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Innate and Adaptive Interferons Suppress IL-1a and IL-1b Production by Distinct Pulmonary Myeloid Subsets during Mycobacterium tuberculosis Infection
Katrin D. Mayer-Barber,1,* Bruno B. Andrade,1 Daniel L. Barber,1 Sara Hieny,1 Carl G. Feng,1 Patricia Caspar,1 Sandy Oland,1 Siamon Gordon,2 and Alan Sher1,* 1Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA 2Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK *Correspondence: [email protected] (K.D.M.-B.), [email protected] (A.S.) DOI 10.1016/j.immuni.2011.12.002
SUMMARY is highlighted by the wide variety of immunopathologies and autoinflammatory diseases that occur in the absence of normal Interleukin-1 (IL-1) receptor signaling is necessary for IL-1 regulation (Dinarello, 2009, 2010; Garlanda et al., 2007). control of Mycobacterium tuberculosis (Mtb) infec- Surprisingly little is known about the expression and processing tion, yet the role of its two ligands, IL-1a and IL-1b, of IL-1 in the context of Mtb infection in vivo. Whereas IL-1b and their regulation in vivo are poorly understood. production in response to Mtb in vitro is dependent on the Here, we showed that both IL-1a and IL-1b are NALP3-ASC inflammasome, we and others have recently re- critically required for host resistance and identified ported that in lungs of Mtb-infected mice, cleaved IL-1b is found even in the absence of the critical inflammasome component two multifunctional inflammatory monocyte-macro- caspase-1. Moreover, although IL-1b is absolutely critical phage and DC populations that coexpressed both for host control of Mtb infection in mice, NLRP3, ASC, or Mtb IL-1 species at the single-cell level in lungs of - caspase-1 play minor roles in host resistance to this pathogen infected mice. Moreover, we demonstrated that (Mayer-Barber et al., 2010; McElvania Tekippe et al., 2010; interferons (IFNs) played important roles in regu- Walter et al., 2010). Although it is not clear what factors mediate lating IL-1 production by these cells in vivo. Type I the processing of IL-1b during Mtb infection in vivo, the inflam- interferons inhibited IL-1 production by both subsets masome independence of the IL-1b response may indicate whereas CD4+ T cell-derived IFN-g selectively sup- that it is produced by an atypical cellular source with unique pressed monocyte-macrophages. These data pro- IL-1b-processing properties. For example, the neutrophils vide a cellular basis for both the anti-inflammatory present in arthritic joints have been shown to be both a key effects of IFNs and probacterial functions of type I producer of the cytokine and to cleave IL-1b in a caspase-1- independent manner (Guma et al., 2009; Joosten et al., 2009). IFNs during Mtb infection and reveal differential The cell populations that produce host-protective IL-1 during regulation of IL-1 production by distinct cell popula- Mtb infection in vivo, however, have not yet been characterized. tions as an additional layer of complexity in the Type II interferon, IFN-g, is the signature cytokine of T helper 1 activity of IL-1 in vivo. (Th1) cells and has well-established protective functions in host resistance to Mtb and other mycobacterial infections in mice and humans. IFN-g is traditionally regarded as a proinflammatory INTRODUCTION cytokine and its protective function during Mtb infection involves the activation of macrophage (Flynn and Chan, 2001). In con- Mycobacterium tuberculosis (Mtb) primarily infects mononuclear trast, the role of type I IFN in the immune response to virulent phagocytes. Host resistance against this important pathogen is Mtb is less clear and even though type I and II IFNs share similar critically dependent on innate immune functions exerted by STAT1-dependent signaling pathways, type I IFNs appear to these cells including the production of inducible nitric oxide syn- promote rather than control infection. Thus, the hypervirulence thase (iNOS), TNF-a, and IL-12,23 p40 (Cooper et al., 2011; of certain Mtb strains correlates with enhanced type I IFN syn- North and Jung, 2004). More recently, it has become clear that thesis and type I IFN receptor-deficient mice infected with Mtb IL-1 is also of critical importance for host control of Mtb infection display lower bacterial loads when compared to WT animals given that mice deficient in IL-1R or its adaptor MyD88 succumb (Manca et al., 2001; Stanley et al., 2007). In addition, Mtb- rapidly to low-dose aerosol infection with Mtb (Fremond et al., infected mice intranasally treated with the type I IFN inducer 2007; Mayer-Barber et al., 2010). Although we have shown that polyinosinic-polycytidylic acid stabilized with poly-L-lysine IL-1R-dependent protection requires IL-1b (Mayer-Barber (pICLC) exhibit exacerbated lung pathology and increased et al., 2010), the contribution of IL-1a, the second major cytokine bacterial burden (Antonelli et al., 2010). The relevance of these agonist for this receptor, has been unclear. observations to human tuberculosis is supported by a recent A major feature of IL-1 is its complex control at the transcrip- study in which the whole blood transcriptional profile of TB tional, posttranscriptional, and signal transduction levels, which patients was found to be dominated by type I and II IFN-induced
Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. 1023 Immunity IFNs Limit IL-1 Production during Tuberculosis
–/– ABFigure 1. Mtb-Infected Il1a Mice Display Acute Percent Percent Lung Mortality and Elevated Bacterial Loads Compa- body weight (%) survival (%) 10 rable to Il1b–/– Animals ) * * À/À À/À À/À 110 9 (A) Body weight and survival of WT, Il1r1 , Il1a Il1b ,
100 10 Il1bÀ/À, and Il1aÀ/À mice after aerosol exposure to Mtb 80 8 100 * * (H37Rv). 60 7 90 (B) Bacterial loads measured 25 days p.i. in lungs of the 40 6 above mouse strains. 80 CFU (log 20 (C) Indicated cytokines were measured by ELISA in BAL WT 5 À/À À/À À/À -/- -/- -/- -/- fluid of WT (white bars), Il1r1 (black bars), Il1a Il1b 70 0 WT À/À À/À Il1r1 Il1b Il1a (dark-gray bars), Il1b (light-gray bars), and Il1a (gray 110 100 C Il1a,Il1b and white striped bars) mice and the means ± SD 80 100 IL-1α (pg/ml) IL-1β (pg/ml) depicted. Dotted lines indicate the limits of detection of the respective assays. Data are representative of a minimum 60 104 * 104 90 of two independent experiments each involving five to ten 40 * 103 103 % 80 mice per group. The asterisk denotes significant (p 0.05) 20 Il1r1-/- 102 102 differences compared to WT controls. 70 0 101 101 110 100 0 0 100 80 IL-1Ra (pg/ml) IL-17 (pg/ml) 104 103 60 ** RESULTS 90 40 102 80 103 20 IL-1a and IL-1b Are Each Critical for Host Il1a,Il1b-/- 101 70 0 ** Resistance to Mtb 102 0 110 To investigate the respective contributions of the 100 TNF-α (pg/ml) IL-12,23 p40 (pg/ml) 5 two ligands IL-1a and IL-1b in IL-1R1-dependent 80 10 104 100 * host resistance to Mtb, we performed a series of 60 104 * 90 experiments directly comparing the suscepti- 40 103 103 À/À À/À À/À À/À 80 bility of Il1r1 , Il1a Il1b , Il1b , and 20 2 À/À Il1b-/- 10 Il1a mice to low-dose aerosol (100–150 CFU) 70 0 101 102 infection. As described previously (Mayer- γ 110 100 IFN- (pg/ml) total nitrite (nmol/L) Barber et al., 2010), IL-1R1- and IL-1b-deficient 105 2 80 10 * * 100 *** mice rapidly lost weight and succumbed within 60 104 40 days (Figure 1A). No further increase in 90 40 101 susceptibility was evident in mice doubly defi- 103 80 -/- 20 cient in both IL-1a and IL-1b. Importantly, mice Il1a 102 0 70 0 deficient in IL-1a alone displayed similar weight -/- -/- -/- -/- -/- -/- -/- À À À À À À -/- 010203040 010203040506070 WT / / / WT loss and mortality as Il1r1 , Il1a Il1b ,or Il1b Il1a Il1b Il1a Il1r1 À À Time post infection (days) Il1r1 Il1b / mice and showed increased bacterial
Il1a,Il1b À À Il1a,Il1b loads equivalent to that of infected Il1b / animals (Figures 1A and 1B). Moreover, we genes, and this signature closely correlated with disease severity observed similar amounts of IL-1b in the lungs of WT and IL-1a (Berry et al., 2010). Nevertheless, the cellular mechanisms by single-deficient mice and conversely comparable amounts of which type I IFNs mediate their probacterial effects in Mtb infec- IL-1a in WT and IL-1b-deficient animals (Figure 1C). IL-1a and tion and whether or not they involve crosstalk with other innate IL-1b singly deficient animals each exhibited lower bacterial cytokine pathways remains unclear. counts than Il1r1À/À or Il1aÀ/ÀIl1bÀ/À mice, highlighting a dual Here, we establish that IL-1a and IL-1b (IL-1a,b) each had requirement for both IL-1 species and suggesting a possible critical functions in murine host resistance against Mtb and synergy in the antibacterial function of the two cytokines. Indeed, identified and characterized the two major myeloid subsets when infected with a lower dose (<50 CFU) of Mtb, both Il1aÀ/À that produce both cytokines in the lungs of Mtb-infected mice and Il1bÀ/À mice survived longer than Il1r1À/À animals (Figure S1 as inflammatory monocyte-macrophages (iMs) and DCs (iDCs) available online). Taken together, these observations argue that with highly polyfunctional properties. Importantly, we demon- IL-1a and IL-1b each have critical nonredundant functions and strated that the endogenous type I IFN was a potent negative cooperate in IL-1R1-mediated host resistance to Mtb. regulator of IL-1a,b production by both of these myeloid cell populations. In addition, our data revealed an anti-inflammatory IL-1a and IL-1b Are Coexpressed by CD11bpos Cells in role for CD4+ T cell-derived IFN-g in dampening IL-1 expression the Lungs of Mtb-Infected Mice by iMs. Together, these findings established polyfunctional Studies in which we measured IL-1a,b proteins in lung homoge- mononuclear phagocytes as the major myeloid source of IL-1 nates of Mtb-infected mice indicated that the cytokines are during Mtb infection and provided a cellular basis for the cross- induced during the acute phase beginning at 2 weeks and talk between IFNs and IL-1 in the immune response to this peak at 3–4 weeks postinfection (p.i., data not shown). For this bacterial pathogen in vivo. reason we chose d25-d28 as the time point for cellular analysis
1024 Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. Immunity IFNs Limit IL-1 Production during Tuberculosis
pos neg neg Lung d28 p.i. Naive lung A CD45 ,TCRβ ,CD19 ,Live IL-1α IL-1β B unst. 5hr Mtb 0.3 WT Il1a-/- Il1b-/- Il1a,Il1b-/- WT 0.2 7 ± 2 25 ± 6 0.2 ± 0 13 ± 7 0.2 ± 0 3.3 ± 2 ng/ml hi 0.1 ,Live hi
Ly6G Ly6G ND
0 Ly6G CD11b unst.Mtb unst.Mtb CD11b IL-1α IL-1β IL-1α IL-1β 7 ± 2 15 ± 5 9.1 ± 4 0.3 0.3 0.1 ± 0
,Live ,Live 15 ± 5 0.2 0.2 neg pos ng/ml ng/ml 0.1 0.1 Ly6G
IL-1 α 31 ± 11 0.1 ± 0 ND ND ND ND
0 0 CD11b unst.Mtb unst.Mtb unst.Mtb unst.Mtb IL-1β
C 2.0 D E F ) total IL-1αpos CD45pos,autofl.neg, CD45pos,autofl.neg, CD45pos,autofl.neg, 6 iM 1.5 pos neg pos, neg pos, neg α,β CD11b ,Ly6G ,Live 21± 5 CD11b Ly6G , 60 CD11b Ly6G , IL-1 DP pos hi ,Live ,Live CD68 ,Live CD68pos,Live
hi 1.0 40 3 23 Ly6Chi CD11cneg Ly6G 0.5 20 IL-1 α CD11b IL-1 α,β DP (%) Ly6C 0 CD68 β 0 CD282 IL-1 unst. IL-1 α 2.0 44 IL-1α CD11c 64 ± 7 Mtb iDC 1.5 30± 6 CD11c CD282 ,Live ,Live 60 2 10 Ly6C Ly6C neg 1.0 pos CD11c 40
Ly6G 0.5 CD11cpos 20 IL-1 α CD13 IL-1 α IL-1 producing cells in lung (x10 CD11b IL-1 α,β DP (%) 65 0 NK1.1 Ly6C 0 0 10 20 30 40 50 75 100 IL-1β unst. Mtb TCRβ CD11c 58 ± 7 CD11c CD13 Time post infection (days)
Figure 2. IL-1a and IL-1b Are Coexpressed by Two Pulmonary CD11bpos Populations, Distinguished by Ly6C, CD11c, CD13 and CD282 Expression during Mtb Infection (A) Pulmonary single-cell suspensions from mice at 4 weeks p.i. were FACS sorted on the basis of Ly6G and CD11b expression into three populations and subsequently stimulated for 18 hr with H37Rv (Mtb) or left unstimulated (unst.). Data shown are means ± SD of IL-1a and IL-1b in culture supernatants. (B) Lung cells from naive mice or mice at 4 weeks p.i. were stimulated for 5 hr with (5hr Mtb) or without (unst.) H37Rv and then stained intracellularly for IL-1a and IL-1b. Numbers indicate mean percentage (±SD) of IL-1 producing cells in depicted gate. (C) Data depict the mean number of IL-1 producing (±SD) cells per lung at various time points after infection. (D) CD68 staining of IL-1 producing cells and use of Ly6C and CD11c for further subsetting (numbers indicate percentage of respective population in depicted gate ± SD). (E) Proportion of IL-1a,b coproducing cells 4 weeks p.i. in each subset after restimulation for 5 hr with (Mtb, dark circles) or without (unst., white circles) H37Rv. Each connecting line depicts an individual animal. (F) CD282 and CD13 expression in correlation with CD11c and IL-1a. Numbers indicate percentage of IL-1-producing cells in depicted gate after restimulation for 5 hr with Mtb. Data in all panels are representative of a minimum of two experiments with three to ten mice each.
of IL-1 production in our studies. Initial experiments utilizing ex vivo restimulation with irradiated (or live) H37Rv (Figure 2B Il1a,Il1bÀ/À/WT BM chimeras confirmed a requirement for and data not shown). The latter observation argues that the hematopoietic cell-derived IL-1 in host resistance (data not potential for IL-1 expression by pulmonary CD11bpos cells is shown). Ex vivo cell sorting experiments from Mtb-infected lungs acquired in the context of Mtb infection in vivo probably via were then performed 4 weeks p.i. for assessing IL-1 production changes in cellular composition, recruitment, and activation by hematopoietic cells of which mononuclear phagocytes and status, rather than through the 5 hr restimulation per se. Consis- neutrophils are the major candidates for production of the tent with the above findings on FACS isolated cell populations, cytokine in vivo. We generated lung single-cell suspensions Ly6Gneg CD11bpos cells expressed the most IL-1a,b by intracel- and FACS sorted non-T and -B cells into CD11bneg cells, lular staining, whereas neutrophils from infected lungs stained Ly6GhiCD11bhi neutrophils, and Ly6GnegCD11bpos cells and weakly for IL-1a but not IL-1b and CD11bneg cells were negative stimulated the cells overnight in the presence or absence of for both cytokines (Figure 2B and data not shown). Importantly, live Mtb. IL-1a,b production was found to be largely confined the ICS analysis revealed that in the Ly6Gneg CD11bpos popula- to the Ly6Gneg CD11bpos sorted fraction, whereas neutrophils tion IL-1a and IL-1b are coexpressed at the single-cell level. generated little and Ly6GnegCD11bneg cells undetectable Moreover, in the early stages of infection, the kinetics of the amounts of IL-1 (Figure 2A). appearance of pulmonary IL-1a,b double-positive (IL-1a, bDP) To analyze IL-1 production at the single-cell level, we per- Ly6Gneg CD11bpos cells closely mirrored the cytokine proteins formed intracellular cytokine staining (ICS) of cells from digested measured by ELISA in lung homogenates of infected mice lung. Importantly, we observed strong IL-1a,b staining in cells (Figures 2C and data not shown). Together, these data show derived from 4 weeks infected but not naive lungs after 5 hr that Ly6Gneg CD11bpos cells and not neutrophils are the major
Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. 1025 Immunity IFNs Limit IL-1 Production during Tuberculosis
hematopoietic source of IL-1 in the lungs of infected mice and IL-12,23p40. In contrast, robust IL-12,23p40 as well as TNFa, that this population coordinately expresses IL-1a,b at the cellular iNOS, and IL-10 production was observed in iDC cells (Figures level. 3A–3D). To determine whether expression of these mediators correlates with IL-1 production, we costained the same popula- The IL-1a,b-Producing Cells in the Lungs of Mtb- tions for IL-1a,b. In both populations, iNOS, TNF-a, and IL-10 Infected Mice Consist of Two Major Populations expression was largely restricted to IL-1a,b DP cells (Figures Distinguishable by Their Expression of CD11c and Ly6C 3B–3D and data not shown). Importantly, within the iDC subset, To discriminate between myeloid versus lymphoid CD11bpos IL-1a,b DP cells failed to make IL-12,23p40, which was instead cells, we included the intracellular macrophage-myeloid marker produced by a separate functionally distinct DC subpopulation CD68 (Figures 2D and S2A) and found that 4 weeks p.i. 30%– (Figures 3B–3D and data not shown). Thus, IL-1-producing 40% (31% ± 8%) of pulmonary Ly6GnegCD11bpos cells were of myeloid cells in the lungs of Mtb-infected mice display a high nonmyeloid origin, such as NK and T cells (Figure 2D). This degree of functional heterogeneity at both the population and analysis revealed that within the CD11bpos population IL-1a,b single-cell level and are key producers of the antimycobacterial expression is restricted to CD68pos cells (Figure 2D and data effector molecules iNOS and TNF-a. Taken together, the above not shown). Moreover, CD68pos, CD11bpos myeloid cells segre- phenotypic and functional analyses provided a platform for gated into two populations based on differential CD11c and investigating the regulation of IL-1a,b cytokine production by Ly6C expression, with the CD11cpos subset expressing varying myeloid cells at the cellular level during Mtb infection in vivo degrees of Ly6C (referred to as iDC cells) while the CD11cneg (Figure S5A). cells displayed uniformly high expression of Ly6C (referred to as iM cells) (Figures 2D–2F). Importantly, the IL-1a,b-coproduc- IFNs Negatively Regulate the IL-1 Pathway in ing cells also dissociated into the same two populations based Mtb-Infected Mouse and Human Mononuclear on CD11c and Ly6C expression (Figures 2D and 2E and data Phagocytes In Vitro not shown). In addition to CD11c itself, we observed that Type I and type II IFNs have been implicated in the regulation of IL-1a,bDP pulmonary iDC cells can also be distinguished from the IL-1 pathway in vitro as well as in autoimmune and infectious iM cells by their high expression of CD13 (aminopeptidase N) diseases (Hu et al., 2005; Schindler et al., 1989; Thacker et al., and CD282 (TLR2) (Figure 2F and data not shown). 2010; Tilg et al., 1993). As noted above, the major IL-1a,b-pro- Further phenotypic characterization revealed that iM cells ducing subsets in Mtb-infected mice exhibited upregulated display striking homogeneity with unimodal expression of all expression of IFN-inducible markers and displayed STAT1 phos- markers screened and thus probably represent a single popula- phorylation. These observations suggested the possible involve- tion (Figure S2). In contrast, expression of most markers by the ment of type I IFN and IFN-g signaling in regulating IL-1 expres- iDC cells was heterogeneous, suggesting the presence of sion in the response of myeloid cells to Mtb infection. multiple subpopulations. iDC cells, when compared to iM cells, To study the effects of type I and type II IFNs on IL-1a,b also displayed higher expression of markers associated with production in the context of Mtb, we first compared the IL-1a,b antigen presentation, suggesting that the iDC subsets con- response of murine and human mononuclear phagocytes sists of multiple DC populations (Figures S2B and S2C). iDC exposed in vitro to Mtb in the presence or absence of IFNs (Fig- IL-1a,b-expressing cells represent one such subpopulation ure 4). We found that pICLC-induced type I IFN and recombinant displaying a unique phenotype characterized by selective upre- IFN-g potently inhibited IL-1a,b cytokine production by murine gulation of CD14, CD206, CD210, CD64, CD30L, CD70, and bone marrow-derived macrophages (BMMFs) after Mtb expo- CD38 among others (Figures 2B–2D). Of note, we found that sure (Figures 4A and 4B). In contrast, in bone marrow-derived both CD11bpos subsets expressed the ligand-binding chain of dendritic cells (BMDCs), IL-1a,b were suppressed by IFN-g but the IFN-g receptor (CD119, IFN-gR1) as well as IFN-inducible not pICLC. As expected, pICLC significantly inhibited IL-1 markers such as Ly6C, PD-L1, MHC class I+II, Sca-1, Fcg recep- production by Ifngr1À/À but not Ifnar1À/À BMMFs and in addi- tors (Figures S2B and S2D), and STAT1 phosphorylated at tion, IFNs did not universally downregulate proinflammatory tyrosine 701 (Figure S2E), arguing that both cell populations cytokines given that TNF-a protein was upregulated in the pres- are subject to IFN-mediated signals in vivo during Mtb infection. ence of pICLC and IFN-g in both cell types (data not shown). Through our findings, we identified two distinct pulmonary IL-27, which also utilizes STAT1 for signal transduction, was CD11bposCD68pos myeloid subsets on the basis of Ly6C and unable to inhibit IL-1 expression, arguing that the suppressive CD11c expression that are capable of coproducing both IL-1a effects of IFNs reflect specific functions of the latter cytokines and IL-1b at the single-cell level after Mtb infection. rather than STAT1 activation per se (Figure 4A). In order to test the relevance of these observations to the Mtb Pulmonary IL-1a,b Expressing Myeloid Cells response of humans, we infected monocyte-derived macro- Are Multifunctional but Distinct from phages (MFs) or monocyte-derived DCs from 21 healthy blood IL-12,23p40-Producing Cells during Mtb Infection donors with Mtb and assessed their ability to generate IL-1a,b We next functionally characterized the two IL-1a,bDP CD11bpos in the presence or absence of IFNs. In agreement with a recent myeloid populations present in the Mtb infected lung for their study from our group focusing on differences in IL-1b regulation ability to produce TNFa, iNOS, IL-10 and IL-12,23p40, mediators in virulent versus avirulent mycobacteria in human monocytes- previously shown to play important roles in host resistance to the macrophages (Novikov et al., 2011), we found that IFN-b inhibits pathogen (Cooper et al., 2011; Redford et al., 2011). The iM production of the former cytokine in macrophages. Here, we cells were found to produce TNFa, iNOS and IL-10 but not show that in Mtb-infected DCs as well as MF pICLC and IFN-b
1026 Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. Immunity IFNs Limit IL-1 Production during Tuberculosis
A 50 60 20 20 suppress both IL-1a and IL-1b and that IFN-g inhibits IL-1b but (%) 40 50 not IL-1a production (Figures 4C). Despite the statistically signif- 15
(%) 15 pos (%) (%) neg
hi 40 30 icant suppression in IL-1b by IFN-g, there was a substantial frac- pos pos 30 pos 10 10 20 20 tion of donors that responded to Mtb infection with lower Ly6C 5 IL-10 5 iNOS 10 TNF α % CD11c 10 amounts of IL-1b (52% 4 ng/ml (MF), 8 ng/ml (DC):low 0 0 0 IL-12,23p40 0 responders; LR), and in these individuals little to no additional unst. Mtb unst. Mtb Mtbunst. Mtbunst. reduction in IL-1b protein was observed after IFN-g treatment 50 60 20 20
50 (%) (MF: 29.6% ± 23.4% reduction in LR; 66.9% ± 13.4% reduction 40 15 15 (%) pos pos (%) 40 (%) 30 in high responders [HRs]; DCs: 38.5% ± 30.1% reduction in
pos 10 10 pos 30 20 pos LR;78.7% ± 0.2% reduction in HR), whereas pICLC or IFN-b 20 5 5
10 IL-10 b iNOS treatment potently suppressed IL-1 by both LR and HR TNF α CD11c 10
0 0 0 IL-12,23p40 0 (>70% ± 5%; Figure 4C and data not shown). This donor varia- unst.Mtb Mtbunst. Mtbunst. Mtbunst. tion may explain the divergent results in our previous study B (Novikov et al., 2011) in which we failed to observe suppression
neg hi of IL-1b by IFN-g in human macrophage. In the murine as well as human systems, we observed upregu- Ly6C
CD11c lation of IL-1R antagonist (IL-1Ra) in a dose-dependent manner by type I and II IFNs in each cell type with the exception of murine DCs (Figure 4D and data not shown). Thus, in both murine and pos human mononuclear phagocytes, IFNs antagonize the IL-1 pathway by dual mechanisms: first by directly suppressing IL-1 α
CD11c IL-1a,b cytokine production and second by upregulation of the iNOS TNFα IL-10 p40 soluble endogenous IL-1Ra, which opposes IL-1 by competing for IL-1R1 binding. C Ly6Chi CD11cneg D Ly6Chi CD11cneg When we investigated possible sources of type I IFN, we found 40 that both murine BMMFs and BMDCs generated detectable 100 lung d28 p.i. 30 5hr Mtb restim (%) amounts of IFN-b in response Mtb infection (Figure 4E). Type I 80 20 IFNs are potent inducers of IL-10 (Chang et al., 2007) and pos 60
positive (%) positive 10 IL-10 has recently been reported to mediate type I IFN’s 40 Percent cytokine Percent 0 suppressive effects on IL-1 (Guarda et al., 2011). In the context 20 IL-1α,β + + + + of Mtb infection in vitro, we observed that IL-10 production is 0 TNFα within IL-1 α + + ++ critically dependent on IFN-aR signaling and that IL-10 is iNOS + + + + Percent cytokine positive Percent
p40 induced by IFN-b and pICLC in a dose-dependent manner in IL-1 β IL-10 iNOS TNF α BMMFs and BMDCs (Figures 4E and 4F and data not shown). pos CD11cpos CD11c To investigate the contribution of IL-10 in type I IFN-mediated 100 20 lung d28 p.i. (%) suppression of IL-1 during Mtb infection in vitro, we added exog- 80 15 5hr Mtb restim enous IL-10 to Mtb-infected BMDMs and observed a significant pos 60 10 inhibition of IL-1b production (Figure 4G). More importantly, the 40 positive (%) positive 5 Mtb-induced IFN-b-mediated suppression of IL-1b was partially 20 cytokine Percent 0 reversed when endogenous IL-10 was neutralized (Figure 4H). IL-1α,β within IL-1 α 0 + + + + Thus in vitro, besides direct suppressive effects, type I IFNs TNFα Percent cytokine positive Percent + + ++
p40 iNOS + + + + can also indirectly inhibit IL-1 production during Mtb infection IL-1 β IL-10 iNOS TNF α via IFN-aR-dependent induction of IL-10. Figure 3. IL-1a,b Coexpressing Cells in the Lungs of Mtb-Infected Mice Are Highly Polyfunctional but Distinct from IL-12,23 p40- Endogenous Type I IFN Suppresses IL-1a,b Production Producing Cells by Both iM and iDC Cells In Vivo (A–D) Quantitative and qualitative analysis of cytokine production in indicated A probacterial role for endogenous type I IFNs has been demon- subsets 28 days after Mtb infection of WT mice. strated previously in experiments in which Ifnar1À/À mice were (A) Frequencies of iNOS-, TNF-a-, IL-10-, and IL-12,23p40-expressing cells within iM (top) and iDC (bottom) cell subsets after restimulation for 5 hr with shown to be less susceptible to Mtb infection (Manca et al., (Mtb, dark circles) or without (unst., white circles) H37Rv determined by ICS. 2005; Stanley et al., 2007). As shown in Figures 5A and 5B, Each connecting line depicts an individual animal. this decrease in bacterial burden was closely associated (B) Costaining of IL-1a with indicated cytokines and iNOS. with an increase in IL-1a,b production by pulmonary myeloid (C) Percentage of cytokine-producing cells within IL-1a-expressing cells of the cells. iM (top) or iDC (bottom) cell subset. (D) Simultaneous analysis of the functional profile of pulmonary iM (top) and iDC (bottom) cell subsets after Mtb infection on the basis of IL-1a, IL-1b, iNOS, and TNF-a expression. All combinations of the possible cytokine expression the proportion of iM or iDC cells expressing a given combination of cytokines patterns are marked on the x axis, whereas the percentages (mean ± SD) of the indicated by the colored boxes at the bottom of the x axis. distinct cytokine-producing subsets within iM or iDC cells are shown on the Data in all panels are representative of a minimum of three independent y axis. The data are summarized in pie charts and each slice corresponds to experiments with three to six animals each.
Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. 1027 Immunity IFNs Limit IL-1 Production during Tuberculosis
A D Figure 4. Type I and II IFNs Negatively Regulate IL-1a and IL-1b Secretion by Murine and Human Myeloid Cell Subsets In- fected with Mtb (A) Murine cytokines measured by ELISA in supernatants of BMMFs and BMDCs from WT mice after exposure to live Mtb (MOI:1) for 48 hr in the presence or absence of recombinant murine IL-27, IFN-g, or pICLC. (B) IL-1b protein in supernatants of BMMFs and BMDCs from WT, Ifnar1À/À,orIfngr1À/À mice incubated with IFN-g or pICLC as indicated after exposure to live Mtb (MOI:1). (C) Human IL-1a and IL-1b measured by ELISA in culture supernatants of human monocyte-derived macrophages [MF]) or monocyte-derived DCs from 21 healthy donors after 24 hr exposure to live Mtb (MOI:5) in the presence or absence of pICLC or the recombinant human cytokines IFN-b and B IFN-g. Horizontal lines indicate the median values. Three asterisks denote significant (p < 0.0001) differences compared to Mtb exposure alone. (D) IL-1Ra protein in culture supernatants of human MFs or DCs (top panels) and murine BMMFs and BMDCs (bottom panels) incubated E as noted in (A) and (B). (E) Cytokines in supernatants of BMMFs derived from WT or Ifnar1À/À mice after exposure to live Mtb (MOI:1) for 24 hr. (F) IL-10 protein in supernatants of WT BMMFs incubated with increasing amounts of pICLC for 40 hr in the presence or absence of Mtb infection. C (G) IL-1b protein in supernatants of WT BMMFs after exposure to live Mtb (MOI:1) for 24 hr incu- bated with recombinant murine IFN-b in the pres- ence or absence of neutralizing IL-10 mAb. F Murine data presented are the means ± SD and representative of two to five independent experi- ments. The asterisk denotes significant (p % 0.05) differences compared to Mtb exposure alone or as indicated with connecting lines.
and 4 weeks later the frequency of G H IL-1a,b DP cells within pulmonary WT or Ifnar1À/À cells was analyzed (Figure 5D). Importantly, in both the iM and the iDC cell subsets, the inability of Ifnar1À/À cells to receive type I IFN signals resulted in a marked increase in the proportion of IL-1a,bDP, even without Mtb restimula- tion (Figures 5D and 5E). In addition, we found that type I IFN signaling sup- pressed iNOS production by iM and iDC To test whether endogenously induced type I IFNs can act cells, and in accordance with the in vitro data presented directly on IL-1a,b-producing cells during Mtb infection in vivo, above, it also induced IL-10 expression by myeloid cells in vivo we generated mixed bone marrow chimeras allowing us to after Mtb infection (Figures 5F, 5G, and S5). In contrast, TNF-a compare Ifnar1À/À and WT myeloid cells in the lungs of the expression was not subject to type I IFN regulation. Furthermore, same animal. This approach controls internally for potential phenotypic analysis revealed that type I IFN signaling influenced differences in bacterial load, inflammatory milieu, and/or cellular expression of only a few IFN inducible and surface markers, microenvironment. WT CD45.1,1 mice were lethally irradiated most notably CD206 and PD-L1 (Figure S3A). Together, these and reconstituted with BM from WT (CD45.1,2) donors mixed data argued that ligation of the type I IFN receptor on each at a 1:1 ratio with Ifnar1À/À CD45.2,2 donors (Figure 5C). The of the two myeloid populations directly suppresses IL-1a,b mixed Ifnar1À/À, WT BM chimeras were then infected with Mtb production in vivo.
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A Lung d28 p.i. B C D pos neg pos pos neg 7.0 CD45 ,autofl. ,CD11b , CD45.1,2 CD45.2,2 CD45 ,autofl. , IL-1α,βDP (%) Ly6Gneg,CD68pos,Live -/- CD11bpos,Ly6Gneg, ) WT B6 BM Ifnar1 BM * CD68pos,Live 70 10 6.5 -/- * WT Ifnar1 60 WT 1:1 53 ± 6 50 6.0 40 30
CFU ( Log
Figure 5. Endogenous Type I IFNs Suppress IL-1a,b Expression by Both iM and iDC Pulmonary Cell Subsets during Mtb Infection (A) Pulmonary bacterial loads measured 4 weeks p.i. in WT or Ifnar1À/À mice. The asterisk denotes significant (p % 0.05) difference compared to WT control. (B) ICS for IL-1a,b by pulmonary myeloid cells in WT or Ifnar1À/À mice 4 weeks p.i. Data are representative of two independent experiments each involving three to five mice per group. Numbers indicate mean percentage (±SD) of IL-1 producing cells in depicted gate. (C) WT CD45.1,1 mice were lethally irradiated and reconstituted with equal ratios of WT (CD45.1,2) and Ifnar1À/À (CD45.2,2) BM cells and infected with Mtb. (D) Analysis of donor BM-derived CD11bpos myeloid cells 4 weeks p.i. in isolated lung cells marked by CD45.1 and CD45.2 expression (percentage ± SD) and frequency of IL-1a,b expression by WT (white circles) or Ifnar1À/À (KO, dark circles) total CD11bpos mononuclear myeloid cells after restimulation for 5 hr with (Mtb) or without (unst.) Mtb. Each connecting line depicts an individual animal. (E) Frequency of IL-1a,b expression by iM and iDC cell subsets in mixed Ifnar1À/À BM chimeric mice. (F and G) Frequencies of iNOS-, TNF-a-, and IL-10-expressing cells within pulmonary WT (white circles) or Ifnar1À/À (KO, dark circles) iM cells (F) and within iDC (G) cells. Data in (A)–(F) are representative of three independent experiments with three to five mice each. The asterisks denote significant (p % 0.05) differences compared to WT controls (ns, not significant).
Host-Protective IFN-g Inhibits IL-1a,b Production abolished in IFN-gR-deficient cells compared to WT cells in the by iM Cells same animal. Moreover, whereas TNF-a expression in both Because in addition to type I IFNs, IFN-g also displayed potent myeloid subset was unaffected by the absence of IFN-gR, suppressive effects on IL-1 expression in vitro, we next used IFN-g potently suppressed IL-10 production in iM but not iDC the same mixed BM chimeric approach to determine whether cells (Figures 6C, 6D, and S5). Thus, while clearly a host-protec- IFN-g signaling also regulates IL-1a,b production by myeloid tive cytokine in Mtb infection, IFNg in common with type I IFN can cells during Mtb infection in vivo. In these experiments, we con- also be anti-inflammatory suppressing IL-1a,b production by structed mixed Ifngr1À/À,WT BM chimeric mice and functionally pulmonary myeloid cells. and phenotypically analyzed gene deficient or WT CD11bpos subsets in the same animal for IFN-inducible surface marker CD4+ T Cells Regulate IL-1 Expression during Mtb expression and their capacity to generate innate cytokines Infection through Their Production of IFN-g (Figures 6 and S3B). Importantly, in the lungs of Mtb infected Th1 cells are a major source of IFN-g during Mtb infection in mice chimeric mice, IL-1 production in the iM cell subset was mark- and therefore could be a key mediator of the suppression of IL-1 edly increased in the absence of IFN-gR, whereas in iDC cells production observed in Ifngr1À/À, WT BM chimeric mice. no difference in IL-1a,b expression was observed between cells In support of this hypothesis, coculture of Mtb-infected of WT or Ifngr1À/À origin (Figures 6A and 6B). In addition, we BMMFs for 3 days with naive TCR transgenic CD4 T cells recog- found that expression of a number of IFN-inducible surface nizing an epitope in the Mtb Ag85b protein (P25) resulted in markers were strongly affected by the absence of IFN-gR reduced IL-1b protein when compared to Mtb-infected cells signaling (Figure S3B). Interestingly, in the iDC cells, expression alone (Figure S4A). This reduction occurred in an IFN-g-depen- of CD206 (macrophage mannose receptor), a surface molecule dent manner and was antigen specific, given that addition of recently shown to be a marker of TLR-induced monocyte- CD4 T cells (OT-II) with an irrelevant specificity failed to suppress derived DCs (Cheong et al., 2010), and IL-10R (CD210) were IL-1b production. Interestingly, whereas addition of exogenous both dependent on IFN-gR signaling (Figure S3B). As expected, IFN-g potently suppressed IL-1b expression by BMDCs (Fig- iNOS expression in both iM and iDC cell subsets was completely ure 4A), coculture with P25 cells did not (Figure S4A). Moreover,
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A Ly6Chi IL-1α,βDP (%) B IL-1α,βDP (%) CD11cneg 70 * CD11cpos 70 60 60 WT
WT 50 50 ns 47 ± 7 39 ± 5 40 40 30 * 30
-/- ns CD45.1 CD45.1 46 ± 4 -/- 20 59 ± 6 20 10 10
CD45.2 0 CD45.2 Ifngr1 0 Ifngr1 IL-1 α IL-1 α WTKO KOWT WTKO KOWT IL-1β unst. Mtb IL-1β unst. Mtb CDLy6Chi CD11cneg CD11cpos iNOSpos TNFαpos IL-10pos iNOSpos TNFαpos IL-10pos 60 60 20 60 60 20 * ns * * 15 * 15 ns 40 40 40 40 ns 10 10 20 * 20 ns 20 20
Percent (%) Percent 5 (%) Percent 5 ns ns ns 0 0 0 0 0 0 WTKO KOWT WTKO KOWT WTKO KOWT WTKO KOWT WTKO KOWT WTKO KOWT unst. Mtb unst. Mtb unst. Mtb unst. Mtb unst. Mtb unst. Mtb
Figure 6. IFN-g Specifically Inhibits IL-1a,b Expression in the iM but not iDC Cell Subset WT CD45.1,1 mice were lethally irradiated and reconstituted with equal parts WT (CD45.1,2) and Ifngr1À/À (CD45.2,2) BM cells and infected with Mtb. (A and B) Distribution of donor BM derived pulmonary CD11bpos myeloid cells 4 weeks p.i. marked by CD45.1 and CD45.2 expression (percentage ± SD) and analysis of IL-1a,b expression by iM (A) and iDC (B) cell subsets gated on WT (WT, white circles) or Ifngr1À/À (KO, dark circles) derived cells after stimulation for 5 hr with (Mtb) or without (unst.) Mtb. Each connecting line depicts an individual animal. (C and D) Frequencies of iNOS-, TNF-a-, and IL-10-expressing cells within pulmonary WT (white circles) or Ifngr1À/À (KO, dark circles), iM (C), and iDC (D) cell subsets. Data in all panels are representative of three independent experiments with three to five mice each. The asterisks denote significant (p % 0.05) differences compared to WT controls (ns = not significant). when IFN-g signaling was disrupted on the BMDCs, P25 cells iNOS expression was critically dependent on IFN-g from this were able to enhance, rather than suppress, IL-1b production source (Figures 7D, S4C, and S5). These findings thus reveal (Figure S4A). This finding suggests that CD4 T cells exert both an anti-inflammatory function for Th1 cell-derived IFN-g in IFN-g-dependent-suppressive as well as IFN-g-independent- modulating IL-1 production by myeloid cells in vivo during Mtb inductive effects on IL-1 production by DCs. infection. We next designed adoptive transfer experiments to test the hypothesis that during Mtb infection, CD4-derived IFN-g is suffi- DISCUSSION cient to mediate the previously observed suppression of IL-1 by iM cells in vivo. The approach utilized allowed us to study Although myeloid cells are the primary targets of mycobacterial APC-CD4 T cell interactions through targeted manipulation of infection (Wolf et al., 2007), they are also pivotal effector cells the genotype of the CD4+ T cells transferred into TcraÀ/À mice that mediate control of intracellular bacterial growth and orches- (Figure S4B). WT or IfngÀ/À CD4+ T cells were adoptively trans- trate the inflammatory response to the pathogen. Previous ferred into TcraÀ/À mice prior to aerosol infection with Mtb and studies have emphasized the roles of myeloid-derived iNOS bacterial loads were measured 4 weeks later (Figures S4B and and TNF-a as important effector molecules for bacterial control 7A). Restimulated lung cells from TcraÀ/À mice reconstituted of Mtb in the mouse model (Saunders et al., 2004; Sko¨ ld and with IfngÀ/À CD4+ T cells (TcraÀ/À+IfngÀ/À CD4) secreted more Behar, 2008). We found that IL-1a and IL-1b each have critical IL-1b when compared to lung cells from TcraÀ/À mice that nonredundant functions in preventing host mortality but appear received WT CD4+ T cells (TcraÀ/À+WT CD4) despite similar to cooperate in mediating IL-1R-dependent bacterial control. pulmonary bacterial loads in the experimental groups at this Indeed, a role for IL-1a in host resistance to Mtb is supported early time point (Figures 7A and 7B). Single-cell analysis of the by a recent study in which the induction of autoantibodies IL-1-producing subsets revealed that iM cells from TcraÀ/À+ against the cytokine resulted in increased mortality during IfngÀ/À CD4 mice produced significantly more IL-1a,b when chronic Mtb infection (Guler et al., 2011). compared to cells from TcraÀ/À+WT CD4, even without restimu- IL-1a and IL-1b are also traditionally regarded as myeloid cell- lation ex vivo (Figure 7C). Moreover, consistent with our findings derived proinflammatory cytokines. Their in vivo source during in mixed BM chimeric mice, IL-1a,b production by iDC cells was Mtb infection had not been examined, and therefore it was unaffected in the absence of CD4+ T cell derived IFN-g whereas unclear whether IL-1 derives from the same cell populations
1030 Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. Immunity IFNs Limit IL-1 Production during Tuberculosis
A Lung d28 p.i. B produce significant amounts of IL-1b. Given our previous find- 7.5 1.8 ings on the caspase-1-independent processing of IL-1b in vivo
) * 7.0 ns 1.5 during Mtb infection (Mayer-Barber et al., 2010), the neutrophil 10 6.5 1.2 represented a logical candidate for the cellular source of IL-1b during Mtb infection. Although not producing pro-IL-1b, neutro- 6.0 0.9 phils could still play a role in the IL-1 response to Mtb in mice by 5.5 0.6 secreting proteases that would cleave extracellular pro-IL-1b IL-1 β (ng/ml) CFU ( Log 5.0 0.3 (Dinarello, 2010). 4.5 0 A key finding of the present study is that in lungs of Mtb- pos -/- -/- infected mice, two major CD11b iM and iDC cell subpopula- WT WT CD4 CD4 tions coproduce IL-1a and IL-1b at the single-cell level. These Tcra -/- Tcra -/- + WT CD4 + WT CD4 cells also produce large amounts of the host-protective media- -/- + Ifng -/- + Ifng hi neg int -/- -/- tors iNOS and TNF-a. The iM (Ly6C , Cd11c , CD282 , and Tcra Tcra neg Tcra Tcra CD13 ) cells represent a single mononuclear phagocyte popu- lation and produce predominantly IL-1a, IL-1b, and TNF-a. They C Ly6Chi CD11cneg IL-1α,βDP (%) display phenotypic markers characteristic of inflammatory 100 Tcra-/- reconstituted with: * monocytes-macrophages previously described in nonlymphoid WT CD4 Ifng-/- CD4 80 tissue (Dunay et al., 2008; Geissmann et al., 2010; Varol et al., 47% 81% 60 2009). In addition they share similar functional properties, e.g., IL-10 and iNOS production, with myeloid-derived suppressor 40 cells that have been extensively studied in long-term unresolved 20 *
IL-1 α pathological conditions such as chronic infections, inflamma- 0 tion, and cancer (Biswas and Mantovani, 2010). β pro-IL-1 unst. Mtb In contrast, the iDC (CD11cpos, Ly6Cneg-int, CD13pos, and CD282int,hi) cells express high levels of MHCII, CD80, and D CD11cpos IL-1α,βDP (%) 100 CD86 as well as additional markers associated with antigen Tcra-/- reconstituted with: -/- presentation and APC-T cell interactions and probably reflect WT CD4 Ifng CD4 80 pulmonary DCs. On the basis of their cytokine expression 36% 41% 60 ns profiles, these cells contain at least two functionally distinct DC hi pos 40 subsets, one producing IL-1a,b (CD282 , Ly6C ) in con- cert with iNOS, TNF-a, and IL-10 and the second producing 20 ns int neg IL-1 α IL-12,23p40 (CD282 , Ly6C ). The IL-1a,b-producing iDC 0 cells closely resemble monocyte-derived inflammatory DCs, an pro-IL-1β unst. Mtb iNOS- and TNF-a-producing DC subset that has been previously implicated in murine resistance to intracellular bacteria, para- + Figure 7. CD4 T Cell-Derived IFN-g Is Sufficient to Suppress IL-1 sites, or viruses and in human psoriasis (De Trez et al., 2009; Production by iM Cells In Vivo À À À À Lin et al., 2008; Lowes et al., 2005; Serbina et al., 2003). In (A) Bacterial loads were measured in lungs of WT, Tcra / , and Tcra / mice reconstituted with WT or IfngÀ/À CD4+ T cells. The asterisk denotes significant contrast, the IL-12,23 p40-expressing iDC cells are similar to (p % 0.05) differences compared to WT controls (ns = not significant). a subset of conventional DCs previously characterized in lung (B) Pulmonary single-cell suspensions from animals in the indicated experi- (GeurtsvanKessel and Lambrecht, 2008). Moreover, both mental groups were incubated with Mtb and IL-1b was measured by ELISA in IL-1a,b- and IL-12,23 p40-producing DC subsets were detected the culture supernatant after 8 hr. Data shown are derived from a pool of three in the lung draining lymph node (data not shown), and it is to five mice per group. possible that these two functionally separate DC populations (C and D) ICS for IL-1a and IL-1b expression by iM (C) and iDC (D) cell subsets in lungs of TcraÀ/À mice reconstituted with WT (left plot, white squares) or play important yet distinct roles in T helper cell differentiation IfngÀ/À (right plot, dark squares) CD4+ T cells and frequencies of IL-1a,b during Mtb infection. Thus, IL-12 is required for the generation expression after stimulation for 5 hr with (Mtb) or without (unst.) Mtb. of IFN-g-producing Th1 cells (Cooper et al., 2011), whereas as Data in all panels are representative of two independent experiments with described here IL-17 protein and Th17 cell responses (data not three to five mice each. The asterisks denote significant (p % 0.05) differences shown) were diminished in lungs of IL-1-deficient animals in- compared to WT controls (ns, not significant). fected with Mtb. Such a functional division of labor between DC populations could play a key factor in determining Th cell that produce the major iNOS and TNF-a effector molecules. lineage fate decisions in lymph nodes and peripheral tissue sites Similarly, the mechanisms that regulate IL-1 expression during during infection. Mtb infection in vivo are poorly understood, particularly the Inflammasome activation is not required for host resistance possible role of other cytokine pathways in controlling IL-1 and IL-1b processing during Mtb infection in vivo (Mayer-Barber production. et al., 2010), so we focused on mechanisms that could regulate In characterizing the innate immune sources of IL-1a and the induction and/or function of both IL-1a and IL-1b. Previous IL-1b, we identified three myeloid cell types (iM cells, iDC cells, in vitro studies had found that type I and type II IFNs modulate and neutrophils) differentially producing the cytokines in the pro-IL-1b expression (Guarda et al., 2011; Guarda et al., 2009; lungs of Mtb-infected mice. Surprisingly, neutrophils did not Masters et al., 2010; Novikov et al., 2011). To investigate the
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role of IFNs in regulating IL-1a,b production in vivo at the cellular that CD4 T cells suppress IL-1b via IFN-g while simultaneously level, we utilized mixed BM chimeras that allowed the side-by- enhancing its expression through an IFN-g-independent mecha- side comparison of WT and gene-deficient populations in the nism, as previously proposed with Tim-3 (Jayaraman et al., same animal. 2010). These two counterregulatory effects mediated by Th1 Type I IFNs had a major suppressive effect on both IL-1a and cells may cancel each other out, thereby explaining the apparent IL-1b production by the iM and iDC cells in the lungs of Mtb- lack of IFN-g-mediated suppression of IL-1a,b production in the infected mice. The precise mechanism by which IFN-aR sig- iDC cell subset in vivo. naling mediates suppression of IL-1a,b expression by these An intriguing paradox raised by our findings concerns the myeloid subsets is unclear. In a recent study by Guarda et al. apparently opposing functions of IFN-g in mediating host resis- (2011), it was shown that type I IFN inhibits LPS induced pro- tance to this pathogen and in suppressing the host-protective IL-1b expression via IL-10. Moreover, IL-10 has been shown to cytokines IL-1a and IL-1b. On the one hand, IFN-g promotes promote susceptibility to Mtb in mice (Redford et al., 2010; Red- inflammation by inducing upregulation of MHC class II and ford et al., 2011; Turner et al., 2002). Here, we implicate IL-10 in iNOS in iM and iDC cells while suppressing IL-10 expression in type I IFN-mediated IL-1 suppression in vitro during Mtb infec- iM cells. On the other hand, IFN-g mediates its anti-inflammatory tion and extend this observation in vivo by showing that IL-10 effects by suppressing IL-1a,b production in iM cells, upregulat- production by pulmonary iM and iDC cells during Mtb infection ing IL-10R expression on iDC cells, and acting as the principal is type I IFN dependent. Furthermore, we show that this IL-10 inducer of the inhibitory ligand PD-L1. Our findings provide a production is limited primarily to IL-1a,b-expressing cells. context for the role of IL-1 in the complex proinflammatory and Although IL-10 may be involved indirectly in the type I IFN suppressive pathways simultaneously induced by IFN-g:in mediated suppression of IL-1 in vivo, our mixed BM chimera contrast to other host-protective factors induced by IFN-g, IL-1 experiments clearly argue that IFN-aR-signals act directly on is actively suppressed. the cytokine-producing cells to inhibit IL-1a,b expression in the Collectively, our data demonstrate that the host detrimental lungs of Mtb-infected mice. type I IFN and host protective IFN-g both downmodulate Although inhibition of IFNgR (CD119) expression has been IL-1a,b production. This situation may have evolved to mutually implicated in the probacterial effects of type I IFNs (Rayamajhi benefit the bacteria and the host by simultaneously inhibiting et al., 2010b), we failed to observe IFN-aR-mediated downregu- IL-1-dependent control of infection while limiting immunopa- lation of CD119 during Mtb infection. Rather, our data reveal that thology. Although the in vivo findings presented here derive type I IFNs act on multiple innate immune cell types to coordinate exclusively from studies in the murine model, their relevance to a multifaceted anti-inflammatory response to Mtb infection, human Mtb infection is supported by the recent work of Berry simultaneously suppressing IL-1a,b and iNOS while promoting et al. (2010) in which a transcriptional profile marked by type I expression of immunosuppressive IL-10 and IL-1Ra. There is and II IFN-induced genes was found to correlate with disease now growing evidence that induction of type I IFN directly serves severity in patients. Our data linking IFN signaling with IL-1 the pathogen as a host evasion mechanism to promote bacterial suppression provide a testable hypothesis for explaining this survival (Rayamajhi et al., 2010a; Trinchieri, 2010), and our find- correlation between IFN expression and human disease. ings argue that this may stem from combined repression of a collection of critical host-protective pathways in which IL-1 EXPERIMENTAL PROCEDURES should now be included as a member. In addition to type I IFNs, we demonstrated that type II IFN Mice and Mtb Infections À À (IFN-g) also modulates IL-1 expression in vivo during Mtb infec- C57BL6 (WT, B6) and Ifngr1 / mice were purchased from Taconic Farms tion. However, we found that IFN-g suppressed IL-1 a,b produc- (Hudson, NY) and Jackson Laboratories (Bar Harbor, ME), respectively. B6.SJL (CD45.1,1), TcraÀ/À, IfngÀ/À, Ifnar1À/À, Il1r1À/À, and OT-II mice were tion in the iM but not the iDC cell population. This observation obtained through a supply contract between the National Institute of Allergy was unexpected given that both subsets not only expressed and Infectious Diseases (NIAID) and Taconic Farms and were backcrossed IFN-gR1 but were also highly responsive to IFN-g during Mtb to B6 for a minimum of ten generations. Ag85b-specific P25 TCR transgenic infection as evidenced by the ablation of iNOS expression in mice, originally generated by Takatsu and colleagues were kindly provided À À À À À À À À the Ifngr1À/À cells. There are several possible explanations for by J. Ernst (NYU). Il1a / , Il1b / , and Il1a / Il1b / mice were originally this differential regulation. First, the iM cell subset represents derived by Y. Iwakura (Tokyo University) and generously supplied by T. Merkel a single cellular population, whereas iDC cells comprise multiple (FDA). All animals were maintained in an AALAC-accredited BSL2 or BSL3 facilities at the NIH and experiments performed in compliance with an animal subpopulations. In the absence of IFN-gR expression, the iDC study proposal approved by the NIAID Animal Care and Use Committee. cell subpopulations underwent major phenotypic changes, Aerosol infection with the H37Rv strain of Mtb (100-150 CFU/mouse unless raising the possibility of selective outgrowth and/or recruitment noted otherwise) and determination of lung bacterial growth were performed of receptor-deficient cells under these conditions. Second, the as previously described (Mayer-Barber et al., 2010). two subsets may integrate IFN-gR-mediated signals in a qualita- tively different fashion (Hu et al., 2006; Masters et al., 2010). Macrophage and DC Differentiation and In Vitro Cultures Lastly, the differential regulation of IL-1 production by inflamma- We cultured murine BM cells for 7 days in either 15% GM-CSF media to tory monocytes-macrophages and DCs could reflect opposing generate BMDC or 30% L929 supernatant media to differentiate BMMFs and then exposed them to H37Rv at a multiplicity of infection of one (MOI:1) activities of Th1 cells on DCs. CD4 T cells have been shown to in the presence or absence of 30 ng/ml IL-27 (Ebioscience, San Diego, CA), be capable of both suppressing and inducing IL-1b expression 200 U/ml IFN-g (Ebioscience), 30 ng/ml IL-10 (Ebioscience), 30 ng/ml IFN-b in myeloid cells (Guarda et al., 2009; Jayaraman et al., 2010). (R&D Systems, Minneapolis, MN), and 20 mg/ml pICLC (kindly provided by Indeed, our in vitro studies with BMDCs support the hypothesis A. Salazar, Oncovir, Inc.) for 24–48 hr and supernatants were harvested. In
1032 Immunity 35, 1023–1034, December 23, 2011 ª2011 Elsevier Inc. Immunity IFNs Limit IL-1 Production during Tuberculosis
some experiments, P25 or OT-II CD4 T cells were enriched from spleen and Statistical Analyses lymph nodes of uninfected mice by magnetic bead separation (Miltenyi The statistical significance of differences between data groups was deter- Biotech, Auburn, CA) and for 60hr coculture at a ratio of 2:1 (T cells:BM- mined with the Mann-Whitney test or the Wilcoxon matched pairs test (mixed derived cells). BM chimera experiments). Human elutriated monocytes were obtained from peripheral blood of 21 healthy CMV negative donors. MFs were generated by culturing monocytes SUPPLEMENTAL INFORMATION with media containing M-CSF (60 ng/ml) for 7 days and DCs after 5 days of culture in the presence of GM-CSF (800 U/ml) and IL-4 (1000 U/ml); the cells Supplemental Information includes five figures and can be found with this were subsequently matured for 48 hr with TNF-a (20 ng/ml). Fresh media article online at doi:10.1016/j.immuni.2011.12.002. with indicated growth factors was added every 48 hr. Cells were exposed to H37Rv (MOI = 5) in the presence or absence IFN-g (10 ng/ml), IFN- b (10 ng/ml), or pICLC (10 mg/ml) for 24 hr. All recombinant human cytokines ACKNOWLEDGMENTS were from Peprotech (Rocky Hill, NJ). We are grateful to the NIAD flow cytometry core facility and in particular B. Cytokine and Nitric Oxide Measurements in Culture Supernatants Hague for assistance with the BSL3 FACS sorting and to the staff of the NIAID and Biological Fluids animal BSL3 facility for excellent technical help. We thank K. Shenderov, G. Murine and human cytokine concentrations in culture supernatants were Trinchieri, Y. Belkaid, and R. Goldzmid for valuable discussion. This research quantitated with commercial ELISA kits (R&D Systems, Minneapolis, MN). was supported by the Intramural Research Program of the NIAID, NIH. S.G.’s Bronchoalveolar lavage (BAL) fluid and cell-free lung homogenates were ob- contributions occurred during a sabbatical supported by a stipend from the tained and cytokines and nitric oxide measured as described previously NIAID and NCI intramural programs. (Mayer-Barber et al., 2010). Received: June 20, 2011 Revised: August 27, 2011 Flow Cytometry Accepted: October 5, 2011 Abs against mouse surface antigens and cytokines were purchased from Published online: December 22, 2011 Ebioscience, Biolegend (San Diego, CA), AbD Serotec (Raleigh, NC), and BD PharMingen (San Diego, CA) and used in 12-color flow cytometry either biotinylated or directly conjugated. 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