Niels A. J. Cremers

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.

Delayed cutaneous wound closure in HO-2 deficient mice despite normal HO-1 expression

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO‐1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO‐2 deficient mice is impaired with exorbitant inflammation and absence of HO‐1 expression. This study addresses the role of HO‐2 in cutaneous excisional wound healing using HO‐2 knockout (KO) mice. Here, we show that HO‐2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO‐2 KO mice compared to WT controls. Surprisingly, wound closure in HO‐2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO‐1 induction in HO‐2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C‐X‐C) ligand‐11 (CXCL‐11) in wounds of HO‐2 KO mice. Abnormal regulation of CXCL‐11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL‐11 expression in HO‐2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.

Mechanical stress changes the complex interplay Between hO-1, inflammation and Fibrosis, During excisional Wound repair

Mechanical stress following surgery or injury can promote pathological wound healing and fibrosis, and lead to functional loss and esthetic problems. Splinted excisional wounds can be used as a model for inducing mechanical stress. The cytoprotective enzyme heme oxygenase-1 (HO-1) is thought to orchestrate the defense against inflammatory and oxidative insults that drive fibrosis. Here, we investigated the activation of the HO-1 system in a splinted and non-splinted full-thickness excisional wound model using HO-1-luc transgenic mice. Effects of splinting on wound closure, HO-1 promoter activity, and markers of inflammation and fibrosis were assessed. After seven days, splinted wounds were more than three times larger than non-splinted wounds, demonstrating a delay in wound closure. HO-1 promoter activity rapidly decreased following removal of the (epi)dermis, but was induced in both splinted and non-splinted wounds during skin repair. Splinting induced more HO-1 gene expression in 7-day wounds; however, HO-1 protein expression remained lower in the epidermis, likely due to lower numbers of keratinocytes in the re-epithelialization tissue. Higher numbers of F4/80-positive macrophages, αSMA-positive myofibroblasts, and increased levels of the inflammatory genes IL-1β, TNF-α, and COX-2 were present in 7-day splinted wounds. Surprisingly, mRNA expression of newly formed collagen (type III) was lower in 7-day wounds after splinting, whereas, VEGF and MMP-9 were increased. In summary, these data demonstrate that splinting delays cutaneous wound closure and HO-1 protein induction. The pro-inflammatory environment following splinting may facilitate higher myofibroblast numbers and increase the risk of fibrosis and scar formation. Therefore, inducing HO-1 activity against mechanical stress-induced inflammation and fibrosis may be an interesting strategy to prevent negative effects of surgery on growth and function in patients with orofacial clefts or in patients with burns.

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

BackgroundMonocytes 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).MethodS100A8 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.ResultsS100A8/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.ConclusionInduction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

BackgroundOsteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA).MethodsAIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR.ResultsFcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity.ConclusionsFcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

Effects of Remote Ischemic Preconditioning on Heme Oxygenase-1 Expression and Cutaneous Wound Repair

Skin wounds may lead to scar formation and impaired functionality. Remote ischemic preconditioning (RIPC) can induce the anti-inflammatory enzyme heme oxygenase-1 (HO-1) and protect against tissue injury. We aim to improve cutaneous wound repair by RIPC treatment via induction of HO-1. RIPC was applied to HO-1-luc transgenic mice and HO-1 promoter activity and mRNA expression in skin and several other organs were determined in real-time. In parallel, RIPC was applied directly or 24h prior to excisional wounding in mice to investigate the early and late protective effects of RIPC on cutaneous wound repair, respectively. HO-1 promoter activity was significantly induced on the dorsal side and locally in the kidneys following RIPC treatment. Next, we investigated the origin of this RIPC-induced HO-1 promoter activity and demonstrated increased mRNA in the ligated muscle, heart and kidneys, but not in the skin. RIPC did not change HO-1 mRNA and protein levels in the wound 7 days after cutaneous injury. Both early and late RIPC did not accelerate wound closure nor affect collagen deposition. RIPC induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection.

Chemokine signaling during midline epithelial seam disintegration facilitates palatal fusion

Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion, and failure results in cleft palate. Palatal fusion and wound repair share many common signaling pathways related to epithelial-mesenchymal cross-talk. We postulate that chemokine CXCL11, its receptor CXCR3, and the cytoprotective enzyme heme oxygenase (HO), which are crucial during wound repair, also play a decisive role in MES disintegration. Fetal growth restriction and craniofacial abnormalities were present in HO-2 knockout (KO) mice without effects on palatal fusion. CXCL11 and CXCR3 were highly expressed in the disintegrating MES in both wild-type and HO-2 KO animals. Multiple apoptotic DNA fragments were present within the disintegrating MES and phagocytized by recruited CXCR3-positive wt and HO-2 KO macrophages. Macrophages located near the MES were HO-1-positive, and more HO-1-positive cells were present in HO-2 KO mice compared to wild-type. This study of embryonic and palatal development provided evidence that supports the hypothesis that the MES itself plays a prominent role in palatal fusion by orchestrating epithelial apoptosis and macrophage recruitment via CXCL11-CXCR3 signaling.

SAT0012 S100A8/A9 increases the mobilization of LY6C high monocytes to the synovium during experimental osteoarthritis

Background It is increasingly recognized that part of the pathology in osteoarthritis (OA) is driven by a low-grade inflammation in the synovium. Pro-inflammatory cytokines released locally during OA, such as S100A8/A9 which are expressed for prolonged periods when compared to IL-1β, IL-6, and TNF-α, may recruit monocytes from the bone marrow (BM) to the joint. In mice, two functionally distinct monocyte populations are described: (i) pro-inflammatory Ly6Chigh monocytes; and (ii) patrolling Ly6Clow monocytes. Objectives The objective of our study is to investigate the role of S100A8/A9 in the recruitment of the different monocyte populations during early collagenase-induced OA (CiOA). Methods S100A8 was intra-articularly injected into the knee joint of naïve wild type C57BL/6 (WT) mice to investigate their direct role in recruitment of monocytes. CiOA was induced by unilateral intra-articular collagenase injection in WT, and S100A9KO mice to investigate the role of S100A8/A9 in more detail. At CiOA day 7, WT and S100A9KO mice were sacrificed together with age-matched saline-injected control mice (n=6/group), and expression of several monocyte cell markers, chemokines, and adhesion molecules were measured in the synovium and BM, in which the cells were also analyzed by FACS. Monocytes were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low, which were further divided into subsets based on their Ly6C expression. Results Injection of S100A8 into the knee joints of naïve mice led to a significantly elevated expression of monocyte-related markers (Ly6C, CCR2, and CX3CR1) and monocyte attracting chemokines (MCP-1, CX3CL1, MIP1α, and KC) within the synovium after 1 and 3 days, suggesting that S100A8/A9 is directly involved in the attraction of monocytes. At CiOA day 7 in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium as compared to saline-injected control joints. In contrast, S100A9KO mice showed a significant increase in Ly6Clow monocytes (2-fold), whereas the number of Ly6Chigh monocytes remained unaffected. Concurrently, a strong upregulation of several chemokines (MCP1, CX3CL1, KC, and MIP1α) was observed locally in the synovium, of which only the Ly6Clow mobilization marker CX3CL1 was significantly higher in S100A9KO mice, corresponding with the increased Ly6Clow monocytes in the synovium of S100A9KO mice. This could however not explain the local increased number of Ly6Chigh monocytes at CiOA day 7 in WT mice, and therefore we next investigated the main source of the monocytes, which is the BM. We observed a decrease of 14% of myeloid cells (consisting partly of Ly6Chigh monocytes) in the BM of WT mice at CiOA day 7, whereas there were no changes in the BM of S100A9KO mice, suggesting that S100A8/A9 affects the release of myeloid populations from the BM. In line with that, expression of adhesion molecules (LFA-1, VCAM, VE-cadherin, PECAM1, and L-selectin) was lower at CiOA day 7 in the BM of S100A9KO mice when compared to WT mice. Conclusions Local induction of OA leads to S100A8/A9 production, and subsequently to the mobilization of Ly6Chigh monocytes into the joint, driving OA pathology. Disclosure of Interest None declared

OP0214 S100A8/A9 Produced Locally during Experimental Osteoarthritis Induces Local and Systemic Changes in Monocyte Populations

Background The etiology of osteoarthritis (OA) is multi-factorial, and is mainly driven by an activated synovium wherein inflammation plays an important role. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) to the site of inflammation. In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (C-C chemokine receptor type 2 (CCR2)high) and patrolling Ly6C-low monocytes (CCR2low). In the BM, monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with CCR2. Objectives The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. Methods CiOA was induced by unilateral-articular collagenase-injection in C57BL/6 mice. At day 7, 21 and 42, mice were sacrificed together with age-matched naive mice (n=6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA and control conditions, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analyzed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9–/– mice during CiOA. Results Expression of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, S100A8 and S100A9 in the synovium were increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM as shown by the decrease in cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day 7 in BM was increased (3.3-fold), suggesting an increased efflux of BM-cells. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA and emphasizes the systemic effects following CiOA. During the course of CiOA increased numbers of Ly6C-high monocytes were also observed in the synovium: 398% at day 7 and 510% at day 42, compared to naïve mice. These effects may be caused by the sustained S100A8/A9 levels; we therefore investigated synovitis in S100A9–/– mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9–/– mice was decreased compared to C57BL/6 mice. Conclusions Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared

OP0146 Locally Administered Adipose Derived Mesenchymal Stem Cells Augment their Anti-Inflammatory Efficacy Through IL-1β Mediated Influx of Neutrophils into Knee Joints with Experimental Osteoarthritis

Background Osteoarthritis (OA) is characterized by cartilage destruction and ectopic bone formation in joints. Several studies have demonstrated that mild synovitis in early phases is conducive to joint damage. This inflammation is reflected by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). Recently we found that adipose derived mesenchymal stem cells (ASCs) exhibit immunosuppressive characteristics (1) and reduce joint pathology after local application into mouse knee joints with experimental OA (2). This anti-inflammatory effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory environment (1). Objectives To examine the effect of IL-1β on the immunosuppressive potency of ASCs in early experimental OA. Methods Experimental OA was induced by injection of collagenase into murine knee joints (CIOA). Total knee joints were stained with haematoxylin/eosin and the PMN specific antibody NIMPR14. ASCs were isolated from adipose tissue and stimulated for 24 hours with IL-1β or interferon-gamma (IFN-γ). Gene expression in synovium and stimulated cells were analyzed using qPCR. Protein levels of chemokines and cytokines were measured in the supernatant and washouts using Luminex. ASCs were co-cultured with MACS isolated bone marrow (BM-) PMNs and analyzed using histology, qPCR and Luminex. Results Injection of ASCs into day 7 CIOA knee joints (when synovitis and IL-1β levels are highest) caused a strong influx of immune cells into the joint cavity shortly after injection (6 hours), which had largely vanished after 24 hours. The attraction of particularly PMN-like cells was confirmed by immunohistochemistry. Synovial gene expression of neutrophil attracting chemokines KC, CXCL5, and CXCL7 were increased. In line with this, IL-1β stimulated ASCs injected in naive knee joints also resulted in a strong influx of PMN-like cells. IL-1β and IFN-γ (as a positive control) stimulation of ASCs in vitro strongly increased gene expression of KC, CXCL5, and CXCL7 as well as protein levels of KC. Finally, we co-cultured ASCs with BM-PMNs in the presence of IL-1β or IFN-γ. After 3 hours, a clear clustering of neutrophils around ASCs was observed which significantly diminished protein levels of KC (-69% after 24h; -76% after 48h). Conclusions Local injection of ASCs into a day 7 CIOA knee-joint expressing low levels of IL-1β causes attraction of PMN-like cells. In vitro, IL-1β stimulated ASCs show an increase in chemokine expression, leading to attraction of and clustering with neutrophils which ultimately results in significantly decreased levels of pro-inflammatory factors like KC. The anti-inflammatory effect of locally applied ASCs into OA joints showing synovitis may be triggered by IL-1β and attraction of PMN-like cells. References ter Huurne M et al. Arthritis Rheum. 64(11):3604-13, 2012. Schelbergen R et al. Osteoarthritis Cartilage. 22(8):1158-66, 2014. Acknowledgements This research was supported by the Dutch Arthritis Association (RF 12-2-405). Disclosure of Interest None declared