Summary 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 FAPα+ fibroblasts suppressed both inflammation and bone erosions in murine 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.

Recent studies have demonstrated that neutrophils are not a homogenous population of cells. Here, we have identified a subset of human neutrophils with a distinct profile of cell-surface receptors [CD54(high), CXC chemokine receptor 1(low) (CXCR1(low))], which represent cells that have migrated through an endothelial monolayer and then re-emerged by reverse transmigration (RT). RT neutrophils, when in contact with endothelium, were rescued from apoptosis, demonstrate functional priming, and were rheologically distinct from neutrophils that had not undergone transendothelial migration. In vivo, 1-2% of peripheral blood neutrophils in patients with systemic inflammation exhibit a RT phenotype. A smaller population existed in healthy donors ( approximately 0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54(low), CXCR1(high)) and naïve cells after activation with formyl-Met-Leu-Phe (CD54(low), CXCR1(low)). It is important that the RT phenotype (CD54(high), CXCR1(low)) is also distinct from tissue-resident neutrophils (CD54(low), CXCR1(low)). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue-experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.

We examined the hypothesis that stromal fibroblasts modulate the ability of endothelial cells (EC) to recruit lymphocytes in a site-specific manner. PBL were perfused over HUVEC that had been cultured with fibroblasts isolated from the inflamed synovium or the skin of patients with rheumatoid arthritis or osteoarthritis, or from normal synovium, with or without exposure to the inflammatory cytokines TNF-α+IFN-γ. Fibroblasts from inflamed synovium, but no others, caused unstimulated HUVEC to bind flowing lymphocytes. This adhesion was supported by α4β1-VCAM-1 interaction and stabilised by activation of PBL through CXCR4–CXCL12. Antibody neutralisation of IL-6 during co-culture effectively abolished the ability of EC to bind lymphocytes. Cytokine-stimulated EC supported high levels of lymphocyte adhesion, through the presentation of VCAM-1, E-selectin and chemokine(s) acting through CXCR3. Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source. In the dermal co-cultures, neutralisation of IL-6 or TGF-β caused partial recovery of cytokine-induced lymphocyte adhesion; this was complete when both were neutralised. Exogenous IL-6 was also found to inhibit response to TNF-α+IFN-γ. Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype. Actions of IL-6 might be pro-inflammatory or anti-inflammatory, depending on the local milieu.

Neutrophils are abundant, short-lived leukocytes, and their death by apoptosis is central to hemostasis and the resolution of inflammation, yet the trigger for their entry into apoptosis is unknown. We show here that death receptor signaling, including CD95 death-inducing signaling complex (DISC) formation and caspase 8 activation, occurred early in neutrophil apoptosis. However, death receptor ligation was not required for apoptosis, suggesting a novel mechanism for caspase 8 activation. We detected ceramide generation and clustering of CD95 in lipid rafts early in neutrophil apoptosis, and neutrophil apoptosis and ceramide generation were both significantly inhibited in acid sphingomyelinase knockout (ASM ؊/؊ ) mice compared to wild-type littermates. Further studies revealed that ceramide generation, CD95 clustering, and neutrophil apoptosis were dependent on reactive oxygen species (ROSs) and were preceded by a fall in reduced glutathione levels. We propose that accumulation of ROSs, as a consequence of altered redox status, initiates ligand-independent death receptor signaling via activation of ASM and clustering of preformed DISC components in lipid rafts and is therefore a primary factor limiting neutrophil life span.

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Mesenchymal stem cells (MSC) possess a range of immunomodulatory properties which they exert through soluble mediators and through direct cell-cell contact. Due to these immune regulatory properties, the safety and clinical efficacy of MSC treatment has been tested in a number of autoimmune disorders. In this review we analyze the current data from early phase trials into Crohn's disease, systemic lupus erythematosus, and rheumatoid arthritis. In general, no adverse side effects were observed in patients treated with MSC; however, their clinical efficacy is difficult to interpret. Systemic or site-specific administration of MSC has been reported to exert potent immunomodulatory effects in 7 of the 11 trials discussed. Nonetheless, the mechanism(s) by which MSC exert their regulatory effects in vivo remain largely unknown. We discuss potential limitations or safety concerns associated with MSC therapy, including the heterogeneity of MSC and their potential contribution to disease pathogenesis, which need to be considered when designing future clinical trials, along with the need to standardize trial design. Although we are bridging the translational gap between scientific observations on MSC function and clinical applications for therapy, our understanding of basic MSC biology is still limited. Despite these issues, large, double-blinded, multicenter clinical trials are already underway. Further research into the endogenous function of MSC is required to elucidate the mechanism by which therapeutic MSC are acting.

The stromal milieu (cellular and matrix components) helps establish tissue "address-codes" that direct leukocyte behavior in inflamed tissue. Coordinated interactions among the stroma, leukocytes, and ECs dictate which leukocytes are recruited, whether they are retained within the inflamed site, and how long they survive. Herein, we discuss how the stromal milieu influences the leukocyte recruitment cascade. Moreover, we explore how corruption of the stromal phenotype in chronic inflammatory diseases contributes to undesired, continuous recruitment of leukocytes. Emerging complex, multicellular, multilayered (co-)culture models are now addressing the molecular circuitry involved in regulating stromal organization during inflammation. Understanding context-specific changes in pro- or anti-inflammatory agents derived from the stroma, such as IL-6 (and its cofactors), is important for the generation of therapeutic strategies that restore the balance between recruitment and clearance of the inflammatory infiltrate in chronic disease.

During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3.ζδ protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type-1-diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to healthy age matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Importantly, control of patient T cell trafficking is re-established by exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic I/R injury, Salmonella infection, Uveitis and Sjögren’s Syndrome, PEPITEM could reduce T cell recruitment into inflamed tissues.