Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

Cutting Edge: IL-6-Driven Immune Dysregulation Is Strictly Dependent on IL-6R α-Chain Expression.

IL-6 binds to the IL-6R α-chain (IL-6Rα) and signals via the signal transducer gp130. Recently, IL-6 was found to also bind to the cell surface glycoprotein CD5, which would then engage gp130 in the absence of IL-6Rα. However, the biological relevance of this alternative pathway is under debate. In this study, we developed a mouse model, in which murine IL-6 is overexpressed in a CD11c-Cre-dependent manner. Transgenic mice developed a lethal immune dysregulation syndrome with increased numbers of Ly-6G+ neutrophils and Ly-6Chi monocytes/macrophages. IL-6 overexpression promoted activation of CD4+ T cells while suppressing CD5+ B-1a cell development. However, additional ablation of IL-6Rα protected IL-6-overexpressing mice from IL-6-triggered inflammation and fully phenocopied IL-6Rα-deficient mice without IL-6 overexpression. Mechanistically, IL-6Rα deficiency completely prevented downstream activation of STAT3 in response to IL-6. Altogether, our data clarify that IL-6Rα is the only biologically relevant receptor for IL-6 in mice.

Balanced Bcl-3 expression in murine CD4+ T cells is required for generation of encephalitogenic Th17 cells.

The function of NF-κB family members is controlled by multiple mechanisms including the transcriptional regulator Bcl-3, an atypical member of the IκB family. By using a murine model of conditional Bcl-3 overexpression specifically in T cells, we observed impairment in the development of Th2, Th1, and Th17 cells. High expression of Bcl-3 promoted CD4+ T-cell survival, but at the same time suppressed proliferation in response to TCR stimulation, resulting in reduced CD4+ T-cell expansion. As a consequence, T-cell-specific overexpression of Bcl-3 led to reduced inflammation in the small intestine of mice applied with anti-CD3 in a model of gut inflammation. Moreover, impaired Th17-cell development resulted in the resistance of Bcl-3 overexpressing mice to EAE, a mouse model of multiple sclerosis. Thus, we concluded that fine-tuning expression of Bcl-3 is needed for proper CD4+ T-cell development and is required to sustain Th17-cell mediated pathology.

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis.

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αß T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis.

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.

Mutated cylindromatosis gene affects the functional state of dendritic cells.

Cylindromatosis gene (CYLD) is a ubiquitously expressed deubiquitinating enzyme, which interacts with members of the NF-κB signaling pathway and attenuates NF-κB and JNK signaling. Here, we report that DC derived from transgenic mice, which solely express a naturally occurring CYLD isoform (CYLD(ex7/8)), display a higher content of nuclear RelB and express elevated levels of NF-κB family members as well as of known NF-κB-target genes comprising costimulatory molecules and pro-inflammatory cytokines, as compared with WT DC. Accordingly, unstimulated CYLD(ex7/8) DC exhibited a significantly higher primary allogenic T-cell stimulatory capacity than WT DC and exerted no tolerogenic activity. Transduction of unstimulated CYLD(ex7/8) DC with relB-specific shRNA reduced their T-cell stimulatory capacity. Treatment with the synthetic glucocorticoid dexamethasone known to inhibit NF-κB and AP-1 activity reverted the pro-immunogenic phenotype and function of CYLD(ex7/8) DC and re-established their tolerogenic function. DC derived from CYLD knockout mice showed no functional alterations compared with WT DC. Therefore, although complete loss of CYLD may be compensated for by other endogenous NF-κB inhibitors, CYLD(ex7/8) acts in a dominant negative manner. Our findings raise the question of whether genetic defects associated with increased NF-κB activity may result in disturbed maintenance of peripheral tolerance.

Naturally occurring short splice variant of CYLD positively regulates dendritic cell function.

Deubiquitination of NF-kappaB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLD(ex7/8) mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow-derived DCs from CYLD(ex7/8) mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205-mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLD(ex7/8) DCs leads to enhanced NF-kappaB activity accompanied by an increased nuclear translocation of the IkappaB molecule Bcl-3, along with nuclear p50 and p65. This suggests that, in contrast to flCYLD, sCYLD is a positive regulator of NF-kappaB activity, and its overexpression induces a hyperactive phenotype in DCs.