The role of suppressor of cytokine signaling (SOCS1 and SOCS3) molecules in determining severity of tuberculosis infection

Introduction: Mycobacterium tuberculosis the causative agent of tuberculosis (TB), down-regulates host immunity and persists within cells. Cytokines are responsible for immune activation but these are characteristically modulated in TB. Expression of cytokine regulatory molecules Suppressor of Cytokine Signaling (SOCS)-1, SOCS3 and T regulatory cells (FOXP3, Tregs marker) molecules are increased in TB but their association with severity and their role in determining disease outcomes TB is unclear. Objective: We investigated the role of SOCS1, SOCS3 and FOXP3 by studying the expression of cytokines across a spectrum of pulmonary (PTB) and extra-pulmonary (ETB) TB patients in comparison with healthy endemic controls (EC). Materials and Methods: This was a cross-sectional study which was approved by the Ethical Review Committee of the AKU. We recruited ECs (n=30) and patients with P113 (n=33) and ETB (n=33). Healthy volunteers were sub-divided according to their tuberculin skin test (TST) reactivity; EC-TST (-), n=15 or positive EC-TST (+), n=15. Or, according to ESAT-6- induced IFN-y responses; EC-IFN-y (-), n=24 (IFN-y median, 0 pg/mL, IQR 0-0 pg/mL) or EC-IFN-y (+), n=5 (IFN-y median, 64.3 pg/mL, IQR 1.1-79.9 pg/mL). PTB was 1111 classified as moderately advanced (PTB-mod, n=20) and far-advanced (PTB-adv, n=13) 1111 disease. ETB was classified as less (L-ETB, n=26) and more severe (D-ETB, n=7) disease. Gene expression of Thl type cytokines (IFN-y, TNF-a, IL-2, IL-6, IL-17A, 1111 CXCL9, CCL2, and CCR2), Th2 type cytokines (IL-4 and IL-10) and regulatory factors 1111 (SOCS1, SOCS3, T-bet, Gata-3, and FOXP3), secretion of Thl (IFN-y, TNF-a, CXCL9, CXCL10 and CCL2) and Th2 cytokines (IL-10) was determined in peripheral blood cells. 1111 In vitro responses to stimulation with live M tuberculosis (Mtb), sonicated (MTB sonicate, MTBs) and Early secreted antigen target-6 (ESAT-6) was determined in each study subject. Immunohistochemical analysis was performed on biopsy specimens of tuberculous lymphadenitis (LNTB) as compared with un-infected reactive lymph nodes. These were classified histologically according to extent of necrosis present into focal (f-LNTB, n=10) or extensive (e-LNTB, n=8) caseous necrosis. Expression of SOCS1, SOCS3 and CXCR3 was performed in each case. Results: SOCS1 mRNA expression was raised in T cells from PTB (p=0.02) as compared with EC-TST (-). In un-stimulated PBMCs, SOCS1 mRNA levels were increased in PTB-adv as compared with PTB-mod (p=0.008). IL-6 secretion levels were increased in PTB-adv (p=0.012) and L-ETB (p=0.036) while, IL-10 secretion levels were increased in PTB-adv (p=0.012), L-ETB (p=0.003) and D-ETB (p=0.026) as compared with EC-TST (-). Compared to EC-TST (-) subjects, PTB patients in response to live Mtb showed increased SOCS1 mRNA (p=0.0067), while decreased SOCS3 mRNA was observed in both PTB (p=0.028) and ETB (p=0.023). Mtb-induced increased FOXP3 mRNA in ETB as compared with EC-TST (-) (p=0.021) and EC-TST (+) (p=0.038). Compared to EC-TST (-) subjects; Mtb—induced decreased SOCS3 mRNA in PTB-adv (p=0.007), increased FOXP3 mRNA (p=0.017) in L-ETB, increased SOCS1 mRNA in PTB-mod (p=0.022), PTB-adv (1)=0.014) and D-ETB (p=0.009). Further, Mtb-stimulated increased SOCS1 mRNA in PTB-adv (p=0.016) and D-ETB (p=0.027) as compared with L-ETB. ESAT-6—stimulation increased SOCS1 (PTB, p=0.023; ETB, p\u3c0.001) mRNA and decreased IFN-y (PTB, p=0.015; ETB, p=0.009) secretion levels in PTB and ETB as compared with EC-IFN-y (+) subjects. ESAT-6-induced increased IL-10 (p\u3c0.001) secretion levels in PTB as compared with EC-1FN-y (-) subjects. MTBs-induced increased SOCS1 (PTB, p\u3c0.001; ETB, p\u3c0.001) and FOXP3 (PTB, p\u3c0.001; ETB, p\u3c0.001) mRNA in PTB and ETB patients as compared with EC-TST (-) subjects. In granulomas, SOCS1, SOCS3 and CXCR3 protein expression was increased in LNTB as compared with controls. SOCS1 and CXCR3 were mainly associated with extensive as compared with focal caseous necrosis. Conclusions: Increased SOCS 1 mRNA in severe forms of PTB and ETB was associated with increase in IL-6 and IL-10 secretion is reflective of un-favourable disease outcomes. ESAT-6- induced SOCS1 could distinguish latent from active TB. In granulomatous lesions, SOCS1 expression in extensive caseous necrosis was indicative of progressive disseminated infection. Overall, we identify an association of SOCS1 with disease severity in TB. As SOCS1 is a key regulator for determining disease outcomes it may be a possible target for new TB treatment