Mycobacterium tuberculosis impairs dendritic cell functions through the serine hydrolase Hip1 - PubMed (original) (raw)

Mycobacterium tuberculosis impairs dendritic cell functions through the serine hydrolase Hip1

Ranjna Madan-Lala et al. J Immunol. 2014.

Abstract

Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope-associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-α, IL-1β, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II- and early secreted antigenic target 6-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies.

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Figures

FIGURE 1

FIGURE 1

Enhanced inflammatory response in hip1 mutant-infected DCs. (A) Purified BMDCs derived from C57BL/6J mice were exposed to medium alone (ui) or infected with the wild type (wt), hip1 mutant or hip1 mutant complemented with Hip1 (comp) Mtb at MOI=5. 24 hours post-infection, cell free supernatants were assayed for IL-12p40 and IL-12p70 by ELISA. Purified C57BL/6J BMDCs were infected with heat-killed wild type (wt), hip1 mutant or comp strain at MOI=5. 24 hours post-infection, cell free supernatants were assayed for IL-12p40, IL-12p70 (B) and IL-23, IL-6, TNF-α, IL-1β and IL-18 (C) by ELISA. Data are representative of three independent experiments. Values are presented as mean +/− SD. *, p <0.05; **, p<0.01; ***, p<0.001.

FIGURE 2

FIGURE 2

Enhanced surface expression of CD40, CD86 and MHC class II on hip1 mutant-infected DCs. C57BL/6J BMDCs were exposed to medium alone (ui), heat-killed wild type (wt) or hip1 mutant at MOI=5 or 1 µg/ml LPS for 24 hours. DCs were labeled with anti-CD11c-APC and anti-CD40-PE, anti-CD86-PE or anti-MHC class II-PE. Representative histograms and median PE fluorescence intensity for CD11c+ cells are shown. Isotype control is shown as gray shaded area. Data are representative of three independent experiments. Values are presented as mean +/− SD, **, p<0.01; ***, p<0.001.

FIGURE 3

FIGURE 3

Wild type Mtb does not block LPS- or hip1 mutant-induced DC maturation. C57BL/6J BMDCs were exposed to 1 µg/ml LPS or heat-killed wild type Mtb (wt) at MOI=5 either independently or together (A) or infected with heat-killed wild type (wt), hip1 mutant or wild type+hip1 mutant (1:1) at MOI=5 for 24 hours (B). DCs were labeled with anti CD11c-APC and anti CD40-PE. Representative histograms and median PE fluorescence intensity for CD11c+ cells is shown. Isotype control is shown as gray shaded area. Data are representative of two independent experiments. Values are presented as mean +/− SD, *, p<0.05; **, p<0.01.

FIGURE 4

FIGURE 4

Mtb impairment of DC activation and maturation requires MyD88- and TLR2/9-dependent pathways. Purified BMDCs from C57BL/6J and MyD88−/− (A) TLR2−/−(B) or TLR2/9−/− (C) mice were exposed to medium alone (ui) or infected with heat-killed wild type (wt) or the hip1 mutant at MOI=5 for 24 hours and cell-free supernatants were assayed for IL-12p40, IL-12p70 and IL-6 by ELISA. (D) Infected BMDCs from MyD88−/− and TLR2/9−/− were labeled with anti-CD11c-APC and anti-CD40-PE. Representative histograms and median PE Fluorescence intensity for CD11c+ cells is shown. Data are representative of three (A and B) or two (C and D) independent experiments. Values are presented as mean +/− SD, *, p<0.05; ***, p<0.001.

FIGURE 5

FIGURE 5

hip1 mutant augments antigen presentation by BMDCs. Purified C57BL/6J BMDCs in medium alone (ui) or infected with heat-killed wild type (wt) or hip1 mutant at MOI=10 for 24 hours were co-cultured with ESAT-61–20 peptide and ESAT-6 specific transgenic CD4 T cells (A) or OVA323–339 peptide and OT-II specific transgenic CD4 T cells for 3 days (B). Cell-free supernatants were collected and assayed for IFN-γ and IL-2 by ELISA. Data are representative of three independent experiments. Values are presented as mean +/− SD. *, p <0.05; **, p<0.01; ***, p<0.001.

FIGURE 6

FIGURE 6

Mtb-DC interactions modulate CD4 T cell differentiation in vitro and in vivo. (A) Purified C57BL/6J BMDCs in medium alone (ui) or infected with heat-killed wild type (wt) or the hip1 mutant at MOI=10 for 24h were co-cultured with CD4 T cells from C57BL/6J mice for 3 days. Cell-free supernatants were collected and assayed for IFN-γ and IL-17 by ELISA. (B) Single-cell suspensions were prepared from lungs of mice aerogenically infected with live wild type (wt) or the hip1 mutant at three weeks post infection, and cells were stimulated with 10 µg/ml ESAT-61–20 peptide for 48 hours. Supernatants were collected and assayed for IFN-γ and IL-17 by ELISA. Data are representative of three (A) or two (B) independent experiments. Values are presented as mean +/− SD. **, p<0.01; ***, p<0.001.

FIGURE 7

FIGURE 7

Mtb interacts with human DCs to impair T cell differentiation. (A) Human MDCs were infected with heat-killed wild type (wt) or hip1 mutant Mtb at MOI=10 for 24 hours. Cell free supernatants were assayed for IL-12p40 and IL-6 by ELISA (B) MDCs infected with heat-killed wild type (wt) or hip1 mutant were co-cultured with autologous lymphocytes isolated from the corresponding donors for 3 days. Cell free supernatants were assayed for IFN-γ and IL-17 by ELISA. Data from 3 healthy donors are represented. Values are presented as mean +/−SD. *, p <0.05; **, p<0.01; ***, p<0.001.

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