Src homology 3-interacting domain of Rv1917c of Mycobacterium tuberculosis induces selective maturation of human dendritic cells by regulating PI3K-MAPK-NF-kappaB signaling and drives Th2 immune responses

Abstract

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-κB signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

FIGURE 1.

Recombinant Rv1917c induces maturation of human DCs. A and B, 7-day-old immature DCs (0.5 × 10⁶ cells/ml) were cultured with GM-CSF and IL-4 alone (Control) or GM-CSF, IL-4, and 5 μg/ml of Rv1917c for 48 h followed by analysis for the expression of surface markers by flow cytometry. The percentage of cells expressing the indicated markers is shown in A, and mean fluorescence intensities (MFI) are shown in B. Data are presented as mean ± S.E. from five independent donors. *, p < 0.05 versus control.

FIGURE 2.

Rv1917c triggers secretion of immunoregulatory cytokines from human DCs. A–E, DCs (0.5 × 10⁶ cells/ml) were cultured with GM-CSF and IL-4 alone (Control) or GM-CSF, IL-4, and 5 μg/ml of Rv1917c for 48 h, and secretion of IL-6 (A), IL-8 (B), TNF-α (C), IL-10 (D), and IL-12 (E) in cell-free culture supernatants was analyzed by cytokine bead array assay. Data are presented as mean ± S.E. from six independent donors. *, p < 0.05 versus control.

FIGURE 3.

Rv1917c-treated DCs stimulate CD4⁺ T cells to produce Th2 cytokines. A and B, DCs were cultured with GM-CSF and IL-4 alone (Control) or GM-CSF, IL-4, and 5 μg/ml of Rv1917c for 48 h. The Rv1917c-matured DCs were co-cultured with allogeneic CD4⁺ T cells at different DC to T cell ratios. After 4 days of co-culture, the cells were pulsed overnight with 0.5 μCi of [³H]thymidine to quantify T cell proliferation (A). Radioactive incorporation was expressed as counts/min (mean ± S.E. of quadruplet values). The data are representative for three independent donors. B, cell-free supernatants from DC:T cell co-cultures were analyzed for the cytokines IL-4, IL-5, IL-10, and IFN-γ. C, PBMCs obtained from tuberculosis patients (TB patients) (n = 11) and healthy control subjects (n = 4) were cultured with or without 5 μg/ml Rv1917c, and cell-free supernatants collected on day 4 were tested for concentrations of secreted IL-5 and IFN-γ. Data are represented as mean ± S.E. *, p < 0.05 versus control; **, p < 0.05 versus Rv1917c (Healthy Controls).

FIGURE 4.

Rv1917c specifically recognizes TLR2 on cell surface. A, cell lysates from DCs were incubated with Rv1917c immobilized on Ni-NTA beads, and bead-bound proteins were analyzed for TLR1, TLR2, TLR4, and TLR6 by immunoblotting. B, DCs were pretreated with either blocking antibodies against TLR1, TLR2, TLR4, TLR6, or isotypic control antibodies followed by incubation with FITC-labeled Rv1917c, and interaction of Rv1917c with DCs was evaluated by confocal microscopy. C, preferential interaction of FITC-labeled Rv1917c with TLR2 cDNA-transfected HEK-293 cells. D, cell lysates from HEK-293 cells transfected with either TLR2 or vector were incubated with Rv1917c immobilized on Ni-NTA beads, and immunoprecipitation of TLR2 was evaluated. BF, Bright Field. Data are representative of two independent experiments.

FIGURE 5.

DC maturation triggered by Rv1917c involves PI3K, ERK1/2, and p38 MAPK-dependent activation of NF-κB. A, DCs were treated with Rv1917c for indicated time points, and phosphorylation of ERK1/2 (pERK1/2), p38 MAPK (pp38), and AKT (pAKT) was analyzed by immunoblotting. B, DCs were pretreated with LY294002 followed by stimulation with Rv1917c for indicated time points, and activation of ERK1/2 and p38 MAPK was analyzed. C, Rv1917c and LPS trigger nuclear translocation of p65 NF-κB. D, DCs were pretreated with ERK1/2 inhibitor (U0126, 10 μm), p38 MAPK inhibitor (SB203580, 20 μm), and PI3K inhibitor (LY294002, 50 μm) followed by treatment with Rv1917c and nuclear translocation of p65 NF-κB was analyzed. Med, Medium. Data are representative of three independent experiments.

FIGURE 6.

Involvement of PI3K, ERK1/2, p38 MAPK, and NF-κB pathways in Rv1917c-induced maturation of DCs. A, DCs were treated with pharmacological inhibitors of ERK1/2 (U0126, 10 μm), p38 MAPK (SB203580, 20 μm), PI3K (LY294002, 50 μm), NF-κB (Bay 11-7082, 20 μm; l-1-tosylamido-2-phenylethyl chloromethyl ketone, 20 μm), or DMSO (vehicle control) for 1 h prior to treatment with Rv1917c for 48 h, and the expression of CD80, CD86, and CD40 was analyzed by flow cytometry. B, DCs were treated as in A, and the level of secreted IL-6, IL-10, and TNF-α was analyzed. Data are presented as mean ± S.E. from three independent donors. *, p < 0.05 versus Rv1917c. MFI, mean fluorescence intensities.

FIGURE 7.

COX-2 activity is critical for Rv1917c-induced differentiation of CD4⁺ T cells into Th2 subtype. A and B, Rv1917c or LPS induces COX-2 expression (A) and PGE₂ secretion (B) in human DCs. C, DCs pretreated with NS-398 followed by stimulation with Rv1917c for 48 h were co-cultured with allogeneic CD4⁺ T cells for 4 days, and cell-free supernatants were analyzed for level of T cell cytokines IL-5 and IL-10. Data in A represent one of the three independent experiments, and data in B and C (mean ± S.E.) are from three independent donors. *, p < 0.05 versus control; **, p < 0.05 versus Rv1917c.