Alessandra Nerviani

Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis

Neutrophils generate chrondroprotective AnxA1-containing microvesicles that enter immune cell–impenetrable cartilage. Microparticles provide protection Neutrophils play an active role in protecting cartilage from damage by dispatching microvesicles (MVs) to do their bidding in this tissue they otherwise can’t access. Headland and colleagues found MVs present in the synovial fluid of patients with rheumatoid arthritis—an autoimmune disease that degrades cartilage in the joints. Cartilage is normally thought of as impenetrable to cells, so neutrophils send MVs, which easily enter the tissue and prevent damage induced by disease through a complex mechanism that involves the proresolving protein annexin A1 and its receptor. In two different mouse models of rheumatoid arthritis, MVs delivered locally entered the cartilage, prevented the loss of proteoglycans, and maintained cartilage integrity. This study suggests that immune cells can provide protection against tissue degradation in inflammatory arthritis and that the MVs may be manipulated to deliver therapeutics to diseased joints. Microvesicles (MVs) are emerging as a new mechanism of intercellular communication by transferring cellular lipid and protein components to target cells, yet their function in disease is only now being explored. We found that neutrophil-derived MVs were increased in concentration in synovial fluid from rheumatoid arthritis patients compared to paired plasma. Synovial MVs overexpressed the proresolving, anti-inflammatory protein annexin A1 (AnxA1). Mice deficient in TMEM16F, a lipid scramblase required for microvesiculation, exhibited exacerbated cartilage damage when subjected to inflammatory arthritis. To determine the function of MVs in inflammatory arthritis, toward the possibility of MV-based therapeutics, we examined the role of immune cell–derived MVs in rodent models and in human primary chondrocytes. In vitro, exogenous neutrophil-derived AnxA1+ MVs activated anabolic gene expression in chondrocytes, leading to extracellular matrix accumulation and cartilage protection through the reduction in stress-adaptive homeostatic mediators interleukin-8 and prostaglandin E2. In vivo, intra-articular injection of AnxA1+ MV lessened cartilage degradation caused by inflammatory arthritis. Arthritic mice receiving adoptive transfer of whole neutrophils displayed abundant MVs within cartilage matrix and revealed that MVs, but not neutrophils themselves, can penetrate cartilage. Mechanistic studies support a model whereby MV-associated AnxA1 interacts with its receptor FPR2 (formyl peptide receptor 2)/ALX, increasing transforming growth factor–β production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs, either directly or loaded with therapeutics, can be harnessed as a unique therapeutic strategy for protection in diseases associated with cartilage degeneration.

Interleukin-27 inhibits ectopic lymphoid-like structure development in early inflammatory arthritis

Decreased interleukin-27 signaling in humans and mice induces the formation of ectopic lymphoid-like structures (ELSs), which are associated with severe disease pathology and resistance to biological therapy in rheumatoid arthritis patients. Increased numbers of podoplanin-expressing Th17 cells in the absence of IL-27R signaling may be involved in driving ELS formation.

Ectopic Lymphoid Structures: Powerhouse of Autoimmunity.

Ectopic lymphoid structures (ELS) often develop at sites of inflammation in target tissues of autoimmune diseases, such as rheumatoid arthritis, Sjögren’s syndrome, multiple sclerosis, myasthenia gravis, and systemic lupus erythematosus. ELS are characterized by the formation of organized T/B cells aggregates, which can acquire follicular dendritic cells network supporting an ectopic germinal center response. In this review, we shall summarize the mechanisms that regulate the formation of ELS in tertiary lymphoid organs, with particular emphasis on the role of lymphoid chemokines in both formation and maintenance of ELS, the role of emerging positive and negative regulators of ELS development and function, including T follicular helper cells and IL-27, respectively. Finally, we shall discuss the main functions of ELS in supporting the affinity maturation, clonal selection, and differentiation of autoreactive B cells contributing to the maintenance and perpetuation of humoral autoimmunity.

Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies

OBJECTIVES Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). METHODS For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. RESULTS Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients. CONCLUSION These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Mast cells in rheumatoid arthritis: friends or foes?

Mast cells are tissue-resident cells of the innate immunity, implicated in the pathogenesis of many autoimmune diseases, including rheumatoid arthritis (RA). They are present in synovia and their activation has been linked to the potentiation of inflammation in the course of RA. However, recent investigations questioned the role of mast cells in arthritis. In particular, animal models generated conflicting results, so that many of their pro-inflammatory, i.e. pro-arthritogenic functions, even though supported by robust experimental evidence, have been labelled as redundant. At the same time, a growing body of evidence suggests that mast cells can act as tunable immunomodulatory cells. These characteristics, not yet fully understood in the context of RA, could partially explain the inconsistent results obtained with experimental models, which do not account for the pro- and anti-inflammatory functions exerted in more chronic heterogeneous conditions such as RA. Here we present an overview of the current knowledge on mast cell involvement in RA, including the intriguing hypothesis of mast cells acting as subtle immunomodulatory cells and the emerging concept of synovial mast cells as potential biomarkers for patient stratification.

Superiority of a High Loading Dose of Cholecalciferol to Correct Hypovitaminosis D in Patients with Inflammatory/Autoimmune Rheumatic Diseases

Objective. To compare 3 different cholecalciferol supplementation regimens in patients with rheumatic diseases. Methods. One hundred fifty-four patients who completed a 6-month course of cholecalciferol supplementation, of whom 111 had an autoimmune/inflammatory rheumatic disease (ARD) and 43 osteoarthritis (NARD), were retrospectively identified from a database of 872 consecutive adult patients who attended a tertiary level immuno-rheumatology clinic from 2007 to 2010. Patients with renal failure or primary hyperparathyroidism were excluded. Plasma 25-hydroxy vitamin D [25(OH)D] and parathyroid hormone (PTH) concentrations were evaluated at baseline and after completion of treatment with (i) a single oral dose of cholecalciferol 300,000 IU, followed by oral cholecalciferol 800–1000 IU daily for 6 months [high-dose loading treatment (HLT) group; n = 40]; (ii) a single oral dose of cholecalciferol 100,000 IU, followed by daily oral cholecalciferol as above [low-dose loading treatment (LLT) group; n = 30]; or (iii) daily oral cholecalciferol as above but without the loading dose [standard therapy (ST); n = 84]. Results. The rates of serum 25(OH)D and PTH normalization (defined as values > 75 nmol/l and < 72.9 pg/ml, respectively) were as follows: HLT, 52.5% (95% CI 37.5–68.5) and 69.2% (95% CI 54.7–83.3); LLT, 36.7% (95% CI 19.7–54.3) and 53.8% (95% CI 36.2–71.8); ST, 31.0% (95% CI 21.1–40.9) and 35.0% (95% CI 14.1–55.9). All regimes increased 25(OH)D (p < 0.001) but only HLT reduced PTH (p < 0.01) in comparison to baseline. The ARD group had a similar 25(OH)D increase but a smaller PTH reduction than the NARD (p < 0.05). Conclusion. An HLT cholecalciferol regimen is needed to correct hypovitaminosis D of patients with rheumatic diseases, with superior 25(OH)D normalization and PTH suppression rates at 6 months.

Role of the IL-23/IL-17 Axis in Psoriasis and Psoriatic Arthritis: The Clinical Importance of Its Divergence in Skin and Joints

Psoriasis is a chronic systemic inflammatory disease causing erythematosus and scaly skin plaques; up to 30% of patients with psoriasis develop Psoriatic Arthritis (PsA), which is characterised by inflammation and progressive damage of the peripheral joints and/or the spine and/or the entheses. The pathogenic mechanisms driving the skin disorder in psoriasis and the joint disease in PsA are sustained by the activation of inflammatory pathways that can be overlapping, but also, at least partially, distinct. Cytokines members of the IL-23/IL-17 family, critical in the development of autoimmunity, are abundantly expressed within the cutaneous lesions but also seem to be involved in chronic inflammation and damage of the synovium though, as it will be here discussed, not in all patients. In this review, we will focus on the state of the art of the molecular features of psoriatic skin and joints, focusing on the specific role of the IL-23/IL-17 pathway in each of these anatomical districts. We will then offer an overview of the approved and in-development biologics targeting this axis, emphasising how the availability of the “target” in the diseased tissues could provide a plausible explanation for the heterogeneous clinical efficacy of these drugs, thus opening future perspective of personalised therapies.

Endothelial Dysfunction in Systemic Lupus Erythematosus: Pathogenesis, Assessment and Therapeutic Opportunities

BACKGROUND Systemic Lupus Erythematosus (SLE) is characterised by increased mortality secondary to Cardiovascular Diseases (CVD). Despite being common in SLE, traditional cardiovascular risk factors cannot entirely justify such increase in CVD-associated mortality. The endothelium is a key regulator of the vascular homeostasis; lupus-associated persistent systemic inflammation may impair endothelium functionality, thus initiating a cascade of events that, in concert with traditional CVD-risk factors, leads to atherosclerosis development and progression. Numerous methods have been used for the in vivo assessment of the endothelial function; among all, Flow- Mediated Dilatation (FMD) has been widely validated in clinical trials. Quantification of the endothelial dysfunction by FMD has been confirmed to be an early predictor of CVD in multiple studies involving both non-CVD and CVD-population and it may therefore represent a likewise efficient biomarker of CVD in SLE. METHODS Research and online content related to endothelial function in SLE is reviewed in this article with special attention to the pathophysiology and therapeutic opportunities. RESULTS To date, the vast majority of the available data, albeit not all, shows that endotheliumdependent FMD values are lower in SLE patients compared to healthy subjects; further studies, however, will be required in order to confirm the usefulness of the endothelial dysfunction quantification as CVD-predictor in the specific clinical setting of lupus. Notably, FMD variations can also be a sensitive marker for assessing specific therapeutic strategies ability of improving endothelial function in SLE patients. CONCLUSION Endothelial function appears to be affected by SLE potentially contributing to the increased cardiovascular risk observed in SLE patients.

Role of chemokines in ectopic lymphoid structures formation in autoimmunity and cancer

Ectopic (or tertiary) lymphoid structures (ELS) are organized aggregates of lymphocytes resembling secondary lymphoid organs and developing in chronically inflamed nonlymphoid tissues during persistent infections, graft rejection, autoimmune conditions, and cancer. In this review, we will first depict the mechanisms regulating ELS generation, focusing on the role played by lymphoid chemokines. We will then characterize ELS forming in target organs during autoimmune conditions, here exemplified by rheumatoid arthritis, and cancer, highlighting the relevance of the tissue‐specific factors. Finally, we will discuss the clinical significance of ELS and the therapeutic potential of their inhibition and/or enhancement depending on the disease considered.

Redox-Mediated Mechanisms Fuel Monocyte Responses to CXCL12/HMGB1 in Active Rheumatoid Arthritis

Chemokine synergy-inducing molecules are emerging as regulating factors in cell migration. The alarmin HMGB1, in its reduced form, can complex with CXCL12 enhancing its activity on monocytes via the chemokine receptor CXCR4, while the form containing a disulfide bond, by binding to TLR2 or TLR4, initiates a cascade of events leading to production of cytokines and chemokines. So far, the possibility that the CXCL12/HMGB1 heterocomplex could be maintained in chronic inflammation was debated, due to the release of reactive oxygen species. Therefore, we have assessed if the heterocomplex could remain active in Rheumatoid Arthritis (RA) and its relevance in the disease assessment. Monocytes from RA patients with active disease require a low concentration of HMGB1 to enhance CXCL12-induced migration, in comparison to monocytes from patients in clinical remission or healthy donors. The activity of the heterocomplex depends on disease activity, on the COX2 and JAK/STAT pathways, and is determined by the redox potential of the microenvironment. In RA, the presence of an active thioredoxin system correlates with the enhanced cell migration, and with the presence of the heterocomplex in the synovial fluid. The present study highlights how, in an unbalanced microenvironment, the activity of the thioredoxin system plays a crucial role in sustaining inflammation. Prostaglandin E2 stimulation of monocytes from healthy donors is sufficient to recapitulate the response observed in patients with active RA. The activation of mechanisms counteracting the oxidative stress in the extracellular compartment preserves HMGB1 in its reduced form, and contributes to fuel the influx of inflammatory cells. Targeting the heterocomplex formation and its activity could thus be an additional tool for dampening the inflammation sustained by cell recruitment, for those patients with chronic inflammatory conditions who poorly respond to current therapies.