Treatment of high fat diet-induced obese pregnant mice with IL-6 receptor antibody does not ameliorate placental function and fetal growth restriction.

PROBLEM: Pregnancy complications and adverse birth outcomes are in part fueled by the rise in obesity and its associated co-morbidities in western societies. Fetal healthy development and placental function are disturbed by an obese, inflammatory environment associated with cytokines, such as interleukin-6, causing inadequate supply of nutrients to the fetus and perinatal programming with severe health consequences. METHOD OF STUDY: Mice received high fat diet (HFD) before and during gestation to induce obesity. We performed an IL-6 receptor antibody (MR16-1) treatment in pregnant obese mice at embryonic days E0.5, E7.5 and E14.5 to investigate whether this could ameliorate HFD-induced and obesity-associated placental dysfunction, evaluated by stereology and western blot, and improve offspring outcome at E15.5 in obese dams. RESULTS: We observed fewer fetuses below the 10th percentile and placental vascularization was less aggravated following MR16-1 treatment of obese dams, showing slight improvements in labyrinth zone (Lz) vascularization. However, placental dysfunction and fetal growth restriction were still apparent in MR16-1 dams compared to lean control dams. Molecular analysis showed significantly elevated IL-6 level in placentas of MR16-1 treated dams. CONCLUSION: Treatment with MR16-1 blocks IL-6 signaling in the placenta, but has only limited effects on preventing HFD-associated placental dysfunction and offspring outcomes in mice, suggesting further mechanisms in the deterioration of placental vascularization and fetal nutrient supply as a consequence of maternal obesity.

Interleukin-6: evolving role in the management of neuropathic pain in neuroimmunological disorders.

BACKGROUND: Neuropathic pain in neuroimmunological disorders refers to pain caused by a lesion or disease of the somatosensory system such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MS and NMOSD are autoimmune disorders of the central nervous system, and ≥ 50% of patients with these disorders experience chronic neuropathic pain. The currently available medications for the management of neuropathic pain have limited effectiveness in patients with MS and NMOSD, and there is an unmet medical need to identify novel therapies for the management of chronic neuropathic pain in these patients. In this review article, we summarize the role of interleukin-6 (IL-6) in the pathogenesis of MS and NMOSD and the ameliorative effects of anti-IL-6 therapies in mouse models of experimental autoimmune encephalomyelitis (EAE). MAIN BODY: Intraperitoneal injection of MR16-1, an anti-IL-6 receptor (IL-6R) antibody, reduced mechanical allodynia and spontaneous pain in EAE mice, which was attributed to a reduction in microglial activation and inhibition of the descending pain inhibitory system. The effect of anti-IL-6 therapies in ameliorating neuropathic pain in the clinical setting is controversial; a reduction in pain intensity has been reported with an anti-IL-6 antibody in four studies, namely a case report, a pilot study, a retrospective observational study, and a case series. Pain intensity was evaluated using a numerical rating scale (NRS), with a lower score indicating lesser pain. A reduction in the NRS score was reported in all four studies. However, in two randomized controlled trials of another anti-IL-6R antibody, the change in the visual analog scale pain score was not statistically significantly different when compared with placebo. This was attributed to the low mean pain score at baseline in both the trials and the concomitant use of medications for pain in one of the trials, which may have masked the effects of the anti-IL-6R antibody on neuropathic pain. CONCLUSION: Thus, anti-IL-6 therapies might have a potential to reduce neuropathic pain, but further investigations are warranted to clarify the effect of inhibition of IL-6 signaling on neuropathic pain associated with MS and NMOSD.

Inhibition of interleukin-6 signaling attenuates aortitis, left ventricular hypertrophy and arthritis in interleukin-1 receptor antagonist deficient mice.

The aim of the present study was to examine whether inhibition of Interleukin (IL)-6 signaling by MR16-1, an IL-6 receptor antibody, attenuates aortitis, cardiac hypertrophy, and arthritis in IL-1 receptor antagonist deficient (IL-1RA KO) mice. Four weeks old mice were intraperitoneally administered with either MR16-1 or non-immune IgG at dosages that were adjusted over time for 5 weeks. These mice were stratified into four groups: MR16-1 treatment groups, KO/MR low group (first 2.0 mg, following 0.5 mg/week, n=14) and KO/MR high group (first 4.0 mg, following 2.0 mg/week, n=19) in IL-1RA KO mice, and IgG treatment groups, KO/IgG group (first 2.0 mg, following 1.0 mg/week, n=22) in IL-1RA KO mice, and wild/IgG group (first 2.0 mg, following 1.0 mg/week, n=17) in wild mice. Aortitis, cardiac hypertrophy and arthropathy were histologically analyzed. Sixty-eight percent of the KO/IgG group developed aortitis (53% developed severe aortitis). In contrast, only 21% of the KO/MR high group developed mild aortitis, without severe aortitis (P<0.01, vs KO/IgG group). Infiltration of inflammatory cells, such as neutrophils, T cells, and macrophages, was frequently observed around aortic sinus of the KO/IgG group. Left ventricle and cardiomyocyte hypertrophy were observed in IL-1RA KO mice. Administration of high dosage of MR16-1 significantly suppressed cardiomyocyte hypertrophy. MR16-1 attenuated the incidence and severity of arthritis in IL-1RA KO mice in a dose-dependent manner. In conclusion, blockade of IL-6 signaling may exert a beneficial effect to attenuate severe aortitis, left ventricle hypertrophy, and arthritis.

Involvement of IL-6 and GSK3ß in impaired sensorimotor gating induced by high-fat diet.

Increased levels of proinflammatory cytokines have been implicated in schizophrenia; however, their pathophysiological roles in abnormal brain dysfunctions remain unclear. We evaluated the effect of proinflammatory cytokines on a high-fat diet (HFD)-induced prepulse inhibition (PPI) deficits in the acoustic startle response. Eight-week-old male C57BL/6J mice were fed a HFD for 3 weeks and then PPI was examined. HFD significantly induced PPI deficits and increased plasma IL-6, but not TNFα, levels. Interestingly, MR16-1 administration during the HFD period ameliorated PPI deficits. Further, in the striatum of HFD-fed mice, phosphorylation of GSK3ß, but not GSK3α, was significantly increased; this increase was attenuated by MR16-1, although the protein levels of GSK3α and ß were not altered. There were no significant differences in either phosphorylation or protein levels of GSK3α, ß in the PFC during the HFD period. These results suggest that increased IL-6 levels during HFD may induce sensorimotor gating deficits, likely through the alteration of striatal GSK3ß phosphorylation. MR16-1 might have a beneficial effect on such HFD-induced sensorimotor gating deficits.

Effects of Rat Anti-mouse Interleukin-6 Receptor Antibody on the Recovery of Cognitive Function in Stroke Mice.

This study aimed to investigate the effects of rat anti-mouse interleukin (IL)-6 receptor antibody (MR16-1) on the recovery of cognitive function in stroke mice. Adult male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO). Mice were randomly assigned into three groups: sham group, model group, and MR16-1 group. After the treatment of MR16-1, spatial learning and memory performance of mice were evaluated by the Morris water maze (MWM) and Y-maze tests. Then, brain slices were obtained and infarct volume and neuronal apoptosis were assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, respectively. Protein expression levels of apoptosis-associated proteins and multiple inflammatory cytokines were determined by Western blot analysis. Real-time quantitative PCR (RT-PCR) was used to examine the mRNA levels of various inflammatory cytokines in brain slices and cerebrospinal fluid (CSF). The results showed that MR16-1 improved performances of stroke mice in MWM and Y-maze tests. Moreover, MR16-1 ameliorated MCAO-induced infarct, neuronal apoptosis, and inflammatory response. Furthermore, MR16-1 promoted the expression of Bcl-2 and inhibited the expression of Bax in stroke mice, which revealed the inhibitory effect of MR16-1 on neuronal apoptosis. IL-6 levels in brain and CSF were both decreased by MR16-1 treatment in stroke mice. MR16-1 ameliorated cognitive dysfunction and apoptosis in stroke mice, involving the inhibition of inflammatory response and pro-apoptotic Bax, and the up-regulation of anti-apoptotic Bcl-2. The data supported that MR16-1 might be a potential therapeutic drug for the treatment of stroke.

Combined Metabolomics and Transcriptomics Approaches to Assess the IL-6 Blockade as a Therapeutic of ALS: Deleterious Alteration of Lipid Metabolism.

In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (p = 0.0041) and decreasing body weight (p < 0.05). A significant increase in regulatory T-cell count (p = 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (p = 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.

Allergen-induced IL-6 trans-signaling activates γδ T cells to promote type 2 and type 17 airway inflammation.

BACKGROUND: A variant in the IL-6 receptor (IL-6R) gene increases asthma risk and is predicted to decrease IL-6 classic signaling and increase IL-6 trans-signaling. This suggests that inhibition of IL-6 trans-signaling, but not classic signaling, might suppress allergic airway inflammation. OBJECTIVES: We sought to determine whether IL-6 signaling contributes to (1) acute experimental asthma induced by clinically relevant allergens and (2) variation in asthma clinical phenotypes in asthmatic patients. METHODS: Mice were sensitized to house dust mite (HDM) or cockroach at day 0, treated with IL-6R inhibitors at day 13, and challenged with the same allergen at days 14 to 17. End points were measured 3 hours after the final challenge. IL-6 and soluble IL-6 receptor (sIL-6R) expression in induced sputum of asthmatic patients was correlated with asthma clinical phenotypes. RESULTS: Both HDM and cockroach induced a type 2/type 17 cytokine profile and mixed granulocytic inflammation in the airways. Both allergens increased IL-6 expression in the airways, but only cockroach induced sIL-6R expression. Therefore HDM challenge promoted IL-6 classic signaling but not trans-signaling; in this model treatment with anti-IL-6R did not suppress airway inflammation. In contrast, cockroach-induced inflammation involved activation of IL-6 trans-signaling and production of IL-17A by γδ T cells. Anti-IL-6R, selective blockade of sIL-6R, or γδ T-cell deficiency significantly attenuated cockroach-induced inflammation. Asthmatic patients with high airway IL-6 and sIL-6R levels were enriched for the neutrophilic and mixed granulocytic subtypes. CONCLUSION: Experimental asthma associated with both high IL-6 and high sIL-6R levels in the airways is attenuated by treatment with IL-6R inhibitors.