Microbial amyloids induce interleukin 17A (IL-17A) and IL-22 responses via Toll-like receptor 2 activation in the intestinal mucosa

Abstract

The Toll-like receptor 2 (TLR2)/TLR1 receptor complex responds to amyloid fibrils, a common component of biofilm material produced by members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. To determine whether this TLR2/TLR1 ligand stimulates inflammatory responses when bacteria enter intestinal tissue, we investigated whether expression of curli amyloid fibrils by the invasive enteric pathogen Salmonella enterica serotype Typhimurium contributes to T helper 1 and T helper 17 responses by measuring cytokine production in the mouse colitis model. A csgBA mutant, deficient in curli production, elicited decreased expression of interleukin 17A (IL-17A) and IL-22 in the cecal mucosa compared to the S. Typhimurium wild type. In TLR2-deficient mice, IL-17A and IL-22 expression was blunted during S. Typhimurium infection, suggesting that activation of the TLR2 signaling pathway contributes to the expression of these cytokines. T cells incubated with supernatants from bone marrow-derived dendritic cells (BMDCs) treated with curli fibrils released IL-17A in a TLR2-dependent manner in vitro. Lower levels of IL-6 and IL-23 production were detected in the supernatants of the TLR2-deficient BMDCs treated with curli fibrils. Consistent with this, three distinct T-cell populations-CD4(+) T helper cells, cytotoxic CD8(+) T cells, and γδ T cells-produced IL-17A in response to curli fibrils in the intestinal mucosa during S. Typhimurium infection. Notably, decreased IL-6 expression by the dendritic cells and decreased IL-23 expression by the dendritic cells and macrophages were observed in the cecal mucosa of mice infected with the curli mutant. We conclude that TLR2 recognition of bacterial amyloid fibrils in the intestinal mucosa represents a novel mechanism of immunoregulation, which contributes to the generation of inflammatory responses, including production of IL-17A and IL-22, in response to bacterial entry into the intestinal mucosa.

Fig 1

Characterization of an S. Typhimurium mutant carrying an unmarked deletion of csgBA genes. Expression of curli fibrils was detected by flow cytometry (A) and Western blotting (B). Cellulose production was monitored on plates containing calcofluor (C). Motility was determined on plates containing 0.3% agar (D). Invasiveness of strains was tested using human colonic epithelial (HT-29) cells (E). Each experiment was repeated three times independently, with similar outcomes, and a representative example is shown.

Fig 2

Bacterial numbers in the organs of streptomycin-pretreated mice after infection with wild-type S. Typhimurium and a csgBA mutant (curli deficient). The numbers of CFU in the colon contents (A), cecum (B), and mesenteric lymph nodes (C) were determined at the indicated time points.

Fig 3

Immune response elicited in the ceca of streptomycin-pretreated mice after infection with wild-type S. Typhimurium and a csgBA mutant (curli deficient). Transcript levels of Tnfa (A), Ifng (B), Il17a (C), Il22 (D), and Lcn2 (E) were measured by quantitative real-time PCR at the indicated time points after infection. Bars represent changes in mRNA levels compared to a group of mock-infected mice from the same time point (geometric means and standard errors). ns, not significant.

Fig 4

Immune response elicited in the cecum of streptomycin-pretreated C57BL/6 and TLR2-deficient (TLR2-/-) mice after infection with wild-type S. Typhimurium and a csgBA mutant (curli deficient). Transcript levels of Il17a (A), Il22 (B), and Ifng (C) were measured by quantitative real-time PCR at 48 h after infection. Bars represent changes in mRNA levels compared to a group of mock-infected mice from the same time point (geometric means and standard errors). ns, not significant.

Fig 5

Splenic naïve CD4⁺ T cells were treated with supernatants collected from bone marrow-derived dendritic cells, which were previously incubated with increasing concentrations of curli fibrils (0.6 μg/ml, 1.2 μg/ml, and 2.5 μg/ml), for 6 h in the presence of anti-CD3 and anti-CD28 antibodies. IL-17 production was determined in the T-cell supernatants after 72 h (A) by ELISA. IL-6 (B) and IL-23 (C) production was determined in the bone marrow-derived dendritic cell supernatants from wild-type and TLR2-deficient mice, which were treated with curli fibrils (0.6 μg/ml, 1.2 μg/ml, and 2.5 μg/ml). Background cytokines levels in the untreated supernatants were marked with a dashed line in the graphs. ns, not significant.

Fig 6

Immune cell infiltration and IL-17 production in C57BL/6 mice infected with wild-type S. Typhimurium and csgBA mutant (curli deficient) bacteria. Cecal tissue-derived cells, depleted of intraepithelial lymphocytes (IELs), were stained with antibodies for CD3, CD4, CD8, B220, γδ-TCR, NKG2D, and IL-17A and analyzed using flow cytometry. The proportion of CD3⁺ CD4⁺, CD3⁺ CD8⁺, or γδ T cells in total tissue-associated lymphocytes at 48 (A) and 72 (C) hours postinfection was determined. The mean fluorescence intensity (MFI) of PE-IL-17A was determined among CD3⁺ CD4⁺, CD3⁺ CD8⁺, and γδ T cells at 48 (B) and 72 (D) hours postinfection. The proportion of LTi cells in tissue-associated lymphocytes (E) and their IL-17A-producing capacity (F) were determined 48 h postinfection by analyzing CD4⁺, CD3⁻, CD8⁻, B220⁻, GR1⁻, CD11b⁻, CD11c⁻, and NK1.1⁻ cells.

Fig 7

IL-6 and IL-23 production in dendritic cells and macrophages in C57BL/6 mice infected with wild-type S. Typhimurium and csgBA mutant (curli-deficient) bacteria 48 h postinfection. (A) The proportion of dendritic cells (CD11b⁺ and DEC-205⁺) and macrophages (F4/80⁺) in cecal tissue-derived cells was analyzed via flow cytometry. The mean fluorescence intensity of IL-23 (B) and IL-6 (C) among dendritic cells and macrophages was also determined. Significant statistical differences are indicated (P < 0.05).