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1.
Fig 4

Fig 4. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

M. tuberculosis-induced expression of host-protective ifng and nos2 is lower in il12rb1−/− mice. mRNA from the lungs of M. tuberculosis-infected C57BL/6 and il12rb1−/− mice was collected at select times postinfection; subsequently generated cDNA was used for real-time quantitation of ifng (A), nos2 (B), irgm1 (C), and tnfa (D) expression levels. Expression of each gene was normalized to that of gapdh in the same sample; data were analyzed via ΔΔCT analysis (7) and is expressed as fold expression over the levels found in genotype-matched uninfected controls. Each data point represents the mean ± SD of the values at each time point and show one experiment representative of a total of three. For the difference between values of il12rb1−/− relative to C57BL/6 mice, P ≤ 0.05 (*) by Student's t test.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
2.
Fig 2

Fig 2. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

The localization of acid-fast bacteria in il12rb1−/− lungs is distinct from that in C57BL/6 mice. Twenty days following M. tuberculosis infection, lungs of C57BL/6 and il12rb1−/− mice were fixed and stained for acid-fast bacilli (AFB) in order to visualize the localization of M. tuberculosis in lungs of either genotype. Shown in panel A, left, is a micrograph at ×10 magnification that is representative of C57BL/6 lungs at this time; at right is a ×100 magnification of the inset indicated in the left panel showing AFB within inflamed alveoli. (B) Representative micrograph at a ×10 magnification of il12rb1−/− lungs collected and acid-fast stained at the same time as those of controls. Magnifications (×100) of select areas (i to iii) showed AFB localizing near the lung vasculature (i), near the airways (iia and b), and in or on alveolar pneumocytes (iii); images in frames iia and iib are serial sections of the same area used to help resolve the morphology of the cell containing AFB. Vs, vessel; Br, bronchiole; Al, alveoli.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
3.
Fig 1

Fig 1. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

il12rb1 is required to limit M. tuberculosis burden in multiple organs. Groups of C57BL/6 (B6) and il12rb1−/− mice were simultaneously infected via aerosol with ∼80 CFU of M. tuberculosis H37Rv. At select times postinfection, M. tuberculosis burdens in the lungs (A), spleen (B), and liver (C) were assessed by plating serial dilutions of homogenized organs on 7H11. Shown for each time is the mean number of CFU (log10) present in four mice per genotype per time point. Error bars represent ±SD; asterisks indicate that a significant difference between C57BL/6 and il12rb1−/− mice was observed at the indicated time point (i.e., P ≤ 0.05 as determined using Student's t test). This experiment was repeated four separate times, each with similar results.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
4.
Fig 8

Fig 8. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

ifng expression by rag1-dependent lineages contributes to M. tuberculosis control. (A) Lethally irradiated rag1−/− mice were reconstituted with either 100% C57BL/6 or 100% ifng−/− bone marrow; 4 weeks later, these chimeras were aerosol infected with M. tuberculosis H37Rv alongside ifng−/− controls. At select times postinfection, lung M. tuberculosis burdens were determined. *, statistically significant difference from C57BL/6 or rag1−/− controls. (B to D) Lethally irradiated rag1−/− mice were reconstituted with one of either of the following bone marrow preparations: 100% C57BL/6, 80% rag1−/− and 20% ifng+/+ cells (T/BifngWT mice), or 80% rag1−/− and 20% ifng−/− (T/BifngKO mice). All chimeras were simultaneously infected via aerosol with M. tuberculosis H37Rv. At indicated times following infection, M. tuberculosis burdens in the lungs (B), spleen (C), and liver (D) were determined. Each data point in panels A to D represents the mean ± SD of the values observed, and panels show one experiment representative of a total of two. *, P ≤ 0.05, between indicated groups as determined by ANOVA of all data points collected at that particular time.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
5.
Fig 3

Fig 3. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

Granulomatous accumulations in il12rb1−/− mice are distinct from those found in wild-type mice. Fifty days following aerogenic infection with M. tuberculosis, lung sections from C57BL/6 and il12rb1−/− mice were stained with Masson's trichrome in order to visually assess the degree of pulmonary pathology. Shown are representative micrographs of features observed in il12rb1−/− mice that were not present in C57BL/6 controls. (A) Micrograph at a ×4 magnification of an il12rb1−/− lung at day 50 postinfection showing the presence of two densely red staining regions, one of which is shown at ×10 magnification in the inset below (i). Shown in inset ii is a ×40 magnification of a pulmonary ossification found in the same il12rb1−/− lung. (B) An acid-fast stain of a serial section located adjacent to the trichrome-stained region shown in panel Ai was used to visualize the localization of AFB relative to the collagenous (i.e., blue) perimeter seen in panel Ai. (C to E) Micrographs at ×100 magnification of select features observed in panel B, namely, the presence of AFB in the necrotic center (C), AFB on or near bronchial epithelium (D), and AFB near the vascular epithelium (E).

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
6.
Fig 7

Fig 7. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

il12rb1 positively regulates T cell ifng expression following M. tuberculosis infection. Lung cell preparations from C57BL/6 and il12rb1−/− mice that were either uninfected (A) or M. tuberculosis infected (50 days postinfection) (B) were stained for T-cell marker Thy1 and intracellular IFN-γ. Isotype control staining (i.e., the top row) was used to discriminate positive staining for IFN-γ. Shown are representative FACS dot plots from each condition; inside the plots of αIFN-γ-stained cells (i.e., the bottom row) are box gates indicating the percentage of Thy1+ cells positive for IFN-γ. (C) Cumulative data demonstrating the percentage of Thy1+ cells expressing IFN-γ, as determined by intracellular cytokine staining, in both C57BL/6 and il12rb1−/− lungs after 50 days of M. tuberculosis infection (Mtb; solid bars). Data from uninfected (UI) lungs (open bars) are also shown. Data are combined from three separate experiments, with four mice per time point per experiment; bars represent the mean percentage ± SD. A significant difference (P ≤ 0.05) between uninfected and infected animals is indicated by an asterisk.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
7.
Fig 6

Fig 6. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

ifng is expressed by multiple hematopoietic subsets during experimental TB. C57BL/6 and IFN-γ–eYFP reporter mice (i.e., Yeti mice) (54) were infected via aerosol with M. tuberculosis. At 50 days postinfection, lung cell preparations were stained with a panel of antibodies for FACS determination of which cells were expressing ifng at that point in time. Using a gating strategy based on their forward scatter and side scatter characteristics, CD45-stained C57BL/6 cells served as a negative control for YFP fluorescence (A) while Yeti cells demonstrated all YFP signal to be found among CD45+ cells (B). (C) Among CD45+ YFP+ cells, we surveyed the extent to which CD4+, CD8+, NK1.1+, GR1+, CD19+, or γδ-TCR+ events were present. Shown are representative histograms for each subset. Shown are data from one experiment that is representative of a total of two (four mice per experiment). (D) Cumulative data demonstrating the percentage of CD45+ YFP+ cells expressing either of the following lineage markers at indicated times following infection: CD4, CD8, NK1.1, GR1, CD19, or γδ-TCR. Data are combined from two separate experiments, with four mice per time point per experiment; bars represent the mean percentage of YFP+ events expressing each marker ± SD. A significant difference (P ≤ 0.05) between two time points (for a given lineage) is indicated by an asterisk. D20, day 20 postinfection; D50, day 50 postinfection; UI, uninfected.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
8.
Fig 5

Fig 5. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

il12rb1 expression by rag1-dependent lineages is required to control M. tuberculosis infection. Lethally irradiated rag1−/− mice were reconstituted with one of either of the following bone marrow preparations: 100% C57BL/6, 80% rag1−/− and 20% il12rb1+/+ cells (T/Bil12rb1WT mice), 100% il12rb1−/−, or 80% rag1−/− and 20% il12rb1−/− (T/Bil12rb1KO mice) cells. All chimeras were simultaneously infected via aerosol with M. tuberculosis H37Rv. Fifty days postinfection with M. tuberculosis, bacterial burdens in the lungs (A), spleen (B), and liver (C) were determined. Each bar represents the mean + SD of the values observed and show one experiment representative of a total of three. *, P ≤ 0.05, between the values of two groups as determined by ANOVA; ns, not significant. (D and E) To confirm that innate lineages retained il12rb1 expression in T/Bil12rb1KO mice, CD45.1 congenic mice were used as il12rb1+/+ donors so as to determine, via flow cytometry of both lung and BAL cell preparations, the percentage of NK1.1+ (D, top row) and CD11c+ (D, bottom row) lineages that were il12rb1 sufficient (CD45.1+) or il12rb1 deficient (CD45.2+). (E) These same lineages (i.e., NK1.1+ and CD11c+), as well as CD45neg, CD45pos, I-Ab+, CD11b+, Ly6G+, and Thy1+ cells from M. tuberculosis-infected T/Bil12rb1KO mice, were magnetically purified and subjected to denaturing SDS-PAGE and Western analysis to visualize IL-12Rβ1 protein expression. Blots were probed with polyclonal anti-mouse IL-12Rβ1; the location of IL-12Rβ1 protein is indicated by an arrow (right) and is consistent with a 100- to 120-kDa glycosylated product as judged by size standards (left).

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
9.
Fig 10

Fig 10. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

il12rb1−/− T cells display defective proliferative capacity and aberrant polarization. T/Bil12rb150/50 mice were infected via aerosol with ∼80 CFU of virulent H37Rv. At 48 days after infection, mice were treated i.p. with BrdU containing saline; 2 days later (day 50 of infection), lung cell preparations were stained with anti-BrdU antibodies as well as the indicated extracellular (i.e., CD4, CD8, and CD44) and intracellular (Tbet, Foxp3, TNF-α, and IL-17) markers of T cell differentiation. Costaining for CD45.2 was used to discriminate between il12rb1+/+ (i.e., CD45.2neg) and il12rb1−/− (i.e., CD45.2pos) populations; shown are representative dot plots of both CD4+ (A to F) and CD8+ (G to L) lung cells. For each dot plot, the numbers in the upper left and right quadrants indicate, respectively, the percentages of il12rb1+/+ and il12rb1−/− T cells expressing either CD44 (A and G), incorporated BrdU (B and H), Tbet (C and I), Foxp3 (D and J), TNF-α (E and K), or IL-17 (F and L). (M and N) Cumulative CD4+ and CD8+ T cell expression data of the indicated markers from two experiments with four T/Bil12rb150/50 mice per experiment. Each bar represents the mean percentage observed ± SD; a statistically significant difference (P ≤ 0.05) between two groups is indicated with an asterisk and was determined by a Student's t test of all data points for an individual marker.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.
10.
Fig 9

Fig 9. From: Early Control of Mycobacterium tuberculosis Infection Requires il12rb1 Expression by rag1-Dependent Lineages.

il12rb1+/+ T cells exhibit dominance over il12rb1−/− T cells during M. tuberculosis infection. Lethally irradiated rag1−/− mice were reconstituted with one of either of the following bone marrow preparations: 80% rag1−/− and 20% CD45.1.il12rb1+/+ (T/Bil12rb1WT), 80% rag1−/− and 20% il12rb1−/− (T/Bil12rb1KO), or 80% rag1−/−, 10% CD45.1.il12rb1+/+, and 10% il12rb1−/− (T/Bil12rb150/50) cells. Following M. tuberculosis infection, we determined the relative percentage of IFN-γ+ cells among cells of either the il12rb1+/+ (i.e., CD45.1pos) or il12rb1−/− (i.e., CD45.1neg) genotype at day 50 postinfection. Shown in panel A is our gating strategy for identification of T lymphocytes. Examination of lung T lymphocytes in M. tuberculosis-infected T/Bil12rb1WT (B) and T/Bil12rb1KO (C) mice established the percentages of T cells expressing IFN-γ in the absence of T cells of a different genotype, whether they be CD45.2neg CD45.1pos il12rb1+/+ cells (F) or CD45.2pos CD45.1neg il12rb1−/− cells (G). (D and E) The same analysis was extended to T/Bil12rb150/50 mice that had been either left uninfected (D) or M. tuberculosis infected (E) at the same time as the controls shown in panels B and C. Box gates in panels B to E indicate the percentages of IFN-γ+ events among T cells of each genotype. (H) IFN-γ expression by lung NK cells 30 days after M. tuberculosis infection in both T/Bil12rb1WT (left) and T/Bil12rb1KO (right) chimeric mice. Dot plots are gated off NK1.1+ Thy1 cells, with numbers indicating the relative percentages of intracellular IFN-γ+ NK cells with either an il12rb1+/+ or il12rb1−/− genotype. CD45.1 congenic mice were used as il12rb1+/+ donors so as to distinguish between NK cells of either genotype. In T/Bil12rb1WT mice (left), both CD45.2+ and CD45.2 cells are il12rb1+/+; in T/Bil12rb1KO mice (right), by definition, CD45.2+ cells are il12rb1−/−, and CD45.2 cells are il12rb1+/+. All dot plots are representative of two separate experiments with four mice per group per experiment. (I) Cumulative data (the experiments performed for panels A to G) demonstrating the percentage of lung CD45.2neg (i.e., il12rb1+/+) and CD45.2pos (i.e., il12rb1−/−) Thy1+ cells expressing intracellular IFN-γ in either of the following groups of chimeras: T/Bil12rb1WT, T/Bil12rb1KO, and T/Bil12rb150/50 mice. Data from both uninfected (UI) and M. tuberculosis-infected (day 50) mice are shown; bars represent the mean ± SD of the percentages observed and are the combined data from eight mice per group (two separate aerosol experiments with four mice/group/time point). A statistically significant difference (P ≤ 0.05) between two groups is indicated with an asterisk and was determined by ANOVA of all data points.

Halli E. Miller, et al. Infect Immun. 2012 November;80(11):3828-3841.

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