The Autoimmune Susceptibility Gene C5orf30 Regulates Macrophage-Mediated Resolution of

This information is current as Emma R. Dorris, Simon J. Tazzyman, John Moylett, of September 25, 2021. Nandhini Ramamoorthi, Jason Hackney, Michael Townsend, Munitta Muthana, Myles J. Lewis, Costantino Pitzalis and Anthony G. Wilson J Immunol 2019; 202:1069-1078; Prepublished online 18

January 2019; Downloaded from doi: 10.4049/jimmunol.1801155 http://www.jimmunol.org/content/202/4/1069

Supplementary http://www.jimmunol.org/content/suppl/2019/01/17/jimmunol.180115 http://www.jimmunol.org/ Material 5.DCSupplemental References This article cites 73 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/202/4/1069.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Autoimmune Susceptibility Gene C5orf30 Regulates Macrophage-Mediated Resolution of Inflammation

Emma R. Dorris,* Simon J. Tazzyman,† John Moylett,* Nandhini Ramamoorthi,‡ Jason Hackney,‡ Michael Townsend,‡ Munitta Muthana,† Myles J. Lewis,x Costantino Pitzalis,x and Anthony G. Wilson*

Genetic variants in C5orf30 have been associated with development of the autoimmune conditions primary biliary cirrhosis and rheumatoid arthritis. In rheumatoid arthritis, C5orf30 expression is -specific, with highest expression found in macrophages and synovial fibroblasts. C5orf30 is highly expressed in inflamed joints and is a negative regulator of tissue damage in a mouse model of inflammatory arthritis. Transcriptomic analysis from ultrasound-guided synovial biopsy of inflamed joints in a well characterized clinical cohort of newly diagnosed, disease-modifying antirheumatic drugs–naive rheumatoid arthritis patients was used to determine the clinical association of C5orf30 expression with disease activity. A combined molecular and computational Downloaded from biology approach was used to elucidate C5orf30 function in macrophages both in vitro and in vivo. Synovial expression of C5orf30 is inversely correlated with both clinical measures of rheumatoid arthritis disease activity and with synovial TNF mRNA expression. C5orf30 plays a role in regulating macrophage phenotype and is differentially turned over in inflammatory and anti-inflammatory macrophages. Inhibition of C5orf30 reduces wound healing/repair–associated functions of macrophages, re- duces signaling required for resolution of inflammation, and decreases secretion of anti-inflammatory mediators. In an animal model of wound healing (zebrafish), C5orf30 inhibition increases the recruitment of macrophages to the wound site. Finally, we http://www.jimmunol.org/ demonstrate that C5orf30 skews macrophage immunometabolism, demonstrating a mechanism for C5orf30-mediated immune regulation. The Journal of Immunology, 2019, 202: 1069–1078.

hromosome 5 open reading frame 30 (C5orf30) is ge- RASF and macrophages (5), two of the cell types implicated in netically associated with two autoimmune disorders: mediating chronic inflammation and tissue damage in RA. Having C rheumatoid arthritis (RA) and primary biliary cirrhosis previously elucidated the role of C5orf30 in RASF, in this article we (1, 2). A single polymorphism in the first intron of characterize the role of C5orf30 in macrophage biology.

C5orf30, rs26232, was first implicated as an autoimmune risk allele Macrophages have a crucial function in innate immunity via their by guest on September 25, 2021 in a genome-wide association study of RA (1) and subsequently role in initiating inflammation, regulating immune responses, and replicated in a British RA population (3). An allele dose association promoting tissue repair (6). Plasticity is a key feature of macro- of the low-risk T allele of rs26232 with reduced radiological phages and facilitates adaptive responses (7). Macrophages are damage of the joints of the hands and feet, a key measure of clinical dynamic, adapting to the microenvironment and reprogramming outcome in RA, was subsequently reported (4). their functional properties in response to stimuli such as those de- We have previously identified C5orf30 as a negative regulator of rived from microbes, activated and resting lymphocytes, and dam- the autoaggressive phenotype of RA synovial fibroblasts (RASF) and aged tissues (7). The M1/M2 paradigm is used to characterize a modulator of tissue damage in the collagen-induced arthritis model macrophage functional diversity (7–9): M1, or classically activated (5). Expression of C5orf30 in the joint is predominantly restricted to macrophages, are protypically proinflammatory; conversely, M2, or

*University College Dublin Centre for Arthritis Research, Conway Institute, Univer- A.G.W. conceived the overall study, supervised all experiments, and wrote and re- sity College Dublin, Dublin D04 W6F6, Ireland; †University of Sheffield, Sheffield viewed the manuscript. S10 2RX, United Kingdom; ‡Biomarker Discovery OMNI, Genentech Research and x The sequences presented in this article have been submitted to ArrayExpress (https:// Early Development, San Francisco, CA 94080; and William Harvey Research Insti- www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6141/) under accession number tute, Barts and The London School of Medicine and Dentistry and Barts Health E-MTAB-6141. National Health Service Trust, Queen Mary University of London, London EC1M 6BQ, United Kingdom Address correspondence and reprint requests to Dr. Emma R. Dorris, University College Dublin Centre for Arthritis Research, Conway Institute, University College ORCIDs: 0000-0003-2014-934X (E.R.D.); 0000-0002-3384-9989 (S.J.T.); 0000- Dublin, Dublin 4, Ireland. E-mail address: [email protected] 0002-8714-0166 (J.M.); 0000-0002-5922-563X (J.H.); 0000-0003-2497- 8903 (M.M.); 0000-0001-9365-5345 (M.J.L.); 0000-0003-1326-5051 (C.P.); 0000- The online version of this article contains supplemental material. 0003-4855-3926 (A.G.W.). Abbreviations used in this article: ActD, actinomycin D; CIAP, calf intestinal alkaline Received for publication August 23, 2018. Accepted for publication December 10, phosphate; C5orf30, chromosome 5 open reading frame 30; CST, Cell Signaling 2018. Technology; DAS, disease activity score; DAS28-ESR, disease activity score in 28 joints–erythrocyte sedimentation rate; 2DG, 2-deoxyglucose; ESR, erythrocyte sedi- This work was supported by Arthritis Ireland and the Health Research Board of mentation rate; KD, knockdown; Mw, macrophage; MDM, monocyte-derived macro- Ireland under the Medical Research Charities Group-Health Research Board (Grant phage; MMP, matrix metalloprotease; OXPHOS, oxidative phosphorylation; p , MRCG-2016-15) joint funding awards for 2017-2020 and by the Irish Research adj adjusted p value; PEAC, Pathobiology of Early Arthritis Cohort; RA, rheumatoid ar- Council (Grant GOIPD/2017/1310). thritis; RASF, RA synovial fibroblast; RNA-seq, RNA sequencing; ROS, reactive oxy- E.R.D. conceived, carried out macrophage and cell line experiments, and wrote and gen species; siC5orf30, small interfering RNA targeting C5orf30; siNTC, nontargeting reviewed the manuscript. S.J.T. carried out zebrafish experiments. J.M. carried out scramble control siRNA; siRNA, small interfering RNA; ST, synovial tissue. mRNA half-life experiments. M.M. conceived human and zebrafish experiments and wrote and reviewed the manuscript. N.R., J.H., M.T., M.J.L., and C.P. conceived, Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 carried out and analyzed the clinical studies, and wrote and reviewed the manuscript. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801155 1070 C5orf30 MEDIATES MACROPHAGE-DRIVEN INFLAMMATION alternatively activated macrophages, have important immunoregu- PE-Cy7-conjugated anti-CD68, FITC-conjugated anti-CD40, PE-conjugated latory and phagocytic activities and can promote angiogenesis, anti-CD80, and PerCP-Cy5.5 anti-CD209 (27, 28). Isotypic control labeling tissue remodeling, and repair (9). The M1/M2 paradigm is a useful was performed in parallel. Values were expressed as the ratio of the mean fluorescence intensity of the marker of interest over the mean fluorescence but oversimplified framework to describe the functional phenotype intensity of the isotype control. of macrophages. In reality, macrophages can adopt a wide array of phenotypes in response to the tissue microenvironment, and polar- RNA extraction and gene expression analysis ization exists on a spectrum between M1 and M2 extremities (10). RNA extraction and reverse were performed as previously Currently, little is known about C5orf30 biology (11, 12) and even described (29). RT-PCR TaqMan for assays C5orf30 (Hs00264241_m1), less about its role in macrophages (5). We report C5orf30 to be a IL-4R (Hs00965056_m1), and 18S rRNA (43108935) were performed in regulator of macrophage function: it is highly expressed in response multiplexed format as previously described (30). to anti-inflammatory stimulants, and inhibition of C5orf30 impedes extraction and Western blot analysis M2-like macrophage functions while enhancing certain M1-like Whole cell lysates were extracted and Western blotting was performed responses. We uncover new insights into C5orf30 physiology and as previously described (30). Primary Abs were as follows: anti-C5orf30 discover differential regulation of C5orf30 in M1- and M2-like [1/500; Prestige/Sigma (5)], anti–p-JNK (1/1000; Cell Signaling Tech- macrophages. An imbalance of macrophage M1–M2 polarization nology [CST]), anti-JNK (1/1000; CST), anti-HIF1a (1/1000; BD Trans- has been associated with RA and multiple inflammatory conditions duction Laboratories), anti–phospho-4E-BP (1/5000; CST), anti-COX2 b (13–17). Identification of the associated with the dynamic (1/1000; CST) and anti– -actin [1/5000; Sigma (31)]. Protein dephosphorylation used 30 mg of protein lysate treated with 30 U changes of macrophages is crucial for revealing the molecular basis of calf intestinal alkaline phosphate (CIAP) in 10 ml of CIAP buffer of disease progression (18, 19) and to fuel the innovation pipeline (Clontech) or buffer alone at 37˚C for 60 min. Untreated, control, and CIAP- Downloaded from for novel therapeutic strategies targeting inflammatory resolution treated lysates were analyzed by Western blot for C5orf30 expression. rather than suppression (20–23). The evidence presented in this t assays study identifies C5orf30 as an immunomodulator in macrophages. 1/2 Our data suggest that reduced C5orf30 expression plays a role in THP1-macrophages (Mw) were polarized for 16 h. Vehicle control was maintaining inflammation in chronic inflammatory conditions. included at all time points. RNA. Actinomycin D (ActD, 10 mg/ml) was added directly to cell cultures without removal of the original stimulant (32, 33). Cells were collected at http://www.jimmunol.org/ Materials and Methods 0, 2, 4, or 8 h after the addition of ActD. Statement of ethics Protein. Cyclohexamide (10 mg/ml) was added to cell cultures, and cells were collected at 6, 24, 30, or 48 h after the addition of cyclohexamide. All experiment protocols on human samples were approved by the insti- Immunoblotting and densitometry were performed using ImageJ as pre- tutional human research ethics committee, and samples were collected with viously described (34, 35). Results were normalized to vehicle control. informed written consent. The Pathobiology of Early Arthritis Cohort (PEAC) study was approved by the King’s College Hospital Research LPS-induced gene expression kinetics Ethics Committee (REC 05/Q0703/198). All zebrafish studies conformed to the institution’s ethical requirements and were performed under Home THP1-Mw were treated with medium only (basal) or LPS (100 ng/ml) for be- tween 30 min and 24 h. Medium containing LPS was removed at 4 h, and cells Office Project Licenses 40/3434. by guest on September 25, 2021 were extensively washed with PBS. Medium (without LPS) was added, and cells PEAC were incubated for 2–20 h. Controls without LPS withdrawal were included for all assays. COX2 was used as a control marker for LPS protein assays (36). Synovial tissue (ST) was obtained from 87 patients recruited at Barts Health National Health Service Trust into the PEAC (http://www.peac-mrc.mds. JNK inhibition qmul.ac.uk) undergoing ultrasound-guided synovial biopsy of the most inflamed joint (knee, wrist, or small joints of hands or feet) (24). All patients THP1-Mw were treated with the anthrapyrazolone JNK inhibitor were disease-modifying antirheumatic drugs– and steroid-naive, had disease SP600125 or vehicle control for 60 min (37). Treatment was removed, and duration ,12 mo, and fulfilled the American College of Rheumatology/ cells were washed. Prewarmed medium (basal) or medium supplemented European League Against Rheumatism 2010 classification criteria for RA. with 100 ng/ml LPS was added to cells and incubated for 4 h. Clinical and laboratory parameters were collected at the time of the biopsy. Small interfering RNA RNA extraction, RNA sequencing, and postsequencing Small interfering RNA (siRNA) targeting C5orf30 (siC5orf30; Dharmacon data preparation siGENOME SmartPool) or a nontargeting scramble control siRNA (siNTC; Dharmacon siGENOME nontargeting pool no.2) transfected at 20 nM Total RNA was extracted from whole ST by TRIzol/chloroform method. Total (primary monocyte-derived macrophages [MDMs]) or 50 nM (THP1-Mw) RNA sequencing (RNA-seq) was performed on an Illumina HiSeq 2500 using Mirus TKO transfection reagent (Mirus Bio) as per the manufac- platform. Raw data were quality-controlled using FastQC and trimmed or turer’s supplied protocol. Gene expression analysis confirmed knockdown removed with cutadapt. Transcript abundance was derived from paired sample (KD) at 48 h, and Western blot analysis confirmed protein KD at 72 h FASTQ files over GENCODE v24/GRCh38 transcripts using Kallisto v0.43.0. (primary MDMs) or 96 h (THP1-Mw) posttransfection. Normalization and analysis of regularized log expression read counts were performed using the DESeq2 package in R statistics. The relationship between Transwell invasion assay C5orf30 expression and biopsy site was analyzed via one-way analysis of means (not assuming equal variances). RNA-seq data are uploaded THP1-Mw transfected with siC5orf30 or siNTC were detached, pelleted, to ArrayExpress and are accessible via accession number E-MTAB-6141 and resuspended in serum-free media supplemented with LPS (100 ng/ml) (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6141/). or TNF (10 ng/ml) 72 h posttransfection. Media-only (basal) controls were included. Invasion assay was performed as previously described (30). Cell culture Phagocytosis assay PBMCs were isolated and macrophages differentiated as previously described (5, 25). Human monocytic THP-1 cells were obtained from the American Type Forty-eight hours posttransfection, MDMs were treated with media only (basal), Culture Collection and differentiated into macrophages as previously described LPS (100 ng/ml), or TNF (10 ng/ml) and incubated overnight. The CytoSelect (26). Reagents: IFN-g (50 ng/ml; PeproTech), LPS (100 ng/ml, 026:B6; Sigma), 96-Well Phagocytosis Assay using opsonized zymosan substrate (Cell Biolabs) IL-4 (20 ng/ml; PeproTech), and TNF (20 ng/ml; R&D Systems). was used as per the manufacturer’s supplied protocol in technical triplicates (38). Flow cytometry Cellular reactive oxygen species detection assay Cells rinsed in PBS were incubated for 1 h in PBS with 5% human serum at Seventy-two hours posttransfection, reactive oxygen species (ROS) was 4˚C. mAbs (BD Biosciences) were used for immunolabeling as follows: measured in MDMs using the 29,79–dichlorofluorescin diacetate Cellular The Journal of Immunology 1071

Reactive Oxygen Species Detection Assay (AbCam) in technical tripli- microscope (Olympus IX81; PerkinElmer) at the time points indicated. Z cates (39). Negative control wells were included in all assays. stack images were taken at 5-mm intervals over the tail fin region and cap- tured using Volocity (PerkinElmer) using the 310 magnification objective. ELISA Computational protein structure prediction Supernatants from siRNA-transfected polarized MDMs were collected 72 h posttransfection. ELISAs were performed with the R&D Systems DuoSet Structural disorder was predicted using PONDR, Predictor of Natural ELISA kits in technical duplicates. Disordered Regions algorithm (46). C5orf30 ubiquitination was predicted using UbPred, predictor of protein ubiquitination sites algorithm (47). Cell viability assay Statistical analysis Cellular MTT Assay Kit I (Roche) was used to monitor cell viability as previously described (29). Anderson–Darling and Shapiro–Wilk tests for normality were performed for all variables. Two-tailed paired Student t test was used for paired Cell metabolism phenotype samples, Student unpaired t test was used to compare two normally dis- 5 tributed independent groups, and ANOVA with Tukey honestly significant THP1 were seeded and subsequently differentiated at 1.25 3 10 cells per well in 24-well XF polystyrene microplates (Seahorse Biosciences). difference post hoc test was used to compare group means. Linear re- t C5orf30 was silenced as described above. LPS (100 ng/ml) was added 12 h gression (of log-transformed data) was used to determine 1/2s. All ex- premeasurement. The Seahorse XFe Extracellular Flux Analyzer assay was periments were performed at least three times unless otherwise stated. performed and analyzed as previously described (40). The experiment was Mean data are expressed normalized to controls. Error bars are SEM. All a p performed on five biological replicates (n = 5) with two technical replicates hypothesis testing assumes an level of 0.05. The values are shown to two decimal places. per condition. All measurements were normalized to cell number using the crystal violet assay.

Data sharing statement Downloaded from Metabolism inhibition assays were performed using 1 mM 2-deoxyglucose (2DG; Sigma) to inhibit glycolysis (41), 1 mg/ml oligomycin (Sigma) to For original data, contact the corresponding author. inhibit oxidative phosphorylation (OXPHOS) (42), and DMSO as a vehicle control. HIF1a expression (43) and phosphorylation of 4E-BP (44) were used to confirm inhibition of glycolysis and OXPHOS, respectively. Results C5orf30 expression is inversely correlated with RA activity Zebrafish

Consistent with our previous finding of C5orf30 as a negative http://www.jimmunol.org/ Colonies of all transgenic zebrafish were maintained under standard aquarium procedures. The line used in this study was Tg(fms:GAL4:UNM), which regulator of the autoaggressive phenotype of RASF, we examined has macrophages labeled in red (45). Morpholino antisense oligonucleotide synovial expression of C5orf30 by RNA-seq of synovial biopsies C5orf30, 59-ACGCTCCACTGGCATCCATTTCCAT-39 (Gene Tools) were from 87 individuals with early RA naive to immune-modulating diluted in phenol red (Sigma) 0.5% solution and injected into the yolk of a treatment (24). Expression of C5orf30 was inversely correlated one- to four-cell embryo at a dose of 1–20 ng. Morpholino was pressure- injected into one- to four-cell embryos at 8 ng. Tail fin was resected. with disease activity as measured by disease activity score (DAS) Embryos or larvae were anesthetized and immobilized in 1% low–melting in 28 joints–erythrocyte sedimentation rate (ESR) (DAS28-ESR) point agarose, and embryos were imaged using a spinning disc confocal (Adjusted p value [padj] = 0.035), inversely correlated with ESR as by guest on September 25, 2021

FIGURE 1. C5orf30 expression is inversely corre- lated with RA activity. Synovial C5orf30 inversely correlates with disease activity in early RA. (A)Sy- novial C5orf30 expression measured by RNA-seq of synovial biopsies from individuals with early RA (,12 mo symptoms and naive to all immune-modu- lating therapies) correlated against clinical markers at baseline, including DAS28-ESR and ESR. (B)Cor- relation between synovial expression of TNF and C5orf30.(C) C5orf30 expression is not significantly different dependent on joint type, categorized as wrist, knee, or small joint (metacarpophalangeal/ proximal interphalangeal). 1072 C5orf30 MEDIATES MACROPHAGE-DRIVEN INFLAMMATION

a general marker of systemic inflammation (padj =0.039),andalso to M1 or M2 phenotype. The t1/2 of C5orf30 mRNA was 3.13 h in inversely correlated with synovial expression of TNF mRNA (padj = M0 cells, which accords very closely with the t1/2 of 3.66 h of the 0.0097) (Fig. 1). C5orf30 expression is uniformly distributed against murine ortholog D1Ert622e (89.16% nt similarity) (50), and did DAS28-ESR by biopsy site and is not significantly different depen- not vary when polarized to M1 (3.17 h) or M2 (3.15 h) (Fig. 2D). dent on joint type, categorized as wrist, knee, or small joint (meta- TNFAIP3 gene expression was used as a control to ensure effec- carpophalangeal/proximal interphalangeal) (F = 2.7229, numerator tive inhibition of transcription (51). df = 2.000, denominator df = 25.193, p =0.085;Fig.1C). Protein turnover is an essential part of cellular homeostasis. t1/2sof proteins often correlate with protein function (52–54). The t of Differential regulation of C5orf30 in pro- and anti- 1/2 C5orf30proteinwas20.13hinM0cells,22.26hinM1cells,and inflammatory macrophages reduced to 12.21 h in M2 cells (Fig. 2E). The increased protein Differentiation of the monocytic cell line THP1 into macrophages levels in M2 cells, combined with increased protein turnover, implies (THP1-Mw) increased expression of C5orf30 protein (2.16 6 more efficient of C5orf30 in M2-like macrophages. 0.56, p = 0.04; Fig. 2A). C5orf30 protein consolidated from Bioinformatic analysis of lysine residues as conical ubiquitination two immunoblot bands into a single band, indicative of dephos- sites was performed using UbPred (47). C5orf30 contains 15 lysines, phorylation. Inflammatory stimulants (INF-g [50 ng/ml] and LPS of which seven are predicted to be ubiquitinated (Supplemental [100 ng/ml]) polarized THP1-Mw to an M1-like phenotype, Table I). Cross-talk between ubiquitination and phosphorylation on whereas IL-4 was used to induce an anti-inflammatory, M2-like the same protein adds specificity and combinatorial logic to cellular phenotype. The model of macrophage polarization was confirmed signaling (34, 35). Two of the predicted ubiquitination sites co-occur by measurement of cell surface markers CD68, CD40, CD80, and with phosphorylation sites (55): S25 and K28 (ubiquitination pre- Downloaded from CD209 (Supplemental Fig. 1). Polarization to M1, but not M2, dicted with high confidence) and K139 (ubiquitination predicted reduced C5orf30 mRNA expression (0.66 6 0.07, p , 0.01) with high confidence) and S140. compared with unpolarized (M0) cells (Fig. 2B). M1 polarization induced C5orf30 phosphorylation at 6 h. M2 macrophages had in- Proinflammatory signals negatively regulate C5orf30 creased C5orf30 protein expression at 24 h (1.67 6 0.23, p = 0.04) LPS reduced C5orf30 expression in both MDMs (0.29 6 0.25, 6

(Fig. 2C). COX2 was included as an M1 marker for immunoblots p = 0.02) and THP1-Mw (0.47 0.05, p = 0.01). TNF down- http://www.jimmunol.org/ (Supplemental Fig. 2) (48). regulated C5orf30 expression in MDMs (0.51 6 0.26, p , 0.01) The stability of mRNA can influence the temporal order of the but not THP1-Mw (0.88 6 0.12, p = 0.41) (Fig. 3A). We inves- gene induction for inflammatory-associated molecules (49). ActD tigated the kinetics of C5orf30 expression in response to LPS: sig- was used to block transcription of mRNA following polarization nificant downregulation was observed at 4 h and maintained at 24 h by guest on September 25, 2021

FIGURE 2. Regulation of C5orf30 in pro- and anti-inflammatory macrophages. (A) Expression of C5orf30 protein was upregulated in differentiated THP1-Mw cells compared with monocyte-like undifferentiated cells (p = 0.04). (B) Stimulation of THP1-Mw for 24 h with IFN-g plus LPS (M1-like) reduced C5orf30 expression compared with unstimulated controls (M0 phenotype). (C) Polarization to M1 for 6 h induced C5orf30 phosphorylation, which was exaggerated by 24 h, whereas polarization to M2 upregulated C5orf30 protein expression by 24 h compared with M0 (densitometry normalized to actin; p = 0.04). (D) Average C5orf30 mRNA t1/2 was 3.13 h (M0, black circles) and did not significantly differ between macrophage phenotypes. (E) Polarization to M2 (green diamonds) greatly reduced average C5orf30 protein t1/2 compared with M0. M1 polarization (red squares) marginally increased C5orf30 t1/2. Averages of n = 3 biological replicates; error bars are SEM. *p , 0.05, ***p , 0.001. The Journal of Immunology 1073 Downloaded from http://www.jimmunol.org/

FIGURE 3. Proinflammatory signals negatively regulate C5orf30 via JNK-dependent and JNK-independent mechanisms. (A) LPS reduced C5orf30 expression in MDMs compared with basal (unstimulated) cells (p =0.02),asdidTNF(p , 0.01). In THP1-Mw, LPS but not TNF stimulation downregulated C5orf30 B expression (p =0.01).( )THP1-Mw were incubated for 4 h with LPS prior to LPS withdrawal. C5orf30 gene expression returned to basal levels 4 h post–LPS by guest on September 25, 2021 withdrawal. (C) LPS reduces C5orf30 protein expression (average densitometry [both bands] normalized to actin [p , 0.01]) and induces phosphorylation of C5orf30. Dephosphorylation treatment of protein lysates removes the upper C5orf30 band and the phosphorylation peak from the densitometry plot. (D) Inhibition of the JNK pathway using SP600125 does not have a direct effect on C5orf30 expression levels; however, JNK inhibition prevents LPS-induced downregulation of C5orf30 protein in a dose-dependent manner but does not inhibit LPS-induced C5orf30 phosphorylation. (E) JNK inhibition prevents LPS-induced downregu- lation of C5orf30. LPS downregulated C5orf30 expression in THP1-Mw without pretreatment (p = 0.04) or with vehicle control (p =0.03)butnotinTHP1-Mw pretreated with SP600125 (p = 0.53). Averages of n = 3 biological replicates; error bars are SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

(p , 0.05). C5orf30 gene expression changes returned to basal levels protein expression is mediated by JNK, but this effect is not de- 4 h post–LPS withdrawal (Fig. 3B), consistent with the observed t1/2 pendent on phosphorylation. of C5orf30 (Fig. 2). Inhibiting C5orf30 skews macrophages away from anti- Immunoblotting indicated LPS-induced C5orf30 phosphorylation inflammatory activities (Fig. 3C). Incubation of lysates with CIAP confirmed LPS-induced phosphorylation, as demonstrated by elimination of the phospho- The production and regulation of proteases that break down and re- band on the immunoblot. Overall C5orf30 protein expression was model tissue are a classic M2 phenotype (7). Using an invasion assay reduced in response to LPS (0.54 6 0.01, p , 0.01) (Fig. 3C). to mimic tissue breakdown and invasion, we found THP1-Mw Active JNK is necessary for maintenance of the M1 phenotype transfected with siC5orf30 displayed reduced invasion compared with (56) and is an important signaling pathway for LPS responsive- transfection controls (0.54 6 0.02, p , 0.01). Exposure to LPS ness. The selective JNK inhibitor SP600125 blocked phospho- or TNF further accentuated this reduction (0.29 6 0.09, p =0.02; JNK (pJNK) in a dose-dependent manner, but total JNK was 0.34 6 0.07, p = 0.01) (Fig. 4A). The matrix metalloproteases unaffected. It had no effect on C5orf30 protein expression when (MMPs) 1, 3, and 9 are central mediators of macrophage-mediated incubated without stimulant; however, inhibition of pJNK pre- tissue turnover; all were all significantly downregulated (MMP1: vented LPS-induced reduction of C5orf30, but not LPS-induced 0.73 6 0.07, p = 0.01; MMP3: 0.70 6 0.03, p = 0.01; MMP9: phosphorylation of C5orf30, in a dose-dependent manner 0.72 6 0.05, p = 0.03; Fig. 4B) in response to C5orf30 inhibition. (Fig. 3D). SP600125 did not alter THP1-Mw viability. SP600125 inhibited LPS-induced downregulation of C5orf30 (Fig. 3E). Cells Inhibiting C5orf30 reduces key mediators of the resolution pretreated with 25 mM SP600125 did not reduce C5orf30 in re- of inflammation sponse to LPS (0.73 6 0.15, p = 0.53); LPS did reduce C5orf30 ROS are important in macrophage biology, both for cell signaling and, gene expression in controls without pretreatment (0.54 6 0.05, at higher concentrations, for pathogen killing. Inhibition of C5orf30 in p = 0.04) or pretreated with DMSO (0.55 6 0.08, p = 0.03). These MDMs reduced ROS production basally (M0 p , 0.01, M1 p , 0.01, data confirm that LPS downregulation of C5orf30 gene and M2 p , 0.01), with TNF stimulation (M0 p , 0.01, M1 p , 0.01, 1074 C5orf30 MEDIATES MACROPHAGE-DRIVEN INFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 4. Regulation of macrophage phenotype by C5orf30. (A) C5orf30 KD reduced the rate of invasion compared with siNTC controls in THP1- Mw. (B) THP1-Mw transfected with siC5orf30 have reduced expression of MMP1, MMP3,andMMP9 compared with nontargeting transfection control cells. (C)At signaling levels of ROS, MDMs transfected with siC5orf30 have reduced ROS expression. C5orf30 does not impact high levels of ROS, such as those induced by the positive control tert-butyl hydroperoxide. (D) In MDMs, C5orf30 silencing reduces IL-6 expression (p = 0.01) and IL-10 (p , 0.01) but not TNF. (E) Il4R gene expression is reduced in C5orf30-silenced MDMs (p , 0.01). (F) siC5orf30-transfected MDMs stimulated with LPS had an increased rate by guest on September 25, 2021 of phagocytosis compared with siNTC controls (p , 0.01). (G)InTHP1-Mw, silencing of C5orf30 upregulates pJNK but not total JNK. Averages of n = 3 biological replicates; error bars are SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

M2 p , 0.01), or LPS stimulation (M0 p , 0.01, M1 p =0.02,M2 Macrophages transfected with siC5orf30 and stimulated with LPS p = 0.04) at lower induced levels of ROS production. However, at had a small but statistically significant increase in phagocytosis of the higher (pathogen-killing) levels of ROS, induced by tert-butyl zymosan (1.15 6 0.05, p = 0.01; Fig. 4F). In THP1-Mw, C5orf30 hydroperoxide, C5orf30 inhibition had no effect (M0 p =0.16,M1 silencing increased pJNK, but not total JNK, a hallmark of M1 p =0.94,M2p = 0.11). Inhibition of C5orf30 reduced signaling levels macrophages (59) (Fig. 4G). of ROS independent of polarization phenotype (one-way ANOVA, C5orf30 regulates macrophage immunometabolism p =0.388).ROSlevelsofM0MDMsareshowninFig.4C;boxplots for M1 and M2 are shown in Supplemental Fig. 2. The recent observations that IL-10 inhibits LPS-induced glucose Cytokines are central mediators of the activities of macrophages uptake and glycolysis, in combination with the Human Protein from pro- to anti-inflammatory phenotypes. In response to siC5orf30 Atlas report of nuclear and mitochondrial-associated C5orf30 inhibition, TNF, a classic inflammatory cytokine, IL-6, a pleiotropic immunostaining (60), are of particular interest in light of our cytokine, and IL-10, a classic M2 cytokine, were measured in MDMs observation that inhibiting C5orf30 reduced IL-10 and increased (Fig. 4D). TNF secretion did not change in response to C5orf30 LPS-induced phagocytosis (60, 61). We hypothesized that the silencing (1.03 6 0.03, p = 0.35); however, secretion of both IL-6 observed C5orf30-mediated effects are underscored by a meta- (0.71 6 0.08, p = 0.01) and IL-10 (0.22 6 0.07, p , 0.01) was reduced. bolic mechanism. The Seahorse Flux Analyzer was used to de- ROS and IL-6 can facilitate resolution of inflammation by in- termine the metabolic phenotype (40) of THP1-Mw in response to ducing upregulation of the IL-4R, thereby amplifying the response C5orf30 silencing. As expected, LPS drove macrophages toward to IL-4 and switching to the M2 phenotype (57, 58). As both ROS a glycolytic phenotype. Interestingly, at baseline, silencing of and IL-6 are reduced in response to siC5orf30, we measured IL4R C5orf30 skewed macrophages toward a glycolytic phenotype both gene expression in response to C5orf30 inhibition. IL4R was basally (p , 0.01) and with LPS treatment (p = 0.01, Fig. 5A). significantly downregulated in MDMs in response to C5orf30 This difference is ablated under stressed conditions (p = 0.10 and inhibition (0.50 6 0.12, p , 0.01, Fig. 4E). p = 0.54, basal and LPS, respectively). Examining the metabolic potential demonstrates that inhibition of C5orf30 reduced the Silencing C5orf30 enhances proinflammatory glycolytic metabolic potential but not the aerobic potential macrophage functions (Fig. 5B). The complete time course for oxygen consumption rate Phagocytosis by macrophages is an early-phase response following and extracellular acidification rate can be found in Supplemental tissue insult and an important function of M1 macrophages. Fig. 3. The Journal of Immunology 1075 Downloaded from http://www.jimmunol.org/

FIGURE 5. C5orf30 regulates macrophage immunometabolism. (A) Silencing of C5orf30 caused a significant increase in glycolytic activity compared with siNTC transfection controls. This is evident in both basal and LPS-stimulated cells at baseline. However, challenging cells with metabolism stressors ablates this difference. (B) Silencing C5orf30 decreases glycolytic potential but not aerobic potential of THP1-derived macrophages. (C) Inhibition of by guest on September 25, 2021 glycolysis (via 2DG) or OXPHOS (via oligomycin) induces increased or slightly decreased C5orf30 protein expression respectively. HIF1a and phospho- 4E-BP are markers of glycolysis and OXPHOS, respectively, and demonstrate effective inhibition of glycolysis and OXPHOS in response to inhibitors. (D) Energy metabolism in macrophages. Top, Inflammatory macrophages are associated with glycolysis, whereas anti-inflammatory (M2-like) macro- phages are associated with OXPHOS. Bottom, Inhibition of glycolysis induces C5orf30 expression, whereas inhibition of OXPHOS causes a decreased expression. Silencing of C5orf30 skews macrophages toward a glycolytic phenotype, which in turn contributes to the observed increase in some inflammatory phenotypes, reduced resolution phase signaling, and reduced repair phase phenotypes. **p , 0.01, ***p , 0.001.

Immunoblotting shows C5orf30 protein expression is increased and disease progression associated with increased local recruit- in response to glycolysis inhibition (via 2DG), and there is a small ment and activation of macrophage and fibroblast cells (17, 18, 65, decrease when OXPHOS is inhibited (via oligomycin). We propose 66). In this study, we show C5orf30 to be inversely correlated with that the observed C5orf30-mediated functional effects (Fig. 4) are disease activity, systemic inflammation, and local TNF levels in due at least in part to the role of C5orf30 in regulating the met- early RA patients. These findings are supported by our experi- abolic phenotype of the cell (Fig. 5D). mental findings, which demonstrate that inflammatory stimulants reduce C5orf30 and, reciprocally, C5orf30 inhibition reduces anti- C5orf30 silencing enhances macrophage recruitment in vivo inflammatory phenotypes and increases certain inflammatory A zebrafish model of inflammation was used to study the role of phenotypes both in vitro and in vivo. We propose that C5orf30- C5orf30 in vivo. As previously described, this model provides mediated regulation of macrophage function is, at least in part, inflammation kinetics similar to those in mammalian systems (62). driven by its ability to inhibit glycolysis, even in the absence of C5orf30 was knocked down in zebrafish with mCherry-labeled inflammatory mediators. The association of low C5orf30 expres- macrophages, and tails were completely transected and imaged sion and the inflammatory metabolic phenotype is of particular 24 h later (Fig. 6A). Quantification of macrophage numbers near interest considering our previous observation that C5orf30 ex- the amplification plane revealed increased recruitment of macro- pression was significantly lower in PBMCs from RA patients KD phages to the wound site in C5orf30 fish (p , 0.01, Fig. 6B). compared with healthy donors (5). Taken together, these are in- dicative of a causative role for C5orf30 in maintaining the in- Discussion flammatory phenotype associated with chronic inflammatory Macrophage-mediated inflammation and tissue damage are fea- diseases such as RA. tures of many chronic inflammatory diseases (63, 64). To date, Macrophages coordinate all phases of the tissue injury and repair C5orf30 has been associated with two autoimmune diseases, RA response (67, 68). Inflammation suppresses tissue repair, and, and primary biliary cirrhosis, which have a shared pathogenesis, therefore, resolution of inflammation is required prior to repair including the skew of macrophages to the inflammatory phenotype and remodeling of tissue (69). Failed resolution in chronic 1076 C5orf30 MEDIATES MACROPHAGE-DRIVEN INFLAMMATION Downloaded from

FIGURE 6. In vivo silencing of C5orf30 increases macrophage recruitment in response to injury. Zebrafish with mCherry-labeled macrophages were microinjected with morpholino (nontargeting scramble control [NTC] or targeting C5orf30). (A) Representative confocal micrographs demonstrate the presence of macrophages (red) at the resection site. Original magnification 310. (B) Quantitation of macrophages near the resection site shows increased recruitment of macrophages to the wound site in C5orf30-deficient zebrafish (n = 10, p , 0.001). http://www.jimmunol.org/ inflammation is typical of diseases such as RA (22, 70, 71). M2 cells, C5orf30 is not phosphorylated at these sites, allowing C5orf30 mediates this resolution phase as evidenced by the for the observed protein degradation. Further experiments are functional data in Figs. 4 and 5. Inhibition of C5orf30 in macro- required to validate this potential regulatory mechanism. phages, even in the absence of inflammatory signals, increases Our data reveal the role of C5orf30 in macrophage biology and, JNK activation and skews macrophages toward a more glycolytic allied with the findings of its role in RA ST, confirm C5orf30 as an phenotype. This reduces signaling mediators required to transition anti-inflammatory and anti-catabolic mediator in RA. The key through the resolution phase. Macrophages are, therefore, not strengths of this study are the important clinical observations in a primed to amplify their response to IL-4, and there is a consequent large cohort of early RA patients, reinforced by both in vitro and reduction in IL-10, MMPs, and tissue remodeling. in vivo experimental evidence, which provides not just an asso- by guest on September 25, 2021 From the initial genetic association of its intronic single nu- ciation between C5orf30 and inflammation but a mechanism to cleotide polymorphism rs26232 with RA (1), very few data have explain how this is mediated. The observations in this study are emerged about its function in macrophages. This may largely be consistent with our previous findings of an inverse correlation of due to its orphan : C5orf30 has no significant homology to C5orf30 expression in the PBMCs from RA patients compared with any other characterized protein, and ∼55% of its amino acid se- healthy controls, which also demonstrated that C5orf30 acts as a quence is predicted to be naturally disordered regions [Predictor of negative regulator of tissue damage in the collagen-induced ar- Natural Disordered Regions prediction (46)]. Increasing C5orf30 thritis mouse model of RA (5). Therapies targeting the resolution could be a feasible therapeutic option. However, to fully estimate rather the suppression of inflammation are an emerging field in the therapeutic potential of C5orf30 as a molecular target, more chronic inflammatory conditions. Future studies will develop our information about its biology and mechanism must be elucidated. understanding of C5orf30 physiology with the goal of identifying We have presented novel biology about the regulation of upstream targetable proteins that can increase C5orf30 and C5orf30 in macrophages. The inflammatory mediator LPS progress the resolution of inflammation. regulates C5orf30 by at least two mechanisms: transcriptionally and via posttranslational modification of the protein. The kinetics Acknowledgments of C5orf30 expression reveals LPS to act quickly to repress C5orf30 We wish to acknowledge Timothy Chicos, University of Sheffield, for transcription. Interestingly, LPS-induced inhibition of C5orf30 assistance with zebrafish studies; Aisling Heeran and Jacintha O’Sullivan, mRNA and protein expression is dependent upon JNK, whereas Trinity College Dublin, for assistance with the Seahorse Flux Analyzer; LPS-mediated protein phosphorylation is independent of JNK. and the staff of the University College Dublin Conway Core Facilities. In contrast, the regulation of C5orf30 in anti-inflammatory We also acknowledge Prof. Denis Shields and Dr. David O’Connell for (M2-like) macrophage phenotypes may be via predominantly guidance during this study. translational mechanisms; it is not upregulated in response to in- flammatory stimulants, nor is there any change in mRNA t1/2. Disclosures However, protein expression is increased, and the protein t1/2 is The authors have no financial conflicts of interest. decreased, indicative of increased protein turnover. The phos- phorylated fraction of C5orf30 is reduced in M2 macrophages. 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