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Metabolic regulation of RA macrophages is distinct from RA fibroblasts and blockade of glycolysis alleviates inflammatory phenotype in both cell types

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Abstract

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.

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Funding

This work was supported in part by awards from the Department of Veteran’s Affairs MERIT Award BX002286, the National Institutes of Health NIH AI147697, the National Psoriasis Foundation (NPF), Pfizer Investigator-Initiated Research (IIR) Program and Chicago Biomedical Consortium (CBC) Accelerator Award.

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Authors and Affiliations

  1. Jesse Brown VA Medical Center, Chicago, IL, USA

    Sadiq Umar, Karol Palasiewicz & Shiva Shahrara

  2. Department of Medicine, Division of Rheumatology, The University of Illinois at Chicago, 840 S Wood Street, CSB suite 1114, Chicago, IL, 60612, USA

    Sadiq Umar, Karol Palasiewicz, Bianca Romay, Rani Rahat, Chandana Tetali, Shiva Arami, Nadera Sweiss, Ryan K. Zomorrodi & Shiva Shahrara

  3. Department of Microbiology and Immunology, Midwestern University, Downers Grove, IL, USA

    Michael V. Volin

  4. Division of Rheumatology, Allergy and Immunology, San Diego, School of Medicine, University of California, La Jolla, CA, USA

    Monica Guma

  5. VA Medical Center, San Diego, CA, USA

    Monica Guma

  6. Division of Pulmonary, Critical Care, Sleep, and Allergy, The University of Illinois at Chicago, Chicago, IL, USA

    Christian Ascoli

  7. School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

    Luke A. J. O’Neill

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Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. SS had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. SU, SS. Acquisition of data. SU, KP, MVV, BR, RR, CT, RZ, SS. Analysis and interpretation of data. SU, KP, MVV, BR, RR, CT, MG, CA, RZ, LO, SS. Providing crucial reagents. SA, NS.

Corresponding author

Correspondence to Shiva Shahrara.

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The authors have declared that no commercial or financial conflict of interest exists.

Ethics approval and consent to participate

All peripheral blood (PB) was collected in accordance with our protocol approved by the University of Illinois at Chicago Institutional Ethics Review Board. All the RA patients have consented to participate in this study and have provided written consent. Furthermore, all animal studies were approved by the University of Illinois at Chicago Animal Care and Use Committee following the legal requirements and guidelines of the state of Illinois in the USA and NIH.

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Supplementary Information

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18_2021_3978_MOESM1_ESM.tif

Supplementary file1 RA monocyte-differentiated MΦs were treated with PBS or LPS/IFNγ (100ng/ml each) for 24h before quantifying the F480+CD86+ cells or F480+CD206+ MΦs, (A1-A2, n=4). RA MΦs were pretreated with DMSO, 2-DG (5mM) or IACS (100nM) o/n. Thereafter cells were untreated (PBS) or stimulated with LPS/IFNγ (100ng/ml each) for 24h before quantifying pyruvate (Sigma MAK332; A3) via colorimetric assay (n=5) or for 6h before measuring HIF1α (A4) transcription by real-time RT-PCR, n=6. The data are shown as mean ± SEM, ** represents p<0.01 and **** denotes p<0.0001 (TIF 199 KB)

18_2021_3978_MOESM2_ESM.tif

Supplementary file2 Western blot density was quantified by Image J in Fig. 1A (B1) and Fig. 2M (B2) and was normalized to equal loading, n=3. The data are shown as mean ± SEM, * represents p<0.05 and ** denotes p<0.01 (TIF 135 KB)

18_2021_3978_MOESM3_ESM.tif

Supplementary file3 Western blot density was quantified by Image J in Fig. 3A (C1) and Fig. 4K (C2) and was normalized to equal loading, n=3. RA FLS were pretreated with DMSO, 2-DG (5mM) or IACS (100nM) o/n. Thereafter cells were untreated (PBS) or stimulated with LPS/IFNγ (100ng/ml each) for 24h before quantifying pyruvate (Sigma MAK332) via colorimetric assay (C3, n=5). To provide a rationale for signaling timepoint selection (30min), RA FLS were treated with LPS/IFNγ (100ng/ml each) for 0-60min and lysates were probed for pERK, pSTAT1and actin (C4). The data are shown as mean ± SEM, * represents p<0.05 and ** denotes p<0.01 (TIF 226 KB)

18_2021_3978_MOESM4_ESM.tif

Supplementary file4 Images with higher magnification are provided for joint F480, iNOS and Arginase staining in CIA mice treated with control, 2-DG or IACS (D1). CIA ankles treated with control, 2-DG or IACS were immunostained with F480 red (1;100, Genetex), iNOS green (Santa Cruz, 1:200), Arginase 1 green (Santa Cruz, 1:200). Each tissue section was analyzed using Image J’s Color threshold function. This function was used to quantify the double-positive cells. 7-20 fields were quantified per tissue, depending upon tissue size and their integrated densities (product of area and mean gray value) and values were averaged (D2 and D3). The data are shown as mean ± SEM, * represents p<0.05 (TIF 2353 KB)

Supplementary file5 All raw Western blots are shown (TIF 485 KB)

Supplementary file6 Demonstrate the gating strategy for effector T cells and macrophages (TIF 403 KB)

Supplementary file7 (JPG 318 KB)

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Umar, S., Palasiewicz, K., Volin, M.V. et al. Metabolic regulation of RA macrophages is distinct from RA fibroblasts and blockade of glycolysis alleviates inflammatory phenotype in both cell types. Cell. Mol. Life Sci. 78, 7693–7707 (2021). https://doi.org/10.1007/s00018-021-03978-5

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  • DOI: https://doi.org/10.1007/s00018-021-03978-5

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