Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Telemedicine with special focus on allergic diseases and asthma-Status 2022: An EAACI position paper.

Efficacious, effective and efficient communication between healthcare professionals (HCP) and patients is essential to achieve a successful therapeutic alliance. Telemedicine (TM) has been used for decades but during the COVID-19 pandemic its use has become widespread. This position paper aims to describe the terminology and most important forms of TM among HCP and patients and review the existing studies on the uses of TM for asthma and allergy. Besides, the advantages and risks of TM are discussed, concluding that TM application reduces costs and time for both, HCP and patients, but cannot completely replace face-to-face visits for physical examinations and certain tests that are critical in asthma and allergy. From an ethical point of view, it is important to identify those involved in the TM process, ensure confidentiality and use communication channels that fully guarantee the security of the information. Unmet needs and directions for the future regarding implementation, data protection, privacy regulations, methodology and efficacy are described.

AllergoOncology: Biomarkers and refined classification for research in the allergy and glioma nexus-A joint EAACI-EANO position paper.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Deciphering the role of platelets in severe allergy by an integrative omics approach.

BACKGROUND: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. METHODS: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC-MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex. RESULTS: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype. CONCLUSIONS: This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies.

Platelet-Derived Extracellular Vesicles as Lipid Carriers in Severe Allergic Inflammation.

The resolution of inflammation is a complex process that is critical for removing inflammatory cells and restoring tissue function. The dysregulation of these mechanisms leads to chronic inflammatory disorders. Platelets, essential cells for preserving homeostasis, are thought to play a role in inflammation as they are a source of immunomodulatory factors. Our aim was to identify key metabolites carried by platelet-derived extracellular vesicles (PL-EVs) in a model of allergic inflammation. PL-EVs were isolated by serial ultracentrifugation using platelet-rich plasma samples obtained from platelet apheresis from severely (n = 6) and mildly (n = 6) allergic patients and non-allergic individuals used as controls (n = 8). PL-EVs were analysed by a multiplatform approach using liquid and gas chromatography coupled to mass spectrometry (LC-MS and GC-MS, respectively). PL-EVs obtained from severely and mildly allergic patients and control individuals presented comparable particle concentrations and sizes with similar protein concentrations. Strikingly, PL-EVs differed in their lipid and metabolic content according to the severity of inflammation. L-carnitine, ceramide (Cer (d18:0/24:0)), and several triglycerides, all of which seem to be involved in apoptosis and regulatory T functions, were higher in PL-EVs from patients with mild allergic inflammation than in those with severe inflammation. In contrast, PL-EVs obtained from patients with severe allergic inflammation showed an alteration in the arachidonic acid pathway. This study demonstrates that PL-EVs carry specific lipids and metabolites according to the degree of inflammation in allergic patients and propose novel perspectives for characterising the progression of allergic inflammation.

AllergoOncology: Danger signals in allergology and oncology: A European Academy of Allergy and Clinical Immunology (EAACI) Position Paper.

The immune system interacts with many nominal 'danger' signals, endogenous danger-associated (DAMP), exogenous pathogen (PAMP) and allergen (AAMP)-associated molecular patterns. The immune context under which these are received can promote or prevent immune activating or inflammatory mechanisms and may orchestrate diverse immune responses in allergy and cancer. Each can act either by favouring a respective pathology or by supporting the immune response to confer protective effects, depending on acuity or chronicity. In this Position Paper under the collective term danger signals or DAMPs, PAMPs and AAMPs, we consider their diverse roles in allergy and cancer and the connection between these in AllergoOncology. We focus on their interactions with different immune cells of the innate and adaptive immune system and how these promote immune responses with juxtaposing clinical outcomes in allergy and cancer. While danger signals present potential targets to overcome inflammatory responses in allergy, these may be reconsidered in relation to a history of allergy, chronic inflammation and autoimmunity linked to the risk of developing cancer, and with regard to clinical responses to anti-cancer immune and targeted therapies. Cross-disciplinary insights in AllergoOncology derived from dissecting clinical phenotypes of common danger signal pathways may improve allergy and cancer clinical outcomes.

Serotonin (5-HT) Shapes the Macrophage Gene Profile through the 5-HT2B-Dependent Activation of the Aryl Hydrocarbon Receptor.

Macrophages can either promote or resolve inflammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine [5-HT]). In fact, pro- and anti-inflammatory macrophages differ in the expression of serotonin receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF-primed monocyte-derived macrophages (M-MØ). 5-HT7 drives the acquisition of profibrotic and anti-inflammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT-related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antiparkinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT-mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B-specific antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics.

Herpes simplex virus 1 proteins can induce skin inflammation in an atopic dermatitis-like mouse model.

Herpes simplex virus type 1 (HSV-1) can induce in certain individuals with atopic dermatitis (AD) severe cutaneous infections that can spread throughout the entire body, a condition named as AD complicated by eczema herpeticum (ADEH). It has been recently found that ADEH patients can produce specific IgE against HSV-1 proteins, which may contribute to lower protection against HSV-1. However, little is known about the capacity of these HSV-1 proteins to produce an inflammatory response at the skin level. In this study, using a mouse model of AD-like dermatitis, three HSV-1 proteins (glycoprotein D -gD-, glycoprotein B -gB- and VP22) were applied on tape-stripped back skin mice in three exposures periods. Ovalbumin (OVA) and 0.9% NaCl were used as positive and negative controls, respectively. Skin samples were obtained for analysis of specific cell components of skin infiltration. The results showed that the viral protein gD induced a statistically significant increase in the number of dermal infiltrating CD3+, CD4+ cells and mast cells compared with the negative control group. gD was also able to induce epidermal thickening and epidermal infiltration of T cells closely related to the one produced in mice sensitized with OVA. However, VP22 and gB contributed to a lesser extent to skin inflammation. These results showed that proteins from HSV-1, especially gD, can have per se an important T cell and mast cell-driven inflammatory potential at the skin level.

Palmitate Conditions Macrophages for Enhanced Responses toward Inflammatory Stimuli via JNK Activation.

Obesity is associated with low-grade inflammation and elevated levels of circulating saturated fatty acids, which trigger inflammatory responses by engaging pattern recognition receptors in macrophages. Because tissue homeostasis is maintained through an adequate balance of pro- and anti-inflammatory macrophages, we assessed the transcriptional and functional profile of M-CSF-dependent monocyte-derived human macrophages exposed to concentrations of saturated fatty acids found in obese individuals. We report that palmitate (C16:0, 200 µM) significantly modulates the macrophage gene signature, lowers the expression of transcription factors that positively regulate IL-10 expression (MAFB, AhR), and promotes a proinflammatory state whose acquisition requires JNK activation. Unlike LPS, palmitate exposure does not activate STAT1, and its transcriptional effects can be distinguished from those triggered by LPS, as both agents oppositely regulate the expression of CCL19 and TRIB3 Besides, palmitate conditions macrophages for exacerbated proinflammatory responses (lower IL-10 and CCL2, higher TNF-α, IL-6, and IL-1ß) toward pathogenic stimuli, a process also mediated by JNK activation. All of these effects of palmitate are fatty acid specific because oleate (C18:1, 200 µM) does not modify the macrophage transcriptional and functional profiles. Therefore, pathologic palmitate concentrations promote the acquisition of a specific polarization state in human macrophages and condition macrophages for enhanced responses toward inflammatory stimuli, with both effects being dependent on JNK activation. Our results provide further insight into the macrophage contribution to obesity-associated inflammation.

Reshaping of Human Macrophage Polarization through Modulation of Glucose Catabolic Pathways.

Macrophages integrate information from the tissue microenvironment and adjust their effector functions according to the prevalent extracellular stimuli. Therefore, macrophages can acquire a variety of activation (polarization) states, and this functional plasticity allows the adequate initiation, regulation, and resolution of inflammatory responses. Modulation of the glucose metabolism contributes to the macrophage adaptation to the surrounding cytokine milieu, as exemplified by the distinct glucose catabolism of macrophages exposed to LPS/IFN-γ or IL-4. To dissect the acquisition of macrophage effector functions in the absence of activating cytokines, we assessed the bioenergetic profile of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ), which do not release pro- or anti-inflammatory cytokines unless subjected to additional activating stimuli. Compared to M-MØ, GM-MØ displayed higher oxygen consumption rate and aerobic glycolysis (extracellular acidification rate [ECAR]), as well as higher expression of genes encoding glycolytic enzymes. However, M-MØ exhibited a significantly higher oxygen consumption rate/ECAR ratio. Surprisingly, whereas aerobic glycolysis positively regulated IL1B, TNF, and INHBA mRNA expression in both macrophage subtypes, mitochondrial respiration negatively affected IL6, IL1B, TNF, and CXCL10 mRNA expression in M-MØ. The physiological significance of these results became evident under low oxygen tensions, as hypoxia enhanced ECAR in M-MØ via HIF-1α and HIF-2α, increased expression of glycolytic enzymes and GM-MØ-specific genes, and diminished expression of M-MØ-associated genes. Therefore, our data indicate that GM-MØ and M-MØ display distinct bioenergetic profiles, and that hypoxia triggers a transcriptomic switch in macrophages by promoting a HIF-1α/HIF-2α-dependent increase in ECAR.

Macrophages from the synovium of active rheumatoid arthritis exhibit an activin A-dependent pro-inflammatory profile.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis and severity correlates with the presence of macrophage-derived pro-inflammatory cytokines within the inflamed synovium. Macrophage-derived cytokines fuel the pathological processes in RA and are targets of clinically successful therapies. However, although macrophage polarization determines cytokine production, the polarization state of macrophages in RA joints remains poorly defined. To dissect the molecular basis for the tissue-damaging effects of macrophages in RA joints, we undertook the phenotypic and transcriptomic characterization of ex vivo isolated CD14(+) RA synovial fluid (RA-SF) macrophages. Flow cytometry and gene profiling indicated that RA-SF macrophages express pro-inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated with homeostatic and anti-inflammatory polarization (IGF1, HTR2B) and exhibit a transcriptomic profile that resembles the activin A-dependent gene signature of pro-inflammatory in vitro-generated macrophages. In fact, high levels of Smad-activating activin A were found in RA-SF and, accordingly, the Smad signalling pathway was activated in ex vivo-isolated RA-SF macrophages. In vitro experiments on monocytes and macrophages indicated that RA-SF promoted the acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2) but led to a significant reduction in the expression of genes associated with homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming the pro-inflammatory polarization ability of RA-SF. Importantly, the macrophage-polarizing ability of RA-SF was inhibited by an anti-activin A-neutralizing antibody, thus demonstrating that activin A mediates the pro-inflammatory macrophage-polarizing ability of RA-SF. Moreover, and in line with these findings, multicolour immunofluorescence evidenced that macrophages within RA synovial membranes (RA-SM) also express pro-inflammatory polarization markers whose expression is activin A-dependent. Altogether, our results demonstrate that macrophages from RA synovial fluids and membranes exhibit an MMP12(+) EGLN3(+) CCR2(+) pro-inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid.

Proteomic profiles of peritoneal fluid-derived small extracellular vesicles correlate with patient outcome in ovarian cancer.

Cancer-derived small extracellular vesicles (sEVs) are capable of modifying the tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid-derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, 2 patient cohorts were enrolled: patients with OvCa who underwent a diagnostic or cytoreductive surgery and nononcological patients, who underwent abdominal surgery for benign gynecological conditions and acted as the control group. Systematic extraction of PFD-sEVs from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage, and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEV proteomic profiles in high-grade serous ovarian carcinomas (HGSOCs) revealed 2 clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of PFD-sEV content provided a prognostic value with potential implications in HGSOC clinical management.

A profibrotic role for thymic stromal lymphopoietin in systemic sclerosis.

OBJECTIVE: [corrected] Systemic sclerosis (SSc) is an autoimmune disease characterised by progressive fibrosis. Although SSc shares pathogenetic features with other autoimmune diseases, the participation of profibrotic Th2 cytokines is unique to SSc, but the mechanisms of Th2 skewing are unknown. We have analysed the expression and function of thymic stromal lymphopoietin (TSLP), a central regulator of Th2-mediated allergic inflammation, in human SSc, primary lung fibrosis and in a mouse model of scleroderma. METHODS: TSLP expression was analysed by immunohistochemistry in human SSc skin, primary lung fibrosis and mouse bleomycin-induced skin fibrosis, and by quantitative RT-PCR in mouse skin and cultured fibroblasts. The regulation of TSLP expression by specific toll-like receptors (TLR)-2, -3 and -4 agonists was analysed in human dermal fibroblast cultures. The role of TSLP in skin fibrosis and local cytokine expression was analysed in TSLP receptor (TSLPR)-deficient mice. RESULTS: TSLP was overexpressed by epithelial cells, mast cells and fibroblasts in human SSc skin and lung fibrosis, and in the bleomycin model of scleroderma. In cultured human and mouse skin fibroblasts, TSLP expression was inducible by activation of TLR, particularly TLR3. In TSLPR-deficient mice, bleomycin-induced fibrosis was significantly reduced in parallel with significantly reduced local expression of IL-13. CONCLUSIONS: These data provide the first evidence of TSLP overexpression in SSc and other non-allergic fibrotic conditions, and demonstrate a profibrotic role that is potentially meditated by specific changes in the local cytokine milieu. Thus, modulating TSLP may have antifibrotic therapeutic implications.

CXCL12γ isoform is expressed on endothelial and dendritic cells in rheumatoid arthritis synovium and regulates T cell activation.

OBJECTIVE: CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation. METHODS: Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti-pan-CXCL12 or anti-CXCL12γ-specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding-defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures. RESULTS: CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN-positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell-stimulatory effects of soluble CXCL12 proteins. CONCLUSION: CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.

Transcriptomics reveals a distinct metabolic profile in T cells from severe allergic asthmatic patients.

The reasons behind the onset and continuation of chronic inflammation in individuals with severe allergies are still not understood. Earlier findings indicated that there is a connection between severe allergic inflammation, systemic metabolic alterations and impairment of regulatory functions. Here, we aimed to identify transcriptomic alterations in T cells associated with the degree of severity in allergic asthmatic patients. T cells were isolated from severe (n = 7) and mild (n = 9) allergic asthmatic patients, and control (non-allergic, non-asthmatic healthy) subjects (n = 8) to perform RNA analysis by Affymetrix gene expression. Compromised biological pathways in the severe phenotype were identified using significant transcripts. T cells' transcriptome of severe allergic asthmatic patients was distinct from that of mild and control subjects. A higher count of differentially expressed genes (DEGs) was observed in the group of individuals with severe allergic asthma vs. control (4,924 genes) and vs. mild (4,232 genes) groups. Mild group also had 1,102 DEGs vs. controls. Pathway analysis revealed alterations in metabolism and immune response in the severe phenotype. Severe allergic asthmatic patients presented downregulation in genes related to oxidative phosphorylation, fatty acid oxidation and glycolysis together with increased expression of genes coding inflammatory cytokines (e.g. IL-19, IL-23A and IL-31). Moreover, the downregulation of genes involved in TGFß pathway together with a decreased tendency on the percentage of T regulatory cell (CD4 + CD25+), suggest a compromised regulatory function in severe allergic asthmatic patients. This study demonstrates a transcriptional downregulation of metabolic and cell signalling pathways in T cells of severe allergic asthmatic patients associated with diminished regulatory T cell function. These findings support a link between energy metabolism of T cells and allergic asthmatic inflammation.

Transcriptome analysis reveals specific changes in osteoarthritis synovial fibroblasts.

OBJECTIVE: Changes in rheumatoid arthritis synovial fibroblast (RASF) gene expression are usually defined by a comparison to osteoarthritis synovial fibroblasts (OASFs). This study was undertaken to analyse the transcriptome of OASFs as compared to RASFs and healthy synovial fibroblasts (HSFs). METHODS: The authors used microarray messenger RNA expression profiling of synovial fibroblasts cultured from osteoarthritis (OA), rheumatoid arthritis and normal synovial tissues. Quantitative real-time PCR of selected genes was performed to validate microarray data. Analysis of variance, Student t test and the Benjamini-Hochberg multiple testing correction method for multiple testing correction were used to determine the statistical significance of the changes between the three groups. RESULTS: Larger numbers of transcripts showed a differential expression in OASFs versus the other groups, rather than in RASFs versus HSFs. Cluster analysis confirmed that the differences between the three groups were mostly due to the differences between OA and the other groups. Functional classification identified a significant number of genes related to growth factor activities, cell adhesion, neurotransmission and Ras signalling that are differentially expressed in OASFs. Classical proinflammatory factors or proteases involved in cartilage degradation were not found to be overexpressed in OASFs. CONCLUSION: Cultured OASFs display a more homogeneous transcriptomic profile than RASFs when compared to HSFs. This supports the participation of synovial fibroblasts in the pathogenesis of OA and may reflect global defects in the mesenchyma-derived lineages of the different tissues in OA joints. These data support individual heterogeneity among RASFs and advise against the use of OASFs as controls.

Synovial fibroblast hyperplasia in rheumatoid arthritis: clinicopathologic correlations and partial reversal by anti-tumor necrosis factor therapy.

OBJECTIVE: Synovial fibroblast (SF) hyperplasia contributes to the pathogenesis of rheumatoid arthritis (RA), but quantitative information on this process is scarce. This study was undertaken to evaluate the fibroblast-specific marker Hsp47 as a quantitative marker for SFs and to analyze its clinicopathologic correlates and evolution after anti-tumor necrosis factor α (anti-TNFα) therapy. METHODS: Synovial biopsy samples were obtained from 48 patients with RA and 20 controls who were healthy or had osteoarthritis (OA). Twenty-five RA patients who had active disease at the time of biopsy underwent a second biopsy after anti-TNFα therapy. Immunolabeling for Hsp47, inflammatory cells, and vascular cell markers was performed. Hsp47-positive lining and sublining fractional areas were quantified, and their correlation with clinicopathologic variables was analyzed. RESULTS: In normal and diseased synovial tissue, Hsp47 was specifically and uniformly expressed by lining, sublining, and perivascular fibroblasts. Lining SF area was significantly increased in both RA and late OA tissue compared to normal tissue. Sublining SF area was increased in RA tissue but not in late OA tissue compared to normal tissue. Lining SF area was positively correlated with macrophage density, Disease Activity Score in 28 joints, and RA disease duration. In contrast, sublining SF area was negatively correlated with RA disease duration and activity. A significant reduction in lining SF area but not sublining SF area was observed after anti-TNFα therapy. CONCLUSION: Our findings indicate that Hsp47 is a reliable marker for quantifying SFs in human synovial tissue. Our data suggest that lining and sublining SFs undergo different dynamics during the course of the disease. Lining SF expansion parallels the activity and temporal progression of RA and can be partially reversed by anti-TNFα therapy.