Background: The development of novel treatment strategies of immune-mediated inflammatory arthritis (IMIA) is still a clinical unmet need. The mitogen-activated protein kinase (MAPK) pathway is activated by environmental stressors, growth factors and inflammatory cytokines. However, the inhibition of central MAPK proteins has so far had undesirable side effects. The MAPK-activated protein kinase 2 (MK2) is a downstream mediator in the MAPK signaling pathway.Objectives: The objective of this study was to explore the effects of a small molecule inhibiting MK2 on synovial fluid mononuclear cells from patients with IMIA.Methods: Synovial fluid mononuclear cells (SFMCs) were obtained from a study population consisting of patients with active rheumatoid arthritis (RA), peripheral spondyloarthritis (SpA) or psoriatic arthritis (PsA) with at least one swollen joint (for obtaining synovial fluid) (n = 11). SFMCs were cultured for 48 h with and without the MK2 inhibitor CC0786512 at 1000 nM, 333 nM and 111 nM and cell free supernatants were harvested and frozen before they were analyzed by the Olink proseek multiplex interferon panel. Results: In SFMCs cultured for 48 h, the MK2 inhibitor decreased the production of chemokine (C-X-C motif) ligand 9 (CXCL9) (P < 0.001), CXCL10 (P < 0.01), hepatocyte growth factor (HGF) (P < 0.01), CXCL11 (P < 0.01), tumor necrosis factor-like weak inducer of apoptosis (TWEAK) (P < 0.05), and interleukin 12B (IL-12B) (P < 0.05) and increased the production of CXCL5 (P < 0.0001), CXCL1 (P < 0.0001), CXCL6 (P < 0.001), transforming growth factor alpha (TGF alpha) (P = 0.01), monocyte-chemotactic protein 3 (MCP-3) (P < 0.01), latency-associated peptide (LAP) TGF beta (P < 0.05) dose-dependently. Conclusions: This study reveals the downstream effects of MK2 inhibition on the secretory profile of SFMCs. Specifically, C-X-C motif chemokine receptors 3 (CXCR3) chemokines were decreased and CXCR2 chemokines were increased. This shift in the chemokine milieu may be one of the mechanisms behind the anti-inflammatory effects of MK2 inhibitors.

Abstract Objective During treatment with immune checkpoint inhibitors (ICI) such as the anti-PD-1 antibody pembrolizumab, half of patients with pre-existing inflammatory arthritis experience disease flares. The underlying immunological mechanisms have not been characterized. Here, we investigate the effect of pembrolizumab on cells involved in inflammation and destruction in the synovial joint and how immunosuppressive treatments affect the pembrolizumab-induced immune reactions. Methods We included synovial fluid mononuclear cells (SFMCs, n = 28) and peripheral blood mononuclear cells (PBMCs, n = 6) from patients with rheumatoid arthritis and peripheral spondyloarthritis and PBMCs from healthy controls (n = 6). Fibroblast-like synovial cells (FLSs) were grown from SFMCs. The in vitro effect of pembrolizumab was tested in SFMCs cultured for 48 h, FLS-PBMC co-cultures and in SFMCs cultured for 21 days (inflammatory osteoclastogenesis). Cells and supernatants were analyzed by ELISA, flow cytometry, and pro-inflammatory multiplex assay. Finally, the effect of the disease-modifying anti-rheumatic drugs (DMARDs) adalimumab (TNFα inhibitor), tocilizumab (IL-6R inhibitor), tofacitinib (JAK1/JAK3 inhibitor), and baricitinib (JAK1/JAK2 inhibitor) on pembrolizumab-induced immune reactions was tested. Results Pembrolizumab significantly increased monocyte chemoattractant protein-1 (MCP-1) production by arthritis SFMCs (P = 0.0031) but not by PBMCs from patients or healthy controls (P = 0.77 and P = 0.43). Pembrolizumab did not alter MMP-3 production in FLS-PBMC co-cultures (P = 0.76) or TRAP secretion in the inflammatory osteoclastogenesis model (P = 0.28). In SFMCs, pembrolizumab further increased the production of TNFα (P = 0.0110), IFNγ (P = 0.0125), IL-12p70 (P = 0.0014), IL-10 (P = 0.0100), IL-13 (P = 0.0044), IL-2 (P = 0.0066), and IL-4 (P = 0.0008) but did not change the production of IL-6 (P = 0.1938) and IL-1 (P = 0.1022). The SFMCs treated with pembrolizumab showed an increased frequency of intermediate monocytes (P = 0.044), and the MCP-1 production increased only within the intermediate monocyte subset (P = 0.028). Lastly, adalimumab, baricitinib, and tofacitinib treatment were able to attenuate the pembrolizumab-induced MCP-1 production (P = 0.0004, P = 0.033, and P = 0.025, respectively), while this was not seen with tocilizumab treatment (P = 0.75). Conclusion Pembrolizumab specifically activated intermediate monocytes and induced the production of several cytokines including TNFα but not IL-6. These findings indicate that flares in patients with pre-existing inflammatory arthritis involve monocyte activation and could be managed with TNFα neutralization.

INTRODUCTION:Immune checkpoint inhibitor-induced inflammatory arthritis (ICI-IA) is a relatively new disease entity caused by ICI agents during cancer therapy. Reactive arthritis (ReA) is a well-known disease entity caused by urogenital or gastrointestinal bacterial infection or pneumonia. In this sense, ICI-IA and ReA are both defined by a reaction to a well-specified causal event. As a result, comparing these diseases may help to determine therapeutic strategies.METHODS:We compared ICI-IA and ReA with special focus on pharmacological management. Specifically regarding treatment, we conducted a literature search of studies published in the PubMed database. Inclusion criteria were studies on treatment with non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GC), or disease modifying antirheumatic drugs (DMARDs) in ICI-IA or ReA. During systematic selection, 21 studies evaluating ICI-IA and 14 studies evaluating ReA were included.RESULTS:In ICI-IA, prospective and retrospective studies have shown effects of non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoid (GC), sulfasalazine (SSZ), methotrexate (MTX), hydroxychloroquine (HCQ) and TNFi. In ReA, retrospective studies evaluated NSAIDs and GC. A randomized controlled trial reported the effect of SSZ, and a retrospective study reported the effect of MTX and SSZ in combination with tumor necrosis factor alpha inhibition (TNFi). For both entities, small case reports show treatment effects of interleukin 6 receptor inhibition (IL-6Ri).DISCUSSION:This literature review identified both similarities and differences regarding the pathogenesis and clinical features of ReA and ICI-IA. Studies on treatment reported effectiveness of NSAIDs, GC, MTX, SSZ and TNFi in both diseases. Further, small case reports showed effects of IL-6Ri.

Background: During cancer treatment with immune checkpoint inhibitors (ICI) such as the anti-PD-1 antibody pembrolizumab, 2-4% of patients develop inflammatory arthritis as an immune related adverse event and half of patients with pre-existing inflammatory arthritis have disease flares. This type of adverse events shows striking similarities with traditional immune mediated inflammatory arthritis. However, the underlying immunological mechanisms of inflammatory arthritis associated with ICI are not fully understood. Objectives: We aimed to develop an in vitro model of inflammatory arthritis associated with ICI, and to use this model to investigate monocyte differentiation and activation following treatment with pembrolizumab. Methods: First, synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from patients with immune mediated inflammatory arthritis (rheumatoid arthritis and peripheral spondyloarthritis, n=22) and PBMCs from healthy controls were incubated with pembrolizumab and assessed for monocyte chemoattractant protein 1 (MCP-1) secretion by ELISA. Then, cytokine production in SFMCs was studied in more detail by the multiplex V-PLEX proinflammatory panel and by intracellular flow cytometry. Finally, pembrolizumab treated SFMCs were incubated with the different disease modifying anti-rheumatic drugs adalimumab, tocilizumab, tofacitinib, and baricitinib. Results: Pembrolizumab significantly increased MCP-1 production in the SFMCcultures (P=0.0031). In contrast, pembrolizumab did not change MCP-1 production by PBMCs from neither patients nor healthy controls (P=0.77 and P=0.43). Pembrolizumab also increased the production of TNFα (P=0.049), IFNγ (P=0.047), and IL-12p70 (P=0.031), but did not change the production of IL-6 (P=0.98). Among SFMCs treated with pembrolizumab there was an increased frequency of intermediate monocytes (P=0.044). Interestingly, pembrolizumab also increased the MCP-1 production within the intermediate monocytes only (P=0.028). In contrast, among SFMCs treated with LPS, only the classical monocyte subset was increased (P=0.0045) and MCP-1 production increased in both intermediate and classical monocyte subsets. Lastly, the TNFα inhibitor adalimumab and the Janus kinase inhibitors baricitinib and tofacitinib attenuated the pembrolizumab-induced MCP-1 production (P=0.0004, P=0.033, and P=0.025, respectively) while this was not seen with the IL-6 inhibitor tocilizumab (P=0.75). Conclusion: We have developed a very simple in vitro model of inflammatory arthritis associated with ICI. Using this model, we found that pembrolizumab specifically activated intermediate monocytes and induced TNFα, IFNγ, and IL-12p70 production, whereas IL-6 was unchanged. These findings were supported by effectful reduction of MCP-1 secretion with TNFα inhibition but not with IL-6 inhibition. This model could potentially be used to further study the effects of ICIs and the underlying immunological mechanisms of inflammatory arthritis associated with ICI. References: None. Disclosure of Interests: Anne Sofie Sørensen: None declared, Morten Nørgaard Andersen: None declared, Kristian Juul-Madsen: None declared, Cæcilie Skejø: None declared, Henrik Schmidt: None declared, Thomas Vorup-Jensen: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB.

Recently, we made the intriguing observation that peripheral blood mononuclear cells (PBMCs) from healthy donors, especially the monocytes, stained slightly positive for the macrophage mannose receptor (CD206). These experiments were performed using a mouse antihuman CD206 PE-Cy7 antibody (BioLegend, clone 15–2). However, it is well established in the literature that monocytes do not express CD206 (1–3). This discrepancy prompted us to perform additional experiments to further investigate the phenomenon. The results from a titration experiment, which initially raised our concern, showed CD206 staining of monocyte-derived macrophages (MDMs) (as expected), but also positive monocytes and lymphocytes (not expected) (Fig. 1A,B). Surprisingly, when repeating the experiment on PBMCs from the same donor using the same anti-CD206 clone (but APC conjugated, BioLegend), the results showed CD206 expression on MDMs, but not on PBMCs (Fig. 1C). Data from another experiment using another clone (anti-CD206 PE, clone 19–2, BD Biosciences) also showed CD206 expression on MDMs, but not on PBMCs (Fig. 1D). This led us to suspect non-specific antibody binding of the PE-Cy7 conjugated anti-CD206 antibody, which was not removed by Fc-receptor blocking with human IgG (100 μg/ml) (4). We investigated whether the phenomenon was caused by antibody aggregates, but centrifugation (5,000xg for 5 min) of the CD206 PE-Cy7 antibody before use did not alleviate the erroneously positive PBMCs (data not shown). Thus, we suspected that the PE-Cy7 fluorochrome was causing the issue. This was in agreement with a previous screening of all murine IgG1 monoclonal isotype controls available in our lab (n = 19) on PBMCs from one healthy donor (2 μg/ml, Supporting Information Table S1). Here, the term “isotype control” is used for murine monoclonal antibodies against a target irrelevant for human samples. This screening showed that all the IgG1 isotype controls displaying high level of non-specific binding to monocytes were conjugated to PE-Cyanine tandems (especially PE-Cy5 and PE-Cy7). Several other IgG1 isotype controls showed limited or no non-specific binding, for example, PerCP-Cy5.5, AlexaFluor 647 (AF647) and BV510, and one PE-Cyanine tandem (PC5, isotype 11) (Supporting Information Fig. S1). Non-specific binding is a well-known challenge in flow cytometry, especially when analyzing myeloid cells. Cyanine acceptors in tandem dyes including PE-Cy5, PE-Cy7, PerCP-Cy5.5, and APC-Cy7 have been shown to bind to monocytes and macrophages (5–7). Studies indicate that this non-specific binding is, at least in part, due to binding to the human high-affinity Fc receptor CD64 (5–7). Importantly, phosphorothioate-oligodeoxynucleotides could block this non-specific binding (5–7), but the mechanism has not been fully understood. Reviews focusing on pitfalls in flow cytometry rarely mention the issue (8,9), even though BioLegend has demonstrated non-specific staining of PE-Cy7, PerCP-Cy5.5, APC-Cy7, PE-Cy5, PE-Dazzle594, and APC-Fire750 conjugated antibodies to monocytes, which could be eliminated by their True-Stain Monocyte Blocker (10). When reviewing all flow cytometry papers published in Cytometry Part A during the past 5 years (2015–2019), excluding nonrelevant papers such as CyTOF studies and non-human cell-based work, we found that of the 74 identified papers, 59 (80%) used at least one Cyanine-tandem conjugate, and 52 (70%) used at least one PE-tandem. Further, of all 74 papers only 15 (20%) used a blocking reagent, which in all cases were Fc receptor blocking reagents, whereas none of the papers mentioned blocking of non-specific binding caused by tandem fluorochromes. Received: 22 September 2020 Revised: 17 November 2020 Accepted: 20 November 2020

This letter describes the potential effect of B cell depletion on immune related adverse events associated with immune checkpoint inhibition. B cell depleting agents such as rituximab reduce B cell to plasma cell differentiation and antibody production. This treatment strategy is used in several immune mediated inflammatory diseases such as rheumatoid arthritis and small vessel vasculitis. The immune related adverse events associated with immune checkpoint inhibition resemble immune mediated inflammatory diseases. Here, we report a lower incidence of hypothyroidism in a trial of combined B cell depletion and immune checkpoint inhibitor treatment compared with studies of immune checkpoint inhibitor monotherapy. This letter aims to increase awareness of the immune related adverse events associated with immune checkpoint inhibition in future clinical trials of immune checkpoint inhibition together with B cell depletion (primarily trials of B cell lymphomas). Hopefully, observations from these clinical trials can guide future treatment strategies to treat or prevent immune related adverse events associated with immune checkpoint inhibition.