Anti-IL-6 cytokine treatment has no impact on elevated hematocrit and splenomegaly in a polycythemia vera mouse model

Somatic mutations in JAK2, MPL and Calreticulin and inflammation play a key role in pathophysiology of chronic myeloproliferative neoplasia (CMN). One of the most prominent cytokines elevated in serum of Polycythemia vera patients is interleukin-6 (IL-6). Currently, it is being discussed whether suppression of inflammation by anti-cytokine approaches as anti-IL-6 treatment may be therapeutically useful in CMN. We here sought to investigate the efficacy of anti-IL-6 treatment on inflammatory cytokines, hematocrit and splenomegaly in CMN like disease. JAK2-V617F knock-in mice (JAK2+/V617F) were treated for three weeks with anti-IL-6 antibody (Ab) or IgG-control. Upon anti-IL-6 Ab treatment, serum levels of CXCL2 and CXCL10 were significantly reduced. In addition, CXCL1, CCL11, M-CSF, G-CSF, IL-17, IL-12p40 and CCL2 were reduced by a factor of 0.3 - 0.8. Partly, this was also achieved by applying high-dose IgG. Hematocrit, erythrocyte and leukocyte counts were elevated in JAK2+/V617F mice but were not reduced by anti-IL6 Ab treatment. In addition, there was no apparent amelioration of splenomegaly and spleen histopathology. In conclusion, anti-IL-6 Ab treatment did not result in improvement of hematological disease parameters but was shown to modulate the serum cytokine signature.

Inflammation induces pro-NETotic neutrophils via TNFR2 signaling

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMN). Here we show that in vitro cytokine treatment leads to the development of a subgroup of human PMN expressing CCR5, termed CCR5+ cytokine-induced PMN (CCR5+ cPMN). Auto/paracrine TNF signaling increases intracellular neutrophil elastase (ELANE) abundance and induces NETosis in CCR5+ cPMN. Triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, a known activator of NETosis. In vivo, we find an increased number of CCR5+ cPMN in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC) but not Crohn's disease (CD). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ cPMN. In conclusion, we identify a phenotype of pro-NETotic, CCR5 positive PMN present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

Clusterin secretion is attenuated by pro-inflammatory cytokines in culture models of cartilage degradation

ABSTRACTProteomic studies have implicated clusterin as a potential biomarker of osteoarthritis (OA). However, there are two isoforms of clusterin with opposing functions, and their roles in OA have not previously been clarified. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone which prevents protein aggregation and enhances cell proliferation and viability, whereas nuclear clusterin (nCLU) acts as a pro-death signal. In this study, we focused on the role of sCLU and used established, pathophysiologically relevant, in vitro culture models to validate this potential biomarker of cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and chondrocytes was analysed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with or without pro-inflammatory cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The amount of sulphated glycosaminoglycans (sGAG) released into the medium was determined by dimethylmethylene blue (DMMB) analysis. Clusterin mRNA expression was quantified by real-time PCR. MMP-3, MMP-13, COMP and sGAG released from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. Release of sCLU was attenuated with cytokine treatment in all three in vitro models. Expression of clusterin mRNA in cartilage explants and chondrocytes was down-regulated 7-days post cytokine stimulation. Cytokine stimulation attenuated expression and secretion of sCLU, therefore potentially limiting the cytoprotection which sCLU provides. These observations further implicate sCLU as having a role in OA, and diagnostic value as a potential biomarker for cartilage degradation.