Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice

Abstract

Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3 resulted in inflammatory cytokine production and development of chronic enterocolitis with enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 deficiency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of Stat1, which is essential for IFN-gamma signaling pathways, Stat3 mutant mice developed chronic enterocolitis. TNF-alpha/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1 cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocolitis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1 responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Furthermore, enterocolitis was significantly improved and IFN-gamma production by T cells was reduced in TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for the development of Th1-dependent enterocolitis.

Figure 1

IBDs in Stat1/Stat3 double-deficient mice. (a-d) Histologic examination of the colons of (a) control, (b) Stat1-deficient, (c) Stat3 mutant, and (d) Stat1/Stat3 double-mutant mice at 8 weeks. H&E staining is shown. ×20. (e)The colitis scores shown for individual mice at 16 weeks of age were total scores for individual sections as described in Methods. The severity of colitis in Stat1/Stat3 double-mutant mice was significantly but only partially improved compared with Stat3 mutant mice (**P < 0.01). *P < 0.001 versus group of control mice.

Figure 2

Enhanced IFN-γ production in Stat1/Stat3 double-deficient mice. (a) Spleen cells were stimulated with PMA and ionomycin for 6 h, then stained for CD4, fixed, and finally stained for IFN-γ and IL-4. Cells were analyzed on FACS by gating on CD4⁺ population. The frequency of cytokine-producing CD4⁺ cells is indicated as a percentage. (b) CD4⁺ T cells were purified from lamina propria and stimulated with plate-bound anti-CD3 Ab. The concentration of IFN-γ was measured by ELISA. (c) Mice were intraperitoneally injected with thioglycollate, and 3 days later peritoneal macrophages were isolated. Macrophages were stimulated with 10 ng/ml LPS for 24 h. Concentrations of TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured.

Figure 3

IBDs in TNF-α/Stat3 double-deficient mice. (a–d) Histologic examination of the colons of (a) control, (b) TNF-α–deficient, (c) Stat3 mutant, and (d) TNF-α/Stat3 double-mutant mice at 16 weeks. H&E staining is shown. ×20. (e) The colitis score of TNF-α/Stat3 double-mutant mice at 16 weeks of age. TNF-α/Stat3 double-mutant mice developed severe chronic colitis, similar to Stat3 mutant mice. *P < 0.001 versus group of control mice. (f) Mice were intraperitoneally injected with thioglycollate, and 3 days later peritoneal macrophages were isolated. Macrophages were stimulated with 10 ng/ml LPS for 24 h. Concentrations of TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured. *Not detected.

Figure 4

IFN-γ production by CD4⁺ T cells in spleen and lamina propria of TNF-α/Stat3 double-mutant mice. CD4⁺ T cells were purified from spleen and lamina propria (LPL), and stimulated with plate-bound anti-CD3 Ab. The concentration of IFN-γ was measured by ELISA. CD4⁺ T cells from TNF-α/Stat3 double-mutant mice produced significantly increased levels of IFN-γ, similar to Stat3 mutant mice.

Figure 5

IL-12/Stat3 double-mutant mice showed no inflammatory change in the colon. (a) Peritoneal macrophages were stimulated with 10 ng/ml LPS for 24 h. Concentrations of TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured. *Not detected. (b–d) Histopathology of the colons of (b) control, (c) IL-12–deficient, and (d) IL-12/Stat3 double-mutant mice at 16 weeks. H&E staining is shown. ×20. (e) The colitis score of IL-12/Stat3 double-mutant mice at 16 weeks of age. None of the IL-12/Stat3 double-mutant mice showed any inflammatory change in the colon. *P < 0.001 between Stat3 mutant mice and control or IL-12/Stat3 double-mutant mice.

Figure 6

Normal production of IFN-γ by CD4⁺ T cells in spleen and lamina propria of IL-12/Stat3 double-mutant mice. CD4⁺ T cells were purified from spleen and lamina propria (LPL) and stimulated with plate-bound anti-CD3 Ab. The concentration of IFN-γ was measured by ELISA. CD4⁺ T cells from IL-12/Stat3 double-mutant mice produced normal levels of IFN-γ, unlike those from Stat3 mutant mice.

Figure 7

RAG2/Stat3 double-mutant mice showed no inflammatory change in the colon. (a) Peritoneal macrophages were stimulated with 10 ng/ml LPS for 24 h. Concentrations of TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured. (b–d) Histopathology of the colon of (b) control, (c) RAG2-deficient, and (d) RAG2/Stat3 double-mutant mice at 16 weeks. H&E staining is shown. ×20. (e) The colitis score of RAG2/Stat3 double-mutant mice at 16 weeks of age. None of the RAG2/Stat3 double-mutant mice showed any inflammatory change in the colon. *P < 0.001 between Stat3 mutant mice and control or RAG2/Stat3 double-mutant mice.

Figure 8

Improved colitis in TLR4/Stat3 double-mutant mice. (a) Peritoneal macrophages were stimulated with 10 ng/ml LPS for 24 h. Concentrations of TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured. LPS-induced production of inflammatory cytokines was abolished in TLR4/Stat3 double-mutant macrophages. *Not detected. (b) CD4⁺ T cells were purified from spleen and stimulated with plate-bound anti-CD3 Ab. The concentration of IFN-γ was measured by ELISA. CD4⁺ T cells from TLR4/Stat3 double-mutant mice produced reduced levels of IFN-γ compared with Stat3 mutant mice. (c–e) Histopathology of the colon of (c) control, (d) TLR4-deficient, and (e) TLR4/Stat3 double-mutant mice at 16 weeks. H&E staining is shown. ×20. (f) The colitis score of TLR4/Stat3 double-mutant mice at 16 weeks of age. TLR4/Stat3 double-mutant mice showed no or moderate inflammatory changes in the colon. *P < 0.001 between Stat3 mutant mice and control or TLR4/Stat3 double-mutant mice.

Figure 9

Impaired IL-10–induced expression of SOCS3 in Stat3 mutant macrophages. (a) Peritoneal macrophages from control and Stat3 mutant mice were stimulated with 10 ng/ml IL-10 for 2 h and analyzed for SOCS3 mRNA expression by Northern blotting. (b) Peritoneal macrophages from control and Stat3 mutant mice were cultured in the presence or absence of 10 ng/ml IL-10 for 24 h and then stimulated with 10 ng/ml LPS for 2 h. Total RNA was extracted and subjected to Northern blot analysis for IL-12p40 mRNA expression. The same membrane was rehybridized with β-actin.

Figure 10

Macrophages and dendritic cells in the large intestine were increased in number and produced IL-12p40 in Stat3 mutant mice with colitis. (a–f) Immunohistochemistry of the colon of (a, c, e) control and (b, d, f) Stat3 mutant mice at 16 weeks. The colon sections were stained with anti-CD11c Ab (green) (a, b, e, f), anti-CD11b Ab (red) (c and d), and anti-CD3 Ab (red) (e and f). Bar: 10 μm. (g) CD11c-positive dendritic cells in the large intestine were stimulated with 100 ng/ml LPS for 24 h. Concentrations of IL-6 and IL-12p40 in the culture supernatants were measured. *Not detected. (h) Immunohistochemistry of the colon of Stat3 mutant mice. The colon sections were stained with anti–IL-12p40 Ab (red) and anti-CD11c Ab (green) oranti-CD11b Ab (green). Bar: 10 μm.