high high Intestinal CX3C receptor 1 (CX3CR1 ) myeloid cells prevent T-cell-dependent colitis

Hisako Kayamaa,b,c, Yoshiyasu Uedaa,b,c, Yukihisa Sawac,d,e, Seong Gyu Jeona,b,c,JiSuMaa,b,c, Ryu Okumuraa,b,c, Atsuko Kuboc,f, Masaru Ishiic,f, Taku Okazakic,g, Masaaki Murakamic,d,e, Masahiro Yamamotoa,b,c, Hideo Yagitah, and Kiyoshi Takedaa,b,c,1

aLaboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan; Laboratories of bMucosal Immunology, eDevelopmental Immunology, and fCellular Dynamics, World Premier International Research Center (WPI) Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan; cCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, 332-0012, Japan; dLaboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan; gDivision of Immune Regulation, Institute for Genome Research, University of Tokushima, Tokushima, 770-8503, Japan; and hDepartment of Immunology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan

Edited by Warren Strober, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board February 1, 2012 (received for review September 12, 2011)

+ + − − Adequate activation of CD4 T lymphocytes is essential for host terized (13–15). CD103 CX3CR1 CD11b DCs have been defense against invading pathogens; however, exaggerated activ- shown to generate and activate gut-tropic CD8+ T cells (16, 17). ity of effector CD4+ T cells induces tissue damage, leading to inflam- These DCs have further been shown to induce development of fl + + + matory disorders such as in ammatory bowel diseases. Several Foxp3 Treg cells (18–20). CX3CR1 CD11b DCs have been unique subsets of intestinal innate immune cells have been identi- shown to mediate inflammatory responses through the induction fied. However, the direct involvement of innate immune cell subsets of Th1 and Th17 cell development (15, 21–24). In addition to fl + − + in the suppression of T-cell-dependent intestinal in ammation is these cell populations, CD103 CX3CR1 CD11b cells and + − poorly understood. Here, we report that intestinal CX3C chemokine CD11b CD11c macrophages have been identified in the in- high high + + receptor 1 (CX3CR1 ) CD11b CD11c cells are responsible for testinal lamina propria (13, 14, 22, 25). Other intestinal myeloid fl prevention of intestinal in ammation through inhibition of T-cell cell populations inducing Treg cells have also been characterized responses. These cells inhibit CD4+ T-cell proliferation in a cell con- (26–28). However, it remains unclear whether cell populations IMMUNOLOGY tact-dependent manner and prevent T-cell-dependent colitis. The other than Treg cells directly contribute to the suppression of suppressive activity is abrogated in the absence of the IL-10/Stat3 inflammatory responses. pathway. These cells inhibit T-cell proliferation by two steps. Ini- In this study, we characterized intestinal CX3C chemokine high + + high high + + tially, CX3CR1 CD11b CD11c cells preferentially interact with receptor 1 (CX3CR1 ) CD11b CD11c cells, which show a T cells through highly expressed intercellular adhesion molecule- cell contact-dependent suppression of T-cell proliferation, leading 1/vascular cell adhesion molecule-1; then, they fail to activate T cells to prevention of intestinal inflammation. because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer Results of wild-type CX CR1high CD11b+ CD11c+ cells prevents development high + + 3 Intestinal CX3CR1 CD11b CD11c Cells Suppress T-Cell Growth. of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells Several unique subsets of innate immune cells in the intestinal lam- are regulatory myeloid cells that are responsible for maintaining ina propria have been identified (12–14, 16–26, 28–30). Among these intestinal homeostasis. + − − + + subsets, CD103 CX3CR1 CD11b CD11c cells and CX3CR1 CD11b+ CD11c+ cells have been reported to bemajor subsets in the mucosal immunology | innate immunity + + + intestine (13, 14). The functions of CX3CR1 CD11b CD11c cells have been characterized in several aspects (13, 14, 17). nflammatory bowel diseases (IBDs), represented by Crohn However, the CD11b+ CD11c+ cell population could be divided Idisease and ulcerative colitis in humans, result from genetic into three subsets based on the expression level of CX3CR1: high intermediate (int) negative (−) abnormalities, as well as uncontrolled intestinal immune respon- CX3CR1 ,CX3CR1 ,andCX3CR1 cells ses toward commensal microflora and dietary antigens (1–3). (Fig. S1A) (13, 17). Although previous studies have indicated the high int Activation of appropriate mucosal immune responses is re- presence of CX3CR1 and CX3CR1 cells, differential func- sponsible for protection against pathogenic microorganisms, tions of these cell subsets in T-cell differentiation have not been whereas excessive immune responses, especially unbalanced characterized (13, 17). Therefore, we examined the effects of T-cell-mediated adaptive immune responses, to commensal mi- three subsets on induction of Th1, Th17, and Treg cells (Fig. 1A). high int − croflora and dietary antigens lead to development of intestinal CX3CR1 ,CX3CR1 ,andCX3CR1 cells were isolated from inflammation. Therefore, T-cell-mediated responses are tightly the colonic lamina propria and cultured with splenic naïve CD4+ + int regulated to suppress aberrant inflammatory responses in the in- T cells for 4 d. CD4 T cells cocultured with CX3CR1 + − testinal mucosa. Over the last few decades, CD4 regulatory T or CX3CR1 cells predominantly produced IL-17 or IFN-γ, (Treg) cells have been demonstrated to prevent T-cell-mediated chronic inflammatory diseases including IBDs (4, 5). Several possible mechanisms for this suppressive effect of Treg cells have Author contributions: H.K. and K.T. designed research; H.K., Y.U., S.G.J., J.S.M., and R.O. been proposed. Regulatory dendritic cells have also been impli- performed research; Y.S., A.K., M.I., T.O., M.M., M.Y., and H.Y. contributed new reagents/ analytic tools; H.K., Y.S., S.G.J., J.S.M., M.I., M.M., and K.T. analyzed data; and H.K. and cated in the immune tolerance by inducing Treg cells (6, 7). In K.T. wrote the paper. tumor models, myeloid-derived suppressor cells (MDSCs) have The authors declare no conflict of interest. been reported to suppress T-cell-mediated responses through This article is a PNAS Direct Submission. W.S. is a guest editor invited by the Editorial several mechanisms (8, 9). Board. Several subsets of intestinal innate phagocytic cells have re- 1To whom correspondence should be addressed. E-mail: [email protected]. cently been identified that modulate intestinal homeostasis ac.jp. – + − − (10 12). In particular, CD103 CX3CR1 CD11b dendritic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + + cells (DCs) and CX3CR1 CD11b DCs have been well charac- 1073/pnas.1114931109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1114931109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 high + + high int − that a CX3CR1 CD11b CD11c subset of intestinal mye- A CX3CR1 CX3CR1 CX3CR1 B # loid cells inhibited T-cell proliferation independently of Treg γ γ 8 ) 3

IFN- induction. 6

4 high CX3CR1 Myeloid Cells Prevent Intestinal Inflammation. We next CPM (x 10 CPM (x 2 high IL-17 assessed the in vivo function of the CX3CR1 subset of in- 0 *** * ++ ++––CD4+ T testinal myeloid cells using a T-cell-dependent colitis model. Se- ––++– + − high CX3CR1 fi CD4 vere combined immunode ciency (SCID) mice given CD45RB ––+ ++ –CX CR1high 3 CD4+ T cells showed severe weight loss with severe intestinal high pathology (Fig. 2 A and B). Cotransfer of CX3CR1 cells Foxp3 dramatically reduced their weight loss and the severity of in- fl high C D testinal in ammation. The cotransfer of CX3CR1 cells did 14 γ 12 not induce any change in the frequency of IL-17-, IFN- -, IL-4-, + ) 3 10 or IL-10-producing CD4 T cells or in the frequency of Foxp3- 8 expressing CD4+ T cells in the colonic lamina propria (Fig. S2). 6 + CPM (x 10 CPM (x 4 However, the total number of CD4 T cells in the lamina propria high CFSE 2 was markedly reduced by CX3CR1 cell coadministration (Fig. (−−−) 0 high − 2C). Assessment of CFSE dilution in transferred CD45RB CX3CR1 − + CX CR1 + CX CR1high high 3 3 T cells : Treg/CX3CR1 CD4 T cells demonstrated robust T-cell proliferation in the − − CX CR1high / 3 colonic lamina propria of Rag2 mice. However, cotransfer of CD4+CD25+ high CX3CR1 cells substantially inhibited CFSE dilution in trans- high + + high Fig. 1. CX3CR1 CD11b CD11c cells in the intestinal lamina propria ferred T cells (Fig. 2D). Transferred CX3CR1 cells were ob- suppress T-cell proliferation. (A) Flow cytometric plots of IL-17-, IFN-γ-, or served just beneath the epithelial cell layers of the intestine but Foxp3-expressing CD4+ T cells cocultured with the indicated cells for 72 h. (B) [3H]thymidine uptake of CD4+ T cells cultured with the indicated cells. #P < 0.022. (C) The fluorescence intensity of CFSE-labeled CD4+ T cells cultured 3 with the indicated cells at a ratio 1:1:1 for 72 h. (D)[ H]thymidine uptake by 130 + − A CD4 T cells cocultured with CX3CR1 DCs in the presence of increasing ratios + + high of splenic CD4 CD25 Treg cells (closed rectangle) or colonic CX3CR1 cells 120 (open circle). All data are representative of two independent experiments (means ± SD of duplicate well measurements). 110

* 100 PBS * % of original weight original of % high high CD45RB + CX3CR1 respectively. In contrast, expression of IFN-γ,IL-17,orFoxp3 CD45RBhigh + high 90 was not induced in CD4 T cells cocultured with the CX3CR1 01 234(weeks) cells. Next, we examined the effects on T-cell proliferation. + high int B 15 CD4 T cells were cocultured with the CX3CR1 ,CX3CR1 , − * or CX3CR1 cells for 72 h, and their proliferation was analyzed 12 3 + by assessing incorporation of [ H]thymidine (Fig. S1B). CD4 T 9 − int cells cocultured with CX3CR1 and CX3CR1 cells showed 6

− Colitis score 3 robust proliferative responses, indicating that the CX3CR1 and PBS CD45RBhigh CD45RBhigh/CX CR1high int 3 CX3CR1 cells had similar properties as DCs in enhancing 0 + PBS T-cell responses. In contrast, CD4 T cells cocultured with CD45RBhigh high high high CX3CR1 cells did not show any enhanced proliferation. CD45RB + CX3CR1 fi high These ndings indicate that CX3CR1 cells are not typical C D E high * DCs. Indeed, CX3CR1 cells express several macrophage- 20 ** 16 ) ) 6 related molecules (CD14, CD68, and F4/80), as well as DC-re- 5 15 lated molecules (CD11c and DEC205) (Fig. S1C). In addition, 12 CD45RBhigh high 10 CX3CR1 cells contain cytoplasmic vacuolar structures char- 8

T cells (x 10 (x T cells high T cells (x 10 CX3CR1 + acteristic of macrophages (Fig. S1D) (14). We further found + 4 5 CD4 high + CD4 that the addition of CX3CR1 cells into a coculture of CD4 CD45RBhigh − 0 0 + high T cells and CX3CR1 DCs profoundly reduced T-cell pro- CX3CR1 CD45RBhigh liferation (Fig. 1B). The suppression of T-cell proliferation by high high CD45RB + CX3CR1 high CFSE (−) the CX3CR1 cells was further confirmed by reduced dilution fl + of the uorescence intensity of CD4 T cells labeled with car- high fl Fig. 2. CX3CR1 myeloid cells alleviate T-cell-dependent intestinal in- boxy uorescein succinimidyl ester (CFSE) (Fig. 1C). We then 5 high + high flammation. (A) SCID mice were injected i.p. with 3 × 10 CD45RB CD4 T compared the suppressive ability of CX3CR1 cells on T-cell × 5 high + cells or PBS (closed circles). After 2 h, 3 10 CX3CR1 cells were trans- proliferation with that of Treg cells. CD4 T cells were cultured ferred (open triangles) or not (closed rectangles). Body weight change was with DCs and anti-CD3 mAb in the presence of various numbers monitored and is presented relative to initial body weight. *P < 0.005 (n =8 high high of Treg cellsorCX3CR1 cells (Fig. 1D). The CX3CR1 per group). (B) Hematoxylin and eosin staining of colon sections at 4 wk cells showed a dose-dependent suppression of T-cell pro- after the transfer described in A (Left) and the colitis score (Right). *P < 0.0012. (Original magnification, 200×.) (C) Numbers of large intestinal lam- liferation in a very similar manner to that induced by Treg cells. high +/GFP ina propria CD4+ Tcellsat2wk(n = 4 per group) (Left) and 4 wk (n = 5 per CX3CR1 cells isolated from CX3CR1 mice, in which group) (Right) after transfer. *P < 0.02; **P < 0.015. (D) CD45RBhigh T cells CX CR1 Ab staining was well correlated with GFP expression − − 3 (3 × 105) were labeled with CFSE and transferred into Rag2 / mice with or (Fig. S1E), also inhibited T-cell proliferation (Fig. S1F). × 5 high + high + + without 3 10 CX3CR1 cells. After 12 d, CFSE dilution in colonic CD4 T CX3CR1 cells were not present in the CD11b CD11c cells was analyzed. (E) Cryosection of the colon from a SCID mouse at 3 d high population in the spleen, mesenteric lymph nodes (MLNs), or after i.v. injection of CFSE-labeled CX3CR1 cells. (Original magnification, thymus (Fig. S1G). Collectively, our in vitro analyses suggested 200×.) Data are representative of three independent experiments (D and E).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114931109 Kayama et al. Downloaded by guest on September 27, 2021 not in MLNs or spleen (Fig. 2E and Fig. S3 A and B). Transferred high A Hpgd Cd163 Hmox1 Cd209f Cd209g Cebpb CX3CR1 cells were in close proximity to T cells in the lamina n.s. + # 0.02 0.012 0.5 propria (Fig. S3C). In addition, the number of CD4 T cells was 0.03 0.04 1.6 high 0.016 0.4 reduced where CX3CR1 cells were present (Fig. S3 C and D). 0.03 1.2 0.008 high 0.02 0.012 0.3 Total number of CX3CR1 cells increased in the colonic lamina 0.02 0.8 0.008 0.2 high 0.004 0.01 0.4 propria of the transferred mice (Fig. S4). Thus, the CX3CR1 0.01 0.004 0.1 relative expression relative * * 0 * * subset of intestinal myeloid cells suppresses T-cell proliferation in 0 0 0 0 0 the intestinal lamina propria, thereby preventing intestinal in- wild-type Il10-/- high B Hpgd Cd163 Hmox1 Cd209f Cd209g Cebpb flammation; hereafter, we call this subset CX3CR1 regulatory 0.045 0.16 3 # 0.09 n.s. myeloid (Mreg) cells. 0.06 0.8 0.12 0.03 2 0.06 0.6 0.04 high 0.08 IL-10/Stat3-Dependent Suppressive Ability of CX3CR1 Mreg Cells. 0.4 0.015 1 0.03 high 0.04 0.02 To determine how CX3CR1 Mreg cells exert their immuno- 0.2 relative expression relative * * * * suppressive function, we performed a comprehensive analysis of 0 0 0 0 0 0 high high wild-type Stat3-/- expression profiles in CX3CR1 Mreg cells. CX3CR1 C n.s. Mreg cells expressed several IL-10-inducible such as Hpgd, 10 * Cd163, Hmox1, Cd209f, and Cd209g (Fig. S5A). Hence, we an- high −/− 8 ) alyzed CX3CR1 Mreg cells in Il10 mice. Because normal 3 high numbers of CX3CR1 Mreg cells were observed in the colon of 6 −/− Il10 mice (Fig. S5B), we isolated these cells and analyzed the 4 expression of the genes that were selectively expressed in wild- 10 CPM (x high 2 type CX3CR1 Mreg cells. Expression of Hpgd, Cd163, Hmox1, − − Cd209f, and Cd209g was severely decreased in Il10 / cells de- 0 spite normal expression of the myeloid cell-related gene Cebpb 15 high fl/fl n.s. (Fig. 3A). CX3CR1 Mreg cells from LysM-cre; Stat3 mice −/− high D 12 (Stat3 CX3CR1 Mreg cells) also showed profoundly de- * creased levels of expression of these genes (Fig. 3B). To evaluate 9 −/− high whether Stat3 CX3CR1 Mreg cells suppress the T-cell IMMUNOLOGY −/− high 6 proliferative response, wild-type or Stat3 CX3CR1 Mreg cells were added to coculture of CD4+ T cells with wild-type PBS CD45RBhigh Colitis score −/− high 3 DCs (Fig. 3C). Stat3 CX3CR1 Mreg cells were not able to −/− high suppress T-cell proliferation. Il10 CX3CR1 Mreg cells were 0 also defective in their suppression of T-cell proliferation (Fig. PBS − − / high CD45RBhigh CD45RBhigh CD45RBhigh 4E). Furthermore, Stat3 CX3CR1 Mreg cells showed im- + + −/− wild-type M Stat3-/- M CD45RBhigh + wild-type M paired prevention of intestinal inflammation in Rag2 mice reg reg reg CD45RBhigh + Stat3−/− M given CD45RBhigh CD4+ T cells (Fig. 3D). Thus, the suppressive reg high function of CX CR1 M cells in vitro and in vivo was im- −/− 3 reg Fig. 3. Defective activity of Stat3 Mreg cells. (A and B) Expression of high paired in the absence of IL-10/Stat3 signaling. Hpgd, Cd163, Hmox1, CD209f, CD209g, and Cebpb mRNA in CX3CR1 Mreg − − fl fl cells from wild-type, Il10 / , and LysM-cre; Stat3 / mice. Data are pre- high CX3CR1 Mreg Cells Inhibit T-Cell Growth by Two Steps. We then sentative of two independent experiments (means ± SD of at least triplicate assessed the mechanism underlying the suppression of T-cell PCRs on the identical sample). *, not detected; #P < 0.025. (C)[3H]thymidine high + − proliferation by CX CR1 M cells. Because MDSCs have uptake by CD4 T cells cultured with CX3CR1 DCs in the presence of wild- 3 reg −/− high been reported to suppress T-cell response through arginase-1, type or Stat3 CX3CR1 Mreg cells. Data are representative of four in- dependent experiments (means ± SD of triplicate well measurements). *P < inducible NOS, and reactive oxygen species (ROS) (8, 9), we − − 0.047. (D) Hematoxylin and eosin staining of colon sections of Rag2 / mice analyzed the effects of inhibitors of these mediators. However, the × 5 high + × 5 high given 3 10 CD45RB CD4 T cells with 3 10 Mreg cells from wild-type inhibitors did not cancel the suppressive activity of CX3CR1 fl/fl fi – or LysM-cre; Stat3 mice (Left) and colitis score (Right). (Original magni - Mreg cells (Fig. S6 A E). Expression of indoleamine 2, 3-dioxy- cation, 200×.) *P < 0.025 (n = 5 per group). genase (IDO) in several regulatory DCs inhibits T-cell responses (6, 20). However, T-cell proliferation was not increased by addi- high high − tion of an IDO inhibitor (Fig. S6F). Moreover, CX3CR1 Mreg considerably higher in CX3CR1 Mreg cells than in CX3CR1 cells did not express Treg-related genes such as Foxp3, Ctla4,and DCs (Fig. 4B). Therefore, we analyzed whether these adhesion high high Folr4 (Fig. S6G). Thus, CX3CR1 Mreg cells possess distinct molecules are involved in the CX3CR1 Mreg suppressive ac- mechanisms from those used by those cells previously shown to high high tivity. Treatment of CX3CR1 Mreg cells with blocking mAbs inhibit T-cell proliferation. Coculture of CX3CR1 Mreg cells + to ICAM-1, ICAM-2, LFA-1, and VCAM-1 canceled the with CD4 T cells in transwell plates did not suppress T-cell high – CX3CR1 Mreg suppressive activity on T-cell proliferation proliferation, indicating that cell cell contact is required for (Fig. 4C). Treatment of CX CR1high M cells with each mAb suppression (Fig. S6H). Addition of CX CR1high M cells to 3 reg 3 reg did not abrogate the suppressive activity (Fig. S7A), but the cocultures of CD4+ T cells and DCs substantially decreased T- combination of ICAM-1 and VCAM-1 mAbs substantially in- cell aggregation around DCs; instead, T cells preferentially as- high high duced T-cell proliferation. In addition, treatment of CX3CR1 sociated with CX3CR1 Mreg cells (Fig. 4A). Inhibition of T-cell − − − − / / high Mreg cells with mAbs to ICAM-1 and VCAM-1 resulted in in- aggregation was not observed in Stat3 and Il10 CX3CR1 + high creased aggregation of CD4 T cells around DCs (Fig. 4D). Mreg cells. Thus, CX3CR1 Mreg cells had a higher affinity to interact with T cells than DCs and, thereby, suppressed T-cell These results indicate that an ICAM-1/VCAM-1-mediated in- responses. These findings prompted us to investigate the ex- teraction is required for suppression. Expression of ICAM-1 and −/− −/− high pression of adhesion molecules that are involved in DC–T-cell VCAM-1 was high in Stat3 and Il10 CX3CR1 Mreg cells, interactions. Surface expression of intercellular adhesion mole- which showed impaired suppressive activity (Fig. 4B and Fig. − − − − cule (ICAM)-1, ICAM-2, lymphocyte function-associated antigen S7B). In addition, T cells aggregated around Stat3 / and Il10 / high (LFA)-1, and vascular cell adhesion molecule (VCAM)-1 was CX3CR1 Mreg cells (Fig. 4A). Therefore, we analyzed how

Kayama et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 A − n.s. ( ) wild-type E 25 120 * n.s. 20 )

100 3 15 80 10

60 10 CPM (x Stat3−/− Il10−/− 5 40 0 20

0

The number of T aggregation cell n.s. 6 ** CD4+ T / DC F B 5 * ) ICAM-1 ICAM-2 3 4 Fig. 4. Two-step mechanism for suppression of T-cell growth 3 by M cells. (A) Green dye-labeled CD4+ T cells were cul- − n.s. reg wild-type CX3CR1 2 − wild-type CX CR1high 10 CPM (x tured with nonlabeled CX3CR1 DCs and red dye-labeled 3 fl fl − − −/− high 1 / / Stat3 CX3CR1 Mreg cells from wild-type, LysM-cre; Stat3 ,orIl10 mice VCAM-1 LFA-1 0 (Left). The number of T-cell aggregation in twenty fields (Right). (Original magnification, 100×.) (B) Expression of ad- hesion molecules on the indicated cells from wild-type and LysM-cre; Stat3fl/fl mice. (C) CD4+ T cells and wild-type − CD80 CD86 CD40 Isotype CX3CR1 DCs were cocultured with Mreg cells pretreated with G the indicated blocking Abs or control Ig for evaluation of T- C high 9 cell proliferation. *P < 0.012. (D) Red dye-labeled CX3CR1 high CX3CR1 n.s. - Mreg cells were treated with blocking Abs to ICAM-1 and ) CX3CR1 3 VCAM-1 then added to the mixture of green dye-labeled * 6 CD80 CD86 CD40 + − H CD4 T cells and nonstained CX3CR1 DCs. (Original magni- fication, 100×.) (E) IL-10 (100 ng/mL) was added to the co-

CPM (x 10 CPM (x − − 3 n.s. wild-type + / Il10−/− culture of CD4 T cells, wild-type DCs, and Il10 Mreg cells. Il10−/− + IL-10 Then, T-cell proliferation was measured. *P < 0.02. (F) Wild- −/− 0 type and Il10 Mreg cells were preincubated with or without 100 ng/mL IL-10 for 72 h. Then, the cells were analyzed for * * 12 # # I 7 the suppressive activity of T-cell proliferation. *P < 0.016; 6 10 **P < 0.034. (G) Surface expression of CD80, CD86, CD40, and ) 3 8 high − 5 MHC class II on CX3CR1 Mreg cells and CX3CR1 DCs. (H) D 4 6 CX CR1high CX CR1high Expression of CD80, CD86, and CD40 on Mreg cells from wild- 3 3 3 −/− (−) (Control IgG) (αα-VCAM-1/ICAM-1) 4 type and Il10 mice cultured for 48 h with or without 100 CPM (x 10 CPM (x 2 − − − − 2 ng/mL IL-10. (I) Stat3 / and Il10 / M cells were pretreated 1 reg 0 with the indicated blocking Abs. Then, Mreg cells were cul- 0 + − tured with CD4 T cells and wild-type CX3CR1 DCs, and T-cell proliferation was measured. *P < 0.025; #P < 0.045. All data are representative of at least two independent experiments CD4+ T /DC (mean values ± SD of triplicate well measurements).

high CX3CR1 Mreg cells with high affinity for T cells show IL-10- was equally and highly expressed in both populations (Fig. 4G high dependent suppression of T-cell proliferation. CX3CR1 Mreg and Fig. S1C). Notably, expression of CD80 and CD86 was −/− −/− high cells produced IL-10 constitutively (Fig. S8 A and B). However, considerably higher in Il10 and Stat3 CX3CR1 Mreg cells + high supplementation of exogenous IL-10 into cocultures of CD4 T than in wild-type CX3CR1 Mreg cells (Fig. 4H and Fig. S9A). −/− high −/− high cells and Il10 CX3CR1 Mreg cells did not induce the re- Furthermore, IL-10 treatment of Il10 CX3CR1 Mreg cells duction of T-cell proliferative responses (Fig. 4E). In addition, decreased the expression of CD80 and CD86 (Fig. 4H). There- high high the suppressive activity of wild-type CX3CR1 Mreg cells was fore, we suspected that CX3CR1 Mreg cells with decreased not blocked in the presence of neutralizing Abs to IL-10 and the expression of costimulatory molecules compete with DCs to − − − − IL-10 receptor (Fig. S8C). Thus, IL-10 is not directly involved in suppress T-cell proliferation. To test this, Stat3 / and Il10 / high the suppression of T-cell proliferation. However, IL-10 pre- CX3CR1 Mreg cells were pretreated with blocking mAbs to −/− −/− high + treatment of Il10 , but not Stat3 ,CX3CR1 Mreg cells CD80 and CD86, and then cocultured with CD4 T cells (Fig. −/− −/− high before coculture led to a substantial reduction of T-cell pro- 4I). Stat3 and Il10 CX3CR1 Mreg cells pretreated with liferative responses, indicating that IL-10 provides a key signal mAbs to CD80 and CD86 suppressed T-cell proliferation. These high high for CX3CR1 Mreg cells to acquire suppressive activity (Fig. 4F findings clearly indicate that CX3CR1 Mreg cells suppress T- high and Fig. S8D). Expression of molecules that transduce coinhi- cell responses via a two-step mechanism: CX3CR1 Mreg cells bitory signals toward T cells, including B7-H4, herpesvirus entry interact with T cells with high affinity through high expression of mediator (HVEM), programmed death ligand (PD-L)1, and PD- adhesion molecules and then show IL-10-dependent suppression high L2 was increased in CX3CR1 Mreg cells compared with of CD80/CD86-mediated costimulatory signals, leading to in- − CX3CR1 DCs (Fig. S8E). However, expression of these coin- hibition of T-cell proliferation. −/− high hibitory molecules remained high in Stat3 CX3CR1 Mreg high cells. Furthermore, the possible involvement of these inhibitory Defective CX3CR1 Mreg Function Results in Development of Colitis. high molecules in the suppressive activity of CX3CR1 Mreg cells The aberrant Th1/Th17-mediated responses attributable to en- was ruled out in experiments using neutralizing Abs and hanced DC activity have been considered to induce intestinal − − knockout mice (Fig. S8 F–H). In contrast, expression of CD80 inflammation in Il10 / mice and innate immune cell-specific high – high and CD86 was severely decreased in CX3CR1 Mreg cells Stat3 mutant mice (31 34). Because the function of CX3CR1 − compared with that in CX3CR1 DCs, although MHC class II Mreg cells was impaired in the absence of IL-10/Stat3, we assessed

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114931109 Kayama et al. Downloaded by guest on September 27, 2021 IFN-γ (ng/ml) IL-17 (ng/ml) inflammation. The IL-10/Stat3 pathway is critically involved in A high # n.s. the suppressive activity of CX3CR1 Mreg cells. n.s. + − + + 30 2.5 To date, CD103 CX3CR1 DCs and CX3CR1 CD11b + 2 CD11c cells have been identified as major DC subsets in the 20 + 1.5 intestine. Previous studies indicated that intestinal CX3CR1 + + 1 10 CD11b CD11c cells are further divided into two subsets based wild-type 0.5 LysM-cre;Stat3fl/fl + PBS on the expression level of CX3CR1, but these studies did not LysM-cre;Stat3fl/fl + wild-type M high int 0 * 0 * * * * reg analyze differential functions of CX3CR1 and CX3CR1 α-CD3 (−) (+) (−) (+)(−) (+) (−)(+) (−) (+)(−) (+) high cells (13, 17). This study clearly demonstrates that CX3CR1 + + 15 CD11b CD11c cells are a regulatory myeloid cell subset B possessing unique functions that directly suppress T-cell pro- 12 * + + liferation. Previous studies indicated that CX3CR1 CD11b 9 CD11c+ cells mediate inflammatory responses such as Th17 cell + 6 induction (21–23). In this regard, when splenic naïve CD4 T Colitis score + + + cells were cocultured with unsorted CX3CR1 CD11b CD11c 3 high int − cells, including CX3CR1 and CX3CR1 cells, T cells did not ( ) M reg 0 vigorously proliferate, although they produced IL-17. In contrast, (−) + int splenic naïve CD4 T cells cocultured with CX3CR1 subset wild-type M reg robustly proliferated and produced higher amounts of IL-17 + compared with T cells cocultured with unsorted CX3CR1 C D + + IFN-γ (pg/ml) IL-17 (ng/ml) CD11b CD11c cells (Fig. S9B). These observations would be # n.s. high 1600 n.s. # 4 ) 20 attributable to suppression of T-cell proliferation by CX3CR1 5 M cells present within the CX CR1+ CD11b+ CD11c+ cell 16 reg 3 1200 3 high int population. Thus, CX3CR1 and CX3CR1 cells are shown to 12 T cells (x 10 (x T cells possess distinct functions suppressing and activating T cells, re- 800 2 + 8 spectively. At present, these functionally distinct populations 400 1 4 can only be separated by expression level of CX3CR1, indicating int IMMUNOLOGY * colonic CD4 that both populations are related. It is possible that CX3CR1 0 0 * 0 high α-CD3 (−)((+)(−) +) −) (+)( (−)((+)(−)(+) −) +)( cells are precursors of CX3CR1 cells, and both cell pop- ulations show plasticity. Several previous reports have shown that wild-type + LysM-cre;Stat3fl/fl + PBS CX3CR1 cells have macrophage-like properties (14, 15, 35, 36). fl/fl LysM-cre;Stat3 + wild-type Mreg high Indeed, CX3CR1 Mreg cells show macrophage-like morphol- ogy and express macrophage-related surface markers. On the Fig. 5. Defective Mreg cell function leads to development of colitis. (A, C, and − − − − fl/fl 4 / / high D)LysM-cre; Stat3 mice were transferred i.p. with 7 × 10 wild-type other hand, we found that Stat3 and Il10 CX3CR1 Mreg high high CX3CR1 Mreg cells at 4 and 6 wk of age. At 2 wk after the last transfer, cells induced T-cell proliferation, indicating that CX3CR1 + splenic (A) or colonic lamina propria (C) CD4 T cells were analyzed for pro- Mreg cells show DC-like properties in the absence of the IL-10/ # + duction of IFN-γ and IL-17A. *, not detected; P < 0.008. Total number of CD4 Stat3 signaling in vitro (Fig. S9C). Therefore, it is possible that T cells in the colonic lamina propria was analyzed (D). #P < 0.0079. Data are int high CX3CR1 cells are precursors of CX3CR1 cells and termi- from two independent experiments with four mice per group. (B) Hema- nally differentiate into CX CR1high M cells sharing some toxylin and eosin staining of colon sections at 2 wk after the last transfer 3 reg (Left) and the colitis score (Right). (Original magnification, 200×.) *P < 0.0002. macrophage-like properties in response to IL-10. Several regulatory subsets of myeloid cells, which might be re- lated to Mreg cells, have been reported. Colonic IL-10-producing high + + the involvement of CX3CR1 Mreg cells in the pathogenesis of F4/80 CD11b myeloid cells have been reported to mediate the fl/fl intestinal inflammation in LysM-cre; Stat3 mice. LysM-cre; maintenance of Foxp3 expression in Treg cells (26). This subset is fl/fl high observed in MLNs, where M cells are not present. Intestinal Stat3 mice were transferred with wild-type CX3CR1 Mreg reg cells and were analyzed for Th1 and Th17 activities. In LysM-cre; macrophages suppress T-cell responses via IL-10-dependent in- fl fl Stat3 / mice, Th1 and Th17 activities, as determined by IFN-γ duction of Treg cells (22). Unlike Mreg cells, these macrophages do and IL-17A production from splenic CD4+ T cells, respectively, not express CD11c, CD14, or DEC205. Most recently, the activity fl/fl of intestinal macrophages has been shown to be regulated by were increased (Fig. 5A). Even in LysM-cre; Stat3 mice fi transferred with wild-type CX CR1high M cells, Th1 and Th17 CX3CR1 (37). Indeed, mice de cient in CX3CR1 were highly 3 reg sensitivetointestinalinflammation induced by dextran sodium activity remained considerably enhanced. However, the severity high sulfate. These intestinal cells might include CX3CR1 Mreg cells. of intestinal inflammation was greatly improved in LysM-cre; high fl/fl high It is interesting to analyze the suppressive activity of CX3CR1 Stat3 mice transferred with wild-type CX3CR1 Mreg cells fi + Mreg cells in CX3CR1-de cient mice. MDSCs are also the cell (Fig. 5B). CD4 T cells in the colonic lamina propria produced population similar to CX CR1high M cells. However, MDSCs increased amounts of IFN-γ and IL-17A even in LysM-cre; 3 reg fl/fl high do not express CD11c or MHC class II and are hardly detected in Stat3 mice transferred with wild-type CX3CR1 Mreg cells; high + healthy mice (9, 38), whereas CX3CR1 Mreg cells express however, the total number of CD4 T cells was markedly de- CD11c and MHC class II and are abundant in the intestinal fi high creased in the lamina propria (Fig. 5 C and D). These ndings lamina propria of healthy mice. Expression of CX CR1 M high 3 reg demonstrate that the defective activity of CX3CR1 Mreg cells cell-related genes (Hpgd, Cd163, Hmox1, Cd209f,andCd209g) is critically involved in the development of spontaneous colitis was severely reduced in MDSCs, whereas MDSC-related genes when T cells are overactivated and that transfer of normal (Arg1, Nos2, Cybb, S100a8,andS100a9) were not expressed in high high CX3CR1 Mreg cells is able to ameliorate the colitis. CX3CR1 Mreg cells (Fig. S9 D and E). + − CD103 CX3CR1 DCs have been shown to promote intestinal Discussion + immune tolerance through the generation of Foxp3 Treg cells (15, high In the present study, we characterized the intestinal CX3CR1 16, 18, 19). Intestinal macrophages are also reported to induce + + + CD11b CD11c Mreg cell subset, which directly inhibits T-cell Foxp3 Treg cells (22). Intestinal epithelial cells have been impli- proliferation and, thereby, prevents T-cell-dependent intestinal cated in promoting differentiation of CD103+ DCs possessing

Kayama et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 high a property to induce Treg cells (39, 40). CX3CR1 Mreg cells Materials and Methods localize very close to intestinal epithelial cells. Therefore, in- Mice. C57BL/6J mice and BALB/c mice at 6–8 wk of age were purchased from testinal epithelial cells might be involved in the final maturation CLEA Japan or Japan SLC. Male 6-wk-old CB17-SCID mice were purchased high fl/fl (or differentiation) of CX3CR1 Mreg cells in the intestinal from CLEA Japan. LysM-cre; Stat3 mice and CX3CR1-EGFP knock-in (het- − − lamina propria through modulation of IL-10 production. erozygous) mice were generated as described (32, 41, 42). Il10 / mice were high In the present study, we characterize intestinal CX3CR1 purchased from The Jackson Laboratory. Each mutant mouse strain was + + high fi CD11b CD11c cells (CX3CR1 Mreg cells) that suppress backcrossed onto a C57BL/6J background for at least ve generations. All intestinal inflammation through direct inhibition of T-cell pro- animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Osaka University. liferation in the intestinal lamina propria. Treg cells with a normal suppressive activity are abundantly present in LysM-cre/Stat3f/f The details of reagents, isolation of lamina propria cells, histopathological high score, and proliferation assay are described in SI Materials and Methods. mice (33), indicating that defective activity of CX3CR1 Mreg cells can cause intestinal inflammation even in the presence of high ACKNOWLEDGMENTS. We thank S. Sakaguchi and T. Hirano for fruitful Treg cells. Therefore, CX3CR1 Mreg cells maintain the intes- discussions; C. Hidaka for secretarial assistance; E. Morii for histological tinal homeostasis together with Treg cells, as well as several innate fi analysis; J. Kikuta and E. Ohata for microscopy analysis; and K. Atarashi, cell subsets that have regulatory functions. Identi cation of an D. Dodd, and Y. Magota for technical assistance. This work was supported by high CX3CR1 Mreg population in the human intestines and char- a grant-in-aid from the Ministry of Education, Culture, Sports, Science and high acterization of human CX3CR1 Mreg function in patients with Technology; the Ministry of Health, Labour and Welfare; The Kato Memorial IBD will be a critical future issue in establishing their role in the Trust for Nambyo Research; the Osaka Foundation for the Promotion of pathogenesis of intestinal inflammation in humans. Clinical Immunology; and the Takeda Science Foundation.

1. Bouma G, Strober W (2003) The immunological and genetic basis of inflammatory 22. Denning TL, Wang YC, Patel SR, Williams IR, Pulendran B (2007) Lamina propria bowel disease. Nat Rev Immunol 3:521–533. macrophages and dendritic cells differentially induce regulatory and interleukin 2. Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of inflammatory bowel 17-producing T cell responses. Nat Immunol 8:1086–1094. disease. Nature 448:427–434. 23. Atarashi K, et al. (2008) ATP drives lamina propria T(H)17 cell differentiation. Nature 3. Strober W, Fuss I, Mannon P (2007) The fundamental basis of inflammatory bowel 455:808–812. disease. J Clin Invest 117:514–521. 24. Manocha M, et al. (2009) Blocking CD27-CD70 costimulatory pathway suppresses – 4. Wing K, Sakaguchi S (2010) Regulatory T cells exert checks and balances on self tol- experimental colitis. J Immunol 183:270 276. erance and autoimmunity. Nat Immunol 11:7–13. 25. Ueda Y, et al. (2010) Commensal microbiota induce LPS hyporesponsiveness in colonic – 5. Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T (2009) Regulatory T cells: macrophages via the production of IL-10. Int Immunol 22:953 962. 26. Murai M, et al. (2009) Interleukin 10 acts on regulatory T cells to maintain expression How do they suppress immune responses? Int Immunol 21:1105–1111. of the transcription factor Foxp3 and suppressive function in mice with colitis. Nat 6. Morelli AE, Thomson AW (2007) Tolerogenic dendritic cells and the quest for trans- Immunol 10:1178–1184. plant tolerance. Nat Rev Immunol 7:610–621. 27. Manicassamy S, et al. (2010) Activation of beta-catenin in dendritic cells regulates 7. Wakkach A, et al. (2003) Characterization of dendritic cells that induce tolerance and immunity versus tolerance in the intestine. Science 329:849–853. T regulatory 1 cell differentiation in vivo. Immunity 18:605–617. 28. Siddiqui KR, Laffont S, Powrie F (2010) E-cadherin marks a subset of inflammatory 8. Gabrilovich DI, Nagaraj S (2009) Myeloid-derived suppressor cells as regulators of the dendritic cells that promote T cell-mediated colitis. Immunity 32:557–567. – immune system. Nat Rev Immunol 9:162 174. 29. Takada Y, et al. (2010) chemoattractant -1 contributes to gut ho- 9. Ostrand-Rosenberg S, Sinha P (2009) Myeloid-derived suppressor cells: Linking in- meostasis and intestinal inflammation by composition of IL-10-producing regulatory fl – ammation and cancer. J Immunol 182:4499 4506. macrophage subset. J Immunol 184:2671–2676. 10. Laffont S, Powrie F (2009) Immunology: Dendritic-cell genealogy. Nature 462: 30. Jung S (2010) Dendritic cells: A question of upbringing. Immunity 32:502–504. 732–733. 31. Kühn R, Löhler J, Rennick D, Rajewsky K, Müller W (1993) Interleukin-10-deficient 11. Strober W (2009) The multifaceted influence of the mucosal microflora on mucosal mice develop chronic enterocolitis. Cell 75:263–274. dendritic cell responses. Immunity 31:377–388. 32. Takeda K, et al. (1999) Enhanced Th1 activity and development of chronic entero- 12. Coombes JL, Powrie F (2008) Dendritic cells in intestinal immune regulation. Nat Rev colitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10:39–49. Immunol 8:435–446. 33. Kobayashi M, et al. (2003) Toll-like receptor-dependent production of IL-12p40 causes 13. Varol C, et al. (2009) Intestinal lamina propria dendritic cell subsets have different chronic enterocolitis in myeloid cell-specific Stat3-deficient mice. J Clin Invest 111: origin and functions. Immunity 31:502–512. 1297–1308. 14. Bogunovic M, et al. (2009) Origin of the lamina propria dendritic cell network. Im- 34. Melillo JA, et al. (2010) Dendritic cell (DC)-specific targeting reveals Stat3 as a nega- munity 31:513–525. tive regulator of DC function. J Immunol 184:2638–2645. 15. Niess JH, Adler G (2010) Enteric flora expands gut lamina propria CX3CR1+ dendritic 35. Niess JH (2010) What are CX3CR1+ mononuclear cells in the intestinal mucosa? Gut cells supporting inflammatory immune responses under normal and inflammatory Microbes 1:396–400. conditions. J Immunol 184:2026–2037. 36. Scott CL, Aumeunier AM, Mowat AM (2011) Intestinal CD103+ dendritic cells: Master – 16. Johansson-Lindbom B, et al. (2005) Functional specialization of gut CD103+ dendritic regulators of tolerance? Trends Immunol 32:412 419. cells in the regulation of tissue-selective T cell homing. J Exp Med 202:1063–1073. 37. Medina-Contreras O, et al. (2011) CX3CR1 regulates intestinal macrophage homeo- 17. Schulz O, et al. (2009) Intestinal CD103+, but not CX3CR1+, antigen sampling cells stasis, bacterial translocation, and colitogenic Th17 responses in mice. J Clin Invest 121:4787–4795. migrate in lymph and serve classical dendritic cell functions. J Exp Med 206: 38. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI (2008) Subsets of myeloid-derived 3101–3114. suppressor cells in tumor-bearing mice. J Immunol 181:5791–5802. 18. Coombes JL, et al. (2007) A functionally specialized population of mucosal CD103+ 39. Iliev ID, Mileti E, Matteoli G, Chieppa M, Rescigno M (2009) Intestinal epithelial cells DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent promote colitis-protective regulatory T-cell differentiation through dendritic cell – mechanism. J Exp Med 204:1757 1764. conditioning. Mucosal Immunol 2:340–350. 19. Sun CM, et al. (2007) Small intestine lamina propria dendritic cells promote de novo 40. Iliev ID, et al. (2009) Human intestinal epithelial cells promote the differentiation of – generation of Foxp3 T reg cells via retinoic acid. J Exp Med 204:1775 1785. tolerogenic dendritic cells. Gut 58:1481–1489. 20. Matteoli G, et al. (2010) Gut CD103+ dendritic cells express indoleamine 2,3-dioxy- 41. Ishii M, et al. (2009) Sphingosine-1-phosphate mobilizes osteoclast precursors and genase which influences T regulatory/T effector cell balance and oral tolerance in- regulates bone homeostasis. Nature 458:524–528. duction. Gut 59:595–604. 42. Jung S, et al. (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeted 21. Uematsu S, et al. (2008) Regulation of humoral and cellular gut immunity by lamina deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20: propria dendritic cells expressing Toll-like receptor 5. Nat Immunol 9:769–776. 4106–4114.

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