# MYSTERIES OF TYPE I IFN RESPONSE: BENEFITS VERSUS DETRIMENTS

EDITED BY: Arno Müllbacher, Yoichi Furuya and Herbert Patrick Ludewick PUBLISHED IN: Frontiers in Immunology and Frontiers in Microbiology

#### *Frontiers Copyright Statement*

*© Copyright 2007-2015 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.*

*The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.*

*Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.*

*Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.*

*As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.*

*All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.*

ISSN 1664-8714 ISBN 978-2-88919-629-6 DOI 10.3389/978-2-88919-629-6

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# **MYSTERIES OF TYPE I IFN RESPONSE: BENEFITS VERSUS DETRIMENTS**

Topic Editors:

**Arno Müllbacher,** Australian National University, Canberra, Australia **Yoichi Furuya,** Albany Medical College, Albany, USA **Herbert Patrick Ludewick,** Albany Medical Center, Albany, USA

Successful containment of an infection is dependent on both innate and adaptive immune response. Cytokines are essential effectors of both of these systems. In particular, type I interferons (IFN-I) are important components of early innate immunity against an infection. However, the production of IFN-I could serve as a double edge sword, either containing an infection or enhancing susceptibility. For example, IFN-I, which is essential for early containment of viral infections, has been shown to be detrimental to the host during bacterial infections. In fact, recent significant reports have shown that influenza virus induced IFN-I responses can enhance the host susceptibility to secondary bacterial infections. These recent reports highlight the expanding immunoregulatory role of IFN-I in the host immunity. With these recent findings in mind, the aim of this research topic is to welcome novel data, opinion and literature reviews on the newly identified dual functions of IFN-I. This research topic wills focus on the following areas of IFN-I: 1) a detrimental role of IFN-I during primary bacterial infection; 2) a detrimental role of viral infection induced IFN-I during secondary bacterial infections; 3) evolutionary pressure that drove detrimental IFN-I response during primary bacterial infection; and 4) does benefit of IFN-I responses during primary viral infections outweigh the adverse consequences of IFN-I mediated enhanced susceptibility to secondary bacterial infections.

**Citation:** Arno Müllbacher, Yoichi Furuya and Herbert P. Ludewick eds. (2015). Mysteries of type I IFN response: benefits versus detriments. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-629-6

# Table of Contents

	- *immunity: a way forward for therapeutics and vaccine design* Danushka Kumara Wijesundara, Yang Xi and Charani Ranasinghe

Elena Tomasello, Emeline Pollet, Thien-Phong Vu Manh, Gilles Uzé and Marc Dalod

## Mysteries of type I IFN response: benefits versus detriments

#### **Yoichi Furuya1\*, Herbert P. Ludewick 1 and Arno Müllbacher 2**

1 Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA

2 Department of Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia

\*Correspondence<EMAIL_ADDRESS>
**Edited and reviewed by:**

Ian Marriott, University of North Carolina at Charlotte, USA

**Keywords: type I interferons, vaccine adjuvant, viral, bacterial**

Successful containment of infection is dependent on both innate and adaptive immune responses. Cytokines are essential components of both of these systems. In particular, type I interferons (IFN-I) are important components of early innate immunity against infections. However, the production of IFN-I could serve as a double-edged sword, in that it could help eliminate infections or in certain instances enhance host's susceptibility to infections. For example, IFN-I provide early resistance against acute viral infections, but are detrimental to the host during certain bacterial infections and chronic viral infections. This Research Topic presents seven articles that address the biological roles of IFN-Is in host immunity and various contexts (e.g., autoimmunity, cancer, viral/bacterial infections, IFN-I therapy, etc.) where IFN-Is could either serve to control or exacerbate disease.

The original research performed by Babb et al. investigates the potential adjuvant activity of gamma-irradiated influenza (1). They demonstrate that co-vaccination with gamma-irradiatedinfluenza virus and poorly immunogenic inactivated Semliki forest virus (SFV) results in enhanced SFV-specific antibody responses without compromising humoral immunity against influenza infections. They have previously shown that gammairradiation destroys the ability of SFV but not influenza A virus to elicit strong IFN-I responses (2). Thus, it is likely that the adjuvant activity of gamma-irradiated influenza virus is attributable to its potency of IFN-I induction. These authors speculate that gamma-irradiated influenza virus may therefore be exploited as an adjuvant to improve the efficacy of poorly immunogenic vaccines owing to its ability to stimulate IFN-I production.

The potent immune activating properties of IFN-I can also be detrimental during viral infection, particularly for viruses that establish chronic infection. This certainly is the case for human or simian immunodeficiency virus (HIV/SIV) infection in nonnatural hosts. As discussed by Tomasello et al. (3) and Furuya et al. (4), IFN-I induced during HIV infections may play a pathogenic role in driving chronic immune activation associated with CD4+ T cell depletion and loss of T cell function. Indeed, a strong correlation exists between chronic low level productions of IFN-I and disease progression in non-natural hosts. Thus, Furuya et al. hypothesize that regulatory mechanisms must exist in natural hosts that actively suppress IFN-I responses despite viral replication (4). It is speculated that understanding how the host can co-exist with HIV without generating an IFN-I response will be crucial in developing therapeutic interventions that can prevent or dampen progression to an acquired immune deficiency syndrome (AIDS). Perhaps, as pointed out by Tomasello et al., the more important question that needs to be addressed first is whether a causal link exists between low levels of IFN-I signaling and the development of AIDS in non-natural hosts or, rather, the upregulation of IFN-stimulated genes simply a marker of disease progression.

The pathogenic role of IFN-I is more widely observed in bacterial infections. For example, the facultative intracellular bacterium *Francisella tularensis* is less virulent in mice deficient in IFN-I receptors (5, 6). Furuya et al. propose in an opinion article that the detrimental role of IFN-I during pulmonary tularemia may be linked to its suppressive effects on neutrophil recruitment, a response that may be protective against respiratory *Francisella tularensis* infection (7). The role of neutrophils during pulmonary tularemia has yet to be fully defined, but it is likely that both the magnitude and timing of cellular recruitment to the lung will determine whether neutrophils promote bacterial clearance or contribute to immunopathology.

In three review articles, the mechanisms that have been proposed to explain the IFN-I-mediated increases in bacterial infection susceptibility are discussed. The review article by Eshleman et al. focuses on the suppressive effects of IFN-I on myeloid cells during intracellular bacterial infections (8). These authors also emphasize that IFN-I can exert a positive anti-inflammatory effect in a number of autoimmune diseases and that this is mediated by the suppression of myeloid cell inflammatory responses. The review article from Dhariwala et al. describes multiple pathways of IFN-I-dependent cell deaths that may aid bacteria escape phagocytosis and thereby contributes to bacterial pathogenesis (9). Indeed, as the author pointed out, bacterial pathogens that benefits from IFN-I signaling are often facultative intracellular bacteria. The final review by Wijesundara et al. highlights pathological contexts in which IFN-I could be exploited in therapy and vaccine design with a particular emphasis on IFN-epsilon (10). IFN-epsilon appears to have both overlapping and distinct functions compared to IFN-alpha and -beta, and therefore, the author urge the need of evaluating contribution of the different members of IFN-I to fully exploit its beneficial effects.

Despite IFN-I being perhaps the most studied cytokine, their immunoregulatory roles are not fully understood. As is evident from the articles presented in this Research Topic, the role played by IFN-I is highly context-dependent and can be both beneficial and detrimental. Further research is clearly required in order for us to selectively harness the protective role of IFN-I while suppressing its damaging effects.

#### **ACKNOWLEDGMENTS**

Funding sources: Yoichi Furuya – Senior Research Fellowship RT-226959-N from the American Lung Association.

#### **REFERENCES**

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

*Received: 06 January 2015; accepted: 09 January 2015; published online: 28 January 2015.*

*Citation: Furuya Y, Ludewick HP and Müllbacher A (2015) Mysteries of type I IFN response: benefits versus detriments. Front. Immunol. 6:21. doi: 10.3389/fimmu.2015.00021*

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

*Copyright © 2015 Furuya, Ludewick and Müllbacher. 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.*

### Gamma-irradiated influenza A virus provides adjuvant activity to a co-administered poorly immunogenic SFV vaccine in mice

#### **Rachelle Babb1 , Jennifer Chan1 , Jasmine E. Khairat 1 ,Yoichi Furuya2† and Mohammed Alsharifi1\***

1 Vaccine Research Laboratory, School of Molecular and Biomedical Science, Centre for Molecular Pathology, The University of Adelaide, Adelaide, SA, Australia 2 Department of Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia

#### **Edited by:**

Murali Krishna Kaja, University of Washington, USA

#### **Reviewed by:**

Ravi Kulkarni, Emory University, USA Suryaprakash Sambhara, Centers for Disease Control, USA

#### **\*Correspondence:**

Mohammed Alsharifi, Vaccine Research Laboratory, School of Molecular and Biomedical Science, Centre for Molecular Pathology, The University of Adelaide, Adelaide, SA 5005, Australia e-mail: mohammed.alsharifi@ adelaide.edu.au

#### **†Present address:**

Yoichi Furuya, Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA

Many currently available inactivated vaccines require "adjuvants" to maximize the protective immune responses generated against the antigens of interest. Recent studies in mice with gamma-irradiated influenza A virus (γ-FLU) have shown its superior efficacy compared to other forms of inactivated FLU vaccines and its ability to induce both potent interferon type-I (IFN-I) responses and the IFN-I-associated partial lymphocyte activation. Commonly, IFN-I responses induced by adjuvants, combined in vaccine preparations, have been shown to effectively enhance the immunogenicity of the antigens of interest.Therefore, we investigated the potential adjuvant activity of γ-FLU and the possible effect on antibody responses against co-administrated antigens, using gamma-irradiated Semliki Forest virus (γ-SFV) as the experimental vaccine in mice. Our data show that co-vaccination with γ-FLU and γ-SFV resulted in enhanced SFV-specific antibody responses in terms of increased titers by sixfold and greater neutralization efficacy, when compared to vaccination with γ-SFV alone. This study provides promising evidence related to the possible use of γ-FLU as an adjuvant to poorly immunogenic vaccines without compromising the vaccine efficacy of γ-FLU.

**Keywords: influenza vaccines, combined vaccine, gamma-irradiation, adjuvants, antibodies**

#### **INTRODUCTION**

Vaccines represent a fundamental aspect of current control strategies against infectious diseases. However, despite the remarkable success of many vaccines, there still remains many challenges in the field of vaccinology, such as generating effective vaccination strategies against notoriously difficult pathogens like hepatitis C virus and human immunodeficiency virus (1). Among the various types of currently used vaccines, live attenuated vaccines can mimic natural infections and consequently have been shown to be very effective in generating long-lived immunity. However, due to the high risk of conversion to their highly pathogenic form and the inability to develop live attenuated vaccines for many pathogens, inactivated vaccines such as inactivated whole viruses and purified antigens have typically been used as the main strategy for vaccine design. However, poor immunogenicity of many inactivated vaccines has severely affected their effectiveness. Therefore, inactivated vaccines often require "adjuvants" to enhance their immunogenicity. Importantly, most immunostimulatory adjuvants have been designed to potently stimulate innate signaling pathways through pattern recognition receptors (PRR) such as toll-like receptors (TLR) and cytosolic receptors (2–5). This ultimately leads to the induction of genes encoding for various immune-modulatory molecules including interferon type-I (IFN-I), which stimulate co-stimulatory molecule expression and antigen presentation (6).

Currently in vaccine design, greater attention is being drawn toward designing adjuvants to effectively boost the immune response toward existing vaccine preparations, which fail to induce sufficient immunity. The influence of IFN-I exogenously or through PRR stimulation has been shown to be very effective (7–9). We have previously reported the superiority of gammairradiated influenza virus (γ-FLU), compared to other flu vaccine formulations, in terms of inducing cross-protective immunity (10–12). We have also shown that γ-FLU similarly to its live form is capable of inducing potent IFN-I responses and the associated partial lymphocyte activation 24 h postchallenge (13, 14). Importantly, in contrast to gamma-irradiation of influenza, we have shown that gamma-irradiation of Semliki Forest virus (SFV) abrogates its ability to induce IFN-I responses (15). Therefore, we investigated the potential adjuvant activity of γ-FLU on co-administered γ-SFV as an experimental model for poor immunogenic vaccines. Here, we report that co-vaccination with γ-FLU and γ-SFV resulted in enhanced SFV-specific antibody titers "by six-folds" when compared to vaccination with γ-SFV alone. This enhancement in antibody titer was also associated with greater SFV neutralization efficacy and importantly; the vaccine efficacy of γ-FLU was not affected.

#### **MATERIALS AND METHODS ETHICS STATEMENT**

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of The University of Adelaide. The protocol was approved by the Animal Ethics Committee at The University of Adelaide (Permit Number: S-2011/119).

#### **VIRUSES AND CELLS**

Avirulent SFV (A7 strain) was grown *in vitro* by infecting Vero cells using multiplicity of infection (MOI) of 0.1, and infected flasks were incubated for 24 h at 37°C in a humidified atmosphere with 5% CO2. Culture supernatants were then collected and clarified to remove cellular debris by centrifugation at 1500 rpm for 5 min. Virus titer was determined by plaque assay on Vero cells to be 3 × 108 PFU/ml. For the vaccine preparation, SFV stock was concentrated using Millipore filtering devices with 100 kDa cutoff (Millipore) and centrifugation at 2000 rpm for 1 h at 4°C using Eppendorf bench top centrifuge. Virus titer of the concentrated SFV was determined by plaque assay on Vero cells to be 5 × 108 PFU/ml.

The influenza type A virus, A/PR/8 [(A/Puerto Rico/8/34 (H1N1)], was grown in 10-day-old embryonated chicken eggs (HiChick, SA, Australia). Each egg was injected with 0.1 ml normal saline containing 1 hemagglutination unit (HAU) of virus, incubated for 48 h at 37°C, and then held at 4°C overnight. The amniotic/allantoic fluids were then harvested, pooled, clarified, and stored at −80°C. Gamma-irradiated A/PR8 vaccine preparations were previously prepared by Dr. Furuya at ANU. Briefly, concentrated virus stocks were prepared using chick erythrocytes as previously described (16). Infectious allantoic fluid was incubated with chicken red blood cells (cRBCs) for 45 min at 4°C allowing the hemagglutinin to bind to erythrocytes, and then centrifuged (4°C, 1500 rpm, 10 min) to remove the allantoic fluid supernatant. The pellets were resuspended in normal saline, incubated for 1 h at 37°C to release the RBCs from the virus, and then centrifuged to remove the erythrocytes and the supernatant containing the virus collected. The titer of the concentrated A/PR8 virus stock (9 × 108 TCID50/ml) was determined by TCID50 assay (17).

#### **VIRUS INACTIVATION**

Concentrated virus stocks were inactivated by exposure to gammairradiation from a 60Co source [Australian Nuclear Science and Technology Organization (ANSTO) at Lucas Heights/NSW]. A/PR8 and SFV received a dose of 10 and 50 kGy, respectively, and they were kept frozen on dry ice during gamma-irradiation. Sterility was tested by two independent methods: plaque assay using MDCK (for A/PR8) or Vero cells (for SFV); and by inoculating embryonated eggs (for A/PR8). The detection limit of our plaque assay is 10 PFU/ml and no plaque forming unit was detected for the irradiated samples. These tests confirmed sterility of inactivated stocks. In addition, we have estimated the minimum inoculum required to cause a positive infection in embryonated eggs and found that the minimum egg infectious dose that causes detectable HA titers in the allantoic fluid after 2 days of incubation is 0.1 TCID50/egg. Embryonated eggs were inoculated with 100 ml of inactivated preparations per egg and incubated for 2 days at 37°C and the allantoic fluid of individual eggs was harvested and tested for virus replication using HA assays. HA titers were negative in the allantoic fluid of these eggs, which illustrates a complete loss of virus infectivity in our inactivated preparations.

#### **MICE AND TREATMENTS**

Wild-type C57B/6 mice (9–10-week-old) were bred under specific pathogen-free conditions and supplied by the Animal Laboratory Services at the University of Adelaide, SA, Australia.

In general, vaccine preparations were diluted using 10-fold serial dilutions and each mouse was injected in the tail vein with 200µl of the relevant virus or vaccine preparation. The following doses were used: live SFV (107 PFU/mouse), γ-SFV (either 106 , 107 , or 108 PFU equivalent/mouse), and γ-FLU (104 , 105 TCID50 equivalent/mouse). Refer to text for specific doses used in each experiment. For co-vaccination, the two vaccine preparations were mixed thoroughly in the same tube and administered as a single injection into experimental animals. Vaccination doses are expressed PFU or TCID50 equivalent. In addition, in some experiments Poly(I:C) was injected intravenously at a dose of 150µg in 200µl of PBS per animal as previously reported (18).

#### **ANTIBODY ANALYSIS**

Semliki Forest virus-specific and FLU-specific antibody responses in serum samples were determined by enzyme-linked immunosorbent assay (ELISA). In brief, Maxisorp plates were coated with concentrated SFV or FLU viral antigen diluted in bicarbonate coating buffer (Na2CO3, NaHCO3, water at pH 9.6) and incubated overnight at room temperature. Non-specific protein binding sites were then blocked with PBS containing 2% skim milk powder for 2 h at room temperature. Fifty microliter volumes of serially diluted serum samples were added to the appropriate wells for 2 h at room temperature followed by the addition of horse radish peroxidase conjugated goat anti-mouse IgG (Thermo Scientific) at room temperature for 2 h. Plates were developed using TMB peroxidase substrate in the dark for 30 min and the reaction was stopped with 2 mol H2SO4. Absorbance was measured at 450 nm using a Microplate ELISA reader (Bio-Tek Instruments).

#### **NEUTRALIZATION ASSAYS**

Plaque reduction assay modified from (19) was used to analyze SFV neutralization. Twenty-four well tissue culture plates were seeded with 1.5 × 105 Vero cells/well and incubated overnight at 37°C in a humidified atmosphere with 5% CO2. Aliquots of serum samples from control and vaccinated animals were incubated at 56°C for 30 min to inactivate complements and serially diluted using EMEM media without FCS. Diluted samples were mixed with equivalent amount of DMEM media containing 100 PFU of SFV. Mixtures (sera and virus) were incubated for 1.5 h at 37°C and then used to infect confluent Vero cell monolayer's (in triplicate) prepared earlier. Initially, culturing media was removed from each well prior to addition of virus/serum mixture. Plates were then incubated for 2 h at 37°C to allow infection of monolayers. Following incubation, the infecting mixture was removed and an agar overlay containing 50% of 1.8% Bacto-Agar, 40% DMEM media, 10% FCS, and 0.002% Fungizone was added to each well and plates were incubated for 3 days at 37°C, 5% CO2. Following incubation, cells were fixed with 5% formalin for 1 h at room temperature. The overlay was then carefully removed and cell monolayers were stained with 0.2% crystal violet. Plaques were enumerated to determine the effect of the serum on virus infectivity.

In addition, a hemagglutination inhibition assay (HAI) was used to test the influenza-specific antibody responses, as previously described (17). Aliquots of sera were incubated at 56°C to inactivate complements for 30 min, then diluted in PBS containing 1% RBCs, and left for another 30 min incubation at room temperature. RBCs within samples were then pelleted by centrifugation at 1400 rpm using a microcentrifuge and supernatants were collected. Twofold serial dilutions were performed using a 96-round well-bottom plate. Fifty microliter of diluted virus (FLU) at a concentration of 80 HAU/ml was then added to each dilution of sera and incubatedfor 30 min at room temperature. PBS containing 1% RBCs was then added to each well and incubated at 4°C. Results were analyzed 24 h later and neutralizing antibodies in each dilution was determined by the presence of a pellet of RBCs at the bottom of the wells.

#### **STATISTICAL ANALYSIS**

Results were expressed as mean ± SEM. Statistical significance among samples was calculated using an unpaired Student's *t*-test. *P* values <0.05 were considered statistically significant.

#### **RESULTS**

#### **ANTIBODY RESPONSES INDUCED BY** γ**-SFV**

Avirulent SFV causes asymptomatic infection characterized by high-titer viremia in adult mice (20, 21), and effective viral clearance has been attributed to rapid antibody responses generated by the host (22). Interestingly, primary cytotoxic T cell responses have been shown to be associated with MHC-I haplotype and restricted to H-2k haplotype expressing mice (23). Particularly, C57/B6 mice (H-2b haplotype) have been classified as cytotoxic T cell non-responders and therefore were used in the study. To confirm that the γ-SFV vaccination strategies used in this study can generate effective antibody responses, serum SFV-specific IgG levels were measured 20 days post-infection with live SFV (107

PFU/mouse) or vaccination with variable doses of γ-SFV (106 ,107 , or 108 PFU equivalent/mouse). Our data illustrate that vaccination with γ-SFV induces high levels of SFV-specific IgG in the serum of vaccinated mice in a dose-dependent manner (**Figure 1A**).

To determine if the detected antibody responses are protective, mice were vaccinated with γ-SFV (107 equivalent PFU/mouse) and challenged 21 days later with live SFV (107 PFU/mouse). Twentyfour hours post-challenge, serum samples were tested for virus infectivity by plaque assay. No viral infectivity was detected in all serum samples from previously vaccinated mice in contrast to the high viremia observed in un-vaccinated control mice following a challenge with live SFV (**Figure 1B**).

It has been previously shown that live SFV infection induces IFN-I and as a consequence promotes Th1 antibody isotype switching (20). Considering the inability of γ-SFV to induce detectable levels of IFN-I (13, 15), the level of IgG isotypes induced by live SFV vs. γ-SFV was investigated. Mice were infected with SFV (107 PFU/mouse) or vaccinated with γ-SFV (107 equivalent PFU/mouse) and serum SFV-specific IgG1 and IgG2c levels were measured over a time course. Both IgG1 and IgG2c levels appeared to be lower following vaccination with γ-SFV in comparison to SFV (**Figures 2A,B**).

#### **THE EFFECT OF CO-ADMINISTRATION OF** γ**-FLU AND** γ**-SFV ON SFV-SPECIFIC ANTIBODY RESPONSES**

Considering the potent IFN-I responses induced by γ-FLU (13, 14), we investigated the effect of γ-FLU and γ-SFV coadministration on SFV-specific antibody responses. Mice were coinjected with γ-SFV (107 PFU equivalent/mouse) and γ-FLU (104 or 105 equivalent TCID50/mouse) and serum SFV-specific IgG levels were measured at day 20 post-vaccination. Our data illustrate the significant enhancement (~six-folds) of SFV-specific IgG levels in mice co-injected with both γ-SFV and γ-FLU compared to mice vaccinated with γ-SFV alone (*p* < 0.05) (**Figures 3A,B**).

In addition, we investigated the kinetics of SFV-specific antibody responses post co-administration of γ-SFV and γ-FLU. Similarly, our data illustrate the consistent enhancement of SFV-specific antibody responses at all tested time points (*p* < 0.05) (**Figure 3C**).

To determine whether enhancement of SFV-specific antibody responses mediated by co-administration of γ-FLU coincides with enhanced SFV neutralization, serial dilutions of the immune sera were tested for their ability to neutralize 100 PFU of live SFV *in vitro* using aVero cells based plaque-inhibition assay. Our results clearly illustrate that while immune sera from γ-SFV alone vaccinated mice have high neutralization activity at 1/100 dilution, this neutralization activity decreased remarkably with every serial dilution to reach ~10% activity at 1/800 serial dilution. In contrast, immune sera from γ-SFV and γ-FLU co-vaccinated mice show significantly higher neutralization values for all tested sera dilutions when compared to sera from γ-SFV alone vaccinated mice (**Figure 3D**).

Next, we investigated the effect of γ-FLU and γ-SFV coadministration on SFV-specific IgG2c and IgG1 levels and whether co-vaccination may promote a particular IgG isotype. Our data illustrates that co-administration of γ-FLU and γ-SFV resulted in significantly enhanced IgG2c levels compared to vaccination with γ-SFV alone (*p* < 0.01). However, co-vaccination did not lead to significant enhancement in IgG1 responses as the detected SFVspecific IgG1 levels appeared similar in both vaccinated groups (**Figures 4A,B**).

#### **THE EFFECT OF CO-VACCINATION ON FLU-SPECIFIC ANTIBODY RESPONSES**

Vaccination with γ-FLU has been shown to elicit homotypic neutralizing antibody responses (10, 11). To determine whether the co-administration of γ-FLU and γ-SFV affects the host's ability to generate effective FLU-specific humoral responses, we investigated FLU-specific IgG responses at day 20 post-vaccination. Our data indicates that co-vaccination did not suppress the induction of FLU-specific IgG responses induced by γ-FLU (**Figure 5A**). We have also tested the neutralizing efficacy of FLU-specific antibodies using HA inhibition assay and our data illustrated that the hemagglutinating activity of 80 HAU of A/PR8 was inhibited at similar levels by immune sera from mice vaccinated with γ-FLU alone or mice co-vaccinated with γ-FLU and γ-SFV (**Figure 5B**).

#### **THE EFFECT OF AN IFN-I INDUCING ADJUVANT ON THE IMMUNOGENICITY OF** γ**-SFV**

In line with the common approaches used for adjuvant design, the adjuvant activity of γ-FLU is expected to be related to its ability to induce potent IFN-I responses and the associated IFN-I-mediated partial lymphocyte activation (13). To illustrate the effect of an IFN-I inducing adjuvant on the immunogenicity of γ-SFV, we evaluated the effect of poly(I:C) and γ-SFV co-administration on SFV-specific antibody responses. The ability of poly(I:C) to induce IFN-I and its potential use as an adjuvant to enhance humoral responses toward poorly immunogenic proteins has been well-documented (4, 7, 24). Therefore, mice were vaccinated with γ-SFV with or without co-injection of poly(I:C) and total SFVspecific IgG levels in the immune sera were analyzed at days 3, 6, 12, and 20 post-vaccination. Our data illustrate that co-injection of poly(I:C) and γ-SFV resulted in a significant enhancement in the level of SFV-specific IgG titers in the serum at all time points compared to the injection of γ-SFV alone (**Figure 6**).

#### **DISCUSSION**

There has always been an increased demand for safe and effective vaccines to reduce the morbidity and mortality associated with particular viral infections. Non-living antigens are often employed in vaccine strategies and many are poor immunogens (2). We have reported previously the efficacy of γ-FLU to generate protective immunity upon homotypic and heterosubtypic influenza A virus challenges (10, 25). In addition, we have demonstrated the ability of the γ-FLU vaccine to induce potent IFN-I responses and the associated partial systemic lymphocyte activation (13, 14). It has been illustrated previously that IFN-I plays a very influential role in the development of B lymphocytes and consequently antibody production (26, 27). In addition, we have reported that γ-SFV, in contrast to live SFV, does not induce detectable levels of IFN-I (13, 15). Therefore, we used γ-SFV vaccine as an experimental model to test the adjuvant activity of γ-FLU.

In general, adjuvants are often used to achieve qualitative/quantitative differences in the immune responses that may include increasing the speed of an immunological response, which

served as negative controls. Data represent mean ± SEM (n = 3) \*\*p < 0.01.

is important especially during pandemic outbreaks (28, 29). Adjuvants have also been employed to promote specific types of immunity,which may not be efficiently generated by the non-adjuvanted antigens, i.e., Th1 vs. Th2 cells, CD8+ vs. CD4+ cells, and specific types of antibody isotypes (2). Our data clearly show that coadministration of γ-SFV with γ-FLU amplified SFV-specific IgG levels with an overall enhancement of ~six-folds. The enhanced titers observed at day 6 post co-vaccination were equal to the titers detected at day 20 following vaccination with γ-SFV alone. Thus, confirming an earlier induction and amplification of SFV humoral responses. We have also shown this enhancement to be associated with increased efficiency in virus neutralization. Furthermore, our data illustrate that γ-FLU promotes enhancement of type-1 antibody response to co-administered γ-SFV, as illustrated by the significant increase in IgG2c but not IgG1. This outcome is commonly desired within vaccine development due to the competent functions of IgG2a during antigen clearance relative to IgG1 (30–32).

In line with the many approaches used for adjuvant design, IFN-I responses induced by γ-FLU may have played an important role in the observed adjuvant activity of γ-FLU. To evaluate the role of IFN-I, the adjuvant activity of Poly IC on antibody responses to γ-SFV was investigated. The enhancement of

humoral responses against poorly immunogenic proteins using Poly I:C is well-documented (4, 7, 24). Poly IC, is a synthetic analog of dsRNA, which is commonly used to stimulate TLR3 and MDA-5 to induce IFN-I and other Th1 priming cytokines such as IL-12 (8, 33, 34). Consistent with previous reports, our data show that co-immunization with Poly IC and γ-SFV resulted in significantly enhanced SFV-specific IgG titers relative to titers observed following vaccination with γ-SFV alone. While this suggest a possible role of IFN-I, more work will be conducted to analyze the underline mechanisms for the adjuvant activity of γ-FLU.

The ultimate goal of using γ-FLU as an adjuvant is to exploit the efficacy of the γ-FLU-mediated immune response toward combined vaccines, in addition to conferring protection against the influenza virus. Our results show that co-administration of γ-FLU and γ-SFV did not affect the titers of FLU-specific IgG and did not affect the neutralizing activity of FLU-specific antibodies. Therefore, humoral responses generated against FLU antigens were not hindered when a second vaccine is present in the environment. Overall, this study is a proof-of-concept illustrating that γ-FLU, a whole virus killed influenza vaccine, can potentially be employed as an adjuvant to increase the quality and magnitude of immune

responses toward co-administered less immunogenic vaccines. Future studies will investigate the clinical relevance of the γ-FLUbased combined vaccination strategy, particularly in relation to intranasal and intramuscular routes of administration. Future studies will also examine the effect of co-administered vaccines on the ability of γ-FLU to induce cross-protective immunity.

#### **REFERENCES**

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

*Received: 02 April 2014; accepted: 24 May 2014; published online: 10 June 2014.*

*Citation: Babb R, Chan J, Khairat JE, Furuya Y and Alsharifi M (2014) Gamma-irradiated influenza A virus provides adjuvant activity to a coadministered poorly immunogenic SFV vaccine in mice. Front. Immunol. 5:267. doi: 10.3389/fimmu.2014.00267*

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

*Copyright © 2014 Babb, Chan, Khairat, Furuya and Alsharifi. 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.*

OPINION ARTICLE published: 23 July 2014 doi: 10.3389/fimmu.2014.00355

## Does type I interferon limit protective neutrophil responses during pulmonary Francisella tularensis infection?

#### **Yoichi Furuya, Donald Steiner and DennisW. Metzger \***

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA \*Correspondence<EMAIL_ADDRESS>
**Edited by:**

Amy Rasley, Lawrence Livermore National Laboratory, USA

**Reviewed by:**

David Weiss, Emory University, USA

**Keywords: Francisella tularensis, mucosal immunity, type I interferon, immune evasion, neutrophil**

#### **FRANCISELLA TULARENSIS**

Host–microorganism co-existence has enabled many pathogens to develop mechanisms to evade the immune system. One prime example is *Francisella tularensis.* This Gram-negative bacterium infects various wild animals such as rodents and rabbits, but also exists in water and soil. In rare cases, humans acquire *F. tularensis* infections through inhalation of particles from infected animals, drinking of contaminated water, ingestion of undercooked infected meat, or a bite from an infected tick or mosquito. Respiratory infection is the most deadly form of disease with a mortality rate as high as 50% if untreated. Due to its extreme virulence and ease of aerosol dissemination, it is classified as a Tier 1 bioterrorism agent by the Centers for Disease Control and Prevention. Given that inhalation of aerosolized *F. tularensis* would be the most likely route of transmission during an act of bioterrorism, recent research has shifted from intravenous/intradermal infection models to respiratory models of tularemia. Upon pulmonary infection, many cell types such as alveolar macrophages, neutrophils, and dendritic cells have been shown to harbor live *F. tularensis* (1, 2). Thus, *F. tularensis* is considered as an intracellular bacterium despite its ability to grow in a culture medium. Currently, whether phagocytes contribute to host defense or promote bacterial replication is an intense focus of biodefense research.

#### **TYPE I INTERFERON (IFN-I)-MEDIATED SUPPRESSION OF NEUTROPHIL RECRUITMENT**

*Francisella tularensis* possesses a remarkable ability to evade the host innate immune response. The first line of defense against most respiratory pathogens is the alveolar macrophage, the predominant cell type found in the airways of naïve hosts. A major role for alveolar macrophages is to efficiently eliminate invading pathogens through phagocytosis. However, *F. tularensis* has evolved a mechanism to escape macrophage killing and to replicate within phagocytes. This, together with the relatively inert properties of the *F. tularensis* LPS, renders alveolar macrophages unable to produce the cytokines and chemokines that are necessary to initiate effective immune responses during the early phases of *F. tularensis* infection (3). However, macrophage infection eventually does trigger a host immune response through bacterial recognition by cytosolic receptors, which in turn, results in type I IFN (IFN-I) production (4, 5). IFN-I signaling stimulates expression of "absent in melanoma 2" (AIM-2), a component of the inflammasome that is required for resistance against *F. tularensis* infection (5, 6). Indeed, mice deficient in AIM-2 are highly susceptible to intradermal or subcutaneous *F. tularensis* challenge (6, 7). However, a contradictory finding is that mice deficient in IFN-I receptors (IFN-IR−/−) exhibit substantially increased resistance to intranasal and intradermal *Francisella* infection (8, 9). This resistance suggests that the detrimental effects of IFN-I outweigh the benefits of IFN-I-mediated AIM-2 expression that is required for the inflammasome response. The detrimental effects of IFN-I were ascribed to its negative influence on expression of γδ T cells that produce IL-17 (8). Increased survival of IFN-IR−/− mice following subcutaneous tularemia was closely associated with an increased

IL-17 response, reduced bacterial burden, and increased influx of neutrophils into the spleen (8). Suppression of IL-17 responses and neutrophil recruitment by IFN-I has similarly been reported in other infectious disease models (10). These various findings have led to the hypothesis that IL-17 mediated neutrophil recruitment is protective against *F. tularensis* infection. Consistent with this report, IL-17A deficient mice were found to be more susceptible to intranasal *F. tularensis* infection (11–13). However, although a role for IL-17 in facilitating neutrophil responses is well established (14), during pneumonic tularemia, recruitment of CD11b+Ly6G/C+ cells was not affected by the absence of IL-17A (13). This suggests that IFN-I may limit neutrophil infiltration during pulmonary *F. tularensis* infection independently from its effects on IL-17 expression. It would be of considerable interest to assess production of neutrophil chemoattractants, such as CXCL1 and CXCL2 in the lungs of IFN-IR−/− mice following intranasal *F. tularensis* infection, to elucidate the mechanisms responsible for IFN-I-mediated suppression of neutrophil recruitment.

An alternative or complementary mechanism that may be responsible for decreased neutrophil recruitment to the site of infection by IFN-I involves host cell death. Immune cells such as lymphocytes, macrophages, and neutrophils can be sensitized by IFN-I toward cell death in a mouse model of *Francisella* and *Listeria monocytogenes* infection (5, 15–17). Consistent with the findings in mice, human neutrophils with upregulated interferon-stimulated gene expression exhibit enhanced cell death following *in vitro* exposure to *Staphylococcus aureus*

(15). This phenomenon, however, seems to be pathogen-specific since enhanced cell death was not observed after exposure to *Pseudomonas aeruginosa* (15). Indeed, spontaneous death of neutrophils in the absence of pathogen can be delayed by *in vitro* IFN-I treatment (1). Thus, the direct effect of IFN-I signaling on neutrophils could be context-dependent. In the case of *Francisella infection*, IFN-I signaling may promote neutrophil death and contribute to the overall decrease in neutrophil numbers at the site of infection.

#### **CONTROVERSIAL ROLE OF NEUTROPHILS DURING PULMONARY TULAREMIA**

Neutrophils make up the first wave of phagocytic cell migration into the lungs during most pulmonary bacterial infections, including respiratory tularemia (2). However, previous studies utilizing the neutrophil depleting antibody, anti-Gr-1, have failed to show an important role for neutrophils during pulmonary *F. tularensis* infection, as assessed by bacterial burden (18). This is in striking contrast to the known importance of neutrophils in host defense against systemic tularemia (18–20). Recently, it has been proposed that neutrophils may even promote immunopathology during pulmonary tularemia. Mice deficient in metalloproteinase-9 (MMP-9), a mediator of leukocyte migration, have reduced neutrophil recruitment into the lungs following intranasal *F. tularensis* infection (21). Surprisingly, these mice exhibit better survival compared to wild-type mice following pulmonary tularemia. The interpretation from this study was that the excessive neutrophil infiltration observed in wild-type mice was detrimental to the host, but the limited recruitment of neutrophils seen in MMP-9−/− mice was beneficial (21). We have recently found that near complete depletion of neutrophils using a neutrophil-specific mAb, anti-Ly6G, significantly reduced survival of intranasal *Francisella*-infected mice, an observation that is consistent with a protective role for low to moderate recruitment of neutrophils (unpublished observations). Overall, the accumulating evidence suggests that neutrophils do play a role during pneumonic tularemia, but it remains uncertain whether neutrophils

exert protective or harmful effects during pneumonic tularemia. It is important to note that some of the discrepancies in results may be due to the differences in experimental conditions such as the type of *in vivo* depleting antibody, timing of antibody administration, and/or mouse strains.

#### **IFN-IR**−**/**− **MICE AS A MODEL TO STUDY PROTECTIVE ROLE OF NEUTROPHILS DURING PULMONARY TULAREMIA**

We propose that IFN-IR−*/*− mice can serve as a valuable tool to better understand the role of IFN-I and neutrophils in defense of mucosal tissues against pulmonary tularemia. To the best of our knowledge, only a few studies have been performed using IFN-IR−/− mice in the pulmonary tularemia model. It would be of considerable interest to determine whether the absence of IFN-I signaling alters the kinetics of pulmonary neutrophil recruitment during respiratory tularemia. If so, an important question to address is whether *in vivo* depletion of neutrophils during the early versus late phases of pulmonary tularemia in IFN-IR−/− mice results in a differential survival outcome. Perhaps in the absence of IFN-I signaling, there is an early influx of neutrophils into the lungs, which promotes early bacterial clearance, but later recruitment is not affected or reduced. This scenario would be consistent with the concept that neutrophils are detrimental if pulmonary infiltration is excessive (21), but their complete absence results in reduced survival. A fine balance may exist between the protective role of neutrophils during the early phase of bacterial infection versus excessive neutrophil recruitment during the recovery phase, which impedes resolution of pulmonary inflammation (depicted in **Figure 1**). An

**FIGURE 1 | A model depicting the proposed roles of IFN-I and neutrophils during F. tularensis infection is shown.** Pulmonary infection with F. tularensis triggers IFN-I production from innate immune cells such as alveolar macrophages. IFN-I may sensitize F. tularensis-infected neutrophils for cell death and contribute to an overall reduction in neutrophil numbers at the site of infection. IFN-I may also directly limit neutrophil recruitment via inhibition of IL-17 producing γδT cells or suppression of other neutrophil chemoattractants. Suppression of neutrophil recruitment may prevent tissue damage associated with excessive infiltration of neutrophils as well as lead to inadequate bacterial clearance, resulting in an unfavorable outcome. An optimal neutrophil response may require moderate recruitment at an early time point to aid in prompt clearance of bacteria without causing significant inflammation later during pulmonary tularemia.

increased understanding of neutrophilmediated mucosal immunity and the role IFN-I signaling in regulating recruitment of these cells to the lung may ultimately facilitate the development of novel therapeutics for protection against tularemia.

#### **ACKNOWLEDGMENTS**

This work was supported by an American Lung Association Senior Research Training Fellowship RT-226959-N to Yoichi Furuya and by NIH grant PO1 AI056320.

#### **REFERENCES**

critical for innate immunity to *Francisella tularensis*. *Nat Immunol* (2010) **11**(5):385–93. doi:10. 1038/ni.1859

**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.

*Received: 16 May 2014; paper pending published: 07 June 2014; accepted: 09 July 2014; published online: 23 July 2014.*

*Citation: Furuya Y, Steiner D and Metzger DW (2014) Does type I interferon limit protective neutrophil responses during pulmonary Francisella tularensis infection? Front. Immunol. 5:355. doi: 10.3389/fimmu.2014.00355*

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

*Copyright © 2014 Furuya, Steiner and Metzger. 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.*

### Unraveling the convoluted biological roles of type I interferons in infection and immunity: a way forward for therapeutics and vaccine design

#### **Danushka KumaraWijesundara1,2\* † ,Yang Xi 3† and Charani Ranasinghe2**

1 Virology Laboratory, Department of Surgery, Basil Hetzel Institute, University of Adelaide, Adelaide, SA, Australia

2 Molecular Mucosal Vaccine Immunology Group, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia

3 Lung and Allergy Research Centre, Translational Research Institute, UQ School of Medicine, The University of Queensland, Woolloongabba, QLD, Australia

#### **Edited by:**

Christine Anne Biron, Brown University, USA

#### **Reviewed by:**

Janis J. Weis, University of Utah, USA Jieliang Li, Temple University, USA

#### **\*Correspondence:**

Danushka Kumara Wijesundara, Virology Laboratory, Department of Surgery, Basil Hetzel Institute, 37a Woodville Road, Woodville, SA 5011, Australia e-mail: danushka.wijesundara@ adelaide.edu.au

†Danushka Kumara Wijesundara and Yang Xi have contributed equally to this work.

It has been well-established that type I interferons (IFN-Is) have pleiotropic effects and play an early central role in the control of many acute viral infections. However, their pleiotropic effects are not always beneficial to the host and in fact several reports suggest that the induction of IFN-Is exacerbate disease outcomes against some bacterial and chronic viral infections. In this brief review, we probe into this mystery and try to develop answers based on past and recent studies evaluating the roles of IFN-Is in infection and immunity as this is vital for developing effective IFN-Is based therapeutics and vaccines. We also discuss the biological roles of an emerging IFN-I, namely IFN-ε, and discuss its potential use as a mucosal therapeutic and/or vaccine adjuvant. Overall, we anticipate the discussions generated in this review will provide new insights for better exploiting the biological functions of IFN-Is in developing efficacious therapeutics and vaccines in the future.

**Keywords: type I interferons, human immunodeficiency virus, IFN-**ε**, vaccine adjuvants, interferon immunity**

### **INTRODUCTION**

Since the initial discovery of type I interferons (IFN-Is) as antiviral agents (1), these cytokines have been extensively studied for their anti-microbial and immune regulatory properties. IFN-I family comprises 13 IFN-α subunits, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-τ, and IFN-δ (in mice only) (2–8). All IFN-Is signal through the IFN-α receptor (IFN-AR) complex to induce synthesis and secretion of IFN-inducible genes or effector proteins with antiviral, pro-apoptotic, and ubiquitination-modifying properties (9– 11). The signaling pathways that IFN-Is utilize to exert various biological effects have been comprehensively reviewed elsewhere and will not be reviewed here [see Ref. (12)]. Numerous cell types produce IFN-Is (e.g., macrophages, myeloid dendritic cells (DCs), fibroblasts, and epithelial cells), but plasmacytoid DCs (pDCs) appear to be the most prolific producers of IFN-Is (13, 14). The production of these cytokines tends to be beneficial to the host particularly against acute viral infections, but there are considerable evidences to suggest that IFN-Is play detrimental roles in autoimmune diseases (15), bacterial and persistent viral infections. Herein, we review how IFN-Is could play beneficial or detrimental roles in pathogen control predominantly with respect to viral infections and discuss how they could be used as therapeutics and vaccine adjuvants. Furthermore, the importance of considering the emerging IFN-εin immunity and vaccine development will be discussed.

#### **THE BENEFITS AND DETRIMENTS OF IFN-Is IN THE CONTROL OF PATHOGENS**

The importance of IFN-Is in protecting hosts against pathogens has been demonstrated in several contexts. Firstly, IFN-AR deficient mice tend to be more susceptible to infection with viruses (particularly acute viral infections) compared to wildtype mice. Some examples include Henipavirus (16), acute Friend virus (17), encephalitic flavivirus (18), lymphocytic choriomeningitis virus (LCMV) Armstrong (19), Hazara virus (20), Dengue virus (21), Respiratory Syncytial Virus (22), and numerous other viral infections (23). Secondly, systemic exhaustion of IFN-Is following a primary viral infection has been shown to increase the host susceptibility to secondary unrelated viral infections in mice (24). Thirdly, therapeutic administration of IFN-Is can reduce viral loads in individuals infected with chronic viruses and promote cancer regression (see below Section "The Use of IFN-Is as Therapeutics and Adjuvants"). Finally, pathogens can attenuate IFN-I responses to promote immune evasion. For instance, human immunodeficiency virus (HIV)-1 can reduce the capacity of IFN producing cells to produce IFN-Is (25–27), induce cytopathic effects on these cells (28–32), and/or block IFN-I mediated intracellular signaling events (33) to help establish a chronic phase infection. Similarly, cancer immune evasion and development could also involve attenuation of IFN-I responses. In agreement with this,Critchley-Thorne et al. (34) have shown that various cancer patients have significantly attenuated expression of interferon stimulate genes in lymphocytes compared to healthy controls.

The benefits of IFN-Is in conferring protection against microbes have been mostly demonstrated using acute viral infection models, but several studies suggest that IFN-Is can also assist in the control of bacterial infections. This was first demonstrated *in vitro* where De la Maza and colleagues (35) showed that IFN-I inhibit *Chlamydia trachomatis* infectivity of human and mouse cell lines. Several subsequent studies have shown that IFN-I could indeed play important roles for inhibiting various stages of bacterial infections. Some examples include replication of *Chlamydophila pneumoniae* (36), recruitment of *Myobacterium tuberculosis* target cells into the lung during early infection (37), and invasion and transmigration of *Streptococcus pneumoniae* in the lungs (38). However, IFN-Is do not always appear to render beneficial outcomes in anti-bacterial immunity. Several studies have reported that IFN-AR deficient mice are better protected than WT controls following bacterial infections such as *Ehrlichia muris* (39), *Chlamydia muridarum* (40), *Listeria monocytogenes* (41, 42), *Myobacterium* species (43, 44), and *Francisella tularensis* (45). Furthermore, induction of IFN-Is following virus infections could make hosts more susceptible to secondary bacterial infections (46–48). The mechanisms as to how IFN-Is exacerbate or make hosts more susceptible to bacterial disease may vary depending on the infection. For instance, IFN-I mediated disease exacerbation has been linked to reduction of interleukin (IL)-17 expressing γδ T cells, increased expression of IL-10 or reduction in cell-mediate immune responses following *F. tularensis*, *M. Leprae*, or *L. monocytogenes*, respectively (42, 44, 45).

Several reports suggest that the detrimental effects of IFN-Is could also support the establishment of persistent viral infections depending on the quantities and duration of IFN-I induction. IFN-Is have been shown to play significant roles in inhibiting various stages (e.g., replication, virus assembly, protein trafficking, and transcription) of HIV-1 life cycle (49–53). However, sustained unlike transient production of IFN-Is resulting from chronic stimulation of pDCs has been proposed to facilitate HIV-1 persistence (54). Similarly following clone 13 LCMV infection transient (within 24 h) hyper-induction of IFN-α and -β has been reported to exacerbate virus pathogenesis and promote viral persistence (19). However, in the same study IFN-Is were crucial for the control of acute Armstrong LCMV infection, which was likely due to lower IFN-I induction following Armstrong compared to clone 13 LCMV infection. In chronic simian immunodeficiency virus (SIV) infection studies, disease free phenotypes of sooty mangabeys have been associated with the abolishment of interferon stimulated gene expression during chronic, but not in acute phase infection (55). Overall, it can be speculated that early, transient yet non-excessive induction of IFN-Is (at least α and β species) are important in the control of acute viral infections. On the contrary, chronic and/or hyper-induction of IFN-Is could provide an environment for enhanced persistence and/or pathogenesis of chronic viral infections.

#### **IFN-Is AND REGULATION OF ADAPTIVE IMMUNITY**

Apart from their most celebrated role as direct anti-viral agents, IFN-Is have also been increasingly recognized as potent regulators of cellular immune responses. Of particular interest to vaccine development has been the ability of these cytokines to regulate adaptive immune responses and this aspect is discussed here.

Dendritic cells are often crucial for initiating adaptive immune responses and serve as important targets for IFN-Is to regulate adaptive immunity. Exposure of IFN-Is facilitates maturation of DCs via increasing the expression of DC-associated chemokine receptors, co-stimulatory molecules, and major histocompatibility complex class I and class II antigen presentation (56–60). Consequently, DCs that mature following IFN-I exposure can effectively prime protective T cell responses (61). A caveat here is that IFN-I responses could operate in a threshold dependent manner where excessive responsiveness is inhibitory to the ability of DCs to prime T cell responses. For instance, following LCMV infection higher induction of IFN-Is has been associated with heightened expression of programed death-ligand 1 (PD-L1) on DCs and PD-L1 interaction with programed death 1 (PD-1) on T cells can inhibit T cell activation (19, 62).

IFN-Is could also act directly on lymphocytes to alter adaptive immune outcomes. Naïve B cells up-regulate the expression of activation markers CD69, CD86, and CD25 following IFN-I exposure *in vitro* (63), but *in vivo* IFN-Is only up-regulate CD69 and CD86 expression on naïve B and T cells (64). The consequences of up-regulating these activation markers are not clear, but *in vitro* studies suggest it could serve to reduce the activation thresholds of naïve B cells unlike T cells (63, 65). Alternatively, CD69 expression resulting from IFN-I exposure can down-regulate sphigosine-1 phosphate receptor-1 on naïve lymphocytes to retain these cells in secondary lymphoid organs (66). This retention mechanism could facilitate a more durable interaction between naïve lymphocytes and DCs for efficient lymphocyte activation to occur. IFN-Is have been reported to represent a distinct third signal for naïve T cell activation to occur and prevent the expansion of regulatory T cells that can inhibit T cell activation (67–69). Furthermore, IFN-Is regulate the functions of lymphocytes even after naïve lymphocyte activation or effector/memory differentiation. Some examples of this include IFN-I mediated enhancement in cell division (63, 70), survival (71, 72), interferon-γ secretion (73), cytotoxicity (74), germinal center formation, and antibody isotype switching (75).

Despite the many studies demonstrating that IFN-Is are capable of boosting adaptive immunity; there have also been several studies in bacterial and chronic viral infection settings suggesting that IFN-I signaling leads to IL-10 production (19, 44, 76, 77). IL-10 is thought to be detrimental to the clearance of these pathogens as has been demonstrated with HIV-1 (78). It is likely that IFN-Is up-regulate PD-1 expression (e.g., on regulatory T cells) and PD-L1 (e.g., on DCs) on cells resulting in a milieu where PD-1/PD-L1 interactions occur; this could facilitate IL-10 production and exhaustion of T cell function during chronic viral infections (19, 76–80). A caveat here is that IFN-Is in some instances can also inhibit IL-10 production and IL-10 production can occur independently of IFN-I signaling (76, 81). Furthermore, IFN-Is up-regulate pro-apoptotic molecules such as Bak on T cells to induce apoptosis independently of T cell exhaustion (82).

Overall, IFN-Is play pivotal roles in boosting adaptive immunity, but the switch from becoming a booster to an inhibitor of adaptive immunity may reflect on how much apoptosis, PD-1/PD-L1 interactions and IL-10 signaling are induced on immune cells due to IFN-Is.

#### **THE USE OF IFN-Is AS THERAPEUTICS AND ADJUVANTS**

The development of efficient methods to purify IFN-I and subsequent high yield purification of IFN-α2 during the late 1970s paved way for the first IFN-I based human clinical trial in 1986 where IFN-α2 was used for treating hairy cell leukemia (83, 84). Since then the therapeutic use of IFN-Is have shown promising outcomes for treatment of several cancers and viral infections. Therapeutic administration of pegylated IFN-α2 have rendered potent anti-viral and immune enhancing effects against hepatitis B virus infection (85, 86). A recent clinical trial has shown that similar outcomes could be achieved even when pegylated IFN-α2 is administered to HIV-infected patients (87). Systemic administration of IFN-α and/or IFN-β has also been reported to reduce viral growth and clinical manifestations of herpes zoster, herpes simplex virus, and cytomegalovirus (CMV) infections (88–91). Furthermore, systemic or intralesional administration of IFN-α and/or IFN-β has been shown to induce a regression of skin-associated wart infections following papilloma virus infections (92–98). IFN-Is have also been used in synergic regimens where administration of IFN-α2 or -β2 and anti-viral drugs (e.g., ribavirin and faldaprevir) could effectively reduce viral loads of certain hepatitis C virus (HCV) genotypes and is currently the best treatment for HCVinfected patients (99–102). A caveat here is that these regimens have also been reported to cause adverse side-effects (103). Apart from treatment of pathogen infections, IFN-Is especially IFN-α2, have also been used for treatment and regression of various cancers (e.g., leukemia, prostrate cancer, and cervical intraepithelial neoplasia) (104–106).

Studies in pre-clinical models suggest that IFN-Is could also be potent vaccine adjuvants for inducing adaptive immune responses. Some examples include when an influenza vaccine adjuvanted with IFN-α/β administered mucosally induced significantly higher IgG2a and IgA antibody responses and protection compared to non-adjuvanted vaccines (107, 108). Interestingly, the species of IFN-Is used as immune adjuvants could have different immune outcomes in terms of enhancing adaptive immunity. Studies in our laboratory suggest that recombinant pox viral vectors encoding IFN-β compared to those encoding IFN-α4 or IFN-ε significantly enhanced systemic T cell immunity against co-encoded antigens in prime-boost vaccination settings (109). However, Xi et al. (110) using similar prime-boost vaccination settings demonstrated that the use of IFN-ε was much more efficient in inducing T cell immunity in mucosal compartments (e.g., lung and gut) compared to IFN-α4 and IFN-β when used as vaccine adjuvants. Another important consideration here is that the vaccine vectors (i.e., pox viruses) used in our studies are acute attenuated viruses and do not chronically induce IFN-Is as is usually the case with persistent virus infections.

There are several confounding factors that could dictate the use of IFN-I in therapy and as vaccine adjuvants. Firstly, unique biological effects have been reported with different members of the IFN-I family and subtypes of IFN-α. Thus, the choice of IFN-I species (e.g., IFN-α2 or IFN-β) could dictate the success of IFN-I treatment or IFN-I based vaccine formulations. Secondly, members of the IFN-I family have different binding affinities and kinetics to the IFN-AR subunits with current comparative studies suggesting that IFN-β has the highest affinity to IFN-AR and antiviral capacity (111–113). A caveat with these studies is that not all members of the IFN-I family were compared. Thirdly, IFN-Is can cause numerous adverse side-effects and induce autoimmunity (e.g., lupus, thyroiditis, diabetes, dermatitis, Sjogren's syndrome, and arthritis) especially in patients with a history of autoimmune manifestations (114). The autoimmune outcomes in these settings

are thought to be a combination of tolerogenic immune function failures and IFN-I mediated maturation of DCs that present autoantigens to activate autoreactive T cells and B cells that make autoantibodies (115).

Collectively, IFN-Is have shown considerable promise for the treatment of cancers and pathogen infections (e.g., chronic viruses) in some clinical settings. IFN-Is are also promising for use as vaccine adjuvants, but the species of IFN-Is used for this purpose could have a significant bearing on adaptive immunity generated at certain immune compartments. For instance, IFN-β could be used to effectively enhance systemic T cell immune responses, whereas IFN-ε is more promising as an adjuvant to enhance mucosal T cell immunity in the lung and the gut mucosae.

#### **IMPORTANCE OF IFN-**ε **IN IMMUNITY AND VACCINE DEVELOPMENT**

Most studies investigating the roles of IFN-Is have done so mainly analyzing the roles of IFN-α and -β. However, investigating the roles of other IFN-I family members is beneficial for effective therapeutic and vaccine development strategies especially given that higher induction of IFN-α and -β could be detrimental to the host as discussed previously. For this purpose, it is indeed intriguing to evaluate the roles of IFN-ε, which unlike other IFN-Is is constitutively expressed and plays various protective roles in reproductive tissues, gut, lung, and the brain (**Table 1**). Since our initial studies characterizing the roles of IFN-ε in inducing antiviral states on cells (109), we have found that this cytokine also possesses potent immune regulatory capacity. Our recent studies indicated that, intranasal immunization of mice with vaccinia virus (VV) encoding murine IFN-ε(VV-HIV-IFN-ε) unlike IFN-α (VV-HIV-IFN-α4) or IFN-β (VV-HIV-IFN-β) could induce rapid clearance of VV in the lung (110). Viral clearance in this instance correlated with several immune outcomes: (i) elevated lung VVspecific CD8+CD107a+IFN-γ + cell population expressing activation markers CD69/CD103,(ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) highly functional CD8+CD4+ double positive T cell subset [CD3highC–C chemokine receptor (CCR)7highCD62Llow] in lung lymph nodes (110). Next when IFN-ε was used in an intranasal/intramuscular

**Table 1 | Site-specific effects of IFN-**ε.

heterologous HIV-1 prime-boost vaccination regimen, elevated HIV-specific effector, but not memory CD8+ T cells responses were detected in spleen, genito-rectal nodes, and Peyer's patches. Furthermore, homing marker α4β7 and CCR9 analysis showed that unlike other IFN-Is, IFN-ε promoted the migration of antigen-specific CD8+ T cells to the gut mucosae (110). These results for the first time established that unlike other IFN-Is, IFNε played a unique role at the mucosae. Another recent study has also further substantiated our findings demonstrating that IFN-ε deficient mice were more susceptible to intra-vaginal herpes simplex virus 2 and *Chlamydia muridarum* infections compared to wild-type mice (117). This suggests that IFN-ε could also be beneficial for the control of certain bacterial infections. A caveat here is that it is unknown whether IFN-ε could cause adverse side-effects in humans as it has not yet been used for treatment or vaccination purposes in humans.

Overall, IFN-ε has great potential to be used as a topical microbicide or a therapeutic to control local lung/gut infections or modulate tissue-specific immunity at sites where pathogens are initially encountered (i.e., mucosal surfaces). Specifically, IFN-ε's ability to enhance CD8+ T cell homing to the gut [gut is the primary site of HIV virus replication and CD4+ T-cell depletion (119)] and also its ability to control infections at the lung mucosae suggest that administration of pegylated forms of IFN-ε or vaccines encoding IFN-ε could be effective for controlling mucosal pathogens such as HIV-1.

#### **CONCLUDING REMARKS**

The dual roles of IFN-Is in providing beneficial and detrimental effects to the host in pathogen control is intriguing for developing IFN-I based vaccines and therapies. Lessons learned from acute viral infection models and studies comparing acute versus chronic infection states suggest that transient, but not sustained and/or excessive induction of IFN-Is is likely to confer protective outcomes. IFN-Is have also proven to be promising therapeutic agents against various pathogens and cancers and could also be used as vaccine adjuvants. The caveat here is that the vaccine vector used should ideally not chronically stimulate the production of IFN-Is, which is expected to be detrimental for the generation of robust adaptive immune responses. Our laboratory and others have demonstrated that IFN-ε has great potential to provide protective outcomes against not only mucosal viral infections, but also certain mucosal bacterial infections. Keeping this in mind, more studies need to evaluate the contribution of the different species of IFN-Is not just IFN-α and -β in immunity against infections. These studies are expected to pave way for the development of novel and effective IFN-I based vaccines/therapies against chronic pathogens and cancers.

#### **ACKNOWLEDGMENTS**

This work was supported by Australian National Health and Medical Research Council project grant award 525431 (Charani Ranasinghe) and ACH2 EOI grants (Charani Ranasinghe).

#### **REFERENCES**

1. Isaacs A, Lindenmann J. Virus interference. I. The interferon. *Proc R Soc Lond B Biol Sci* (1957) **147**(927):258–67. doi:10.1098/rspb.1957.0049

chronic hepatitis B. *N Engl J Med* (2005) **352**(26):2682–95. doi:10.1056/ NEJMoa043470

**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.

*Received: 30 May 2014; accepted: 13 August 2014; published online: 29 August 2014. Citation: Wijesundara DK, Xi Y and Ranasinghe C (2014) Unraveling the convoluted biological roles of type I interferons in infection and immunity: a way forward for therapeutics and vaccine design. Front. Immunol. 5:412. doi: 10.3389/fimmu.2014.00412 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Wijesundara, Xi and Ranasinghe. 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.*

## The curious case of type I IFN and MxA: tipping the immune balance in AIDS

#### **Andrea Kinga Marias Furuya, Hamayun J. Sharifi and Carlos M. C. de Noronha\***

Albany Medical Center, Center for Immunology and Microbial Disease, Albany, NY, USA \*Correspondence<EMAIL_ADDRESS>
#### **Edited by:**

Blossom Damania, University of North Carolina at Chapel Hill, USA

#### **Reviewed by:**

Dietmar Fuchs, Innsbruck Medical University, Austria Lishan Su, The University of North Carolina at Chapel Hill, USA

#### **Keywords: HIV, SIV, IFN, MxA**

Human immunodeficiency virus (HIV) remains a significant public health challenge. According to the World Health Organization, there were approximately 35.3 million people living with HIV/AIDS worldwide in 2012, with Sub-Saharan Africa being the most affected region (1). HIV has claimed 27 million lives. It is estimated that two million people die from HIV/AIDS each year.

Highly active antiretroviral therapies (HAART) now allow HIV-infected individuals to live longer and healthier lives. HAART is, however, burdened with side effects and high costs, and threatened by evolving viral resistance. A hallmark of both HIV and simian immunodeficiency virus (SIV) infection is chronic immune activation together with uncontrollable viremia and lymphocyte apoptosis that progresses to acquired immunodeficiency syndrome (AIDS) (2, 3). The deleterious role for immune activation in HIV infection is supported by animal studies. Although the exact mechanism underlying AIDS resistance of natural hosts for SIV, like sooty mangabey (SM) monkeys, is unknown, reports support the idea that disease progression in non-natural hosts such as rhesus macaques (RM) is mainly due to immune system dysfunction triggered by HIV/SIV infections (4, 5). In non-natural hosts, SIV causes progressive impairment of the immune system characterized by high viremia, CD4+ T cell depletion, and loss of T cell function. Continuous CD4+ T cell loss eventually leads to AIDS as the regenerative capacity of the immune system gradually decreases despite the excess of homeostatic cytokines (6). This exhaustion and increased T cell

apoptosis, a phenomenon seen in pathogenic infections, may be due to direct and indirect killing by the virus. Indirector bystander-killing, is the loss of uninfected and abortively infected T cells possibly due to generalized immune activation (7, 8). It is widely accepted that type I interferon-producing (IFN I) plasmacytoid dendritic cells (pDC) play a central role in this generalized immune activation (9–11). On the other hand, natural simian hosts preserve T lymphocyte populations despite high viremia and show attenuated immune activation that favors the maintenance of CD4+ T cells; hence, SMs do not develop AIDS (7). Although HIV preferentially infects CD4+ T cells and macrophages, efficient binding and infection of pDCs by HIV have been demonstrated and may contribute to AIDS pathogenesis (12, 13).

Plasmacytoid dendritic cells are specialized cells found in blood and lymphoid tissues. The main function of pDCs is to produce IFN in response to bacterial and viral DNA. Following activation, these produce 1000-fold more IFN I than other IFN producing cells (11). Upon HIV infection, pCDs become activated and express CCR7 and CXCR3, migration markers that induce redistribution to the lymph nodes (LN) (14–16). Further, recent reports suggest that HIV-induced pathogenesis occurs mainly in LNs where the IFN I that pDCs produce elevates serum levels during both acute and chronic infections (13, 17–19).

Type I IFNs are powerful cytokines and adaptive immune system modulators. They are produced upon viral infection, replication, and/or the introduction of doublestranded RNA (20). IFNs trigger antiviral activity and induce the maturation of effector T cells. Therefore, an interesting immunoregulatory role for type I IFN is in lymphocyte activation during viral infection (21). There is, however, evidence that this otherwise beneficial interferon can become detrimental to the host during chronic HIV infection. Persistent levels of IFN I induce apoptosis in both HIV-infected CD4+ T cells and in those that do not become productively infected (bystander-killing) leading to accelerated depletion of CD4+ T cells during pathogenic infection (22). This effect is due to type I IFN triggered apoptosis of uninfected CD4+ T cells via TNF-related apoptosis inducing ligand (TRAIL) (17). Originally, SM, which are natural SIV hosts, were thought to have reduced immune system activation during both acute and chronic SIV infections. Moreover, SM pDCs produce less IFN I *ex vivo* in response to SIV, which leads to less immune activation during chronic infection (23). Other works, however, have shown that natural hosts exhibit an initially strong, but rapidly controlled, IFN I response (24–26). Therefore, both natural and non-natural hosts mount strong type I IFN responses during the acute stage of infection but only natural hosts suppress the response by the chronic stage, 4–16 months after infection. This downregulation occurs despite sustained high levels of viremia. Unlike natural hosts, non-natural hosts maintain high levels of IFN I production at all times. It is important to note that cells from SIV-infected natural hosts can be repeatedly stimulated *in vitro* to produce type I IFN and to upregulate interferon-stimulated genes (ISG) during the chronic stage of SIV infections suggesting that these cells are neither more

refractory nor resistant to re-stimulation (26). Therefore, the downmodulation of IFN I in natural hosts is likely due to negative control mechanisms. The exact mechanism underlying the downregulation of IFN I is, however, not fully understood. The regulatory complexity of the IFN pathway and its overall effects on target cells make pinpointing a resolution mechanism challenging. A role for immunomodulatory proteins, negative regulation of IFN responses, and other mechanisms have been suggested [reviewed in Ref. (2)]. Efforts have been made to discover the IFN I downregulation pathway by examining the regulation of ISGs as well as immunosuppressive genes (24, 26). One common denominator is the myxovirus resistance protein (MxA or Mx1) gene. This finding was supported by a recent clinical study in which Chang et al. found that persistent higher expression of type I IFN and ISGs, including MxA, may explain, at least in part, the increased immune activation and more rapid disease progression in females with chronic HIV infections when compared to males with similar viral loads (27). Harris et al. convincingly showed that natural hosts African green monkeys (AGM) and SM begin to downregulate MxA responses by 28 dpi whereas nonnatural hosts (RM) maintained high levels of MxA in LN. This work, however, did not reveal a mechanism of IFN I resolution (24). Further, it is unclear whether IFN is directly downregulated or whether downstream signaling negatively regulates IFN. Understanding the mechanism of Mx regulation in the transition phase, from acute to chronic infection of natural hosts, should reveal new targets for therapies to block the chronic immune system activation associated with disease progression.

MxA is an IFN induced protein expressed in cells like macrophages and hepatocytes. It is best known for its antiviral activity against orthomyxoviruses (28). MxA was first described in 1962 when Lindenmann showed that A2G inbred mice were resistant to doses of mouse-adapted influenza virus that were lethal to other inbred mice. This resistance was dependent on a single dominant locus named Mx1 and was exquisitely specific for orthomyxoviruses (29). Later, it was discovered that Mx1 was the first member of a small gene family and that the spectrum of antiviral activity is in fact much larger than originally thought. Most species have one to three Mx protein isoforms with different antiviral activity depending on their intracellular localization (28). Mouse Mx1 protein is found primarily in the nucleus whereas human MxA is cytoplasmic. Hence, each protein blocks influenza virus at a different stage of the viral replication cycle (30). Of note, Mx2, an interferon-induced protein, has recently been shown to inhibit HIV infection after entry (31, 32). Mx2, however, differs from Mx1 in that Mx2 localizes to the nucleus and its antiviral activity relies on a nuclear localization signal (32).

What are Mx proteins and how do they exert their antiviral activity? Most importantly, how are Mx proteins involved in SIV/HIV infections? Mx proteins belong to the superfamily of GTPases, which includes dynamins, dynamin-like proteins, and mitofusins [described in more detail in Ref. (33)]. These proteins are involved in endocytosis, intracellular vesicle transport, and mitochondria distribution. Mx proteins are mainly characterized by three conserved domains: an N-terminal GTPase domain (GTP binding), a middle domain responsible for interaction with the GTPase effector domain (GED), and the Cterminus GED domain, which recognizes the virus. Two amphipatic α-helices form leucine zippers in the C-terminus. Furthermore, Mx proteins can self-assemble into higher order ring-like structures to form a helical stack (28). The higher order structures may represent a storage form whereas the monomers are likely the active form of MxA (34). Human *MxA* can be induced by IFN or directly by the virus through different pathways. Activation of Mx by IFN involves the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and the formation of an IFNstimulated gene factor 3 (ISGF3) multimeric complex, which in turn migrates into the nucleus and binds an IFN-stimulated response element (ISRE) upstream of the Mx gene (35, 36). It is not yet clear how viral infection activates Mx but it seems to be independent of ISGF3, involving STAT1 instead and possibly the IFN-regulatory factors 1 and 3 (IRF1 and IRF3) (37–39). Mx gene activation is fast. Its protein product is detectable within 4 h (40). It is thus likely that Mx gene activation in response

to both virus and IFN through two different pathways is evolutionarily advantageous to the host. Indeed HIV can induce MxA transcription and activate ISGs independently of IFN (41). As mentioned previously, in contrast to non-natural hosts, natural SIV hosts downregulate MxA responses during the transition to chronic infection despite high viremia (24). Moreover with every upregulation/activation, there must be a downregulation of the response in order to maintain homeostasis. Indeed overexpression of MxA is a common pathogenic link in Fanconi Anemia (FA), which consists of a group of at least five autosomal recessive disorders. Overexpression of MxA can lead to cancer susceptibility, apoptosis, bone marrow failure, and abnormal instability in cells (42). One potential mechanism for regulation of Mx gene expression relies on the IRF-1 and IRF-2 proteins. The most probable mediator of Mx induction is IRF-1, which is increased in the presence of virus and after IFN treatment (43, 44). This suggests that the functional synergy between the two independent pathways converges in upregulation of Mx. Additionally, the virus may synergize with IFN to increase Mx levels. IRF-1 induces IRF-2 production to repress ISG transcription. This in turn inhibits IRF-1 function. IRF-1 and IRF-2 share homology in their DNA binding domain; however, IRF-2 has higher affinity for binding and a longer half-life (45). Interestingly, IRF-2 also protects quiescent hematopoietic stem cells (HSC) from type I IFN exhaustion (46). In order to avoid cancer susceptibility, increased apoptosis, bone marrow failure, and HSC depletion, natural SIV hosts apparently evolved to control the IFN responses (**Figure 1**); however, the exact mechanism is not yet clear. Studying variations in the Mx genes, simultaneously with standardized screening of their antiviral properties in natural and non-natural SIV hosts, could explain why natural hosts can downregulate MxA responses with the onset of the chronic stage of infection. Additionally, mining for polymorphisms in regulatory genes, such as IRF-2, which correlate with enhanced DNA binding or half-life of the activated protein may offer an avenue for designing new therapeutics for controlling the immune system hyperactivation that is associated with AIDS progression.

#### **CONCLUSION**

Studies in non-human primates have revealed the importance of immune activation in HIV/SIV pathogenesis. Chronic immune activation, associated with CD4+ T cell depletion and loss of T cell function, is likely due to increased serum levels of type I IFN. Although type I IFN is necessary to control viral infections, its detrimental effects during the chronic stage of HIV/SIV infection has been well documented. Natural SIV hosts, in sharp contrast to nonnatural SIV hosts, control IFN responses with active regulatory mechanisms. Currently, it is not clear how this regulation is achieved on the molecular level. Understanding the basis of active IFN response repression is crucial as these may play an important role in protection against disease progression. Regulation of the type I IFN pathway is very complex making it hard to narrow down the exact factor or factors responsible for repression of this pathway. Evidence supporting a role for MxA genes, warrants studying polymorphisms in MxA genes, which may shed light on the mechanism of active IFN downregulation. Additionally, better understanding of how Mx genes are regulated will further broaden our perspective for understanding the evolution of host–virus interactions.

#### **REFERENCES**

immunodeficiency virus-infected monocytes. *J Leukoc Biol* (1994) **55**(3):299–309.

**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.

*Received: 30 May 2014; accepted: 18 August 2014; published online: 02 September 2014.*

*Citation: Furuya AKM, Sharifi HJ and de Noronha CMC (2014) The curious case of type I IFN and MxA: tipping the immune balance in AIDS. Front. Immunol. 5:419. doi: 10.3389/fimmu.2014.00419*

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

*Copyright © 2014 Furuya, Sharifi and de Noronha. 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: taming of myeloid cells and possible implications for autoimmunity

### **Emily M. Eshleman1 and Laurel L. Lenz 1,2\***

1 Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, USA

2 Department of Biomedical Research, National Jewish Health, Denver, CO, USA

#### **Edited by:**

Yoichi Furuya, Albany Medical College, USA

#### **Reviewed by:**

Birgit Strobl, University of Veterinary Medicine Vienna, Austria Yubin Zhang, Fudan University, China

#### **\*Correspondence:**

Laurel L. Lenz, Department of Immunology and Microbiology, University of Colorado School of Medicine, Mail Stop 8333, Room P18-9130, 12800 E. 19th Avenue, Aurora, CO 80045-2537, USA e-mail<EMAIL_ADDRESS>
Type I interferons (IFNs) were first described for their ability to protect the host from viral infections and may also have beneficial effects under specific conditions within some bacterial infections.Yet, these pleiotropic cytokines are now known to exacerbate infections by numerous life-threatening bacteria, including the intracellular pathogens Listeria monocytogenes and Mycobacterium tuberculosis. The evidence that such detrimental effects occur during bacterial infections in both animals and humans argues for selective pressure. In this review, we summarize the evidence demonstrating a pro-bacterial role for type I IFNs and discuss possible mechanisms that have been proposed to explain such effects.The theme emerges that type I IFNs act to suppress myeloid cell immune responses. The evolutionary conservation of such anti-inflammatory effects, particularly in the context of infections, suggests they may be important for limiting chronic inflammation. Given the effectiveness of type I IFNs in treatment of certain autoimmune diseases, their production may also act to raise the threshold for activation of immune responses to self-antigens.

**Keywords: interferons, interferon receptors, bacterial pathogens, macrophage activation, immune suppression, autoimmunity**

#### **INTRODUCTION**

Type I interferons (IFNs) are a class of cytokines that includes numerous IFNα subtypes, IFNβ, IFNδ, IFNε, IFNκ, IFNτ, and IFNω (1, 2). These secreted factors are predominantly produced by innate immune and non-immune cells of humans and other animals in response to recognition of conserved microbial products, rather than specific antigens. The different type I IFNs vary in their sequences but bind and signal using a common, ubiquitously expressed, heteromeric cell surface receptor (IFNAR) comprised of IFNAR1 and IFNAR2 chains. Ligation of IFNAR in diverse cell types activates a canonical JAK/STAT signaling cascade primarily involving JAK1, Tyk2, STAT1, and STAT2 proteins. Activation of these factors leads to induced transcription of numerous type I IFN-stimulated genes (ISGs), the protein products of which largely act to disrupt various stages of viral replication (3, 4). Type I IFNs are thus important for resistance to several viral infections and are used in the clinic for effective antiviral therapy (2). However, type I IFNs also exert a variety of other effects on cellular functions and immune responses. For example, they up or down regulate production of and responsiveness to other cytokines, chemokines, and can stimulate cell growth, cell survival, or apoptosis (2, 5). Consequently, these cytokines are also used for treatment of melanomas, leukemias, and other cancers (6), and as immune modulatory agents to suppress neuroinflammation in patients suffering from relapse-remitting multiple sclerosis (MS) (2). Hence, type I IFNs exert seemingly opposing pro- or antiinflammatory effects and pro- or anti-apoptotic effects. It is likely that these opposing effects reflect cell type-specific differences in the activation of secondary or "non-canonical" signaling events and/or variations in the dominance of a specific type I IFN species.

Indeed, individual type I IFN proteins vary in their ability to elicit specific responses and stimulation of different cell types can cause distinct signaling events (7, 8).

The second class of IFN protein (type II or IFNγ) is more critical for host defense against intracellular bacterial pathogens, such as *Mycobacterium tuberculosis* and *Listeria monocytogenes*. IFNγ signals through its own ubiquitously expressed heteromeric receptor (IFNGR), which utilizes IFNGR1 and IFNGR2 chains to activate a canonical JAK/STAT pathway primarily involving JAK1, JAK2, and STAT1. In contrast to type I IFNs, which are broadly expressed, IFNγ is produced primarily by lymphocytes. Antigenspecific IFNγ production occurs when appropriate T lymphocyte populations respond to specific microbial antigens, while antigen non-specific production of IFNγ is stimulated by cytokines such as interleukin (IL)-12 and IL-18. Studies using *L. monocytogenes* and other bacterial infection models indicate that both T and natural killer (NK) cells are capable of this antigen non-specific IFNγ production (9–11). Myeloid cells such as macrophages and dendritic cells (DCs) are key targets of IFNγ, as shown by the increased susceptibility to *L. monocytogenes* infection in mice selectively defective for functional IFNGR1 in myeloid cells (12, 13). The expression of numerous IFNγ activated genes (GAGs) is induced by the cytokine. Some of these genes are identical to ISGs and have antiviral effects. However, IFNγ is unique in its ability to elicit a potent anti-microbial state of activation in macrophages. This "M1-type" activation is associated with increased expression of GAGs such as nitric oxide synthase 2 (NOS2) and NADPH oxidase subunits. These enzymes generate nitric oxide (NO) and reactive oxygen species (ROS) that alter cell signaling and under appropriate circumstances can mediate direct killing of bacteria

(14, 15). IFNγ-inducible GTPases also promote macrophage resistance to bacterial and parasite infections by increasing the ability of phagosomal compartments to contain and kill engulfed microbes (16–18). IFNγ also upregulates myeloid cell expression of MHC II and other factors important for antigen presentation and T cell activation (19). In addition, IFNγ impacts maintenance and proliferation of hematopoietic stem cells (HSC). Specifically, basal production of IFNγ in the absence of infection drives HSC cycling and the elevated levels occurring during infection can activate HSC proliferation and myelopoiesis to replenish monocytes and other immune cells (20, 21).

Despite the antiviral effects of type I IFNs, and in the context of the antibacterial effects of IFNγ, it is increasingly evident that host responsiveness to type I IFNs correlates with increased host susceptibility to infections by *L. monocytogenes*, *M. tuberculosis*, *Francisella tularensis*, and several other intracellular bacterial pathogens (22–24). Here, relying heavily on the *L. monocytogenes* model, we review the pathways involved in the induction of type I IFNs by intracellular bacteria and various mechanisms proposed to account for the suppressive effects of type I IFN signaling. A theme that emerges from these studies is that type I IFNs have suppressive effects on anti-microbial and antigen-presenting function of myeloid cells. Such effects may contribute to both the observed ability of these cytokines to increase host susceptibility during bacterial infections and to their effectiveness in therapy of neuroinflammatory disease.

#### **BACTERIAL FACTORS CONTRIBUTING TO TYPE I IFN PRODUCTION DURING L. MONOCYTOGENES INFECTION**

*Listeria monocytogenes* is a Gram-positive facultative intracellular bacterium that causes the systemic disease Listeriosis. The mortality rate of Listeriosis is quite high even in hospitalized patients; hence, *L. monocytogenes* remains a leading cause of death from foodborne illnesses within the United States. *L. monocytogenes* can infect hematopoietic and non-hematopoietic cell types through phagocytosis or cellular mediated uptake (25, 26). Following systemic infection in the murine model *L. monocytogenes* localizes to the liver and spleen where resident phagocytes, primarily macrophages and DCs, engulf the bacteria. *L. monocytogenes* that escape from phagosomal compartments in these cells can replicate within the cell cytosol and further propagate the infection into neighboring cells. To facilitate vacuolar escape, the bacteria secrete a pore-forming hemolysin (hly) known as listeriolysin O (LLO) (27).

Like many other bacteria, *L. monocytogenes* induces production of pro-inflammatory cytokines such as TNFα and type I IFNs when engulfed by professional phagocytes. Studies of infection in bone marrow derived macrophages (BMM) suggest that there are two waves of the cellular response to *L. monocytogenes* infection (28, 29). An "early phase" gene expression profile is seen at 1–2 h post infection by both virulent wild-type *L. monocytogenes* and avirulent ∆hly or heat-killed bacteria that cannot escape from vacuolar compartments into the host cytosol (28, 29). Several of these "early phase" genes, including *il1b*, *tnfa*, and those encoding several chemokines are induced through the activation of Toll-like receptor (TLRs) and the ensuing activation of NFκB (28, 29). A subsequent "late-phase" response is

observed at 4–8 h after infection by wild-type, but not killed or ∆hly, *L. monocytogenes* strains (28, 29). "Late-phase" genes include IFNβ, multiple subtypes of IFNα, and several ISGs (28, 29). The fact that killed and ∆hly *L. monocytogenes* strains fail to induce this late-phase IFN-dominated response supports the interpretation that products from bacteria replicating within the BMM cytosol stimulate cytosolic pathogen recognition receptors (PRR), though TLR stimulation can augment the induction of type I IFNs during *L. monocytogenes* infection (29, 30). There has been considerable interest in identifying the cytosolic PRRs responsible for type I IFN production during *L. monocytogenes* infection.

#### **PATHWAYS LEADING TO THE PRODUCTION OF TYPE I IFNs DURING BACTERIAL INFECTION**

Pathways known to be important for induction of type I IFN within *L. monocytogenes*-infected phagocytes are diagrammed in **Figure 1**. Amongst the earliest identified cytosolic PRRs were the nucleotide-binding oligomerization domain (NOD)-containing proteins; members of the nucleotide-binding domain, leucine-rich repeat (LRR) protein family referred to as NLRs. NOD1 and NOD2 proteins sense distinct muropeptide fragments from the cell wall from *L. monocytogenes* and other bacteria (31–33). Recognition of appropriate muropeptides activates a serine/threonine kinase receptor interacting protein (RIP2) to initiate downstream signaling and activation of NFκB (31). With regards to triggering of type I IFN production, NOD1, NOD2, and RIP2 seem to play an ancillary role. They augment the induction of type I IFN and other cytokine expression in response to *L. monocytogenes* (29, 31–33), but mice and BMM deficient for any one of these proteins retain the ability to mount inflammatory responses and synthesize type I IFNs in response to *L. monocytogenes* (31, 34, 35). Thus, the recognition of bacterial cell wall components by the cytosolic NOD1 and NOD2 proteins is not crucial for the induction of type I IFNs during *L. monocytogenes* infection.

Nucleic acids are potent inducers of type I IFN production and it was shown that extracts from *L. monocytogenes* induce IFNβ production in a manner sensitive to DNAse treatment of the extracts (36). *L. monocytogenes* was also reported to actively secrete both RNA and DNA during infection of macrophages (37). Cytosolic RNA is detected by the RNA helicase retinoic acid inducible gene 1 protein (RIG-I), related RIG-I-like (RLR) proteins including melanoma differentiation-associated gene 5 (MDA5), as well as other non-RLR helicases and PRRs (38–40). RNA recognition by RIG-I and MDA5 induces their recruitment to mitochondria, where they encounter an adaptor protein [mitochondrial antiviral signaling (MAVS)] that regulates downstream signaling to induce type I IFNs (41–43). Secreted *L. monocytogenes* DNA could also be detected using these RNA receptor systems if it is transcribed into RNA by host cell RNA polymerase III (37). However, deficiency in RIG-I, MDA5, or MAVS fails to ablate IFNβ production by *L. monocytogenes*-infected BMM (37, 44, 45). Thus, it does not appear that RNA sensing is crucial for recognizing cytosolic *L. monocytogenes* infection in this cell type, although it may play a more important role in sensing *L. monocytogenes* infection of other cell types and for sensing infection by other bacteria, namely *Legionella pneumophila* (46, 47).

DNA present in the host cell cytosol can also be detected and trigger the production of type I IFNs (40). Several putative receptors have been identified that might mediate such recognition, including the DNA-dependent activator of IRFs (DAI), IFNinducible gene (IFI)-16, and LRR flightless-interacting protein (LRRFIP1). Deficiency or knockdown of DAI, IFI16, or LRRFIP1 fails to completely ablate type I IFN production by infected murine BMM (48–50). However, recently IFI16 was shown to have a larger role in the recognition of *L. monocytogenes* DNA in the human macrophage cell line, THP1. Knockdown of IFI16 in these cells drastically reduced the production of IFNβ in response to *L. monocytogenes* DNA (51). The DNA-binding DEAD-box helicase DDX41 has also been shown to bind *L. monocytogenes* DNA. DDX41 elicits type I IFN production through a mechanism requiring the stimulator of interferon genes protein (STING), also called MITA, MPYS, or ERIS (52). STING induces type I IFN production by activating the TNFR-associated NF-κB kinase (TANK) binding kinase 1 (TBK1), which phosphorylates a C-terminal serine residue on the transcription factor IFN regulatory factor 3 (IRF3) to induce IRF3 dimerization and nuclear translocation (34, 53). IRF3 and other IRF family members bind to the promoters of type I IFN genes and ISGs to regulate and initiate their

transcription. TBK1 and IRF3 are thus not surprisingly essential for the production of type I IFNs during *L. monocytogenes* infection (34, 54). Likewise, deficiency or knockdown of STING significantly decreases IRF3 activation and IFNβ production in BMM, DC, or fibroblasts infected with *L. monocytogenes* (55–57).

It appears that STING does not respond to intact DNA, but rather to endogenous or exogenous cyclic-di-nucleotides (40). In the presence of cytosolic dsDNA, the enzyme cGAS synthesizes an endogenous cyclic-di-nucleotide, cGAMP (58). Binding of cGAMP to STING occurs with a very high affinity (~4 nM) and induced conformational changes that presumably initiate the downstream events that culminate in type I IFN production (59). Exogenous cyclic-di-nucleotides can also activate STING (56, 57, 60). Both cyclic-di-GMP (cdGMP) and cyclic-di-AMP (cdAMP) are produced by bacteria and function as second messengers. There is evidence that cdAMP is released from replicating *L. monocytogenes* (61, 62), thus it is conceivable that cdAMP secreted by the bacterium mediates the STING-dependent production of type I IFNs in *L. monocytogenes*-infected macrophages. However, while the affinity of cdAMP for STING is not known, STING binds cdGMP ~300-fold lower affinity (~1µM) than cGAMP DNA (59). Thus, it is also conceivable that cGAMP produced by cGAS

in response to secreted bacterial DNA contributes to STINGdependent type I IFN production. Regardless, it is important to keep in mind that STING deficient mice showed significantly reduced serum IFNβ only very early (8 h) after *L. monocytogenes* infection (56, 57). Thus, systemic *L. monocytogenes* infection can trigger type I IFN through multiple pathways and the impact of STING on overall type I IFN production in this model is limited.

#### **TYPE I IFN SIGNALING AND INCREASED SUSCEPTIBILITY TO BACTERIAL INFECTION**

In certain bacterial infection models, protective effects of type I IFNs have been reported. For example, type I IFN can reduce bacterial burdens in cultured cells infected with *L. pneumophila* or *Chlamydia trachomatis* and survival of mice is increased in sepsis models with group B *Streptococcus* and *E. coli* (63–65). Mice lacking expression of IFNε, which is abundantly expressed within the female reproductive tract, were also reported to be highly susceptible to urogenital infection by *C. muridarum* (66). The precise mechanisms are not clear in these cases, but the observed protective effects appear to reflect unique aspects of the models and/or pathogens studied since there is considerable evidence to indicate that type I IFNs instead play a deleterious role during infections by numerous other bacterial pathogens (22–24). Specifically, studies with mice lacking IFNAR1 report that bacterial burdens are significantly reduced and survival increased following systemic or mucosal infections with intracellular bacteria that infect the cytosol of host cells, such as *L. monocytogenes* (54, 67–70) and *F. tularensis* (71) as well as bacteria like *M. tuberculosis* (72–74) and *C. muridarum* (75, 76) that reside within vacuolar compartments. In addition, heightened type I IFN production correlates with increased host susceptibility to several bacterial infections. In mice, examples of this include the correlation of increased type I IFN production in mice with a mutated ubiquitin specific peptidase (USP18) and sensitivity to *Salmonella typhimurium* (77). Furthermore, isolates of *L. monocytogenes* and *M. tuberculosis* that hyper-induce type I IFN production have heightened pathogenicity in animal models (78, 79). The administration of type I IFNs or agents that induce these cytokines also causes increased susceptibility to *L. monocytogenes* and *M. tuberculosis* in model infections (54, 78, 80). Type I IFN production is also increased during viral infections. In mice, lymphocytic choriomeningitis virus (LCMV) infection potently induces type I IFNs and leads to ~1000-fold increased susceptibility to a secondary *L. monocytogenes* infection as measured by bacterial burdens (81). In humans, a similar situation occurs following infection with influenza virus. Influenza infections are often associated with secondary bacterial infections and secondary bacterial pneumonias are estimated to account for up to 25% of the more than 250,000 annual deaths attributed to influenza (82, 83). Such secondary infections are also thought to have caused most of the deaths from the 1918 influenza pandemic (84). *Streptococcus pneumoniae* is a prevalent bacterial cause of pneumonias and a model of influenza and secondary *S. pneumoniae* infection showed that increased bacterial burdens and mortality was dependent on IFNAR expression (85). Severe bacterial infections have also been noted in patients receiving prolonged IFNα2 therapy for chronic hepatitis C virus infection (86–88). Moreover, in the absence of obvious viral infections, signatures of type I IFN responses correlate with disease progression in human tuberculosis and leprosy patients (89, 90). Thus, despite numerous differences in the receptors and cytokines themselves, the association of type I IFNs with exacerbated bacterial infections appears to have been conserved in murine and human systems. An improved understanding how these cytokine responses are deleterious to their hosts and what has driven their conservation across this evolutionary span are important questions to address.

#### **MECHANISMS PROPOSED TO ACCOUNT FOR THE PRO-BACTERIAL EFFECTS OF TYPE I IFNs**

A summary of the proposed mechanisms for the deleterious effects of type I IFN signaling during bacterial infections is outlined in **Figure 2**.

#### **INDUCTION OF HOST CELL DEATH**

It has long been known that bacterial infections can induce death of multiple cell types within tissues of murine hosts. In the systemic *L. monocytogenes* infection model, this cell death is exacerbated by type I IFNs. O'Connell et al. observed that expression of proapoptotic genes such as TNF-related apoptosis-inducing ligand (TRAIL), promyelocytic leukemia (PML), and death-associated protein 6 (Daxx) were increased in the spleens of *L. monocytogenes*-infected wild-type, but not IFNAR1 deficient, mice (54). Consistent with increased apoptosis in these tissues, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining is also increased in the spleens of infected wildtype mice, when compared to infected IFNAR1−/− or IRF3−/− mice (54, 69). This TUNEL staining was localized to lymphocyte rich follicles within the spleens, suggesting that type I IFN might induce apoptosis of lymphocytes and that such apoptosis could itself be detrimental (54, 69). The possibility that lymphocyte apoptosis is deleterious to the host is also consistent with the observation that mice deficient in lymphocytes or T cells alone are resistant to acute systemic *L. monocytogenes* infection (91–93). Resistance in T cell-deficient hosts is thought to reflect constitutively heightened macrophage activation (94), and correlates with reduced production of the anti-inflammatory cytokine IL-10 (91). It was thus proposed that in mice responsive to type I IFNs, the uptake of apoptotic cells by macrophages triggers their production of IL-10, which in turn inhibits host resistance (91). Myeloid cells are also sensitive to apoptosis in response to type I IFN and IFNAR expression has also been correlated with increased apoptosis of splenic and pulmonary macrophages during *L. monocytogenes* and pulmonary *C*. *muridarum* infections, respectively (54, 76). As mentioned above, these type I IFNs increase expression of the pro-apoptotic factor TRAIL during *L. monocytogenes* infection (54). Similar to IFNAR1−/− mice, mice lacking TRAIL demonstrate reduced TUNEL staining and increased resistance during *L. monocytogenes* infection (95). These effects were further correlated with increased numbers of splenic lymphocytes and monocytes. Type I IFN production induced by LCMV infection also correlates with granulocyte apoptosis and impaired control of *L. monocytogenes* infection (81). Thus, there is a clear association between type I IFNs, cellular death, and impaired myeloid cell responses during bacterial infections. Nonetheless, it remains

unclear whether apoptosis of T or myeloid cells is a primary cause of the increased host susceptibility.

#### **SUPPRESSION OF PRO-INFLAMMATORY CYTOKINE AND CHEMOKINE PRODUCTION**

Type I IFN production during viral infection is known to suppress production of IL-12 and other pro-inflammatory cytokines (96). Similarly, type I IFN production was associated with reduced secretion of IL-12 and TNFα in both *L. monocytogenes* and *M. tuberculosis* infection models (68, 72). Type I IFNs also suppress IL-1β production by inhibiting inflammasome activation (97), and reduced IL-1β secretion correlated with increased host susceptibility in *M. tuberculosis* infection models (98, 99). Expression of chemokines such as CCL2 is also regulated by type I IFNs (100, 101). CCL2 and its chemokine receptor CCR2 are critical for migration of inflammatory monocytes to sites of infection by *L. monocytogenes* and other bacteria (102–104). Spleens of IFNAR1−/− mice have increased accumulation of inflammatory monocytes during *L. monocytogenes* infection (68), however, type I IFNs upregulate CCL2 and recruitment of monocytes into the lung during *M. tuberculosis* infection (80). In the latter study, accumulation of monocytes correlated with more severe infection. By contrast, type I IFNs were reported to impair production of CXCL1, CXCL2, and neutrophil accumulation in lungs and more severe infection in mice infected with *S. pneumoniae* (85). Moderately, impaired neutrophil recruitment was also correlated with reduced IL-17 production and increased disease severity in mice in response to type I IFNs during *F. tularensis* and *L. monocytogenes* infections (71). However, it is debated whether or not neutrophils are protective during infections by *L. monocytogenes* and other intracellular bacteria (105, 106). Moreover, the neutropenia seen in patients treated with type I IFNs fails to correlate with their susceptibility to bacterial infections (86, 87). Thus, type I IFNs can alter production of cytokines and chemokines involved in neutrophil or inflammatory macrophage recruitment.

#### **SUPPRESSION OF MYELOID CELL RESPONSIVENESS TO IFN**γ

IFNγ is critical for the pro-inflammatory anti-microbial (M1) type activation of macrophages and transgenic mice lacking responsiveness to IFNγ selectively in myeloid cells are highly susceptible to *L. monocytogenes* and other intracellular pathogens (12, 13, 107). The macrophages activated by IFNγ have increased expression of molecules involved in both MHC class I and MHC class II antigen presentation, as well as enzymes producing reactive oxygen and nitrogen species with potential anti-microbial functions and proinflammatory cytokines and chemokines, including IL-12 (19). Expression of some, but not all, of these genes can also be induced when macrophages are stimulated with type I IFNs. In contrast, only IFNγ stimulates macrophages to express or upregulate MHC class II molecules (108). Indeed, stimulation of macrophages with type I IFNs suppresses their induction of MHC II expression in response to IFNγ. As mentioned above, type I IFNs also suppress production of IL-12 and CXCL1 and 2 (85, 96). These data suggest that type I IFNs are able to prevent or dampen classical M1-type anti-microbial macrophage activation in response to IFNγ. Consistent with this interpretation, a recent report revealed an inverse correlation between IFNβ and IFNγ gene expression patterns in

lesions of human leprosy patients (90). The IFNβ-driven response also correlated with IL-10 production, and IFNβ production contributed to IL-10 secretion, leading the authors to conclude that the impaired IFNγ responses in *M. leprae* infected macrophages is due to IL-10 production (90). Indeed, IFNβ and IL-10 treatments both impaired the ability of IFNγ to induce expression of the vitamin D receptor, the vitamin D-1a-hydroxylase, and the anti-microbial peptides cathelicidin and DEFB4 in macrophages (90).

Type I IFNs were also associated with the induction of IL-10 secretion and Programed death-ligand 1 (PD-L1) expression by myeloid cells during chronic LCMV infection (109, 110). Experiments using antibody blockade of IFNAR showed reduced expression of these immune suppressive factors and increased clearance of persistent viral infections (109, 110). Interestingly, the blockade of IFNAR also suppressed production of IL-1β and IL-18, arguing against the notion that improved viral clearance was due to increased inflammasome activation. Rather, the improved viral clearance was associated with increased serum IFNγ and blockade of IFNAR failed to improve viral clearance in mice treated with antibody to block IFNγ (109, 110). The therapeutic effects of blocking type I IFN signaling also correlated with an improved ratio of stimulatory versus immune regulatory antigen-presenting cells (APCs) and enhanced antiviral T cell responses. Although the authors of one study further suggested that the suppression of inflammatory and immune responses in these studies reflected chronic type I IFN signaling (109), a second study observed that type I IFNs increased IL-10 secretion and PD-L1 expression by DCs as early as 1 day post infection (110).

Leading up to these recent studies,prior efforts had also demonstrated suppressive effects of type I IFNs on myeloid cell activation during systemic *L. monocytogenes* infection of mice (70). In this model, the suppressive effects of type I IFNs correlate with reductions in myeloid cell surface IFNGR1. Similarly, surface IFNGR1 staining is significantly reduced on myeloid cells from *M. tuberculosis* infected patients compared to healthy control and effective treatment of these patients correlates with restored myeloid surface expression of IFNGR1 (111). IFNAR expression is necessary and recombinant type I IFNs are sufficient to trigger IFNGR1 down regulation in mouse and human myeloid, but not T cells (70, 112), suggesting this mechanism might contribute to a selective inhibition of myeloid cell responsiveness to IFNγ. Indeed, the reduced expression of IFNGR1 correlates with decreased responsiveness to IFNγ as indicated by reduced STAT1 phosphorylation and impaired induction of MHC class II expression in the context of *L. monocytogenes*, *M. tuberculosis*, and *F. novicida* infections (70, 111, 113).

Additional mechanistic studies have revealed that type I IFNs suppress myeloid cell surface IFNGR1 within hours of stimulating the IFNAR and that this effect is due to transcriptional silencing of the otherwise constitutively expressed *ifngr1* gene (70, 111, 112). The rapid reductions in *ifngr1* transcript abundance following IFNβ stimulation are preceded by loss of activated RNA polymerase II at the *ifngr1* transcriptional start site and the accumulation of epigenetic marks on nearby histones that are indicative of condensed chromatin (112). The reduction in *ifngr1* transcription is also associated with recruitment of the early growth response 3 (Egr3) transcription factor shortly after IFNβ treatment (112). Egr3 is a member of the Egr family of zinc finger transcription factors originally defined for their role in regulation of cell growth and differentiation (114, 115). Egr3 can act as an activator or repressor in response to various stimuli, depending on post-translational modifications and association with various adapter proteins (114– 117). One such adaptor protein, the NGFI-A binding protein Nab1 is a known corepressor and is also recruited to the *ifngr1* promoter shortly after Egr3 (112). Knockdown of Nab1, but not Nab2, prevented IFNGR1 down regulation in macrophages treated with IFNβ, suggesting that recruitment of a repressive Egr3/Nab1 complex is responsible for rapid silencing of *ifngr1* transcription (112). Given that the half-life of IFNGR1 protein is estimated at 3–4 h (118), such transcriptional silencing is sufficient to rapidly reduce myeloid cell responsiveness to IFNγ. Nonetheless, type I IFN stimulation does not appear to cause a complete loss of myeloid cell surface IFNGR1, possibly due to the induction of SOCS proteins and other endogenous negative feedback circuits that attenuate cellular responses to IFNAR signaling. These results suggest that down regulation of IFNGR1 expression might be an early step in the cascade of events leading to the suppression of myeloid cell responses that result in increased bacterial burdens and disease severity during acute and chronic bacterial infections, and the establishment or maintenance of chronic viral infections.

#### **COMMON FEATURES OF PROPOSED MECHANISMS**

Given the numerous effects of type I IFNs on various cells of the immune system and on non-immune cells, it is plausible that their pro-pathogen effects vary for different pathogens. This seems particularly likely for pathogens infect different tissue or cell types, where the responses to type I IFNs may differ. For instance, a recent study demonstrated that IFNAR expression on non-hematopoietic cells was required to increase host susceptibility to the intracellular bacterial pathogen *Ixodes ovatus Ehrlichia* (119). The effects of these cytokines may also differ depending on the route of infection and the presence or nature of competing commensal microbes. For example, Kerbauer et al. suggested that type I IFNs might not be as detrimental to the host following gastric infection of mice with *L. monocytogenes* (120). Regardless, results from the studies highlighted above clearly implicate myeloid cells/APCs as key targets of type I IFNs in settings where these cytokines are deleterious to the host. Precisely, how these cytokines act to dampen myeloid cell immunity and what selective advantage this confers on the host remains to be discerned.

#### **APPOSING EFFECTS OF TYPE I IFNs IN AUTOIMMUNE DISEASES**

The role of type I IFN signaling during autoimmune disease remains controversial,possibly indicating that these cytokines have opposing effects in different disease settings. For example, chronic type I IFN production is a hallmark of systemic lupus erythematosus (SLE) and several groups have reported a subset of ISGs upregulated in SLE patients compared to healthy controls (121– 124). ISG signatures were also associated with disease severity and progression in SLE patients (121–123). It has thus been suggested that type I IFNs promote SLE pathology through the activation of effector cells. Type I IFNs can paradoxically promote not just death of lymphocytes, but also T cell survival, proliferation, cytotoxicity, and B cell differentiation and antibody production. Any of these effects might conceivably contribute to increased tissue damage and disease progression in SLE patients.

In contrast to the exacerbation of SLE by type I IFNs, these same cytokines confer therapeutic benefits in certain other autoimmune diseases. The most obvious example of this is the neuroinflammatory disease MS. IFNβ is a common therapy and has been shown to reduce the frequency of clinical exacerbations in patients with relapse-remitting MS (125). The mechanisms for these beneficial effects remain uncertain. However, type I IFNs also suppress disease in the murine experimental autoimmune encephalomyelitis (EAE) model of MS. As for MS, IFNβ is therapeutic in the EAE model and deletion of the *ifnb* gene or IFNAR1 robustly increased EAE pathogenesis in mice (126, 127). Furthermore, using conditional knockouts, it was shown that IFNAR1 expression on myeloid cells was specifically required for the therapeutic effects of IFNβ during EAE (127). Deficiencies in IFNAR1 expression on myeloid cells also severely exacerbated disease and correlated with increased secretion of TNFα and CCL2 as well as increased expression of MHC II (127). These immunosuppressive effects of IFNβ treatment in humans may likewise target myeloid cells.

Other autoimmune diseases where type I IFNs appear to play a protective role include collagen type II induced arthritis in nonhuman primates (128). Treatment with double-stranded RNA species or recombinant IFNα also lowered the frequency and severity of arthritic symptoms in the murine model of antigeninduced arthritis (129). The pharmacokinetics of IFNβ therapies have shown to be a barrier is translating many of these treatments from animal models to clinical use in humans. However, Mullen et al. engineered a latent form of IFNβ that can only become activated when cleaved by aggrecanase (130). Aggrecanases are highly expressed within the joints and synovial fluid of rheumatoid arthritis and osteoarthritis patients and are responsible for the cleavage of aggrecan, an important component of joint tissue (130). This delivery method allows for temporal and tissue specific release of IFNβ that resulted in a significantly increased half-life of IFNβ as well as reduced pathology and joint swelling from collagen induced arthritis (130).

Humans with autoimmunity often carry a single nucleotide mutation in protein tyrosine phosphatase non-receptor type 22 (PTPN22) (131). PTPN22 is an intracellular protein tyrosine phosphatase that is exclusively found in immune cells (131), and was recently associated with TLR signaling for type I IFN synthesis in myeloid cells (132). Functional PTPN22 was also shown to suppress inflammatory arthritis and promote gut homeostasis (132). Mice deficient in PTPN22 demonstrate increased susceptibility in the dextran sodium sulfate (DSS) mouse model of acute colitis (132). TLR stimulation by microbiota also induced immunosuppressive effects that correlated with type I IFN production and decreased progression of experimental colitis in mice (133). Treatment with recombinant IFNβ phenocopied the decreased colitis achieved through TLR stimulation (133). Moreover, in a randomized placebo controlled study of active ulcerative colitis, a significant clinical response, and in some cases, disease remission, was seen in patients that received IFNβ therapy compared to the patients that received placebo (134, 135). It was noted that the therapeutic effects of IFNβ treatment in this disease correlated with reduced production of IL-13, an effector cytokine driving intestinal inflammation (134). Mice deficient in type I IFN signaling have been shown to have exacerbated DSS-induced acute colitis (136, 137). Furthermore, mice with IFNAR1 deletion specifically in myeloid cells demonstrated significantly increased weight loss and colitis disease activity score when treated with DSS (136). These data suggest that type I IFN signaling specifically in myeloid cell is protective during DSS-induced acute colitis. Interestingly, the authors further showed that IFNAR1−/− mice recovered from DSS treatment more quickly than wild-type mice, suggesting a deleterious role of type I IFNs during the recovery phase of colitis (136).

#### **CONCLUSION**