STAT3 expression by myeloid cells is detrimental for the T- cell-mediated control of infection with <i>Mycobacterium tuberculosis</i>

<div><p>STAT3 is a master regulator of the immune responses. Here we show that <i>M</i>. <i>tuberculosis</i>-infected <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, <i>socs3</i>^<i>fl/fl</i> <i>cd11c cre</i> mice deficient of SOCS3 in DCs showed increased susceptibility to <i>M</i>. <i>tuberculosis</i> infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with <i>M</i>. <i>tuberculosis</i>.</p></div

SOCS3 and STAT3 in antigen-specific T cells are important regulators of IL-17 and IFN-γ secretion.

<p><i>Stat</i>3^<i>fl/fl</i><i>lck cre p25-tg</i> T cells (A, B), <i>socs3</i>^<i>fl/fl</i> <i>lck cre p25-tg</i> T cells (C, D) or control <i>p25-tg</i> T cells were incubated for 3 days with either BCG, peptide 25 and LPS-stimulated or untreated BMDCs. The mean concentration of IL-17 (A, C) and IFN-γ (B, D) ± SEM in supernatants from triplicate cultures is shown (**p<0.01 and ***p<0.001 Student’s t test). <i>Graphical summary</i> (E): Mice deficient in STAT3 in myeloid cells show increased resistance while <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> mice displaying augmented STAT3 activation had impaired resistance against infection with <i>M</i>. <i>tuberculosis</i>. The differential control of infection in excess or deficiency of STAT3 is not due to the intrinsic regulation of bacterial control by macrophages but rather to differences in the ability of DCs to regulate the differentiation of specific T-cells. <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs release higher levels of IL-6 and IL-23 after stimulation with mycobacterial or other TLR agonists, while secretion of these cytokines is reduced in <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs. <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs show improved ability to trigger IL-17 release by mycobacteria-specific T cells while the opposite is observed when using <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs. The IL-17 secretion by T cells is also controlled via gp130R signalling, indicating an autocrine or paracrine loop by IL-6 family cytokines. The increased resistance to <i>M</i>. <i>tuberculosis</i> infection of <i>stat3</i>^<i>fl/fl</i> lysm cre mice was IL-17 dependent. <i>Socs3</i>^<i>fl/fl</i> <i>lysm cr</i>e (but not <i>stat3</i>^<i>fl/fl</i> <i>lysm cre)</i> DCs improved capacity of mycobacteria-specific T cells priming <i>in vivo</i>.</p

<i>Stat3</i>^<i>fl/fl</i><i>lysm cre</i> mice are resistant to infection with <i>M</i>. <i>tuberculosis</i>.

<p><i>Stat3</i>^<i>fl/fl</i><i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> littermate controls were sacrificed at indicated time points after aerosol infection with <i>M</i>. <i>tuberculosis</i> and colony forming units (CFU) per lung (A) and spleen (B) were assessed. The CFU per organ of individual mice and the median per group at the indicated time points after infection are depicted. For each time point 8–10 control and 8–10 mutant mice were infected simultaneously. We performed separated experiments for 4 and 8 weeks post infection. Only one representative of the two experiments for 4 as well as 8 weeks post infection is depicted. Differences in CFU are significant (*p<0.05, **p<0.01, ***p<0.001 Mann Whitney U test). Histopathological scoring of hematoxylin-eosin stained paraffin lung sections from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice measured 4 and 8 weeks after infection with <i>M</i>. <i>tuberculosis</i>. The mean ± SEM % lung area with granulomas (C) and the relative neutrophil density score (D) are depicted. Differences are significant (*p<0.05, ***p<0.001 Student t test). A representative dot plot (E) and the frequency (F) of CD11b⁺CD11c^-Ly6C^dim Ly6G⁺ neutrophils in lungs <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 3 and 4 weeks after infection with <i>M</i>. <i>tuberculosis</i> are shown. Differences between 4 mice at each time point are significant (*p<0.05 Student’s t test). Total RNA was extracted from the lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice at the indicated time points after infection with <i>M</i>. <i>tuberculosis</i>. The relative concentration of neutrophil elastase (<i>elane</i>) (G) and myeloperoxidase (<i>mpo)</i> transcripts (H) in relation to <i>hprt</i> mRNA levels in the same sample was determined by real time PCR. The mean fold induction of these transcripts ± SEM is depicted. Differences with control mice are significant (*p<0.05, **p<0.01 Student t-test).</p

Role of STAT3 and SOCS3 in APCs in the regulation of T cell priming.

<p>The median fluorescent intensity (MFI) of MHCII, CD80 and CD86 on CD11c⁺ <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> or <i>stat3</i>^<i>fl/fl</i> (A-C) BMDCs was determined by FACS analysis 24 h after infection with BCG or non-infected controls. The mean MFI ± SEM are depicted. Experimental scheme of <i>M</i>. <i>tuberculosis</i> infection followed 17 days after by transfer of 3. 10⁶ <i>p25-tg</i> naïve T cells. Mice were sacrificed 3 days later (D). The mean MFI of CD62L (E) and CD69 (F) ± SEM on MLN CD4⁺ <i>p25-tg</i> or recipient T cells from either <i>stat3</i>^<i>fl/fl</i> lysm cre or <i>stat3</i>^<i>fl/fl</i> <i>M</i>. <i>tuberculosis-</i>infected mice. <i>Socs3</i>^<i>fl/fl</i> <i>cd11c cre and socs3</i>^<i>fl/fl</i> littermate controls were sacrificed 4 weeks after aerosol infection with <i>M</i>. <i>tuberculosis</i> and colony forming units (CFU) per lung (G) and spleen (H) were assessed. The CFU per organ of individual mice and the median per group at the indicated time points after infection are depicted. Differences in CFU are significant (**p<0.01, ***p<0.001 Mann Whitney U test). Representative FACS histograms and the mean MFI ± SEM of CD80, CD86 and MHC-II on <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>socs3</i>^<i>fl/fl</i> BMDCs infected or not with BCG for 24 hs (I-L). Differences with <i>socs3</i>^<i>fl/fl</i> BMDCs (n = 4 cultures per group) are significant (*p<0.05, ***p<0.001 Student <i>t</i> test). Representative FACS histograms and mean MFI levels of CD62L (M, O) and CD69 (N, P) ± SEM on <i>p25-tg</i> and host CD4⁺ MLN T cells before or 21 days after infection with <i>M</i>. <i>tuberculosis</i>. Differences with <i>socs3</i>^<i>fl/fl</i> BMDCs are significant (*p<0.05 and **p<0.01 Student t test).</p

STAT3 and SOCS3 regulate the secretion of IL-17 by antigen-specific T cells.

<p>Total RNA was extracted from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC cultures 24 h after <i>M</i>. <i>tuberculosis</i> infection. <i>Il6</i> and <i>il23p19</i> mRNA were measured by real time PCR and normalized to the <i>hprt</i> mRNA levels in the same samples. The mean and <i>il6</i> (A) <i>il-23p19</i> (B) mRNA fold increase ± SEM levels in triplicate independent cultures are depicted (*p<0.05, **p<0.01 and ***p<0.001 Student’s t test). The mean fold increase of <i>il6</i> (C) and <i>il23p19</i> (D) ± SEM were measured by real-time PCR in triplicate cultures of stat3^<i>fl/fl</i> <i>lysm</i> cre and <i>stat3</i>^<i>fl/fl</i> BMDCs 24 h after stimulation with either LPS, CpG or Pam3K (*p<0.05 and **p<0.01 Student’s t test). <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC (E-H) or BMM (H) were stimulated with either BCG (E), heat killed BCG (F), <i>M</i>. <i>tuberculosis</i> (G), or with LPS and the peptide 25 of Ag85b (H) and incubated 6 h after with <i>p25-tg</i> CD4+ naïve T cells (at a ratio of 4:1 BMDC). The concentration of IL-17 in the culture supernatants was measured by ELISA 72h after co-incubation. The mean IL-17 ng/ ml ± SEM from triplicate cultures is depicted (* p<0.05; **p<0.01 and ***p<0.001 Student’s t test). Total RNA was extracted from <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>socs3</i>^<i>fl/fl</i> BMDC cultures at different times after infection with mycobacteria. The mean <i>Il6</i> (I) and <i>IL23p19</i> (J) mRNA levels ± SEM levels determined by real time PCR are depicted (**p<0.01 Student’s t test). The concentration of IL-17 was measured 72h supernatants of co cultures of <i>p25-tg</i> naïve T cells incubated with either BCG (K), <i>M</i>. <i>tuberculosis</i> (L) or pept25 and LPS (M)-stimulated socs3^<i>fl/fl</i> <i>lysm cre</i> and socs3^<i>fl/fl</i> BMDCs. The mean IL-17 ng/ ml ± SEM from triplicate independent cultures were determined by ELISA (*p<0.05; **p<0.01 and ***p<0.001 Student’s t test). IL-17 was measured in 72 h culture supernatants from control or BCG-infected <i>gp130</i>^<i>F/F</i> BMDC and <i>p25-tg</i> T cells. The mean IL-17 ng/ ml ± SEM from triplicate cultures from infected or control BMDCs is shown in (N). Differences are significant at ***p<0.001 Student’s t test. The mean fold increase of <i>socs3</i> transcript ± SEM in total RNA from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC at different time points after <i>M</i>. <i>tuberculosis</i> infection as compared to uninfected controls were measured by real time PCR are depicted (O).</p

Myeloid cell expression of STAT3 hamper IL-17 secretion by T cells during M. tuberculosis infection.

<p>The frequency of IL-17-secreting PPD and PMA and ionomycin (P.I) -stimulated CD4+ pulmonary T cells from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 4 (A) and 8 (B) weeks after infection with <i>M</i>. <i>tuberculosis</i> was measured by FACS. A representative graph plot from PPD stimulated lungs at 4 w after infection (C) and the mean percentage of IL-17-secreting CD4+ cells ± SEM (A, B) are displayed (n = 6 per group, *p<0.05, **p<0.01 and ***p<0.001 Mann Whitney U test). The mean fold increase of <i>il17a</i> (D), <i>il22</i> (E), <i>cxcxl5</i> (F), <i>il23p19</i> (G) and <i>il6</i>(H) mRNA ± SEM was measured by real time PCR in the total RNA from lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice at different time points after <i>M</i>. <i>tuberculosis</i> infection (n = 8 per group **p<0.01 Student’s t test). The mean frequency of PPD (I) and PMA and ionomycin (J) -stimulated CD4+ pulmonary T cells from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 8 weeks after infection with <i>M</i>. <i>tuberculosis</i> co-secreting or not IFN-γ was measured by FACS (n = 4 per group, *p<0.05, **p<0.01 and ***p<0.001 Mann Whitney U test). A representative graph plot from CD3+CD4+ gated PPD and P.I. stimulated IFN-γ and / or IL-17+ lung cells (K) is depicted. The frequency of both IL-17 and IFN-γ and only IL-17+ secreting cells from <i>stat3</i>^<i>fl/fl</i><i>lysm cre</i> is higher as compared to <i>stat3</i>^<i>fl/fl</i> controls; the frequency of IL-17+/IFN-γ- is higher than IL-17+/IFN-γ+ cells. This was determined after either PPD or PMA/ ionomycin stimulation (Two-way ANOVA *p<0.05 and *** p<0.0001). <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> littermate controls were treated i.p. 1 day before and once per week after aerosol infection with <i>M</i>. <i>tuberculosis</i> with 500 μg anti-IL-17RA M751or left untreated. Mice were sacrificed 4 weeks after the infection and colony forming units (CFU) per lung (L) and spleen (M) were assessed. The CFU per organ of individual mice and the median per group at the indicated time points after infection are depicted. Differences in CFU are significant (*p<0.05, **p<0.01 Mann Whitney U test). The mean fold increase of <i>mpo</i> mRNA ± SEM was measured by real time PCR in lysates from lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 4 weeks after infection with <i>M</i>. <i>tuberculosis</i> treated or not with anti-IL-17RA as described above (n≥ 4 per group, *p<0.05 Student’s t test) (N).</p

STAT3 in myeloid cells impairs IFN-γ secretion by mycobacteria-specific T cells in vitro.

<p>The concentration of IL-12 p40 in supernatants from mycobacteria-infected <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDCs (A) or BMM (B) at different times after incubation were determined by ELISA. The mean IL-12p40 ± SEM pg/ ml from triplicate cultures is depicted. Differences with <i>stat3</i>^<i>fl/fl</i> BMDCs are significant (**p<0.01, ***p<0.001 Student t test). Total RNA was extracted from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC cultures 24 h after <i>M</i>. <i>tuberculosis</i> infection. The mean <i>Il-12p35</i> mRNA levels ± SEM levels measured by real time PCR are depicted (C) (**p<0.01 Student t test). <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC were infected with either BCG (D), <i>M</i>. <i>tuberculosis</i> (E) or stimulated with heat killed BCG (F) or with LPS and peptide 25 of Ag85b (G) washed and incubated 6 h after with <i>p25-tg</i> CD4⁺ naïve T cells (at a ratio of 4:1 BMDC) (G). The concentration of IFN-γ in the culture supernatants was measured by ELISA 72h after co-incubation. The mean IFN-γ ± SEM from triplicate cultures is depicted (***p<0.001 Student t test). The concentration of IL-12p40 in supernatants from mycobacteria-infected <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>socs3</i>^<i>fl/fl</i> BMDCs was determined by ELISA (H). The mean IL-12p40 ± SEM ng/ ml from triplicate cultures is depicted (**p<0.01 and ***p<0.001 Student t test). <i>Socs3</i>^<i>fl/fl</i> <i>lysm cre</i> (I), socs3^<i>fl/fl</i> <i>cd11 cre</i> (J) and <i>socs3</i>^<i>fl/fl</i> BMDC were infected with BCG and incubated 6 h after with <i>p25-tg</i> T cells. The concentration of IFN-γ in the supernatants was measured by ELISA 72h after co-incubation. The mean IFN-γ ± SEM from triplicate cultures is depicted (**p<0.01 Student t test). <i>Il12p40</i>^<i>-/-</i> (K), <i>gp130</i>^<i>F/F</i> (M) and WT BMDC were infected with BCG and co-incubated with <i>p25-tg</i> T cells as described. Mycobacterial-infected <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>socs3</i>^{<i>fl/fl</i>.} were cultured in presence of recombinant IL-12p70 or left untreated (K) and co-incubated with p25Tg-T cells (L). The mean IFN-γ ± SEM in supernatants from triplicate cultures was measured by ELISA (**p<0.01, ***p<0.001 Student t test). The frequency of IFN-γ-secreting cells in PPD-stimulated pulmonary T cells from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 4 and 8 weeks after infection with <i>M</i>. <i>tuberculosis</i> was analysed by ICS (N, O). The mean frequency of IFN-γ-secreting within CD4+ cells (N) and CD8+ (O) ± SEM is displayed (n = 5 per group). The levels of <i>ifng</i> (P), <i>inos</i> (Q)and <i>cxcl9</i> (R) mRNA in the lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice before and at the indicated time points after aerosol infection with <i>M</i>. <i>tuberculosis</i> were determined by real time PCR.</p

Stat3 deficient and control BMM show similar control of the intracellular growth of <i>M</i>. <i>tuberculosis</i>.

<p>The levels of <i>tnf</i> mRNA in the lungs from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice at the indicated time points after infection with <i>M</i>. <i>tuberculosis</i> (A), and in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM incubated with <i>M</i>. <i>tuberculosis</i> (C) or BCG (D) were determined by real time PCR. The mean fold increase of mRNA level ± SEM in 8 mice per group (A) or in triplicate independent cultures per condition compared to non-infected cultures (C, D) of one of two independent experiments is depicted (*p<0.05, **p<0.01, ***p<0.001 Student t test). The mean concentration of TNF in the supernatants of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM at different times after <i>M</i>. <i>tuberculosis</i> infection were measured by ELISA (B). The mean % of infected BMM (E), the bacteria number per BMM (F) and the bacteria per infected BMM (G) ± SEM at 0 and 3 days after infection of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM were determined after staining with auramin-rhodamin T for mycobacterial and DAPI for nuclei (E-G). One out of 2 independent experiments performed is depicted. Bacterial CFU were determined in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM after infection with BCG (H) or <i>M</i>. <i>tuberculosis</i> (I) at a MOI of 5:1. The mean CFU ± SEM from triplicate cell cultures is shown. Three independent experiments for each panel were performed.</p

Detection of <i>mecC-</i>Positive <i>Staphylococcus aureus</i> (CC130-MRSA-XI) in Diseased European Hedgehogs (<i>Erinaceus europaeus</i>) in Sweden

<div><p>Recently, a novel <i>mec</i> gene conferring beta-lactam resistance in <i>Staphylococcus aureus</i> has been discovered. This gene, <i>mecC</i>, is situated on a SCC<i>mec</i> XI element that has to date been identified in clonal complexes 49, 130, 425, 599 and 1943. Some of the currently known isolates have been identified from animals. This, and observations of <i>mecA</i> alleles that do not confer beta-lactam resistance, indicate that <i>mec</i> genes might have a reservoir in <i>Staphylococcus</i> species from animals. Thus it is important also to screen wildlife isolates for <i>mec</i> genes. Here, we describe <i>mecC</i>-positive <i>Staphylococcus aureus</i> (ST130-MRSA-XI) and the lesions related to the infection in two diseased free-ranging European hedgehogs (<i>Erinaceus europaeus</i>). One was found dead in 2003 in central Sweden, and suffered from <i>S. aureus</i> septicaemia. The other one, found on the island of Gotland in the Baltic Sea in 2011, showed a severe dermatitis and was euthanised. ST130-MRSA-XI isolates were isolated from lesions from both hedgehogs and were essentially identical to previously described isolates from humans. Both isolates carried the complete SCC<i>mec</i> XI element. They lacked the <i>lukF-PV/lukS-PV</i> and <i>lukM/lukF-P83</i> genes, but harboured a gene for an exfoliative toxin homologue previously described from <i>Staphylococcus hyicus</i>, <i>Staphylococcus pseudintermedius</i> and other <i>S. aureus</i> of the CC130 lineage. To the best of our knowledge, these are the first reported cases of CC130-MRSA-XI in hedgehogs. Given that one of the samples was taken as early as 2003, this was the earliest detection of this strain and of <i>mecC</i> in Sweden. This and several other recent observations suggest that CC130 might be a zoonotic lineage of <i>S. aureus</i> and that SCC<i>mec</i> XI/<i>mecC</i> may have originated from animal pathogens.</p></div

Novel primer and probe sequences and PCR conditions.

<p>Novel primer and probe sequences and PCR conditions.</p