The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.

Tocilizumab is a humanized monoclonal antibody that is approved for the treatment of different human inflammatory diseases, including rheumatoid arthritis and cytokine release syndrome. Tocilizumab binds to the interleukin-6 receptor (IL-6R) and thereby blocks signaling of the pro-inflammatory cytokine IL-6. Initial studies and all authority assessment reports state that tocilizumab is effective in humans, but cannot bind to the murine or rat IL-6R and thus not block IL-6 signaling in the mouse. However, several recent studies described the use of tocilizumab in mice and reported biological effects that were attributed to IL-6 blockade. In this study, we investigate the capability of tocilizumab to block IL-6 signaling using different human and murine cell lines. Our results unequivocally confirm the original state of the art that tocilizumab blocks signaling via the human IL-6R, but does not block IL-6 signaling in murine cells.

IL-6 is required for the response of mice against Listeria monocytogenes. Control of infection depends on classical IL-6 signaling via membrane IL-6Rα, but IL-6 target cells and protective mechanisms remain unclear. We used mice with IL-6Rα-deficiency in T cells (Il6rafl/fl×CD4cre) or myeloid cells (Il6rafl/fl×LysMcre) to define the role of these cells in IL-6-mediated protection. Abrogation of IL-6Rα in T cells did not interfere with bacteria control and induction of TH1 and CD8+ T-cell responses. IL-6Rα-deficiency in myeloid cells caused significant defects in listeria control. This defect was not associated with reduced recruitment of granulocytes and inflammatory monocytes, and both cell populations were activated and not impaired in cytokine production. However, IL-6Rα-deficient inflammatory monocytes displayed diminished expression of IL-4Rα and of CD38, a protein required for phagocytosis and innate control of listeria. In vitro studies revealed that IL-4 and IL-6 cooperated in induction of CD38. In listeria-infected mice, phagocytic activity of inflammatory monocytes correlated with CD38 expression levels on cells and inflammatory monocytes of Il6rafl/fl×LysMcre mice were significantly impaired in phagocytosis. In conclusion, we demonstrate that inhibition of classical IL-6 signaling in myeloid cells causes alterations in differentiation and function of these cells, which subsequently prevent effective control of L. monocytogenes.

Non-alcoholic steatohepatitis (NASH) is a liver disease with the potential to lead to cirrhosis and hepatocellular carcinoma. Interleukin-6 (IL-6) has been implicated in the pathogenesis of NASH, with the so-called IL-6 'trans-signaling' cascade being responsible for the pro-inflammatory actions of this cytokine. We aimed to block IL-6 'trans-signaling', using a transgenic mouse that overexpresses human soluble glycoprotein130 (sgp130Fc Tg mice) fed a commonly used dietary model of inducing NASH (methionine and choline deficient-diet; MCD diet) and hypothesized that markers of NASH would be ameliorated in such mice. Sgp130Fc Tg and littermate control mice were fed a MCD or control diet for 4 weeks. The MCD diet induced many hallmarks of NASH including hepatomegaly, steatosis, and liver inflammation. However, in contrast with other mouse models and, indeed, human NASH, the MCD diet model did not increase the mRNA or protein expression of IL-6. Not surprisingly, therefore, markers of MCD diet-induced NASH were unaffected by sgp130Fc transgenic expression. While the MCD diet model induces many pathophysiological markers of NASH, it does not induce increased IL-6 expression in the liver, a key hallmark of human NASH. We, therefore, caution the use of the MCD diet as a viable mouse model of NASH.

ADAM17 is a member of the A Disintegrin And Metalloproteinase family of proteases. It is ubiquitously expressed and causes the shedding of a broad spectrum of surface proteins such as adhesion molecules, cytokines and cytokine receptors. By controlled shedding of these proteins from leukocytes, ADAM17 is able to regulate immune responses. Several ADAM17 targets on T cells have been implicated in T-cell migration, differentiation and effector functions. However, the role of ADAM17 in T-cell responses is still unclear. To characterize the function of ADAM17 in T cells, we used Adam17fl/fl×CD4cre+ mice with a T-cell restricted inactivation of the Adam17 gene. Upon stimulation, ADAM17-deficient CD4+ and CD8+ T cells were impaired in shedding of CD62L, IL-6Rα, TNF-α, TNFRI and TNFRII. Surprisingly, we could not detect profound changes in the composition of major T-cell subsets in Adam17fl/fl×CD4cre+ mice. Following infection with Listeria monocytogenes, Adam17fl/fl×CD4cre+ mice mounted regular listeria-specific CD4+ TH1 and CD8+ T-cell responses and were able to control primary and secondary infections. In conclusion, our study indicates that ADAM17 is either not required in T cells under homoeostatic conditions and for control of listeria infection or can be effectively compensated by other mechanisms.