Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen‐induced arthritis

Abstract Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune‐modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen‐induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen‐specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of TH1 and TH17 cells in the spleens of CIA mice. WKYMVm attenuated TH1 and TH17 differentiation in a dendritic cell (DC)‐dependent manner. WKYMVm‐induced beneficial effects against CIA and WKYMVm‐attenuated TH1 and TH17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL‐10 production from lipopolysaccharide‐stimulated DCs and WKYMVm failed to suppress TH1 and TH17 differentiation in the presence of anti‐IL‐10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of TH1 and TH17 cells via IL‐10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.

In the centre of RA pathogenesis, dendritic cells (DCs) play crucial roles in activation of CD4 lymphocytes by presenting proper T-cell receptor stimulatory and co-stimulatory signalling cues, and context-dependent cytokines, polarizing them into several subsets of CD4 T cells including T H 1 and T H 17. 8 Meanwhile, tolerogenic DC has immuno-suppressive properties and sustain peripheral tolerance by preventing excessive lymphocytes activation with antiinflammatory surface molecules and cytokines such as TGFβ and IL-10. 9 Since the progress of RA is associated with dysregulated activation of T H 1 and T H 17 lymphocytes, it is therefore crucial to identify molecular targets for switching the DC responses to tolerogenic states while suppressing excessive immune activation.
Formyl peptide receptors (FPRs), well-known chemoattractant receptors for leukocyte recruitment, are expressed in diverse immune system 10,11 and can regulate immune cell activation and differentiation. 12 FPRs can recognize a diverse range of agonists that include formyl peptides derived from bacteria or mitochondria and host-derived agonists (serum amyloid A, LL-37) and regulate immune cell response in a ligand-specific manner. 10,13,14 WKYMVm, a surrogate agonist for FPRs, 15,16 shows therapeutic effects against several infectious and inflammatory diseases such as polymicrobial sepsis, ulcerative colitis and respiratory disease, [17][18][19][20][21] implying the important roles of FPRs in immune modulation. Previously, the function of FPRs was investigated in autoimmune arthritis, 22,23 and it was known that serum amyloid A, an another endogenous FPR2 agonist, mediates synovial hyperplasia and angiogenesis via FPR2 of synovial fibroblasts during progress of RA. 24,25 However, the function of FPRs in adaptive immunity remains unclear in autoimmune disease.
In this study, we investigated the roles of FPR in autoimmune disease with a well-known FPR agonist WKYMVm in a CIA mouse model by focusing on DC-mediated CD4 T-cell differentiation.   26 and each score from individuals were combined.

| CIA mouse model
Vehicle (1× phosphate-buffered saline) or WKYMVm, synthesized by Anygen (Gwangju, Korea) with a purity >99.6%, was subcutaneously injected into the CIA mice model daily following the secondary boosting. Cyclosporin H (CsH) (Enzo Life Sciences, Farmingdale, New York, USA) and WRW4 (Anygen, Gwangju, Korea) were subcutaneously injected 30 min before WKYMVm injection. For the therapeutic administration, WKYMVm was subcutaneously injected into the CIA mice model daily after onset of the clinical signs of CIA.
After monitoring CIA, mice were sacrificed at the CIA peak after secondary boosting for analysis.

| Enzyme-linked immunosorbent assay (ELISA)
The levels of IgG1 and IgG2a reactive to immunized collagen in the peripheral blood serum were determined by using a mouse antibovine CII IgG1 and IgG2a antibody assay kit with tetramethylbenzidine (TMB) substrate (Chondrex, Redmond, WA, USA). Cytokine ELISA and antibody detection were carried out according to the manufacturer's instructions.

| Histology of arthritic joints
One leg was randomly selected from each mouse and dissected for histology. The knee joints were collected and fixed in 4% para- ples were dehydrated in 50%, 70%, 90% and 100% ethanol and xylene and mounted by using balsam. They were observed by using DM750 microscope (Leica, Wetzlar, Germany).

| Intracellular cytokine staining and flow cytometry
For the detection of intracellular cytokines, cells were reactivated by PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) with protein transport inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) for 5 h. Cells were blocked by anti-mouse CD16/32 antibodies before surface staining. Surface proteins expressed on cells were stained with fluorescence-conjugated antibodies diluted in FACS buffer (1× phosphate-buffered saline with 0.5% bovine serum albumin) for 30 min. Intracellular cytokine staining was performed using intracellular fixation and permeabilization buffer set (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's recom-

| Ex vivo collagen stimulation
Splenocytes were isolated from CIA mice at 35 days after immunization. Cells were stimulated by CII (50 μg/ml) with vehicle or 1 μM

| Generation of bone marrow-derived DCs (BMDCs) and maturation
Mouse bone marrow cells were isolated from 8-to 12-week-old

| Statistical analysis
All results are expressed as the mean ± SEM for the data obtained from the indicated number of experiments. Statistical analysis was performed using Student's t test or two-way ANOVA. A p value ≤ 0.05 was considered statistically significant.

| An immune-modulating peptide, WKYMVm, elicits beneficial effects against CIA
First, we examined the effects of WKYMVm, a surrogate agonist for FPRs, on CIA according to a previous report. 27

| FPR1 mediates WKYMVm-induced beneficial effects against CIA
Previous reports demonstrated that WKYMVm acts on three different FPR members (FPR1, FPR2 and FPR3) in human leukocytes and at least two FPR members (FPR1 and FPR2) in mouse leukocytes. 14,29 In this study, we examined which FPR subtype is involved in the beneficial effects of WKYMVm against CIA by using FPR1 or FPR2 antagonists, CsH or WRW4, respectively. Administration of CsH, an FPR1 antagonist, blocked WKYMVm-elicited beneficial effects against CIA, showing increased paw thickness compared to the WKYMVm-alone group ( Figure 2A). However, WRW4, an FPR2 antagonist, did not affect WKYMVm-induced beneficial effects against CIA (Figure 2A). Histological analysis showed that WKYMVm-induced joint damage reduction was blocked by CsH but not by WRW4 ( Figure 2B). Through H&E and safranin O staining, we also found that WKYMVm-induced cartilage restoration and inhibition of immune cell infiltration were blocked by CsH but not by WRW4 ( Figure 2B). Taken together, our results suggest that WKYMVm-induced beneficial effects against CIA are mediated by FPR1 but not by FPR2.

| WKYMVm inhibits IFNγ-or IL-17Aproducing CD4 T cells via FPR1 in CIA mice
Previously, several cytokines were reported to mediate the pathogenesis of RA. 30 Among these, IFNγ and IL-17 are the major contributors in RA progression. 14,15,31 The major sources of these cytokines are effector CD4 T cells differentiated into T H 1 and T H 17. IFNγ and IL-17A are known to induce the expression of cell-to-cell interaction molecules and activate fibroblast-like synoviocytes to mediate inflammation in the synovium. 32 In this study, we observed that the establishment of CIA in mice could augment the levels of IFNγ + CD4 T cells and IL-17A + CD4 T cells ( Figure 3A,B). We then examined Splenocytes isolated from CIA mice were restimulated by CII and simultaneously treated with vehicle or WKYMVm during activation.
WKYMVm treatment significantly decreased IL-17 and IFNγ production ( Figure 3C). Taken together, WKYMVm effectively blocked CII specific T H 1-and T H 17-mediated immune reactions and this effect was mediated by FPR1.

| WKYMVm-induced decrease of T H 1 and T H 17 cells is dependent on FPR1 expressed on DCs
Since WKYMVm administration elicited beneficial effects against CIA by downregulating T H 1 cells in the spleen (Figures 1 and 3A),    in T-cell differentiation was also controlled by DCs presenting CII, BMDCs matured by LPS plus CII were co-cultured with CD4 T cells from CIA mice which were sensitized to CII. As in the previous results, the generation of T H 1 and T H 17 was also suppressed by WKYMVm in co-culture conditions, showing an FPR1 dependency ( Figure 4D). In

conclusion, WKYMVm suppresses T H 1 and T H 17 cell differentiation
in the presence of DCs by working on FPR1 expressed on the surface of DCs, affecting the interaction between the DCs and CD4 T cells.

| WKYMVm further increases IL-10 production from LPS-stimulated DCs which has a role in suppressing T-cell differentiation
DCs are matured in pathogenic conditions, and mature DCs produce several cytokines, present antigens and provide stimulatory signals to T cells. 8 Mature DCs express high levels of surface molecules such as major histocompatibility complex (MHC) and CD80/86 which have a role in DC-to-cell interaction with T cells. 8 Depending on the surrounding environment, DCs can be stimulatory or regulatory, which may stimulate or suppress T-cell activation, respectively, leading to polarization of T cells into various effector or regulatory subtypes. 9 Since we found that WKYMVm suppresses T H 1 and T H 17 cell generation in the presence of DCs, we examined whether WKYMVm can affect regulatory cytokine production in CIA mice.
And we found that administration of WKYMVm significantly increased IL-10 producing DCs ( Figure 5A). We then tested the effects of WKYMVm on the production of IL-10 from mature DCs. Being matured by LPS, DCs produced several cytokines such as IL-10, IL-6, IL-12 and IL-1β. Addition of WKYMVm significantly increased the production of IL-10 from mature DCs, which was inhibited by CsH but not by WRW4 (Figure 5B), suggesting a crucial role of FPR1.
However, WKYMVm did not affect the levels of IL-6, TNFα, IL-12 and IL-1β (data not shown). Furthermore, in the ex vivo stimulation of CII, WKYMVm significantly increased IL-10 from the splenocytes of CIA mice ( Figure 5C). Thus, we next focused on the expected function of IL-10 from DCs to regulate T-cell differentiation in vitro.
Previously, it was reported that IL-10 produced from DCs can suppress T-cell expansion. 9,33,34 Here, we aimed to see whether IL-

| WKYMVm shows therapeutic effects in experimental CIA
We also examined whether WKYMVm shows therapeutic effects against CIA. For this, we administered vehicle or WKYMVm daily after onset of the clinical signs of CIA. As shown in Figure 6A

| DISCUSS ION
In this study, we found that that the immune-modulating peptide DCs can be classified into two distinct subsets, the stimulatory and regulatory (or tolerogenic) types. 9 Regulatory DCs produce IL-10, which show suppressive responses against active immune responses. 9 In this study, we attempted to test the effects of WKYMVm on the stimulatory and tolerogenic phenotype of DCs.
Although WKYMVm failed to suppress the expression of proteins that provide stimulatory signals such as the CD40 ligand (CD40L), MHC II, CD80/86 (data not shown) and any other cytokines, WKYMVm augmented the production of IL-10 in response to LPS from DCs ( Figure 5B). Since IL-10 is the representative cytokine produced by regulatory DCs, 9 and IL-10 produced from DCs can suppress T-cell expansion, 33,34 our results suggest that WKYMVm stimulates the generation of regulatory DCs. In a previous report, we demonstrated that WKYMVm inhibits human monocytederived DC maturation caused by LPS, showing a decrease of IL-12, decrease of CD86/HLA-DR and decrease of allostimulatory activity. 35 Collectively from our previous report and current findings, we suggest that WKYMVm may have more complex effects on DC maturation and differentiation in human monocyte-derived DCs and mouse BMDCs.   Another previous report demonstrated that FPR signalling initiated by Cpd43, a dual agonist for FPR1 and FPR2, makes CD4 T cells more apoptotic and inhibits the proliferation of fibroblast-like synoviocytes, then attenuating the CIA mouse RA model via FPR2. 36 The functional role of FPR2 in the regulation of RA pathogenesis was also demonstrated by showing that deletion of annexin A1, an endogenous FPR2 agonist, exacerbates arthritis severity in K/BxN serum-injected mice. 22 However, the functional role of FPR1 on RA pathogenesis and mode of action thereof remained to be resolved.
Since we found that WKYMVm showed beneficial effects against CIA (Figure 1), and WKYMVm is a surrogate agonist for mouse FPR family members such as FPR1 and FPR2, 14  WKYMVm is a useful material to control RA. We also suggest FPR1 as a major target to control DCs for the treatment of autoimmune diseases.  The data that support the findings of this study are available from the corresponding author upon reasonable request.