M.tuberculosis bloqueia IL-1 produzida pelas células mielóides do Pulmão - Immunity

Innate and Adaptive Interferons Suppress IL-1α and IL-1β Production by Distinct Pulmonary Myeloid Subsets during Mycobacterium tuberculosis Infection

Katrin D. Mayer-Barber^1, , , Bruno B. Andrade¹, Daniel L. Barber¹, Sara Hieny¹, Carl G. Feng¹, Patricia Caspar¹, Sandy Oland¹, Siamon Gordon², Alan Sher^1, , 

1	Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

2	Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK

Received 20 June 2011; revised 27 August 2011; Accepted 5 October 2011. Published online: December 22, 2011. Available online 22 December 2011.

Summary

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1β, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1β are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4⁺ T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Graphical Abstract

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Highlights

► Besides IL-1β, IL-1α is also required for IL-1R1-mediated host resistance to Mtb ► Both IL-1 species are coproduced by two subsets of inflammatory myeloid cells ► Type I IFN suppresses IL-1α and IL-1β production by inflammatory monocytes and DCs ► CD4 T cell-derived IFN-γ inhibits IL-1α and IL-1β production by inflammatory monocytes

Introduction

Mycobacterium tuberculosis (Mtb) primarily infects mononuclear phagocytes. Host resistance against this important pathogen is critically dependent on innate immune functions exerted by these cells including the production of inducible nitric oxide synthase (iNOS), TNF-α, and IL-12,23 p40 ( [Cooper et al., 2011] and [North and Jung, 2004] ). More recently, it has become clear that IL-1 is also of critical importance for host control of Mtb infection given that mice deficient in IL-1R or its adaptor MyD88 succumb rapidly to low-dose aerosol infection with Mtb ( [Fremond et al., 2007] and [Mayer-Barber et al., 2010] ). Although we have shown that IL-1R-dependent protection requires IL-1β (Mayer-Barber et al., 2010), the contribution of IL-1α, the second major cytokine agonist for this receptor, has been unclear.

A major feature of IL-1 is its complex control at the transcriptional, posttranscriptional, and signal transduction levels, which is highlighted by the wide variety of immunopathologies and autoinflammatory diseases that occur in the absence of normal IL-1 regulation ( [Dinarello, 2009] , [Dinarello, 2010] and [Garlanda et al., 2007] ). Surprisingly little is known about the expression and processing of IL-1 in the context of Mtb infection in vivo. Whereas IL-1β production in response to Mtb in vitro is dependent on the NALP3-ASC inflammasome, we and others have recently reported that in lungs of Mtb-infected mice, cleaved IL-1β is found even in the absence of the critical inflammasome component caspase-1. Moreover, although IL-1β is absolutely critical for host control of Mtb infection in mice, NLRP3, ASC, or caspase-1 play minor roles in host resistance to this pathogen ( [Mayer-Barber et al., 2010] , [McElvania Tekippe et al., 2010] and [Walter et al., 2010] ). Although it is not clear what factors mediate the processing of IL-1β during Mtb infection in vivo, the inflammasome independence of the IL-1β response may indicate that it is produced by an atypical cellular source with unique IL-1β-processing properties. For example, the neutrophils present in arthritic joints have been shown to be both a key producer of the cytokine and to cleave IL-1β in a caspase-1-independent manner ( [Guma et al., 2009] and [Joosten et al., 2009] ). The cell populations that produce host-protective IL-1 during Mtb infection in vivo, however, have not yet been characterized.

Type II interferon, IFN-γ, is the signature cytokine of T helper 1 (Th1) cells and has well-established protective functions in host resistance to Mtb and other mycobacterial infections in mice and humans. IFN-γ is traditionally regarded as a proinflammatory cytokine and its protective function during Mtb infection involves the activation of macrophage (Flynn and Chan, 2001). In contrast, the role of type I IFN in the immune response to virulent Mtb is less clear and even though type I and II IFNs share similar STAT1-dependent signaling pathways, type I IFNs appear to promote rather than control infection. Thus, the hypervirulence of certain Mtb strains correlates with enhanced type I IFN synthesis and type I IFN receptor-deficient mice infected with Mtb display lower bacterial loads when compared to WT animals ( [Manca et al., 2001] and [Stanley et al., 2007] ). In addition, Mtb-infected mice intranasally treated with the type I IFN inducer polyinosinic-polycytidylic acid stabilized with poly-L-lysine (pICLC) exhibit exacerbated lung pathology and increased bacterial burden (Antonelli et al., 2010). The relevance of these observations to human tuberculosis is supported by a recent study in which the whole blood transcriptional profile of TB patients was found to be dominated by type I and II IFN-induced genes, and this signature closely correlated with disease severity (Berry et al., 2010). Nevertheless, the cellular mechanisms by which type I IFNs mediate their probacterial effects in Mtb infection and whether or not they involve crosstalk with other innate cytokine pathways remains unclear.

Here, we establish that IL-1α and IL-1β (IL-1α,β) each had critical functions in murine host resistance against Mtb and identified and characterized the two major myeloid subsets that produce both cytokines in the lungs of Mtb-infected mice as inflammatory monocyte-macrophages (iMs) and DCs (iDCs) with highly polyfunctional properties. Importantly, we demonstrated that the endogenous type I IFN was a potent negative regulator of IL-1α,β production by both of these myeloid cell populations. In addition, our data revealed an anti-inflammatory role for CD4⁺ T cell-derived IFN-γ in dampening IL-1 expression by iMs. Together, these findings established polyfunctional mononuclear phagocytes as the major myeloid source of IL-1 during Mtb infection and provided a cellular basis for the crosstalk between IFNs and IL-1 in the immune response to this bacterial pathogen in vivo.

Results

IL-1α and IL-1β Are Each Critical for Host Resistance to Mtb

To investigate the respective contributions of the two ligands IL-1α and IL-1β in IL-1R1-dependent host resistance to Mtb, we performed a series of experiments directly comparing the susceptibility of Il1r1^−/−, Il1a^−/−Il1b^−/−, Il1b^−/−, and Il1a^−/− mice to low-dose aerosol (100–150 CFU) infection. As described previously (Mayer-Barber et al., 2010), IL-1R1- and IL-1β-deficient mice rapidly lost weight and succumbed within 40 days (Figure 1A). No further increase in susceptibility was evident in mice doubly deficient in both IL-1α and IL-1β. Importantly, mice deficient in IL-1α alone displayed similar weight loss and mortality as Il1r1^−/−, Il1a^−/−Il1b^−/−, or Il1b^−/− mice and showed increased bacterial loads equivalent to that of infected Il1b^−/− animals (Figures 1A and 1B). Moreover, we observed similar amounts of IL-1β in the lungs of WT and IL-1α single-deficient mice and conversely comparable amounts of IL-1α in WT and IL-1β-deficient animals (Figure 1C). IL-1α and IL-1β singly deficient animals each exhibited lower bacterial counts than Il1r1^−/− or Il1a^−/−Il1b^−/− mice, highlighting a dual requirement for both IL-1 species and suggesting a possible synergy in the antibacterial function of the two cytokines. Indeed, when infected with a lower dose (<50 CFU) of Mtb, both Il1a^−/− and Il1b^−/− mice survived longer than Il1r1^−/− animals (Figure S1 available online). Taken together, these observations argue that IL-1α and IL-1β each have critical nonredundant functions and cooperate in IL-1R1-mediated host resistance to Mtb.

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Figure 1.
Mtb-Infected Il1a^−/− Mice Display Acute Mortality and Elevated Bacterial Loads Comparable to Il1b^−/− Animals
(A) Body weight and survival of WT, Il1r1^−/−, Il1a^−/−Il1b^−/−, Il1b^−/−, and Il1a^−/− mice after aerosol exposure to Mtb (H37Rv).
(B) Bacterial loads measured 25 days p.i. in lungs of the above mouse strains.
(C) Indicated cytokines were measured by ELISA in BAL fluid of WT (white bars), Il1r1^−/− (black bars), Il1a^−/−Il1b^−/− (dark-gray bars), Il1b^−/− (light-gray bars), and Il1a^−/− (gray and white striped bars) mice and the means ± SD depicted. Dotted lines indicate the limits of detection of the respective assays. Data are representative of a minimum of two independent experiments each involving five to ten mice per group. The asterisk denotes significant (p ≤ 0.05) differences compared to WT controls.

IL-1α and IL-1β Are Coexpressed by CD11b^pos Cells in the Lungs of Mtb-Infected Mice

Studies in which we measured IL-1α,β proteins in lung homogenates of Mtb-infected mice indicated that the cytokines are induced during the acute phase beginning at 2 weeks and peak at 3–4 weeks postinfection (p.i., data not shown). For this reason we chose d25-d28 as the time point for cellular analysis of IL-1 production in our studies. Initial experiments utilizing Il1a,Il1b^−/−→WT BM chimeras confirmed a requirement for hematopoietic cell-derived IL-1 in host resistance (data not shown). Ex vivo cell sorting experiments from Mtb-infected lungs were then performed 4 weeks p.i. for assessing IL-1 production by hematopoietic cells of which mononuclear phagocytes and neutrophils are the major candidates for production of the cytokine in vivo. We generated lung single-cell suspensions and FACS sorted non-T and -B cells into CD11b^neg cells, Ly6G^hiCD11b^hi neutrophils, and Ly6G^negCD11b^pos cells and stimulated the cells overnight in the presence or absence of live Mtb. IL-1α,β production was found to be largely confined to the Ly6G^neg CD11b^pos sorted fraction, whereas neutrophils generated little and Ly6G^negCD11b^neg cells undetectable amounts of IL-1 (Figure 2A).

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Figure 2.
IL-1α and IL-1β Are Coexpressed by Two Pulmonary CD11b^pos 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-1α and IL-1β 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-1α and IL-1β. 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-1α,β 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-1α. 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.

To analyze IL-1 production at the single-cell level, we performed intracellular cytokine staining (ICS) of cells from digested lung. Importantly, we observed strong IL-1α,β staining in cells derived from 4 weeks infected but not naive lungs after 5 hr ex vivo restimulation with irradiated (or live) H37Rv (Figure 2B and data not shown). The latter observation argues that the potential for IL-1 expression by pulmonary CD11b^pos cells is acquired in the context of Mtb infection in vivo probably via changes in cellular composition, recruitment, and activation status, rather than through the 5 hr restimulation per se. Consistent with the above findings on FACS isolated cell populations, Ly6G^neg CD11b^pos cells expressed the most IL-1α,β by intracellular staining, whereas neutrophils from infected lungs stained weakly for IL-1α but not IL-1β and CD11b^neg cells were negative for both cytokines (Figure 2B and data not shown). Importantly, the ICS analysis revealed that in the Ly6G^neg CD11b^pos population IL-1α and IL-1β are coexpressed at the single-cell level. Moreover, in the early stages of infection, the kinetics of the appearance of pulmonary IL-1α,β double-positive (IL-1α, βDP) Ly6G^neg CD11b^pos cells closely mirrored the cytokine proteins measured by ELISA in lung homogenates of infected mice (Figures 2C and data not shown). Together, these data show that Ly6G^neg CD11b^pos cells and not neutrophils are the major hematopoietic source of IL-1 in the lungs of infected mice and that this population coordinately expresses IL-1α,β at the cellular level.

The IL-1α,β-Producing Cells in the Lungs of Mtb-Infected Mice Consist of Two Major Populations Distinguishable by Their Expression of CD11c and Ly6C

To discriminate between myeloid versus lymphoid CD11b^pos cells, we included the intracellular macrophage-myeloid marker CD68 (Figures 2D and S2A) and found that 4 weeks p.i. ∼30%–40% (31% ± 8%) of pulmonary Ly6G^negCD11b^pos cells were of nonmyeloid origin, such as NK and T cells (Figure 2D). This analysis revealed that within the CD11b^pos population IL-1α,β expression is restricted to CD68^pos cells (Figure 2D and data not shown). Moreover, CD68^pos, CD11b^pos myeloid cells segregated into two populations based on differential CD11c and Ly6C expression, with the CD11c^pos subset expressing varying degrees of Ly6C (referred to as iDC cells) while the CD11c^neg cells displayed uniformly high expression of Ly6C (referred to as iM cells) (Figures 2D–2F). Importantly, the IL-1α,β-coproducing cells also dissociated into the same two populations based on CD11c and Ly6C expression (Figures 2D and 2E and data not shown). In addition to CD11c itself, we observed that IL-1α,βDP pulmonary iDC cells can also be distinguished from iM cells by their high expression of CD13 (aminopeptidase N) and CD282 (TLR2) (Figure 2F and data not shown).

Further phenotypic characterization revealed that iM cells display striking homogeneity with unimodal expression of all markers screened and thus probably represent a single population (Figure S2). In contrast, expression of most markers by the iDC cells was heterogeneous, suggesting the presence of multiple subpopulations. iDC cells, when compared to iM cells, also displayed higher expression of markers associated with antigen presentation, suggesting that the iDC subsets consists of multiple DC populations (Figures S2B and S2C). iDC IL-1α,β-expressing cells represent one such subpopulation displaying a unique phenotype characterized by selective upregulation of CD14, CD206, CD210, CD64, CD30L, CD70, and CD38 among others (Figures 2B–2D). Of note, we found that both CD11b^pos subsets expressed the ligand-binding chain of the IFN-γ receptor (CD119, IFN-γR1) as well as IFN-inducible markers such as Ly6C, PD-L1, MHC class I+II, Sca-1, Fcγ receptors (Figures S2B and S2D), and STAT1 phosphorylated at tyrosine 701 (Figure S2E), arguing that both cell populations are subject to IFN-mediated signals in vivo during Mtb infection.

Through our findings, we identified two distinct pulmonary CD11b^posCD68^pos myeloid subsets on the basis of Ly6C and CD11c expression that are capable of coproducing both IL-1α and IL-1β at the single-cell level after Mtb infection.

Pulmonary IL-1α,β Expressing Myeloid Cells Are Multifunctional but Distinct from IL-12,23p40-Producing Cells during Mtb Infection

We next functionally characterized the two IL-1α,βDP CD11b^pos myeloid populations present in the Mtb infected lung for their ability to produce TNFα, iNOS, IL-10 and IL-12,23p40, mediators previously shown to play important roles in host resistance to the pathogen ( [Cooper et al., 2011] and [Redford et al., 2011] ). The iM cells were found to produce TNFα, iNOS and IL-10 but not IL-12,23p40. In contrast, robust IL-12,23p40 as well as TNFα, iNOS, and IL-10 production was observed in iDC cells (Figures 3A–3D). To determine whether expression of these mediators correlates with IL-1 production, we costained the same populations for IL-1α,β. In both populations, iNOS, TNF-α, and IL-10 expression was largely restricted to IL-1α,β DP cells (Figures 3B–3D and data not shown). Importantly, within the iDC subset, IL-1α,β DP cells failed to make IL-12,23p40, which was instead produced by a separate functionally distinct DC subpopulation (Figures 3B–3D and data not shown). Thus, IL-1-producing myeloid cells in the lungs of Mtb-infected mice display a high degree of functional heterogeneity at both the population and single-cell level and are key producers of the antimycobacterial effector molecules iNOS and TNF-α. Taken together, the above phenotypic and functional analyses provided a platform for investigating the regulation of IL-1α,β cytokine production by myeloid cells at the cellular level during Mtb infection in vivo (Figure S5A).

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Figure 3.
IL-1α,β Coexpressing Cells in the Lungs of Mtb-Infected Mice Are Highly Polyfunctional but Distinct from IL-12,23 p40-Producing Cells
(A–D) Quantitative and qualitative analysis of cytokine production in indicated subsets 28 days after Mtb infection of WT mice.
(A) Frequencies of iNOS-, TNF-α-, IL-10-, and IL-12,23p40-expressing cells within iM (top) and iDC (bottom) cell subsets after restimulation for 5 hr with (Mtb, dark circles) or without (unst., white circles) H37Rv determined by ICS. Each connecting line depicts an individual animal.
(B) Costaining of IL-1α with indicated cytokines and iNOS.
(C) Percentage of cytokine-producing cells within IL-1α-expressing cells of the 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-1α, IL-1β, iNOS, and TNF-α expression. All combinations of the possible cytokine expression patterns are marked on the x axis, whereas the percentages (mean ± SD) of the distinct cytokine-producing subsets within iM or iDC cells are shown on the y axis. The data are summarized in pie charts and each slice corresponds to the proportion of iM or iDC cells expressing a given combination of cytokines indicated by the colored boxes at the bottom of the x axis.
Data in all panels are representative of a minimum of three independent experiments with three to six animals each.

IFNs Negatively Regulate the IL-1 Pathway in Mtb-Infected Mouse and Human Mononuclear Phagocytes In Vitro

Type I and type II IFNs have been implicated in the regulation of the IL-1 pathway in vitro as well as in autoimmune and infectious diseases ( [Hu et al., 2005] , [Schindler et al., 1989] , [Thacker et al., 2010] and [Tilg et al., 1993] ). As noted above, the major IL-1α,β-producing subsets in Mtb-infected mice exhibited upregulated expression of IFN-inducible markers and displayed STAT1 phosphorylation. These observations suggested the possible involvement of type I IFN and IFN-γ signaling in regulating IL-1 expression in the response of myeloid cells to Mtb infection.

To study the effects of type I and type II IFNs on IL-1α,β production in the context of Mtb, we first compared the IL-1α,β response of murine and human mononuclear phagocytes exposed in vitro to Mtb in the presence or absence of IFNs (Figure 4). We found that pICLC-induced type I IFN and recombinant IFN-γ potently inhibited IL-1α,β cytokine production by murine bone marrow-derived macrophages (BMMΦs) after Mtb exposure (Figures 4A and 4B). In contrast, in bone marrow-derived dendritic cells (BMDCs), IL-1α,β were suppressed by IFN-γ but not pICLC. As expected, pICLC significantly inhibited IL-1 production by Ifngr1^−/− but not Ifnar1^−/− BMMΦs and in addition, IFNs did not universally downregulate proinflammatory cytokines given that TNF-α protein was upregulated in the presence of pICLC and IFN-γ in both cell types (data not shown). IL-27, which also utilizes STAT1 for signal transduction, was unable to inhibit IL-1 expression, arguing that the suppressive effects of IFNs reflect specific functions of the latter cytokines rather than STAT1 activation per se (Figure 4A).

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Figure 4.
Type I and II IFNs Negatively Regulate IL-1α and IL-1β Secretion by Murine and Human Myeloid Cell Subsets Infected with Mtb
(A) Murine cytokines measured by ELISA in supernatants of BMMΦs 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-γ, or pICLC.
(B) IL-1β protein in supernatants of BMMΦs and BMDCs from WT, Ifnar1^−/−, or Ifngr1^−/− mice incubated with IFN-γ or pICLC as indicated after exposure to live Mtb (MOI:1).
(C) Human IL-1α and IL-1β measured by ELISA in culture supernatants of human monocyte-derived macrophages [MΦ]) 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-β and IFN-γ. 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 MΦs or DCs (top panels) and murine BMMΦs and BMDCs (bottom panels) incubated as noted in (A) and (B).
(E) Cytokines in supernatants of BMMΦs derived from WT or Ifnar1^−/− mice after exposure to live Mtb (MOI:1) for 24 hr.
(F) IL-10 protein in supernatants of WT BMMΦs incubated with increasing amounts of pICLC for 40 hr in the presence or absence of Mtb infection.
(G) IL-1β protein in supernatants of WT BMMΦs after exposure to live Mtb (MOI:1) for 24 hr incubated with recombinant murine IFN-β in the presence or absence of neutralizing IL-10 mAb.
Murine data presented are the means ± SD and representative of two to five independent experiments. The asterisk denotes significant (p ≤ 0.05) differences compared to Mtb exposure alone or as indicated with connecting lines.

In order to test the relevance of these observations to the Mtb response of humans, we infected monocyte-derived macrophages (MΦs) or monocyte-derived DCs from 21 healthy blood donors with Mtb and assessed their ability to generate IL-1α,β in the presence or absence of IFNs. In agreement with a recent study from our group focusing on differences in IL-1β regulation in virulent versus avirulent mycobacteria in human monocytes-macrophages (Novikov et al., 2011), we found that IFN-β inhibits production of the former cytokine in macrophages. Here, we show that in Mtb-infected DCs as well as MΦ pICLC and IFN-β suppress both IL-1α and IL-1β and that IFN-γ inhibits IL-1β but not IL-1α production (Figures 4C). Despite the statistically significant suppression in IL-1β by IFN-γ, there was a substantial fraction of donors that responded to Mtb infection with lower amounts of IL-1β (52% ≤ 4 ng/ml (MΦ), 8 ng/ml (DC):low responders; LR), and in these individuals little to no additional reduction in IL-1β protein was observed after IFN-γ treatment (MΦ: 29.6% ± 23.4% reduction in LR; 66.9% ± 13.4% reduction in high responders [HRs]; DCs: 38.5% ± 30.1% reduction in LR;78.7% ± 0.2% reduction in HR), whereas pICLC or IFN-β treatment potently suppressed IL-1β by both LR and HR (>70% ± 5%; Figure 4C and data not shown). This donor variation may explain the divergent results in our previous study (Novikov et al., 2011) in which we failed to observe suppression of IL-1β by IFN-γ in human macrophage.

In the murine as well as human systems, we observed upregulation 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 human mononuclear phagocytes, IFNs antagonize the IL-1 pathway by dual mechanisms: first by directly suppressing IL-1α,β cytokine production and second by upregulation of the soluble endogenous IL-1Ra, which opposes IL-1 by competing for IL-1R1 binding.

When we investigated possible sources of type I IFN, we found that both murine BMMΦs and BMDCs generated detectable amounts of IFN-β in response Mtb infection (Figure 4E). Type I IFNs are potent inducers of IL-10 (Chang et al., 2007) and IL-10 has recently been reported to mediate type I IFN's suppressive effects on IL-1 (Guarda et al., 2011). In the context of Mtb infection in vitro, we observed that IL-10 production is critically dependent on IFN-αR signaling and that IL-10 is induced by IFN-β and pICLC in a dose-dependent manner in BMMΦs and BMDCs (Figures 4E and 4F and data not shown). To investigate the contribution of IL-10 in type I IFN-mediated suppression of IL-1 during Mtb infection in vitro, we added exogenous IL-10 to Mtb-infected BMDMs and observed a significant inhibition of IL-1β production (Figure 4G). More importantly, the Mtb-induced IFN-β-mediated suppression of IL-1β was partially reversed when endogenous IL-10 was neutralized (Figure 4H). Thus in vitro, besides direct suppressive effects, type I IFNs can also indirectly inhibit IL-1 production during Mtb infection via IFN-αR-dependent induction of IL-10.

Endogenous Type I IFN Suppresses IL-1α,β Production by Both iM and iDC Cells In Vivo

A probacterial role for endogenous type I IFNs has been demonstrated previously in experiments in which Ifnar1^−/− mice were shown to be less susceptible to Mtb infection ( [Manca et al., 2005] and [Stanley et al., 2007] ). As shown in Figures 5A and 5B, this decrease in bacterial burden was closely associated with an increase in IL-1α,β production by pulmonary myeloid cells.

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Figure 5.
Endogenous Type I IFNs Suppress IL-1α,β Expression by Both iM and iDC Pulmonary Cell Subsets during Mtb Infection
(A) Pulmonary bacterial loads measured 4 weeks p.i. in WT or Ifnar1^−/− mice. The asterisk denotes significant (p ≤ 0.05) difference compared to WT control.
(B) ICS for IL-1α,β by pulmonary myeloid cells in WT or Ifnar1^−/− 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 Ifnar1^−/− (CD45.2,2) BM cells and infected with Mtb.
(D) Analysis of donor BM-derived CD11b^pos myeloid cells 4 weeks p.i. in isolated lung cells marked by CD45.1 and CD45.2 expression (percentage ± SD) and frequency of IL-1α,β expression by WT (white circles) or Ifnar1^−/− (KO, dark circles) total CD11b^pos 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-1α,β expression by iM and iDC cell subsets in mixed Ifnar1^−/− BM chimeric mice.
(F and G) Frequencies of iNOS-, TNF-α-, and IL-10-expressing cells within pulmonary WT (white circles) or Ifnar1^−/− (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).

To test whether endogenously induced type I IFNs can act directly on IL-1α,β-producing cells during Mtb infection in vivo, we generated mixed bone marrow chimeras allowing us to compare Ifnar1^−/− and WT myeloid cells in the lungs of the same animal. This approach controls internally for potential differences in bacterial load, inflammatory milieu, and/or cellular microenvironment. WT CD45.1,1 mice were lethally irradiated and reconstituted with BM from WT (CD45.1,2) donors mixed at a 1:1 ratio with Ifnar1^−/− CD45.2,2 donors (Figure 5C). The mixed Ifnar1^−/−, WT BM chimeras were then infected with Mtb and 4 weeks later the frequency of IL-1α,β DP cells within pulmonary WT or Ifnar1^−/− cells was analyzed (Figure 5D). Importantly, in both the iM and the iDC cell subsets, the inability of Ifnar1^−/− cells to receive type I IFN signals resulted in a marked increase in the proportion of IL-1α,βDP, even without Mtb restimulation (Figures 5D and 5E). In addition, we found that type I IFN signaling suppressed iNOS production by iM and iDC cells, and in accordance with the in vitro data presented above, it also induced IL-10 expression by myeloid cells in vivo after Mtb infection (Figures 5F, 5G, and S5). In contrast, TNF-α expression was not subject to type I IFN regulation. Furthermore, phenotypic analysis revealed that type I IFN signaling influenced expression of only a few IFN inducible and surface markers, most notably CD206 and PD-L1 (Figure S3A). Together, these data argued that ligation of the type I IFN receptor on each of the two myeloid populations directly suppresses IL-1α,β production in vivo.

Host-Protective IFN-γ Inhibits IL-1α,β Production by iM Cells

Because in addition to type I IFNs, IFN-γ also displayed potent suppressive effects on IL-1 expression in vitro, we next used the same mixed BM chimeric approach to determine whether IFN-γ signaling also regulates IL-1α,β production by myeloid cells during Mtb infection in vivo. In these experiments, we constructed mixed Ifngr1^−/−,WT BM chimeric mice and functionally and phenotypically analyzed gene deficient or WT CD11b^pos subsets in the same animal for IFN-inducible surface marker expression and their capacity to generate innate cytokines (Figures 6 and S3B). Importantly, in the lungs of Mtb infected chimeric mice, IL-1 production in the iM cell subset was markedly increased in the absence of IFN-γR, whereas in iDC cells no difference in IL-1α,β expression was observed between cells of WT or Ifngr1^−/− origin (Figures 6A and 6B). In addition, we found that expression of a number of IFN-inducible surface markers were strongly affected by the absence of IFN-γR signaling (Figure S3B). Interestingly, in the iDC cells, expression of CD206 (macrophage mannose receptor), a surface molecule recently shown to be a marker of TLR-induced monocyte-derived DCs (Cheong et al., 2010), and IL-10R (CD210) were both dependent on IFN-γR signaling (Figure S3B). As expected, iNOS expression in both iM and iDC cell subsets was completely abolished in IFN-γR-deficient cells compared to WT cells in the same animal. Moreover, whereas TNF-α expression in both myeloid subset was unaffected by the absence of IFN-γR, IFN-γ potently suppressed IL-10 production in iM but not iDC cells (Figures 6C, 6D, and S5). Thus, while clearly a host-protective cytokine in Mtb infection, IFNγ in common with type I IFN can also be anti-inflammatory suppressing IL-1α,β production by pulmonary myeloid cells.

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Figure 6.
IFN-γ Specifically Inhibits IL-1α,β 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 Ifngr1^−/− (CD45.2,2) BM cells and infected with Mtb.
(A and B) Distribution of donor BM derived pulmonary CD11b^pos myeloid cells 4 weeks p.i. marked by CD45.1 and CD45.2 expression (percentage ± SD) and analysis of IL-1α,β expression by iM (A) and iDC (B) cell subsets gated on WT (WT, white circles) or Ifngr1^−/− (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-α-, and IL-10-expressing cells within pulmonary WT (white circles) or Ifngr1^−/− (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).

CD4⁺ T Cells Regulate IL-1 Expression during Mtb Infection through Their Production of IFN-γ

Th1 cells are a major source of IFN-γ during Mtb infection in mice and therefore could be a key mediator of the suppression of IL-1 production observed in Ifngr1^−/−, WT BM chimeric mice.

In support of this hypothesis, coculture of Mtb-infected BMMΦs for 3 days with naive TCR transgenic CD4 T cells recognizing an epitope in the Mtb Ag85b protein (P25) resulted in reduced IL-1β protein when compared to Mtb-infected cells alone (Figure S4A). This reduction occurred in an IFN-γ-dependent manner and was antigen specific, given that addition of CD4 T cells (OT-II) with an irrelevant specificity failed to suppress IL-1β production. Interestingly, whereas addition of exogenous IFN-γ potently suppressed IL-1β expression by BMDCs (Figure 4A), coculture with P25 cells did not (Figure S4A). Moreover, when IFN-γ signaling was disrupted on the BMDCs, P25 cells were able to enhance, rather than suppress, IL-1β production (Figure S4A). This finding suggests that CD4 T cells exert both IFN-γ-dependent-suppressive as well as IFN-γ-independent-inductive effects on IL-1 production by DCs.

We next designed adoptive transfer experiments to test the hypothesis that during Mtb infection, CD4-derived IFN-γ is sufficient to mediate the previously observed suppression of IL-1 by iM cells in vivo. The approach utilized allowed us to study APC-CD4 T cell interactions through targeted manipulation of the genotype of the CD4⁺ T cells transferred into Tcra^−/− mice (Figure S4B). WT or Ifng^−/− CD4⁺ T cells were adoptively transferred into Tcra^−/− mice prior to aerosol infection with Mtb and bacterial loads were measured 4 weeks later (Figures S4B and 7A). Restimulated lung cells from Tcra^−/− mice reconstituted with Ifng^−/− CD4⁺ T cells (Tcra^−/−+Ifng^−/− CD4) secreted more IL-1β when compared to lung cells from Tcra^−/− mice that received WT CD4⁺ T cells (Tcra^−/−+WT CD4) despite similar pulmonary bacterial loads in the experimental groups at this early time point (Figures 7A and 7B). Single-cell analysis of the IL-1-producing subsets revealed that iM cells from Tcra^−/−+Ifng^−/− CD4 mice produced significantly more IL-1α,β when compared to cells from Tcra^−/−+WT CD4, even without restimulation ex vivo (Figure 7C). Moreover, consistent with our findings in mixed BM chimeric mice, IL-1α,β production by iDC cells was unaffected in the absence of CD4⁺ T cell derived IFN-γ whereas iNOS expression was critically dependent on IFN-γ from this source (Figures 7D, S4C, and S5). These findings thus reveal an anti-inflammatory function for Th1 cell-derived IFN-γ in modulating IL-1 production by myeloid cells in vivo during Mtb infection.

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Figure 7.
CD4⁺ T Cell-Derived IFN-γ Is Sufficient to Suppress IL-1 Production by iM Cells In Vivo
(A) Bacterial loads were measured in lungs of WT, Tcra^−/−, and Tcra^−/− mice reconstituted with WT or Ifng^−/− CD4⁺ T cells. The asterisk denotes significant (p ≤ 0.05) differences compared to WT controls (ns = not significant).
(B) Pulmonary single-cell suspensions from animals in the indicated experimental groups were incubated with Mtb and IL-1β was measured by ELISA in the culture supernatant after 8 hr. Data shown are derived from a pool of three to five mice per group.
(C and D) ICS for IL-1α and IL-1β expression by iM (C) and iDC (D) cell subsets in lungs of Tcra^−/− mice reconstituted with WT (left plot, white squares) or Ifng^−/− (right plot, dark squares) CD4⁺ T cells and frequencies of IL-1α,β expression after stimulation for 5 hr with (Mtb) or without (unst.) Mtb.
Data in all panels are representative of two independent experiments with three to five mice each. The asterisks denote significant (p ≤ 0.05) differences compared to WT controls (ns, not significant).

Discussion

Although myeloid cells are the primary targets of mycobacterial infection (Wolf et al., 2007), they are also pivotal effector cells that mediate control of intracellular bacterial growth and orchestrate the inflammatory response to the pathogen. Previous studies have emphasized the roles of myeloid-derived iNOS and TNF-α as important effector molecules for bacterial control of Mtb in the mouse model ( [Saunders et al., 2004] and [Sköld and Behar, 2008] ). We found that IL-1α and IL-1β each have critical nonredundant functions in preventing host mortality but appear to cooperate in mediating IL-1R-dependent bacterial control. Indeed, a role for IL-1α in host resistance to Mtb is supported by a recent study in which the induction of autoantibodies against the cytokine resulted in increased mortality during chronic Mtb infection (Guler et al., 2011).

IL-1α and IL-1β are also traditionally regarded as myeloid cell-derived proinflammatory cytokines. Their in vivo source during Mtb infection had not been examined, and therefore it was unclear whether IL-1 derives from the same cell populations that produce the major iNOS and TNF-α effector molecules. Similarly, the mechanisms that regulate IL-1 expression during Mtb infection in vivo are poorly understood, particularly the possible role of other cytokine pathways in controlling IL-1 production.

In characterizing the innate immune sources of IL-1α and IL-1β, we identified three myeloid cell types (iM cells, iDC cells, and neutrophils) differentially producing the cytokines in the lungs of Mtb-infected mice. Surprisingly, neutrophils did not produce significant amounts of IL-1β. Given our previous findings on the caspase-1-independent processing of IL-1β in vivo during Mtb infection (Mayer-Barber et al., 2010), the neutrophil represented a logical candidate for the cellular source of IL-1β during Mtb infection. Although not producing pro-IL-1β, neutrophils could still play a role in the IL-1 response to Mtb in mice by secreting proteases that would cleave extracellular pro-IL-1β (Dinarello, 2010).

A key finding of the present study is that in lungs of Mtb-infected mice, two major CD11b^pos iM and iDC cell subpopulations coproduce IL-1α and IL-1β at the single-cell level. These cells also produce large amounts of the host-protective mediators iNOS and TNF-α. The iM (Ly6C^hi, Cd11c^neg, CD282^int, and CD13^neg) cells represent a single mononuclear phagocyte population and produce predominantly IL-1α, IL-1β, and TNF-α. They display phenotypic markers characteristic of inflammatory monocytes-macrophages previously described in nonlymphoid tissue ( [Dunay et al., 2008] , [Geissmann et al., 2010] and [Varol et al., 2009] ). In addition they share similar functional properties, e.g., IL-10 and iNOS production, with myeloid-derived suppressor cells that have been extensively studied in long-term unresolved pathological conditions such as chronic infections, inflammation, and cancer (Biswas and Mantovani, 2010).

In contrast, the iDC (CD11c^pos, Ly6C^neg-int, CD13^pos, and CD282^int,hi) cells express high levels of MHCII, CD80, and CD86 as well as additional markers associated with antigen presentation and APC-T cell interactions and probably reflect pulmonary DCs. On the basis of their cytokine expression profiles, these cells contain at least two functionally distinct DC subsets, one producing IL-1α,β (CD282^hi, Ly6C^pos) in concert with iNOS, TNF-α, and IL-10 and the second producing IL-12,23p40 (CD282^int, Ly6C^neg). The IL-1α,β-producing iDC cells closely resemble monocyte-derived inflammatory DCs, an iNOS- and TNF-α-producing DC subset that has been previously implicated in murine resistance to intracellular bacteria, parasites, or viruses and in human psoriasis ( [De Trez et al., 2009] , [Lin et al., 2008] , [Lowes et al., 2005] and [Serbina et al., 2003] ). In contrast, the IL-12,23 p40-expressing iDC cells are similar to a subset of conventional DCs previously characterized in lung (GeurtsvanKessel and Lambrecht, 2008). Moreover, both IL-1α,β- and IL-12,23 p40-producing DC subsets were detected in the lung draining lymph node (data not shown), and it is possible that these two functionally separate DC populations play important yet distinct roles in T helper cell differentiation during Mtb infection. Thus, IL-12 is required for the generation of IFN-γ-producing Th1 cells (Cooper et al., 2011), whereas as described here IL-17 protein and Th17 cell responses (data not shown) were diminished in lungs of IL-1-deficient animals infected with Mtb. Such a functional division of labor between DC populations could play a key factor in determining Th cell lineage fate decisions in lymph nodes and peripheral tissue sites during infection.

Inflammasome activation is not required for host resistance and IL-1β processing during Mtb infection in vivo (Mayer-Barber et al., 2010), so we focused on mechanisms that could regulate the induction and/or function of both IL-1α and IL-1β. Previous in vitro studies had found that type I and type II IFNs modulate pro-IL-1β expression ( [Guarda et al., 2011] , [Guarda et al., 2009] , [Masters et al., 2010] and [Novikov et al., 2011] ). To investigate the role of IFNs in regulating IL-1α,β production in vivo at the cellular level, we utilized mixed BM chimeras that allowed the side-by-side comparison of WT and gene-deficient populations in the same animal.

Type I IFNs had a major suppressive effect on both IL-1α and IL-1β production by the iM and iDC cells in the lungs of Mtb-infected mice. The precise mechanism by which IFN-αR signaling mediates suppression of IL-1α,β expression by these myeloid subsets is unclear. In a recent study by Guarda et al. (2011), it was shown that type I IFN inhibits LPS induced pro-IL-1β expression via IL-10. Moreover, IL-10 has been shown to promote susceptibility to Mtb in mice ( [Redford et al., 2010] , [Redford et al., 2011] and [Turner et al., 2002] ). Here, we implicate IL-10 in type I IFN-mediated IL-1 suppression in vitro during Mtb infection and extend this observation in vivo by showing that IL-10 production by pulmonary iM and iDC cells during Mtb infection is type I IFN dependent. Furthermore, we show that this IL-10 production is limited primarily to IL-1α,β-expressing cells. Although IL-10 may be involved indirectly in the type I IFN mediated suppression of IL-1 in vivo, our mixed BM chimera experiments clearly argue that IFN-αR-signals act directly on the cytokine-producing cells to inhibit IL-1α,β expression in the lungs of Mtb-infected mice.

Although inhibition of IFNγR (CD119) expression has been implicated in the probacterial effects of type I IFNs (Rayamajhi et al., 2010b), we failed to observe IFN-αR-mediated downregulation of CD119 during Mtb infection. Rather, our data reveal that type I IFNs act on multiple innate immune cell types to coordinate a multifaceted anti-inflammatory response to Mtb infection, simultaneously suppressing IL-1α,β and iNOS while promoting expression of immunosuppressive IL-10 and IL-1Ra. There is now growing evidence that induction of type I IFN directly serves the pathogen as a host evasion mechanism to promote bacterial survival ( [Rayamajhi et al., 2010a] and [Trinchieri, 2010] ), and our findings argue that this may stem from combined repression of a collection of critical host-protective pathways in which IL-1 should now be included as a member.

In addition to type I IFNs, we demonstrated that type II IFN (IFN-γ) also modulates IL-1 expression in vivo during Mtb infection. However, we found that IFN-γ suppressed IL-1 α,β production in the iM but not the iDC cell population. This observation was unexpected given that both subsets not only expressed IFN-γR1 but were also highly responsive to IFN-γ during Mtb infection as evidenced by the ablation of iNOS expression in the Ifngr1^−/− cells. There are several possible explanations for this differential regulation. First, the iM cell subset represents a single cellular population, whereas iDC cells comprise multiple subpopulations. In the absence of IFN-γR expression, the iDC cell subpopulations underwent major phenotypic changes, raising the possibility of selective outgrowth and/or recruitment of receptor-deficient cells under these conditions. Second, the two subsets may integrate IFN-γR-mediated signals in a qualitatively different fashion ( [Hu et al., 2006] and [Masters et al., 2010] ). Lastly, the differential regulation of IL-1 production by inflammatory monocytes-macrophages and DCs could reflect opposing activities of Th1 cells on DCs. CD4 T cells have been shown to be capable of both suppressing and inducing IL-1β expression in myeloid cells ( [Guarda et al., 2009] and [Jayaraman et al., 2010] ). Indeed, our in vitro studies with BMDCs support the hypothesis that CD4 T cells suppress IL-1β via IFN-γ while simultaneously enhancing its expression through an IFN-γ-independent mechanism, as previously proposed with Tim-3 (Jayaraman et al., 2010). These two counterregulatory effects mediated by Th1 cells may cancel each other out, thereby explaining the apparent lack of IFN-γ-mediated suppression of IL-1α,β production in the iDC cell subset in vivo.

An intriguing paradox raised by our findings concerns the apparently opposing functions of IFN-γ in mediating host resistance to this pathogen and in suppressing the host-protective cytokines IL-1α and IL-1β. On the one hand, IFN-γ promotes inflammation by inducing upregulation of MHC class II and iNOS in iM and iDC cells while suppressing IL-10 expression in iM cells. On the other hand, IFN-γ mediates its anti-inflammatory effects by suppressing IL-1α,β production in iM cells, upregulating IL-10R expression on iDC cells, and acting as the principal inducer of the inhibitory ligand PD-L1. Our findings provide a context for the role of IL-1 in the complex proinflammatory and suppressive pathways simultaneously induced by IFN-γ: in contrast to other host-protective factors induced by IFN-γ, IL-1 is actively suppressed.

Collectively, our data demonstrate that the host detrimental type I IFN and host protective IFN-γ both downmodulate IL-1α,β production. This situation may have evolved to mutually benefit the bacteria and the host by simultaneously inhibiting IL-1-dependent control of infection while limiting immunopathology. Although the in vivo findings presented here derive exclusively from studies in the murine model, their relevance to human Mtb infection is supported by the recent work of Berry et al. (2010) in which a transcriptional profile marked by type I and II IFN-induced genes was found to correlate with disease severity in patients. Our data linking IFN signaling with IL-1 suppression provide a testable hypothesis for explaining this correlation between IFN expression and human disease.

Experimental Procedures

Mice and Mtb Infections

C57BL6 (WT, B6) and Ifngr1^−/− mice were purchased from Taconic Farms (Hudson, NY) and Jackson Laboratories (Bar Harbor, ME), respectively. B6.SJL (CD45.1,1), Tcra^−/−, Ifng^−/−, Ifnar1^−/−, Il1r1^−/−, and OT-II mice were obtained through a supply contract between the National Institute of Allergy and Infectious Diseases (NIAID) and Taconic Farms and were backcrossed to B6 for a minimum of ten generations. Ag85b-specific P25 TCR transgenic mice, originally generated by Takatsu and colleagues were kindly provided by J. Ernst (NYU). Il1a^−/−, Il1b^−/−, and Il1a^−/−Il1b^−/− mice were originally derived by Y. Iwakura (Tokyo University) and generously supplied by T. Merkel (FDA). All animals were maintained in an AALAC-accredited BSL2 or BSL3 facilities at the NIH and experiments performed in compliance with an animal study proposal approved by the NIAID Animal Care and Use Committee. Aerosol infection with the H37Rv strain of Mtb (100-150 CFU/mouse unless noted otherwise) and determination of lung bacterial growth were performed as previously described (Mayer-Barber et al., 2010).

Macrophage and DC Differentiation and In Vitro Cultures

We cultured murine BM cells for 7 days in either 15% GM-CSF media to generate BMDC or 30% L929 supernatant media to differentiate BMMΦs and then exposed them to H37Rv at a multiplicity of infection of one (MOI:1) in the presence or absence of 30 ng/ml IL-27 (Ebioscience, San Diego, CA), 200 U/ml IFN-γ (Ebioscience), 30 ng/ml IL-10 (Ebioscience), 30 ng/ml IFN-β (R&D Systems, Minneapolis, MN), and 20 μg/ml pICLC (kindly provided by A. Salazar, Oncovir, Inc.) for 24–48 hr and supernatants were harvested. In some experiments, P25 or OT-II CD4 T cells were enriched from spleen and lymph nodes of uninfected mice by magnetic bead separation (Miltenyi Biotech, Auburn, CA) and for 60hr coculture at a ratio of 2:1 (T cells:BM-derived cells).

Human elutriated monocytes were obtained from peripheral blood of 21 healthy CMV negative donors. MΦs were generated by culturing monocytes 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 were subsequently matured for 48 hr with TNF-α (20 ng/ml). Fresh media with indicated growth factors was added every 48 hr. Cells were exposed to H37Rv (MOI = 5) in the presence or absence IFN-γ (10 ng/ml), IFN-β (10 ng/ml), or pICLC (10 μg/ml) for 24 hr. All recombinant human cytokines were from Peprotech (Rocky Hill, NJ).

Cytokine and Nitric Oxide Measurements in Culture Supernatants and Biological Fluids

Murine and human cytokine concentrations in culture supernatants were quantitated with commercial ELISA kits (R&D Systems, Minneapolis, MN). Bronchoalveolar lavage (BAL) fluid and cell-free lung homogenates were obtained and cytokines and nitric oxide measured as described previously (Mayer-Barber et al., 2010).

Flow Cytometry

Abs against mouse surface antigens and cytokines were purchased from 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. The Abs used were directed against Ly6C (clone AL-21), Ly6G (1A8), CD13 (R3-63), CD11c (HL3 and N418), CD282 (6C2), CD68 (FA-11), CD45.1 (A20), CD45.2 (104), I-A^b (M5/114.15.2), IL-1α PE (ALF-161), IL-1β APC (polyclonal and replacement pro-IL-1β monoclonal NJTEN3), iNOS (polyclonal), IL-12,23 p40 (C15.6), TNF-α (MP6-XT22), IL-10 (JES5-16E3), CD11b (M1/70), TCR-β (H57-597), CD19 (6D5), NK1.1 (PK136), 7/4 (7/4), IL10R/CD210 (1B1.3a), CD206 (MR5D3), IFN-γRI/CD119 (GR20), CD14 (Sa14-2), PDL-1/CD274 (MIH5), Sca-1(D7), and CD16/32 (93). Biotinylated antibodies were detected with streptavidin-conjugated Qdot 605 from Molecular Probes-Invitrogen (Carlsbad, CA). Ultraviolet fixable live-dead cell stain was purchased from Molecular Probes-Invitrogen and used in accordance with the manufacturer's protocol. All samples were acquired on a LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Three Star, Ashland, OR).

Isolation and Restimulation of Myeloid Cells from Lung Tissue

Perfused lungs from infected mice were cut into 1–2 mm pieces and subsequently digested with Liberase Cl (0.4 mg/ml in PBS; Roche-Diagnostics, Indianapolis, IN). The reaction was stopped after 30–45 min with an equal volume of fetal calf serum. Digested lung was fully dispersed by passage through a 100 μm pore size cell strainer and an aliquot was removed for bacterial load measurements. Isolated cells were then washed, counted, and resuspended in media containing monensin (0.1%, Ebioscience) in the presence or absence of 100 ug/ml irradiated Mtb H37Rv at 1 × 10⁶ cells per well in 96-well plates and incubated at 37°C, 5% CO₂. Five hours later, cells were surface stained, fixed, permeabilized, and ICS performed.

Preparation of Mixed BM Chimeric Mice

B6.SJL (CD45.1,1) mice were lethally irradiated (950 rad) and reconstituted with a total of 10⁷ donor BM cells from C57BL/6 CD45.1,2 wild-type (WT) mice mixed at equal parts with BM cells from CD45.2,2 mice deficient (KO) in either IFN-γR1 or IFN-αR1. Mice were allowed to reconstitute for 8–10 weeks before aerosol infection with H37Rv.

T Cell Isolation and Adoptive Transfer

CD4 T cells for adoptive transfer were enriched from the spleen and lymph nodes of uninfected B6 or Ifng^−/− mice by magnetic bead separation to ∼90%–95% purity, according to the manufacturer's protocol (Miltenyi Biotec) and injected intravenously into Tcra^−/− mice (4 × 10⁶ cells/animal) 1–7 days prior to aerosol infection with H37Rv.

Statistical Analyses

The statistical significance of differences between data groups was determined with the Mann-Whitney test or the Wilcoxon matched pairs test (mixed BM chimera experiments).

Acknowledgments

We are grateful to the NIAD flow cytometry core facility and in particular B. Hague for assistance with the BSL3 FACS sorting and to the staff of the NIAID animal BSL3 facility for excellent technical help. We thank K. Shenderov, G. Trinchieri, Y. Belkaid, and R. Goldzmid for valuable discussion. This research was supported by the Intramural Research Program of the NIAID, NIH. S.G.'s contributions occurred during a sabbatical supported by a stipend from the NIAID and NCI intramural programs.

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