Stimulator of IFN Genes (STING) is a cytosolic DNA sensor that plays a central role in host protection against pathogens upon binding of DNA-derived ligands. STING primarily acts by controlling the transcription of type I interferons (IFNs) and pro-inflammatory cytokines. Notably, STING can be inhibited or activated pharmacologically to control STING-associated pathologies. 5, 6-Dimethylxanthenone-4-acetic Acid (DMXAA) is a pharmacological activator of murine STING that induces IFN-β and its affected genes. Here, we report that macrophages from DMXAA-treated mice engulfed significantly higher numbers of apoptotic cells ex vivo, and exhibited enhanced reprogramming reflected by an increased IL-10 and reduced inflammatory cytokine secretion upon LPS exposure. Macrophage reprogramming was significantly hampered in STING and IFN-β -deficient macrophages. Furthermore, we used virtual docking and batch screening to identify biased STING agonists (BiSTs) that enhanced IL-10 and IFN-β production by splenocytes while inhibiting TNFα. One of these compounds, termed BiST 2.1, also induced the murine STING pathway in vivo and human macrophages. Finally, we found BiST 2.1 to enhance the resolution of liver fibrosis induced by CCl4. Thus, our findings indicate that STING can be harnessed to drive IFN-β-mediated IL-10 secretion by resolution phase macrophages and consequently shape macrophage function to enhance the resolution of inflammation and treat fibrotic disorders.

Purpose: The adiponectin-PEPITEM pathway negatively regulates leukocyte infiltration into inflamed tissues, but this immunomodulation is lost in patients with established treated RA. Here we investigate the status of the adiponectin-PEPITEM pathway in early treatment naïve arthritis patients and whether replacing it may have clinical benefit. Methods: Adiponectin receptors were measured by qPCR or flow cytometry on peripheral blood lymphocytes (PBLs) from healthy donors or patients presenting to early arthritis clinics with undifferentiated arthritis or treatment naïve RA. Mice with collagen-induced (CIA), antigen-induced (AIA) or gouty arthritis where treated daily with PEPITEM or vehicle control. Disease severity and joint swelling were assessed daily. Joints were digested and assessed by flow cytometry to determine leukocyte infiltration or by scRNAseq of sorted CD45+ cells, ELISA or western blot analysis for gene or protein expression. Results: Adiponectin receptor gene and protein is significantly reduced in leukocytes from patients with untreated RA compared to patients with resolving arthritis or healthy controls. In vivo, PEPITEM significantly reduced disease severity, joint swelling and leukocyte infiltration (including T-cells and monocyte/macrophages) in CIA, AIA and gouty arthritis models, when compared to vehicle treated mice. Using the inflamed AIA joints, scRNAseq analysis revealed notable decrease in genes linked with chemokine activity and cell chemotaxis in M1-like macrophages in PEPITEM treated animals compared to vehicle controls, whilst the converse was true for the M2-like macrophage cells. These data were confirmed at protein level, where significant reductions in several pro-inflammatory cyto/chemokines (e.g., JE (CCL2), KC (IL-8), TNF, IL-6) were seen in the joints of PEPITEM treated mice compared to controls. Moreover, significant modulation of COX2 and NF-B signalling pathways were also detected in animals treated with PEPITEM. In vitro, PEPITEM significantly reduced TNF and IL-6 production by macrophages. Conclusions: Patients with RA with defects in the adiponectin-PEPITEM pathway would potentially benefit from PEPITEM replacement therapy. Preclinical murine studies demonstrate PEPITEM modulates leukocyte trafficking within inflamed joints by switching the synovium microenvironment from a pro-inflammatory to a more pro-resolution state. Thus, PEPITEM may offer an alternative therapy alone or in combination with other biologics for early RA patients to reset the synovial microenvironment.

Rheumatoid arthritis (RA) patients face a two-fold increased risk of cardiovascular diseases, including heart failure [Lancet 2022; 400: 733-43]. Notably, they are particularly prone to heart failure with preserved ejection fraction, marked by diastolic dysfunction. The causes of diastolic dysfunction in RA remain unknown, while existing medications provide limited cardioprotection. Addressing this clinical gap, we characterised the first mouse model mirroring left ventricular diastolic dysfunction in RA patients, the K/BxN F1 mice [PNAS 2021; 118(38):e2020385118], which present diastolic dysfunction ~4 weeks after development of arthritis. Our focus centered on the annexin A1 (AnxA1)/formyl-peptide receptor type 2 (FPR2) pro-resolving pathway. Echocardiography-monitored cardiac diastolic dysfunction correlated with immune cell infiltration and fibroblast proliferation. Prophylactic or therapeutic treatment with 1 µg s.c. hrAnxA1 daily halted or reversed cardiomyopathy, respectively. These effects correlated with decreased fibroblast numbers and activation, and macrophage skewing toward an M2-like polarisation. Given that both Fpr1 and Fpr2 mRNA expression are elevated in K/BxN F1 mouse hearts, we tested here relevant agonists, addressing also if selective Fpr2 agonism adds value as compared to a dual Fpr1/Fpr2 agonist. To this end, we tested two small molecules: BMS986235 (Fpr2 selective) and Compound43 (dual Fpr1/Fpr2 agonist) at 3 mg/kg and 10 mg/kg per os, daily from week 4. Both compounds prevented cardiac dysfunction (monitored from week 4 to 8) but targeted distinct cellular types (quantified at end of the experiment). BMS986235 reduced activated T cells, M1-like macrophage proportion, and fibroblast numbers. Conversely, Compound43 reduced galectin-3 expression in fibroblasts mitigating pro-fibrotic activity, and decreased pro-inflammatory monocyte and macrophage infiltration. Neither treatment impacted on the degree of arthritis. In summary, these findings suggest a potential for FPR-based pharmacological interventions in treating inflammatory arthritis-associated cardiomyopathy.

Background: Cancer cachexia is a devastating syndrome characterized by progressive muscle wasting and hyperinflammation. While the underlying mechanisms remain poorly characterized, there are no effective treatments for cancer cachexia. Cancer cachexia is driven by systemic inflammation and pro-inflammatory cytokines. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are anti-inflammatory lipid mediators which stimulating inflammation resolution. EETs are rapidly metabolized by the enzyme soluble epoxide hydrolase (sEH). Thus, we hypothesized that pharmacological inhibition of the sEH may prevent cancer cachexia via the resolution of inflammation. Methods: We investigated murine cancer cachexia models using genetically engineered pancreatic (KPC) and prostate (TRAMP C1) tumor cell lines, and genetically engineered mice (transgenic adenocarcinoma of the mouse prostate-TRAMP). Cachectic mice were treated with human sEH inhibitor EC5026. Results: An increase in sEH expression was observed in the spleen, liver, heart, gastrocnemius (GA), and tibialis anterior (TA) muscles compared to healthy controls by IHC and qPCR (n = 5/group). KPC cells injected intraperitoneally induced a reduction in the weights of the spleen, liver, heart, brain, GA, and TA compared to healthy controls (n = 15/group). KPC mice treated with EC5026 showed increased and comparable organ weights to healthy controls. In addition, treatment with EC5026 significantly improved survival rates in both KPC and TRAMP models. A sustained survival of over 250 days was observed post-injection in the KPC model (n=15 mice/group). In the TRAMP model, 5/5 of mice treated with EC5026 survived 230 days post-treatment compared to no survival of vehicle-treated mice (n=5). Mice treated with EC5026 showed a reduction in sEH expression in all tissues. Additionally, qPCR analysis of gastrocnemius tissue indicated that sEH inhibition attenuated pro-inflammatory markers and eicosanoid enzymes, evidenced by reduced expression of IL-6, NF-kB, LTB4R, Cox-2, Alox5, Alox12, Cyp2j5, and Cyp2c65 comparatively (n=5) and increased pro-resolving receptors (e.g. RvD2/GPR18). Immunoprofiling by flow cytometry (n=15/group) revealed a significant increase in macrophages, CD8+ T cells, and NK cells, and a decrease in CD4+ T cells in EC5026-treated KPC mice across various cachexia tissues. KPC mice treated with EC5026 expressed similar percentages of immune cell populations compared to healthy controls in each tissue type. Conclusions: Thus, sEH inhibition may be a novel host-directed therapeutic approach to cancer cachexia by targeting the host immune response via stimulation of resolution of inflammation without toxicity or immunosuppression.