MPYS/STING-mediated TNF-α, not type I IFN, is essential for the mucosal adjuvant activity of (3'-5')-cyclic-di-guanosine-monophosphate in vivo

Abstract

The bacterial second messenger (3'-5')-cyclic-di-guanosine-monophosphate (CDG) is a promising mucosal adjuvant candidate that activates balanced Th1/Th2/Th17 responses. We showed previously that CDG activates stimulator of IFN genes (STING)-dependent IFN-I production in vitro. However, it is unknown whether STING or IFN-I is required for the CDG adjuvant activity in vivo. In this study, we show that STING(-/-) mice (Tmem173(<tm1Camb>)) do not produce Ag-specific Abs or Th1/Th2/Th17 cytokines during CDG/Ag immunization. Intranasal administration of CDG did not induce TNF-α, IL-1β, IL-6, IL-12, or MCP-1 production in STING(-/-) mice. Surprisingly, we found that the cytokine and Ab responses were unaltered in CDG/Ag-immunized IFNAR(-/-) mice. Instead, we found that CDG activates STING-dependent, IFN-I-independent TNF-α production in vivo and in vitro. Furthermore, using a TNFR1(-/-) mouse, we demonstrate that TNF-α signaling is critical for CDG-induced Ag-specific Ab and Th1/Th2 cytokine production. This is distinct from STING-mediated DNA adjuvant activity, which requires IFN-I, but not TNF-α, production. Finally, we found that CDG activates STING-dependent, but IRF3 stimulation-independent, NF-κB signaling. Our results established an essential role for STING-mediated TNF-α production in the mucosal adjuvant activity of CDG in vivo and revealed a novel IFN-I stimulation-independent STING-NF-κB-TNF-α pathway.

FIGURE 1.

STING is essential for the mucosal adjuvant activity of CDG. (A) Outline of the study. Mice were vaccinated i.n. with three doses (14 d apart) of OVA (20 μg) together with CDG (5 μg) or OVA alone (20 μg). Blood and nasal wash samples were collected 14 d after the last immunization (day 42). (B–F) Each group has four mice. Sera or nasal washes from the four mice in the same group were pooled. OVA-specific Abs in the sera and nasal washes were measured by ELISA, as described in Materials and Methods (n = 3). (G) Splenocytes were collected 14 d after the last immunization and were activated in vitro with OVA (100 μg/ml) for 4 d. Supernatants from the same group were pooled together. Th1 (IFN-γ), Th2 (IL-4), and Th17 (IL-17) cytokines were measured by ELISA (n = 3). Data are mean ± SE from three independent experiments. *p, 0.05.

FIGURE 2.

STING is essential for CDG-induced cytokine and chemokine production in vivo. (A) Mice were administered CDG (10 μg) or PBS i.n. After 15 h, cytokine production was measured in BALF by ELISA (n = 3). Data are mean ± SE from three independent experiments. (B) Cells from BALF were analyzed by FACScan with the indicated Abs. Live cells were gated (n = 3). *p < 0.05.

FIGURE 3.

IFN-I signaling is dispensable for the mucosal adjuvant activity of CDG. (A–E) WT and IFNAR^−/− mice were vaccinated i.n. as in Fig. 1. Abs in the blood and nasal wash samples were determined as in Fig. 1 (n = 3). (F) Splenocytes from WT and IFNAR^−/− mice were collected and activated in vitro with OVA (100 μg/ml) as in Fig. 1. (G) Th1/Th2/Th17 cytokines were determined in supernatants as in Fig. 1 (n = 3). Data are mean ± SE from three independent experiments. *p, 0.05. n.s, Nonsignificant

FIGURE 4.

IFN-I signaling is dispensable for CDG-induced cytokine and chemokine production in vivo. (A) WT, STING^−/−, and IFNAR^−/− mice were given CDG (10 μg) or PBS i.n. Cytokine production was measured in BALF as in Fig. 2 (n = 3). Data are mean ± SE from three independent experiments. (B) Live cells from BALF were analyzed by FACScan with the indicated Abs as in Fig. 2 (n = 3). (C) BMDCs from WTand IFNAR^−/− μiχε were activated by CDG (10 μg/ml) or medium alone (mock) for 18 h. Cells were stained with Abs against CD80 and CD86. Live cells were analyzed by flow cytometry (n = 3). *p, 0.05. n.s., Nonsignificant.

FIGURE 5.

STING differentially regulates CDG- and ISD-induced TNF-α production in vitro. BMDMs (A) or BMDCs (B) from WT, STING^−/−, or IFNAR^−/− mice were activated by CDG (10 μg/ml), LPS (200 ng/ml), ISD (10 μg/ml), or medium alone (mock) for 6 h. TNF-α production was measured in the supernatant by ELISA (n = 3). Data are mean ± SE from three independent experiments. *p <, 0.05. N.D., Not detected.

FIGURE 6.

TNF-α signaling is critical for the mucosal adjuvant activity of CDG. (A–F) WTand TNFR1^−/− mice were vaccinated i.n. as in Fig. 1. Abs in the blood and nasal wash samples were determined as in Fig. 1 (n = 3). (G) Splenocytes from WTand TNFR1^−/− mice were collected and activated in vitro with OVA (100 μg/ml) as in Fig. 1. Th1/Th2/Th17 cytokines were determined in supernatants as in Fig. 1 (n = 3). Data are mean ± SE from three independent experiments. (H) WT, STING^−/−, and TNFR1^−/− mice were treated as in Fig. 2. Cytokine production was measured in BALF as in Fig. 2 (n = 3). Data are mean ± SE from three independent experiments. (I) BMDCs from WT, STING^−/−, or TNFR1^−/− mice were activated by TNF (600 ng/ml), CDG (10 μg/ml), or medium alone (mock) for 18 h. Cells were stained with Abs against CD86. Live cells were analyzed by flow cytometry (n = 2). *p<,0.05. N.D., Not detected; n.s, nonsignificant.

FIGURE 7.

STING is required for CDG-induced NF-κB activation. BMDCs (A) or BMDMs (B) from C57BL/6, STING^2/+, or STING^−/− mice were activated by CDG (10 μg/ml) for 4 h. Nuclear fractions were isolated as described in Materials and Methods. Samples were run on SDS-PAGE gel and probed with the indicated Abs (n = 3). n.s, Nonspecific.

FIGURE 8.

Uncoupling STING-mediated NFkB activation and IRF3 activation. BMDCs (A) or BMDMs (B) from C57BL/6 mice were treated with Bx-795 (1 μM), MG-132 (4 μg/ml), or DMSO (mock) for 2 h. Cells were then activated by CDG (10 μg/ml) for 4 h. Nuclear fractions were isolated and processed as in Fig. 7 (n = 3). BMDCs (C) or BMDMs (D) were treated with Bx-795 (1 μM) or DMSO (mock) as in (A) and (B). Cells were then activated by CDG (10 μg/ml) for the indicated time. WCLs were run on SDS-PAGE gel and probed with the indicated Abs (n = 3). BMDCs (E) or BMDMs (F) were treated with MG-132 (4 μg/ml) or DMSO (mock) as in (A) and (B). Cells were then activated by CDG (10 μg/ml) for the indicated time. WCLs were run on SDS-PAGE gel and probed with the indicated Abs (n = 3). n.s, Nonspecific.

FIGURE 9.

Uncoupling STING-mediated TNF-α and IFN-I production. BMDMs (A) or BMDCs (B) from C57BL/6 mice were treated with Bx-795 (1 mM) or DMSO (untreated) for 2 h. Cells were then activated by CDG (10 μg/ml) for 5 h. TNF-α and IFN-α were measured in supernatant (n = 3). Data are mean ± SE from three independent experiments. BMDMs (C) or BMDCs (D) were treated with MG-132 (4 μg) or DMSO (untreated) for 2 h. Cells were then activated by CDG (10 μg/ml) for 5 h. TNF-α and IFN-α were measured in supernatant (n = 3). Data are mean ± SE from three independent experiments. (E) Uncoupling of CDG–STING–TBK1–IRF3–IFN-I with CDG–STING–NF-κB–TNF-α in BMDMs and BMDCs. *p<, 0.05. N.D., Not detected.