Cell autonomous expression of CXCL-10 in JAK2V617F-mutated MPN.

PURPOSE: Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem- and progenitor cells. Mutation of Janus-Kinase 2 (JAK2) is the most frequent genetic event detected in Philadelphia-negative MPN. In advanced phases, the clinical hallmark of the disease is a striking inflammatory syndrome. So far, the cellular and molecular basis of inflammation is not fully understood. We, therefore, sought to investigate the relationship of activating JAK2 mutation and aberrant cytokine expression in MPN. METHODS: Cytokine array was performed to identify Jak2V617F-related cytokine expression and secretion. CXCL10 mRNA expression was analyzed by qPCR in peripheral blood cells. To exclude paracrine/autocrine stimulation as a potential mechanism, we generated Ba/F3-EpoR-JAK2WT or EpoR-JAK2V617F cells lacking CXCL10 receptor. Pharmacologic inhibition of JAK2 kinase was achieved by JAK-Inhibitor treatment. Signaling pathways and downstream effectors were characterized by Western blotting, immunofluorescence microscopy, luciferase reporter assays, qPCR, and chromatin-immunoprecipitation studies. RESULTS: We identified CXCL10 as the most highly induced cytokine in JAK2-mutated cell lines. In MPN patients, CXCL10 is highly expressed in JAK2V617F but not JAK2WT MPN or healthy donor controls. Moreover, CXCL10 expression correlates with JAK2V617F allelic burden. High CXCL10 correlates with the presence of clinical risk factors but not with clinical symptoms and quality of life. Pharmacologic inhibition of mutated JAK2 kinase inhibits CXCL10 expression. NFκB signaling is activated downstream of JAK2V617F receptor and directly induces CXCL10 expression. CONCLUSIONS: Our data provide first evidence for a link between oncogenic JAK2V617F signaling and cell intrinsic induction of CXCL10 induced by activated NFkB signaling.

The PPE18 protein of Mycobacterium tuberculosis inhibits NF-κB/rel-mediated proinflammatory cytokine production by upregulating and phosphorylating suppressor of cytokine signaling 3 protein.

Mycobacterium tuberculosis bacteria are known to suppress proinflammatory cytokines like IL-12 and TNF-α for a biased Th2 response that favors a successful infection and its subsequent intracellular survival. However, the signaling pathways targeted by the bacilli to inhibit production of these cytokines are not fully understood. In this study, we demonstrate that the PPE18 protein of M. tuberculosis inhibits LPS-induced IL-12 and TNF-α production by blocking nuclear translocation of p50, p65 NF-κB, and c-rel transcription factors. We found that PPE18 upregulates the expression as well as tyrosine phosphorylation of suppressor of cytokine signaling 3 (SOCS3), and the phosphorylated SOCS3 physically interacts with IκBα-NF-κB/rel complex, inhibiting phosphorylation of IκBα at the serine 32/36 residues by IκB kinase-ß, and thereby prevents nuclear translocation of the NF-κB/rel subunits in LPS-activated macrophages. Specific knockdown of SOCS3 by small interfering RNA enhanced IκBα phosphorylation, leading to increased nuclear levels of NF-κB/rel transcription factors vis-a-vis IL-12 p40 and TNF-α production in macrophages cotreated with PPE18 and LPS. The PPE18 protein did not affect the IκB kinase-ß activity. Our study describes a novel mechanism by which phosphorylated SOCS3 inhibits NF-κB activation by masking the phosphorylation site of IκBα. Also, this study highlights the possible mechanisms by which the M. tuberculosis suppresses production of proinflammatory cytokines using PPE18.