M2‐like macrophages polarized by Foxp3− Treg‐of‐B cells ameliorate imiquimod‐induced psoriasis

Abstract Our group have demonstrated that splenic B cells contributed to the CD4+CD25− naive T cells conversion into CD4+CD25+Foxp3− regulatory T cells without adding appended cytokines, named Treg‐of‐B cells which were potent suppressors of adaptive immunity. We like to investigate whether Treg‐of‐B cells could promote alternatively activated macrophage (M2 macrophages) polarization and alleviate inflammatory disease, psoriasis. In this study, we co‐cultured the bone marrow‐derived macrophages (BMDMs) with Treg‐of‐B cells under LPS/IFN‐γ stimulation and analyzed the M2‐associated gene and protein using qPCR, western blotting, and immunofluorescence staining. We also examined the therapeutic effect of Treg‐of‐B cell‐induced M2 macrophage for skin inflammation using imiquimod (IMQ)‐induced psoriatic mouse model. Our results showed that BMDMs co‐cultured with Treg‐of‐B cells upregulated typical M2‐associated molecules, including Arg‐1, IL‐10, Pdcd1lg2, MGL‐1, IL‐4, YM1/2 and CD206. In an inflammatory environment, TNF‐α and IL‐6 production by macrophages co‐cultured with Treg‐of‐B cells was decreased significantly. The molecular mechanism revealed that Treg‐of‐B cells promoted M2 macrophage polarization via STAT6 activation in a cell contact‐dependent manner. Moreover, the treatment with Treg‐of‐B cell‐induced M2 macrophages attenuated the clinical manifestations of psoriasis, such as scaling, erythema and thickening in the IMQ‐induced psoriatic mouse model. T cell activation in draining lymph nodes was decreased in the Treg‐of‐B cell‐induced M2 macrophage group after IMQ application. In conclusion, our findings suggested that Foxp3− Treg‐of‐B cells could induce alternatively activated M2 macrophages through STAT6 activation, providing a cell‐based therapeutic strategy for psoriasis.

contrast, M2 (alternatively activated) macrophage polarization was originally observed in response to Th2 cytokines, which exert antiinflammatory, immunomodulatory and tissue repair effects. 1 M2 macrophages are subdivided into M2a, M2b and M2c. Interleukin (IL)-4 and IL-13 activate M2a and M2b macrophages by immune complexes, and M2c macrophages are induced by transforming growth factor beta (TGFβ), IL-10 and glucocorticoids. 2 M2 macrophages express low levels of inflammatory markers, such as tumour necrosis factor-alpha (TNFα) and inducible nitric oxide (iNOS), and high levels of anti-inflammatory factors, such as IL-10, Arg-1 and CD206. 3 A previous study showed that monocytes co-cultured with expanded regulatory T cells (Treg) had reduced major histocompatibility complex class II expression accompanied by upregulation of M2 markers, CD206 and IL- 10. 4 Treg cells are important in maintaining immune tolerance and preventing autoimmunity. 5 Naturally occurring Treg cells are broadly classified into thymus-derived Treg (tTreg) cells and peripheral Treg cells (pTreg cells), which exert forkhead box p3 (Foxp3)-dependent immunosuppressive effects. 6 Treg cells suppress the activation and proliferation of immune cells via multiple mechanisms. For example, the release of TGFβ and IL-10 enables Treg cells to suppress the proliferative response of effector T cells. 7 Moreover, Treg cells modulate antigen-presenting cells (APCs) function via interfering its maturation via the interaction of lymphocyte-activation gene 3 (LAG3)/major histocompatibility complex class II (MHC II) and cytotoxic T lymphocyte antigen-4 (CTLA4) /CD80/CD86-mediated induction of indoleamine 2, 3 dioxygenase (IDO). 7 In contrast, Foxp3 − type 1 regulatory T cells do not express Foxp3, which regulates innate immunity. 8 A previous study showed that type 1 regulatory T cells suppress macrophage inflammasome activation, resulting in reduced caspase-1 activation and IL-1β production. 8 B cells have been found to play a critical role in maintaining and inducing immune tolerance. B cell-deficient mice developed worsening and delayed recovery from EAE disease, suggesting the role of B cells in immune tolerance. 9 Previous studies revealed that resting B cells could expand the Treg population via TGFβ or in an allogenic condition. 10,11 In addition, thymus-derived B cells played a critical role in the development of thymic Treg cell precursors and the proliferation of mature thymic Treg cells. 12 A previous study indicated that splenic B cells induced regulatory T cells in the presence of a mature immunological synapse. 13 Our group demonstrated that Peyer's patch B cells could convert naïve T cells into Foxp3 − Treg cells, referred to as Treg-of-B cells. 14 After that, we also found that peritoneal B-1a cells and splenic B2 cells could generate functional CD4 + Foxp3 − Treg cells, which have similar Treg-associated characteristics, such as the expression of PD-1, LAG3, ICOS, GITR, CTLA4 and OX40, and produced IL-10. [15][16][17] With regard to in vivo application, Treg-of-B cells could suppress Th2-mediated allergic asthma, joint inflammation in collageninduced arthritis and Th1/Th17-mediated intestinal inflammation and gouty inflammation. 14,[16][17][18][19] In addition, other study indicated that adoptive transfer of B cells into the B cell-deficient mice (μMT mice) contributed to the restoration of Treg cells number in these mice and prevent DSS-induced colitis. 20 Furthermore, we reported that Treg-of-B cells could modulate innate immunity via inhibiting LPS/ATP and monosodium urate (MSU)-induced NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages. 19 Psoriasis is a chronic inflammatory skin disorder characterized by erythema, scaling, thickening and systemic inflammation and it affects 2-4% of the general population. 21 Although the pathogenic mechanisms are still unclear, recent studies have indicated that macrophages play a crucial role in psoriasis-like skin inflammation.
A previous study showed that patients with severe psoriasis have a high ratio of M1/M2 macrophages. 22 Epithelium/dermis-lining macrophages have been observed in human psoriasis. 23 Other studies have also indicated that activated macrophage recruitment to skin lesions is critical for the maintenance of psoriasis. 24 M1 macrophages can activate Th1/Th17 cells and suppress M2 macrophages by producing IFNγ, IL-23 and IL-17. 25 In psoriasis, dysregulation of Th1 and Th17 infiltration into the dermis of inflamed skin lesions contributes to activation of the IL-23/IL-17 axis, which stimulates keratinocytes to amplify the inflammatory response. 26 Therefore, targeting M1 macrophage polarization is a potential therapeutic strategy for the treatment of psoriasis.
A previous study indicated that human CD4 + CD25 + FoxP3 + Tregs induce alternative CD163 + /CD206 + human monocytes/macrophages in both a cell contact-and soluble factor-dependent manner. 27 We indicated that Treg-of-B cells suppressed LPS/ATP and MSU-induced NLRP3 inflammasome activation in macrophages by inhibiting nuclear factor-kappa B (NF-κB) signalling. 19 It has been demonstrated that the induction of NLRP3-associated protein and mRNA expression is inhibited in M2 macrophages. 28,29 We investigated whether Treg-of-B cells could induce M2 macrophage polarization, and studied the molecular mechanism underlying Tregof-B-induced M2 macrophages. Moreover, we used topical application of the TLR7/8 ligand, imiquimod (IMQ), to induce psoriasis-like skin inflammation in a mouse model. We also further explored the therapeutic potential of M2 macrophages in an animal model of psoriasis and provided a novel cell-based therapeutic approach for inflammation disease.

| Animals
Six-to-eight weeks old male BALB/c mice were purchased from the National Animal Center. STAT6 −/− mice were obtained from the Jackson Laboratory. All mice were maintained at the Laboratory

| Induction of Treg-of-B cells
Splenic CD4 + CD25 − T cells and B220 + B cells were isolated from BALB/c mice. Splenic B220 + B cells were immunomagnetically purified using a BD IMag Cell Separation Magnet. Splenic CD4 + CD25 − T cells were purified by negative immunomagnetic selection using the EasySep Mouse CD4 + T Cell Isolation Kit (STEMCELL Technologies).

| Differentiation of bone marrow-derived macrophages (BMDMs)
Bone marrow cells were harvested from the femurs and tibias of BALB/c and STAT6 −/− mice and cultured in 10% fetal bovine serum-RMPI medium supplemented with 15 ng/mL macrophage colonystimulating factor (MCSF, Peprotech) for 6 days to differentiate into macrophages.

| IMQ model and adoptive transfer of Tregof-B cell-induced M2 macrophages
Mice were treated with 62.5 mg of 5% IMQ cream (Aldara; 3M Pharmaceuticals) on the shaved dorsal skin daily for 4 consecutive days. On day 5, the mice were sacrificed, and the skin tissue, axillary lymph nodes (ALNs), inguinal lymph nodes (ILNs) and spleen were

| Haematoxylin and eosin staining and scoring severity
Dorsal skin specimens were fixed with 10% formalin solution prior to paraffin embedding. Sections were stained with hematoxylin and eosin to examine immune cell infiltration into subcutaneous tissues and epidermal thickness. Epidermal thickness in the skin section was further determined using H and E staining.
To score the severity of skin inflammation in mice with psoriasis, the scoring system was based on the clinical Psoriasis Area and Severity Index. Scaling and erythema of the dorsal skin were scored on a scale of 0 (none) to 4 (very marked), as previously described. 30 Scoring was performed in a double-blind manner.

| Western blotting of Arg-1, p-STAT6 and total STAT6
Western blotting was performed, as previously described. 31 Cells were lysed with Triton X-100-based lysis buffer supplemented with phosphatase and protease inhibitors. Protein lysates were incubated at 95°C for 10 min. Samples were subjected to 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (PVDF, millipore) and incubated with antibodies against Arg-1(Cell signalling #93668), p-STAT6 (BD #558241), total STAT6 (BD, #612290) and β-actin (Merck Millipore, AB_2223041). The ratio of phosphorylated STAT6 to the total basal STAT6 protein was quantified by performing densitometry on the immunoblots and analyzed using Image J.

| Flow cytometry
To characterize the surface markers, the isolated cells were stained with fluorescence-conjugated antibodies, which are listed in Table 2. Data acquisition and analysis were performed using the BD FACSLyric system (BD Biosciences) and FlowJo software (BD Biosciences).

| T cell proliferation
Purified CD4 + CD25 − (responder) T cells were co-cultured with different suppressor T cells, including Treg-of-B cells and control T cells, in the presence of mitomycin C-treated splenocytes upon Con A stimulation (2 μg/mL) for 3 days. The suppressive effect was determined by [ 3 H] thymidine incorporation, which was added to the cultures for 18 h.

| Statistical analysis
Data were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison tests, to calculate between-group TA B L E 1 The sequences of primers used for Q-PCR.  differences, using GraphPad Prism 6.0. Statistical significance was set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001.

| Characterization of splenic Foxp3 − Treg cells induced by splenic B cells
To culture splenic Treg cells induced by splenic B cells, we examined the expression of Treg-associated markers. The results showed that Treg-of-B cells expressed CD25, LAG3, GITR, ICOS, OX40, PD1 and negligibly expressed Foxp3 ( Figure 1A-C).
The gating strategy can be found in (Appendix S1: Figure S1).
Treg-of-B cells produced higher levels of IL-4, IFNγ and IL-10 than tTreg cells ( Figure 1D). We have further confirmed Il10 mRNA expression using qPCR, which revealed that Treg-of-B cells expressed higher levels of Il10 mRNA than those of tTregs or CD4 + CD25 − T cell subsets ( Figure 1E).
To examine the suppressive effect of Treg-of-B cells, we performed a suppression assay. Although Foxp3 expression was absent, Treg-of-B significantly inhibited T cell proliferation ( Figure 1F). Therefore, splenic B cells effectively induce Foxp3 − Treg cells.

| Treg-of-B cells induce M2 macrophage polarization
We Western blotting showed that BMDMs co-cultured with Tregof-B cells produced high levels of Arg-1 protein ( Figure 3D). In contrast, the production of Arg-1 in BMDMs in the Transwell system disappeared. Moreover, inhibition of the contact between Treg-of-B cells and BMDMs significantly reversed TNFα production compared to co-cultures ( Figure 3E). Hence, Treg-of-B cell-mediated modulation of macrophage function, including its induction of M2 macrophage polarization, requires cell-to-cell contact.

| Treg-of-B cells induced tolerogenic M2-like macrophage programming by activating STAT6
A previous study showed that STAT6 activation promotes M2 macrophage polarization. 32 Therefore, we investigated whether ( Figure 4B). We also found that blocking IL-4 did not affect the suppression of TNFα production by Treg-of-B cells ( Figure 4C). Next, we found that disruption of cell contact between BMDMs and Treg-

of-B cells downregulated the phosphorylation of STAT6 in BMDMs
( Figure 4D). These results indicated that Treg-of-B cells promote M2 macrophage polarization via STAT6 activation, rather than via IL-4, in a cell contact-dependent manner.

| STAT6 in BMDMs was critical for the induction of M2 macrophages by Treg-of-B cells, but no effect on macrophage activation
To confirm the role of STAT6 activation for M2 macrophage po-

| Treg-of-B-induced M2 macrophages ameliorate skin inflammation in a mouse model of psoriasis
In vitro, we found that Treg-of-B cells drove macrophages toward an anti-inflammatory M2 phenotype. Next, we examined whether Treg-of-B-induced M2 macrophages could be used as immunotherapy for skin inflammation. Mice were treated with 5% IMQ cream on their back for 4 consecutive days, which mimics human psoriasis.
The dorsal skin displayed typical features of psoriasis-like dermatitis, including scaling, erythema and thickening, beginning 2-3 days after IMQ application, with the greatest severity on day 5 ( Figure 6A,B).
Compared to the IMQ group, skin inflammation, such as erythema and scaling, was significantly attenuated in the Treg-of-B-induced M2-treated group ( Figure 6B).
Histological analysis showed that epidermal thickening and immune cell infiltration were significantly increased in IMQ-treated mice compared with controls ( Figure 6C). In contrast, the Treg-of-B- The production of Arg-1 protein in BMDMs was detected by western blotting. (E) TNFα production by BMDMs co-cultured with Treg-of-B cells in a Transwell system was measured by ELISA. '-'(control) group was BMDMs. The data are representative of two or three independent experiments. The values are expressed as mean ± SEM *p < 0.05, **p < 0.01 and ***p < 0.001, ****p < 0.0001, by one-way analysis of variance (anova) with Bonferroni's multiple comparison test.

F I G U R E 4 Treg-of-B cells induced programming of M2 macrophages via STAT6 activation. (A) Phosphorylated STAT6 and total STAT6
in LPS/IFNγ-stimulated BMDMs co-cultured with anti-CD3/CD28 stimulated Treg-of-B cells were detected by western blotting. The ratio of phosphorylated STAT6 to the total basal STAT6 protein was quantified by performing densitometry on the immunoblots and analyzed using Image J. (B) IL-4 neutralizing antibodies were added to the Treg-of-B cell-BMDMs co-culture system. The mRNA expression in BMDMs was analyzed by Q-PCR. (C) Inflammatory cytokine production was measured by ELISA. (D) Phosphorylated STAT6 and total STAT6 were detected in BMDMs co-cultured with Treg-of-B cells in the presence or absence of a Transwell system. The ratio of phosphorylated STAT6 to the total basal STAT6 protein was quantified by performing densitometry on the immunoblots and analyzed using Image J. (E) The expression of downstream molecules of the STAT6 pathway in BMDMs co-cultured with Treg-of-B cells was analyzed. The data are representative of two or three independent experiments. '-'(control) group was BMDMs. The values are expressed as mean ± SEM *p < 0.05, **p < 0.01 and ****p < 0.0001; n.s. = not significant. The data were analyzed by one-way analysis of variance (anova) with Bonferroni's multiple comparison test.   are stimulated by IL-10. [37][38][39] In addition, a previous study indicated that human CD4 + CD25 + FoxP3 + Treg cells induce alternative CD163 + /CD206 + human monocytes/macrophages in both a cell contact-and soluble factor-dependent manner. 27 It was suggested that Treg-mediated induction of CD206 + alternative macrophages occurred in a cytokine-independent manner, while induction of CD163 + expression in human macrophages was partially dependent on IL-10. In our study, Treg-of-B cell-mediated induction of M2 macrophages mainly occurred in a cell contact-dependent manner, partially via IL-4 ( Figure 3 and Figure 4). In addition, Treg-of-B

| DISCUSS ION
cell-mediated suppression of macrophages is dependent on cell contact, and involves decreased TNFα production. It has been suggested that Treg-of-B cells could modulate innate immunity by inducing M2 macrophages and maintaining immune homeostasis, which may involve a novel cell contact mechanism.
Previous studies indicated that activation of STAT6 by IL-4/IL- Macrophages are heterogeneous innate immune cells that exhibit high plasticity. A previous study indicated that the distribution of activated macrophages in psoriatic skin plays a key role in uncontrolled cutaneous inflammation. 44,45 Increased M1 macrophage polarization is associated with psoriasis severity. 22 Therefore, the modulation of macrophage polarization may be a novel therapeutic target for psoriasis.
In the pathogenesis of psoriasis, the activated APCs, such as macrophages and dendritic cells lead to Th cells differentiation into IFNγ-producing T cells and IL-17-producing Th17 cells. 46 The dysregulated T cells infiltrated into the dermis of inflamed skin, which contributed to amplifying the immune response and causing the hyperproliferation of keratinocytes. 47 In addition, the downstream ef-  In conclusion, we demonstrated that Treg-of-B cells induced

ACK N O WLE D G E M ENTS
This work was supported by grants from National Taiwan University (NTU111L893401) and National Taiwan University Hospital (NTUH-110 L-1006). We would like to thank the staff of the Immune Research Core, the 7th Lab and the Second Core Lab at the Department of Medical Research, National Taiwan University Hospital, for providing technical support.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have declared no competing interests exist.