Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis.

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

Nuclear Receptor Subfamily 4A Signaling as a Key Disease Pathway of CD1c+ Dendritic Cell Dysregulation in Systemic Sclerosis.

OBJECTIVE: This study was undertaken to identify key disease pathways driving conventional dendritic cell (cDC) alterations in systemic sclerosis (SSc). METHODS: Transcriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 patients with SSc, including all major disease subtypes. We performed differential expression analysis for the different SSc subtypes and healthy donors to uncover genes dysregulated in SSc. To identify biologically relevant pathways, we built a gene coexpression network using weighted gene correlation network analysis. We validated the role of key transcriptional regulators using chromatin immunoprecipitation (ChIP) sequencing and in vitro functional assays. RESULTS: We identified 17 modules of coexpressed genes in cDCs that correlated with SSc subtypes and key clinical traits, including autoantibodies, skin score, and occurrence of interstitial lung disease. A module of immunoregulatory genes was markedly down-regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted nuclear receptor 4A (NR4A) subfamily genes (NR4A1, NR4A2, NR4A3) as the key transcriptional regulators of inflammation. Indeed, ChIP-sequencing analysis indicated that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T cell activation. CONCLUSION: NR4A1, NR4A2, and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDCs. Thus, the NR4A family represents novel potential targets to restore cDC homeostasis in SSc.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α.

OBJECTIVE: SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production. METHODS: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays. RESULTS: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2α (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs. CONCLUSION: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.

Extracellular SPARC cooperates with TGF-ß signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts.

OBJECTIVES: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. METHODS: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-ß1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. RESULTS: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-ß and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-ß signalling. CONCLUSION: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-ß dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS: In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre:Runx3f/f mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS: Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS: We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease.

Induction of Inflammation and Fibrosis by Semaphorin 4A in Systemic Sclerosis.

OBJECTIVE: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc). METHODS: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay. RESULTS: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors. CONCLUSION: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.

mTOR inhibition by metformin impacts monosodium urate crystal-induced inflammation and cell death in gout: a prelude to a new add-on therapy?

OBJECTIVE: Gout is the most common inflammatory arthritis worldwide, and patients experience a heavy burden of cardiovascular and metabolic diseases. The inflammation is caused by the deposition of monosodium urate (MSU) crystals in tissues, especially in the joints, triggering immune cells to mount an inflammatory reaction. Recently, it was shown that MSU crystals can induce mechanistic target of rapamycin (mTOR) signalling in monocytes encountering these crystals in vitro. The mTOR pathway is strongly implicated in cardiovascular and metabolic disease. We hypothesised that inhibiting this pathway in gout might be a novel avenue of treatment in these patients, targeting both inflammation and comorbidities. METHODS: We used a translational approach starting from ex vivo to in vitro and back to in vivo. RESULTS: We show that ex vivo immune cells from patients with gout exhibit higher expression of the mTOR pathway, which we can mimic in vitro by stimulating healthy immune cells (B lymphocytes, monocytes, T lymphocytes) with MSU crystals. Monocytes are the most prominent mTOR expressers. By using live imaging, we demonstrate that monocytes, on encountering MSU crystals, initiate cell death and release a wide array of proinflammatory cytokines. By inhibiting mTOR signalling with metformin or rapamycin, a reduction of cell death and release of inflammatory mediators was observed. Consistent with this, we show that patients with gout who are treated with the mTOR inhibitor metformin have a lower frequency of gout attacks. CONCLUSIONS: We propose mTOR inhibition as a novel therapeutic target of interest in gout treatment.

Hypoxia potentiates monocyte-derived dendritic cells for release of tumor necrosis factor α via MAP3K8.

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Characterization of CD30/CD30L(+) Cells in Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis.

The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30(+) T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L(+) T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells.

Gene Expression Profiling in Peripheral Blood Cells and Synovial Membranes of Patients with Psoriatic Arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is an inflammatory arthritis whose pathogenesis is poorly understood; it is characterized by bone erosions and new bone formation. The diagnosis of PsA is mainly clinical and diagnostic biomarkers are not yet available. The aim of this work was to clarify some aspects of the disease pathogenesis and to identify specific gene signatures in paired peripheral blood cells (PBC) and synovial biopsies of patients with PsA. Moreover, we tried to identify biomarkers that can be used in clinical practice. METHODS: PBC and synovial biopsies of 10 patients with PsA were used to study gene expression using Affymetrix arrays. The expression values were validated by Q-PCR, FACS analysis and by the detection of soluble mediators. RESULTS: Synovial biopsies of patients showed a modulation of approximately 200 genes when compared to the biopsies of healthy donors. Among the differentially expressed genes we observed the upregulation of Th17 related genes and of type I interferon (IFN) inducible genes. FACS analysis confirmed the Th17 polarization. Moreover, the synovial trascriptome shows gene clusters (bone remodeling, angiogenesis and inflammation) involved in the pathogenesis of PsA. Interestingly 90 genes are modulated in both compartments (PBC and synovium) suggesting that signature pathways in PBC mirror those of the inflamed synovium. Finally the osteoactivin gene was upregulared in both PBC and synovial biopsies and this finding was confirmed by the detection of high levels of osteoactivin in PsA sera but not in other inflammatory arthritides. CONCLUSIONS: We describe the first analysis of the trancriptome in paired synovial tissue and PBC of patients with PsA. This study strengthens the hypothesis that PsA is of autoimmune origin since the coactivity of IFN and Th17 pathways is typical of autoimmunity. Finally these findings have allowed the identification of a possible disease biomarker, osteoactivin, easily detectable in PsA serum.

Crossreactive autoantibodies directed against cutaneous and joint antigens are present in psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is a chronic inflammatory disease of unknown origin, characterized by erosions and new bone formation. Diagnosis of PsA is mainly clinical and there are no biomarkers available. Moreover in PsA autoantibodies have not been described so far. Indeed an autoimmune origin has been suggested but never proven. Aim of the study was to investigate the possible presence of autoantibodies typically associated with PsA. METHODS: We used pooled IgG immunoglobulins derived from 30 patients with PsA to screen a random peptide library in order to identify disease relevant autoantigen peptides. RESULTS: Among the selected peptides, one was recognised by nearly all the patients' sera. The identified peptide (PsA peptide: TNRRGRGSPGAL) shows sequence similarities with skin autoantigens, such as fibrillin 3, a constituent of actin microfibrils, desmocollin 3, a constituent of the desmosomes and keratin 78, a component of epithelial cytoskeleton. Interestingly the PsA peptide shares homology with the nebulin-related anchoring protein (N-RAP), a protein localized in the enthesis (point of insertion of a tendon or ligament to the bone), which represents the first affected site during early PsA. Antibodies affinity purified against the PsA peptide recognize fibrillin, desmocollin, keratin and N-RAP. Moreover antibodies directed against the PsA peptide are detectable in 85% of PsA patients. Such antibodies are not present in healthy donors and are present in 13/100 patients with seroposive rheumatoid arthritis (RA). In seronegative RA these antibodies are detectable only in 3/100 patients. CONCLUSIONS: Our results indicate that PsA is characterized by the presence of serum autoantibodies crossreacting with an epitope shared by skin and joint antigens.

Gene expression profiling in peripheral blood mononuclear cells of patients with common variable immunodeficiency: modulation of adaptive immune response following intravenous immunoglobulin therapy.

BACKGROUND: Regular intravenous immunoglobulin treatment is used to replace antibody deficiency in primary immunodeficiency diseases; however the therapeutic effect seems to be related not only to antibody replacement but also to an active role in the modulation of the immune response. Common variable immunodeficiency is the most frequent primary immunodeficiency seen in clinical practice. METHODS: We have studied the effect of intravenous immunoglobulin replacement in patients with common variable immunodeficiency by evaluating the gene-expression profiles from Affimetrix HG-U133A. Some of the gene array results were validated by real time RT-PCR and by the measurement of circulating cytokines and chemokines by ELISA. Moreover we performed FACS analysis of blood mononuclear cells from the patients enrolled in the study. RESULTS: A series of genes involved in innate and acquired immune responses were markedly up- or down-modulated before therapy. Such genes included CD14, CD36, LEPR, IRF-5, RGS-1, CD38, TNFRSF25, IL-4, CXCR4, CCR3, IL-8. Most of these modulated genes showed an expression similar to that of normal controls after immunoglobulin replacement. Real time RT-PCR of selected genes and serum levels of IL-4, CXCR4 before and after therapy changed accordingly to gene array results. Interestingly, serum levels of IL-8 remained unchanged, as the corresponding gene, before and after treatment. FACS analysis showed a marked decrease of CD8+T cells and an increase of CD4+T cells following treatment. Moreover we observed a marked increase of CD23⁻CD27⁻IgM⁻IgG⁻ B cells (centrocytes). CONCLUSIONS: Our results are in accordance with previous reports and provide further support to the hypothesis that the benefits of intravenous immunoglobulin therapy are not only related to antibody replacement but also to its ability to modulate the immune response in common variable immunodeficiency.