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. 2009 Aug 11;106(32):13463-8.
doi: 10.1073/pnas.0906988106. Epub 2009 Jul 29.

IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells

Liying Guo  1 , Gang Wei, Jinfang Zhu, Wei Liao, Warren J Leonard, Keji Zhao, William Paul
Affiliations

IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells

Liying Guo et al. Proc Natl Acad Sci U S A. .

Abstract

Expression of T1ST2, the IL-33R, by Th2 cells requires GATA3. Resting Th2 cells express little GATA3, which is increased by IL-33 and a STAT5 activator, in turn increasing T1ST2 from its low-level expression on resting Th2 cells. Th2 cells that have upregulated T1ST2 produce IL-13, but not IL-4, in response to IL-33 plus a STAT5 activator in an antigen-independent, NF-kappaB-dependent, cyclosporin A (CsA)-resistant manner. Similarly, Th17 cells produce IL-17A in response to IL-1beta and a STAT3 activator and Th1 cells produce IFNgamma in response to IL-18 and a STAT4 inducer. Thus, each effector Th cell produces cytokines without antigenic stimulation in response to an IL-1 family member and a specific STAT activator, implying an innate mechanism through which memory CD4 T cells are recruited by an induced cytokine environment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
STAT5 is required for T1ST2 expression. (A) Naïve CD4 T cells purified from 5C.C7 transgenic Rag2−/− mice were cultured under Th2 conditions for indicated periods. D1 indicates cells that had just completed Th2 priming. D5 indicates cells cultured in IL-2-containing cRPMI for 5 days after completion of Th2 priming. (B) 3xTh2 cells that had been maintained in IL-2 medium for 2 weeks were cultured in medium containing indicated cytokines for 48 h and then stained for T1ST2. (C) 3xD14 Th2 cells were cultured in the cytokines for the indicated periods and then stained for T1ST2. The experiments were performed 3 times.
Fig. 2.
Fig. 2.
T1ST2 expression is GATA3 dependent. (A) 2xTh2 cells derived from Gata3“floxed” or C57BL/6 mice were retrovirally infected with GFP-Cre during the third round of Th2 priming. GFP-Cre positive cells were sorted and cultured under Th2 conditions for an additional round and cells were collected to measure T1st2 mRNA by QPCR. Four-round primed Th1 (4xTh1) cells derived from C57BL/6 mice were cultured and examined in parallel. (B) 3xTh2 cells from Gata3“floxed” or C57BL/6 mice were retrovirally infected with GFP-Cre. GFP-Cre positive and negative cells were sorted and cultured under Th2 conditions for an additional round and then maintained in IL-2-containing medium. Two weeks later, the cells were divided into 3 portions, cultured in medium containing IL-2 alone, IL-33 alone, or IL-2 plus IL-33. Two days later, cells were collected to measure T1st2 mRNA. (C) CD4 T cells were cultured under Th2 conditions and 2 days later the cells were retrovirally infected with Gata3-NGFR or NGFR. After an additional 2 days, cells were stained to measure NGFR and T1ST2 expression immediately (D1) or after 7 days of culture in IL-2-containing medium (D7). (D) 1xTh1, 1xTh17, and 1xTh2 cells were fixed immediately after completion of priming and immunoprecipitated with anti-GATA3. D7 1xTh2 cells were cultured in IL-2 plus IL-33 for 2 days and ChIP with anti-GATA3 or control mouse IgG was performed. (E) MFI of GATA3 staining for D1 2xTh2 cells; D8 2xTh2 cells; D8 cells cultured in IL-2, IL-33, or IL-2 plus IL-33 for an additional 48 h (D8 + 2 days IL-2, D8 + 2 days IL-33, D8 + 2 days IL-2 + 33). The experiments were performed two times.
Fig. 3.
Fig. 3.
STAT5 is directly involved in GATA3 and T1ST2 upregulation. (A and B) 2xTh2 cells from Stat5“floxed” or C57BL/6 mice were retrovirally infected with GFP-Cre. GFP-Cre positive cells were sorted and cultured in IL-4-containing-cRPMI for 6 days. Cells were then cultured in IL-2, IL-33, or IL-2 plus IL-33 for 48 h, with IL-4 in the medium, and then collected to measure T1st2 mRNA (A) or stained for GATA3 expression (B). (C) D7 1xTh2 cells were cultured in IL-2 plus IL-33 for an additional 2 days, and ChIP with anti-STAT5A, anti-STAT5B was performed. (D) D7 1xTh2 cells were cultured in IL-2, IL-33, or IL-2 plus IL-33 for 24 h and ChIP with anti-STAT5A, anti-STAT5B, or control mouse IgG was performed. The experiments were performed twice.
Fig. 4.
Fig. 4.
IL-33, together with activated STAT5, induces IL-13 and IL-5 but not IL-4 or IL-2. (A) D1 1xTh2 cells (activated) and D10 1xTh2 cells (rested) were cultured in medium containing IL-2, IL-33, or IL-2 plus IL-33 for the indicated periods and supernatants collected to measure IL-13 production by ELISA. (B) Cells in A were stimulated for 4 h to measure IL-13 production by intracellular staining. (C) Relative amount of IL-13 production was calculated by multiplying the MFI and the percentage of IL-13-producing cells. (D) 3xTh2 cells were stimulated with P+I, TSLP, IL-33, or TSLP+IL-33 for 48 h. The supernatants were collected. (E) 3xTh2 cells were stimulated with P+I, TSLP, IL-33, or TSLP+IL-33 for indicated periods and collected to measure Il13, Il5, Il4, and Il2 mRNA. (F) 3xTh2 cells were stimulated with P+I or IL-2 plus IL-33 for 4 h to test IL-4 and IL-13 production. The experiments were performed 3 times.
Fig. 5.
Fig. 5.
IL-33 plus IL-2-induced IL-13 production is NF-κB and p38 dependent but NFAT independent. (A) 3xTh2 cells were pretreated with the indicated inhibitors for 30 min and then stimulated with IL-33 plus IL-2 or P+I for 4 h to test IL-4 and IL-13 production. (B) 3xTh2 cells were pretreated with DMSO (mock) or CsA for 30 min and then stimulated with PMA or PB-anti-CD3/CD28 for 6 h to determine IL-4 and IL-13 production. The experiments were performed 3 times.
Fig. 6.
Fig. 6.
IL-1, together with IL-23, IL-21, or IL-6 induces IL-17A production. (A) 1xTh1, 1xTh2, and 1xTh17 cells were collected after 4-day priming and mRNA for Tbet, Gata3, Rorc, Il18r1, T1st2, and Il1r1 measured. (B) Rested 1xTh17 and 1xTh1 cells were cultured in cRPMI with indicated cytokines for 24 h and then collected to test expression of indicated mRNAs. (C) Rested 1xTh1 cells were cultured in cRPMI with the indicated cytokines for 24 h and then stained for IL-18Rα expression. (D) 1xTh17 cells were cultured in cRPMI for 2 days and then stimulated with the indicated combination of cytokines for 48 h. Supernatants were collected and ELISA was performed to test IL-17A production. The experiments were performed 3 times.
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