Osteopontin Regulates Development and Function of Invariant Natural Killer T Cells

Osteopontin regulates development and function of invariant natural killer T cells

Hongyan Diao*†‡, Kazuya Iwabuchi§, Lanjuan Li†, Kazunori Onoe§, Luc Van Kaer¶, Shigeyuki Konʈ, Yoshinari Saitoʈ, Junko Morimotoʈ, David T. Denhardt**, Susan Rittling††, and Toshimitsu Uede‡ʈ

*State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Afﬁliated Hospital of Medical College, Zhejiang University, Hangzhou, Zhejiang 310003, China; †Department of Matrix Medicine, §Division of Immunobiology and ʈMolecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan; ¶Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; **Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854; and ††The Forsyth Institute, Boston, MA 02115

Edited by Harvey Cantor, Dana–Farber Cancer Institute, Boston, MA, and approved September 4, 2008 (received for review June 24, 2008) Invariant natural killer T (iNKT) cells belong to a subset of lympho- Osteopontin (OPN) skews T cell differentiation toward Th1 (13). cytes bridging innate and acquired immunity. We demonstrated OPN gene expression is regulated by T-bet, and, importantly, that osteopontin (OPN) is involved in the activation of iNKT cells. T-bet-dependent expression of OPN in T cells is essential for In the present work, we examined whether OPN affects develop- skewing of T cells toward Th1 cell differentiation (14). T-bet is also ment and function of iNKT cells. We found that the number of critical for OPN expression in plasmacytoid dendritic cells (pDC) peripheral iNKT cells was significantly reduced in OPN-deficient (15). Intracellular OPN acts as an integral part of the signal mice compared with wild-type mice. Although the number of transduction machinery in pDC. OPN has been implicated in the thymic iNKT cells was not different between WT and OPN-deficient pathogenesis of various immunological disorders (16, 17). There- mice, intrathymic iNKT cell maturation was impaired in OPN- fore, it is important to examine whether OPN affects the develop- deficient mice. iNKT cell function was also significantly altered in ment and function of immune cells beside T cells and pDC. In this OPN-deficient mice, as evidenced by (i) deficient down-regulation regard, iNKT cells are involved in the pathogenesis of hepatic injury of iNKT cell receptor, (ii) reduction of IL-4 production while pre- (18). We demonstrated that OPN-deficient mice were protected serving production of IFN-␥, and (iii) reduction of Fas ligand (FasL) from Con A-induced hepatic injury and OPN is critically involved expression, leading to reduced Fas/FasL-dependent cytotoxicity in iNKT cell activation (17). Therefore, we attempted to clarify how against hepatocytes. Importantly, activation of the transcription OPN affects the development and function of iNKT cells. Here, we factors NFAT2 (nuclear factor of activated T cells 2) and GATA-3 was show that iNKT cell number in the liver and spleen of OPN- impaired, whereas activation of T-bet was preserved in iNKT cells deficient mice, compared with WT mice, is significantly reduced. of OPN-deficient mice. These data collectively indicate that OPN Although the thymic iNKT cell number was not different between plays a pivotal role not only in the development, but also in the wild-type (WT) and OPN-deficient mice, intrathymic development function of iNKT cells. of iNKT cells was blocked in OPN-deficient mice. iNKT cell function was also significantly altered in OPN-deficient mice, as nuclear factor of activated T cells (NFAT) ͉ GATA-3 ͉ Fas ligand (FasL) evidenced by (i) deficient down-regulation of iNKT cell receptor upon activation; (ii) reduction of IL-4 secretion and production, while preserving production of IFN-␥; and (iii) reduction of Fas nvariant natural killer T (iNKT) cells constitute a subset of T cells ligand (FasL) expression, leading to reduced Fas/FasL-dependent Iand specifically recognize glycolipid antigens presented by CD1d cytotoxicity against hepatocytes. Importantly, activation of the (1), regulating T helper (Th)1 and Th2 immune responses by transcription factors NFAT2 and GATA-3 was impaired, whereas producing various cytokines (1, 2). iNKT cells are involved in the activation of T-bet was preserved in iNKT cells of OPN-deficient development of several human diseases such as type I diabetes, mice. These data collectively indicate that OPN plays a pivotal role multiple sclerosis, and systemic lupus erythematosus (3–5). not only in the development but also in the function of iNKT cells. It has been shown that specific transcription factors are critical for the development of polarized Th1 and Th2 responses (6) in Results and Discussion conventional T cells. The T-bet is a master coordinator of gene Involvement of OPN in iNKT Cell Development. We isolated iNKT expression in T cells and is critical for the development of Th1 T ϩ cells from normal WT mice and studied OPN expression at the cells (7). T-bet-deficient CD4 T cells exhibit a marked decrease in mRNA level. Resident intrahepatic iNKT cells clearly expressed IFN-␥ production. Interestingly, mature iNKT cells cannot be OPN, whereas iNKT cells from OPN-deficient mice did not (Fig. formed in the absence of T-bet (8). GATA-3 is a key transcription 1A). OPN is involved in the survival of cells (16) and differentiation factor that controls Th2 cell development and Th2 cytokine pro- and development of dendritic cells (DCs) (19). Thus, we examined ϩ duction in conventional T cells (9). CD4 T cell numbers are whether OPN deficiency impacted the development of iNKT cells. markedly reduced in GATA-3-deficient mice, and in vitro differ- In our previous preliminary examination, we detected only a slight entiated CD4ϩ T cells from these animals have defects in Th2 cytokine production. Similarly, mature iNKT cells are drastically reduced in GATA-3-deficient mice. In addition, GATA-3 mRNA Author contributions: H.D., L.L., L.V.K., and T.U. designed research; H.D., K.I., K.O., S.K., Y.S., and J.M. performed research; D.T.D. and S.R. contributed new reagents/analytic tools; H.D. expression correlates with IL-4 expression in iNKT cells (10). analyzed data; and H.D. and T.U. wrote the paper. STAT6 is critical for the development of Th2 responses in conven- The authors declare no conflict of interest. tional T cells, yet iNKT cells do not require STAT6 for IL-4 This article is a PNAS Direct Submission. production (11). The nuclear factor of activated T cells (NFAT) family is also involved in regulating the Th1/Th2 balance (6). NFAT Freely available online through the PNAS open access option. ‡To whom correspondence may be addressed at: Institute for Genetic Medicine, Hokkaido activation leads to the production of a variety of cytokines in University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail: [email protected] or conventional T cells. In contrast, NFAT2 activation leads to the [email protected]. ␣ ␥ up-regulation of IL-4 expression, but not TNF- or IFN- in NKT This article contains supporting information online at www.pnas.org/cgi/content/full/ cells (12). Thus, T-bet, STAT6, GATA-3, and NFAT2 behave 0806089105/DCSupplemental. differently between iNKT and conventional T cells. © 2008 by The National Academy of Sciences of the USA

15884–15889 ͉ PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806089105 Downloaded by guest on September 27, 2021 Fig. 1. Deﬁcient intrathymic maturation of iNKT cells in OPN-deﬁcient mice. (A) RT-PCR analyses of mRNA expression of OPN in iNKT cells, isolated from liver of WT and OPNϪ/Ϫ adult mice. Data are representative of three independent experiments. (B) Percentage of iNKT cells in various organs of WT and OPNϪ/Ϫ mice at the indicated time points after birth. Data are presented as mean Ϯ SEM, n ϭ 5 per group. (C) iNKT cells from B were further fractionated by CD44-FITC and NK1.1-PerCP-Cy5.5. (Upper) Data are representative of three independent experiments. (Lower) Data represent mean Ϯ SEM. n ϭ 4 per group. *, P Ͻ 0.05; **, P Ͻ 0.005.

decrease in the numbers of hepatic iNKT cells in OPN-deficient 1C). The maturation of thymic iNKT cells was altered greatly in the mice (17). However, in those experiments OPN-deficient mice were absence of OPN. The percentage of immature stage 1 iNKT cells studied at early backcrosses to C57BL/6 mice. In the present work, gradually decreased as mice aged, which contrasts with the gradual we used OPN-deficient mice, backcrossed to C57BL/6 at least 11 increase of stage 3 iNKT cells in WT mice. At 1 week, Ϸ6% and generations. Then, we compared the number of CD1d-restricted 33% of iNKT are at stage 3 and stage 1, respectively, and at 4 weeks, iNKT cells in liver, spleen, and thymus between adult OPN- 32% and 7% of iNKT are at stage 3 and stage 1, respectively, in WT deficient and WT mice. The percentages of CD1d-restricted iNKT mice. In contrast, 20% of thymic iNKT cells remained in stage 1 at cells in the liver and spleen of OPN-deficient mice were significantly 4 weeks in OPN-deficient mice. The number of stage 3 iNKT cells lower than those in WT mice. In the liver and spleen, there was an was significantly reduced at 3 and 4 weeks in OPN-deficient mice. Ϸ2-fold reduction compared with WT iNKT cells, but there was no It is known that stage 1 thymic iNKT cells cannot exit the thymus significant difference in thymic iNKT cell prevalence between (20). Thus, our data indicate that the reduction of peripheral iNKT OPN-deficient and WT mice [supporting information (SI) Fig. S1]. cells in OPN-deficient mice can be partly explained by deficient To characterize the involvement of OPN during iNKT cell devel- intrathymic iNKT cell maturation. After exiting the thymus, iNKT opment further, we examined the total numbers of iNKT cells (Fig. cells undergo further maturation by up-regulating CD69 expression S2). There was no significant difference in terms of iNKT cell (20). Therefore, we compared the expression of CD69 on periph- numbers in thymus between OPN-deficient and WT mice from 1 eral iNKT cells between WT and OPN-deficient mice. We found no to 4 weeks (Fig. S2 and Fig. 1B). iNKT cells in the spleen of significant difference in terms of CD69 expression (Fig. S3), OPN-deficient mice were lower than those in WT mice at 4 weeks, indicating that the iNKT cell maturational step involving CD69 but not from 1 to 3 weeks. The cell number of iNKT cells in the liver up-regulation does not depend on the presence of OPN. One may was significantly reduced in OPN-deficient mice, compared with argue that reduction of peripheral iNKT cells can be explained by those in WT mice at 3 and 4 weeks, but not at 1 and 2 weeks. Thus, an increase in apoptosis of peripheral iNKT cells in OPN-deficient the number of peripheral iNKT cells is significantly reduced, mice. Thus, we compared apoptosis of iNKT cells in peripheral whereas that of intrathymic iNKT cells is not reduced in OPN- organs and found that the number of apoptotic iNKT cells was not deficient mice. This reduction of peripheral iNKT cell could be significantly different between WT and OPN-deficient mice caused by a blockade in iNKT cell maturation. This possibility (Fig. S4). prompted us to examine whether intrathymic maturation of iNKT cells in OPN-deficient mice was impaired. Immature iNKT cells Impairment of Activation-Induced T Cell Receptor (TCR) Down- undergo several well defined developmental stages in the thymus. Regulation on Peripheral iNKT Cells in OPN-Deficient Mice. We The most immature iNKT cells are at CD44ϪNK1.1Ϫ (stage 1), next investigated whether OPN is involved in iNKT cell function. which differentiate through the CD44ϩNK1.1Ϫ (stage 2) to become Stimulation of iNKT cells with anti-CD3 or specific ligand, ␣- ϩ ϩ CD44 NK1.1 (stage 3) cells (20). At 1 week, Ϸ33, 60, and 6% of galactosylceramide (␣-GalCer), results in the rapid disappearance IMMUNOLOGY iNKT cells are at stage 1, 2, and 3, respectively, in WT mice (Fig. of these cells in vivo (21). In addition, iNKT cell activation by Con

Diao et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15885 Downloaded by guest on September 27, 2021 Fig. 2. Deficient TCR-mediated signaling in OPN-deficient mice. (A) OPNϪ/Ϫ and WT mice were injected with Con A (10 mg/kg) or PBS, and percentages of NKT cells were analyzed at 2 h after injection. (Left) Cells from liver. (Right) Cells from spleen. Data represent mean Ϯ SEM, n ϭ 5 per group. (B) OPNϪ/Ϫ and WT mice were injected with Con A (10 mg/kg) or PBS, and iNKT cell numbers in liver and spleen were analyzed at the indicated time points. Data represent mean Ϯ SEM, n ϭ 5 per group. (C) Serum cytokine levels were measured after ␣-GalCer injection at the indicated time points. Data represent mean Ϯ SEM, n ϭ 8 per group. (D) Production of cytokines by iNKT cells from WT or OPNϪ/Ϫ mice. iNKT cells were stimulated for 3 days in vitro with ␣-GalCer. Cytokine production in the culture supernatant was measured by ELISA. Data represent mean Ϯ SEM of three independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.005; NS, not significant.

A leads to a rapid reduction in iNKT cell numbers, caused by IL-4, in response to ␣-GalCer both in vivo and in vitro (1). To profound down-regulation of iNKT cell receptors (18, 22). There- determine whether deficiency of OPN also affects cytokine secre- fore, we used ␣-GalCer-loaded CD1d-dimer or NK1.1 and TCR␤ tion by iNKT cells in vivo, we injected i.v. ␣-GalCer into OPN- to trace the response of iNKT cells to Con A and examined whether deficient and WT mice. IL-4 levels reached a peak in the serum of down-regulation of the iNKT cell receptor is impaired in OPN- WTmiceat2hafterinjection (Fig. 2C). In OPN-deficient mice, deficient mice. As shown in Fig. 2A, injection of Con A led to a IL-4 levels were significantly lower than those in WT mice. In significant reduction of percentage of iNKT cells in the liver and contrast, there was no significant difference in IFN-␥ levels between spleen of WT mice at 2 h. In contrast, such a reduction of WT and OPN-deficient mice. Nevertheless, neither IL-4 nor IFN-␥ ␣ percentage of splenic iNKT cells was not observed in OPN- was detected in serum of -GalCer-treated CD1d-deficient (thus, deficient mice. A significant reduction of percentage of hepatic iNKT cell-deficient) mice, indicating that those cytokines are iNKT cells was observed in OPN-deficient mice, but this was much derived from iNKT cells. However, it is possible that other cells milder compared with WT mice. To characterize the down- were activated by iNKT cell-derived cytokines, leading to the production of IL-4 or IFN-␥. Thus, purified iNKT cells were regulation of iNKT cells in the presence or absence of OPN further, ␣ we monitored the actual number of iNKT cells at various time stimulated in vitro with -GalCer. Consistent with our in vivo work, points after Con A injection. After an initial reduction of iNKT cell significantly lower amounts of IL-4 were secreted by iNKT cells from OPN-deficient mice than WT mice, but similar levels of IFN-␥ number at 24 h, there was a significant increase in iNKT cell number were secreted (Fig. 2D). Thus, it appears that the lack of OPN at 48 and 72 h in spleen and liver of WT mice (Fig. 2B). In contrast, results in reduced IL-4 secretion by iNKT cells. To confirm this although iNKT cell number increased at 48 and 72 h in spleen and finding further, purified iNKT cells were stimulated in vitro with liver of OPN-deficient mice, this increase was substantially lower ␣-GalCer in the presence or absence of exogenous recombinant than in WT mice. It should be noted that at 24 h, iNKT cell numbers OPN. We found that treatment of iNKT cells of OPN-deficient in OPN-deficient mice were much higher than in WT mice. This mice with exogenous OPN resulted in significant augmentation of finding is explained by the lack of significant receptor down- IL-4, but not IFN-␥ production (Fig. S5). Nevertheless, iNKT cells regulation in OPN-deficient mice. Thus, our data demonstrated derived from WT mice secreted similar amounts of IL-4 in the that iNKT cell function, as judged by TCR down-regulation, is absence or presence of exogenous OPN. We then tested whether impaired in OPN-deficient mice. These data led us to hypothesize the lack of OPN simply affected secretion or also production of that other iNKT cell functions might be impaired in the absence of IL-4. Therefore, purified iNKT cells were stimulated in vitro with OPN as well. ␣-GalCer, and cytokine production was examined by real-time PCR. Results showed impaired production of IL-4 but not IFN-␥ Impaired Secretion of IL-4 by OPN-Deficient iNKT Cells. One unique in OPN-deficient mice (Fig. S6). feature of iNKT cells is the rapid secretion of cytokines, including IFN-␥ and IL-4, upon activation. Cytokine secretion by iNKT cells Reduced Cytotoxicity of OPN-Deficient iNKT Cells Against Hepato- plays a critical role in the pathogenesis of various diseases (23, 24). cytes. iNKT cells exhibit cytotoxicity against hepatocytes in a iNKT cells produce high levels of cytokines, especially IFN-␥ and Fas/FasL-dependent manner in a Con A-induced hepatic injury

15886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806089105 Diao et al. Downloaded by guest on September 27, 2021 Fig. 3. Reduced cytotoxic ability of iNKT cells in OPN-deficient mice. (A) iNKT cells of WT or OPNϪ/Ϫ mice were stimulated for 3 days in vitro as described in Materials and Methods. The cells were subjected to a standard cytotoxic assay with target TLR2 cells. Data represent mean Ϯ SEM of three independent experiments. (B) Fas expression of TLR2 was analyzed by flow cytometry. Data are representative of three independent experiments. (C) iNKT cells of WT mice were cultured as described above. The target cells were treated with Fas-siRNA or control siRNA. A standard cytotoxic assay against TLR2 cells was performed. Cont siRNA, control siRNA. Data represent mean Ϯ SEM of three independent experiments. (D) FasL expression of iNKT cells from WT or OPNϪ/Ϫ mice. FasL mRNA expression was determined by quantitative real-time PCR. The relative value of gene expression was normalized against the gene expression levels of G3PDH. Data represent mean Ϯ SEM of three independent experiments. (E) Up- regulation of FasL mRNA expression on iNKT cells by exogenous IL-4. iNKT cells of OPNϪ/Ϫ mice or WT mice were cultured with or without exogenous recombinant IL-4 (200 ng/ml) for 5 h. FasL expression by iNKT cells was determined by quantitative real-time PCR. The relative value of gene expression was normalized against the gene expression levels of G3PDH. Data represent mean Ϯ SEM of three independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.005; NS, not significant.

model (18, 25, 26). In addition, we demonstrated that hepatic injury Defective Activation of NFAT2 in iNKT Cells of OPN-Deficient Mice. in this model is significantly reduced in OPN-deficient mice (17). Our work demonstrated that signaling through the iNKT cell We reasoned that reduced hepatic injury after Con A injection in receptor was impaired in OPN-deficient mice, as judged by im- OPN-deficient mice is caused by reduced OPN-mediated neutro- paired IL-4 secretion and defective receptor down-regulation. It is phil infiltration into the liver and thus reduced neutrophil-mediated known that the transcription factor NFAT2 plays a critical role in hepatic injury (17). However, it is also possible that Fas/FasL- IL-4 production by iNKT cells (12). Therefore, we examined dependent iNKT cell cytotoxicity is impaired in OPN-deficient whether activation of NFAT2 is defective in ␣-GalCer-activated mice, and thus, we assessed the cytotoxic activity of iNKT cells iNKT cells from OPN-deficient mice. NFAT2 protein was clearly against a murine hepatocyte cell line (TLR2). iNKT cells derived up-regulated in nuclear extracts of spleen obtained from WT mice ␣ from WT mice exhibited significant cytotoxicity (Fig. 3A). There at 30 min after -GalCer injection (Fig. 4A), indicating that iNKT was a statistically significant reduction in the cytotoxicity of cell stimulation in vivo leads to activation of NFAT2. However, in iNKT cells from OPN-deficient mice. We then tested whether the OPN-deficient mice, NFAT2 protein was not increased, demon- iNKT cell cytotoxicity against TLR2 cells was Fas/FasL-dependent. strating that NFAT2 activation is defective in the absence of OPN. TLR2 cells expressed high levels of Fas (Fig. 3B). The knockdown Consistent with our Western blot analyses, NFAT2 gene expression ␣ ␣ of Fas expression on TLR2 cells by small interference RNA (Fas in -GalCer-treated WT mice was 2-fold higher than in -GalCer- siRNA) significantly reduced cytotoxicity of WT iNKT cells against treated OPN-deficient mice (Fig. 4B). As expected, augmentation TLR2 cells (Fig. 3C). Importantly, iNKT cells derived from OPN- of NFAT2 gene expression was not detected in OPN-deficient mice. In conventional T cells, GATA-3 is also involved in Th2 cytokine deficient mice expressed significantly lower amounts of FasL expression (9), whereas T-bet is involved in Th1 cytokine expression mRNA compared with those from WT mice (Fig. 3D). Collectively, (7). We examined GATA-3 and T-bet gene expression before and these data suggested that reduced cytotoxicity of iNKT cells from after ␣-GalCer injection and found that GATA-3 expression was OPN-deficient mice against hepatocytes is caused by reduced also impaired in OPN-deficient mice, whereas T-bet expression was expression of FasL, although we could not eliminate the possible preserved in these animals (Fig. 4C). It is interesting to note that involvement of other factors. To address directly whether OPN is T-bet expression is essential for OPN expression (14), whereas OPN critical for FasL expression in iNKT cells, we used iNKT cell expression is not required for T-bet expression. To confirm further hybridoma 1B6, which has been described in ref. 27. The expression that OPN is involved in NFAT activation in iNKT cells, iNKT cell of OPN by 1B6 cells was silenced by OPN-specific siRNA. FasL hybridoma 1B6 cells were stimulated in vitro with ␣-GalCer. The protein expression on 1B6 cells was significantly reduced by OPN- expression of OPN was silenced by OPN-specific siRNA. The specific siRNA (Fig. S7). It is known that IL-4 regulates FasL intranuclear NFAT2 protein expression was clearly increased in expression and augments the cytotoxicity of iNKT cells (28). In the the 1B6 cells after stimulation, whereas silencing of OPN gene present study, we found that activated iNKT cells derived from expression by OPN-specific siRNA led to suppression of NFAT2 OPN-deficient mice secrete reduced amounts of IL-4. Therefore, transcription, thus reduced expression of intranuclear NFAT2 by we reasoned that reduced expression of FasL on iNKT cells is 1B6 cells (Fig. 4D). We also found that NFAT2 mRNA expression caused by reduced secretion of IL-4 by iNKT cells in OPN-deficient in the 1B6 cells was augmented upon stimulation by ␣-GalCer and mice. To test this hypothesis, we treated iNKT cells from WT or that the augmentation of NFAT2 mRNA expression was abrogated OPN-deficient mice with exogenous IL-4 and studied FasL mRNA by OPN gene silencing (Fig. S8). Importantly, production of IL-4 by expression. Expression of FasL mRNA by iNKT cells of OPN- activated 1B6 cells was significantly reduced by OPN gene silencing,

deficient mice, but not WT mice, was significantly increased upon whereas production of IFN-␥ remained unaltered (Fig. 4D). IMMUNOLOGY stimulation with IL-4 (Fig. 3E). In conclusion, our proposed model for the role of OPN in iNKT

Diao et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15887 Downloaded by guest on September 27, 2021 Fig. 4. Deficient activation of NFAT2 by iNKT cells in OPN-deficient mice. (A) Western blot analysis of splenic nuclear extracts. Nuclear extracts were prepared from WT and OPN-deficient mice 30 min after ␣-GalCer injection. Data are representative of three independent experiments. (B and C) Quantitative real-time PCR analyses of gene expression of NFAT2, GATA-3, and T-bet. The relative value of gene expression was normalized against the gene expression levels of G3PDH. Data represent mean Ϯ SEM of three independent experiments. (D) NFAT2 expression by 1B6 hybridoma cells. 1B6 hybridoma cells were treated with OPN-siRNA and stimulated with ␣-GalCer and irradiated CD1d-transfectants. IL-4 and IFN-␥ production in the culture supernatant was measured by ELISA. (Upper) Data are representative of three independent experiments. (Lower) Data represent mean Ϯ SEM of three independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.005; NS, not significant.

cells is illustrated in Fig. 5. The absence of OPN results in impaired Biosciences). Cells were stained with a primary antibody (␣-GalCer-loaded mouse iNKT cell development and defective peripheral iNKT cell activa- CD1d-IgG1 fusion protein DimerX) for 60 min at 4°C, followed by a secondary tion, as evidenced by deficient receptor down-regulation and IL-4 anti-mouse IgG1-phycoerythrin (PE) and anti-TCR␤-antigen-presenting cell (H57– ␣ production. iNKT cell antigen receptor-mediated signaling path- 597) antibody for 30 min at 4°C. iNKT cells were defined as -GalCer-loaded CD1d ϩ ␤ϩ ways, such as activation of NFAT2, was also impaired. Reduced dimer TCR cells. Apoptosis was assayed by annexinV-FITC apoptosis detection kit I. Other antibodies used for staining were anti-NK1.1-PerCP-Cy5.5 (PK136), IL-4 production caused by defective activation of NFAT2 led to anti-FasL-PE (MFL3), anti-Fas-PE (Jo2), anti-CD69-FITC (H1.2F3), and anti-CD44- reduced FasL expression on iNKT cells, which resulted in attenu- FITC (IM7) (all from BD Biosciences). All analyses were performed on a FACSCali- ated cytotoxic activity of iNKT cells against Fas-positive hepato- bur (BD Biosciences) with CellQuest software. cytes. Collectively, our findings have revealed a critical contribution of OPN in the development and function of iNKT cells, which might In Vitro and in Vivo iNKT Cell Stimulation. iNKT cells (gated as ␣-GalCer-loaded be exploited for treatment of diseases that are controlled by OPN CD1d-dimerϩ TCR␤ϩ) were isolated by a FACS Vantage instrument (Becton Dick- or iNKT cells. inson). The sorted iNKT cells were cultured overnight with recombinant IL-2. NKT cells were further stimulated with 100 ng/ml ␣-GalCer (Pharmaceutical Research Materials and Methods Laboratories, Kirin Brewery) in the presence of 100-Gy-irradiated CD11cϩ DCs. In Animals. Specific pathogen-free female C57BL/6 (B6) mice were purchased from some experiments, mice were injected i.v. with 100 ␮g/kg ␣-GalCer or vehicle. Japan SLC. OPN-deficient (OPNϪ/Ϫ) mice (29), backcrossed 11 times to B6 mice at CD1d-transfected cells were provided by Albert Bendelac (University of Chicago, the Institute for Genetic Medicine, Hokkaido University, were used. All mice were Chicago, IL). maintained under specific pathogen-free conditions and used according to in- stitutional guidelines. Analysis of mRNA Expression. Total RNA was isolated by using TRIzol (Invitrogen). The specific primers used were as follows: glyceraldehyde-3-phosphate dehydro- Flow Cytometric Analysis. Hepatic lymphocytes were isolated as described in ref. genase (G3PDH), 5Ј-ACCACAGTCCATGCCATCAC-3Ј (sense), 5Ј-TCCACCACCCCT- 17. Liver, spleen, and thymus cells were first incubated with anti-Fc␥R (2.4G2; BD GTTGCTGTA-3Ј (antisense); Fas, 5Ј-GAGAATTGCTGAAGACATGACAATCC-3Ј

15888 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806089105 Diao et al. Downloaded by guest on September 27, 2021 Fig. 5. Schematic representation of the link between OPN, NFAT activation, IL-4 production, and FasL expression in iNKT cells.

(sense), 5Ј-GTAGTTTTCACTCCAGACATTGTCC-3Ј (antisense); FasL, 5Ј-ATCCCTCT- CAAACCAGACdTdT-3Ј (sense) and 5Ј-GUCUGGUUUGCACUUGCACdTdT-3Ј (anti- GGAATGGGAAGA-3Ј (sense) and 5Ј-CCATATCTGTCCAGTAGTGC-3Ј (antisense); sense); mOPN siRNA, 5Ј-GCCAUGACCACAUGGACGAdTdT-3Ј (sense) and 5Ј- GATA-3, 5Ј-AGAACCGGCCCCTTATCAA-3Ј (sense) and 5Ј-AGTTCGCGCAGGAT- dTdTCGGUACUGGUGUACCUGCU-3Ј (antisense). Irrelevant control siRNA GTCC-3Ј (antisense); T-bet, 5Ј-CAACAACCCCTTTGCCAAAG-3Ј (sense) and 5Ј- (nonspecific control VIII) was purchased from Fisher Scientific. TCCCCCAAGCAGTTGACAGT-3Ј (antisense). Quantitative real-time PCR analysis of mRNA expression was carried out by using LightCycler Fast Start DNA Master 51Cr-Release Cytotoxic Assay. The cytotoxic activity of iNKT cells was assayed by SYBR Green I systems (Roche Diagnostics). The expression of mRNA was calculated a 4-h 51Cr-release cytotoxic assay with the TLR2 hepatocyte cell line (RIKEN Cell by LightCycler software version 3. Data were standardized by G3PDH. Bank) as described in ref. 30. The cells were harvested and seeded at the indicated E/T ratios. Protein Extraction and Western Blot Analyses. Transcription factors were analyzed by extracting nuclear protein with reagent NE-PER (nuclear and cytoplasmic Statistics. Data are presented as means Ϯ SEM and are representative of at least extraction reagents; Pierce) according to the manufacturer’s instructions. After two independent experiments. The significance of differences between two ␮ calibration of protein content, 15 g of each of protein extract was electropho- groups was determined by using Student’s t test. resed through 10–20% polyacrylamide Tris⅐HCl Ready Gels (Bio-Rad) and probed with polyclonal anti-NFAT2 (7A6) as described in ref. 17. ACKNOWLEDGMENTS. We thank Drs. M. Taniguchi and K. Seino (Research Center for Allergy and Immunology, Institute of Physical and Chemical Research, siRNA Design. siRNA against Fas was procured from B-Bridge, Inc.. The sequences Yokohama, Japan) for TLR2 cells. This work was supported by Japan Society for of the selected region to be targeted by siRNA for Fas were: 5Ј-GUGCAAGUG- the Promotion of Science Grant 17790315 (to H.D.).

1. Kawano T, et al. (1997) Cd1d-restricted and TCR-mediated activation of V␣14 NKT cells 17. Diao H, et al. (2004) Osteopontin as a mediator of NKT cell function in T cell-mediated by glycosylceramides. Science 278:1626–1629. liver diseases. Immunity 21:539–550. 2. Bendelac A, Rivera MN, Park SH, Roark JH (1997) Mouse CD1-specific NK1 T cells: 18. Takeda K, et al. (2000) Critical contribution of liver natural killer T cells to a murine Development, specificity, and function. Annu Rev Immunol 15:535–562. model of hepatitis. Proc Natl Acad Sci USA 97:5498–5503. 3. Wilson SB, et al. (1998) Extreme Th1 bias of invariant V␣24J␣Q T cells in type 1 diabetes. 19. Kawamura K, et al. (2005) Differentiation, maturation, and survival of dendritic cells Nature 391:177–181. by osteopontin regulation. Clin Diagn Lab Immunol 12:206–212. 4. Illes Z, et al. (2000) Differential expression of NK T cell V␣24J␣Q invariant TCR chain in 20. Godfrey DI, Berzins SP (2007) Control points in NKT cell development. Nat Rev Immunol the lesions of multiple sclerosis and chronic inflammatory demyelinating polyneurop- 7:505–518. athy. J Immunol 164:4375–4381. 21. Harada M, et al. (2004) Down-regulation of the invariant V␣14 antigen receptor in NKT ␣ 5. Sumida T, Maeda T, Taniguchi M, Nishioka K, Stohl W (1998) TCR V24 gene expression cells upon activation. Int Immunol 16:241–247. in double-negative T cells in systemic lupus erythematosus. Lupus 7:565–568. 22. Tiegs G, Hentschel J, Wendel A (1992) A T cell-dependent experimental liver injury in 6. Ho IC, Glimcher LH (2002) Transcription: Tantalizing times for T cells. Cell 109:S109–S120. mice inducible by concanavalin A. J Clin Invest 90:196–203. 7. Lugo-Villarino G, Maldonado-Lopez R, Possemato R, Penaranda C, Glimcher LH (2003) 23. Akbari O, et al. (2003) Essential role of NKT cells producing IL-4 and IL-13 in ␥ T-bet is required for optimal production of IFN- and antigen-specific T cell activation the development of allergen-induced airway hyperreactivity. Nat Med 9:582– by dendritic cells. Proc Natl Acad Sci USA 100:7749–7754. 588. 8. Townsend MJ, et al. (2004) T-bet regulates the terminal maturation and homeostasis 24. Kinjo Y, Kronenberg M (2005) V␣14I NKT cells are innate lymphocytes that participate of NK and V␣14I NKT cells. Immunity 20:477–494. in the immune response to diverse microbes. J Clin Immunol 25:522–533. 9. Pai SY, Truitt ML, Ho IC (2004) GATA-3 deficiency abrogates the development and 25. Seino K, et al. (1997) Contribution of Fas ligand to T cell-mediated hepatic injury in maintenance of T helper type 2 cells. Proc Natl Acad Sci USA 101:1993–1998. mice. Gastroenterology 113:1315–1322. 10. Kim PJ, et al. (2006) GATA-3 regulates the development and function of invariant NKT cells. J Immunol 177:6650–6659. 26. Saito T, et al. (2004) Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 11. Kaplan MH, Wurster AL, Smiley ST, Grusby MJ (1999) STAT6-dependent and -indepen- 2-deficient mice contributes to severe concanavalin A-induced hepatitis. J Immunol dent pathways for IL-4 production. J Immunol 163:6536–6540. 173:579–585. 12. Wang ZY, et al. (2006) Regulation of Th2 cytokine expression in NKT cells: Unconven- 27. Nyambayar D, et al. (2007) Characterization of NKT cell hybridomas expressing invari- tional use of STAT6, GATA-3, and NFAT2. J Immunol 176:880–888. ant T cell antigen receptors. J Clin Exp Hematop 47:1–8. ␣ 13. Weber GF, Ashkar S, Glimcher MJ, Cantor H (1996) Receptor–ligand interaction be- 28. Kaneko Y, et al. (2000) Augmentation of V 14 NKT cell-mediated cytotoxicity by tween CD44 and osteopontin (ETA-1). Science 271:509–512. interleukin 4 in an autocrine mechanism resulting in the development of concanavalin 14. Shinohara ML, et al. (2005) T-bet-dependent expression of osteopontin contributes to A-induced hepatitis. J Exp Med 191:105–114. T cell polarization. Proc Natl Acad Sci USA 102:17101–17106. 29. Rittling SR, et al. (1998) Mice lacking osteopontin show normal development and bone 15. Shinohara ML, et al. (2006) Osteopontin expression is essential for interferon-␣ pro- structure but display altered osteoclast formation in vitro. J Bone Miner Res 13:1101– duction by plasmacytoid dendritic cells. Nat Immunol 7:498–506. 1111. 16. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS (2001) Osteopontin as a 30. Yanai N, Suzuki M, Obinata M (1991) Hepatocyte cell lines established from transgenic means to cope with environmental insults: Regulation of inflammation, tissue remod- mice harboring temperature-sensitive simian virus 40 large T-antigen gene. Exp Cell

eling, and cell survival. J Clin Invest 107:1055–1061. Res 197:50–56. IMMUNOLOGY

Diao et al. PNAS ͉ October 14, 2008 ͉ vol. 105 ͉ no. 41 ͉ 15889 Downloaded by guest on September 27, 2021