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

STAT3 is a master regulator of the immune responses. Here we show that M. tuberculosis-infected stat3fl/fl lysm cre 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 stat3fl/fl lysm cre 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 stat3fl/fl lysm cre 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, socs3fl/fl cd11c cre mice deficient of SOCS3 in DCs showed increased susceptibility to M. tuberculosis 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 M. tuberculosis.

Introduction Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, remains a leading public health problem worldwide. TB causes 9 million new cases and 1.5 million deaths each year [1]. However, host factors determining the outcome of infection are not completely understood.
The transcription factor STAT3 is a central regulator of immunity, mediating inflammatory but also anti-inflammatory responses [10,11]. The functions of STAT3 are pleiotropic. STAT3 is activated by phosphorylation in response to cytokines of the IFN-receptor family (such as IL-10) and by some members of the IL-2 receptor family that uses the common γ chain receptor or after stimulation of several receptor tyrosine kinases (EGF, CSF-1, and PDGF). Additionally, STAT3 is activated by the common signal transducing molecule gp130 utilized by the IL-6 receptor family [12], and in response to G-CSF and leptin as their receptors are homologous to gp130.
STAT3 is critical for defense against bacterial and fungal infections. Low IL-17 secreting Tcell proportions were reported in patients bearing STAT3 mutations. These patients were prone to chronic candidiasis and staphylococcal diseases [13]. Chronic candidiasis is frequently present in patients deficient in IL-17 receptor A [14]. STAT3 deficient patients may also display impaired immunity against chronic viral infections [15,16].
In mice, knockout of STAT3 is lethal, so in vivo studies on STAT3 functions have been performed using conditional knock out mice. Stat3 fl/fl lysm cre mice, deficient in STAT3 in myeloid cells, display enhanced susceptibility to endotoxic shock and develop chronic enterocolitis with age [17]. The phenotype of these animals is similar to IL-10 -/mice, including increased expression of TNF and other inflammatory cytokines, since IL-10 suppresses induction of TNF-α via STAT3 [18]. Recently, STAT3 was shown to favour intracellular growth of M. tuberculosis in human macrophages [19]. Moreover, the presence of pSTAT3+ monocytes associated with the progression of the disease in M. tuberculosis infected non-human primates [20].
We have previously analysed the role of SOCS3, a molecule that inhibits STAT3 activation after triggering of several cytokine and growth factor receptors, and found that mice devoid in SOCS3 in myeloid or lymphoid cells showed increased susceptibility to M. tuberculosis [21].
The role of STAT3 during infection with M. tuberculosis in vivo is still unknown. We here examine the role of STAT3 in M. tuberculosis by using stat3 fl/fl lysm cre mice. We highlight that STAT3 expression in APCs inhibits T H 17 associated responses resulting in an increased susceptibility to infection with M. tuberculosis.

Stat3 fl/fl lysm cre mice are resistant to infection with M. tuberculosis
First, the role of STAT3 expression in myeloid cells in the control of infection with M. tuberculosis was examined using stat3 fl/fl lysm cre mice. Lungs and spleens from stat3 fl/fl lysm cre mice after 4 and 8 weeks of infection showed significantly lower M. tuberculosis burden than stat3 fl/fl littermates (Fig 1A and 1B). A smaller area of the lung parenchyma of stat3 fl/fl lysm cre mice was occupied by granulomas when compared to control lungs 4 but not at 8 weeks after infection ( Fig 1C).
The density of granulocytes in the lung parenchyma was determined either by H&E staining of sections (Fig 1D) or by labelling of CD11b + CD11c -Ly6C dim Ly6G + neutrophils (Fig 1E  and 1F) in lung suspensions from stat3 fl/fl lysm cre and stat3 fl/fl mice 3 and 4 weeks after M. tuberculosis infection. The neutrophil density (Fig 1D-1F) and the levels of neutrophil myeloperoxidase (mpo) and elastase (elane) mRNAs (Fig 1G and 1H) were also higher in lungs from stat3 fl/fl lysm cre mice at 4 and 8 but not at 14 weeks after infection with M. tuberculosis-compared to controls (Fig 1D-1H and S1A-S1C Fig).

Stat3-deficient and control BMM show similar control of the growth of intracellular M. tuberculosis
Activated STAT3 hampers TNF expression [22,23]. Lungs from stat3 fl/fl lysm cre mice infected with M. tuberculosis (Fig 2A) as well as BMM infected with M. tuberculosis or BCG (Fig 2B-2D) showed higher TNF protein and mRNA levels than controls. Since TNF has been shown to mediate M. tuberculosis control in macrophages [24], we speculated that stat3 fl/fl lysm cre macrophages could display a better control of intracellular mycobacteria.
A higher frequency of stat3 fl/fl lysm cre BMMs were infected when measured 4 h after coincubation with the M. tuberculosis, although the number of bacteria per infected cell was similar ( Fig 2E-2G). Three days after infection M. tuberculosis infected mutant and WT BMM showed similar numbers of infected cells and bacteria per total or infected cell (Fig 2E-2G). Stat3 fl/fl lysm cre BMM showed no improved control of M. tuberculosis or BCG growth in vitro 6 days after infection as measured by CFU in lysates (Fig 2H and 2I). Altogether, we observed no indication of an improved bacterial growth control or reduced bacterial uptake in stat3 fl/fl lysm cre BMM.

Role of STAT3 and SOCS3 in APCs in the regulation of T cell priming
Several cytokines controlled by STAT3 are potent regulators of the expression of co-stimulatory molecules on APCs. Therefore, we studied whether STAT3 played a role in regulation of T cell priming. As expected, the density of co-stimulatory molecules CD80 and CD86 as well as MHC-II levels increased on BMDCs after mycobacterial stimulation. The expression of MHCII, CD80 and CD86 in either control or stat3 fl/fl lysm cre BMDCs before or after mycobacterial stimulation was similar (Fig 3A-3C). To investigate if the expression of STAT3 by myeloid cells could modulate T cell priming during infection with M. tuberculosis T cell receptor transgenic T cells specific for the immunodominant mycobacterial Ag85B 240-254 peptide (p25-tg) cells were inoculated i.v. into stat3 fl/fl lysm cre or stat3 fl/fl mice 17 days after infection with M. tuberculosis (Fig 3D). Three days after transfer, the expression of CD69 (which increases after T cell receptor triggering) and CD62L (the L-selectin ligand that hampers T cells to traffic to the periphery) was measured on p25-tg T cells and host T cells from the mediastinal lymph nodes (MLN). The expression of CD69 was increased and the expression of CD62L was reduced in p25-tg T cells from MLN of infected mice when compared to as well as 8 weeks post infection is depicted. Differences in CFU are significant ( Ã p<0.05, ÃÃ p<0.01, ÃÃÃ p<0.001 Mann Whitney U test). Histopathological uninfected control mice. Similar levels of the CD69 and CD62L were expressed by p25-tg or host T cells from stat3 /fl lysm cre or stat3 fl/fl infected mice (Fig 3E and 3F). SOCS3 inhibits STAT3 activation by different cytokine receptors, e.g. those of the IL-6 receptor family [10]. In accordance with the results obtained with socs3 fl/fl lysm cre mice [21], socs3 fl/fl cd11c cre mice showed higher bacterial levels in lungs and spleens after infection with M. tuberculosis than control animals (Fig 3G and 3H). The cd11c cre transgene has been shown to be expressed in the majority of conventional and plasmacytoid DCs [25] Mycobacteria-stimulated BMDC from socs3 fl/fl lysm cre showed lower levels of MHCII, CD80 and CD86 than control cells (Fig 3I-3L). When T cell priming in vivo was studied by transfering p25-tg naïve T cells into M. tuberculosis infected animals, the density of CD62L on donor p25-tg T cells and on host MLN T cells was lower in WT mice as compared to socs3 fl/fl lysm cre mice recipients (Fig 3M and 3O). The p25-tg T cells in the MLN of socs3 fl/fl lysm cre mice also showed lower surface density of CD69 as compared to those from WT-infected mice (Fig 3N and 3P). However, the expression of CD69 in host MLN T cells from M. tuberculosisinfected WT and socs3 fl/fl lysm cre mice was similar ( Fig 3P).
Thus, deficiency of SOCS3 but not STAT3 in APCs regulates T cell priming during M. tuberculosis infection in vivo.

STAT3 in myeloid cells impairs IFN-γ secretion by mycobacteria-specific T cells
IFN-γ is required for protection against M. tuberculosis [2,3]. STAT3 has been shown to inhibit the transcription of IL-12, a potent inducer of IFN-γ secretion by T cells [23]. We then analysed if the increased resistance to M. tuberculosis of stat3 fl/fl lysm cre mice is associated with higher IFN-γ secretion by T cells. Higher levels of IL-12p40 (the α-chain of IL-12 and IL-23) in supernatants and il12p40 mRNA in cell lysates of BCG-infected stat3 fl/fl lysm cre BMM or BMDC compared to controls were measured (Fig 4A and 4B). The il12p35 mRNA coding for the β-chain of the IL-12 heterodimer was also expressed in higher amounts by M. tuberculosis-or BCG-stimulated stat3 fl/fl lysm cre BMDC compared to controls ( Fig 4C).
Thus, whether STAT3-deficient APCs are better stimulators of IFN-γ secretion by mycobacteria-specific T cells than WT APCs was investigated. To test this hypothesis, p25-tg T cells were incubated with either BCG-or M. tuberculosis-infected stat3 fl/fl lysm cre or stat3 fl/fl BMDCs and the IFN-γ titers in the supernatants measured. IFN-γ levels in supernatants were elevated compared to those incubated with WT APCs (Fig 4D and 4E). Supernatants from cultures of p25-tg T cells incubated with heat-killed BCG-stimulated stat3 fl/fl BMDC or BMM also contained higher levels of IFN-γ than those using control APCs (Fig 4F), indicating that infection is not required for such responses. In line with this, IFN-γ levels were higher in supernatants from p25-tg T cells co-incubated with stat3 fl/fl lysm cre BMDC or BMM stimulated with oligopeptide p25 (amino acids 240-254) from Ag85b, a major immunodominant H2 b epitope [26] recognized by the p25-tg T cells, in presence of LPS ( Fig 4G). scoring of hematoxylin-eosin stained paraffin lung sections from stat3 fl/fl lysm cre and stat3 fl/fl mice measured 4 and 8 weeks after infection with M. tuberculosis. The mean ± SEM % lung area with granulomas (C) and the relative neutrophil density score (D) are depicted. Differences are significant ( Ã p<0.05, ÃÃÃ p<0.001 Student t test). A representative dot plot (E) and the frequency (F) of CD11b + CD11c -Ly6C dim Ly6G + neutrophils in lungs stat3 fl/fl lysm cre and stat3 fl/fl mice 3 and 4 weeks after infection with M. tuberculosis are shown. Differences between 4 mice at each time point are significant ( Ã p<0.05 Student's t test). Total RNA was extracted from the lungs of stat3 fl/fl lysm cre and stat3 fl/fl mice at the indicated time points after infection with M. tuberculosis. The relative concentration of neutrophil elastase (elane) (G) and myeloperoxidase (mpo) transcripts (H) in relation to hprt 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 ( Ã p<0.05, ÃÃ p<0.01 Student t-test). https://doi.org/10.1371/journal.ppat.1006809.g001 Confirming previous results [21,27], socs3 fl/fl lysm cre BMDC showed diminished IL-12 secretion after mycobacterial stimulation ( Fig 4H). Furthermore, IFN-γ secretion by p25-tg Tcells incubated with mycobacteria-infected socs3 fl/fl lysm cre or socs3 fl/fl cd11 cre BMDCs was reduced (Fig 4I and 4J).
In line with these results, mycobacteria-infected il12p40 -/-BMDCs showed reduced ability to trigger IFN-γ secretion by p25-tg T cells than controls ( Fig 4K). Moreover, the addition of rec IL-12 restored the capacity of socs3 fl/fl lysm cre BMDC to stimulate IFN-γ secretion by p25-tg T cells (Fig 4L).
Cells derived from gp130 F/F mice, harbouring a mutation that ablates SOCS3 binding to the gp130, show exaggerated gp130-mediated STAT3 responses [28]. Mycobacteria-infected gp130 F/F BMDCs also showed a reduced ability to stimulate IFN-γ secretion p25-tg T cells compared to WT cells ( Fig 4M).
The In addition, levels of ifng, and the IFN-γ-regulated inos and cxcl9 transcripts were increased in lungs after infection as compared to uninfected controls, but the titers of these transcripts in lungs from stat3 fl/fl lysm cre and stat3 fl/fl -infected mice were comparable (Fig 4P-4R).

Myeloid STAT3 hamper T H 17 responses during M. tuberculosis infection
The neutrophil density and the levels of neutrophil transcripts were enhanced in the lungs of M. tuberculosis-infected stat3 fl/fl lysm cre as compared to control mice (Fig 1D-1H). IL-17 has been shown to stimulate granulopoiesis via G-CSF production and to induce the expression of CXC chemokines involved in granulocyte recruitment [29]. Thus, we investigated whether the increased neutrophil levels in lungs from M. tuberculosis-infected stat3 fl/fl lysm cre was associated with augmented T H 17 responses. The frequency of IL-17-secreting, PPD-stimulated CD4 + T cells from lungs from stat3 fl/fl lysm cre mice 4 and 8 weeks after infection with M. tuberculosis were elevated when compared to stat3 fl/fl controls (Fig 5A-5C). Instead, the frequency of γδ T cells in lungs and the frequency of IL-17 secreting pulmonary γδ + T cells from WT or stat3 fl/fl lysm cre infected mice was similar (S3A- S3C Fig).
In addition, levels of il17a and il22, transcripts that code for T H 17 cytokines were higher in lungs from stat3 fl/fl lysm cre mice than in those from littermate controls when measured at 4 and 8 weeks after infection (Fig 5D and 5E). Higher levels of il17 mRNA were also observed in stat3 fl/fl lysm cre mice 14 weeks after infection with M. tuberculosis, while the increase of Il22 mRNA did not reach statistical significance (S4A and S4B Fig). CXCL5 is a neutrophil chemotactic protein stimulated by IL-17 [30]. The level of cxcl5 mRNA was increased in lungs from M. tuberculosis-infected stat3 fl/fl lysm cre mice ( Fig 5F).
Substantial in vivo data support the notion that IL-6 and IL-23 are required at different stages of T H 17-cell differentiation [31,32]. Levels of il6 and il23 mRNA were elevated in the lungs of M. tuberculosis-infected stat3 fl/fl lysm cre mice when compared to levels in lungs from WT mice (Fig 5G and 5H). T H 17 cells show a high degree of developmental flexibility, and when exposed to IL-12 or IL-23, they can rapidly acquire effector functions that are normally associated with T H 1 responses such as IFN-γ production [33]. These IFN-γ and IL-17 secreting cells were shown to be pathogenic in murine models of autoimmune diseases, and were also associated with murine colitis and human IBDs [34]. The majority of PPD or PMA/ ionomycin-stimulated IL-17 secreting CD4 + T cells in lungs from M. tuberculosis-infected stat3 fl/fl lysm cre or stat3 fl/fl mice were not IFN-γ co-producers (Fig 5I-5K). We next studied whether IL-17 played a role in the increased control of infection of stat3 fl/fl lysm cre mice. For these experiments, mice were treated with neutralizing IL-17RA mab (M751) before and during infection with M. tuberculosis. Similar bacterial levels were found in lungs and spleens from stat3 fl/fl lysm cre and stat3 fl/fl animals treated with anti-IL17RA mAb. As expected, stat3 fl/fl lysm cre mice from untreated mice showed reduced bacterial numbers in lungs and spleens than those from stat3 fl/fl controls (Fig 5L and 5M).
The levels of mpo mRNA was measured in lungs from anti-IL-17RA to control for the IL17RA neutralization. As expected, levels of mpo mRNA were increased in lungs from M. tuberculosis infected stat3 fl/fl lysm cre mice as compared to WT. In contrast, levels of mpo mRNA in lungs from infected or anti-IL17RA treated stat3 fl/fl lysm cre and stat3 fl/fl mice was similar. Lower titters of mpo mRNA were found in stat3 fl/fl lysm cre infected mice treated with anti-IL-17RA compared to untreated infected controls, while mpo mRNA levels in anti-IL17RA treated or untreated infeceted stat3 fl/fl mice were similar ( Fig 5N).
Hence, increased M. tuberculosis control during infection of stat3 fl/fl lysm cre mice is dependent on IL-17, but IL-17 neutralization did not increase the susceptibility to M. tuberculosis of mice with normal STAT3 function.

STAT3 expression in antigen presenting cells inhibit the generation of IL-17 secreting mycobacteria-specific T cells
The role by which STAT3 in activated APCs regulates IL-17 secretion by specific T cells was then studied. The mRNA levels of Il6 and il23p19 were both increased in BMM and BMDCs co-incubated with mycobacteria in vitro (Fig 6A and 6B and S5A Fig). An increased accumulation of il6 and il23p19 mRNA was also observed after stimulation with the TLR agonists LPS, CpG or Pam3K of stat3 fl/fl lysm cre as compared to stat3 fl/fl BMM at 6 and 24 h after stimulation

STAT3 in myeloid cells impairs IFN-γ secretion by mycobacteria-specific T cells in vitro.
The concentration of IL-12 p40 in supernatants from mycobacteria-infected stat3 fl/fl lysm cre and stat3 fl/fl 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 stat3 fl/fl BMDCs are significant ( ÃÃ p<0.01, ÃÃÃ p<0.001 Student t test). Total RNA was extracted from stat3 fl/fl lysm cre and stat3 fl/fl BMDC cultures 24 h after M. tuberculosis infection. The mean Il-12p35 mRNA levels ± SEM levels measured by real time PCR are depicted (C) ( ÃÃ p<0.01 Student t test). Stat3 fl/fl lysm cre and stat3 fl/fl BMDC were infected with either BCG (D), M. tuberculosis (E) or stimulated with heat killed BCG (F) or with LPS and peptide 25 of Ag85b (G) washed and incubated 6 h after with p25-tg 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 ( ÃÃÃ p<0.001 Student t test). The concentration of IL-12p40 in supernatants from mycobacteria-infected socs3 fl/fl lysm cre and socs3 fl/fl BMDCs was determined by ELISA (H). The mean IL-12p40 ± SEM ng/ ml from triplicate cultures is depicted ( ÃÃ p<0.01 and ÃÃÃ p<0.001 Student t test). Socs3 fl/fl lysm cre (I), socs3 fl/fl cd11 cre (J) and socs3 fl/fl BMDC were infected with BCG and incubated 6 h after with p25-tg T cells. The concentration of IFN-γ in the supernatants was measured by ELISA 72h after coincubation. The mean IFN-γ ± SEM from triplicate cultures is depicted ( ÃÃ p<0.01 Student t test). ( Fig 6C and 6D and S5B Fig). This indicates that STAT3-mediated inhibition of the expression of IL-6 and IL-23 is not restricted to mycobacterial infection or stimulation with mycobacterial molecules. Supernatants from cultures of mycobacteria-infected stat3 fl/f lysm cre BMM or BMDC co-incubated with naïve p25-tg T cells contained higher titers of IL-17 than those using stat3 fl/fl controls (Fig 6E-6G). IL-17 levels were also higher in supernatants from p25-tg T cells co-incubated with stat3 fl/fl lysm cre BMDC stimulated with either live or heat-killed BCG, M. tuberculosis or peptide 25 from Ag85b in presence of LPS (Fig 6F-6H).
Whether SOCS3 in mycobacteria-infected APCs also regulated IL-17 secretion by antigenspecific T cells was then measured. We found that mycobacteria-infected socs3 fl/fl lysm cre BMM contained lower levels of il6 and il23p19 mRNA than their WT counterparts (Fig 6I and  6J). Moreover, p25-tg T cells incubated with socs3 fl/fl lysm cre BMM stimulated either with BCG, M. tuberculosis or the cognate p25 peptide secreted lower levels of IL-17 than those stimulated by WT BMDCs (Fig 6K-6M). Similarly, supernatants from co-cultures of p25-tg T cells with mycobacteria-infected gp130 F/F BMDC contained higher levels of IL-17 than those using wild type BMDCs (Fig 6N).
We then asked whether STAT3 deficiency regulated the levels of socs3 mRNA transcripts in mycobacteria-infected macrophages. M. tuberculosis-infected stat3 fl/fl lysm cre and stat3 fl/fl BMMs showed similar levels of socs3 mRNA (Fig 6O).

STAT3 and SOCS3 in T cells show a differential regulation of IL-17 and IFN-γ secretion
Different to the inhibitory role of STAT3 in myeloid cells we here showed, STAT3 expression in T cells has been indicated to be required for T H 17 cell differentiation in vitro and in vivo [31]. SOCS3, via hyperactivation of STAT3, has been shown to increase IL-17 secretion [35]. In order to compare the role of STAT3 and SOCS3 in T cells and APCs in the regulation of cytokine secretion by T cells, stat3 fl/fl lck cre p25-tg and socs3 fl/f lck cre p25-tg mice were generated. The culture supernatants of stat3 fl/fl lck cre p25-tg T cells stimulated with BCG-infected or Ag85b peptide-pulsed BMDCs showed low or undetectable levels of IL-17 as compared to controls (lck cre p25-tg T cells) (Fig 7A). Instead IL-17 levels in supernatants from socs3 fl/fl lck cre p25-tg T cells co-incubated with BCG or peptide loaded BMDCs were higher than controls (Fig 7C).
The titters of IFN-γ in the supernatants of stat3 fl/fl p25-tg T cells incubated with BCG-(but not with Ag85b peptide-) stimulated BMDCs were higher than those incubated with control T cells (Fig 7B). IFN-γ levels in supernatants from mycobacteria or peptide pulsed BMDCs incubated with socs3 fl/fl lck cre p25-tg were instead lower than those using control p25-tg T cells ( Fig 7D).
Thus, STAT3 in APCs and T cells, has a dissimilar ability to regulate IL-17 secretion by agspecific T cells, while SOCS3 in APCs and T cells promote T cell mediated IFN-γ secretion.

Discussion
We report here that stat3 fl/f lysm cre mice show reduced M. tuberculosis load in lungs and spleens, indicating that STAT3 expression in myeloid cells is detrimental for the control of infection with M. tuberculosis. Despite reduced area of the lung occupied by granuloma the area with inflammation was not reduced and the numbers of infiltrating pulmonary neutrophils were elevated in stat3 fl/fl lysm cre mice. Neutrophil accumulation late during infection have been associated with susceptibility to M. tuberculosis, whereas early after infection neutrophils play a protective role and contribute to early priming of T cells in the draining lymph node [36][37][38][39].
Although mice lacking STAT3 expression in bone marrow progenitors display peripheral neutrophilia under resting conditions [40], the pathways involved in neutrophil mobilization and response to chemokines during inflammation have been shown to be STAT3 dependent [41,42]. Thus, we consider it unlikely that the increase in pulmonary neutrophils observed during infection occurs as a direct consequence of STAT3 deficiency in these cells. Rather, increased levels of IL-17 and IL-22, cytokines that stimulate the expression of neutrophil recruiting chemokines [29], might contribute to the accumulation of granulocytes in lungs from M. tuberculosis-infected stat3 fl/fl lysm cre mice. In agreement with this hypothesis, the frequency of IL-17 secreting mycobacteria-specific CD4 + T cells, but not of γδ + T cells, were elevated in the lungs from stat3 fl/fl lysm cre mice compared to controls.
STAT3 expression in APCs proved to be a major regulator of the expression of cytokines that control T cell differentiation. This was shown in STAT3-and in SOCS3-deficient APC, which display higher levels of activated STAT3 when stimulated with mycobacteria or other innate receptor agonists [21,43]. Thus, while mycobacteria-infected STAT3 deficient APCs showed an improved ability to trigger IL-17 secretion by antigen-specific T cells, the opposite was observed using socs3 fl/fl lysm cre or gp130 F/F BMDCs or macrophages as APCs. We observed an increased IFN-γ secretion by antigen-specific T cells incubated with STAT3-deficient, mycobacteria-infected APCs. However, IFN-γ responses were not increased in vivo. Whether this is due to the present of cytokines that might stimulate IL-17 responses while antagonizing T H 1 cells (such as for example TGF-β) remains to be explored. Whereas in vitro cultures used provide a proper tool to gain mechanistic insights, the diversity of populations, the tissue localization and the balance between the host immune responses and mycobacteria in the chronic infection might account for differences observed between in vitro responses and the control of infection in mice.
The increased resistance to M. tuberculosis in stat3 fl/fl lysm cre mice is mirrored by data showing that mice with SOCS3 deficiency in myeloid cells display reduced resistance to TB and toxoplasmosis [21,44]. Here we show that socs3 fl/fl cd11c cre mice. CD11c cre in these mice has been shown to be expressed in ca 90% of splenic DCs, compared with <10% of lymphocytes and <1% of myeloid cells such as granulocytes [25]. Thus, these animals in which SOCS3 is deleted in DCs but not in inflammatory macrophages and neutrophils [25] are also more susceptible to infection with M. tuberculosis. This supports that the major role of STAT3 and SOCS3 in myeloid cells in the control of infection with M. tuberculosis is not due to an altered ability of SOCS3 or STAT3-deficient macrophages to control the growth of the intracellular mycobacteria in vitro, as shown here and ref [21].
To our knowledge, this is the first report showing that STAT3 deficiency in myeloid cells promotes IL-17 secretion by antigen-specific T cells in vitro and in vivo. Such a role was related to the increased secretion of T H 17 inducing IL-6 and IL-23 by STAT3-deficient APCs. The increased expression of IL-6 and IL-23 in stat3 fl/fl lysm cre APCs was not restricted to the infection with attenuated or virulent mycobacteria since, it was observed after incubating mutant APCs with different TLR agonists or bacterial lysates, confirming previous data [45]. The opposite effect was observed using socs3 fl/fl lysm cre BMM, which were poor inducers of IL-17 secretion by mycobacteria-specific T cells. In relation to this, DC that secrete IL-12p40 (required for T cell differentiation into Th17 or Th1) in the lymph nodes of mycobacteria infected mice are primarily uninfected [46].
Since IL-6 can be produced by various hematopoietic and non-hematopoietic cells, we suggest that APCs are relevant cellular sources of IL-6 for the differentiation of IL-17 secreting cells during infection. Moreover, our data using gp130 F/F BMDCs indicate that DC-derived IL-6 acts in an autocrine/ paracrine manner on DCs to regulate their ability to stimulate IL-17 secretion by T cells. A role for gp130/ IL6/ STAT3 pathway in susceptibility to M. tuberculosis has been previously determined. The high susceptibility of gp130 F/F mice to infection with M. tuberculosis was not observed in gp130 F/F il6 -/or gp130 F/F stat3 +/mice [21]. Our observations on stat3 fl/fl lysm cre mice are reminiscent of those seen in il10 -/or anti-IL-10R mAb treated mice that resulted in enhanced lung T H 1 and T H 17 responses after BCG vaccination [47]. Depletion of IL-10 resulted in elevated protection to M. tuberculosis in some studies but not others [47][48][49][50]. However different to our model, IL-10 might not only impair the functions of APCs but is also secreted by T cells and has a direct inhibitory effect on T H 1 or T H 17 cells [51].
We showed that the improved M. tuberculosis control in stat3 fl/fl lysm cre is IL-17-mediated, since administration of neutralization anti-IL-17RA antibodies abrogated differences in bacterial burden between mutant and control mice. IL-17 might contribute to long term protection, control of infection after vaccination or control of hypervirulent strains of M. tuberculosis [52][53][54][55]. IL-17 has been suggested to induce of protective T H 1 responses against mycobacterial infection [52,56]. IL-17 has been also shown to mediate CXCL13 induction in the lung, a chemokine that contributes to the localization of pro-inflammatory cytokine-producing CXCR5+ T cells within lymphoid structures, promoting those macrophage activation and mycobacterial control [53,57].
However, other studies have indicated that IL-17 is dispensable after primary infection with M. tuberculosis [58]. In line with the later observations, we observed similar M. tuberculosis load in lungs or spleens of WT mice treated or not with anti-IL-17RA.
Levels of MHCII and CD80 and CD86 were lower on socs3 fl/fl lysm cre BMDCs after mycobacterial stimulation confirming previous findings showing reduced MHCII and co-stimulatory molecules after LPS stimulation of SOCS3-deficient BMDCs [59]. Furthermore, the activation of mycobacteria specific p25-tg T cells was also diminished in MLN from M. tuberculosis-infected socs3 fl/fl lysm cre mice as compared to controls. Of importance, p25-tg T cell proliferation was not detectable in the MLN of mice infected with an Ag85b deficient strain of M. tuberculosis indicating the specificity of p25-tg T cell priming [60]. The Ag85b KO M. tuberculosis strain grew in the lungs and disseminated to the MLN at a rate equivalent to that of wild-type bacteria. Instead, stat3 fl/fl lysm cre and control BMDCs expressed similar levels of MHC-II and co-stimulatory molecules after mycobacterial stimulation in vitro and similar levels of activated antigen-specific T cells in vivo. Different to these results, STAT3 deficient APCs have been shown increased MHCII levels after IL-6 stimulation [61].
Finally, the role of STAT3 in T cells in regulation of antigen-specific IFN-γ and IL-17 T cell responses was investigated. Contrary to the role of STAT3 in APCs, IL-17 secretion was hampered in mycobacteria-specific STAT3-deficient T cells. STAT3 is required for the responses to both IL-6, IL-21 and IL-23 and for the expression of RORγt by T cells [62]. Instead, SOCS3-deficient antigen-specific T cells secreted higher IL-17 levels as previously reported in other systems [35], while IFN-γ responses were inhibited. Thus, while the role of STAT3 in T cells in the control of M. tuberculosis remains to be studied, these results illustrate the pleiotropic effect of STAT3 in regulation of infection-induced immune responses in different cell types.
In summary, we here showed using SOCS3-and STAT3-deficient mice that STAT3 in myeloid cells is detrimental for the control of infection with M. tuberculosis. Surprisingly, this occurs via impairing secretion of IL-17 by antigen-specific T cells (Fig 7E).

Ethics statement
The animals were housed and handled at the Dept.

Mice
Mice containing loxP-flanked stat3 and socs3 alleles have been described before [43]. For a myeloid-specific deletion these were bred with transgenic lysm cre mice [63]. Socs3 fl/fl mice were also bred with cd11c cre transgenic animals. Stat3 fl/fl or socs3 fl/fl littermates negative for cre expression were used as controls for all experiments. Gp130 F/F mice with a homozygous substitution of tyrosine (Y) 757 to phenylalanine (F) within the common IL-6 family receptor gp130 abrogating the SOCS3 binding site have been described before [64]. Transgenic T cell receptor p25-tg mice with a T-cell receptor specific for peptide 25 (aa 240-254) of mycobacterial Ag85B on H2 b haplotype were used [65]. p25-tg rag2 -/mice expressing ECFP were generated by crossing p25-tg with rag1 -/mice [65] with ECFP mice on a rag2 -/background (kindly provided by Dr. Ronald Germain, NIAID, NIH). The ECFP expression co-localized with Vβ11 used by p25tg T cells [65]. Socs3 fl/fl lck cre and stat3 fl/fl lck cre mice deficient in SOCS3 and STAT3 in T cells were crossed with p25-tg mice to generate p25-tg socs3 fl/fl lck cre and p25-tg stat3 fl/fl lck cre mice. P25-tg lck cre mice were also obtained and used as controls.
Bacteria were quantified on Middlebrook 7H11 agar containing 10% enrichment of oleic acid, albumin, dextrose, catalase, 5 μg of amphotericin B per ml and 8 μg/ ml polymyxin B grown for 3 weeks at 37˚C.

Generation of mouse bone marrow-derived macrophages
Bone marrow was extracted from tibia and femurs of mice and resuspended in DMEM containing glucose and supplemented with 10% FCS and 30% L929 cell-conditioned medium (as a source of macrophage-colony stimulating factor). Bone marrow cells were passed through a 70 μm cell strainer, plated and incubated for 6 days at 37˚C, 5% CO 2 . Bone marrow-derived macrophage (BMM) cultures were then washed vigorously to remove non-adherent cells, trypsinized, counted and cultured for one day at 37˚C in 24, 12 or 6 well plates. We have previously shown that these BMM are F4/80 + , CD14 + and Mac-3 + [67].

Quantification of intracellular mycobacteria
In order to quantify intracellular M. tuberculosis uptake and growth, BMM cells were plated on glass slides at 2.10 5 cells per well in 24 well plates, incubated for 4 h with M. tuberculosis (MOI 2) and washed with PBS for 3 times to remove the extracellular bacteria before either fixation or replacing the medium. Three days after infection cells were washed with PBS, fixed with 2% PFA and stained with phalloidin to label F-actin (Life technologies, 1:100), DAPI (1:500) and auramine-rhodamine T to label mycobacteria (BD). Micrographs from infected macrophages (400X) were obtained and a total of at least 1000 BMM from 3 independent cultures and categorized as infected or uninfected. The intracellular M. tuberculosis were enumerated. BMM harboring 5 or more bacteria were considered as containing 5. In some cultures, mycobacterial CFU from BMM 6 days after infection were determined.

Generation of mouse bone marrow-derived dendritic cells
Mouse bone marrow-derived dendritic cells (BMDC) were differentiated as previously described [68]. Briefly, bone marrow was extracted from tibia and femurs and cell suspensions cultured in RPMI-1640 medium containing 10% FCS and 2 ng/ ml GM-CSF (Peprotech, Rocky Hill, NJ). Fresh medium and cytokine were replaced after 3 days. After six days of culture, loosely adherent cells were harvested and seeded in concentrations for infection. Harvested cells were further selected for CD11c expression with magnetic beads (Miltenyi Biotech) before seeding.

T cell priming in vitro
BMDC or BMM were stimulated with either live or heat killed BCG, M. tuberculosis or Ag85b peptide in presence of LPS for 6 h. Then, cells were washed and co-incubated with p25-tg CD4+ lymph node transgenic T cells from rag2 -/-p25-Tg mice (at a ratio of 4:1 BMDC). The cultures were further incubated for 24-48 hs at 37C 5% CO 2 . At these time points the concentration of IFN-γ and IL-17 in the supernatants was measured by ELISA.

Real time PCR
Transcripts were quantified by real time PCR as previously described [66]. Hprt was used as a control gene to calculate the ΔC t values for individual samples. The relative amount of cytokine/ hprt transcripts was calculated using the 2 -(ΔΔCt) method. These values were then used to calculate the relative expression of cytokine mRNA in uninfected and infected cells and tissues.

Flow cytometry and intracellular cytokine staining
Lungs were perfused with PBS through the heart before removal from mice. Lungs were mechanically minced into small pieces and digested with 3 mg/ ml Collagenase D and 30 μg/ ml DNase I for 1 h at 37˚C, and single-cell suspensions prepared by filtering lung tissue through 70-μm nylon cell strainers. To further remove impurities cells were loaded in 40/ 70% Percoll gradient in PBS and centrifuged 30 min room temperature. The cells at the interphase were collected and washed. Single spleen cell suspensions were obtained by mechanical disruption, lysis of erythrocytes and straining over a 70-μm nylon mesh. Lung, lymph node and spleen cells were stained for CD3, CD4, CD8, γδ-TCR, CD62L, CD69, CD44, CD11b, CD11c, Ly6C and Ly6G (all eBioscience) and fixed before acquisition.
For determination of IFN-γ and IL-17-producing cells, lung cells were incubated with PPD or with 50 ng/ml phorbol myristate acetate (PMA) and 2 μg/ml ionomycin (Sigma) for 6 or 18 h at 37 o C. Brefeldin (10 μg/ ml) was added to the cultures the last 4 h of stimulation. Cells were then stained with cell population-specific antibodies, and live/ dead staining, fixed, permeabilized using leukocyte permeabilization reagent IntraPrep™ (Immunotech, Marseille, France) and further stained with anti-IL-17a or anti-IFN-γ (eBioscience).
Data were acquired in a CyAn™ ADP (Beckman Coulter) or an LSRII Flow cytometry and analyzed with FlowJo software (Tree star Inc., Ashland, OR).

Histopathological analysis
Formalin fixed left lungs of mice experimentally inoculated with M. tuberculosis were blocked on paraffin. From each lung sample 4 sections were obtained, one longitudinal along the long axis of the lobe and 3 across/transversal of the remaining piece of lung.
The blocks were processed and sections were stained with haematoxylin-eosin. All sections were interpreted by the same pathologist (D. G-W.) and scored semi-quantitatively, blinded to the variables of the experiment.
The following features were scored: • Lung area occupied with granulomas (% of the total area of the section) • Lung area free of lesions or area of healthy lung (% of the total area of the section)

Statistics
The Mann Whitney test for the bacterial CFU load in vivo and of the ICS analysis. For each experiment, 8-10 control and 8-10 mutant mice were infected. We performed separated experiments for 4 and 8 weeks post infection. One of two independent experiments showing similar results is shown. The analysis of cytokine secretion or mRNA, histopathological scores and frequencies was done using the Student's t test for unpaired samples. All in vitro experiments were performed at least twice. A two-way ANOVA was used to compare the differences in IL-17 secretion between genotypes, as well as between cells that co-secrete IFN-γ or not.