S1PR4‐dependent CCL2 production promotes macrophage recruitment in a murine psoriasis model

The sphingolipid sphingosine‐1‐phosphate (S1P) fulfills distinct functions in immune cell biology via binding to five G protein‐coupled receptors. The immune cell‐specific sphingosine‐1‐phosphate receptor 4 (S1pr4) was connected to the generation of IL‐17‐producing T cells through regulation of cytokine production in innate immune cells. Therefore, we explored whether S1pr4 affected imiquimod‐induced murine psoriasis via regulation of IL‐17 production. We did not observe altered IL‐17 production, although psoriasis severity was reduced in S1pr4‐deficient mice. Instead, ablation of S1pr4 attenuated the production of CCL2, IL‐6, and CXCL1 and subsequently reduced the number of infiltrating monocytes and granulocytes. A connection between S1pr4, CCL2, and Mϕ infiltration was also observed in Zymosan‐A induced peritonitis. Boyden chamber migration assays functionally linked reduced CCL2 production in murine skin and attenuated monocyte migration when S1pr4 was lacking. Mechanistically, S1pr4 signaling synergized with TLR signaling in resident Mϕs to produce CCL2, likely via the NF‐κB pathway. We propose that S1pr4 activation enhances TLR response of resident Mϕs to increase CCL2 production, which attracts further Mϕs. Thus, S1pr4 may be a target to reduce perpetuating inflammatory responses.


Introduction
The lipid mediator sphingosine-1-phosphate (S1P) is the ligand for five G-protein coupled receptors [1]. Of these, S1pr4 is predominantly expressed on cells of the immune system. S1P coupling to its receptors affects survival, proliferation, activation and, most cytokines act on the local epithelium to produce chemokines that recruit myeloid cells to the skin leading to chronic disease [3]. The role of S1P in general and S1pr4 in particular during psoriasis is not known and current literature suggests conflicting roles for S1P. Some propose that its application might be beneficial [4]. Others showed that S1P enhanced psoriasis in patients via S1PR1 [5]. The S1PR1 inhibitor ponesimod, however, did not progress to phase 3, since its oral application caused lymphopenia, directly affecting T cell migration [6]. Targeting S1PR4 may attenuate IL-17 production, while effects on T cell migration will be unlikely. This may hold potential for the treatment of psoriasis, a hypothesis we tested using S1pr4 −/− mice in a murine psoriasis model.

S1pr4-deficiency does not attenuate IL-17 production in psoriasis
To analyze whether S1pr4 promotes psoriasis through regulating IL-17 production, WT versus S1pr4 −/− mice were subjected to the IMQ psoriasis model. Quantifying the severity of the inflammatory response, using the previously described simplified PASI score [7], we observed reduced inflammation in S1pr4 −/− animals, significantly at day 3 ( Fig. 1A), which was accompanied by reduced epidermal thickening and dedifferentiation (Fig. 1B). We therefore focused on the early, inflammatory phase of the model for further studies and chose experimental endpoints after 6, 24, and 72 h. LC-MS/MS analysis of ground skin samples confirmed increased S1P levels in psoriatic versus healthy skin 24 h after IMQ application (Fig. 1C). Unexpectedly, mRNA expression of IL-23 (Fig. 1D) and IL-17A (Fig. 1E) were similar between psoriatic WT and S1pr4 −/− animals. IL-23 was upregulated after 6 h followed by IL-17A upregulation at 24 h. Using cytometric bead arrays (CBA), we did not observe a decrease of IL-17A protein in extracellular fluid of S1pr4 −/− animal skin (Fig. 1F). IL-23 protein was not detected. Analyzing the immune infiltrate in psoriatic skin via FACS revealed unaltered T cell populations comparing WT and S1pr4 −/− animals 72 h after disease induction ( Fig. 1G; Supporting Information Fig. 1). S1pr4 in other models promoted IL-6-dependent IL-17 production from CD4 + T cells [2]. In IMQinduced psoriasis, γδ T cells are a major source of IL-17 and depend on IL-23 rather than IL-6 [7], which was not affected by S1pr4 KO.

S1pr4 KO reduces Mφ infiltration and CCL2 production
We noticed that S1pr4 ablation caused an unexpected reduction in infiltrating neutrophils and Mφs 72 h after IMQ application ( Fig.  1G and H). This was confirmed via histology, where differences in monocyte/Mφ numbers were already detected after 24 h ( Fig.  1I-K). In order to understand reduced myeloid cell infiltration, we analyzed mRNA expression of chemokines and inflammatory cytokines and found reduced levels of CCL2 ( Fig. 2A), CXCL1 (Fig. 2B), and IL-6 ( Fig. 2C) in the skin of S1pr4 −/− mice. CCL2 and CXCL1 are chemotactic agents for monocytes and neutrophils, respectively, while IL-6 is an important mediator in psoriasis [8]. While all three cytokines were upregulated 6 h after induction of inflammation, the expression of CCL2 decreased rapidly and was undetectable after 24 h. CXCL1 expression increased persistently for 72 h. IL-6 expression was highest at 24 h and returned to basal levels at 72 h. CCL2 protein peaked 6 h after IMQ application, with less protein in S1pr4 −/− animals compared to the WT (Fig. 2D), closely following its mRNA profile. CXCL1 protein also followed its mRNA expression pattern 6 and 24 h after disease onset (Fig. 2E). IL-6 protein was present in large amounts after 24 h in WT animals that persisted until 72 h, with S1pr4 −/− mice producing drastically reduced levels (Fig. 2F). We then validated findings in a Zymosan-induced acute peritonitis model. Zymosaninduced peritonitis is defined by rapid granulocyte influx within the first 24 h, followed by a delayed influx/return of monocytes and Mφs into the peritoneal cavity. In S1pr4 −/− mice, we again saw significantly reduced Mφ numbers 72 h after Zymosan injection ( Fig. 2G and H). This was preceded by reduced levels of CCL2 in the peritoneal lavage after 24 h. Neither IL-6 nor CXCL1 was changed by the ablation of S1pr4 in this model (Fig. 2I). Thus, S1pr4 deficiency reduced CCL2 production and Mφ numbers in two independent inflammatory mouse models. While CCL2 is a potent inducer of monocyte migration [9], we aimed to confirm that reduced CCL2 production in S1pr4 −/− animals sufficed to reduce monocyte migration. In a transwell migration assay, supernatants of WT skin explants induced migration of primary BM monocytes, while supernatants of S1pr4 −/− explants did not. However, recombinant CCL2 restored monocyte migration toward S1pr4 −/− skin explant supernatants (Fig. 2J).

Concluding remarks
In summary, we show that S1pr4 is negligible for IL-17 production during psoriasis but promotes CCL2 production in resident Mφs during early inflammation. Our data suggest that attenuated CCL2 production translates to reduced infiltration of Mφs into the inflamed tissue. Mφs and monocytes are critically involved during auto-immune diseases such as psoriatic arthritis or MS [11,12]. Increased CCL2 production in resident Mφs after activation of S1pr4 suggests that S1pr4 acts as an enhancer of the initial inflammatory response, which fits to the observation that S1P levels are often elevated in inflammatory diseases [13]. S1pr4 might therefore be a promising target in inflammatory diseases that are driven by CCL2 and Mφ recruitment. The reduction of T H 17 cells upon S1pr4 ablation observed before [2] may add to an antiinflammatory potential of blocking S1pr4. This may not only be interesting for auto-immune diseases but also for cancer, where CCL2 production correlates with increased numbers of tumorassociated Mφs, which promote the formation of metastasis [14].

Animal experiments
For animal experiments, the guidelines of the Hessian animal care and use committee were followed (FU/1059). Psoriasis-like skin inflammation was induced as described by van der Fits et al. and severity evaluated according to their PASI scoring system [15]. Mice were euthanized and skin samples harvested after 6, 24, or 72 h for further analysis. For induction of Zymosan A-dependent peritonitis, WT and S1pr4 −/− mice were injected i.p. with 10 mg/kg Zymosan A (Sigma-Aldrich, St. Louis, USA). After up to 72 h, peritoneal lavage was obtained for further analysis.

Flow cytometry analysis
Flow cytometry experiments followed the "Guidelines for the use of flow cytometry and cell sorting in immunological studies" [16].

Quantitative PCR
Total RNA from skin and tumor samples was extracted using peg-GOLD RNAPure (Peqlab Biotechnologie, Erlangen, Germany) and reverse transcribed with the Maxima First Strand cDNA Synthesis kit (Thermo Fisher, Waltham, USA). Quantitative real-time PCR reactions were conducted with the iQ SYBR Green Supermix (Bio-Rad, Hercules, USA) on a CFX96 Connect system (Bio-Rad). Relative mRNA expression was analyzed based on the Ct method and normalized to Rps27A.

In vitro activation of peritoneal Mφs
Peritoneal Mφs from healthy WT or S1pr4 −/− mice were cultured in high attachment 24-well plates for 4 h followed by washing and stimulation with Zymosan A (1 μg/mL) and S1pr4 agonist Cym50308 (200 nM) for 24 h.

Immunoblotting
Peritoneal Mφs were collected in lysis buffer, incubated for 5 min at 95°C with SDS buffer, and resolved on polyacrylamide gels followed by transfer onto nitrocellulose membranes. Nonspecific binding was blocked with 5% milk, followed by incubation with antibodies against iκBα (Cell Signaling, Danvers, USA; #4814), and actin (Sigma, St. Louis, USA, A2066). Proteins were visualized by IRDye secondary antibodies using the Li-Cor Odyssey imaging system (all from LICOR Bioscience, Bad Homburg, Germany). Supporting information Figure 3 shows an uncropped blot.

Migration assay
Full skin explants were extracted as described before [18] and were individually placed into 24-well untreated tissue culture dishes (Falcon, Becton Dickinson, Franklin Lakes, USA). After 5 min to allow adherence, 200 μL medium was added. Plates were incubated at 37°C, 5% CO 2 . After 24 h, 1.5 mL medium was added to each well. Supernatants were used for Boyden chamber migration assays after another 24 h. A total of 3.5 × 10 5 total BM cells were added in 96-transwell inserts (5 μm, Corning) and allowed to migrate toward explant supernatant for 2 h. Rm-CCL2 (10 ng/mL) was used as a positive control. Migrated and non-migrated cells were counted using flow cytometry with Flow-Count Fluorospheres (Beckman Coulter, Krefeld, Germany) as internal counting standard.