Macrophages In Systemic Sclerosis Research Articles

Efferocytosis dysfunction in CXCL4-induced M4 macrophages: phenotypic insights in systemic sclerosis in vitro and in vivo.

Systemic sclerosis (SSc) is an autoimmune disease characterized by antinuclear antibody production, which has been linked to an excess of apoptotic cells, normally eliminated by macrophages through efferocytosis. Additionally, circulating levels of CXCL4, a novel SSc biomarker, correlate with more severe fibrotic manifestations of the disease. Considering the defective efferocytosis of macrophages in SSc and the CXCL4-related M4 macrophage phenotype, we hypothesized that CXCL4 could be involved in the alteration of phagocytic functions of macrophages in SSc, including LC3-associated phagocytosis (LAP), another phagocytic process requiring autophagy proteins and contributing to immune silencing. In this study, CXCL4 levels were measured by ELISA in vitro in the serum of SSc patients, and also in vivo in the serum and lungs of C57BL/6J SSc mice induced by intradermal injections of hypochloric acid (HOCl) or Bleomycin (BLM), with evaluation of M4 markers. Circulating monocytes from healthy donors were also differentiated in vitro into M4 monocytes-derived macrophages (MDMs) in the presence of recombinant CXCL4. In M4-MDMs, phagocytosis of fluorescent beads and expression level of efferocytic receptors were evaluated by flow cytometry in vitro, while efferocytosis of pHrodo-stained apoptotic Jurkat cells was evaluated by real-time fluorescence microscopy. LAP quantification was made by fluorescence microscopy in M4-MDMs exposed to IgG-coated beads as well as apoptotic Jurkat cells. Our results demonstrated that efferocytosis was significantly reduced in M0-MDMs from healthy donors exposed to the CXCL4-rich plasma of SSc patients. In vivo, CXCL4 expression was increased in the lungs of both SSc-mouse models, along with elevated M4 markers, while efferocytosis of BLM-mice alveolar macrophages was decreased. In vitro, M4-MDMs exhibited reduced efferocytosis compared to M0-MDMs, notably attributable to lower CD36 receptor expression and impaired phagocytosis capacities, despite enhanced LAP. Autophagic gene expression was increased both in vitro in SSc MDMs and in vivo in BLM mice, thus acting as a potential compensatory mechanism. Altogether, our results support the role of CXCL4 on the impaired efferocytosis capacities of human macrophages from SSc patients and in SSc mice.

Periostin-An inducer of pro-fibrotic phenotype in monocytes and monocyte-derived macrophages in systemic sclerosis.

Enhanced circulating blood periostin levels positively correlate with disease severity in patients with systemic sclerosis (SSc). Monocytes/macrophages are predominantly associated with the pathogenesis of SSc, but the effect of periostin on immune cells, particularly monocytes and macrophages, still remains to be elucidated. We examined the effect of periostin on monocytes and monocyte-derived macrophages (MDM) in the pathogenesis of SSc. The modified Rodnan total skin thickness score in patients with dcSSc was positively correlated with the proportion of CD80-CD206+ M2 cells. The proportion of M2 macrophages was significantly reduced in rPn-stimulated MDMs of HCs compared to that of SSc patients. The mRNA expression of pro-fibrotic cytokines, chemokines, and ECM proteins was significantly upregulated in rPn-stimulated monocytes and MDMs as compared to that of control monocytes and MDMs. A similar trend was observed for protein expression in the respective MDMs. In addition, the ratio of migrated cells was significantly higher in rPn-stimulated as compared to control monocytes. These results suggest that periostin promotes inflammation and fibrosis in the pathogenesis of SSc by possible modulation of monocytes/macrophages.

Epigenetic Regulation of Profibrotic Macrophages in Systemic Sclerosis-Associated Interstitial Lung Disease.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)-expressing macrophages in SSc-ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc-ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype. We performed single-cell RNA sequencing (scRNA-Seq) on 11 explanted SSc-ILD and healthy control lung samples, as well as single-cell assay for transposase-accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single-cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat. We identified distinct macrophage subpopulations using scRNA-Seq analysis in healthy and SSc-ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia-associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix-loop-helix family member E40, Kruppel-like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation. Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc-ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc-ILD, specifically targeting profibrotic macrophages.

Contribution of monocytes and macrophages to the pathogenesis of systemic sclerosis: recent insights and therapeutic implications.

To discuss recent studies addressing the role of monocytes and macrophages in the pathogenesis of systemic sclerosis (SSc) based on human and mouse models. Studies indicate that monocyte adhesion could be increased in SSc secondary to an interferon-dependent loss of CD52, and chemotaxis up-regulated through the CCR3/CCL24 pathway. Beyond the conventional M1/M2 paradigm of macrophage subpopulations, new subpopulations of macrophages have been recently described in skin and lung biopsies from SSc patients. Notably, single-cell ribonucleic acid sequencing has provided evidence for SPP1+ lung macrophages or FCGR3A+ skin macrophages in SSc. Impaired pro-resolving capacities of macrophages such as efferocytosis, i.e. the ability to phagocyte apoptotic cells, could also participate in the inflammatory and autoimmune features in SSc. Through their potential pro-fibrotic and pro-inflammatory properties, macrophages are at the cross-road of key SSc pathogenic processes and associated manifestations. Investigative drugs targeting macrophage polarization, such as pan-janus kinase inhibitors (tofacitinib or ruxolitinib) impacting both M1 and M2 activations, or Romilkimab inhibiting IL-4 and IL-13, have shown promising results in preclinical models or phase I/II clinical trials in SSc and other fibro-inflammatory disorders. Macrophage-based cellular therapy may also represent an innovative approach for the treatment of SSc, as initial training of macrophages may modulate the severity of fibrotic and autoimmune manifestations of the disease.

Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis.

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood.Methods and ResultsImmunohistochemistry analysis showed accumulation of CD14+ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68+ and mannose-R+ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14+ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-β signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14+ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14+ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-β, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14+ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-β signalling pathway with SD208 (TGF-β receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-β-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion.ConclusionsOur findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14+ monocytes and CD14+ pulmonary macrophages in SSc and highlighted the capability of CD14+ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14+ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.

Profibrotic Activation of Human Macrophages in Systemic Sclerosis

Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts. Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates. The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05). These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

Pro-fibrotic Activation of Human Macrophages in Systemic Sclerosis

Abstract Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in Systemic sclerosis (SSc), and we have shown that macrophages constitute the dominant inflammatory signature in SSc tissue. However, little is known about how these cells contribute to fibrotic activation in SSc. Using a bioinformatics approach, we show that the gene expression profile of blood-derived human SSc macrophages is significantly enriched in SSc patient skin. We characterize the immunophenotype of human SSc macrophages as pro-fibrotic, demonstrating that these cells are activated under basal conditions, expressing elevated levels of surface markers associated with activation and releasing CCL2, IL-6, and TGF-beta. We also show that STAT3 is phosphorylated in macrophage from SSc patient compared to the healthy donor, and the phosphorylation is attenuated after IL-6 blockade, suggesting that increased expression of IL-6 by SSc macrophages accounts, at least in part, for increased signaling. Moreover, our results suggest that activation of SSc macrophages arises from soluble factors, as differentiation of healthy donor monocytes in SSc patient-derived plasma confers the immunophenotype of SSc patient macrophages. For the first time, we show that co-culture of human SSc macrophages with SSc fibroblasts induces fibroblast activation. Collectively, these studies implicate macrophages as likely drivers of fibrosis in SSc and suggest therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

Circulating classical monocytes share a common transcriptional signature with skin macrophages in Systemic Sclerosis

Abstract The etiology and pathogenesis of systemic sclerosis (SSc) are poorly understood. Circulating monocytes likely play a critical role in SSc progression through secretion of proinflammatory molecules and as precursors of macrophages that can reorganize the extracellular matrix, thereby leading to development of end-organ fibrosis. Here, we evaluate the transcriptional similarities between circulating classical monocytes (CMo) and skin macrophages of SSc patients. Bulk RNA-seq was performed on sorted CMo from SSc blood obtained through the Prospective Registry of Early Systemic Sclerosis (PRESS) cohort, and transcriptional profiles were analyzed along with profiles from matched control samples. Additionally, sorted CD45+ cells from skin biopsies of one SSc patient and one control patient were prepared for single-cell RNA-seq. There was a numerical expansion of skin macrophages from the control to the SSc sample, but no significant difference in the quantity of circulating CMo between control and SSc patients. Of the 152 significantly upregulated genes (DESeq2, p&amp;lt;0.05, fold change&amp;gt;2) observed in circulating CMo population and the 290 upregulated genes (fold change&amp;gt;2) in the skin macrophage cluster compared to their respective controls, we found 23 genes in common (p&amp;lt;1.23×10−8, hypergeometric distribution test). These shared genes were involved in various immune and inflammatory regulation processes. These data indicate that a common transcriptional signature may exist between circulating CMo and macrophages present in the skin of SSc patients, potentially suggesting that macrophage-specific pathways at the site of fibrosis can be detected in a circulating precursor population.

Macrophages and cadherins in fibrosis and systemic sclerosis.

Macrophages are key players in systemic sclerosis (SSc) and fibrosis. The mechanism by which macrophages regulate fibrogenesis is unclear and understanding the origin and function of macrophages is critical to developing effective therapeutics. Novel targets on macrophages are under investigation and recently, cadherins have emerged as a potential therapeutic target on macrophages. The current review will discuss the importance of macrophages in SSc and fibrosis and summarize recent studies on the role of cadherin-11 (Cdh11) on macrophages and fibrosis. Genome-wide expression studies demonstrate the importance of macrophages in SSc and fibrosis. Although M2 macrophages are associated with fibrosis, the presence of a mixed M1/M2 phenotype in fibrosis has recently been reported. Several studies aiming to identify macrophage subsets involved in fibrogenesis suggest that monocyte-derived alveolar macrophages are key players in the development of murine lung fibrosis. Recent functional studies show that Cdh11 regulates macrophages, fibroblast invasion, and adhesion of macrophages to myofibroblasts. Macrophages play an important role in SSc and fibrosis. New insights into the mechanisms by which macrophages regulate fibrogenesis have been discovered on the basis of Cdh11 studies and suggest that targeting Cdh11 may be an effective target to treat fibrosis.

Macrophages in Systemic Sclerosis: Novel Insights and Therapeutic Implications.

Macrophages play key roles in tissue homeostasis and immune surveillance, mobilizing immune activation in response to microbial invasion and promoting wound healing to repair damaged tissue. However, failure to resolve macrophage activation can lead to chronic inflammation and fibrosis, and ultimately to pathology. Activated macrophages have been implicated in the pathogenesis of systemic sclerosis (SSc), although the triggers that induce immune activation in SSc and the signaling pathways that underlie aberrant macrophage activation remain unknown. Macrophages are implicated in fibrotic activation in SSc. Targeted therapeutic interventions directed against SSc macrophages may ameliorate inflammation and fibrosis. While current studies have begun to elucidate the role of macrophages in disease initiation and progression, further work is needed to address macrophage subset heterogeneity within and among SSc end-target tissues to determine the disparate functions mediated by these subsets and to identify additional targets for therapeutic intervention.

Efferocytosis capacities of blood monocyte-derived macrophages in systemic sclerosis.

A defect in the apoptotic cell clearance (efferocytosis) by phagocytic cells may participate in autoimmunity and chronic inflammation. The mechanisms leading to the emergence of autoimmunity in systemic sclerosis (SSc) are still to be determined. In this study, the efferocytosis capacities of blood monocyte-derived macrophages (MDM) from patients with SSc were evaluated. Blood monocytes obtained from patients with SSc and healthy donors (HD) were differentiated in vitro into macrophages. The capacities of MDM to engulf CFSE+ apoptotic Jurkat human T lymphocytes were compared between SSc MDM and HD using flow cytometry. The expression of classical engulfing receptors in SSc MDM and HD MDM was also evaluated and their involvement in the modulation of efferocytosis was confirmed using a siRNA approach. The mean phagocytic index (PI) reflecting efferocytosis capacities of SSc MDM (PI = 19.3 ± 3.0; n = 21) was significantly decreased in comparison with the PI of HD MDM (PI = 35.9 ± 3.0; n = 31; P < 0.001). In comparison with HD, SSc MDM exhibited a downregulated expression of scavenger receptor (SR)-B1, SR-A1 and integrin β5 (ITGβ5). In HD MDM, the extinction of these receptors was followed by a reduction of efferocytosis only for the repression of ITGβ5, suggesting a possible selective role of this integrin in the impaired efferocytosis observed in SSc. As efferocytosis may be at the crossroads of inflammation, autoimmunity and fibrosis, in showing impaired efferocytosis capacities of blood MDM in SSc, our study offers new pathogenesis considerations for the involvement of macrophages in the autoimmune processes driving this disorder.

200 Evaluating novel peptide therapeutics targeting pathogenic macrophages in systemic sclerosis

200 Evaluating novel peptide therapeutics targeting pathogenic macrophages in systemic sclerosis

The cytokine language of monocytes and macrophages in systemic sclerosis

Many important observations suggest monocyte/macrophage involvement in systemic sclerosis (SSc). A high concentration of immune mediators, such as IL-6, IL-10 and IL-13, the infiltration of mononuclear cells in affected organs and the production of autoantibodies suggest that immune system dysfunction drives SSc pathogenesis. The recently reported study by Higashi-Kuwata and colleagues, in light of other observations, provides further insight into activation of macrophages/monocytes in SSc patients, suggesting that these cells undergo distinct activation pathways. These results emphasize the need for more detailed analyses of the several markers now defined in SSc peripheral blood mononuclear cells and tissues to better define the cytokine language speaking to monocytes/macrophages in SSc that promote vascular injury and tissue fibrosis.

Effect of N-acetyl- l-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O 2 −) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO −) and O 2 − production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO − and O 2 − was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5×10 −5 M, 24 h) significantly reduced (−21%) the ONOO − production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O 2 − both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO −, NAC administration reduces ONOO − production and can be an useful treatment to alleviate SSc symptoms.

Endothelin-1 secretion by alveolar macrophages in systemic sclerosis.

Endothelin-1 (ET-1), a potent fibroblast/smooth muscle cells mitogen, has been implicated in the pathogenesis of systemic sclerosis lung disease (SSc). Since monocytes and macrophages are thought to be activated in SSc, we hypothesized that alveolar macrophages (AM) and their precursors blood monocytes from patients with SSc produced more ET-1 than cells from healthy subjects. ET-1 and big ET-1 concentrations were measured in plasma, in bronchoalveolar lavage (BAL) fluids and in cell culture supernatants from monocytes and alveolar macrophages derived from 13 patients with definite SSc with lung involvement and from 10 control subjects. Plasma and BAL fluid ET-1 and big ET-1 levels were similar in both controls and patients with SSc. ET-1 and big ET-1 concentrations in unstimulated alveolar macrophage supernatants were similar in both groups. In contrast, LPS-stimulated alveolar macrophages from patients with SSc secreted higher amounts of ET-1 and big ET-1 than control subjects. ET-1 and big ET-1 concentrations in monocyte supernatants (either LPS-stimulated or not) were not different in patients and controls. These results show that AM from patients with SSc are hyperresponsive to LPS in vitro in terms of ET-1 and big ET-1 production and suggest that AM could participate in vivo in the overproduction of this potentially profibrotic mediator in patients with SSc.

Interleukin 6 secretion by monocytes and alveolar macrophages in systemic sclerosis with lung involvement.

Interleukin-6 (IL-6) has been involved in the pathogenesis of inflammatory and/or autoimmune diseases. We characterized the production of IL-6 by blood monocytes and alveolar macrophages (AM) in 11 patients with definite systemic sclerosis (SSc) with lung involvement and in eight normal control subjects. IL-6 levels were determined in serum, bronchoalveolar lavage (BAL) supernatants, monocytes, and AM cell culture supernatants using an ELISA kit. BAL cell analysis evidenced an alveolitis with hypercellularity and increased neutrophils and mast cells absolute counts. Serum and BAL IL-6 levels were low and similar in SSc and control groups. The monocytes of the group with SSc secreted more IL-6 than did the control group, both spontaneously (p = 0.01) and after LPS stimulation (p = 0.0007). Spontaneous secretion of IL-6 by AM tended to be higher in the SSc group than in the control group (p = 0.22). LPS-induced IL-6 secretion by AM was similar in both groups. Our study demonstrates that during SSc lung disease, spontaneous and stimulated IL-6 secretion by blood monocytes is increased, compared with secretion by healthy control subjects. By contrast, IL-6 secretion by AM is normal despite evidence of mild alveolitis.