Role of SOCS proteins during mycobacterial infections

Mycobacterium tuberculosis is the world’s most successful bacterial killer. During infection, mycobacteria reside inside host cells encapsulated within a granuloma structure in the latent, asymptomatic phase of infection. Only 10% of latently infected develop active, infectious tuberculosis months or years after the initial infection. The mechanisms that determine latency and bacterial control as well as protection during active tuberculosis are still not fully understood. Members of the SOCS protein family are regulators of cytokine signaling via inhibition of JAK-STAT activation and their expression is increased during different kinds of infections. Therefore, the purpose of this thesis was to study the role of SOCS1, SOCS2 and SOCS3 during mycobacterial infections. We demonstrated that infection with M. tuberculosis in vitro and in mice strongly upregulated SOCS1 expression. Interestingly, SOCS1 reduced IFN-γ secretion by macrophages in response to IL-12 rather than responses to IFN-γ itself. In line, SOCS1-deficient macrophages showed improved growth control of mycobacteria in vitro. Furthermore, in a mouse model of infection, we demonstrated that SOCS1 expression by macrophages impaired bacterial clearance before the onset of protective adaptive immune responses. However, SOCS1 did not hamper adaptive immunecontrolled bactericidal mechanisms at later time points. At this stage of infection, SOCS1 expression by non-macrophage cells protected mice from severe immunopathology. Additionally, we showed that SOCS2 expression was induced in an IRF3- dependent manner after infection with M. bovis BCG or incubation with LPS in vitro. However, SOCS2-deficient and control mice infected with M. tuberculosis displayed similar bacterial burdens in the lungs. In studying the role of SOCS3 in different mouse models, we found that the lack of SOCS3 in either myeloid or T cells dramatically increased susceptibility to M. tuberculosis infection. During infection, SOCS3 expression in macrophages and dendritic cells was required to prevent an inhibitory effect of IL-6 on TNF and IL-12 secretion and elevated IFN-γ expression by CD4+ T cells. More detailed studies revealed that the lack of SOCS3 in myeloid cells could be mimicked by mutating the SOCS3 binding site of the gp130 receptor. This indicates that among the receptors, which can be regulated by SOCS3, the control over the IL-6 family gp130 receptor is fundamental for proper immune responses. Surprisingly, mice bearing SOCS3-deficient T cells were not susceptible to BCG infection. Moreover, a proper defense against challenge with M. tuberculosis infection was restored if mice deficient for SOCS3 in T cells had been BCG-vaccinated. In conclusion, we demonstrated a pivotal role of SOCS1 and SOCS3 on the outcome of infection with M. tuberculosis. SOCS1 expression allows fast bacterial growth during the early phase of infection and protects from severe inflammation during later stages. SOCS3 expression in myeloid and T cells independently mediates resistance to M. tuberculosis infection by modulating T cell functions. Based on the obtained data, we suggest that SOCS3-regulated pathways are promising targets for future therapies as well as vaccination strategies

SOCS3 Expression by Thymic Stromal Cells Is Required for Normal T Cell Development

The suppressor of cytokine signaling 3 (SOCS3) is a major regulator of immune responses and inflammation as it negatively regulates cytokine signaling. Here, the role of SOCS3 in thymic T cell formation was studied in Socs3fl/fl Actin-creER mice (Δsocs3) with a tamoxifen inducible and ubiquitous Socs3 deficiency. Δsocs3 thymi showed a 90% loss of cellularity and altered cortico-medullary organization. Thymocyte differentiation and proliferation was impaired at the early double negative (CD4-CD8-) cell stage and apoptosis was increased during the double positive (CD4+CD8+) cell stage, resulting in the reduction of recent thymic emigrants in peripheral organs. Using bone marrow chimeras, transplanting thymic organoids and using mice deficient of SOCS3 in thymocytes we found that expression in thymic stromal cells rather than in thymocytes was critical for T cell development. We found that SOCS3 in thymic epithelial cells (TECs) binds to the E3 ubiquitin ligase TRIM 21 and that Trim21−/− mice showed increased thymic cellularity. Δsocs3 TECs showed alterations in the expression of genes involved in positive and negative selection and lympho-stromal interactions. SOCS3-dependent signal inhibition of the common gp130 subunit of the IL-6 receptor family was redundant for T cell formation. Together, SOCS3 expression in thymic stroma cells is critical for T cell development and for maintenance of thymus architecture.publishedVersio

Expression of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis

Background Appropriate immune activation of T cells and macrophages is central for the control of Mycobacterium tuberculosis infections. IFN-γ stimulated responses are lowered in tuberculosis (TB), while expression of Suppressor of Cytokine Signaling (SOCS) molecules – 1 and 3 and CD4+CD25+FoxP3+T regulatory cells is increased. Here we investigated the association of these molecules in regard to clinical severity of TB. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from patients with pulmonary TB (PTB, n = 33), extra-pulmonary TB (ETB, n = 33) and healthy endemic controls (EC, n = 15). Cases were classified as moderately advanced or far advanced PTB, and less severe or severe disseminated ETB. M. tuberculosis -stimulated IFN-γ, SOCS1, SOCS3 and FoxP3 gene expression and secretion of Th1 and Th2 cytokines was measured. Statistical analysis was performed using Mann–Whitney U, Wilcoxon Rank and Kruskal Wallis non-parametric tests. Results In un-stimulated PBMCs, IL-6 (p = 0.018) and IL-10 (p = 0.013) secretion levels were increased in PTB while IL-10 was also increased in ETB (p = 0.003), all in comparison with EC. M. tuberculosis-stimulated IL-6 (p = 0.003) was lowered in ETB as compared with EC. SOCS1 mRNA expression in M. tuberculosis stimulated PBMCs levels in moderately advanced PTB (p = 0.022), far advanced (p = 0.014) PTB, and severe ETB (p = 0.009) were raised as compared with EC. On the other hand, SOCS1 mRNA titers were reduced in less severe ETB, in comparison with severe ETB (p = 0.027) and far advanced PTB (p = 0.016). SOCS3 mRNA accumulation was reduced in far advanced PTB (p = 0.007) and FoxP3 mRNA expression was increased in less severe ETB as compared with EC (p = 0.017). Conclusions The lowered SOCS1 mRNA levels in patients with less severe extra-pulmonary TB as compared to those with more severe ETB and PTB may lead to elevated IFN-γ pathway gene expression in the latter group. As localized ETB has shown to be associated with more effective Th1 immunity and adaptive responses, this suggests a role for SOCS1 in determining disease outcome in extra-pulmonary TB

LPS Regulates SOCS2 Transcription in a Type I Interferon Dependent Autocrine-Paracrine Loop

Recent studies suggest that SOCS2 is involved in the regulation of TLR signaling. In this study, we found that the expression of SOCS2 is regulated in human monocyte-derived DC by ligands stimulating TLR2, 3, 4, 5, 8 and 9 signaling. SOCS2 induction by LPS was dependent on the type I IFN regulated transcription factors IRF1 and IRF3 as shown by using silencing RNAs for IRFs. Blocking endogenous type I IFN signaling, by neutralizing antibodies to the receptor IFNAR2, abolished SOCS2 mRNA expression after TLR4 stimulation. Transcription factors STAT3, 5 and 6 displayed putative binding sites in the promoter regions of the human SOCS2 gene. Subsequent silencing experiments further supported that STAT3 and STAT5 are involved in LPS induced SOCS2 regulation. In mice we show that SOCS2 mRNA induction is 45% lower in bone marrow derived macrophages derived from MyD88−/− mice, and do not increase in BMMs from IRF3−/− mice after BCG infection. In conclusion, our results suggest that TLR4 signaling indirectly increases SOCS2 in late phase mainly via the production of endogenous type I IFN, and that subsequent IFN receptor signaling activates SOCS2 via STAT3 and STAT5

Immune mapping of human tuberculosis and sarcoidosis lung granulomas

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas multifocal granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40-60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients

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