Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Polymyalgia rheumatica shows metabolomic alterations that are further altered by glucocorticoid treatment: Identification of metabolic correlates of fatigue.

OBJECTIVE: In polymyalgia rheumatica (PMR), glucocorticoids (GCs) relieve pain and stiffness, but fatigue may persist. We aimed to explore the effect of disease, GCs and PMR symptoms in the metabolite signatures of peripheral blood from patients with PMR or the related disease, giant cell arteritis (GCA). METHODS: Nuclear magnetic resonance spectroscopy was performed on serum from 40 patients with untreated PMR, 84 with new-onset confirmed GCA, and 53 with suspected GCA who later were clinically confirmed non-GCA, and 39 age-matched controls. Further samples from PMR patients were taken one and six months into glucocorticoid therapy to explore relationship of metabolites to persistent fatigue. 100 metabolites were identified using Chenomx and statistical analysis performed in SIMCA-P to examine the relationship between metabolic profiles and, disease, GC treatment or symptoms. RESULTS: The metabolite signature of patients with PMR and GCA differed from that of age-matched non-inflammatory controls (R2 > 0.7). There was a smaller separation between patients with clinically confirmed GCA and those with suspected GCA who later were clinically confirmed non-GCA (R2 = 0.135). In PMR, metabolite signatures were further altered with glucocorticoid treatment (R2 = 0.42) but did not return to that seen in controls. Metabolites correlated with CRP, pain, stiffness, and fatigue (R2 ≥ 0.39). CRP, pain, and stiffness declined with treatment and were associated with 3-hydroxybutyrate and acetoacetate, but fatigue did not. Metabolites differentiated patients with high and low fatigue both before and after treatment (R2 > 0.9). Low serum glutamine was predictive of high fatigue at both time points (0.79-fold change). CONCLUSION: PMR and GCA alter the metabolite signature. In PMR, this is further altered by glucocorticoid therapy. Treatment-induced metabolite changes were linked to measures of inflammation (CRP, pain and stiffness), but not to fatigue. Furthermore, metabolite signatures distinguished patients with high or low fatigue.

The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse.

The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.

PEPITEM modulates leukocyte trafficking to reduce obesity-induced inflammation.

Dysregulation of leukocyte trafficking, lipid metabolism, and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in lifestyle choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B cells were observed in the secondary lymphoid tissues (e.g. spleen and inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimize the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through lifestyle modifications.

New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function.

OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

The Ying and Yang of Sphingosine-1-Phosphate Signalling within the Bone.

Bone remodelling is a highly active and dynamic process that involves the tight regulation of osteoblasts, osteoclasts, and their progenitors to allow for a balance of bone resorption and formation to be maintained. Ageing and inflammation are risk factors for the dysregulation of bone remodelling. Once the balance between bone formation and resorption is lost, bone mass becomes compromised, resulting in disorders such as osteoporosis and Paget's disease. Key molecules in the sphingosine-1-phosphate signalling pathway have been identified for their role in regulating bone remodelling, in addition to its more recognised role in inflammatory responses. This review discusses the accumulating evidence for the different, and, in certain circumstances, opposing, roles of S1P in bone homeostasis and disease, including osteoporosis, Paget's disease, and inflammatory bone loss. Specifically, we describe the current, often conflicting, evidence surrounding S1P function in osteoblasts, osteoclasts, and their precursors in health and disease, concluding that S1P may be an effective biomarker of bone disease and also an attractive therapeutic target for disease.

Non-pharmacological interventions for vascular health and the role of the endothelium.

The most common non-pharmacological intervention for both peripheral and cerebral vascular health is regular physical activity (e.g., exercise training), which improves function across a range of exercise intensities and modalities. Numerous non-exercising approaches have also been suggested to improved vascular function, including repeated ischemic preconditioning (IPC); heat therapy such as hot water bathing and sauna; and pneumatic compression. Chronic adaptive responses have been observed across a number of these approaches, yet the precise mechanisms that underlie these effects in humans are not fully understood. Acute increases in blood flow and circulating signalling factors that induce responses in endothelial function are likely to be key moderators driving these adaptations. While the impact on circulating factors and environmental mechanisms for adaptation may vary between approaches, in essence, they all centre around acutely elevating blood flow throughout the circulation and stimulating improved endothelium-dependent vascular function and ultimately vascular health. Here, we review our current understanding of the mechanisms driving endothelial adaptation to repeated exposure to elevated blood flow, and the interplay between this response and changes in circulating factors. In addition, we will consider the limitations in our current knowledge base and how these may be best addressed through the selection of more physiologically relevant experimental models and research. Ultimately, improving our understanding of the unique impact that non-pharmacological interventions have on the vasculature will allow us to develop superior strategies to tackle declining vascular function across the lifespan, prevent avoidable vascular-related disease, and alleviate dependency on drug-based interventions.

Podoplanin regulates the migration of mesenchymal stromal cells and their interaction with platelets.

Mesenchymal stromal cells (MSCs) upregulate podoplanin at sites of infection, chronic inflammation and cancer. Here, we investigated the functional consequences of podoplanin expression on the migratory potential of MSCs and their interactions with circulating platelets. Expression of podoplanin significantly enhanced the migration of MSCs compared to MSCs lacking podoplanin. Rac-1 inhibition altered the membrane localisation of podoplanin and in turn significantly reduced MSC migration. Blocking Rac-1 activity had no effect on the migration of MSCs lacking podoplanin, indicating that it was responsible for regulation of migration through podoplanin. When podoplanin-expressing MSCs were seeded on the basal surface of a porous filter, they were able to capture platelets perfused over the uncoated apical surface and induce platelet aggregation. Similar microthrombi were observed when endothelial cells (ECs) were co-cultured on the apical surface. Confocal imaging shows podoplanin-expressing MSCs extending processes into the EC layer, and these processes could interact with circulating platelets. In both models, platelet aggregation induced by podoplanin-expressing MSCs was inhibited by treatment with recombinant soluble C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b). Thus, podoplanin may enhance the migratory capacity of tissue-resident MSCs and enable novel interactions with cells expressing CLEC-2.

Fine wine or sour grapes? A systematic review and meta-analysis of the impact of red wine polyphenols on vascular health.

PURPOSE: Red wine polyphenols (RWP) are plant-based molecules that have been extensively studied in relation to their protective effects on vascular health in both animals and humans. The aim of this review was to quantify and compare the efficacy of RWP and pure resveratrol on outcomes measures of vascular health and function in both animals and humans. METHODS: Comprehensive database searches were carried out through PubMed, Web of Science and OVID for randomised, placebo-controlled studies in both animals and humans. Meta-analyses were carried out on acute and chronic studies of RWP in humans, alongside sub-group analysis where possible. Risk-of-bias assessment was carried out for all included studies based on randomisation, allocation, blinding, outcome data reporting, and other biases. RESULTS: 48 animal and 37 human studies were included in data extraction following screening. Significant improvements in measures of blood pressure and vascular function following RWP were seen in 84% and 100% of animal studies, respectively. Human studies indicated significant improvements in systolic blood pressure overall (- 2.6 mmHg, 95% CI: [- 4.8, - 0.4]), with a greater improvement in pure-resveratrol studies alone (- 3.7 mmHg, 95% CI: [- 7.3, - 0.0]). No significant effects of RWP were seen in diastolic blood pressure or flow-mediated dilation (FMD) of the brachial artery. CONCLUSION: RWP have the potential to improve vascular health in at risk human populations, particularly in regard to lowering systolic blood pressure; however, such benefits are not as prevalent as those observed in animal models.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

Monocyte Subsets Coregulate Inflammatory Responses by Integrated Signaling through TNF and IL-6 at the Endothelial Cell Interface.

Two major monocyte subsets, CD14+CD16- (classical) and CD14+/dimCD16+ (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6hi) was added to nonclassical/intermediate monocyte cocultures (TNFhi), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes.

Adipogenic Differentiation of Mesenchymal Stem Cells Alters Their Immunomodulatory Properties in a Tissue-Specific Manner.

Chronic inflammation is associated with formation of ectopic fat deposits that might represent damage-induced aberrant mesenchymal stem cell (MSC) differentiation. Such deposits are associated with increased levels of inflammatory infiltrate and poor prognosis. Here we tested the hypothesis that differentiation from MSC to adipocytes in inflamed tissue might contribute to chronicity through loss of immunomodulatory function. We assessed the effects of adipogenic differentiation of MSC isolated from bone marrow or adipose tissue on their capacity to regulate neutrophil recruitment by endothelial cells and compared the differentiated cells to primary adipocytes from adipose tissue. Bone marrow derived MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated endothelial cells (EC), but MSC-derived adipocytes were no longer able to suppress neutrophil adhesion. Changes in IL-6 and TGFß1 signalling appeared critical for the loss of the immunosuppressive phenotype. In contrast, native stromal cells, adipocytes derived from them, and mature adipocytes from adipose tissue were all immunoprotective. Thus disruption of normal tissue stroma homeostasis, as occurs in chronic inflammatory diseases, might drive "abnormal" adipogenesis which adversely influences the behavior of MSC and contributes to pathogenic recruitment of leukocytes. Interestingly, stromal cells programmed in native fat tissue retain an immunoprotective phenotype. Stem Cells 2017;35:1636-1646.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1ß2 in Newly Formed Tertiary Lymphoid Structures.

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)ßR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1ß2/LTßR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.

Mesenchymal Stem Cells as Endogenous Regulators of Inflammation.

This chapter discusses the regulatory role of endogenous mesenchymal stem cells (MSC) during an inflammatory response. MSC are a heterogeneous population of multipotent cells that normally contribute towards tissue maintenance and repair but have garnered significant scientific interest for their potent immunomodulatory potential. It is through these physicochemical interactions that MSC are able to exert an anti-inflammatory response on neighbouring stromal and haematopoietic cells. However, the impact of the chronic inflammatory environment on MSC function remains to be determined. Understanding the relationship of MSC between resolution of inflammation and autoimmunity will both offer new insights in the use of MSC as a therapeutic, and also their involvement in the pathogenesis of inflammatory disorders.

Identification of a transitional fibroblast function in very early rheumatoid arthritis.

OBJECTIVES: Synovial fibroblasts actively regulate the inflammatory infiltrate by communicating with neighbouring endothelial cells (EC). Surprisingly, little is known about how the development of rheumatoid arthritis (RA) alters these immunomodulatory properties. We examined the effects of phase of RA and disease outcome (resolving vs persistence) on fibroblast crosstalk with EC and regulation of lymphocyte recruitment. METHODS: Fibroblasts were isolated from patients without synovitis, with resolving arthritis, very early RA (VeRA; symptom ≤12 weeks) and established RA undergoing joint replacement (JRep) surgery. Endothelial-fibroblast cocultures were formed on opposite sides of porous filters. Lymphocyte adhesion from flow, secretion of soluble mediators and interleukin 6 (IL-6) signalling were assessed. RESULTS: Fibroblasts from non-inflamed and resolving arthritis were immunosuppressive, inhibiting lymphocyte recruitment to cytokine-treated endothelium. This effect was lost very early in the development of RA, such that fibroblasts no longer suppressed recruitment. Changes in IL-6 and transforming growth factor beta 1 (TGF-ß1) signalling appeared critical for the loss of the immunosuppressive phenotype. In the absence of exogenous cytokines, JRep, but not VeRA, fibroblasts activated endothelium to support lymphocyte. CONCLUSIONS: In RA, fibroblasts undergo two distinct changes in function: first a loss of immunosuppressive responses early in disease development, followed by the later acquisition of a stimulatory phenotype. Fibroblasts exhibit a transitional functional phenotype during the first 3 months of symptoms that contributes to the accumulation of persistent infiltrates. Finally, the role of IL-6 and TGF-ß1 changes from immunosuppressive in resolving arthritis to stimulatory very early in the development of RA. Early interventions targeting 'pathogenic' fibroblasts may be required in order to restore protective regulatory processes.

Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk.

Stromal cells actively modulate the inflammatory process, in part by influencing the ability of neighboring endothelial cells to support the recruitment of circulating leukocytes. We hypothesized that podocytes influence the ability of glomerular endothelial cells (GEnCs) to recruit neutrophils during inflammation. To address this, human podocytes and human GEnCs were cultured on opposite sides of porous inserts and then treated with or without increasing concentrations of TNF-α prior to addition of neutrophils. The presence of podocytes significantly reduced neutrophil recruitment to GEnCs by up to 50% when cultures were treated with high-dose TNF-α (100 U/ml), when compared with GEnC monocultures. Importantly, this phenomenon was dependent on paracrine actions of soluble IL-6, predominantly released by podocytes. A similar response was absent when HUVECs were cocultured with podocytes, indicating a tissue-specific phenomenon. Suppressor of cytokine signaling 3 elicited the immunosuppressive actions of IL-6 in a process that disrupted the presentation of chemokines on GEnCs by altering the expression of the duffy Ag receptor for chemokines. Interestingly, suppressor of cytokine signaling 3 knockdown in GEnCs upregulated duffy Ag receptor for chemokines and CXCL5 expression, thereby restoring the neutrophil recruitment. In summary, these studies reveal that podocytes can negatively regulate neutrophil recruitment to inflamed GEnCs by modulating IL-6 signaling, identifying a potential novel anti-inflammatory role of IL-6 in renal glomeruli.

The autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and function in patients with rheumatoid arthritis and healthy individuals.

OBJECTIVES: A genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it is most highly expressed in neutrophils. We have investigated the effects of the variant on neutrophil function in health and in patients with RA. METHODS: Healthy individuals and patients with RA were genotyped for PTPN22 (R620W) and neutrophils isolated from peripheral blood. Neutrophil adhesion and migration across inflamed endothelium were measured. Calcium (Ca(2+)) release and reactive oxygen species (ROS) production in response to fMLP stimulation were also assessed. RESULTS: Expression of R620W enhanced neutrophil migration through cytokine activated endothelium (non-R620W=24%, R620W=45% migrating cells, p<0.001). Following fMLP stimulation, neutrophils that were heterozygous and homozygous for R620W released significantly more Ca(2+) when compared to non-R620W neutrophils, in healthy individuals and patients with RA. fMLP stimulation, after TNF-α priming, provoked more ROS from neutrophils heterozygous for R620W in patients with RA (non-R620W vs R620W=∼1.75-fold increase) and healthy individuals (non-R620W vs R620W=fourfold increase) and this increase was statistically significant in healthy individuals (p<0.001) but not in patients with RA (p<0.25). CONCLUSIONS: Expression of PTPN22 (R620W) enhanced neutrophil effector functions in health and RA, with migration, Ca(2+) release and production of ROS increased. Neutrophils are found in large numbers in the RA joint, and this hyperactivity of R620W cells may directly contribute to the joint damage, as well as to the initiation and perpetuation of the chronic immune-mediated inflammatory processes driving the disease.

Shear stress regulated gene expression and angiogenesis in vascular endothelium.

The behavior of vascular EC is greatly altered in sites of pathological angiogenesis, such as a developing tumor or atherosclerotic plaque. Until recently it was thought that this was largely due to abnormal chemical signaling, i.e., endothelial cell chemo transduction, at these sites. However, we now demonstrate that the shear stress intensity encountered by EC can have a profound impact on their gene expression and behavior. We review the growing body of evidence suggesting that mechanotransduction, too, is a major regulator of pathological angiogenesis. This fits with the evolving story of physiological angiogenesis, where a combination of metabolic and mechanical signaling is emerging as the probable mechanism by which tight feedback regulation of angiogenesis is achieved in vivo.

Crosstalk between mesenchymal stem cells and endothelial cells leads to downregulation of cytokine-induced leukocyte recruitment.

Mesenchymal stem cells (MSC) have immunomodulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow-derived MSC with EC and found that MSC could downregulate adhesion of flowing neutrophils or lymphocytes and their subsequent transendothelial migration. This applied for EC treated with tumor necrosis factor-α (TNF), interleukin-1ß (IL-1), or TNF and interferon-γ combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL-6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralizing antibody against IL-6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL-6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knockdown of IL-6-receptor expression in MSC as well as EC inhibited anti-inflammatory effects. This was explained when we detected soluble IL-6R receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralization of transforming growth factor-ß indicated that activation of this factor in coculture contributed to IL-6 production. Thus, crosstalk between MSC and EC caused upregulation of production of IL-6 by MSC which in turn downregulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL-6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease.