1. Volume 35, Issue 6, Pages 1023-1034 (December 2011)
Innate and Adaptive Interferons Suppress IL-1α and IL-1β Production by Distinct Pulmonary Myeloid Subsets during Mycobacterium tuberculosis Infection 
Katrin D. Mayer-Barber, Bruno B. Andrade, Daniel L. Barber, Sara Hieny, Carl G. Feng, Patricia Caspar, Sandy Oland, Siamon Gordon, Alan Sher 
Immunity 
Volume 35, Issue 6, Pages (December 2011)
DOI: /j.immuni
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Immunity 2011 35, 1023-1034DOI: (10.1016/j.immuni.2011.12.002)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. 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.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
IL-1α and IL-1β 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-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.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. 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.
Immunity  , DOI: ( /j.immuni )
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6. 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 < ) 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.
Immunity  , DOI: ( /j.immuni )
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7. 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 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-1α,β expression by WT (white circles) or Ifnar1−/− (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-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).
Immunity  , DOI: ( /j.immuni )
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8. 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 CD11bpos 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).
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions

9. 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).
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions