Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint ...homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTK
TREM2
and MerTK
LYVE1
) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTK
STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTK
STM subpopulations could therefore be a potential treatment strategy for RA.
MicroRNA-155 (miR-155) is an important regulator of B cells in mice. B cells have a critical role in the pathogenesis of rheumatoid arthritis (RA). Here we show that miR-155 is highly expressed in ...peripheral blood B cells from RA patients compared with healthy individuals, particularly in the IgD
CD27
memory B-cell population in ACPA
RA. MiR-155 is highly expressed in RA B cells from patients with synovial tissue containing ectopic germinal centres compared with diffuse synovial tissue. MiR-155 expression is associated reciprocally with lower expression of PU.1 at B-cell level in the synovial compartment. Stimulation of healthy donor B cells with CD40L, anti-IgM, IL-21, CpG, IFN-α, IL-6 or BAFF induces miR-155 and decreases PU.1 expression. Finally, inhibition of endogenous miR-155 in B cells of RA patients restores PU.1 and reduces production of antibodies. Our data suggest that miR-155 is an important regulator of B-cell activation in RA.
Current treatments for rheumatoid arthritis (RA) do not reverse underlying aberrant immune function. A genetic predisposition to RA, such as HLA-DR4 positivity, indicates that dendritic cells (DC) ...are of crucial importance to pathogenesis by activating auto-reactive lymphocytes. Here we show that microRNA-34a provides homoeostatic control of CD1c
DC activation via regulation of tyrosine kinase receptor AXL, an important inhibitory DC auto-regulator. This pathway is aberrant in CD1c
DCs from patients with RA, with upregulation of miR-34a and lower levels of AXL compared to DC from healthy donors. Production of pro-inflammatory cytokines is reduced by ex vivo gene-silencing of miR-34a. miR-34a-deficient mice are resistant to collagen-induced arthritis and interaction of DCs and T cells from these mice are reduced and do not support the development of Th17 cells in vivo. Our findings therefore show that miR-34a is an epigenetic regulator of DC function that may contribute to RA.
The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like ...domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.
Inflammation can be protective or pathogenic depending on context and timeframe. Acute inflammation, including the accumulation of CD4 T cells, accompanies protective immune responses to pathogens, ...but the presence of activated CD4 T cells at sites of inflammation is associated with chronic inflammatory disease. While significant progress has been made in understanding the migration of CD4 T cells into inflamed sites, the signals that lead to their persistence are poorly characterized. Using a murine ear model of acute inflammation and intravital two-photon imaging, we have dissected the signals that mediate CD4 T cell persistence. We report the unexpected finding that the bioactive lipid, sphingosine-1-phosphate (S1P), is both necessary and sufficient for the persistence of activated CD4 T cells at peripheral tissues in acute inflammation. S1P mediated the enhanced motility of CD4 T cells at inflamed tissues but did not affect their migration to the downstream draining lymph node. We found that sphingosine kinase-1, which regulates S1P production is increased at inflamed sites in mice and in patients with the chronic inflammatory disease, rheumatoid arthritis. Together, these data suggest that S1P, or its regulators, may be key targets to promote or disrupt accumulation of CD4 T cells at inflamed tissues.
We explored the potential link between chronic inflammatory arthritis and COVID-19 pathogenic and resolving macrophage pathways and their role in COVID-19 pathogenesis. We found that bronchoalveolar ...lavage fluid (BALF) macrophage clusters FCN1+ and FCN1+SPP1+ predominant in severe COVID-19 were transcriptionally related to synovial tissue macrophage (STM) clusters CD48hiS100A12+ and CD48+SPP1+ that drive rheumatoid arthritis (RA) synovitis. BALF macrophage cluster FABP4+ predominant in healthy lung was transcriptionally related to STM cluster TREM2+ that governs resolution of synovitis in RA remission. Plasma concentrations of SPP1 and S100A12 (key products of macrophage clusters shared with active RA) were high in severe COVID-19 and predicted the need for Intensive Care Unit transfer, and they remained high in the post-COVID-19 stage. High plasma levels of SPP1 were unique to severe COVID-19 when compared with other causes of severe pneumonia, and IHC localized SPP1+ macrophages in the alveoli of COVID-19 lung. Investigation into SPP1 mechanisms of action revealed that it drives proinflammatory activation of CD14+ monocytes and development of PD-L1+ neutrophils, both hallmarks of severe COVID-19. In summary, COVID-19 pneumonitis appears driven by similar pathogenic myeloid cell pathways as those in RA, and their mediators such as SPP1 might be an upstream activator of the aberrant innate response in severe COVID-19 and predictive of disease trajectory including post-COVID-19 pathology.
To test the hypothesis that miR-155 regulates monocyte migratory potential via modulation of chemokine and chemokine receptor expression in RA, and thereby is associated with disease activity.
The ...miR-155 copy-numbers in monocytes from peripheral blood (PB) of healthy (n = 22), RA (n = 24) and RA SF (n = 11) were assessed by real time-PCR using synthetic miR-155 as a quantitative standard. To evaluate the functional impact of miR-155, human monocytes were transfected with control or miR-155 mimic, and the effect on transcript levels, and production of chemokines was evaluated by Taqman low-density arrays and multiplex assays. A comparative study evaluated constitutive chemokine receptor expression in miR-155
and wild-type murine (CD115
Ly6C
Ly6G
) monocytes.
Compared with healthy monocytes, the miR-155 copy-number was higher in RA, peripheral blood (PB) and SF monocytes (PB P < 0.01, and SF P < 0.0001). The miR-155 copy-number in RA PB monocytes was higher in ACPA-positive compared with ACPA-negative patients (P = 0.033) and correlated (95% CI) with DAS28 (ESR), R = 0.728 (0.460, 0.874), and with tender, R = 0.631 (0.306, 0.824) and swollen, R = 0.503 (0.125, 0.753) joint counts. Enforced-expression of miR-155 in RA monocytes stimulated the production of CCL3, CCL4, CCL5 and CCL8; upregulated CCR7 expression; and downregulated CCR2. Conversely, miR155
monocytes showed downregulated CCR7 and upregulated CCR2 expression.
Given the observed correlations with disease activity, these data provide strong evidence that miR-155 can contribute to RA pathogenesis by regulating chemokine production and pro-inflammatory chemokine receptor expression, thereby promoting inflammatory cell recruitment and retention in the RA synovium.
Objective
We previously reported an increased expression of microRNA‐155 (miR‐155) in the blood monocytes of patients with rheumatoid arthritis (RA) that could be responsible for impaired monocyte ...polarization to anti‐inflammatory M2‐like macrophages. In this study, we employed two preclinical models of RA, collagen‐induced arthritis and K/BxN serum transfer arthritis, to examine the therapeutic potential of antagomiR‐155‐5p entrapped within PEGylated (polyethylene glycol PEG) liposomes in resolution of arthritis and repolarization of monocytes towards the anti‐inflammatory M2 phenotype.
Methods
AntagomiR‐155‐5p or antagomiR‐control were encapsulated in PEG liposomes of 100 nm in size and −10 mV in zeta potential with high antagomiR loading efficiency (above 80%). Mice were injected intravenously with 1.5 nmol/100 μL PEG liposomes containing antagomiR‐155‐5p or control after the induction of arthritis.
Results
We demonstrated the biodistribution of fluorescently tagged PEG liposomes to inflamed joints one hour after the injection of fluorescently tagged PEG liposomes, as well as the liver's subsequent accumulation after 48 hours, indicative of hepatic clearance, in mice with arthritis. The injection of PEG liposomes containing antagomiR‐155‐5p decreased arthritis score and paw swelling compared with PEG liposomes containing antagomiR‐control or the systemic delivery of free antagomiR‐155‐5p. Moreover, treatment with PEG liposomes containing antagomiR‐155‐5p led to the restoration of bone marrow monocyte defects in anti‐inflammatory macrophage differentiation without any significant functional change in other immune cells, including splenic B and T cells.
Conclusion
The injection of antagomiR‐155‐5p encapsulated in PEG liposomes allows the delivery of small RNA to monocytes and macrophages and reduces joint inflammation in murine models of RA, providing a promising strategy in human disease.
Introduction We previously reported an increased expression of miR‐155 in rheumatoid arthritis (RA) patients blood monocytes that could be responsible for impaired monocyte polarization to ...anti‐inflammatory M2‐like macrophages. In this study, we employed two pre‐clinical models of RA: the Collagen‐Induce‐Arthritis (CIA) and the K/BxN Serum‐Transfer‐Arthritis (STA), to examine the therapeutic potential of antagomiR‐155‐5p entrapped within PEGylated (PEG)‐liposomes in resolution of arthritis and re‐polarization of monocytes towards anti‐inflammatory M2 phenotype. Methods AntagomiR‐155‐5p or antagomiR‐control were encapsulated in PEG‐liposomes of 100 nm in size and ‐10mV in zeta potential with high antagomiR loading efficiency (above 80%). Mice were injected intravenously with 1,5nmol/100μL PEG‐liposomes containing antagomiR‐155‐5p or control after induction of arthritis. Results We demonstrated the biodistribution of fluorescently tagged‐PEG‐liposomes to inflamed joints 1 hour after injection of fluorescently tagged‐PEG‐liposomes and as well as their subsequent liver's accumulation after 48 hours, indicative of hepatic clearance, in arthritic mice. Injection of PEG‐liposomes containing antagomiR‐155‐5p decreased arthritis score and paw swelling as compared to PEG‐liposomes containing antagomiR‐control or systemic delivery of free antagomiR‐155‐5p. Moreover, treatment with PEG‐liposomes containing antagomiR‐155‐5p lead to the restoration of bone marrow monocytes defect in anti‐inflammatory macrophage differentiation without any significant functional change in other immune cells including splenic B and T cells. Conclusion The injection of antagomiR‐155‐5p encapsulated in PEG‐liposomes allows delivering small RNA to monocytes/macrophages, reduced joint inflammation in murine models of RA, providing a promising strategy in human disease. image
Abstract
Background
Dendritic cells (DCs) direct immune responses against pathogens while maintaining tolerance to self-antigens. However, their aberrant activation can lead to autoimmunity and ...inflammation. DCs consist of two subtypes: plasmacytoid and myeloid DCs. Recently, single-cell sequencing has revealed complex heterogeneity within myeloid DCs. These cells can be divided into CD141pos (DC1), DC2 defined as CD1highCD163neg and DC3 defined as CD1clowCD163high. In addition, a population of CD1cnegCD141negCD16pos (DC4), which share some gene expression with CD16pos monocytes, has been identified. To date, most studies of RA investigated DC precursors in circulation or synovial fluid (SF). However, DCs perform their functions within lymphoid or peripheral tissue. Therefore, in this study, we sought to characterise myeloid DC subsets in synovial tissue (ST) with the prospect of better understanding their role in driving autoimmunity in RA.
Methods
We developed a flow sorting strategy to characterise the phenotype of distinct myeloid DC subsets in multiple biological compartments (PB, SF, and ST). Ultrasound-guided ST biopsies (RA n = 9; Psoriatic arthritis n = 3 with active disease) were digested with liberase prior to analysis. PB DCs (RA n = 19, Psoriatic arthritis n = 16, healthy donors n = 12), and SF DC (n = 3) were used as comparators. ST, SF and PB DCs were sorted then microRNA, pro-inflammatory and regulatory cytokine expression analysed by amplified qPCR. To evaluate the role of miR-155 in RA CD1cpos DC activation, CD1cpos cells were computationally sub-setted from a scRNAseq synovial dataset comparing synovial tissues of healthy individuals (n = 4) with patients with active RA (n = 7) and those in sustained clinical and ultrasound remission (n = 7). To elucidate the role of miR-155, CD1cpos DC from RA patients (n = 10) were sorted then transfected with miR-155 mimic or control mimic and the cytokines expression and produced were evaluated using quantitative RT-PCR and Luminex, respectively.
Results
Our data revealed that whilst all myeloid DCs subsets can be present in inflamed RA synovium they occur with variable frequencies. CD1cpos (DC2/3) being the most abundant, followed by CD16pos DCs (DC4), whilst CD141pos DCs (DC1) occur in low numbers or are absent. CD1cpos DC sorted from RA ST express high levels of epigenetic regulators of inflammatory response miR-155, IL-6, TNF-a and IL-23 as compared to circulating cells.Unsupervised clustering of synovial CD1c cells identified 4 separate clusters including a distinctive miR155 positive CD1c cluster in RA patients. These cells showed significantly higher expression of pro-inflammatory cytokines and co-stimulatory molecules compared to other synovial CD1c clusters. Overexpression of miR-155 in CD1cpos leads to increased production of TNF-a and IL-23.
Conclusion
Our data show that miR-155 is strongly implicated as an epigenetic regulator of the pro-inflammatory synovial CD1cpos DCs sub-cluster and contributes to exacerbating RA.
Disclosures
A. Elmesmari None. L. MacDonald None. D. Vaughan None. B. Tolusso None. L. Bui None. M. Gigante None. F. Federico None. G. Ferraccioli None. E. Gremese None. I. B McInnes None. S. Alivernini None. M. Kurowska-Stolarska None.
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