Mycobacterium avium subsp . paratuberculosis MAP1889c Protein Induces Maturation of Dendritic Cells and Drives Th2-biased Immune Responses

Abstract

:Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of chronic granulomatous bowel disease in animals and is associated with various autoimmune diseases in humans including Crohn's disease. A good understanding of the host-protective immune response and antibacterial immunity controlled by MAP and its components may contribute to the development of effective control strategies. MAP1889c was identified as a seroreactive antigen in Crohn's disease patients. In this study, we investigated the immunological function of MAP1889c in dendritic cells (DCs). MAP1889c stimulated DCs to increase expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) class molecules and to secret higher interleukin (IL)-10 and moderate IL-6, tumor necrosis factor (TNF)-α, and IL-12p70 levels through the Toll-like receptor (TLR) 4 pathway. MAP1889c-induced DC activation was mediated by mitogen-activated protein kinases (MAPKs), cAMPp-response element binding protein (CREB), and nuclear factor kappa B (NF-κB). In particular, the CREB signal was essential for MAP1889c-mediated IL-10 production but not TNF-α and IL-12p70. In addition, MAP1889c-matured DCs induced T cell proliferation and drove the Th2 response. Production of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokines and anti-inflammatory cytokines was suppressed and enhanced respectively by MAP1889c pretreatment in DCs and T cells. Furthermore, treatment of MAP1889c in M. avium-infected macrophages promoted intracellular bacterial growth and IL-10 production. These findings suggest that MAP1889c modulates the host antimycobacterial response and may be a potential virulence factor during MAP infection.

Keywords: MAP1889c protein; Mycobacterium avium subsp. paratuberculosis; dendritic cells; interleukin-10.

Figure 1

Preparation of the MAP1889c protein: (A) MAP1889c purified from E. coli extracts was subjected to SDS-PAGE and Western blot analysis using a mouse anti-His antibody. All proteins were analyzed by SDS-PAGE with Coomassie blue staining. (B) The cytotoxic effects of MAP1889c were analyzed by flow cytometry. Bone marrow-derived dendritic cells (BMDCs) were stimulated with MAP1889c (1 to 10 μg/mL), lipopolysaccharide (LPS) (100 ng/mL), or staurosporine (STS, 100 nM) for 24 h and then stained with Annexin V and propidium iodide (PI). The percentage of positive cells in each quadrant is indicated. The results are representative of three experiments.

Figure 2

Endocytic activity of MAP1889c-treated dendritic cells (DCs): Bone marrow-derived dendritic cells (BMDCs) were treated with 100 ng/mL Lipopolysaccharide (LPS) or 1 or 10 μg/mL MAP1889c for 24 h, incubated with dextran- fluorescein isothiocyanate (FITC) at 37 °C or 4 °C for 1 h, and then stained with a phycoerythrin (PE)-conjugated anti-CD11c⁺ antibody. Endocytic activity was assessed by flow cytometric analysis of dextran-FITC uptake. The percentage of CD11c⁺dextran⁺ cells is indicated. The bar graphs depict the mean values ± SD (n = 3). * p < 0.05 and *** p < 0.001 for treatment compared with the untreated controls (CON) or for the difference between treatment data. n.s., no significant difference.

Figure 3

MAP1889c induces BMDC maturation and high levels of IL-10 production. The BMDCs were stimulated with MAP1889c (1, 5, or 10 μg/mL) and LPS, 100 ng/mL for 24 h. (A) The expressions of surface markers were analyzed by two-color flow cytometry. The cells were gated to exclude CD11c⁺ cells. BMDCs were stained with anti-CD80, anti-CD86, anti-major histocompatibility complex (MHC) class I, or anti-MHC class II antibodies. The histograms are representative of five experiments. The bar graphs show the percentage (mean ± SD of five experiments) for each surface molecule on CD11c⁺ cells. (B) Interleukin (IL)-10, IL-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-12p70 cytokines from the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All data are expressed as the mean ± SD (n = 3). All data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 for treatment compared with untreated controls (CON) or for the difference between treatment data.

Figure 4

MAP1889c induces DC activation via Toll-like receptor (TLR) 4 pathways. BMDCs derived from wild-type (WT), TLR2^–/–, and TLR4^–/– mice were treated with MAP1889c (10 μg/mL), LPS (100 ng/mL), or Pam3CSK4 (Pam3) (100 ng/mL) for 24 h. (A) MAP1889c-treated BMDCs for 1 h were fixed and then stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue) and Alexa Fluor-488-conjugated anti-MAP1889c antibody. Representative images out of three independent experiments are shown. Scale bar, 10 μm. (B) The cell lysates from BMDCs treated with MAP1889c for 6 h were used for immunoprecipitation with anti-mouse IgG and anti-His or with anti-TLR2 and anti-TLR4 antibodies. Thereafter, proteins were detected using immunoblotting with anti-His, anti-TLR2, or anti-TLR4 antibodies. The total is shown as the mean total cell lysates (input). (C) Production of IL-10, TNF-α, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as the mean ± SD (n = 3). (D) Expression of CD80, CD86, and MHC class I molecules on BMDCs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on CD11c⁺ cells across three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 for treatment values in BMDCs from TLR2^–/– or TLR4^–/– mice compared with MAP1889c-, LPS-, or Pam3CSK4-treated BMDCs from WT mice.

Figure 5

MAP1889c induces DC maturation via mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-κB), and cAMPp-response element binding protein (CREB) pathways, and the CREB signal is involved in IL-10 production. BMDCs stimulated with MAP1889c for the indicated times were lysed, and the proteins in the total cell lysate were separated by SDS-PAGE followed by immunoblot analysis using antibodies against (A) phospho-extracellular signal-regulated kinases (ERK)1/2, phospho-p38, phospho-IκB-α, IκB-α, and β-actin. This image is representative of three experiments showing similar results. (B) BMDCs were plated in covered glass chamber slides and treated with MAP1889c for 1 h, and the immunoreactivity of the p65 subunit of NF-κB in cells was determined by immunofluorescence. Scale bar, 10 μm. (C,D) BMDCs were pretreated with pharmacological inhibitors of ERK (U0126, 10 μM), p38 (SB203580, 20 μM), NF-κB (BAY11-7082, 5 μM), CREB (666-15, 5 μM), or dimethyl sulfoxide (DMSO, Sigma) (vehicle control) for 1 h prior to treatment with MAP1889c (10 μg/mL). After 24 h, the amounts of IL-10, TNF-α, and IL-12p70 in the culture medium were measured by ELISA (C). The mean ± SD is shown for three independent experiments. The expression levels of CD80 and MHC-I were analyzed by flow cytometry (D). Bar graphs show percentages (mean ± SD of three separate experiments) for each surface molecule on CD11c⁺ cells. ** p < 0.01 or *** p < 0.001 for each inhibitor treatment compared with MAP1889c-treated controls. (E) Total lysates of BMDCs stimulated with MAP1889c for the indicated times were separated by SDS-PAGE, followed by immunoblot analysis using phospho-CREB and CREB. (F,G) BMDCs were transfected with CREB siRNA (siCREB) or nonspecific siRNA as a control (siCON) and then with MAP1889c (10 μg/mL) for 30 min (F) or 24 h (G). (F) Immunoblot analysis using antibodies against phospho-CREB and CREB. (G) IL-10, TNF-α, and IL-12p70 production in the culture supernatant were measured by ELISA. All data are expressed as the mean ± SD (n = 3). All data are expressed as the mean ± SD (n = 3). *** p < 0.001 for siRNA transfection compared with MAP1889c-treated cells.

Figure 6

MAP1889c-activated BMDCs induce T cell proliferation and a Th2 response. (A,B) BMDCs from C57BL/c were activated with MAP1889c (10 ug/mL) or LPS- (100 ng/mL), pulsed with Ag85B (10 ug/mL) for 24 h, and then co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-stained splenocytes isolated from Ag85B-immunized BALB/c (A) or C57BL/6 (B) mice for 72 h. The proliferation of CD4⁺ and CD8⁺ T cells was then assessed by flow cytometry. CON, T cells co-cultured with untreated DCs. (C–E) Unstimulated, LPS-stimulated, and MAP1889c-stimulated DCs were co-cultured with naïve splenocytes of C57BL/6 mice at a DC to splenocyte ratio of 1:10. After 72 h, the levels of T-bet and GATA-3 expression in the T cells were assessed by immunoblotting (C). The cytokine levels in the culture supernatants were measured by ELISA (D). Subsequently, IL-10-producing CD4⁺ T cells (CD4⁺IL-10⁺) and IL-10-producing CD8⁺ T cells (CD8⁺IL-10⁺) were gated as shown (E). Gating strategies are shown in Figure S2. All data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 for the treatment compared with untreated controls (CON) or for the difference between treatment data. n.s., no significant difference.

Figure 7

The frequencies of T-bet⁺ T cells and IFN-γ production are suppressed in restimulated splenocytes from MAP1889c-immunized mice. (A) Splenocytes isolated from Ag85B- and MAP1889c-immunized mice were stimulated with Ag85B and MAP1889c, respectively. After 72 h, the expression levels of T-bet and GATA-3 in T cells were assessed by immunoblotting. One representative blot out of five independent experiments is shown. (B) The frequency of GATA-3⁺ or T-bet⁺ T cells in splenocytes stimulated specifically with each Ag were analyzed by flow cytometry, and (C) production of IFN-γ, IL-2, and IL-4 in culture supernatants was measured by ELISA. Gating strategies are shown in Figure S3. All data are expressed as the mean ± SD (n = 3). *** p < 0.001 for the treatment compared with untreated controls (CON) or for the difference between treatment data. n.s., no significant difference.

Figure 8

MAP1889c suppresses the LPS-induced pro-inflammatory response and promotes intracellular bacterial survival. (A) BMDCs were incubated with LPS (100 ng/mL) with or without pretreatment with MAP1889c (10 μg/mL) for 1 h. After 24 h, TNF-α, IL-12p70, IL-10, and IL-1β productions were analyzed by ELISA in culture supernatants. CON, medium control. (B) Unstimulated (CON)-, LPS-, MAP1889c-, and LPS-stimulated DCs pretreated with MAP1889c were co-cultured with splenocytes of naïve mice for 72 h at a DC to splenocytes ratio of 1:10. The cytokine levels in the culture supernatants were measured by ELISA. All data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 for the treatment compared with untreated controls (CON) or for the difference between treatment data. (C,D) Bone marrow-derived macrophages (BMDMs) were infected with Mycobacterium avium at a multiplicity of infection (MOI) of 1 for 4 h and then further treated with amikacin to kill extracellular bacteria for 2 h, washed three times, and incubated with or without 10 μg/mL MAP1889c, or 100 ng/mL LPS for 72 h. Intracellular bacterial growth was determined by plating the cell lysates on 7H10 agar for 0 to 72 h (C). The amounts of IL-10, TNF-α, and IL-12p70 in the culture medium were measured by ELISA (D). The mean ± SD is shown for three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 for the treatment compared with untreated controls (CON) or for the difference between treatment data. n.s., no significant difference.