

with virus replication in autocrine and paracrine ways (40). It is now known that type I IFNs are cytokines produced in response to viral, bacterial, and fungal pathogens, as well as parasites. The effector mechanisms of type I IFNs mainly derive from products of genes which are transcriptionally regulated by type I IFN signaling. Type I IFNs induce hundreds of interferon-stimulated genes (ISGs) through activation of the homodimeric STAT1 and the heterotrimeric STAT1–STAT2–IRF9 (i.e., ISGF3) transcription factors (41). Bacteria trigger type I IFN production mostly following the recognition of bacterial nucleic acids or the Gram-negative cell wall component lipopolysaccharide (LPS) by innate immune receptors (12, 42). The induction mechanisms have been best studied for IFN-β which belongs together with IFNα4 to the first type I IFNs produced during infection and is the driver of other type I IFN genes (43, 44). The induction of IFN-β by bacterial DNA is complex and involves different pathways. The most common mode of IFN-β induction by bacterial DNA is through the cytosolic DNA sensor cyclic GMP-AMP synthase, as described for *Francisella novicida*, group B streptococcus (GBS) (*Streptococcus agalactiae*), *Legionella pneumophila*, *Listeria monocytogenes*, *Mycobacterium tuberculosis*, and *Neisseria gonorrhoeae* (45–51). The cytosolic DNA sensor Ifi204 (IFI16 in humans) contributes to IFN-β induction in the course of *F. novicida* and *L. monocytogenes* (48, 49). The signaling events downstream of these DNA sensors involve the STING– TBK1–IRF3 pathway driving the IFN-β gene transcription. *L. monocytogenes* can activate this pathway also independently of DNA sensing. This alternative mechanism is driven by binding of the bacterial product c-di-AMP to STING (51, 52). The recognition of bacterial DNA through unmethylated CpG motif-containing DNA by the endosomal Toll-like receptor 9 can also contribute to IFN-β induction, although the importance and relevance of this pathway in the context of the overall IFN-β production and host response have not been entirely clarified (42). Bacterial RNA has been recently established as another key IFN-β inducer. A short and highly conserved sequence found in the bacterial 23S rRNA is recognized by the mouse TLR13 leading to Myd88- and IRF5-dependent IFN-β induction (9, 53–56). Human cells employ TLR8 as IFN-β-inducing RNA sensor rather than TLR13 which is missing in humans (9, 53, 57–59). The precise nature of bacterial RNA triggering the human TLR8 remains to be identified.

IFN-β induction by LPS occurs after binding of this ligand to the TLR4 and the following internalization into the endosome (60). Subsequently, a TRIF-dependent activation of the kinase TBK1 causes phosphorylation of the transcription factor IRF3 to stimulate IFN-β gene transcription. Endosomal signaling has been also implicated in IFN-β induction by TLR2 in response to Gram-positive bacteria, although this mechanism appears to be restricted to specific immune cells and/or pathogens (61–63). The cytosolic receptors NOD1 and NOD2 were reported to trigger IFN-β production following infection with *Helicobacter pylori* and *M. tuberculosis*, respectively (20, 64). NOD2 engagement can induce IFN-β also in responses to *Staphylococcus aureus* (14).

While the pathways causing IFN-β induction by bacteria are relatively well understood, more studies are needed to assess the importance of individual pathways for the overall IFN-β production in whole organism rather than cells. One of the rare studies on this topic revealed that the dominant IFN-β-inducing pathway during infection with *Streptococcus pyogenes* (group A streptococcus) is the TLR13-mediated RNA recognition pathway (9).

Additional work is also needed to clarify the key IFN-β producing cells, as investigated during, e.g., *L. monocytogenes* infections (65–67) and the reported cell type-specific features of IFN-β induction (12, 15, 16).

# TIPPING THE BALANCE I: BENEFITS OF IMMUNOMODULATORY EFFECTS OF TYPE I IFN SIGNALING DURING BACTERIAL INFECTIONS

Type I IFNs' ability to stimulate immune responses against viruses has been established very early after their discovery but it soon became clear that these cytokines exhibit also immunosuppressive activities. The first evidence for such immunosuppressive activities was provided in a study showing that type I IFNs were able to reduce carrageenin-induced footpad swelling (68). Thus, type I IFNs are now regarded as immunomodulatory cytokines capable of enhancing or dampening the immune response, depending on the context. This ambiguousness contributes to the disparate and still incompletely understood roles of type I IFNs during bacterial infections. Importantly, no unifying principles have been found to date: neither the beneficial nor detrimental effects of type I IFN signaling correlate with the broad pathogen classification into Gram-positive and -negative, extra- and intracellular pathogens, or the route of infection (**Table 1**; **Figure 1**).

Immunosuppressive effects of type I IFN signaling are beneficial during infection with the Gram-positive largely extracellular human pathogen *S. pyogenes* (9). *S. pyogenes* is the causative agent of mild (e.g., pharyngitis and scarlet fever) but also invasive and life-threatening infections (e.g., cellulitis, necrotizing fasciitis, and streptococcal toxic shock syndrome). Mice deficient in the type I IFN receptor IFNAR1 are more susceptible to subcutaneous *S. pyogenes* infection, which is a relevant model of severe invasive infection of the soft tissue (15). Type I IFN signaling promotes resistance against *S. pyogenes* by suppressing the transcription of the *Il1b* gene (9). The absence of type I IFN signaling results in an unrestricted production of IL-1β thereby causing a lethal hyperinflammation and organ damage. Importantly, type I IFN signaling balances rather than prevents *Il1b* transcription so that a controlled and life-saving IL-1β production is achieved (9). The key IFN-β producer and effector cells in this infection model are both LysM+ and CD11c+ myeloid cells (9).

Immunomodulation by type I IFN signaling is protective during infection with the human Gram-positive extracellular pathogen GBS (16, 17). GBS is regarded as commensal microbe asymptomatically colonizing the skin and mucosal tissues of 30% people, yet it is the leading cause of severe neonatal pneumonia and meningitis in developed countries. The absence of IFNAR1 results in increased bacterial loads during both subcutaneous GBS infection of neonate mice and intravenous infection of adult animals (16, 17). Similarly, type I IFN signaling is protective against uncontrolled bacteremia during infection with the Gram-negative intracellular bacterium *L. pneumophila*, which is a frequent cause of the severe pneumonia, Legionnaire's disease (18). Type I IFN signaling inhibits in a cell-autonomous way replication of *L. pneumophila* inside the infected cell (18, 19). The organismal physiology of the protective effects of type I IFN signaling during infection with GBS and *L. pneumophila* remains to be elucidated so that it is presently unclear whether immunosuppressive or immunostimulatory effects of type I IFNs drive the resistance against these two pathogens.

Stimulation of the immune response by type I IFN signaling is advantageous in defense against the Gram-negative pathogen *H. pylori* (20). *H. pylori* is a frequent cause of chronic gastritis and is associated with increased risk of gastric ulcers and stomach cancer. Deficiency in type I IFN signaling causes increased *H. pylori* loads in the stomach of orally infected mice. The lack of type I IFN responses is associated with decreased levels of the chemokine CXCL10 suggesting that type I IFNs promote defense against *H. pylori* by stimulating CXCL10-driven inflammation (20). Immunostimulatory effects of type I IFNs are beneficial also during gastric infection with the food-borne Gram-positive intracellular pathogen *L. monocytogenes* (28). *L. monocytogenes* infects the gastrointestinal tract, where it traverses the epithelial barrier and spreads into distant organs. Deficiency in type I IFN signaling results in an increased bacterial dissemination and is accompanied by diminished production of several proinflammatory cytokines, including TNF and IL-6 upon gastric infection using oral gavage (28). Interestingly, type I IFN signaling plays no role in an infection model using food contaminated with *L. monocytogenes* (30).

#### TIPPING THE BALANCE II: DISADVANTAGES OF IMMUNOMODULATORY EFFECTS OF TYPE I IFN SIGNALING DURING BACTERIAL INFECTIONS

Immunosuppression by type I IFN signaling is detrimental during infection with the intracellular pathogen and causative agent of tuberculosis, *M. tuberculosis* (22–24). Type I IFN signalingmediated inhibition of IL-1 cytokines during *M. tuberculosis* lung infection blunts the antimicrobial defense and results in increased local as well as systemic bacterial loads (24). The key type I IFN effector cells are transplantable inflammatory monocyte-macrophage cells and DCs (24). The precise mechanism of IL-1 inhibition by type I IFN signaling in this infection model is not resolved but includes both direct as well as indirect mechanisms (24). The indirect IL-1 inhibition appears to be mediated by the anti-inflammatory cytokine IL-10 which is known to be upregulated by type I IFNs (69). The importance of type I IFN signaling in *M. tuberculosis* infections is underlined by type I IFN signaling-associated gene expression pattern found in blood cells of patients with active tuberculosis (70). The detrimental function of type I IFN signaling during *M. tuberculosis* lung infection is converted into a protective one if IFN-γ signaling is missing (71). Under such conditions, type I IFNs inhibit the polarization of macrophages into infection-permissive alternatively activated macrophages.

Inhibition of immune response by type I IFNs is deleterious during infection with the facultative intracellular Gramnegative bacterium *F. novicida* (31). *F. novicida* is a subspecies of *Francisella tularensis* which infects humans through the skin or aerosol droplets and causes ulceroglandular or pneumonic tularemia, respectively. IFNAR1-deficient mice infected intradermally with *F. novicida* respond by an increased IL-17 production compared to WT animals and, correspondingly, are more resistant against infection (31). Similar increase in resistance is also observed during lung infection with *F. tularensis* (31). The key IL-17 producers during *F. novicida* infection are IL-17A+ γδ T cells which show enhanced expansion in the absence of type I IFN signaling.

Interferon-β exacerbates infection with *S. typhimurium* by reducing the ability of the host to launch a complete immune response (34). *S. typhimurium* is Gram-negative intracellular pathogen associated with gastroenteritis in humans and a severe disease resembling typhoid fever in mice. Mice deficient in IFNβ are more resistant against oral infection with *S. typhimurium* and display enhanced expression of IL-1β and the neutrophil chemoattractants, CXCL1 and CXCL2 (34). These changes are attributable to IFN-β-mediated inhibition of these genes in macrophages and are independent of *S. typhimurium*-induced macrophage death.

*Listeria monocytogenes*-induced type I IFN signaling downregulates the expression of both type II IFN receptor subunits, IFNGR1 and IFNGR2, thereby decreasing the responsiveness of macrophages and DCs to IFN-γ (29). The suppression of the IFN-γ signaling results in an increased susceptibility to *L. monocytogenes* infection. Increased susceptibility to *L. monocytogenes* infection is also caused by type I IFN-mediated induction of anti-inflammatory IL-10 (72).

Immunosuppressive effects of type I IFNs are harmful during postinfluenzal bacterial pneumonia (36–39). The immune response during a secondary postinfluenzal infection with the Gram-positive extracellular pathogen *S. pneumoniae*, a key causative agent of pneumonia, is impaired. This is caused by the ability of type I IFNs to suppress production of the neutrophil chemoattractants CXCL1 and CXCL2, the macrophage chemoattractant CCL2, and the inflammation-promoting cytokine IL-17 (36–38). The resulting reduction of leukocyte infiltration in the lung diminishes the capability of the host to control bacterial growth. Similar alterations in the immune response appear to be responsible for the increased susceptibility of mice to postinfluenzal infection with *S. aureus* and *Pseudomonas aeruginosa* (39). The mechanisms of the immunosuppressive effects of type I IFN signaling during postinfluenzal bacterial infection are not well understood but they might act downstream of the type I IFN-mediated inhibition of IL-1 cytokines.

Enhancement of the inflammatory response is associated with detrimental effects of type I IFN signaling during lung infection with the Gram-positive extracellular pathogen *S. aureus* (21). IFNAR1-deficient mice exhibit a lower TNF and IL-6 production and decreased leukocyte infiltration in the lung compared to WT animals suggesting that type I IFN signaling causes an exacerbated tissue damage (21). The pathogenicity of *S. aureus* strains correlates with the levels of type I IFNs induced during infection with differently virulent strains (14).

#### TIPPING THE BALANCE III: REGULATION OF TISSUE AND CELL INTEGRITY BY TYPE I IFNs DURING BACTERIAL INFECTIONS

Type I IFN signaling plays an indispensable role in the preservation of the epithelial barrier and epithelial integrity during lung infection with *S. pneumoniae* (10, 11). Type I IFN signaling promotes the maintenance of lung epithelial tight junctions during *S. pneumoniae* infection thereby reducing the passage of the pathogen from alveoli into the lung parenchyma (10). IFNAR1-deficient mice display increased permeability of the lung epithelium and enhanced invasiveness of *S. pneumoniae* infection associated with higher bacterial burden in distant organs. Type I IFN signaling protects the barrier function of the lung during *S. pneumoniae* infection also by promoting survival of the alveolar epithelial type II cells as revealed by IFNAR1 deletion specifically in this subtype of the barrier epithelium (11).

A common detrimental effect of type I IFN signaling during bacterial infections is the induction of various types of leukocyte cell death. Type I IFN-facilitated apoptosis of macrophages and lymphocytes appears to contribute to the increased susceptibility of WT mice compared to IFNAR1-deficient animals to intravenous and intraperitoneal infection with *L. monocytogenes* (25–27, 73). Type I IFN-facilitated death of macrophages is associated also with harmful effects of type I IFNs during infection with *F. novicida* and *S. typhimurium* (32, 33). *F. novicida* promotes macrophage death by type I IFN-mediated inflammasomeactivation whereas *S. typhimurium* employs type I IFN induction to stimulate RIP-dependent macrophage necroptosis.

# CONCLUSION AND FUTURE DIRECTIONS

Ample evidence exists for the pivotal role of type I IFNs in regulation of defense against bacterial pathogens. The complex and often disparate effects of type I IFNs on the outcome of different bacterial infections provide chances to exploit type I IFNs and their inducers as well as effectors for adjuvant therapies tailored to specific infectious diseases. A prerequisite for the development of such therapies is a detailed understanding of the molecular, cellular, and organismal physiology of type IFNs in the course of bacterial infections. The following topics appear particularly important since they represent rather underexplored yet critically important areas.

#### Pathogen Species

The inconsistent roles of type I IFNs during infections with different bacteria remain a significant and challenging topic in the current research. Only few common principles of type I IFN action have been found to date. They include the cell death-promoting effects of type I IFNs which contribute mostly to detrimental functions of type I IFN signaling. Another frequent observation is the suppression of IL-1β and neutrophil chemoattractants—these effects are, however, associated with both beneficial and harmful consequences for the infection outcome. Future studies employing pathogens which have not yet been analyzed in detail, such as *Klebsiella pneumoniae*, uropathogenic *E. coli*, or *Clostridium difficile*, might reveal novel common principles.

# Infection Route and Tissue-Specific Features of Type I IFN Signaling

The complexity of type I IFN function in bacterial infections is further increased by the distinct effects of type I IFNs in response to the same but differently administered pathogen. Type I IFNs are harmful followed intraperitoneal or intravenous infection with *L. monocytogenes* but protective in a physiologically more relevant intragastric infection (25–28). In contrast, type I IFN signaling has no impact on the overall outcome of *L. monocytogenes* infection after ingesting pathogen-contaminated food (30). These observations suggest that type I IFN signaling has, with regard to bacterial infections, distinct functions in different tissues/organs. This implication is supported by a recent study showing that exogenous type I IFN has, depending on the site of administration, disparate effects on the course of lung infection with *Coxiella burnetii* (35). Infections with *C. burnetii*, a Gram-negative intracellular bacterium, in humans occur after inhalation of bacteria and result in Q fever which can develop into an atypical pneumonia. Lung infection with *C. burnetii* in mice has a more severe course in WT compared to IFNAR1-deficient animals indicating that type I IFN signaling is disadvantageous in this infection model (35). Consistently, intraperitoneally administered type I IFN exacerbates *C. burnetii* infection. However, intratracheal delivery of type I IFN ameliorates the course of *C. burnetii* infection. The mechanisms of these distinct effects of type I IFN signaling in different tissues remain to be elucidated. Future studies should investigate other pathogens known of using various routes of infection and focus on physiologically most relevant routes.

#### REFERENCES

# Most Significant Type I IFN Inducers and Effectors

Modulation of type I IFN production during bacterial infection might represent a powerful approach in therapy of infectious diseases. Therapeutic targeting of type I IFN production requires the knowledge of the most important type I IFN-inducing pathway in a given infection. As most bacterial pathogens employ more than one pathway to stimulate type I IFN production, future efforts should focus on the identification of the most crucial bacterial and cellular components involved in type I IFN induction. These studies will need to use a combination of bacterial and animal host genetics for functional assessment and suitable reporter as well as imaging systems for type I IFN detection *in vivo*. On the effector side, recent studies provided a number of novel type I IFN-induced factors which interfere with pathogen replication and survival. Notably, various type I IFN-inducible small GTPbinding proteins have recently been showed to significantly contribute to the effects of type I IFNs [e.g., Ref. (74–77)]. These and yet to be discovered effectors represent potential targets for therapeutic intervention.

#### Human versus Mouse Systems

While many host defense mechanisms are well conserved among mice and men, important differences exist. For example, the mouse type I IFN inducer TLR13 is not expressed in humans (9, 53). Conversely, the human but not mouse TLR8 appears to be involved in type I IFN induction by bacterial RNA (59). Some antimicrobial functions of human neutrophils are enhanced by type I IFNs (78) whereas such stimulatory effects have so far not been described in mouse neutrophils. Future work should put more emphasis on studies of common and distinct features of type I IFN functions in bacterial infections.

# AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

# FUNDING

This work was supported by the Austrian Science Fund (FWF) grants P24540-B21 and I1621-B22 to PK, by funding from the European Union Seventh Framework Programme Marie Curie Initial Training Networks (FP7-PEOPLE-2012-ITN) for the project INBIONET under grant agreement PITN-GA-2012-316682, and by a joint research cluster initiative of the University of Vienna and the Medical University of Vienna.

**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 © 2016 Kovarik, Castiglia, Ivin and Ebner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Host and viral Modulation of RiG-i-Mediated Antiviral immunity

#### *Yiliu Liu1,2, David Olagnier1,2† and Rongtuan Lin1,2,3\**

*1 Jewish General Hospital, Lady Davis Institute, McGill University, Montreal, QC, Canada, 2Division of Experimental Medicine, McGill University, Montreal, QC, Canada, 3Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada*

Innate immunity is the first line of defense against invading pathogens. Rapid and efficient detection of pathogen-associated molecular patterns *via* pattern-recognition receptors is essential for the host to mount defensive and protective responses. Retinoic acid-inducible gene-I (RIG-I) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific RNA structures. Upon viral RNA recognition, RIG-I recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type I interferons (IFNs), as well as a large variety of antiviral interferon-stimulated genes. The RIG-I activation is tightly regulated *via* various posttranslational modifications for the prevention of aberrant innate immune signaling. By contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from RIG-I detections and targeting receptor or signaling molecules for degradation. These virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the RIG-I pathway. In this review, we summarize the recent advances regarding RIG-I pathogen recognition and signaling transduction, cell-intrinsic control of RIG-I activation, and the viral antagonism of RIG-I signaling.

Keywords: innate immunity, antiviral, infection, RIG-I, type I IFNs, virus–host interaction

# INTRODUCTION

Eukaryotic organisms rely on the host innate immune system to defend against viruses or other pathogenic microbes in early phases of infection. The innate antiviral immune response starts with the detection of evolutionarily conserved structures, termed pathogen-associated molecular patterns (PAMPs), by a set of germline-encoded pattern-recognition receptors (PRRs). With respect to their cellular localization, ligand specificity, and functions, PRRs are categorized into distinct families including the toll-like receptors, nucleotide-binding oligomerization domain-like receptors, C-type lectin receptors, retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) (1–5), as well as cytosolic viral DNA sensors such as cyclic GMP-AMP synthase (6, 7). Following the detection of specific viral PAMPs, PRRs trigger the activation of intracellular signaling cascades, ultimately leading to the production of type I interferons (IFNs), as well as pro-inflammatory cytokines. Secreted IFNs are crucial for the induction of numerous interferon-stimulated genes (ISGs); the products of which are major forces in controlling and restricting viral infections, thereby establishing a cellular antiviral state as well as helping to shape the adaptive immune response (8). Recent studies showed that viruses have evolved complex strategies to affect multiple stages of the host antiviral defense, from inhibiting the viral detection to manipulating components of the signaling pathways (9, 10).

#### *Edited by:*

*Uday Kishore, Brunel University London, United Kingdom*

#### *Reviewed by:*

*Winfried Barchet, University of Bonn, Germany Nicola Tamassia, University of Verona, Italy*

> *\*Correspondence: Rongtuan Lin*

####<EMAIL_ADDRESS>†Present address:*

*David Olagnier, Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark*

#### *Specialty section:*

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

*Received: 07 October 2016 Accepted: 16 December 2016 Published: 03 January 2017*

#### *Citation:*

*Liu Y, Olagnier D and Lin R (2017) Host and Viral Modulation of RIG-I-Mediated Antiviral Immunity. Front. Immunol. 7:662. doi: 10.3389/fimmu.2016.00662*

To ensure successful antiviral defenses and to avoid aberrant or dysregulation of host immune signaling, antiviral pathways need to be tightly regulated at each level. In this review, we will summarize the cell-intrinsic regulation of RIG-I receptor activity, as well as the viral strategies to subvert the RIG-I signaling machinery.

# RIG-I STRUCTURE AND LIGAND INTERACTIONS

The three members of the RLR family: RIG-I, MDA5 (melanoma differentiation factor 5), and LGP2 (laboratory of genetics and physiology 2) are expressed in most cell and tissue types. They function as cytoplasmic sensors for the recognition of a variety of RNA viruses and subsequent activation of downstream signaling to drive type I IFN production and antiviral gene expressions. These three RLR proteins are RNA-dependent ATPases belonging to the DExD/H-box family of helicases (11). Structurally, RLRs have a similar central helicase core that is comprised of two helicase domains, Hel1 and Hel2 with an insertion termed Hel2i. In addition, they all have a C-terminal domain (CTD). However, only RIG-I and MDA5 contain two N-terminal caspase activation and recruitment domains (CARDs) (3) (**Figure 1A**). Among these three, RIG-I is the founding member and hence the most intensively studied member of this family. Each domain of RIG-I plays unique roles during RIG-I autorepression and activation. In brief, the CTD and helicase domain are involved in RNA ligand binding and ATP hydrolysis-involved conformational changes (12–14), whereas the RIG-I CARDs facilitate interaction with other downstream CARD containing molecules (15).

Retinoic acid-inducible gene-I has been shown to be involved in the recognition of a variety of RNA viruses in the cytoplasm, such as the Sendai virus, influenza A and B viruses (IAV, IBV), vesicular stomatitis virus, measles virus (MV), Newcastle disease virus, Ebola virus (EBOV), dengue virus (DENV), and hepatitis C virus (HCV) (16–19). The short double-stranded (ds) RNA with a triphosphate (ppp) motif at the 5′-end, as found in these viral genomes, were shown to be a key signature recognized by RIG-I (20, 21). The 5′ppp dsRNA of viral nucleocapsids has also been characterized as stimulating RIG-I (22). 5′-Diphosphate-bearing RNA (5′ppRNA), either naturally contained in viruses, produced by *in vitro* transcription, or *via* chemical synthesis, were all shown to bind to RIG-I and were sufficient to activate RIG-I (20, 23). Physiologically, the control of *in vitro* and *in vivo* infections of reoviruses, which bear the 5′ppRNA genome, relies on RIG-I functionality (24). It is worth noting that the *in vitro-*synthesized 5′pppRNA sequences also trigger RIG-I activation (25). These agonists have demonstrated their therapeutic potential as broadspectrum antiviral agents and could be optimized as vaccine adjuvant candidates (26–30). Furthermore, the recognition of several DNA viruses, including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus (EBV), vaccinia virus (VACV), and adenovirus, *via* the RNA polymerase III were found to be RIG-Idependent (31, 32). Interestingly, the RIG-I-mediated upregulation of STING is required for protection against the HSV-1 by the RIG-I agonist, offering new evidence of the overlapping between RIG-I signaling and the host response to DNA viral infection (33). Notably, viral RNA triggered RIG-I signaling also mediates the inflammatory response *via* distinct pathways. The first involves the formation of the RIG-I inflammasome through interactions between RIG-I, ASC, and caspase-1 and the stimulation of IL-1β release. The second involves the adaptor proteins CARD9, Bcl-10, mitochondrial antiviral signaling protein (MAVS), and the activation of nuclear factor-κB (NF-κB) (34, 35). Upon RNA ligand binding, RIG-I undergoes a series of conformational changes and posttranslational modifications (PTMs) to achieve full activation (further detail below).

# RIG-I SIGNALING TRANSDUCTION

Activated RIG-I recruits its downstream adaptor molecule MAVS (also known as IPS-1, CARDIF, and VISA) through CARD–CARD-mediated interactions (36, 37). The oligomeric RIG-I CARD assembly and the polymeric formation of MAVS, together serve as a signaling platform for protein complexes that mediate the bifurcation of signaling into two branches. One branch recruits tumor necrosis factor receptor-associated factors (TRAF)-2/6 and the receptor-interacting protein 1 to subsequently activate the IKK complex, resulting in NF-κB activation (38). The other branch signals through TRAF3 and activates the TANK/IKKγ/IKKϵ/TBK1 complex, leading to the phosphorylation and dimerization of interferon regulator factors (IRF)-3 and -7 (39, 40). Activated IRF3/7 and NF-κB then translocate to the nucleus, together with ATF2, c-Jun, and the transcription coactivator CREB-binding protein/p300, to coordinate the IFN and pro-inflammatory gene expressions (41). Once secreted, IFNs bind to specific cell surface receptors and activate the JAK–STAT pathway. The activated transcription factors STAT1, STAT2, and IRF9 form the interferon-stimulated gene factors (ISGF3) complex. ISGF3 then translocates to the nucleus and coordinates the transcription of hundreds of ISGs including RIG-I, thus generating an amplifying loop leading to the accumulation of RIG-I during several types of infections (8) (**Figure 1B**).

# MECHANISMS OF RIG-I ACTIVATION

#### RIG-I Autorepression

Structural and biochemical studies have demonstrated that the activation of RIG-I is a multi-step process and is primarily regulated by conformational changes and PTMs. When initially identified as a dsRNA sensor, it was hypothesized that RIG-I was under negative regulation in physiological conditions. The over expression of the CARD domain of RIG-I alone demonstrated superior signaling activity than full length RIG-I in absence of viral PAMPs (2). Studies by Saito et al. showed that the deletion of CARD was dominant-negative for RIG-I signaling. By contrast, the deletion of repressor domain (RD) resulted in constitutive signaling, whereas RD expression alone ablated RIG-I signaling actions. Together, these findings provided the model of RIG-I autoregulation in which the RD is predicted to mask CARDs for signaling transduction in uninfected cells (42). The crystal structural analysis further delineated the models of autorepressed and ligand activated states of RIG-I, respectively. In a

recognizing RNA viruses with a 5′ppp signature. Upon viral RNA recognition, RIG-I recruits the adaptor protein mitochondrial antiviral signaling protein to activate the TBK1–IKKϵ complex and IKKα–IKKβ complex, which are responsible for the activation of transcription factors interferon regulator factor (IRF) 3, IRF7, and nuclear factor-κB. These transcription factors then translocate to the nucleus and coordinate the induction of type I interferons (IFNs). This is followed by the binding of the IFNs α and β to their cognate receptor, which will lead to the transcriptional activation of interferon-stimulated genes (ISGs) by the JAK/STAT signaling pathway. The products of ISGs are key factors in limiting pathogen spreading.

ligand-free state, CARDs and Hel2i interactions hinder dsRNA binding and inactivate RIG-I (14). The binding of 5′ppp dsRNA to RD leads to a conformational switch of RIG-I, which releases the autorepressed CARDs and exposes the helicase domain for ATP binding (14, 43). ATP hydrolysis is essential for RIG-I signaling. It enables RIG-I to translocate along the dsRNA, and further promotes the oligomerization of RIG-I CARDs. These processes assemble RIG-I into a filamentous architecture which facilitates the CARD–CARD interactions with the mitochondrial MAVS, leading to the subsequent signaling transduction for IFN production (44, 45). Importantly, RIG-I ATPase activity also plays a role in distinguishing self-RNA from non-self-RNA (46). It was reported that RIG-I ATP hydrolysis increases the binding affinity of RIG-I and dsRNA ligands; whereas the RIG-I mutants deficient in ATP hydrolysis promotes the interaction of RIG-I and self-dsRNA and results in unintentional immune signaling (47).

#### Posttranslational Control of RIG-I Ubiquitination

One of the first PTMs of RIG-I following the initial ligand recognition is performed by the robust ubiquitination machinery (**Figure 2**). Mass spectrometry analysis revealed that TRIM25, a member of the tripartite motif (TRIM) protein family possessing E3 ligase activity, induces the covalent Lys63-linked ubiquitination of RIG-I. Mechanistically, the C-terminal SPRY domain of TRIM25 interacts with CARD1 and facilitates the ubiquitination of CARD2 at K172 (48). The RIG-I–TRIM25 ubiquitination complex, associates with the adaptor protein 14-3-3ϵ and translocates to mitochondria for MAVS binding (49). Mutation

of K172 disrupts the interaction between RIG-I and MAVS thus abrogating downstream signaling and IFNs production (50). Furthermore, a RIG-I splice variant which lacks the TRIM25 interaction domain acts as a feedback inhibitor of RIG-I signaling transduction upon viral infections (48). In addition, Riplet (RING-finger protein leading to RIG-I activation, also named RNF135 or REUL), another E3 ubiquitin ligase, also promotes RIG-I ubiquitination. Multiple sites within the CARDs, as well as within the CTD of RIG-I, were identified as the crucial ubiquitin anchoring residues (51–53). Among which, K63-linked polyubiquitination (pUb) at Lys788, is demonstrated as being critical for RIG-I activation. However, unlike TRIM25-induced ubiquitination, Riplet induced RIG-I pUb is dispensable for RIG-I-RNA binding but is essential for releasing CARD from its autorepressed state. This enhances TRIM25 functionality as well as promoting the oligomerization of RIG-I and the activation of MAVS (54). MEX3C (Mex-3 RNA binding family member C), another recently identified E3 ligase, also mediates Lys63-Ub at K99 and K169 of CARD, playing a critical role in RIG-I activation (55). In addition, the oligoadenylate synthetases-like (OASL) protein, although not an E3 ubiquitin ligase itself, contains a dsRNA-binding groove and enhances RIG-I activation by mimicking the K63-linked pUb through its ubiquitin-like (UBL) domain (56, 57). Non-covalent binding of K63-ubiquitin chains to CARDs also potently activates RIG-I (58). Recent structural analysis suggests that covalent and non-covalent binding of ubiquitin synergistically stabilize RIG-I tetramerization and enhance polymerization of MAVS CARDs (59).

On the other hand, several deubiquitinating enzymes (DUBs) were identified to remove K63-linked pUb chains from RIG-I, thus dampening RIG-I signaling. The tumor suppressor protein cylindromatosis (CYLD) removes K63-linked pUb chains from RIG-I as well as TBK1 and IKKϵ to inhibit the IRF3 response, serving as a pathway negative regulator (60). Syndecan-4, a newly identified negative regulator of RIG-I, functions through attracting CYLD to RIG-I complex, thus potentiating the K63-mediated deubiquitination of RIG-I (61). In addition, the ubiquitin-specific protease (USP) family members, such as USP3 and USP21, were also identified as inhibitors of RIG-I activation by deubiqutinating RIG-I (62, 63).

In contrast to K63-linked ubiquitination, which promotes protein activation, K48-linked ubiquitination triggers proteasomal degradation of its target. For instance, the RING-finger protein 125 (RNF125), together with the ubiquitin E2 ligase UbcH5, conjugate K48-linked ubiquitin to RIG-I and MAVS, targeting them for proteasomal degradation and thereby inhibiting downstream signaling (64). Similarly, RNF122 was recently demonstrated to mediate the proteasomal degradation of RIG-I by delivering the K48-linked ubiquitin to RIG-I CARDs (65). The linear ubiquitin assembly complex (LUBAC) has been shown to promote K48 pUb of TRIM25, leading to its degradation (66). Conversely, the deubiquitinase USP15 antagonizes LUBAC by removing K48 linked ubiquitin from TRIM25, leading to its stabilization and thereby promoting RIG-I-mediated antiviral signaling (67).

#### Phosphorylation

In parallel with ubiquitination, phosphorylation has emerged in the past several years as a critical regulator of the RIG-I signaling transduction (**Figure 2**). Protein purification and mass spectrometry analysis identified that phosphorylation of Thr170 in the CARDs antagonizes RIG-I signaling by inhibiting TRIM25-mediated Lys172 ubiquitination and MAVS binding (68). Ser8 phosphorylation of CARDs also serves as a negative regulator of RIG-I (69). In addition, the CTD of RIG-I is constitutively phosphorylated at Thr770 and Ser854/855 by casein kinase II to promote intermolecular interactions between CTD and CARDs, thereby maintaining RIG-I at an autorepressive state to prevent premature downstream signaling (70). A recent mass spectrometry analysis revealed that IKK phosphorylates RIG-I at Ser855, thereby providing a negative feedback regulation of RIG-I (71). Furthermore, conventional protein kinase C-α (PKC-α) and PKC-β have also been shown to phosphorylate CARDs, thus suppressing RIG-I–TRIM interaction and subsequent antiviral responses (72). In fact, RIG-I signaling activity is controlled by a dynamic balance between phosphorylation and dephosphorylation. Dephosphorylation of RIG-I occurs rapidly with the presence of viral RNA. A functional siRNA screen identified phosphoprotein phosphatase 1-α (PP1α) and PP1γ as essential phosphatases responsible for CARDs dephosphorylation at Ser8 and Thr170, leading to RIG-I signal activation and viral inhibition (73).

#### Acetylation

In addition to the ubiquitination and phosphorylation described above, acetylation modulation has recently started to gain more acknowledgment for controlling RIG-I activity (**Figure 2**). Mass spectrometry has identified the acetylation of two lysine residues (K858 and K909) in the CTD of RIG-I at its inactivate state and are deacetylated during viral infection (74). The mutation of these two sites restricts RIG-I from undergoing the virusinduced interaction with MAVS. K858 and K909 acetylation of RIG-I has also been shown to control the PAMP RNA-induced RIG-I oligomerization (75). The cytoplasmic deacetylase HDAC6-mediated removal of K909 acetylation has been shown as critical for RIG-I binding to dsRNA during viral infections (76). Furthermore, HDAC6-dependent RIG-I deacetylation also regulates RIG-I oligomerization upon ligand binding, thus facilitating RIG-I activation (75).

#### Other Regulatory Mechanisms

RIG-I signal transduction is further regulated by additional PTMs, regulatory proteins, and other cellular processes (**Figure 2**). It is worth noting that a number of UBL proteins including SUMO, ISG15, FAT10, and Atg8–Atg12 are involved in these positive or negative regulatory mechanisms (77). SUMOylation serves as a positive regulator of RIG-I by enhancing the RIG-I and MAVS binding (78). On the contrary, the HLA-F adjacent transcription 10 (FAT10), an UBL modifier protein, was shown to negatively regulate RIG-I by modulating RIG-I solubility through a non-covalent association with CARDs (79). In addition, IFN-induced ISG15 negatively regulates the RIG-I mediated signaling in a feedback-loop control manner (80). SEC14L1 has been observed competing with MAVS for RIG-I CARD binding (81). Furthermore, autophagy has been reported to be involved in RIG-I modulation through its key regulator, the Atg5–Atg12 conjugate. Atg5–Atg12 has been found to suppress RIG-I–MAVS interaction, thereby inhibiting downstream signaling (82). Recently, deamidation of CTD has been described as a distinct means to induce RIG-I activation. For examples, vGAT (glutamine amidotransferase), from KSHV (kaposi's sarcomaassociated herpesvirus) and γHV68 (murine gamma herpesvirus 68), recruits cellular phosphoribosylformyglycinamide synthase to deamindate and activate RIG-I (83, 84).

# VIRAL ANTAGONISM OF RIG-I SIGNALING

In order to establish infections, viruses have developed sophisticated mechanisms to counteract host immune responses. With regard to RIG-I signaling, these include mechanisms such as altering viral genomes and their intermediate transcripts to avoid detection, manipulating the activation and degradation of RIG-I and MAVS, as well as modulating downstream signaling cascades. Studying these antagonistic viral strategies has greatly broadened our understanding of RIG-I activation and regulation.

#### Sequestration of Viral RNAs

Since 5′ triphosphate (5′ppp) is an important feature recognized by RIG-I, modification of this motif has long been described as one of the major mechanisms for viruses to antagonize RIG-I signaling. Crimean–Congo hemorrhagic fever virus, Borna disease virus (BDV), and hantavirus (HTNV) remove the 5′ppp group on their genome posttranscriptionally, make RIG-I unable to bind to viral RNA, and therefore incapable of triggering RIG-I activation (85). Mechanistically, HTNV uses the "prime and realign" strategy to generate a 5′-terminal monophosphorylate (86, 87). BDV on the other hand, employs genome trimming to form a 3′-terminal overhang as well as convert 5′ppp to 5′p to avoid detection by RIG-I (88). The arenavirus presents an unpaired 5′ppp-nucleotide overhang to evade recognition by RIG-I (89). The 5′-end of viral RNA can also be modified through RNA-capping pathways. For example, the genomic RNA of polioviruses linked to Vpg (viral protein genome-linked) to cap the 5′-end from exposure to RIG-I (90). The 5′-end capping with 7-methyl guanosine and methylation of 5′ppp dsRNA at the 2′-*O* position makes viral RNA non-distinguishable from the host mRNAs, and therefore does not stimulate RIG-I (91, 92).

By contrast, some viruses encode viral proteins to prevent RNA recognition. The EBOV utilizes its VP35 protein to sequester viral RNA (18). The crystal structural analysis indicates that the VP35 interferon inhibitory domain competes with RIG-I for dsRNA binding by forming an "end-cap" complex with dsRNA, resulting in substantially diminished activation of RIG-I (93). Similarly, the marburg virus VP35 spirals around the dsRNA backbone and end-caps the dsRNA to escape from RIG-I detection (94, 95). The IAV non-structural protein 1 (NS1) possesses dsRNA-binding properties to shield viral RNA from RIG-I (96). IAV has also been shown to antagonize RIG-I activation *via* its viral polymerase subunit PB2. PB2 position 627K in the mammalian strain increases PB2-nucleocapids binding affinity, thus inhibiting RIG-I interaction with the nucleoprotein-encapsidated 5′ppp RNA (22, 97).

In addition to altering and concealing their genome to prevent RNA binding, viruses also re-localize viral RNA to specific cellular compartments, such as mitochondria, endoplasmic reticulum (ER), and Golgi, to avoid cytosolic surveillance by RIG-I. For instance, the DENV conceals dsRNA in the intracellular membrane as an escape strategy (98). ER is an important organelle for viral entry, replication, and assembly. The severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) has been shown to induce a modified ER to hide its replicating RNA from detection (99). These viral antagonism strategies highlight the importance of cellular organelle localization in viral–host interactions during innate antiviral responses.

#### Manipulation of RIG-I–MAVS Signaling Modulation of the PTMs

As reviewed above, ubiquitination represents one critical PTM mechanism of RIG-I activation and, not surprisingly, is an attractive target for viral manipulation (**Figure 3A**). Viruses have evolved ways to inhibit K63-linked ubiquitination of RIG-I by interacting with the E3 ligases TRIM25 and Riplet. For instance, IAV NS1 from various strains has been shown to suppress TRIM25-mediated RIG-I CARDs ubiquitination. Among all the TRIM25 binding amino acids identified in NS1, R38/K41 and E96/E97 were described as critical in interfering with the coil-coiled domain of TRIM25. These interactions resulted in an inhibition of TRIM25 multimerization and therefore blocked the RIG-I CARDs ubiquitination (100). Intriguingly, NS1-TRIM25 binding is found to be preserved in human and avian, but lost in mouse, indicating a species-specific manner of inhibition. This study further demonstrates that the NS1 suppression of RIG-I ubiquitination in mouse is Riplet-dependent (101). Conversely, phosphorylation of NS1 at Thr49 was recently identified as impairing the NS1–TRIM25 interaction, thereby suppressing its antagonistic activity of RIG-I signaling (102). Phosphorylation of another site on NS1, Thr80, has also been reported to disrupt NS1 binding affinity with RIG-I (103). Similar to IAV, the IBV non-structural NS protein (NS1-B) has recently been described as inhibiting RIG-I ubiquitination, which involves TRIM25-NS1 C-terminal effector domain interaction and the RIG-I/TRIM25/ NS1-B complex formation (104). By contrast, the protease NS3-4A of HCV functions differently, rather than inhibiting TRIM25, it is thought to target the E3 ligase Riplet. NS3-4A directly disrupts Riplet, abolishes Riplet-mediated RIG-I ubiquitination, and further reduces the interaction between TRIM25 and RIG-I (54).

On the other hand, some viruses encode enzymes that directly deubiquitinate RIG-I. For instance, KSHV encoded deubiquitinase ORF64 cleaves Lys63-ubiquination chains on CARDs, blocks CARDs interaction between RIG-I and MAVS, thereby downregulating RIG-I signaling (105). Other viruses including arterivirus, nairovirus, SARS-CoV, and foot-and-mouth disease virus (FMDV) have also been reported to downregulate RIG-I ubiquitination through their viral encoded DUBs (106, 107).

Few viruses have been shown to manipulate RIG-I regulation with regards to targeting the phosphorylation or dephosphorylation process of RIG-I. Nevertheless, it was reported that MV efficiently escapes antiviral response *via* suppressing RIG-I dephosphorylation in dendritic cells (DCs). In this study, the growth arrest and DNA damage protein (GADD34) was shown to form complexes with PP1 to facilitate RIG-I activation. The MV infection induced DC-SIGN signaling results in an inhibition of GADD34-PP1 phosphatases activity and thereby impairs RIG-I activation (108).

# Degradation of RIG-I and MAVS

Another distinct strategy used by viruses to antagonize RIG-I signaling is the direct cleavage or degradation of the receptor and multiple members of the signaling cascade (**Figure 3A**). RIG-I has been reported in some studies to be cleaved by the proteinase 3Cpro during infections with picornavirus, coxsackievirus B3 (CVB3), and enterovirus 71 (EV71) (109, 110). The encephalomyocarditis virus directs both caspase- and proteasome-dependent degradation of RIG-I (111). Intriguingly, the NS1–NS2 degradasome of the respiratory syncytial virus (RSV) has been shown to mediate the proteasomal degradation of RIG-I (112).

Mitochondrial antiviral signaling protein is also a well-studied molecule which is often targeted by many types of viral-induced cleavage. For example, the hepatitis A virus (HAV) cleaves MAVS for proteolysis by its protease 3Cpro (113). Both CVB3 proteinase 2Apro and 3Cpro trigger MAVS cleavage at different sites during infection, and the cleavage of MAVS by EV71 is accomplished *via* its 2Apro activity (110, 114). In addition, serine protease NS3-4A of HCV cleaves MAVS, removing it from the mitochondria, thereby inhibiting downstream signaling (36, 115). In a parallel fashion, many viruses mediate cellular proteolytic degradation of MAVS to attenuate RIG-I antiviral responses. Hepatitis B virus viral protein HBx triggers the proteasome-mediated degradation of MAVS through Lys136 ubiquitination (116). Another study

Figure 3 | (A) Viral inhibition of retinoic acid-inducible gene-I (RIG-I)– mitochondrial antiviral signaling protein (MAVS) signaling. Numerous viral proteins have been described as RIG-I signaling antagonists which inhibit the activation of RIG-I and its adaptor protein MAVS. Influenza A virus (IAV) NS1 and influenza B virus NS1 were both shown to suppress the TRIM25-mediated ubiquitination of RIG-I caspase activation and recruitment domains. The protease NS3-4A of hepatitis C virus functions by targeting Riplet. A number of virus-encoded deubiquitination enzymes from various viruses remove Lys63-linked ubiquitin from RIG-I, resulting in signaling suppression. The measles virus efficiently blocks RIG-I dephosphorylation to prevent RIG-I activation. Dengue virus (DENV), respiratory syncytial virus, and thrombocytopenia syndrome virus (SFTSV) were involved in disrupting RIG-I translocation to the mitochondria. Numerous virus-encoded proteases were shown to directly cleave or degrade RIG-I and MAVS. Additionally, the PB1-F2 of IAV decreases the mitochondrial membrane potential thereby inhibiting RIG-I–MAVS interaction. (B) Viral modulation of downstream signaling components. Ubiquitination- and phosphorylation-mediated TBK1 activation were shown be antagonized by a number of viruses, including foot-and-mouth disease virus, mouse hepatitis virus A59, molluscum contagiosum virus, and DENV. IAV NS1, herpes simplex virus type 1, severe acute respiratory syndrome coronavirus (SARS-CoV), and several paramyxoviruses have been shown to interfere with interferon regulator factor (IRF) 3 phosphorylation. Epstein–Barr virus suppresses IRF7 transcriptional activity. The rotavirus targets both IRF3 and IRF7 for degradation. The binding of transcription factors to the IFNB promoter is also challenged by herpes simplex virus and the thogoto virus. Other viruses subvert the JAK–STAT signaling to inhibit the production of interferon-stimulated genes. STAT1 and STAT2 are therefore often targets by a number of viruses.

reported that the HAV cysteine protease ABC targets MAVS for proteolysis at mitochondrial membrane (113). Additionally, viral modulation of cellular organelles such as mitochondria also affects RIG-I–MAVS signaling. The PB1-F2 of IAV, for instance, has been described as decreasing the mitochondrial membrane potential, resulting in the acceleration of mitochondrial fragmentation, thereby inhibiting RIG-I–MAVS signaling (117–119).

It is important to note that the proper localization of RIG-I and MAVS is a prerequisite for effective signaling transduction. MAVS resides on the mitochondrial membrane, peroxisomes, and mitochondria-associated membranes for antiviral signaling. In fact, a RIG-I translocon has been identified to direct RIG-I redistribution from cytosol to membranes during viral infection (49). Studies have shown that several viruses encode proteins to disrupt the proper localization of RIG-I or MAVS as a novel mechanism of regulation, such as NS3 of DENV (113), nucleoprotein of RSV (120), and non-structural proteins of thrombocytopenia syndrome virus (SFTSV) (121).

#### Modulation of Downstream Signaling Components

To ensure successful RIG-I signaling transduction, the kinase activities of TBK1 and IKKϵ are tightly controlled *via* various regulatory mechanisms and are common targets of viruses (**Figure 3B**). For example, both the leader proteinase (Lpro) of FMDV (122) and the non-structural protein 3 (ns3) of the mouse hepatitis virus A59 (123) inhibit ubiquitination of TBK1. Dengue virus serotype4 non-structural proteins, NS2A and NS4B, as well as the FLIPs proteins encoded by the molluscum contagiosum virus (MCV), all reduce TBK1 phosphorylation, thereby preventing its activation (124, 125). Several viruses have been shown to prevent the formation of functional TBK1-containing complexes. The K7 protein of the VACV prevents TBK1/IKKϵ complex-induced IRF activation by targeting host DEAD box protein 3 (DDX3) (126). Two other viruses, the NY-1 HTNV and SARS-CoV, disrupt the TBK1–TRAF3 and TANK–TBK1/IKKϵ complex, respectively (127, 128). Moreover, SFTSV has been shown to irreversibly re-localize TBK1 and IKK from mitochondria and sequester the TBK1/IKKϵ/IRF3 complex *via* the formation of inclusion bodies, causing signaling cascade termination (129).

Viral regulation of the transcription factors, IRFs and NF-κB, further serve as points of control in RIG-I signaling (**Figure 3B**). One of the best studied examples is the inhibition of IRF3 activity by the IAV NS1 protein (130). Besides this, the HSV-1, rabies virus, SARS-CoV, as well as several paramyxoviruses have been demonstrated to interfere with the phosphorylation state of IRF3, thereby blocking IFN induction (131–134). The EBV conjugates SUMO to IRF7 at lysine 452 to decrease IRF7 transcriptional activity (135). The rotavirus NS1, targets both IRF3 and IRF7 for degradation to prevent IRFs from undergoing dimerization (136). Viruses have also developed various means to suppress the IRF3 DNA binding ability. Herpes simplex virus, thogoto virus, and KSHV, all developed strategies to downregulate IRF3 transcriptional activity by either disrupting IRF3 binding complex formations or competing binding regions on the IFNB promoter (137–139). Viral strategies in inhibiting cytoplasmic or transcriptional activities of NF-κB have been extensively studied during the VACV infection. Studies reported that multiple proteins encoded by VACV and HSV-1 suppress NF-κB activation (140–143).

Viruses have also developed multiple inhibitory mechanisms to counteract the IFN stimulation of ISGs by targeting STAT1 and/or STAT2 (**Figure 3B**). For example, the langat virus was shown to inhibit the phosphorylation of both STAT1 and STAT2 (144). Varicella viruses and the Japanese encephalitis virus, both block the JAK/STAT1 pathway through multiple mechanisms including inhibiting STAT proteins phosphorylation and nucleotranslocation (145, 146). The non-structural protein NS5 of

#### REFERENCES

several flaviviruses, have been shown to target STAT proteins *via* distinct mechanisms. For example, MNV NS5 inhibits STAT1 phosphorylation, whereas DENV NS5 interacts with UBR4 to promote STAT2 degradation (147, 148). By contrast, the Zika virus NS5 induced proteasomal degradation of STAT2 was recently identified as UBR4 independent (149). Furthermore, other viruses, such as HCV (150), RSV (151), and paramyxovirus (152), also demonstrate negative regulation of the JAK–STAT pathway.

#### CONCLUDING REMARKS

Studies from the past decade have well established RIG-I as one of the principal PRRs for the recognition of cytoplasmic viral RNA, as well as defining its critical role in the induction of IFNs during viral infections. Our understanding of the RIG-I-mediated antiviral response has been greatly expanded with the key discoveries made regarding the molecular mechanism of RIG-I regulation, such as ubiquitination, phosphorylation, and acetylation. Meanwhile, investigating viral strategies to manipulate RIG-I responses not only allow us to understand the viral pathogenesis, but also significantly contributed to our knowledge of how RIG-I is activated and regulated. These new insights into the viral-mediated RIG-I regulations are important for vaccine and drug development aiming to suppress infectious diseases and enhance immune responses.

#### AUTHOR CONTRIBUTIONS

YL wrote the manuscript. RL and DO revised and approved the manuscript.

#### ACKNOWLEDGMENTS

The authors would like to thank Alexandre Sze for his critical reading and editing of the manuscript. This research was supported by grant from Canadian Institutes of Health Research (MOP130401) to RL; DO was supported by a Peter Quinlan McGill postdoctoral fellowship. The authors would like to acknowledge all the colleagues in the field and apologies to those whose important contributions could not be included in the review due to space constraints. The figures of the review were illustrated using the Servier Medical Art library, http://www. servier.com/Powerpoint-image-bank.

antibody-enhanced dengue virus infection. *MBio* (2015) 6:e1316–1315. doi:10.1128/mBio.01316-15

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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.

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

# Protozoan Parasites and Type i iFNs

*Sasha Silva-Barrios and Simona Stäger\**

*INRS-Institut Armand Frappier, Center for Host-Parasite Interactions, Laval, QC, Canada*

For many years, the role of interferon (IFN)-I has been characterized primarily in the context of viral infections. However, regulatory functions mediated by IFN-I have also been described against bacterial infections and in tumor immunology. Only recently, the interest in understanding the immune functions mediated by IFN-I has dramatically increased in the field of protozoan infections. In this review, we discuss the discrete role of IFN-I in the immune response against major protozoan infections: *Plasmodium*, *Leishmania*, *Trypanosoma*, and *Toxoplasma*.

Keywords: protozoan infections, IFN-I, *Leishmania*, *Toxoplasma*, *Plasmodium*, *Trypanosoma*

#### INTRODUCTION

#### *Edited by:*

*Joao P. B. Viola, Instituto Nacional de Cancer (INCA), Brazil*

#### *Reviewed by:*

*Claudia Ida Brodskyn, Centro de Pesquisas Gonçalo Moniz (Fiocruz Bahia), Brazil Maria Regina D'Império Lima, University of São Paulo, Brazil*

#### *\*Correspondence:*

*Simona Stäger<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 27 September 2016 Accepted: 05 January 2017 Published: 19 January 2017*

#### *Citation:*

*Silva-Barrios S and Stäger S (2017) Protozoan Parasites and Type I IFNs. Front. Immunol. 8:14. doi: 10.3389/fimmu.2017.00014*

Innate and adaptive immune responses are key factors in the control of infectious and chronic diseases; the balance between these two systems is mainly orchestrated by cytokines. Interferons (IFNs) are a large family of cytokines that were first discovered in 1957 in the context of viral infections. The name IFN is due to the capacity of these antiviral factors to interfere with viral replication in mammalian cells (1). Numerous studies have been carried out since their discovery, which allowed the identification of several related molecules. Based on their structural characteristics and the restricted affinity by the receptor molecule with which they directly interact, IFNs are classified into three main groups: type I (IFN-I), type II (IFN-II), and the recently identified type III (IFN-III) (2).

The IFN-I family includes two main classes of related cytokines: IFN-α, which comprises 13 different subtypes encoded by 13/14 different genes; and IFN-β, a product encoded by a single gene and a group of other less studied IFNs (IFN-ϵ, IFNδ, IFNκ, IFNτ, IFNω) (2). The ability to produce and respond to IFN-I is distributed in a wide variety of cells. This confers several autocrine and paracrine effects that have been extensively characterized mainly in viral infections. IFN-I signaling is mediated through a common cell surface receptor, the IFN-I receptor (IFNAR) (3, 4).

The IFN-II family is represented by a single gene product, IFN-γ, and is mainly produced by T lymphocytes and natural killer (NK) cells. IFN-II responses are mediated by the binding of IFN-γ to a heterodimeric molecule, the IFN-γ receptor (IFNGR), ubiquitously expressed in a wide range of cells. IFNGR is involved in the modulation of different cell functions and is a key factor for host defence to intracellular pathogens in various infection models (5).

Finally, the IFN-III family, also known as IFN-λ, comprises four different subtypes: IFN-λ1, IFNλ2, IFN-λ3, and IFN-λ4. The members of this novel IFN family interact through a unique receptor, the IFN-λ receptor (IFN-λR). In contrast to IFNAR and IFNGR, the expression of IFN-λR is mainly restricted to cells of epithelial origins. The role of IFN-III has yet to be better characterized; however, they appear to induce similar responses to IFN-I (6).

The crosstalk between IFNs and their specific receptors elicits an intracellular signaling cascade that mainly enhances inflammatory responses. The well-characterized signaling cascades of IFN-I and IFN-II are fairly similar. In both cases, Janus kinase 1 (JAK1) and tyrosine kinase 2, associated with IFNAR and IFNGR, are activated. This results in activation and following formation of a heterodimer complex comprised by the cytoplasmic transcription factor signal transducer and activator of transcription 1 and 2 (STAT1/STAT2). STAT1/STAT2 dimers can be translocated to the nucleus and interact with the IFN regulatory factor 9 to form the IFN-stimulated gene factor 3 complex, leading to the transcription of IFN-stimulated genes (ISGs). By contrast, IFN-II signaling through IFNGR activates the JAK/ STAT pathway leading to the transcription of pro-inflammatory targets downstream of γ-activated sequences (2, 7).

Interferon (IFN)-I production is mainly induced in response to the activation of receptors on the membrane and/or cytosol, such as pattern recognition receptors (PRRs). PRRs can be activated by conserved pathogens component and endogenous molecules. In most of the cases, the production of IFN-I is related to the activation of PRRs that recognize xenogeneic or autologous nucleic acid, such as toll-like receptors (TLRs) (8).

Interferon (IFN)-I is historically best known for their capacity to elicit antiviral responses; however, they also play a role in bacterial infections and autoimmune diseases (4). The role of IFN-I in regulating the immune response against pathogens is fairly complicated. IFN-I can have enhancing or suppressive effects depending on the disease, the stage of infection, and the amount produced. For instance, IFN-I enhances the antigenpresenting capacity of DCs (9–11), favors the development of T cell responses (12–14), and promotes antibody responses (15, 16) during acute viral infections. By contrast, type I IFNs play an immunosuppressive role during chronic viral infections (17–19), reduce IFN-γ responsiveness in macrophages (20, 21), block B cell functions at high concentrations (22, 23), and can promote the expression of immunosuppressive factors such as IL-10 and PDL-1 (24–27). This duality is also observed in the context of autoimmune diseases, where IFN-I plays a pathogenic role in systemic lupus erythematosus and Sjogren's syndrome (28, 29), whereas it has therapeutic effects in multiple sclerosis (30).

While IFN-γ has been widely characterized in the modulation of the immune response against protozoan infections, the contribution of IFN-I to host defence against parasites is less clear. In the past few years, a growing body of literature suggests an important role for IFN-I during protozoan infections, particularly in the innate immune response.

In this review, we provide a brief overview of IFN-I mediated effects on the host response in various protozoan infection models and the possible mechanisms involved.

#### PROTOZOAN PARASITES AND IFN-I

Interferon (IFN)-I is involved in the modulation of innate immune responses promoting antigen presentation and NK cell functions. They are also known to play a role in the regulation of the adaptive immune system, promoting the development of antigen-specific T and B lymphocytes against numerous pathogens and inducing immunological memory (7). In most of the cases, these key features are important factors that limit pathogen proliferation; however, IFN-I may also lead to disease exacerbation. Protozoan parasites such as *Plasmodium*, *Leishmania*, *Trypanosoma*, and *Toxoplasma* are causing diseases that are among the most lethal and widespread around the world, primarily affecting populations of developing countries. The contribution of IFN-I in the host immune response to these pathogens will be discussed below.

#### *Plasmodium*

*Plasmodium* parasites are the causative agents of malaria, one of the most widespread diseases in the world. The infection presents itself in a wide range of pathologies that can degenerate into severe anemia and the high-risk cerebral malaria (CM). Members of the *Plasmodium* genus have a complex life cycle between an invertebrate (female mosquitoes of the *Anopheles* genus), in which the sexual cycle occurs, and a mammalian host. During the mosquito blood meal, sporozoites are inoculated into the dermis of the mammalian host. In the initial phase of infection, circulating sporozoites can reach lymph nodes, where the priming of B and T cells occurs, or migrate to the liver (31, 32). Within the liver, sporozoites transform first into schizonts within hepatocytes and then into merozoites. This phase is asymptomatic and is known as the pre-erytocytic stage (33). Merozoites are then released into the blood stream. Once they reach the blood, merozoites invade red blood cells, where they undergo cyclic asexual replication initiating the typical symptomatic manifestations of blood-stage malaria, which are caused by the exponential growth of the parasite and massive destruction of erythroid cells (34).

Most of the current knowledge about the immune response to *Plasmodium* parasites has been derived from a combination of *in vitro* and *in vivo* observations in human patients (e.g., *P. falciparum*, *P. vivax*, *P. malariae*, *P. knowlesi*, and *P. ovale*) and murine models of infections (e.g., *P. berghei*, *P. yoelii*, *P. chabaudi*, and *P. vinckei*) (34).

During the pre-erythrocytic stage, sporozoite invasion of hepatocytes and subsequent development into merozoites can be blocked by sporozoite-specific antibodies generated by previous exposure to malaria or by immunization; however, this stage is not completely efficient because sporozoites remain in circulation for a short period of time. When T cell priming takes place, infected hepatocytes can be eliminated by cytotoxic CD8 T cells. CD8 T cells, IFNγ, and TNF are critical components required for elimination of infected hepatocytes in humans and the mouse model (35). However, the immune response at this stage is insufficient and released merozoites can reach erythrocytes giving rise to blood-stage malaria (35).

In the erythrocytic stage, early interaction between merozoites and innate immune cells such as dendritic cells, monocytes, macrophages, NK cells, NKT cells, and γδT cells is important for the control of parasite replication and the resolution of infection (33). This phase is characterized by a strong pro-inflammatory response, mediated by the activation of NK, NKT, CD8, and CD4 T cells that produce large amounts of IFNγ and other proinflammatory cytokines. IFNγ activates phagocytic cells, such as macrophages, enhancing the secretion of pro-inflammatory cytokines and promoting phagocytosis of circulating parasites and infected red blood cells, which results in the control of parasitemia (36). Polyreactive and specific antibodies against blood-stage malaria can limit parasite propagation between erythrocytes by opsonization and agglutination of parasites and infected erythrocytes; however, humoral responses during the infection are dependent on the presence of circulating merozoites (37). Infected erythrocytes on the surface express parasitic protein which allows them to bind to vascular endothelial cells and avoid clearance. This event induces obstructions in the blood flow and is associated with a strong inflammatory response and the development of CM (33).

Although IFN-γ is the most extensively studied IFN in malaria infection, part of the attention has now been diverted to type I IFNs. IFN-I can have a host-protective or detrimental effect, depending on the stage of the infection or the species of *Plasmodium* involved.

One of the first reports involving type I IFNs demonstrated that administration of mouse serum containing high levels of IFN-I protected mice from *P. berghei* infection by reducing blood parasitemia (38). Similar protective responses were observed after treatment with IFN-β, which prevented death related to CM in *P. berghei*-infected mice (39). By contrast, treatment with recombinant IFN-α during the hepatic cycle in mice infected *P. yoelii* sporozoites did not alter the hepatic parasite burden. However, mice showed reduced parasitemia and decreased signs of immunopathology (40).

*Plasmodium* parasites were reported to induce IFN-I responses. Transcriptomic analysis carried out in mice with blood-stage infection with *P. berghei* revealed that IFN regulatory factors were upregulated during the acute phase (41). Induction of a typical type I IFN signature was also observed in hepatocytes from mice infected with *P. berghei* and *P. chabaudi* sporozoites, where genes such as *Mda*, *Irf3*, *Irf7*, and *Stat1* were upregulated (42–44). Similar results were observed in humans. Patients infected with *P. vivax* and *P. falciparum* showed a predominantly IFN-I transcriptional signature during the mild and the severe phase of infection (44, 45).

Recently, Liehl et al. showed that induction of IFN-I during liver stages of the infection is required for host defence against *P. berghei*. Recognition of *P. berghei* nucleic acids by Mda5 induced IFN-I and consequently, the recruitment of leukocytes necessary for parasite elimination in the liver (42). In *P. yoelii*infected mice, recruitment and expansion of CD49b+CD3+NKT and CD8+T cells to the liver were mediated by IFN-I signaling (43). Migration of neutrophils to the liver is also modulated by IFN-I in mice infected with *P. chabaudi* (44). These studies suggest that functionality of the innate immune response in the liver relies on both IFN-I and IFN-II.

In contrast to the protective effects discussed above, a pathogenic role for IFN-I in *Plasmodium* infections has also been described. For instance, impaired IFN-I signaling has been linked to a protective effect in human patients. Polymorphism in the human gene encoding for IFNAR1 are strongly associated with protection against CM (46). This observation is in agreement with results obtained in a murine model, where the lack of IFN-I signaling led to strong resistance to CM and reduced parasite load during *P. berghei* infection (47, 48). Moreover, in *P. chabaudi*-infected mice, IFN-I appear to suppress Th1 responses that are crucial in the control of hyperparasitemia, by modulating dendritic cell functions (49). In addition, IFN-I and Myd88 signaling are responsible for a decreased recruitment of conventional DCs to the spleen during experimental *P. berghei* or *P. yoelii* infection (50).

Perhaps a better approach for truly understanding the role and function of IFN-I during malaria consists in the identification of modulator molecules that could act in the IFN-I signaling cascade. Recently, regulators of IFN-I response have been identified through genome-wide analysis (Trans-species expression quantitative trait locus, ts-eQLT) during *P. yoelii* infection. Eight genes (*Ak3*, *Fc*γ*r1*, *Fosl1*, *Havcr2*, *Sipr5*, *Parp14*, *Selenbp2*, and *Helb*) had an effect on IFN-I activation. For example, *Fc*γ*r1*−/− mice infected with *P. yoelii* showed significantly higher mRNA and protein levels of IFN-β than wild-type mice, suggesting a negative regulation in the IFN-β response (51).

Future experiments are granted to clarify the spatio-temporal role of IFN-I during malaria.

The role of IFN-I during *Plasmodium* infections is summarized in **Table 1**.

#### *Toxoplasma*

*Toxoplasma gondii* is an obligate intracellular protozoan parasite that can infect a wide range of vertebrates and cause a zoonotic disease called toxoplasmosis. *T. gondii* could be considered one of the most successful parasites worldwide; at least 50% of the human population is infected with *Toxoplasma*. The parasite success is mainly due to its ability to invade any nucleated cell and to survive outside the mammalian host (52). *T. gondii* strains are classified in three main lineages, based on the virulence of the strain in the mouse model. This virulence profile does not necessarily correlate to the degree of human infection.  By contrast, type II and III strains are less virulent and lead to the establishment of chronic infection (53). *T. gondii* can undergo both asexual (schizogony) and sexual (gametogony) replication. Gametogony and oocyst formation is restricted to feline species that act as a definitive hosts; sexual reproduction of sporozoites occurs within intestinal epithelial cells. Asexual stages of *T. gondii* are not host-specific. Many mammals and birds can act as intermediate hosts. After ingestion of *T. gondii* oocysts by an intermediate host, the parasite transforms into tachyzoites that rapidly undergo multiplication

within the parasitophorous vacuole inside various cell types. If the infection is controlled, parasites are retained in tissue cysts; if not, they can cause a systemic lethal disease (54, 55).

Humans are considered as an accidental intermediate host for *Toxoplasma*. In immune-competent individuals, the infection with *T. gondii* is mostly clinically silent, but cause severe diseases in immune-suppressed patients in particular with an impaired T cell and IFNγ response (55). Protective immunity is typically achieved by inducing an IL-12-driven Th1 immune response (56, 57).

In the mouse model, IFN-I can already be detected in the serum of *T. gondii*-infected animals during the acute phase (58–60); IFN-I levels gradually increase with the progression of the infection (60). IFN-I was also detected in the brain and spleen of infected mice (61). These results demonstrate that *T. gondii* not only induces IFN-γ, but also IFN-I.

Recently, inflammatory monocytes were identified as the major source of IFN-β in mesenteric lymph nodes. IFN-β production by inflammatory monocytes required three fundamental events: parasite internalization, TLR activation (mainly TLR4 and 2), and efficient MyD88 signaling. Interestingly, heat killed parasites induced higher levels of IFN-β in inflammatory monocytes (62), suggesting that *Toxoplasma* might limit IFN-I responses (62), possibly by blocking STAT1 (63).

As for many other infection models, the first studies carried out during the 1960s on the role of IFN-I in toxoplasmosis evaluated the impact of a treatment with IFN-I on infected cells *in vitro*. Pre-treatment of mouse fibroblast with IFNs conferred protection to *T. gondii* infection (64). In agreement with this observation, human neonatal and adult macrophages treated with IFN-I were able to control parasite multiplication, even if less effectively than IFN-γ treated cells (65). Moreover, human monocyte-derived macrophages treated with human IFN-β in combination with *Escherichia coli* lipopolysaccharides (LPS), but not with murine IFN-β (MuIFN-β) or rHuIFN-β alone (66), were more resistant to *T. gondii* infection (67).

In the mouse model of toxoplasmosis, treatment with HuIFN-β showed a protective effect, which was enhanced by the combination of rHuIFN-β and LPS and was IFN-γ dependent (66). In agreement with these results, it was shown that *Ifnar*−/− mice orally infected with *T. gondii* have an increased parasite load compared to wild-type mice; higher parasite burdens correlated with a decrease in survival (68).

These results suggest that IFN-β may be produced at the onset of infection to enhance the IFN-γ responses.

A study using human fibroblasts as host cells revealed that treatment of *T. gondii*-infected cells with IFN-I had no effect on parasite replication (69), suggesting that the protective effect of IFN-I depends on cell type and/or timing of exposure to the cytokine (prior to or after infection).

During *T. gondii* infection regulation of tryptophan metabolism is a key component for parasite survival. Indeed, tryptophan degradation inhibits parasite replication. In *T. gondii*-infected mice, indoleamine 2,3-dioxygenase (IDO), a tryptophan catalyzer (70, 71), is enhanced by IFN-II (72). However, it has also been reported that IFN-I can regulate IDO in human retinal pigment epithelial cells, inhibiting therefore *T. gondii* replication (73). Together, these results demonstrate that IFN-I also contribute to the regulation of protective immunity against *T. gondii* (**Table 2**).

#### *Leishmania*

*Leishmania* is a complex genus of obligate intracellular protozoan parasites that cause a widespread disease collectively known as Leishmaniasis. The life cycle of these parasites takes place between a mammalian host and a sandfly vector (genus *Lutzomyia* and *Phlebotomus*). Once in the hosts, the promastigote form of the parasite preferentially infects macrophages, but can also be found in other cell types, such as dendritic cells, neutrophils, and fibroblasts. Promastigotes then transform into the non-flagellated form called amastigotes within the host's cell. The *Leishmania* spp. involved and the mammalian host immune status determine the clinical manifestation of the disease. Parasites can either reside in the skin and/or mucosal surfaces, which results in cutaneous (i.e., *Leishmania major*) or mucocutaneous (i.e., *Leishmania braziliensis*) Leishmaniasis; or disseminate to internal organs such as liver, spleen, and bone marrow, causing visceral Leishmaniasis (VL), the most severe form of the disease (i.e., *Leishmania donovani*) (74).

*Leishmania* immunity is mostly mediated by T lymphocytes. In experimental models, control of infection is mediated by a polarized Th1 response, induced by an initial production of IL-12 by DCs (75). IFN-γ secreting CD4 and CD8 T cells contribute to parasite control by enhancing the ability of phagocytic cells to kill intracellular *Leishmania* (74, 76).

As for many other protozoan models, IFN-II is the main mediators of the cellular immune response. However, IFN-I and IFN-I inducible genes are gradually gaining importance in the *Leishmania* field. One of the pioneer work on the role of IFN-I in Leishmaniasis described the prophylactic treatment with synthetic double-stranded RNA (Poly I:C) prior to *L. donovani* infection. Injection of Poly I:C triggered a burst of IFN-I and led to the control of the hepatic parasite burden (77). The role of endogenous IFN-I was studied for the first time using strains causing cutaneous Leishmaniasis. The induction of IFN-I was observed in macrophages infected *in vitro* with *L. major* promastigotes (78, 79) and in skin macrophages from infected animals (79), showing that promastigotes could enhance IFN-I expression in the host cell. The combination of exogenous IFN-I with *L. major* promastigotes was shown to activate macrophages, inducing type 2 nitric oxide synthase (NOS2). NOS2 is required for parasite elimination; mice deficient in this enzyme are more susceptible to *L. major* infection (80). As for *T. gondii*, the timing of the host cell's exposure to IFN-I determines the effect on parasite control. Indeed, pre-treatment of macrophages with

#### TABLE 2 | Role of interferon (IFN)-I in *Toxoplasma* infection.

exogenous IFN-I failed to induce NOS2. Similar results were obtained with high doses of exogenous IFN-I, while a low IFN-I dose in combination with *L. major* enhanced leishmanicidal activity (78, 81). These results suggest that the design of *in vitro* experiments greatly influences the outcome of IFN-I treatment in infected macrophages and that the role of IFN-I should be better studied in *in vivo* models.

The protective role of endogenous IFN-I during infection was confirmed by neutralizing IFN-I in mice experimentally infected with *L. major*. In fact, IFN-I neutralization rendered *L. major*infected mice more susceptible to infection and enhanced parasite multiplication. IFN-I blockade led to abolishment of NOS2 function and reduced cytotoxic activity and IFNγ production by NK cells at early stages of infection (79).

Opposite results were obtained in human macrophages infected *in vitro* with New World *Leishmania* spp. IFN-β treatment of *L. braziliensis* and *Leishmania amazonensis*-infected macrophages enhanced the parasite burden through a superoxidedependent, NO-independent mechanism (82). In this model, it was shown that IFN-β can regulate the superoxide dismutase SOD1 activity. SOD1 is responsible for catalyzing the disproportionation of superoxide to hydrogen peroxide and dioxygen and is an important constituent in apoptotic signaling and oxidative stress. It has been observed that biopsies from cutaneous Leishmaniasis patients express high levels of SOD1 (82).

The importance of endogenous IFN-I during chronic infection has been investigated using IFNAR-deficient mice in the context of *L. amazonensis* infection. *L. amazonensis* infected *Ifnar*−/− mice developed attenuated cutaneous lesions and displayed a decreased parasite load. This effect appeared to be STAT1 independent, a key protein in the IFN signaling (83). Furthermore, *L. amazonensis*-infected *Ifnar*−/− mice exhibited high levels of neutrophils and lower inflammatory monocytes recruitment at early times post infection. This unique profile was also observed in *L. major* and *L. braziliensis* infections (83). *In vitro* coculture of infected WT macrophages with *Ifnar*−/− neutrophils revealed that IFNAR-deficient neutrophils promote parasite killing (83). This evidence supports the pathogenic role of IFN-I signaling in cutaneous Leishmaniasis caused by New World *Leishmania* species.

We also observed a negative role for IFN-I in an experimental model of VL. *L. donovani* amastigotes were shown to induce IFN-I expression in B cells in an endosomal TLR-dependent manner. This cytokine was involved in a positive regulatory loop that led to the upregulation of endosomal TLRs and to IL-10 production in B cells (84). B cell-derived IL-10 was shown to suppress protective T cells responses and increase disease susceptibility (85). B cells are known to play a detrimental role during VL (86), not only by secreting IL-10 but also for their excessive antibody production (87). Indeed, hypergammaglobulinemia is a hallmark of VL. Interestingly, IFN-I seems to be regulating antibody production during VL. Specific ablation of endosomal TLRs or IFN-I signaling in B cells was shown to severely reduce the Ig titer in the serum of *L. donovani*-infected mice, suggesting that parasite activation of B cells *via* endosomal TLRs and IFN-I are involved in the induction if hypergammaglobulinemia (84). Furthermore, mice with a B cell-specific deficiency in endosomal TLR or IFNAR were more resistant to *L. donovani* infection than their wild-type counterpart.

Very little is known about the function of IFN-I in VL patients. It was reported that human mononuclear phagocytes can be activated by IFN-β, but less efficiently than IFN-γ (88). Exogenous treatments with IFN-I and IFN-II but not IL-2, failed to restore the cytotoxic activity of NK isolated from VL patients (89). Also, treatment of the cutaneous lesion in patients with IFN-I did not improve healing, compared with IFN-γ treatment (72, 90).

Because dendritic cells can also be infected by *Leishmania*, it is important to consider the induction of IFN-I by the parasite and its possible effect in these cells as well. Transcriptomic analysis of human DCs infected *in vitro* with *L. major* or *L. donovani* showed a differential expression pattern for IL-12 associated genes, the NF-KB pathway, and IFN regulatory factors (91). IFN-β produced by *L. major*-infected DCs seems to be required for IL-12 secretion by the infected DC, suggesting that protective Th1 responses, which are IL-12 depended, may also depend on IFN-I (92).

A summary on the role of IFN-I during Leishmaniasis can be found in **Table 3**.

#### Trypanosomes

Trypanosomes are digenetic protozoan parasites that infect domestic and wild animals, as well humans. Although many species of trypanosomes cause important veterinary disease, mainly two species cause significant human morbidity: *Trypanosoma brucei* and *Trypanosoma cruzi*. These two species are responsible for causing the sleeping sickness (African trypanosomiasis) and the Chagas disease (American trypanosomiasis), respectively.

The life cycle of these parasites takes place between the invertebrate vector and the vertebrate host. *T. brucei* and other African's trypanosomes are transmitted to the mammalian host by a tsetse fly bite. In the blood stream, metacyclic trypomastigotes differentiate into bloodstream trypomastigotes. In humans, trypanosomes proliferate in the blood and lymphatic system at early stages of the infection. This stage is associated with an anti-inflammatory response. At chronic stages, parasites can pass through the blood–brain barrier and enter the central nervous

system. This stage is associated with inflammatory changes in the brain and is characterized by a neurological disturbance (93, 94).

In *T. cruzi* (American trypanosomiasis), metacyclic trypomastigotes are released in the feces/urine of the triatomine vector after a blood meal. Trypomastigotes can successfully infect the mammalian host if they are able to reach the mucosa or injured skin areas. In contrast to African trypanosomes, *T. cruzi* is an intracellular parasite that has the capacity to invade, differentiate into amastigotes, and replicate within a wide range of nucleated cells. This characteristic is one of the most important features of *T. cruzi* within the host. Amastigotes differentiate into infective bloodstream trypomastigotes, before being released upon cell lysis. The released parasites can then infect neighboring cells or enter the bloodstream (95).

During the acute phase, the innate immune response against *T. cruzi* is characterized by the induction of a cell-mediated response that involves the production of IFN-γ and TNF (by NK and T cells), required for enhancing iNOS activity by phagocytic cells and for priming the adaptive immune response. iNOS activation is critical for controlling parasite growth during the infection (95). *T. cruzi* elicits a prominent IFN-I response at early times of infection (96–99). As mentioned before for *Plasmodium*, the role of IFN-I in *T. cruzi* infection is controversial. Some studies ascribe a protective role to IFN-I; others demonstrate that IFN-I induces pathology. The effect of IFN-I mainly depends on the dose, amount of parasites, and the inoculation route used to set up the infection.

The first studies on the role of IFN-I investigated the outcome of exogenous IFN-I treatment in *T. cruzi*-infected mice. The results showed that administration of IFN-I increased resistance to infection by stimulating T and NK cell activities, which are essential for protection (100, 101).

In an intradermal model of infection, transcriptomic analysis of excised skin from the inoculation site revealed that *T. cruzi* upregulated the expression of ISGs as early as 24 h after infection. Induction of ISGs was dependent on IFN-I signaling, suggesting that IFN-I is an important component of the innate immune response to *T. cruzi* (99). In agreement with the above mentioned literature, studies carried out in *Ifnar*−/− mice infected with *T. cruzi* revealed that efficient IFN-I signaling was required for controlling parasites growth during the acute phase of infection (102, 103). IFN-I was necessary for enhancing NO production in phagocytic cells (102). NO is considered the major effector molecule for intracellular amastigotes elimination within infected cells, being important for the control of parasite multiplication (95).

By contrast, another group reported a potential pathogenic role for IFN-I. In this work, a lethal dose of parasites inoculated intradermally was used to set up the infection in WT and *Ifnar*−/− mice. Surprisingly, *T. cruzi*-infected *Ifnar*−/− mice survived the challenge and were able to control parasite replication (104). Besides the fact that splenocytes from *Ifnar*−/− mice produced higher levels of IFN-II, plasma cytokine profile in *T. cruzi*infected *Ifnar*−/− mice were not different to control mice (104). Additionally, T cells populations were not inherently different compared with control mice (104), and IFN-γ production by CD8+T cells was not affected by impaired IFN-I signaling (105), suggesting that, in this model, endogenous IFN-I is not the only relevant signal in host defense against *T. cruzi*.

Taken together, the role of IFN-I in *T. cruzii* infection differs from one experimental model to the other, depending on the dose and the route of infection (106). This could explain the controversy about the observations on the role of IFN-I in the *T. cruzi* model of infection (**Table 4**).

The immune response to African trypanosomes is quite different than that to *T. cruzi*. First, parasites never enter the host cell at any stage of their development. The success of these parasites is mainly due to their ability to change the composition of the variant surface glycoprotein (VSG) by switching genes. This confers them the capacity to evade B- and T-cell-mediated immune responses and results in fluctuating waves of parasitemia that characterize African trypanosomiasis (94). VSG is a strong antigen that induces Th1 responses and promotes autoantibody and cytokines production, in particular TNF. Other trypanosome proteins and soluble factors, such as a trypanosomereleased triggering factor, also trigger IFN-γ production by T and NK cells and are involved in macrophage activation toward an M1 phenotype, which is required for the control of parasite multiplication during the acute phase of infection. However, sustained activation of M1 macrophages is associated with disease exacerbation. The progression of the infection toward the development of an acute fatal disease or a prolonged chronic infection is determined by the balance between a type I and type II immune responses and the switch from the early type I immune response (dominated by M1 macrophage activation) from a type II (M2 macrophages) regulatory response that controls the inflammation (107).

The literature on the role of IFN-I in African trypanosomiasis is scarce. A study involving *Trypanosoma brucei rhodesiense* reported a beneficial effect of IFN-I during the acute phase of infection. Indeed, *Ifnar*−/− mice displayed delayed control of parasite burden during the first week of infection and died earlier than wild-type controls. Moreover, mice hyperresponsive for IFN-I (*Ubp43*−/−) exhibited a significant defect in Th1 responses and IFN-γ production, suggesting that IFN-I plays a role in the early stages of disease. Nevertheless, IFN-I contributes to the downregulation of IFN-γ production and loss of host resistance during chronic infection (108).

No effects of IFN-I signaling were observed in *Trypanosoma brucei brucei*-infected *Ifnar*−/− mice, which showed similar levels of parasitemia to wild-type mice, suggesting that in this model parasite control is independent of IFN-I (109). However, IFN-I

regulates T cell infiltration to the brain parenchyma at chronic stages of the infection (109).

In conclusion, the contribution of IFN-I to protective immunity against several protozoan parasites is still unclear. Variations in parasite numbers used for infections, the site of inoculation, and the dose of IFN-I all seem to influence the outcome and the interpretation of the results. A spatio-temporal analysis of the role of IFN-I integrated with a more detailed investigation of cell-specific signaling pathways elicited by the cytokine could help to better dissect the involvement of IFN-I in the immune response.

#### REFERENCES

# AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

# FUNDING

This work was supported by Canadian Institutes of Health Research grant MOP 123293 (to SS) and the Natural Science and Engineering Research Council of Canada (to SS). SS-B was partly supported by a studentship from the Fondation Armand-Frappier.

the control of *Leishmania donovani*. *Eur J Immunol* (1998) 28(2):669–80. doi:10.1002/(SICI)1521-4141(199802)28:02<669::AID-IMMU669> 3.0.CO;2-N

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

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

# Canonical and Non-Canonical Aspects of JAK–STAT Signaling: Lessons from interferons for Cytokine Responses

*Andrea Majoros1 , Ekaterini Platanitis1 , Elisabeth Kernbauer-Hölzl1 , Felix Rosebrock1 , Mathias Müller2 and Thomas Decker1 \**

*1Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria, 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria*

Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signal transduction mediates cytokine responses. Canonical signaling is based on STAT tyrosine phosphorylation by activated JAKs. Downstream of interferon (IFN) receptors, activated JAKs cause the formation of the transcription factors IFN-stimulated gene factor 3 (ISGF3), a heterotrimer of STAT1, STAT2 and interferon regulatory factor 9 (IRF9) subunits, and gamma interferon-activated factor (GAF), a STAT1 homodimer. In recent years, several deviations from this paradigm were reported. These include kinase-independent JAK functions as well as extra- and intranuclear activities of U-STATs without phosphotyrosines. Additionally, transcriptional control by STAT complexes resembling neither GAF nor ISGF3 contributes to transcriptome changes in IFN-treated cells. Our review summarizes the contribution of non-canonical JAK–STAT signaling to the innate antimicrobial immunity imparted by IFN. Moreover, we touch upon functions of IFN pathway proteins beyond the IFN response. These include metabolic functions of IRF9 as well as the regulation of natural killer cell activity by kinase-dead TYK2 and different phosphorylation isoforms of STAT1.

Keywords: signal transduction, JAK–STAT, non-canonical, interferon, innate immunity

# INTRODUCTION

Since their discovery in the late 1950s (1), interferons (IFNs) have been assigned various functions that extend far beyond the initially observed antiviral activity. Three families of IFNs have been described and are known as type I (IFN-I, mainly IFNα/β), type II (IFN-II or IFNγ), and type III (IFN-III or IFNλ). In canonical IFN signaling, all types of IFNs produce a transcriptionally active signal transducer and activator of transcription 1 (STAT1) through receptor-bound Janus kinase (JAK)-mediated phosphorylation of tyrosine (Y) 701. The IFNγ receptor employs JAK1 and JAK2 to phosphorylate exclusively STAT1, causing its homodimerization. STAT1 dimers, also called gamma interferon-activated factor (GAF), translocate to the nucleus and promote gene expression by binding to gamma interferon-activated site (GAS) of interferon-stimulated genes (ISG). On the other hand, stimulation with IFN-I or IFN-III leads to TYK2- and JAK1-mediated phosphorylation of STAT1 and STAT2. After forming heterodimers, these two proteins associate with interferon regulatory factor 9 (IRF9) to form a transcriptionally active IFN-stimulated gene factor 3 (ISGF3)

#### *Edited by:*

*Claudia U. Duerr, McGill University, Canada*

#### *Reviewed by:*

*Laura Eugenia Velazquez, French Institute of Health and Medical Research, France Uwe Vinkemeier, University of Nottingham, UK*

*\*Correspondence:*

*Thomas Decker<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 28 September 2016 Accepted: 09 January 2017 Published: 26 January 2017*

#### *Citation:*

*Majoros A, Platanitis E, Kernbauer-Hölzl E, Rosebrock F, Müller M and Decker T (2017) Canonical and Non-Canonical Aspects of JAK–STAT Signaling: Lessons from Interferons for Cytokine Responses. Front. Immunol. 8:29. doi: 10.3389/fimmu.2017.00029*

that controls gene expression by binding to interferon-stimulated response elements (ISRE) in a different set of ISGs (2, 3). Although a large majority of IFN-induced gene expression is mediated by canonical pathways, it has become clear that the components of these pathways are able to exert non-canonical activity: tyrosine kinase-independent action of JAKs, transcriptional complexes other than ISGF3 and GAF, and pathways building on U-STATs that are not phosphorylated on tyrosine. This review will focus on these non-canonical functions of JAK–STAT signaling components.

## IFNs AND THEIR ROLE IN RESISTANCE TO VIRUSES AND BACTERIA

As major components of the innate immune system against viral infections, all type I interferons (IFN-I) stimulate cell-autonomous antiviral activity (4). In addition, they increase cellular immunity through contributions to natural killer (NK) and T cell activation (5). IFN-I act as modulators of cellular immunity by selectively enhancing clonal expansion and survival of CD8+ T cells (6), directing the immune response toward Th1-dominace (7) and activating NK cells (8).

In the context of antibacterial defense, genes activated by IFN-I enhance inflammation and the death of infected cells (9). In addition, they impact on cells at the interface of the innate and adaptive immune systems, such as macrophages and dendritic cells, to increase antigen presentation and trigger the adaptive response (10). Immunostimulatory activities of IFN-I also contribute to immunosurveillance against cancer (11). On the other hand, their proinflammatory activity renders them driving forces behind a group of interferonopathies, such as the Aicardi-Goutieres syndrome (12, 13).

While IFN-I are generally protective against viral infections, they can be both friend and foe in the defense of bacterial pathogens (14, 15). For example, IFN-I exert protective effects in the case of *Chlamydia pneumoniae* (16), *Legionella pneumophila* (17), *Salmonella typhimurium* (18), and both group A and group B *Streptococcus* infections (19–21). However, in infections with *Listeria monocytogenes*, *Francisella tularensis*, and *Mycobacterium tuberculosis*, production of IFN-I is associated with decreased innate immunity (22–27).

Major target cells of IFNγ are macrophages and T cells. Many of the genes induced by IFNγ are transcription factors, which amplify the transcriptional response and, as in Th cells, influence cell differentiation (28–30). IFNγ is particularly important in macrophage biology, where it provides cellautonomous antimicrobial activity through the upregulation of microbicidal gene products (31). Further, impact of IFNγ on macrophage activation results from its ability to synergize with or to antagonize the effects of different cytokines, growth factors, and pathogen-associated molecular pattern-signaling pathways (e.g., TNFα, IL-4, CSF-1, IFNα/β, LPS, and CpG DNA). Through these mechanisms, IFNγ activates macrophages to express antimicrobial and antitumor effects. It upregulates chemokines and adhesion molecules, directing cells to the sites of inflammation. In the adaptive immunity, IFNγ plays an important role in Th1 responses, repressing the development of Th2 and Th17 T cell responses (30) and acting directly on B cells to promote class switching from IgG2 to IgG3 (32). Mice and humans deficient in IFNγ or IFNGR1 show a decrease in natural resistance to bacterial, parasitic, and viral infections (33–35). Mice and cells lacking IFNγ display compromised tumor rejection, underlining its importance in tumor surveillance (11).

Discovered in the year 2003, IFN-III, better known as IFNλ, are the most recently described members of the IFN family (36). Although signaling through a different receptor complex with IFNλR/IL10R2 chains, IFNλ also stimulate formation of the ISGF3 complex. Given the similarities between the IFN-I and IFNλ signaling pathways, some of the non-canonical signals described below for IFN-I may apply to IFNλ as well. While IFNλ produce similar biological changes of their target cells as IFN-I, including antiviral, antiproliferative, and antitumor activity, the key to different organismic responses to IFN-III lies in their receptor distribution, which is prevalent on cells of epithelial origin (37). In line with that, defects in IFN-III production or signaling cause reduced innate immunity to viral pathogens replicating in epithelia of the lung and the gut. Mice lacking the IFN-I receptor are resistant to all IFN-I subtypes, but retain their sensitivity to IFN-III (38). However, studies performed in IFNAR1−/−, IL-28Rα−/−, and IFNAR1/IL-28Rα double knockout mice show that, in primary airway epithelia, upon influenza infection, IFN-I and IFN-III mediate parallel amplification loops that lead to the induction of fully overlapping groups of ISGs (39).

#### KINASE-INDEPENDENT JAK ACTIVITY

Janus kinases are non-receptor tyrosine kinases which have essential roles in cytokine and growth factor signaling (40, 41). There are four different JAKs (JAK1, JAK2, JAK3, and TYK2) that cross-phosphorylate and activate each other when ligandassociated cytokine receptor chains come in close proximity. Canonical, i.e., kinase-dependent functions of JAKs include tyrosine phosphorylation of receptor chains and of STATs at a single tyrosine residue near the C-terminal end. STAT phosphorylation is thought to require SH2 domain-mediated docking to the modified receptor chains, consistent with impaired phosphorylation at mutant receptors lacking the critical tyrosine for JAK-mediated phosphorylation (2).