Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases.

Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases. Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here, we describe a first-in-class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4, which inhibits NETosis and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. We provide a detailed understanding of the epitope selectivity of CIT-013. Detection of CIT-013 epitopes in rheumatoid arthritis (RA) synovium provides evidence that RA is an autoimmune disease with excessive citrullinated NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NETosis, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhance their phagocytosis by macrophages in an Fc-dependent manner. This is confirmed using a murine neutrophilic airway inflammation model where a mouse variant of CIT-013 reduced tissue NET burden with significant anti-inflammatory consequences. CIT-013's therapeutic activity provides new insights for the development of NET antagonists and indicates the importance of a new emerging therapy for NET-driven diseases with unmet therapeutic needs.

IL-1ß-Mediated Activation of Adipose-Derived Mesenchymal Stromal Cells Results in PMN Reallocation and Enhanced Phagocytosis: A Possible Mechanism for the Reduction of Osteoarthritis Pathology.

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

S100A8/A9 increases the mobilization of pro-inflammatory Ly6Chigh monocytes to the synovium during experimental osteoarthritis.

BACKGROUND: Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). METHOD: S100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS. RESULTS: S100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM. CONCLUSION: Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

Branchial nitrogen cycle symbionts can remove ammonia in fish gills.

Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.

Voluntary timing of food intake increases weight gain and reduces basal plasma cortisol levels in common carp (Cyprinus carpio L.).

We investigated the effect of timing of food intake on growth in common carp (Cyprinus carpio L.). Juvenile carp were demand-fed for 22 days using a computerized pendulum feeder that registered meal requests. Controls were pair-fed at 10:00 h, both groups were kept at 12L:12D (lights on at 06:30 h). Demand-fed fish displayed highest food intake at 22:00 h, and the lowest at 10:00. After 22 days, demand-fed fish had grown by 20% of their initial body weight, compared to 4% of the pair-fed control. Plasma cortisol levels in demand-fed fish were remarkably low and stable, whereas in the control group levels had increased 60-fold at 10:00 h compared to 22:00 h. Hepatic mRNA expression of leptin-a1 and leptin-a2 also differed markedly between groups and time points, with leptin-a2 expression being lowest in the demand-fed group at the time point of lowest food intake. We conclude that timing of food intake is an important determinant of endocrine status, growth and welfare.