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• W2296304057 abstract "•LysM+ and CD11c+ myeloid cells are major sources of IFN-I in S. pyogenes-infected mice•S. pyogenes-infected Ifnar1−/− mice display hyperinflammation and tissue damage•Systemic levels of IL-1β are increased in S. pyogenes-infected Ifnar1−/− mice•IFN-I is required to control IL-1β levels and ensure a balanced inflammatory response Type I interferons (IFN-Is) are fundamental for antiviral immunity, but their role in bacterial infections is contradictory and incompletely described. Streptococcus pyogenes activates IFN-I production in innate immune cells, and IFN-I receptor 1 (Ifnar1)-deficient mice are highly susceptible to S. pyogenes infection. Here we report that IFN-I signaling protects the host against invasive S. pyogenes infection by restricting inflammation-driven damage in distant tissues. Lethality following infection in Ifnar1-deficient mice is caused by systemically exacerbated levels of the proinflammatory cytokine IL-1β. Critical cellular effectors of IFN-I in vivo are LysM+ and CD11c+ myeloid cells, which exhibit suppression of Il1b transcription upon Ifnar1 engagement. These cells are also the major source of IFN-β, which is significantly induced by S. pyogenes 23S rRNA in an Irf5-dependent manner. Our study establishes IL-1β and IFN-I levels as key homeostatic variables of protective, yet tuned, immune responses against severe invasive bacterial infection. Type I interferons (IFN-Is) are fundamental for antiviral immunity, but their role in bacterial infections is contradictory and incompletely described. Streptococcus pyogenes activates IFN-I production in innate immune cells, and IFN-I receptor 1 (Ifnar1)-deficient mice are highly susceptible to S. pyogenes infection. Here we report that IFN-I signaling protects the host against invasive S. pyogenes infection by restricting inflammation-driven damage in distant tissues. Lethality following infection in Ifnar1-deficient mice is caused by systemically exacerbated levels of the proinflammatory cytokine IL-1β. Critical cellular effectors of IFN-I in vivo are LysM+ and CD11c+ myeloid cells, which exhibit suppression of Il1b transcription upon Ifnar1 engagement. These cells are also the major source of IFN-β, which is significantly induced by S. pyogenes 23S rRNA in an Irf5-dependent manner. Our study establishes IL-1β and IFN-I levels as key homeostatic variables of protective, yet tuned, immune responses against severe invasive bacterial infection. Infectious diseases are a consequence of insufficient immune responses, but deleterious effects of pathogens can also be caused by hyperstimulation and/or chronic activation of the immune system. Some pathogens can take advantage of both inadequate and exaggerated immune responses, as exemplified by the highly versatile gram-positive human pathogen Streptococcus pyogenes (Group A Streptococcus) (Cole et al., 2011Cole J.N. Barnett T.C. Nizet V. Walker M.J. Molecular insight into invasive group A streptococcal disease.Nat. Rev. Microbiol. 2011; 9: 724-736Crossref PubMed Scopus (291) Google Scholar). The most common condition associated with S. pyogenes infection is streptococcal pharyngitis, which is a largely self-limiting disease with substantial economic burden (Wessels, 2011Wessels M.R. Clinical practice. Streptococcal pharyngitis.N. Engl. J. Med. 2011; 364: 648-655Crossref PubMed Scopus (176) Google Scholar). In addition, S. pyogenes infections account for 650,000 annual cases of the life-threatening necrotizing fasciitis and streptococcal toxic shock syndrome, with mortality exceeding 20% despite therapy (Carapetis et al., 2005Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. The global burden of group A streptococcal diseases.Lancet Infect. Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1947) Google Scholar). Human host factors determining susceptibility to S. pyogenes infections are not well characterized. Immunogenetics analyses suggest that a complex and precisely balanced immune response is required for successful protection. Mutations in the signaling adaptor Myd88 result in a dramatic susceptibility to pyogenic infections in children but not in adults (von Bernuth et al., 2008von Bernuth H. Picard C. Jin Z. Pankla R. Xiao H. Ku C.L. Chrabieh M. Mustapha I.B. Ghandil P. Camcioglu Y. et al.Pyogenic bacterial infections in humans with MyD88 deficiency.Science. 2008; 321: 691-696Crossref PubMed Scopus (622) Google Scholar), indicating that a robust stimulation of the innate immune system is fundamental for defense against S. pyogenes under conditions in which adaptive immunity is still underdeveloped. Several human leukocyte antigen class II haplotypes are associated with strong protection against severe invasive S. pyogenes infections, whereas others increase the risk of life-threatening disease (Kotb et al., 2002Kotb M. Norrby-Teglund A. McGeer A. El-Sherbini H. Dorak M.T. Khurshid A. Green K. Peeples J. Wade J. Thomson G. et al.An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections.Nat. Med. 2002; 8: 1398-1404Crossref PubMed Scopus (306) Google Scholar). The high-risk haplotypes are often associated with an augmented cytokine storm. Experiments employing mice deficient in Myd88, Tnf, Toll-like receptor (Tlr) 2, or Unc93b1 or mice depleted of macrophages or dendritic cells (DCs) corroborate the fundamental role of the innate immune response (Fieber et al., 2015Fieber C. Janos M. Koestler T. Gratz N. Li X.D. Castiglia V. Aberle M. Sauert M. Wegner M. Alexopoulou L. et al.Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.PLoS ONE. 2015; 10: e0119727Crossref Scopus (32) Google Scholar, Fieber and Kovarik, 2014Fieber C. Kovarik P. Responses of innate immune cells to group A Streptococcus.Front. Cell. Infect. Microbiol. 2014; 4: 140Crossref PubMed Scopus (39) Google Scholar, Goldmann et al., 2004Goldmann O. Rohde M. Chhatwal G.S. Medina E. Role of macrophages in host resistance to group A streptococci.Infect. Immun. 2004; 72: 2956-2963Crossref PubMed Scopus (96) Google Scholar, Loof et al., 2010Loof T.G. Goldmann O. Gessner A. Herwald H. Medina E. Aberrant inflammatory response to Streptococcus pyogenes in mice lacking myeloid differentiation factor 88.Am. J. Pathol. 2010; 176: 754-763Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, Loof et al., 2007Loof T.G. Rohde M. Chhatwal G.S. Jung S. Medina E. The contribution of dendritic cells to host defenses against Streptococcus pyogenes.J. Infect. Dis. 2007; 196: 1794-1803Crossref PubMed Scopus (36) Google Scholar, Mishalian et al., 2011Mishalian I. Ordan M. Peled A. Maly A. Eichenbaum M.B. Ravins M. Aychek T. Jung S. Hanski E. Recruited macrophages control dissemination of group A Streptococcus from infected soft tissues.J. Immunol. 2011; 187: 6022-6031Crossref PubMed Scopus (37) Google Scholar, Tsatsaronis et al., 2014Tsatsaronis J.A. Walker M.J. Sanderson-Smith M.L. Host responses to group a streptococcus: cell death and inflammation.PLoS Pathog. 2014; 10: e1004266Crossref PubMed Scopus (38) Google Scholar). Adaptive interleukin (IL)-17-mediated immunity contributes to defense in the upper respiratory tract epithelium (Dileepan et al., 2011Dileepan T. Linehan J.L. Moon J.J. Pepper M. Jenkins M.K. Cleary P.P. Robust antigen specific th17 T cell response to group A Streptococcus is dependent on IL-6 and intranasal route of infection.PLoS Pathog. 2011; 7: e1002252Crossref PubMed Scopus (72) Google Scholar). However, cellular mechanisms ensuring protective yet balanced immune responses remain to be characterized. Type I interferons (IFN-Is) are cytokines produced in response to viral, bacterial, and fungal pathogens. The function of IFN-I in viral infections is well established and is always protective. The IFN-I role in the context of bacterial infections is less clear, because both beneficial and detrimental effects have been reported, depending on the pathogen (Stifter and Feng, 2015Stifter S.A. Feng C.G. Interfering with immunity: detrimental role of type I IFNs during infection.J. Immunol. 2015; 194: 2455-2465Crossref PubMed Scopus (65) Google Scholar, Trinchieri, 2010Trinchieri G. Type I interferon: friend or foe?.J. Exp. Med. 2010; 207: 2053-2063Crossref PubMed Scopus (632) Google Scholar). Of particular relevance for bacterial infections appear to be immunosuppressive effects of IFN-Is. For example, immunosuppression by virus-induced IFN-Is compromises immunity against post-influenza bacterial pneumonia (Nakamura et al., 2011Nakamura S. Davis K.M. Weiser J.N. Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice.J. Clin. Invest. 2011; 121: 3657-3665Crossref PubMed Scopus (208) Google Scholar, Shahangian et al., 2009Shahangian A. Chow E.K. Tian X. Kang J.R. Ghaffari A. Liu S.Y. Belperio J.A. Cheng G. Deng J.C. Type I IFNs mediate development of postinfluenza bacterial pneumonia in mice.J. Clin. Invest. 2009; 119: 1910-1920Crossref PubMed Scopus (378) Google Scholar). Similarly, attenuation of immune responses has been linked with the detrimental role of IFN-I signaling in Mycobacterium tuberculosis infection models (Mayer-Barber et al., 2011Mayer-Barber K.D. Andrade B.B. Barber D.L. Hieny S. Feng C.G. Caspar P. Oland S. Gordon S. Sher A. Innate and adaptive interferons suppress IL-1α and IL-1β production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.Immunity. 2011; 35: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). A key question pertains to the protective mechanism of IFN-I in infections with important pathogens such as S. pyogenes or Group B Streptococcus (Gratz et al., 2011Gratz N. Hartweger H. Matt U. Kratochvill F. Janos M. Sigel S. Drobits B. Li X.D. Knapp S. Kovarik P. Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.PLoS Pathog. 2011; 7: e1001345Crossref PubMed Scopus (98) Google Scholar, Mancuso et al., 2009Mancuso G. Gambuzza M. Midiri A. Biondo C. Papasergi S. Akira S. Teti G. Beninati C. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.Nat. Immunol. 2009; 10: 587-594Crossref PubMed Scopus (262) Google Scholar). It is well established that S. pyogenes activates IFN-I production in innate immune cells and that IFN-I receptor 1 (Ifnar1)-deficient mice are highly susceptible to subcutaneous S. pyogenes infection, a model of severe invasive cellulitis in humans (Gratz et al., 2008Gratz N. Siller M. Schaljo B. Pirzada Z.A. Gattermeier I. Vojtek I. Kirschning C.J. Wagner H. Akira S. Charpentier E. Kovarik P. Group A Streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9.J. Biol. Chem. 2008; 283: 19879-19887Crossref PubMed Scopus (74) Google Scholar, Mancuso et al., 2009Mancuso G. Gambuzza M. Midiri A. Biondo C. Papasergi S. Akira S. Teti G. Beninati C. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.Nat. Immunol. 2009; 10: 587-594Crossref PubMed Scopus (262) Google Scholar). Our current study shows that IFN-I signaling plays a key role in homeostatic mechanisms required for an efficient yet not destructive immune reaction against invasive bacterial infection of soft tissue. S. pyogenes was found by us and others to stimulate expression of IFN-β, the primary IFN-I, in murine bone marrow-derived macrophages (BMDMs) and granulocyte-macrophage colony stimulating factor-differentiated conventional dendritic cells (cDCs) (Gratz et al., 2011Gratz N. Hartweger H. Matt U. Kratochvill F. Janos M. Sigel S. Drobits B. Li X.D. Knapp S. Kovarik P. Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.PLoS Pathog. 2011; 7: e1001345Crossref PubMed Scopus (98) Google Scholar, Mancuso et al., 2009Mancuso G. Gambuzza M. Midiri A. Biondo C. Papasergi S. Akira S. Teti G. Beninati C. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.Nat. Immunol. 2009; 10: 587-594Crossref PubMed Scopus (262) Google Scholar). To identify cells producing IFN-I in vivo, we employed reporter mice allowing Cre-Lox-regulated cell type-specific monitoring of the Ifnb promoter activity (Lienenklaus et al., 2009Lienenklaus S. Cornitescu M. Zietara N. Łyszkiewicz M. Gekara N. Jabłónska J. Edenhofer F. Rajewsky K. Bruder D. Hafner M. et al.Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.J. Immunol. 2009; 183: 3229-3236Crossref PubMed Scopus (171) Google Scholar). Bioluminescence imaging of global reporter mice (Ifnb+/Δβ-luc) revealed increased Ifnb promoter activity at the site of infection and in inguinal lymph nodes 24 and 48 hr after infection (Figures 1A and 1B ). Reporter mice for the LysM+ (Ifnb+/fl β-luc-LysMCre) or CD11c+ (Ifnb+/fl β-luc-CD11cCre) lineages exhibited an overall lower luciferase signal before infection when compared to the uninfected global reporter mice, but the signal increased upon infection (Figures 1A and 1B). The induction of luciferase by S. pyogenes at the site of infection was comparable in the three reporter mice (Figure 1B, right panel), indicating that LysM+ and CD11c+ myeloid cells are the major IFN-β producers in vivo. Because certain myeloid populations express both LysM and CD11c markers (Abram et al., 2014Abram C.L. Roberge G.L. Hu Y. Lowell C.A. Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice.J. Immunol. Methods. 2014; 408: 89-100Crossref PubMed Scopus (279) Google Scholar), the luciferase activity in Ifnb+/fl β-luc-LysMCre and Ifnb+/fl β-luc-CD11cCre mice might originate from the same or distinct cell populations. Neither basal nor infection-stimulated Ifnb promoter activity was detected in T cell-specific reporter mice (Ifnb+/fl β-luc-CD4Cre) (Figures 1A and 1B). The relatively high luciferase signal in uninfected global reporter mice (Figures 1A and 1B) results from basal Ifnb gene expression, which, for unknown reasons, occurs in various cells and tissues (Lienenklaus et al., 2009Lienenklaus S. Cornitescu M. Zietara N. Łyszkiewicz M. Gekara N. Jabłónska J. Edenhofer F. Rajewsky K. Bruder D. Hafner M. et al.Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.J. Immunol. 2009; 183: 3229-3236Crossref PubMed Scopus (171) Google Scholar, Sato et al., 2000Sato M. Suemori H. Hata N. Asagiri M. Ogasawara K. Nakao K. Nakaya T. Katsuki M. Noguchi S. Tanaka N. Taniguchi T. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction.Immunity. 2000; 13: 539-548Abstract Full Text Full Text PDF PubMed Scopus (1094) Google Scholar). BMDMs employ the transcription factor Irf3 for IFN-β induction by S. pyogenes, while cDCs use Irf5 (Gratz et al., 2011Gratz N. Hartweger H. Matt U. Kratochvill F. Janos M. Sigel S. Drobits B. Li X.D. Knapp S. Kovarik P. Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.PLoS Pathog. 2011; 7: e1001345Crossref PubMed Scopus (98) Google Scholar). To determine the roles of Irf3 and Irf5 in vivo, we investigated the activity of the IFN-β promoter-driven luciferase reporter in mice lacking Irf3 (Ifnb+/Δβ-luc-Irf3−/−) or Irf5 (Ifnb+/Δβ-luc-Irf5−/−). The induction of the Ifnb promoter at the site of S. pyogenes infection in mice lacking Irf3 was comparable to Ifnb+/Δβ-luc control reporter mice (Figures 1C and 1D). In contrast, mice lacking Irf5 exhibited severe impairment of Ifnb promoter induction (Figures 1C and 1D). These experiments showed that Irf5 is the major IFN-β activator in vivo, thereby resembling IFN-β production by cDCs in vitro. Irf5-dependent IFN-β induction in cDCs can be triggered by S. pyogenes-derived RNA (Gratz et al., 2011Gratz N. Hartweger H. Matt U. Kratochvill F. Janos M. Sigel S. Drobits B. Li X.D. Knapp S. Kovarik P. Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.PLoS Pathog. 2011; 7: e1001345Crossref PubMed Scopus (98) Google Scholar). A short conserved sequence of the bacterial 23S rRNA, including that of S. pyogenes, was shown to induce pro-inflammatory cytokines (e.g., Tnf and Il-6) by triggering Tlr13 (Fieber et al., 2015Fieber C. Janos M. Koestler T. Gratz N. Li X.D. Castiglia V. Aberle M. Sauert M. Wegner M. Alexopoulou L. et al.Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.PLoS ONE. 2015; 10: e0119727Crossref Scopus (32) Google Scholar, Li and Chen, 2012Li X.D. Chen Z.J. Sequence specific detection of bacterial 23S ribosomal RNA by TLR13.eLife. 2012; 1: e00102Crossref Scopus (96) Google Scholar, Oldenburg et al., 2012Oldenburg M. Krüger A. Ferstl R. Kaufmann A. Nees G. Sigmund A. Bathke B. Lauterbach H. Suter M. Dreher S. et al.TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification.Science. 2012; 337: 1111-1115Crossref PubMed Scopus (312) Google Scholar). To test whether S. pyogenes 23S rRNA is also capable of inducing IFN-β, we treated cDCs with total S. pyogenes RNA, purified S. pyogenes rRNA fractions, or oligoribonucleotides comprising the Tlr13-stimulating 23S rRNA sequence (SA19) (Oldenburg et al., 2012Oldenburg M. Krüger A. Ferstl R. Kaufmann A. Nees G. Sigmund A. Bathke B. Lauterbach H. Suter M. Dreher S. et al.TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification.Science. 2012; 337: 1111-1115Crossref PubMed Scopus (312) Google Scholar) and its mutated version (mut-SA19). Both 23S rRNA and SA19 stimulated IFN-β production, whereas 16S and 5S rRNAs and mut-SA19 did not (Figure 1E, left panel). Induction of IFN-β by 23S rRNA was entirely Irf5 dependent (Figure 1E), in agreement with the requirement of Irf5 for IFN-β induction by S. pyogenes in vivo (Figures 1C and 1D). Consistent with the reported broader role of Irf5 in Tlr signaling (Takaoka et al., 2005Takaoka A. Yanai H. Kondo S. Duncan G. Negishi H. Mizutani T. Kano S. Honda K. Ohba Y. Mak T.W. Taniguchi T. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors.Nature. 2005; 434: 243-249Crossref PubMed Scopus (797) Google Scholar), Tnf induction was also strongly dependent on Irf5 (Figure 1E). Finally, by using Tlr13-deficient cDCs, we were able to show that S. pyogenes and S. pyogenes RNA induced IFN-β in a Tlr13-dependent way (Figure 1F). In summary, our data reveal that myeloid cells expressing LysM and/or CD11c are the major source of IFN-β in S. pyogenes-infected mice. Our observation that IFN-β induction is largely Irf5 dependent in vivo implies that CD11c+ DCs are the main IFN-β source. Furthermore, our data identify S. pyogenes 23S rRNA to be the IFN-β-inducing Tlr13 ligand in cDCs. To identify the effector cells of IFN-I signaling, we infected mice lacking Ifnar1 in CD11c+ (Ifnar1fl/fl-CD11cCre) or LysM+ (Ifnar1fl/fl-LysMCre) cells and compared them with control mice (Ifnar1fl/fl), as well as full Ifnar1 knockout mice (Ifnar1−/−) (Figure 2A). Ifnar1 ablation in both CD11c+ and LysM+ cells resulted in significantly decreased survival, akin to the susceptibility of Ifnar1−/− mice (Figure 2A). In general, animals surviving longer than 5 days recovered regardless of the genotype. Thus, Ifnar1 signaling in CD11c+ and LysM+ cells is critically contributing to the protective effects of IFN-I in S. pyogenes infection. To examine whether increased bacterial loads caused higher susceptibility of Ifnar1-deficient mice, we counted bacterial numbers at the site of primary infection (i.e., skin lesion), in the blood, and in distant organs (spleen and liver) at 24 and 48 hr post-infection (p.i.)—that is, before occurrence of the first moribund animals. No differences in bacterial burden between wild-type (WT) and Ifnar1−/− mice were detected at any location or time point (Figures 2B and 2C). Therefore, the beneficial function of IFN-I must be exerted by mechanisms other than those involved in restricting bacterial burden. Given that the increased severity of infection in Ifnar1−/− mice was not caused by higher bacterial burden, we assessed markers of liver and kidney injury. Serum levels of bilirubin, aspartate transaminase (AST), and glutamate dehydrogenase (GLDH) were significantly higher in S. pyogenes-infected Ifnar1−/− when compared to control animals 48 hr p.i., indicative of exacerbated hepatocellular injury (Figure 3A). Alanine transaminase (ALT) was also increased, albeit the difference was not significant (Figure 3A). Serum levels of creatinine and urea were also significantly elevated in Ifnar1−/− mice, suggesting a more severe impairment of renal function (Figure 3A). To evaluate leukocyte infiltration and pathological changes in organ architecture, H&E-stained sections of the infected skin, liver, spleen, and kidney were examined. Necrotic areas, which were frequently observed in all layers of the infected skin, were more often surrounded by areas of substantial leukocytic infiltrates in Ifnar1−/− mice than in WT controls (Figure 3B). Analysis of spleen sections revealed massive infiltration of myeloid cells with expansion of the white pulp and retraction of the red pulp in Ifnar1−/− mice (Figure 3C). The liver damage histopathology score in Ifnar1−/− mice was significantly higher for hepatocellular vacuolation but not for inflammatory infiltrates (Figure 3D). Kidney sections did not reveal any major pathological change except for rare tubular casts in both genotypes (Figure 3E). The lack of major morphological signs of kidney damage, together with elevated blood levels of urea and creatinine in Ifnar1−/− mice, suggest a pre-renal kidney failure in the course of infection-triggered hypotension. Exacerbated organ damage and leukocyte infiltration in Ifnar1−/− mice demonstrated that the absence of IFN-I signaling results in hyperinflammation both at the site of infection and in distant tissues. To directly determine the inflammatory milieu, we examined the amounts of the pro-inflammatory cytokines Tnf, IL-1α, and IL-1β; the neutrophil chemoattractant Cxcl1; and the anti-inflammatory cytokine IL-10 in the infected skin, liver, and spleen 48 hr after the infection. The levels of IL-1β were considerably increased at all locations in Ifnar1−/− animals when compared to WT controls (Figure 4A). In contrast, IL-1α was increased in the Ifnar1−/− spleen but not at other sites, and Tnf and IL-10 levels were similar in all tissues in both genotypes (Figures 4B–4D). Elevated IL-1α amounts in Ifnar1−/− spleens might result from more excessive damage in this organ, because damaged tissue is a rich source of this cytokine (Chen and Nuñez, 2010Chen G.Y. Nuñez G. Sterile inflammation: sensing and reacting to damage.Nat. Rev. Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2046) Google Scholar). Cxcl1 amounts were in general slightly higher in Ifnar1−/− mice, but the difference from WT controls was only significant in infected skin lesions (Figure 4E). Cytokine amounts were low in uninfected animals of each genotype (Figure 4F). The basal IL-1β levels appeared higher in uninfected Ifnar1−/− mice, suggesting that IFN-I signaling was attenuating IL-1β production also in untreated mice. Collectively, these data demonstrate that the absence of IFN-I signaling in S. pyogenes-infected animals causes massive tissue damage and hyperinflammation associated with a profound and selective failure to control IL-1β production. Tissue sections showed that IFN-I signaling attenuated leukocyte infiltration both at the site of infection and in distant organs. To examine the role of IFN-I signaling in recruiting immune cells, we employed an air pouch infection model, which allows qualitative and quantitative analysis of infiltrating cells at the site of infection (Loof et al., 2010Loof T.G. Goldmann O. Gessner A. Herwald H. Medina E. Aberrant inflammatory response to Streptococcus pyogenes in mice lacking myeloid differentiation factor 88.Am. J. Pathol. 2010; 176: 754-763Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Air pouch exudates of Ifnar1−/− and WT mice were analyzed by flow cytometry and ELISA 24 hr p.i. In agreement with previous studies (Loof et al., 2010Loof T.G. Goldmann O. Gessner A. Herwald H. Medina E. Aberrant inflammatory response to Streptococcus pyogenes in mice lacking myeloid differentiation factor 88.Am. J. Pathol. 2010; 176: 754-763Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), more than 90% of infiltrating cells were Ly6G+Ly6C+ neutrophils (Figure 5A). These percentages were similar in both Ifnar1−/− and WT mice (Figure 5A). However, the total numbers of all infiltrating cells and neutrophils were more than 2-fold higher in Ifnar1−/− exudates (Figure 5A, lower right panel). IL-1β was markedly elevated in air pouches of Ifnar1−/− animals (Figure 5B). IL-1α and Cxcl1 were also elevated in Ifnar1−/− samples, albeit to a lesser extent than IL-1β, whereas Tnf and IL-10 levels were comparable in both genotypes (Figure 5B). Thus, the cytokine profiles in air pouches were similar to those in infected skin (Figures 4 and 5B). To assess whether elevated IL-1β levels in Ifnar1−/− exudates resulted from higher leukocytes numbers or from augmented IL-1β production, we employed cell type-specific Ifnar1 deletions. Air pouches generated in animals lacking Ifnar1 in neutrophils and macrophages (Ifnar1fl/fl-LysMCre) or cDCs (Ifnar1fl/fl-CD11cCre) contained numbers of leukocytes comparable to those of Ifnar1fl/fl controls (Figure 5C, left panel). Hence, the increased leukocyte infiltration observed in Ifnar1−/− mice (Figure 5A) was caused by the absence of IFN-I signaling in multiple lineages. However, IL-1β was increased in air pouches of Ifnar1fl/fl-LysMCre but not Ifnar1fl/fl-CD11cCre animals (Figure 5C, right panel), indicating that higher IL-1β levels resulted from uncontrolled production by neutrophils and/or macrophages. Because neutrophils represent by far the largest population of recruited cells, they probably contribute most significantly to the augmented IL-1β production at the infection site of Ifnar1−/− mice. To understand the mechanism of IL-1β inhibition by IFN-I, we analyzed effects of exogenous and endogenous IFN-β on processing the IL-1β precursor (pro-IL-1β) in S. pyogenes-infected BMDMs. S. pyogenes-infected Ifnar1−/− BMDMs secreted 2-fold more IL-1β than WT BMDMs (Figure 6A). Exogenous IFN-β suppressed IL-1β secretion by WT but not Ifnar1−/− BMDMs (Figure 6A). Tnf levels were not affected by IFN-I signaling (Figure 6A). Western blot analysis of supernatants confirmed that IL-1β measured by ELISA was the mature cytokine (Figure 6B). The analysis confirmed inhibition of IL-1β but not Tnf by IFN-I signaling (Figures 6B and S1). Pro-IL-1β was higher in cell lysates of infected Ifnar1−/− BMDMs compared to WT cells, and exogenous IFN-β suppressed pro-IL-1β in WT but not Ifnar1−/− BMDMs (Figures 6C and S2). Thus, pro-IL-1β was suppressed by IFN-I, as was mature IL-1β release into cell supernatants. The levels of the IL-1β-processing enzyme caspase-1 (both procaspase-1 and the processed p20 form) were similar in infected WT and Ifnar1−/− BMDMs, although a slight decrease was detected upon treatment of WT BMDMs with IFN-β (Figure 6C). Together, these results indicate that IFN-I signaling is suppressing IL-1β largely before translation rather than at the posttranslational processing steps. Il1b mRNA induced by S. pyogenes was also upregulated in Ifnar1−/− BMDMs when compared to WT cells, and exogenous IFN-β suppressed Il1b mRNA in WT but not Ifnar1−/− BMDMs (Figure 6D). Tnf mRNA induction was not affected by the genotype or IFN-β treatment (Figure 6D). Il1b mRNA levels are strongly influenced by 3′ UTR-mediated regulation of mRNA stability (Kratochvill et al., 2011Kratochvill F. Machacek C. Vogl C. Ebner F. Sedlyarov V. Gruber A.R. Hartweger H. Vielnascher R. Karaghiosoff M. Rülicke T. et al.Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation.Mol. Syst. Biol. 2011; 7: 560Crossref PubMed Scopus (89) Google Scholar). However, mRNA decay assays did not show any effect of IFN-β on Il1b mRNA stability (Figure 6E), suggesting that IFN-I signaling regulated the transcription of Il1b. To measure changes in transcription, we examined the levels of nascent transcripts (pre-mRNA). S. pyogenes-mediated Il1b pre-mRNA induction was higher in Ifnar1−/− BMDMs than in WT cells, and exogenous IFN-β suppressed Il1b mRNA in WT but not Ifnar1−/− BMDMs (Figure 6F), confirming that IFN-I signaling inhibited Il1b gene transcription. Il1a induction was not affected by the lack of IFN-I signaling (Figure S3). IFN-I reduced Il1b gene transcription also in cDCs (Figure 6G). Similarly, IFN-β suppressed IL-1β but not Tnf production by peritoneal neutrophils (Figure 6H). Because S. pyogenes is a human-specific pathogen, we asked whether IFN-I signaling suppressed IL-1β production in human cells. Primary human monocytes and macrophages have been shown to produce IFN-β in response to bacterial RNA and infection, among others with S. pyogenes, in a TLR8-dependent way (Bergstrøm et al., 2015Bergstrøm B. Aune M.H. Awuh J.A. Kojen J.F. Blix K.J. Ryan L. Flo T.H. Mollnes T.E. Espevik T. Stenvik J. TLR8 senses Staphylococcus aureus RNA in human primary monocytes and macrophages and induces IFN-β production via a TAK1-IKKβ-IRF5 signaling pathway.J. Immunol. 2015; 195: 1100-1111Crossref PubMed Scopus (91) Google Scholar, Eigenbrod et al., 2015Eigenbrod T. Pelka K. Latz E. Kreikemeyer B. Dalpke A.H." @default.
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• W2296304057 title "Type I Interferon Signaling Prevents IL-1β-Driven Lethal Systemic Hyperinflammation during Invasive Bacterial Infection of Soft Tissue" @default.
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