IL-36γ Protects against Severe Influenza Infection by Promoting Lung Alveolar Macrophage Survival and Limiting Viral Replication This information is current as of October 3, 2018. Alexander N. Wein, Paul R. Dunbar, Sean R. McMaster, Zheng-Rong Tiger Li, Timothy L. Denning and Jacob E. Kohlmeier J Immunol 2018; 201:573-582; Prepublished online 30 May 2018; Downloaded from doi: 10.4049/jimmunol.1701796 http://www.jimmunol.org/content/201/2/573 Supplementary http://www.jimmunol.org/content/suppl/2018/05/29/jimmunol.170179 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 45 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/201/2/573.full#ref-list-1 Why The JI? Submit online. by guest on October 3, 2018 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-36g Protects against Severe Influenza Infection by Promoting Lung Alveolar Macrophage Survival and Limiting Viral Replication Alexander N. Wein,* Paul R. Dunbar,* Sean R. McMaster,* Zheng-Rong Tiger Li,* Timothy L. Denning,† and Jacob E. Kohlmeier* Although influenza virus infection remains a concerning disease for public health, the roles of individual cytokines during the im- mune response to influenza infection are not fully understood. We have identified IL-36g as a key mediator of immune protection during both high- and low-pathogenesis influenza infection. Il36g mRNA is upregulated in the lung following influenza infection, and mice lacking IL-36g have greatly increased morbidity and mortality upon infection with either H1N1 or H3N2 influenza. The increased severity of influenza infection in IL-36g–knockout (KO) mice is associated with increased viral titers, higher levels of proinflammatory cytokines early in infection, and more diffuse pathologic conditions late in the disease course. Interestingly, the Downloaded from increased severity of disease in IL-36g–KO mice correlates with a rapid loss of alveolar macrophages following infection. We find that the alveolar macrophages from naive IL-36g–KO mice have higher expression of M2-like surface markers compared with wild-type (WT) mice and show increased apoptosis within 24 h of infection. Finally, transfer of WT alveolar macrophages to IL- 36g–KO mice restores protection against lethal influenza challenge to levels observed in WT mice. Together, these data identify a critical role for IL-36g in immunity against influenza virus and demonstrate the importance of IL-36g signaling for alveolar macrophage survival during infection. The Journal of Immunology, 2018, 201: 573–582. http://www.jimmunol.org/ espite the broad availability of licensed vaccines, influ- and chemotactic factors (3–6). IFNs serve to limit protein trans- enza viral illness remains a major threat to public health, lation within infected cells and decrease the permissibility of D particularly among children and the elderly (1, 2). In uninfected cells to the virus during the mobilization and recruit- severe cases, influenza infection can cause primary viral pneu- ment of immune cells from the bone marrow and circulation, monia or sensitize the host to secondary bacterial infection and which can take 4–5 d (7–9). Several innate cell types have been can also exacerbate underlying health problems, including con- shown to be key to controlling influenza infection. NK cells can gestive heart failure, chronic obstructive pulmonary disease, and recognize infected cells via NKp46 binding to surface hemag- by guest on October 3, 2018 asthma. One reason for the continued public health burden of glutinin, leading to lysis of cells that display viral proteins (8). influenza is the imperfect antigenic match between the vaccine Neutrophils can also serve to promote viral clearance in severe and circulating influenza strains that results in only 50–80% cases of influenza, but neutrophils, along with monocytes, have protection in the best of years (2). In the absence of effective been shown to contribute to immune pathologic condition during cellular or humoral memory responses, the innate defenses of the some models of influenza infection (10, 11). In contrast, among respiratory tract serve as the only mechanisms to control the the earliest responders to influenza infection in the lung are tissue- spread of virus in the early phases of infection. resident alveolar macrophages. In mice, these cells have been Influenza infection of the respiratory epithelium leads to the shown to decrease the infection of type I alveolar epithelial cells activation of antiviral responses via the extracellular and endo- during the initial 48 h of infection, before other innate cell types somal TLR receptors or the intracellular receptors RIG-I and arrive in the lung (12). Depletion of alveolar macrophages by NLRP3, causing cells to release IFNs as well as proinflammatory either genetic knockout (KO) or liposomal clodronate treatment increases morbidity, mortality, and viral load during influenza infection (12, 13). Therefore, lung alveolar macrophages play a *Department of Microbiology and Immunology, Emory University School of Med- icine, Atlanta, GA 30322; and †Institute for Biomedical Sciences, Georgia State critical role as one of the few lung-resident innate immune cell University, Atlanta, GA 30303 types capable of responding to influenza infection during the ORCIDs: 0000-0002-8813-3523 (A.N.W.); 0000-0002-9082-6636 (P.R.D.). initial stages of infection, prior to immune recognition that leads Received for publication January 2, 2018. Accepted for publication May 8, 2018. to the initial inflammatory burst alerting the systemic immune This work was supported by National Institutes of Health Grant R01HL122559 system to a lung infection. (to J.E.K.) and Emory University. P.R.D. was supported by National Institutes of Health Severe influenza infections are characterized by viral infection Grant F31AI124611 and S.R.M. was supported by National Institutes of Health Grant F30HL118954. throughout the upper and lower respiratory tract (14). Conse- quences of increased viral titers throughout the respiratory tract Address correspondence and reprint requests to Dr. Jacob E. Kohlmeier, Emory University School of Medicine, 1510 Clifton Road, RRC, Room 3133, Atlanta, include hyperactive cytokine responses, and patients hospitalized GA 30322. E-mail address: [email protected] with severe influenza showed significant increases in proin- The online version of this article contains supplemental material. flammatory cytokines (15). Elevated expression of type I IFNs, Abbreviations used in this article: BAL, bronchoalveolar lavage; EID50, 50% egg TNF-a, IL-1, IL-6, IL-18, and IL-33 have been observed in infectious dose; KO, knockout; NP, nuclear protein; qPCR, quantitative PCR; WT, pathogenic influenza infections, suggesting that inhibition of these wild-type. cytokines may ameliorate influenza pathologic conditions and Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 improve host survival (16). However, deletion or blockade of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701796 574 IL-36g IN INFLUENZA INFECTION proinflammatory cytokine pathways, including IFN-a,IFN-g,TNF, TGC CAT TCT GCC GCA TT-39, x31 NP reverse: 59-GCT GAT TTG IL-1, and IL-6, has shown mixed results in mice and humans (9, 17– GCC CGC AGA TG-39, probe: 59-/5HEX/TA+G T+CT +CCA TAT TTT 24). General immunosuppression with corticosteroids has also shown CAT T+G+G AA+G C/3BHQ_1/-39. Expression levels of IL-36 family members were determined by real-time quantitative PCR (qPCR) on a Bio- little to no effect on patient outcome in clinical trials (24–26). These Rad CFX96 Real-Time system and CFX Manager v3.1 using primers failures have renewed efforts to identify novel proinflammatory host against Il36a, Il36b, Il36g, and Hprt. Sequences were Il36a forward: 59- mediators that contribute to influenza pathogenesis. One such family TAG TGG GTG TAG TTC TGT AGT GTG-39, Il36a reverse: 59-GTT of proinflammatory mediators is the IL-36 family of cytokines, CGT CTC AAG AGT GTC CAG ATA T-39, Il36b forward: 59-ACA AAA AGC CTT TCT GTT CTA TCA T-39, Il36b reverse: 59-CCA TGT ATT consisting of IL-36a,-36b,and-36g, which are recently discovered TAC TTC TCA GAC T-39, Il36g forward: 59-AGA GTA ACC CCA GTC members of the IL-1 family. These proteins have been shown to be AGC GTG-39, Il36g reverse: 59-AGG GTG GTG GTA CAA ATC CAA-39, important in several different inflammatory conditions, including Hprt forward: 59-CAC AGG ACT AGA ACA CCT GC-39, Hprt reverse: asthma, colitis, and psoriasis (27–29). In addition, IL-36g adminis- 59-GCT GGT GAA AAG GAC CTC T-39. For Il36g expression in sorted tration has been shown to increase mucus production and neutrophil immune cells, cDNA was synthesized with the iScript cDNA synthesis kit before real-time qPCR (Bio-Rad). Real-time qPCR data were analyzed migration into the lungs of mice (28). A recent study showed that IL- using CFX Manager before export to Microsoft Excel. 36g–KO mice were more susceptible to pulmonary Streptococcus pneumoniae infection, demonstrating an important role for IL-36g in Lung harvest for pathological examination lung immunity (30). In addition, deletion of IL-36R has been shown Mice were euthanized as above, and the ribcage was carefully removed.