Lucy Victoria Norling

The cellular mechanisms of omega-3 polyunsaturated fatty acid (ω3 PUFA) supplementation were investigated in inflammatory arthritis. Mice fed a prophylactic ω3 PUFA-enriched diet displayed reduced arthritis and paw oedema, with a discrete regulation of innate and stromal cell phenotypes in arthritic joints. Non-classical monocytes and VCAM-1 ⁺ synovial fibroblasts were lower at peak arthritis while pro-resolution MerTK ⁺ CD206 ⁺ macrophages were increased at resolution onset. Lipid mediator profiling of arthritic paws identified a distinct repertoire of molecules in ω3-fed mice, with elevated EPA-derived resolvin E2 (RvE2) and RvE4, compared with increased TXB ₂ and PGF _2α with a normal diet. Pharmacological treatment of mice with RvE2 and RvE4 concomitant with arthritis induction, attenuated arthritis and increased MerTK ⁺ CD206 ⁺ macrophages in arthritic joints. Synovial explant treatment revealed reduced pro-inflammatory mediator and enzyme release including IL-6, CCL2 and MMP-9. In arthritic patient blood, RvE2 and RvE4 (100 nM) lowered monocyte ICAM-1 expression. Collectively, these data identify cellular and molecular determinants of an ω3 diet in pre-clinical and human settings, highlighting therapeutic potential.

Rheumatoid arthritis (RA) is associated with heart and lung dysfunction. Current therapies fail to attenuate such complications. Here, we identify formyl-peptide receptor type 2 (FPR2) as a therapeutic target to treat heart and lung dysfunction associated with inflammatory arthritis. Arthritic mice on high levels of dietary homocysteine develop cardiac diastolic dysfunction and reduced lung compliance, mirroring two comorbidities in RA. Therapeutic administration of a small molecule FPR2 agonist (BMS986235) to hyper-homocysteine arthritic mice prevented diastolic dysfunction (monitored by echocardiography) and restored lung compliance. These tissue-specific effects were secondary to reduced neutrophil infiltration, modulation of fibroblast activation and phenotype (in the heart) and attenuation of monocyte and macrophage numbers (in the lung). A dual FPR1/2 agonist (compound 43) failed to prevent the reduction in lung compliance of arthritic mice and promoted the accumulation of inflammatory monocytes and pro-fibrotic macrophages in lung parenchyma. This cellular response lies downstream of FPR1-mediated potentiation of CCL2-dependent monocyte chemotaxis and activation. This finding supports the therapeutic development of selective FPR2 agonists to mitigate two impactful comorbidities associated with inflammatory arthritides.

Rheumatoid arthritis (RA) is associated with heart and lung dysfunction. Current therapies fail to attenuate such complications. Here, we identify formyl-peptide receptor type 2 (FPR2) as therapeutic target to treat heart and lung dysfunction associated with inflammatory arthritis. Arthritic mice on high levels of dietary homocysteine develop cardiac diastolic dysfunction and reduced lung compliance, mirroring two comorbidities in RA. Therapeutic administration of a small molecule FPR2 agonist (BMS986235) to hyper-homocysteine arthritic mice prevented diastolic dysfunction (monitored by echocardiography) and restored lung compliance. These tissue-specific effects were secondary to reduced neutrophil infiltration, modulation of fibroblast activation and phenotype (in the heart) and attenuation of monocyte and macrophage numbers (in the lung). A dual FPR1/2 agonist (compound 43) failed to prevent the reduction in lung compliance of arthritic mice and promoted accumulation of inflammatory monocytes and pro-fibrotic macrophages in lung parenchyma. This cellular response lies downstream of FPR1-mediated potentiation of CCL2-dependent monocyte chemotaxis and activation. This finding supports therapeutic development of selective FPR2 agonists to mitigate two impactful comorbidities associated with inflammatory arthritides.

Objective: This study was undertaken to establish the potential therapeutic profile of neutrophil‐derived extracellular vesicles (EVs) in experimental inflammatory arthritis and associate pharmacological activity with specific EV components, focusing on microRNAs.Methods: Neutrophil EVs were administered intra‐articularly through a prophylactic or therapeutic protocol to male C57BL/6 mice undergoing serum‐transfer–induced inflammatory arthritis. Transcriptomic analysis of knees was performed on joints following EV administration, naive and arthritic mice (untreated; n = 4/group) and EV‐treated diseased mice (intra‐articular administration) with contralateral (vehicle‐treated; n = 8/group). Comparison of healthy donor and patients with rheumatoid arthritis (RA) neutrophil EVs was performed.Results: EVs afforded cartilage protection with an increase in collagen‐II and reduced collagen‐X expression within the joint. To gain mechanistic insights, RNA sequencing of the arthritic joints was conducted. A total of 5,231 genes were differentially expressed (P < 0.05), with 257 unique to EV treatment. EVs affected key regenerative pathways involved in joint development, including Wnt and Notch signaling. This wealth of genomic alteration prompted to identify microRNAs in EVs, 10 of which are associated with RA. As a proof of concept, we focused on miR‐455‐3p, which was detected in both healthy donor and RA EVs. EV addition to chondrocyte cultures elevated miR‐455‐3p and exerted anticatabolic effects upon interleukin‐1β stimulation; these effects were blocked by actinomycin or miR‐455‐3p antagomir.Conclusion: Neutrophils from patients with RA yielded EVs with composition, efficacy, and miR‐455‐3p content similar to those of healthy volunteers, suggesting that neutrophil EVs could be developed as an autologous treatment to protect and repair joint tissue of patients affected by inflammatory arthritides. image

Osteoarthritis (OA) is the most prevalent age-related degenerative disorder, which severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), and alterations in chondrocyte EVs during OA have previously been reported. Herein, we investigated factors influencing chondrocyte EV release and the functional role that these EVs exhibit. Both 2D and 3D models of culturing C28I/2 chondrocytes were used for generating chondrocyte EVs. We assessed the effect of these EVs on chondrogenic gene expression as well as their uptake by chondrocytes. Collectively, the data demonstrated that chondrocyte EVs are sequestered within the cartilage ECM and that a bi-directional relationship exists between chondrocyte EV release and changes in chondrogenic differentiation. Finally, we demonstrated that the uptake of chondrocyte EVs is at least partially dependent on β1-integrin. These results indicate that chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work.

Lucy V Norling, Dianne Cooper; Being old and female is an inflammatory combination, Journal of Leukocyte Biology, , qiad077, https://doi.org/10.1093/jleuko/qiad

Using a global formyl peptide receptor (Fpr) 2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony, bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome, and impaired bacterial clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 upregulation was absent in myeloid cells, and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages toward an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2-competent septic mice.

Background: Rapid and accurate new biomarkers to predict risk of cardiovascular disease (CVD) are essential. The utility of extracellular vesicles in predicting the CVD risk is postulated, yet it remains unknown whether their expression is altered in response to statin therapy. Methods: We performed in-vitro studies with human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (hVSMC), and conducted a nested case–control study (nCCS) in hypertensive patients (n = 40) randomized to either atorvastatin or placebo in the ASCOT-LLA. Cases had a major adverse cardiovascular event or death (MACE) during 3.5 years of follow-up (median) from the time of extracellular vesicle characterization while controls, matched for age and duration of treatment, remained event-free. Conditional logistic regression models determined the risk of MACE. Additionally, the relationship of extracellular vesicle levels with statin therapy was assessed. Results: Added to HUVEC, extracellular vesicles increased neutrophil recruitment, and to hVSMC, aggravated calcification and proliferation. In the nCCS, compared with controls, cases (i.e. with MACE) had preceding higher levels of CD14+ and CD14+/CD41+ extracellular vesicles (P = 0.009 and P = 0.012, respectively) and a significant reduction in the median size of the vesicles (P = 0.037). On matched analysis, higher CD14+ extracellular vesicles were associated with a 3.7-fold increased risk of MACE (P = 0.032). Patients treated with atorvastatin (vs. placebo) had both reduced size of extracellular vesicles and the proportion of CD146+ extracellular vesicles (P = 0.034 and P = 0.020, respectively). Conclusion and relevance: These pilot analyses suggest a mechanistic role for extracellular vesicles in the development of CVD, with significant and differential changes in extracellular vesicles amongst those at risk of MACE, and those on atorvastatin therapy.