<i>Socs3fl/fl LysM cre</i> mice show higher susceptibility to infection with <i>M. tuberculosis</i>.

<p><i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> 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&lt;0.05 and **p&lt;0.01 Mann Whitney U test).Gross-pathology photograph of the lungs from Socs3^fl/fl and Socs3^fl/fl LysM cre mice 8 weeks after infection with M. tuberculosis (C). Histopathological scoring of hematoxylin-eosin stained paraffin lung sections from <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> mice measured 4 weeks after infection with <i>M. tuberculosis</i> (D). The mean % lung area with granulomas or free of lesions ± SEM is displayed. Differences with controls are significant (n = 8 per group, *p&lt;0.05 Student t test). The cumulative mortality of <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n = 10) after aerosol infection with <i>M. tuberculosis</i> is depicted (E). Survival curves are different (Log-rank test p&lt;0.005). CFU per lung, spleen and liver in <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> 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&lt;0.01 Mann Whitney U test). The mean percentage of Gr1+F4/80- neutrophils in the lung of <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n = 5 per group) 3 weeks after infection with <i>M. tuberculosis</i> ± 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 <i>M. tuberculosis</i> infection (n≥5 per group) was determined by real time PCR. The mean fold <i>Mpo</i> mRNA increase ± SEM in is depicted (H).</p

SOCS3-deficient γδ+ T cells secrete IL-17 during <i>M. tuberculosis</i> infection.

<p><i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> 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 <i>Il-17a</i> and <i>Hprt</i> transcripts was measured by real time PCR (A). The mean fold increase of <i>IL-17a</i> mRNA ± SEM in lungs from infected mice (n≥5 per group) was calculated. One out of two independent experiments is depicted. Differences with infected Socs3^fl/fl mice are significant (*p&lt;0.05 Student t test). <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice were sacrificed 2.5 weeks after aerosol infection with <i>M. tuberculosis</i>. 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&lt;0.05, **p&lt;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 <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> 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&lt;0.01, ***p&lt;0.001 Student t test). The presence of IL-17-secreting cells in PMA/ionomycin-stimulated lung cell suspensions from <i>Socs3^fl/fl lck cre</i> or <i>Socs3^fl/fl</i> mice before or 16 days after infection with <i>M. tuberculosis</i> 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&lt;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). <i>Rag1</i>^−/− mice were infected with <i>M. tuberculosis</i> 2 weeks after inoculation with either 1.2×10⁶ CD4+ or 2×106 CD90+ <i>Socs3^fl/fl lck cre</i> 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&lt;0.01 Student t test).</p

<i>Gp130^F/F</i> mice display dramatic susceptibility to infection with <i>M. tuberculosis</i>.

<p><i>Gp130^F/F</i>, <i>gp130^F/F Il-6^−/−</i>, <i>gp130^F/F/Stat3^+/−</i> and control mice were sacrificed 4 weeks after aerosol infection with <i>M. tuberculosis</i>, and CFU per lung assessed (A). The CFU in lungs of individual mice and the median per group (n≥6) are depicted. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003442#s2" target="_blank">Results</a> are pooled from two independent experiments. Differences in CFU are significant (*p&lt;0.05, ***p&lt;0.001 Mann Whitney U test). A gross-pathology photograph of the lungs from <i>gp130^F/F</i>, <i>gp130^F/F Il-6^−/−</i> and control mice 4 weeks after infection with <i>M. tuberculosis</i> is shown (B). <i>Gp130^F/F</i>, <i>gp130^F/F Il-6^−/−</i> and control mice were sacrificed at 4 weeks after <i>M. tuberculosis</i> infection and the total RNA was extracted from lungs. The accumulation of <i>Il-6</i> (C) and <i>Il-12 p40</i> (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&lt;0.01 Student t test). 2×10⁶ CD90+ <i>gp130^F/F</i> and control splenic T cells were inoculated i.v. into <i>Rag1^−/−</i> mice. Two weeks after transfer, mice were infected via the aerosol route with <i>M. tuberculosis</i>. Mice were sacrificed 4 weeks after infection and the CFU in lungs determined. The median CFU (n≥10) in lungs, quartiles and the 99^th percentiles are depicted (E). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003442#s2" target="_blank">Results</a> are pooled from two independent experiments. Differences in CFU are significant (**p&lt;0.01, ***p&lt;0.001 Mann Whitney U test). The CFU in lungs of <i>gp130^F/F</i> bone marrow→WT and WT bone marrow→ WT radiation chimeric mice were measured one month after infection with <i>M. tuberculosis</i>. A box and whisker diagram showing the median CFU (n≥8), quartiles and the 99^th percentiles is depicted (F). Differences in CFU are significant (***p&lt;0.001 Mann Whitney U test).</p

Reduced IL-12 secretion in <i>Socs3^fl/fl LysM cre</i> macrophages and DCs after mycobacterial infection.

<p>The levels of <i>Il-12 p40</i> mRNA were measured in triplicate cultures of <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMM infected with BCG (A) or <i>M. tuberculosis</i> (B) or treated with Pam3CSK4 for 24 h (A). Additionally, 50 ng/ml recombinant IL-6 was added to <i>M. tuberculosis</i>-infected samples (B). One out of two independent experiments is shown (*p&lt;0.05 and ***p&lt;0.001 Student t test). The concentration of IL-12 in supernatants from BCG-infected <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> 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&lt;0.05, **p&lt;0.01, ***p&lt;0.001 Student t test). The levels of <i>Il-12 p40</i> mRNA were measured in triplicate cultures of <i>gp130^F/F</i>, <i>gp130^F/F Il-6^−/−</i> or WT BMM infected with <i>M. tuberculosis</i> (D), (**p&lt;0.05 and ***p&lt;0.001 Student t test). The levels of <i>Socs3</i> (E) and <i>Il-12 p40</i> mRNA (F) in triplicate cultures of <i>M. tuberculosis-</i>infected <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMDC were determined by real time PCR. One of two independent experiments is shown, (*p&lt;0.05 Student t test). The concentration of IL-12 was determined by ELISA in supernatants from triplicate cultures of <i>M. tuberculosis</i>-infected <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMDC co-incubated or not with 50 ng/ml IL-6 (G), (*p&lt;0.05, Student t test). Total RNA was extracted from <i>M. tuberculosis</i>-infected CD11c+ and CD11c- <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> BMDC cultures 24 h after <i>M. tuberculosis</i> infection. The mean <i>Il-12p40</i> mRNA levels ± SEM levels measured by real time PCR are depicted (H). The concentration of IL-12p40 in supernatants from <i>M. tuberculosis</i>-infected <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> splenic DCs was determined by ELISA (I). The mean IL-12p40 ± SEM pg/ml from triplicate cultures is depicted (*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 Student t test). The fold increase of <i>Il-12 p40</i> mRNA in the lungs of <i>M. tuberculosis</i>-infected <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> 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&lt;0.05, Student t test). Total RNA was isolated from the lungs of <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> mice (n≥7 per group) treated or not with CD4 cell-depleting antibodies 2.5 weeks after infection with <i>M. tuberculosis</i> (K). The mean <i>Ifn</i>-<i>γ</i> mRNA ± SEM is depicted, (*p&lt;0.05, Student t test). Bacterial loads in lungs from <i>Socs3^fl/fl</i> and CD4+ cell-depleted <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> mice (n≥5) 2.5 weeks after <i>M. tuberculosis infection</i> are shown (L). A box and whisker diagram showing the median CFU, quartiles and the 99^th percentiles is depicted, (*p&lt;0.05, **p&lt;0.01 Mann Whitney U test).</p

SOCS3-deficient macrophages do not display increased <i>M. tuberculosis</i> growth.

<p>Mouse BMM were infected with <i>M. tuberculosis</i> 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 <i>MyD88^−/−</i> (A) WT (C57Bl/6) (A, B), <i>Socs3^fl/fl</i> (C) or <i>Socs3^fl/fl LysM cre</i> (C) BMM at the indicated time points after infection. The mean fold <i>Socs3</i> 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&lt;0.05, **p&lt;0.01, ***p&lt;0.001 Student t test). Phosphorylated STAT3, total STAT3 and actin in lysates <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMM after infection with <i>M. tuberculosis</i> was detected by Western Blot (D). Bacterial levels were determined in <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMM after infection <i>M. tuberculosis</i> 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&lt;0.05 Student t test) <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> pulmonary macrophages were infected with <i>M. tuberculosis</i> at a MOI of 1. The CFU were determined at the indicated time points in triplicate cell cultures (*p&lt;0.05 Student t test)(G). <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMM were infected with <i>M. tuberculosis</i> 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 <i>Socs3^fl/fl</i> BMM are significant (*p&lt;0.05 Student t test). Total RNA was extracted from <i>Socs3^fl/fl LysM cre</i>, <i>Socs3^fl/fl</i> (I, L), WT and gp130^F/F (K) BMM at the indicated times after infection with <i>M. tuberculosis</i> at a MOI of 5. The relative accumulation of <i>iNos</i>, <i>Cxcl10</i> and <i>Hprt</i> mRNA was measured by real time PCR. The mean fold increase of <i>iNos</i> (I, K), or <i>Cxcl10</i> (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&lt;0.05 and **p&lt;0.01Student t test). Nitrite concentrations in supernatants of <i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> BMM at different times after infection with <i>M. tuberculosis</i>. The mean NO₂⁻ concentration ± SEM in triplicate cultures per condition from one of two independent experiments is depicted (***p&lt;0.001 Student t test)(J).</p

γδ+ T cell numbers are increased in organs of <i>Socs3^fl/fl lck cre</i> mice.

<p>The frequency of γδ+ T cells within CD3+ cells in the thymus (A), spleen (B) and lung (C) of <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> 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 <i>Socs3^fl/fl</i> mice (n = 4 per group) are significant (*p&lt;0.05, ***p&lt;0.001 Student t test). The gating strategy and representative dot-plots for spleens of infected <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice are shown (E). Two million <i>Socs3^fl/fl lck cre</i> or <i>Socs3^fl/fl</i> CD90+ T cells positively selected from spleens using magnetic beads were inoculated i.v. into <i>Rag1^−/−</i> mice. A group of animals was also inoculated with both <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl T</i> cells (F). <i>Rag1^−/−</i> mice were alternatively transferred with 1.2×10⁶ CD4+ or 2×10⁶ CD90+ <i>Socs3^fl/fl lck cre</i> spleen cells (G) or in a different experiment with 2×10⁶ FACS-sorted CD3+ depleted of γδ+ T cells or total CD3+ <i>Socs3^fl/fl lck cre</i> spleen cells (H). Two weeks after transfer, mice were infected with <i>M. tuberculosis</i> 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 99^th percentiles in lungs and spleens are depicted (F–H). Differences in CFU are significant (*p&lt;0.05, **p&lt;0.001, ***p&lt;0.001 Mann Whitney U test).</p

Proposed model of SOCS3-mediated roles during infection with <i>M. tuberculosis</i>.

<p>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 <i>M. tuberculosis</i> infection.</p

SOCS3-deficient BMM show diminished TNF secretion after infection with <i>M. tuberculosis</i>.

<p><i>Socs3^fl/fl LysM cre</i> and <i>Socs3^fl/fl</i> mice were infected with <i>M. tuberculosis</i> 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 <i>Il-6</i> and <i>Hprt</i> transcripts was measured by real time PCR. The mean fold <i>Il-6</i> mRNA increase ± SEM in lungs from infected mice (n≥5 per group) was calculated (*p&lt;0.05 Student t test)(A). The levels of <i>Il-6</i> mRNA in <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> (B) or <i>gp130^F/F</i> and WT (D) BMM infected with <i>M. tuberculosis</i> 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&lt;0.05 Student t test). The mean IL-6 concentration ± SEM in supernatants of <i>M. tuberculosis</i>-infected <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> pulmonary macrophages as determined by ELISA is depicted. IL-6 secretion by BCG-infected <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> peritoneal macrophages is shown (C). The concentration of TNF was measured in supernatants of <i>M. tuberculosis</i>-infected <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> BMM co-incubated with or without 50 ng/ml of recombinant IL-6 (E). The levels of <i>Tnf</i> mRNA in <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl LysM cre</i> (F) or <i>gp130^F/F</i>, <i>gp130^F/F Il-6^−/−</i> and WT (G) BMM infected with <i>M. tuberculosis</i> 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&lt;0.05, **p&lt;0.01, ***p&lt;0.001 Student t test).</p

BCG-immunization protects <i>Socs3^fl/fl</i> and <i>Socs3^fl/fl lck cre</i> mice equally well against <i>M. tuberculosis</i> challenge.

<p><i>M. tuberculosis</i>-infected <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice were sacrificed 4 weeks after infection and the total RNA was extracted from lungs. The accumulation of <i>Ifn-γ</i> (A) or <i>Hprt</i> 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&lt;0.05, **p&lt;0.01 Student t test). <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice were sacrificed 2.5 weeks after infection with <i>M. tuberculosis</i>. 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&lt;0.001 ANOVA with Bonferroni correction). Total RNA was extracted from lungs of <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice 6 weeks after BCG infection. Levels of <i>Il-17a</i> (C) mRNA expression was determined by real time PCR (n = 6 mice per group). <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> 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. <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> 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). <i>Socs3^fl/fl lck cre</i> and <i>Socs3^fl/fl</i> mice were infected with 10⁶ BCG i.v. and were challenged with <i>M. tuberculosis</i> 10 weeks post BCG infection. Four weeks after <i>M. tuberculosis</i> 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&lt;0.05, ***p&lt;0.001 Mann Whitney U test). Wild type mice were either infected with 10⁶ BCG i.v. (I, J) or via aerosol with <i>M. tuberculosis</i> Harlingen (H, J). The total RNA was isolated from lungs or pulmonary CD90+ T cells at the indicated time points. The mean fold <i>Socs3</i> mRNA increase ± SEM (n = 5 per group) was determined by real-time PCR (H–J). Differences with between groups are significant (*p&lt;0.05 Student t test).</p

SARS-CoV-2 evolution influences GBP and IFITM sensitivity.

SARS-CoV-2 spike requires proteolytic processing for viral entry. A polybasic furin-cleavage site (FCS) in spike, and evolution toward an optimized FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show interferon-inducible restriction factors Guanylate-binding proteins (GBP) 2 and 5 interfere with furin-mediated spike cleavage and inhibit the infectivity of early-lineage isolates Wuhan-Hu-1 and VIC. By contrast, VOCs Alpha and Delta escape restriction by GBP2/5 that we map to the spike substitution D614G present in these VOCs. Despite inhibition of spike cleavage, these viruses remained sensitive to plasma membrane IFITM1, but not endosomal IFITM2 and 3, consistent with a preference for TMPRSS2-dependent plasma membrane entry. Strikingly, we find that Omicron is unique among VOCs, being sensitive to restriction factors GBP2/5, and also IFITM1, 2, and 3. Using chimeric spike mutants, we map the Omicron phenotype and show that the S1 domain determines Omicron's sensitivity to GBP2/5, whereas the S2' domain determines its sensitivity to endosomal IFITM2/3 and preferential use of TMPRSS2-independent entry. We propose that evolution of SARS-CoV-2 for the D614G substitution has allowed for escape from GBP restriction factors, but the selective pressures on Omicron for spike changes that mediate antibody escape, and altered tropism, have come at the expense of increased sensitivity to innate immune restriction factors that target virus entry