Figure 1.
Socs3fl/fl LysM cre mice show higher susceptibility to infection with M. tuberculosis.
Socs3fl/fl LysM cre and Socs3fl/fl littermate controls were sacrificed at indicated time points after aerosol infection with M. tuberculosis and colony forming units (CFU) per lung (A) and spleen (B) were assessed. The CFU per lung of individual mice and the median per group (n≥4) at the indicated time points after infection are depicted. Differences in CFU are significant (*p<0.05 and **p<0.01 Mann Whitney U test).Gross-pathology photograph of the lungs from Socs3fl/fl and Socs3fl/fl LysM cre mice 8 weeks after infection with M. tuberculosis (C). Histopathological scoring of hematoxylin-eosin stained paraffin lung sections from Socs3fl/fl LysM cre and Socs3fl/fl mice measured 4 weeks after infection with M. tuberculosis (D). The mean % lung area with granulomas or free of lesions ± SEM is displayed. Differences with controls are significant (n = 8 per group, *p<0.05 Student t test). The cumulative mortality of Socs3fl/fl and Socs3fl/fl LysM cre mice (n = 10) after aerosol infection with M. tuberculosis is depicted (E). Survival curves are different (Log-rank test p<0.005). CFU per lung, spleen and liver in Socs3fl/fl and Socs3fl/fl LysM cre mice (n≥5 per group) were assessed 6 weeks after infection with 106 BCG i.v. (F). The median CFU and interquartile range per group are depicted. Differences in CFU are significant (**p<0.01 Mann Whitney U test). The mean percentage of Gr1+F4/80- neutrophils in the lung of Socs3fl/fl and Socs3fl/fl LysM cre mice (n = 5 per group) 3 weeks after infection with M. tuberculosis ± SEM was determined by FACS analysis (G). The accumulation of myeloid peroxidase (Mpo) transcripts in lungs from mice at 0, 3 or 8 weeks after M. tuberculosis infection (n≥5 per group) was determined by real time PCR. The mean fold Mpo mRNA increase ± SEM in is depicted (H).
Figure 2.
SOCS3-deficient macrophages do not display increased M. tuberculosis growth.
Mouse BMM were infected with M. tuberculosis at a MOI of 5∶1 (A–C). BMM were treated with the indicated concentrations of BAY-117082 1 h before infection (B). Total RNA was isolated from MyD88−/− (A) WT (C57Bl/6) (A, B), Socs3fl/fl (C) or Socs3fl/fl LysM cre (C) BMM at the indicated time points after infection. The mean fold Socs3 mRNA induction ± SEM measured by real time PCR is depicted. A representative of 3 experiments is shown (C). Differences with WT (A, C) or untreated (B) BMM are significant (*p<0.05, **p<0.01, ***p<0.001 Student t test). Phosphorylated STAT3, total STAT3 and actin in lysates Socs3fl/fl LysM cre and Socs3fl/fl BMM after infection with M. tuberculosis was detected by Western Blot (D). Bacterial levels were determined in Socs3fl/fl LysM cre and Socs3fl/fl BMM after infection M. tuberculosis H37Rv at a MOI of 0.5∶1 (E) or 5∶1 (F). The mean CFU ± SEM from triplicate cell cultures is shown. Two independent experiments for each panel were performed. (*p<0.05 Student t test) Socs3fl/fl LysM cre and Socs3fl/fl pulmonary macrophages were infected with M. tuberculosis at a MOI of 1. The CFU were determined at the indicated time points in triplicate cell cultures (*p<0.05 Student t test)(G). Socs3fl/fl LysM cre and Socs3fl/fl BMM were infected with M. tuberculosis at a MOI of 5. One hundred U/ml recombinant IFN-γ were added 24 h after infection. The CFU were determined in triplicate cell cultures (H). One out of two independent experiments is shown. Differences with Socs3fl/fl BMM are significant (*p<0.05 Student t test). Total RNA was extracted from Socs3fl/fl LysM cre, Socs3fl/fl (I, L), WT and gp130F/F (K) BMM at the indicated times after infection with M. tuberculosis at a MOI of 5. The relative accumulation of iNos, Cxcl10 and Hprt mRNA was measured by real time PCR. The mean fold increase of iNos (I, K), or Cxcl10 (L) mRNA ± SEM in triplicate samples for each genotype and time point in one out of two independent experiments is depicted. Differences with control BMM are significant (*p<0.05 and **p<0.01Student t test). Nitrite concentrations in supernatants of Socs3fl/fl LysM cre and Socs3fl/fl BMM at different times after infection with M. tuberculosis. The mean NO2− concentration ± SEM in triplicate cultures per condition from one of two independent experiments is depicted (***p<0.001 Student t test)(J).
Figure 3.
SOCS3-deficient BMM show diminished TNF secretion after infection with M. tuberculosis.
Socs3fl/fl LysM cre and Socs3fl/fl mice were infected with M. tuberculosis Harlingen via the aerosol route. Animals were sacrificed at the indicated time points after infection and the total RNA was extracted from lungs. The accumulation of Il-6 and Hprt transcripts was measured by real time PCR. The mean fold Il-6 mRNA increase ± SEM in lungs from infected mice (n≥5 per group) was calculated (*p<0.05 Student t test)(A). The levels of Il-6 mRNA in Socs3fl/fl and Socs3fl/fl LysM cre (B) or gp130F/F and WT (D) BMM infected with M. tuberculosis were determined by real time PCR. The mean fold increase of mRNA level ± SEM in triplicate independent cultures per condition compared to non-infected cultures in one out of two independent experiments is depicted (*p<0.05 Student t test). The mean IL-6 concentration ± SEM in supernatants of M. tuberculosis-infected Socs3fl/fl and Socs3fl/fl LysM cre pulmonary macrophages as determined by ELISA is depicted. IL-6 secretion by BCG-infected Socs3fl/fl and Socs3fl/fl LysM cre peritoneal macrophages is shown (C). The concentration of TNF was measured in supernatants of M. tuberculosis-infected Socs3fl/fl and Socs3fl/fl LysM cre BMM co-incubated with or without 50 ng/ml of recombinant IL-6 (E). The levels of Tnf mRNA in Socs3fl/fl and Socs3fl/fl LysM cre (F) or gp130F/F, gp130F/F Il-6−/− and WT (G) BMM infected with M. tuberculosis were determined by real time PCR. The mean fold increase of mRNA level ± SEM in triplicate independent cultures per condition compared to non-infected cultures of one of two independent experiments is depicted (*p<0.05, **p<0.01, ***p<0.001 Student t test).
Figure 4.
Reduced IL-12 secretion in Socs3fl/fl LysM cre macrophages and DCs after mycobacterial infection.
The levels of Il-12 p40 mRNA were measured in triplicate cultures of Socs3fl/fl LysM cre and Socs3fl/fl BMM infected with BCG (A) or M. tuberculosis (B) or treated with Pam3CSK4 for 24 h (A). Additionally, 50 ng/ml recombinant IL-6 was added to M. tuberculosis-infected samples (B). One out of two independent experiments is shown (*p<0.05 and ***p<0.001 Student t test). The concentration of IL-12 in supernatants from BCG-infected Socs3fl/fl and Socs3fl/fl LysM cre BMM co-incubated with 50 ng/ml IL-6 was determined by ELISA (C). The mean IL-12 concentration ± SEM from triplicate cultures per condition in one of two independent experiments is depicted, (*p<0.05, **p<0.01, ***p<0.001 Student t test). The levels of Il-12 p40 mRNA were measured in triplicate cultures of gp130F/F, gp130F/F Il-6−/− or WT BMM infected with M. tuberculosis (D), (**p<0.05 and ***p<0.001 Student t test). The levels of Socs3 (E) and Il-12 p40 mRNA (F) in triplicate cultures of M. tuberculosis-infected Socs3fl/fl LysM cre and Socs3fl/fl BMDC were determined by real time PCR. One of two independent experiments is shown, (*p<0.05 Student t test). The concentration of IL-12 was determined by ELISA in supernatants from triplicate cultures of M. tuberculosis-infected Socs3fl/fl LysM cre and Socs3fl/fl BMDC co-incubated or not with 50 ng/ml IL-6 (G), (*p<0.05, Student t test). Total RNA was extracted from M. tuberculosis-infected CD11c+ and CD11c- Socs3fl/fl and Socs3fl/fl LysM cre BMDC cultures 24 h after M. tuberculosis infection. The mean Il-12p40 mRNA levels ± SEM levels measured by real time PCR are depicted (H). The concentration of IL-12p40 in supernatants from M. tuberculosis-infected Socs3fl/fl and Socs3fl/fl LysM cre splenic DCs was determined by ELISA (I). The mean IL-12p40 ± SEM pg/ml from triplicate cultures is depicted (*p<0.05, **p<0.01, ***p<0.001 Student t test). The fold increase of Il-12 p40 mRNA in the lungs of M. tuberculosis-infected Socs3fl/fl LysM cre and Socs3fl/fl mice relative to uninfected mice is displayed (J). The data is pooled from 2 independent experiments with n≥5 animals per group in each one (*p<0.05, Student t test). Total RNA was isolated from the lungs of Socs3fl/fl LysM cre and Socs3fl/fl mice (n≥7 per group) treated or not with CD4 cell-depleting antibodies 2.5 weeks after infection with M. tuberculosis (K). The mean Ifn-γ mRNA ± SEM is depicted, (*p<0.05, Student t test). Bacterial loads in lungs from Socs3fl/fl and CD4+ cell-depleted Socs3fl/fl and Socs3fl/fl LysM cre mice (n≥5) 2.5 weeks after M. tuberculosis infection are shown (L). A box and whisker diagram showing the median CFU, quartiles and the 99th percentiles is depicted, (*p<0.05, **p<0.01 Mann Whitney U test).
Figure 5.
Mice with SOCS3-deficient T cells are susceptible to infection with M. tuberculosis.
Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed at the indicated time points after aerosol infection with M. tuberculosis, and CFU in lungs assessed (A). The CFU per lung of individual mice and the median per group (n≥5) are depicted. Results are pooled from three independent experiments. Differences in CFU are significant (***p<0.001 Mann Whitney U test). The cumulative mortality of Socs3fl/fl lck cre and Socs3fl/fl mice (n = 10 mice per group) after aerosol infection with M. tuberculosis is depicted (B). Differences in survival curves are significant (Log-rank test p<0.0005) with a median survival of 38 days for Socs3fl/fl lck cre mice. Hematoxylin-eosin stained paraffin lung sections from Socs3fl/fl (C) and Socs3fl/fl lck cre mice (D) and their histopathological scoring (E, F) determined 4 weeks after infection with M. tuberculosis. The mean % lung area with granulomas or free of lesions ± SEM and the mean score of lymphocytes within the granuloma or in perivascular spaces ± SEM is shown (F). Differences with controls are significant (n = 5 per group, *p<0.05 Student t test). Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed 4 weeks after M. tuberculosis infection and the total RNA was extracted from lungs. The accumulation of Mpo and Hprt transcripts was measured by real time PCR (G). The mean fold increase of Mpo mRNA ± SEM in lungs from infected mice (n≥5 per group) was calculated. Differences with infected Socs3fl/fl mice are significant (*p<0.05 Student t test). The mean frequency of FoxP3+ within CD4+ pulmonary lymph node CD3+ T cells from Socs3fl/fl lck cre and Socs3fl/fl mice (n = 5 per group) was determined by FACS analysis 4 weeks after infection with M. tuberculosis (H).
Figure 6.
γδ+ T cell numbers are increased in organs of Socs3fl/fl lck cre mice.
The frequency of γδ+ T cells within CD3+ cells in the thymus (A), spleen (B) and lung (C) of Socs3fl/fl lck cre and Socs3fl/fl mice obtained before or 2.5 weeks after M. tuberculosis infection were determined by FACS. Mean percentage (A–C) and the total numbers (D) of γδ+ within CD3+ T cells in the lungs ± SEM are depicted. Differences with Socs3fl/fl mice (n = 4 per group) are significant (*p<0.05, ***p<0.001 Student t test). The gating strategy and representative dot-plots for spleens of infected Socs3fl/fl lck cre and Socs3fl/fl mice are shown (E). Two million Socs3fl/fl lck cre or Socs3fl/fl CD90+ T cells positively selected from spleens using magnetic beads were inoculated i.v. into Rag1−/− mice. A group of animals was also inoculated with both Socs3fl/fl lck cre and Socs3fl/fl T cells (F). Rag1−/− mice were alternatively transferred with 1.2×106 CD4+ or 2×106 CD90+ Socs3fl/fl lck cre spleen cells (G) or in a different experiment with 2×106 FACS-sorted CD3+ depleted of γδ+ T cells or total CD3+ Socs3fl/fl lck cre spleen cells (H). Two weeks after transfer, mice were infected with M. tuberculosis via the aerosol route. Mice (n≥6 per group) were sacrificed 4 weeks after infection. Box and whisker diagrams showing the median CFU, quartiles and the 99th percentiles in lungs and spleens are depicted (F–H). Differences in CFU are significant (*p<0.05, **p<0.001, ***p<0.001 Mann Whitney U test).
Figure 7.
SOCS3-deficient γδ+ T cells secrete IL-17 during M. tuberculosis infection.
Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed before and at 2.5 and 4 weeks after M. tuberculosis infection and the total RNA was extracted from lungs. The accumulation of Il-17a and Hprt transcripts was measured by real time PCR (A). The mean fold increase of IL-17a mRNA ± SEM in lungs from infected mice (n≥5 per group) was calculated. One out of two independent experiments is depicted. Differences with infected Socs3fl/fl mice are significant (*p<0.05 Student t test). Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed 2.5 weeks after aerosol infection with M. tuberculosis. Lung cell suspensions were stimulated or not with 20 µg/ml PPD for 48 h. The IL-17 level in supernatants was determined by a cytokine bead assay (CBA) (B). The mean IL-17 concentration ± SEM (n≥6 animals per group) is depicted. Differences in cytokine concentrations are significant (*p<0.05, **p<0.01 ANOVA with Bonferroni correction). CD90+ naïve spleen T cells were co-cultured either with uninfected, BCG-infected BMDCs (DC) or with their 48 h supernatants (SN). After 72 h, the IL-17 levels in culture supernatants were measured by ELISA. A representative out of three independent experiments is shown (C). 105 γδ+, CD4+ or CD90+ FACS sorted T cells from Socs3fl/fl lck cre and Socs3fl/fl mice were co-cultured with supernatants from BCG-infected BMDCs for 72 h. The mean IL-17 concentration in supernatants from triplicate cultures ± SEM is depicted (D). Differences in cytokine concentrations are significant (**p<0.01, ***p<0.001 Student t test). The presence of IL-17-secreting cells in PMA/ionomycin-stimulated lung cell suspensions from Socs3fl/fl lck cre or Socs3fl/fl mice before or 16 days after infection with M. tuberculosis was measured by FACS as described in materials and methods. Representative FACS dot plots from CD3+ gated infected lung cells before or after PMA/ionomycin stimulation are shown (E). The frequency of IL-17-secreting γδ+ within CD3+ cells in uninfected or infected mice is displayed (n = 6, *p<0.05 Mann Whitney U test) (F). The mean frequency of IL-17-secreting CD4+, CD8+ and γδ+ within CD3+ cells in lungs of infected mice (5 mice per group) ± SEM is depicted (G). Rag1−/− mice were infected with M. tuberculosis 2 weeks after inoculation with either 1.2×106 CD4+ or 2×106 CD90+ Socs3fl/fl lck cre spleen cells. Mice were sacrificed 4 weeks after infection and lung cell suspensions incubated for 48 h. The mean concentration of IL-17 in supernatants ± SEM (n = 6) is depicted (H). Differences in cytokine concentrations are significant (**p<0.01 Student t test).
Figure 8.
BCG-immunization protects Socs3fl/fl and Socs3fl/fl lck cre mice equally well against M. tuberculosis challenge.
M. tuberculosis-infected Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed 4 weeks after infection and the total RNA was extracted from lungs. The accumulation of Ifn-γ (A) or Hprt transcripts was measured by real time PCR. The mean fold cytokine mRNA increase ± SEM in lungs from infected mice (n≥5 per group) was calculated. One out of two independent experiments is depicted. Differences with controls are significant (*p<0.05, **p<0.01 Student t test). Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed 2.5 weeks after infection with M. tuberculosis. Lung cell suspensions were stimulated with 20 µg/ml PPD and concentrations of IFN-γ (B) in supernatants after 48 h were measured by cytokine bead assay (CBA). The mean cytokine concentration ± SEM (n≥6 animals per group) is depicted. Differences in cytokine concentrations are significant (***p<0.001 ANOVA with Bonferroni correction). Total RNA was extracted from lungs of Socs3fl/fl lck cre and Socs3fl/fl mice 6 weeks after BCG infection. Levels of Il-17a (C) mRNA expression was determined by real time PCR (n = 6 mice per group). Socs3fl/fl lck cre and Socs3fl/fl mice were immunized s.c. with 5×106 heat-killed BCG and boosted after 2 weeks with 2.5×106 heat-killed BCG. Mice were sacrificed four weeks after the priming, and spleen cell suspensions from immunized or non-immunized control mice were co-incubated with 15 µg/ml PPD for 72 h. The concentration of IL-17 (D) and IFN-γ (E) in the culture supernatants was determined by ELISA. The mean cytokine concentration ± SEM (n = 6 animals per group) is depicted. Socs3fl/fl lck cre and Socs3fl/fl mice were sacrificed 6 weeks after i. v. infection with 106 BCG, and the CFU per lung, spleen and liver were quantified. The individual and median CFU per group (n≥6) are depicted (F). Socs3fl/fl lck cre and Socs3fl/fl mice were infected with 106 BCG i.v. and were challenged with M. tuberculosis 10 weeks post BCG infection. Four weeks after M. tuberculosis infection, mice were sacrificed and the CFU in the lungs were quantified. The individual and median CFU per group (n≥6) are shown (G). Differences in CFU are significant (**p<0.05, ***p<0.001 Mann Whitney U test). Wild type mice were either infected with 106 BCG i.v. (I, J) or via aerosol with M. tuberculosis Harlingen (H, J). The total RNA was isolated from lungs or pulmonary CD90+ T cells at the indicated time points. The mean fold Socs3 mRNA increase ± SEM (n = 5 per group) was determined by real-time PCR (H–J). Differences with between groups are significant (*p<0.05 Student t test).
Figure 9.
Gp130F/F mice display dramatic susceptibility to infection with M. tuberculosis.
Gp130F/F, gp130F/F Il-6−/−, gp130F/F/Stat3+/− and control mice were sacrificed 4 weeks after aerosol infection with M. tuberculosis, and CFU per lung assessed (A). The CFU in lungs of individual mice and the median per group (n≥6) are depicted. Results are pooled from two independent experiments. Differences in CFU are significant (*p<0.05, ***p<0.001 Mann Whitney U test). A gross-pathology photograph of the lungs from gp130F/F, gp130F/F Il-6−/− and control mice 4 weeks after infection with M. tuberculosis is shown (B). Gp130F/F, gp130F/F Il-6−/− and control mice were sacrificed at 4 weeks after M. tuberculosis infection and the total RNA was extracted from lungs. The accumulation of Il-6 (C) and Il-12 p40 (D) transcripts was measured by real time PCR. The mean fold cytokine mRNA increase ± SEM in lungs from infected mice (n = 5 per group) are depicted. Differences with controls are significant (**p<0.01 Student t test). 2×106 CD90+ gp130F/F and control splenic T cells were inoculated i.v. into Rag1−/− mice. Two weeks after transfer, mice were infected via the aerosol route with M. tuberculosis. Mice were sacrificed 4 weeks after infection and the CFU in lungs determined. The median CFU (n≥10) in lungs, quartiles and the 99th percentiles are depicted (E). Results are pooled from two independent experiments. Differences in CFU are significant (**p<0.01, ***p<0.001 Mann Whitney U test). The CFU in lungs of gp130F/F bone marrow→WT and WT bone marrow→ WT radiation chimeric mice were measured one month after infection with M. tuberculosis. A box and whisker diagram showing the median CFU (n≥8), quartiles and the 99th percentiles is depicted (F). Differences in CFU are significant (***p<0.001 Mann Whitney U test).
Figure 10.
Proposed model of SOCS3-mediated roles during infection with M. tuberculosis.
SOCS3 expression in antigen-presenting cells prevented IL-6-mediated inhibition of IL-12 secretion SOCS3 expression in T cells reduces the frequency of γδ+ T cells in different organs and the secretion of IL-17 by γδ+ T cells in response to infection in a gp130-independent manner. Expression of Socs3 in myeloid and lymphoid cell populations is critical for a proper control of M. tuberculosis infection.