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This study was designed to delineate the functional significance of CCL21 in metabolic reprogramming in experimental arthritis and differentiated rheumatoid arthritis (RA) macrophages (MΦs). To characterize the influence of CCL21 on immunometabolism, its mechanism of action was elucidated by dysregulating glucose uptake in preclinical arthritis and RA MΦs. In CCL21 arthritic joints, the glycolytic intermediates hypoxia-inducible factor 1α (HIF1α), cMYC and GLUT1 were overexpressed compared with oxidative regulators estrogen-related receptor γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1)-α. Interestingly, 2-deoxy-D-glucose (2-DG) therapy mitigated CCL21-induced arthritis by restraining the number of joint F4/80 + iNOS + MΦs without impacting F4/80 + Arginase + MΦs. Similar to the preclinical findings, blockade of glycolysis negated CCL21-polarized CD14 + CD86 + GLUT + MΦ frequency; however, CD14 + CD206 + GLUT + MΦs were not implicated in this process. In CCL21-induced arthritis and differentiated RA MΦs, the inflammatory imprint was uniquely intercepted by 2-DG via interleukin-6 (IL-6) downregulation. Despite the more expansive inflammatory response of CCL21 in the arthritic joints relative to the differentiated RA MΦs, 2-DG was ineffective in joint tumor necrosis factor-α, IL-1β, CCL2 and CCL5 enrichment. By contrast, disruption of glycolysis markedly impaired CCL21-induced HIF1α and cMYC signaling in arthritic mice. Notably, in RA MΦs, glycolysis interception was directed toward dysregulating CCL21-enhanced HIF1α transcription. Nonetheless, in concurrence with the diminished IL-6 levels, CCL21 differentiation of CD14 + CD86 + GLUT1 + MΦs was reversed by glycolysis and HIIF1α inhibition. Moreover, in the CCL21 experimental arthritis or differentiated RA MΦs, the malfunctioning metabolic machinery was accompanied by impaired oxidative phosphorylation because of reduced PGC1α or peroxisome proliferator-activated receptor-γ expression. CCL21 reconfigures naïve myeloid cells into glycolytic RA CD14 + CD86 + GLUT + IL-6 high HIF1α high MΦs. Therefore, inhibiting the CCL21/CCR7 pathway may provide a promising therapeutic strategy., (© 2021 Australian and New Zealand Society for Immunology, Inc.)

Recent studies show a connection between glycolysis and inflammatory response in rheumatoid arthritis (RA) macrophages (MΦs) and fibroblasts (FLS). Yet, it is unclear which pathways could be targeted to rebalance RA MΦs and FLS metabolic reprogramming. To identify novel targets that could normalize RA metabolic reprogramming, TLR7-mediated immunometabolism was characterized in RA MΦs, FLS and experimental arthritis. We uncovered that GLUT1, HIF1α, cMYC, LDHA and lactate were responsible for the TLR7-potentiated metabolic rewiring in RA MΦs and FLS, which was negated by IRAK4i. While in RA FLS, HK2 was uniquely expanded by TLR7 and negated by IRAK4i. Conversely, TLR7-driven hypermetabolism, non-oxidative PPP (CARKL) and oxidative phosphorylation (PPARγ) were narrowly dysregulated in TLR7-activated RA MΦs and FLS and was reversed by IRAK4i. Consistently, IRAK4i therapy disrupted arthritis mediated by miR-Let7b/TLR7 along with impairing a broad-range of glycolytic intermediates, GLUT1, HIF1α, cMYC, HK2, PFKFB3, PKM2, PDK1 and RAPTOR. Notably, inhibition of the mutually upregulated glycolytic metabolites, HIF1α and cMYC, was capable of mitigating TLR7-induced inflammatory imprint in RA MΦs and FLS. In keeping with IRAK4i, treatment with HIF1i and cMYCi intercepted TLR7-enhanced IRF5 and IRF7 in RA MΦs, distinct from RA FLS. Interestingly, in RA MΦs and FLS, IRAK4i counteracted TLR7-induced CARKL reduction in line with HIF1i. Whereas, cMYCi in concordance with IRAK4i, overturned oxidative phosphorylation via PPARγ in TLR7-activated RA MΦs and FLS. The blockade of IRAK4 and its interconnected intermediates can rebalance the metabolic malfunction by obstructing glycolytic and inflammatory phenotypes in RA MΦs and FLS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recent studies have shown the significance of metabolic reprogramming in immune and stromal cell function. Yet, the metabolic reconfiguration of RA macrophages (MΦs) is incompletely understood during active disease and in crosstalk with other cell types in experimental arthritis. This study elucidates a distinct regulation of glycolysis and oxidative phosphorylation in RA MΦs compared to fibroblast (FLS), although PPP (Pentose Phosphate pathway) is similarly reconfigured in both cell types. 2-DG treatment showed a more robust impact on impairing the RA M1 MΦ-mediated inflammatory phenotype than IACS-010759 (IACS, complexli), by reversing ERK, AKT and STAT1 signaling, IRF8/3 transcription and CCL2 or CCL5 secretion. This broader inhibitory effect of 2-DG therapy on RA M1 MΦs was linked to dysregulation of glycolysis (GLUT1, PFKFB3, LDHA, lactate) and oxidative PPP (NADP conversion to NADPH), while both compounds were ineffective on oxidative phosphorylation. Distinctly, in RA FLS, 2-DG and IACS therapies constrained LPS/IFNγ-induced AKT and JNK signaling, IRF5/7 and fibrokine expression. Disruption of RA FLS metabolic rewiring by 2-DG or IACS therapy was accompanied by a reduction of glycolysis (HIF1α, PFKFB3) and suppression of citrate or succinate buildup. We found that 2-DG therapy mitigated CIA pathology by intercepting joint F480 + iNOS + MΦ, Vimentin + fibroblast and CD3 + T cell trafficking along with downregulation of IRFs and glycolytic intermediates. Surprisingly, IACS treatment was inconsequential on CIA swelling, cell infiltration, M1 and Th1/Th17 cytokines (IFN-γ/IL-17) and joint glycolytic mediators. Collectively, our results indicate that blockade of glycolysis is more effective than inhibition of complex 1 in CIA, in part due to its effectiveness on the MΦ inflammatory phenotype., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objective: In rheumatoid arthritis (RA), elevated serum interleukin-34 (IL-34) levels are linked with increased disease severity. IL-34 binds to 2 receptors, macrophage colony-stimulating factor receptor (M-CSFR) and syndecan 1, which are coexpressed in RA macrophages. Expression of both IL-34 and syndecan 1 is strikingly elevated in the RA synovium, yet their mechanisms of action remain undefined. This study was undertaken to investigate the mechanism of action of IL-34 in RA., Methods: To characterize the significance of IL-34 in immunometabolism, its mechanism of action was elucidated in joint macrophages, fibroblasts, and T effector cells using RA and preclinical models., Results: Intriguingly, syndecan 1 activated IL-34-induced M-CSFR phosphorylation and reprogrammed RA naive cells into distinctive CD14+CD86+GLUT1+ M34 macrophages that expressed elevated levels of IL-1β, CXCL8, and CCL2. In murine M34 macrophages, the inflammatory phenotype was accompanied by potentiated glycolytic activity, exhibited by transcriptional up-regulation of GLUT1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α) and amplified pyruvate and l-lactate secretion. Local expression of IL-34 provoked arthritis by expanding the glycolytic F4/80-positive, inducible nitric oxide synthase (iNOS)-positive macrophage population, which in turn attracted fibroblasts and polarized Th1/Th17 cells. The cross-talk between murine M34 macrophages and Th1/Th17 cells broadened the inflammatory and metabolic phenotypes, resulting in the expansion of IL-34 pathogenicity. Consequently, IL-34-instigated joint inflammation was alleviated in RAG -/- mice compared to wild-type mice. Syndecan 1 deficiency attenuated IL-34-induced arthritis by interfering with joint glycolytic M34 macrophage and osteoclast remodeling. Similarly, inhibition of glycolysis by 2-deoxy-d-glucose reversed the joint swelling and metabolic rewiring triggered by IL-34 via HIF-1α and c-Myc induction., Conclusion: IL-34 is a novel endogenous factor that remodels hypermetabolic M34 macrophages and facilitates their cross-regulation with T effector cells to advance inflammatory bone destruction in RA., (© 2021, American College of Rheumatology.)

The molecular mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein was characterized to identify novel therapies. The impact of tofacitinib, IL-6R Ab, or TNFi therapy was determined on Spike protein or LPS/IFN-γ-induced signaling, inflammation, and metabolic reprogramming in MΦs and/or rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS). ACE2 frequency was markedly expanded in MΦs compared to T cells and RA FLS. Tofacitinib suppresses Spike protein potentiated STAT1 signaling, whereas this function was unchanged by TNFi. Tofacitinib impairs IL-6/IFN/LPS-induced STAT1 and STAT3 phosphorylation in RA MΦs and FLS. Interestingly, tofacitinib had a broader inhibitory effect on the monokines, glycolytic regulators, or oxidative metabolites compared to IL-6R Ab and TNFi in Spike-protein-activated MΦs. In contrast, all three therapies disrupted IFN-α and IFN-β secretion in response to Spike protein; nonetheless, the IFN-γ was only curtailed by tofacitinib or IL-6R Ab. While tofacitinib counteracted MΦ metabolic rewiring instigated by Spike protein, it was inconsequential on the glycolysis expansion mediated via HK2 and/or LDHA in the activated RA MΦ and FLS. Nevertheless, the potentiated inflammatory response and the diminished oxidative phosphorylation modulated by Spike protein and/or LPS/IFN-γ stimulation in MΦs or RA FLS were reversed by tofacitinib. In conclusion, tofacitinib suppresses MΦ inflammation and immunometabolism triggered by Spike protein and may provide a promising strategy for COVID-19 patients., (© 2021 Wiley-VCH GmbH.)

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation., (© 2021 Wiley-VCH GmbH.)

Thirty percent of psoriasis patients develop psoriatic arthritis (PsA), nevertheless the mechanism remains unknown. Endogenous GU-rich miRNAs activate endosomal TLR7 that plays a critical role in autoimmune diseases. We found that endogenous TLR7 ligands, miR-29 and miR-Let7b, were markedly increased in PsA compared to osteoarthritis (OA) synovial fluid (SF)s. We showed that intradermal (i.d.) miR-Let7b injection promoted skin inflammation, which was characterized by amplified Th1 cells, CD68 + M1 macrophages, and transcriptional upregulation of glycolytic mediators, GLUT1, C-MYC, and HIF1α. Expansion of skin Th1 cells driven by miR-Let7b was also linked to elevated M1-associated IRFs. Interestingly, i.d. miR-Let7b administration exacerbated suboptimal joint inflammation along with metabolic reconfiguration of the PsA-like preclinical model. Moreover, TLR7 agonist, R837, potentiated metabolic reprogramming and expression of IL-1β, IL-6, and IL-12 in murine macrophages, enabling myeloid-to-T-cell crosstalk. Consistently, treatment with glycolytic inhibitors, 2-DG and/or HIF1αi, reversed R837-induced metabolic remodeling and disrupted the TLR7-driven inflammatory phenotype in myeloid and lymphoid cells. Similar to miR-Let7b, R837 also differentiates progenitor cells into mature osteoclasts, primarily through RANKL induction. Taken together, this study indicates that TLR7-instigated metabolic rewiring of macrophages and their cross-regulation of T cells connects skin immunopathology to joint inflammation., (© 2020 Wiley-VCH GmbH.)

Synovial macrophages are crucial in the development of joint inflammation and bone damage; however, the pathways that control macrophage remodeling in inflammatory M1 cells or bone-eroding osteoclasts are not fully understood. We determined that elevated IL-7R/CD127 expression is the hallmark of rheumatoid arthritis (RA) M1 macrophages and that these cells are highly responsive to interleukin-7 (IL-7)-driven osteoclastogenesis. We established that lipopolysaccharide (LPS), interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), the classic M1 macrophage mediators, enhance IL-7R expression in RA and murine macrophages. The local expression of IL-7 provokes arthritis, predominantly through escalating the number of F480 + iNOS + cells rather than CD3 + T cells. Ectopic LPS injection stabilizes IL-7-induced arthritis by increasing myeloid IL-7R expression, in part via IFNγ induction. Hence, in RAG-/- mice, IL-7-mediated arthritis is suppressed because of the reduction in myeloid IL-7R expression due to the lack of IFNγ. Moreover, the amelioration of IL-7-induced arthritis by anti-TNF therapy is due to a decrease in the number of cells in the unique F480 + iNOS + IL-7R + CCL5 + subset, with no impact on the F480 + Arginase + cell or CD3 + T cell frequency. Consistent with the preclinical findings, the findings of a phase 4 study performed with RA patients following 6 months of anti-TNF therapy revealed that IL-7R expression was reduced without affecting the levels of IL-7. This study shifts the paradigm by discovering that IL-7-induced arthritis is dependent on F480 + iNOS + IL-7R + CCL5 + cell function, which activates TH-1 cells to amplify myeloid IL-7R expression and disease severity.

In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.

Immunosenescence is a state of unbalanced immune responsiveness, characterized by a diverse repertoire of seemingly discreet and paradoxical alterations in all aspects of immunity arising in an aging-associated manner. We asked whether aging-associated alterations in the ability of apoptotic cells to elicit immunomodulatory responses (innate apoptotic immunity; IAI) or in IAI responses themselves might underlie the confounding aging-associated anomalies of immunosenescence. We explored this question by examining, as a function of animal age, responsiveness of murine macrophages on the single cell level. We monitored the expression of pro- and anti-inflammatory cytokines cytofluorimetrically in response to pro-inflammatory Toll-like receptor (TLR) stimulation and anti-inflammatory treatment with apoptotic cells. While we found no alterations with age in the potency of apoptotic cells or in the initiation and magnitude of IAI responses, we did identify a cell-intrinsic deficiency in anti-inflammatory IAI response termination linked with age and preceding manifestations of immunosenescence. Further, we found that an aging-associated deficiency in response termination also is evident following TLR stimulation. These surprising observations reveal that a loss of homeostatic immune control with animal age results from the dysregulation of response termination (as distinct from response initiation) and is exerted on the level of transcription. We suggest that, with advancing age, cells become locked into relatively longer-lived response states. Aging-associated immune dysfunctions may reflect a diminution in the cellular nimbleness of immune responsiveness., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Aging is associated with a waning of normal immune function. This "immunosenescence" is characterized by a diverse repertoire of seemingly discreet and unbalanced immune alterations. A number of studies have suggested that aging-associated alterations in innate immune responsiveness, especially responsiveness dependent on Toll-like Receptor (TLR) engagement, are causally involved. We find, however, that the magnitude and dose-dependency of responsiveness to TLR engagement (assessed with respect to cytokine production) in distinct populations of murine macrophages are not altered generally with animal age or as a consequence of immunosenescence. Responses elicited with a wide array of TLR agonists were examined by extensive functional analyses, principally on the level of the individual cell. These studies reveal an intriguing "all-or-nothing" response behavior of macrophages, independent of animal age. Although reports to the contrary have been cited widely, aging-associated immune decline cannot be attributed to widespread alterations in the extents of TLR-dependent innate immune macrophage responses., Competing Interests: The authors declare that they have no conflicts or competing commercial interests in relation to this work., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)