Mycobacterium tuberculosis conserved hypothetical protein rRv2626c modulates macrophage effector functions

Abstract

Secretory proteins of Mycobacterium tuberculosis are the major immunomodulators of the host immune response. Open reading frame (ORF) Rv2626c, encoding a conserved hypothetical protein eliciting a strong humoral immune response in patients with tuberculosis (TB), was shown to be up-regulated upon infection in mice under hypoxic conditions. We now show that recombinant Rv2626c protein (rRv2626c) can bind to the surface of murine macrophages and elicit the type-1 immune response, as manifested by nitric oxide (NO) secretion and expression of inducible nitric oxide synthase (iNOS). Significant induction of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha] was evident upon stimulation of murine macrophages, as well as peripheral blood mononuclear cells (PBMCs) isolated from patients with active TB disease, with rRv2626c. Stimulation with rRv2626c also enhanced the expression of costimulatory molecules such as B7-1, B7-2 and CD40 on murine macrophages. We further show that the production of NO and pro-inflammatory cytokines in response to rRv2626c is mediated by the transcription factor nuclear factor (NF)-kappaB, and this was further confirmed using pyrrolidine dithiocarbamate (PDTC), a specific pharmacological inhibitor of NF-kappaB. Rv2626c therefore appears to modulate macrophage effector functions by eliciting both innate and adaptive immune responses, suggesting its possible use as a vaccine candidate.

Figure 1

Purification of recombinant Rv2626c. His-tagged Rv2626c was over-expressed in the Escherichia coli pLys strain and purified by Co⁺⁺-affinity chromatography. Lanes 1–8 show the successive elution fractions of the recombinant protein. M is the protein molecular size marker. Tris-Tricine sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) fractionation and subsequent Coomassie blue staining revealed 99% homogeneity of the purified recombinant protein.

Figure 2

Recombinant Rv2626c (rRv2626c) binds stably to the murine macrophage surface with high affinity. RAW 264.7 macrophages were incubated with rRv2626c (3 μg) at 4° for 60 min and with normal mouse serum or anti-Rv2626c antibody (raised in a mouse), followed by incubation with anti-mouse fluorescein isothiocyanate (FITC) conjugate. The fluorescence was monitored by fluorescence-activated cell sorter (FACS) analysis.

Figure 3

Recombinant Rv2626c (rRv2626c) induces nitric oxide (NO)/inducible nitric oxide synthase (iNOS) in murine macrophages. (a) The dose-dependent effect of rRv2626c on NO production in RAW 264.7 macrophages. RAW 264.7 macrophages (3 × 10⁵) were treated with either rRv2626c or proteinase K (200 ng/ml) treated rRv2626c for 48 hr and the supernatants were assayed for NO by the Griess reagent assay. A combination of lipopolysaccharide (LPS) (1 μg/ml) and interferon (IFN)-γ (1 ng/ml) was used as a positive control. Data represent the mean ± standard deviation (SD) of at least three triplicate experiments. Statistical significance was determined using Student’s t-test and the effect was found to be significant at P < 0·001. (b) RAW 264·7 macrophages were stimulated with rRv2626c. The positive control group received LPS plus IFN-γ. The whole-cell extracts were prepared from these cells to measure iNOS expression by immunoblotting using polyclonal iNOS antibody. Equal loading of protein was confirmed by Ponceau S stain. The blot represents one of the three immunoblots showing similar results.

Figure 4

Recombinant Rv2626c (rRv2626c)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression is mediated by nuclear factor (NF)-κΒ. (a) RAW 264·7 macrophages were treated with rRv2626c in the absence or presence of pyrrolidine dithiocarbamate (PDTC) (10 μm). The positive control group received lipopolysaccharide (LPS) plus interferon (IFN)-γ. Nuclear extracts prepared from these cells were used to measure NF-κB activation by electrophoretic mobility shift assay (EMSA). (b) In a different experiment, RAW 264.7 macrophages were treated with rRv2626c in the presence or absence of PDTC (10 μm). The positive control group received LPS plus IFN-γ. Nuclear extracts prepared from these cells were used to examine the nuclear translocation of the p50 and p65 subunits of NF-κΒ by immunoblotting using specific antibody. Ponceau S staining of the membrane confirmed equal loading of the nuclear extract. The blot represents one of three immunoblots showing similar results. (c) In another experiment, RAW 264.7 macrophages were treated with rRv2626c in the presence or absence of PDTC (10 μm). The positive control group received LPS plus IFN-γ. The whole-cell extracts were prepared from these cells to measure iNOS expression by immunoblotting. Ponceau S staining of the membrane was used as a protein loading control. The blot represents one of three immunoblots showing similar results. (d) RAW 264·7 macrophages were treated with different concentrations of rRv2626c in the presence or absence of PDTC (10 μm). The LPS plus IFN-γ treatment served as a positive control. After 48 hr, culture supernatants were harvested to measure NO production by the Griess assay. Data represent the mean ± standard deviation (SD) of three experiments. Statistical significance was determined using Student’s t-test and the effect was found to be significant at P < 0·001.

Figure 5

Recombinant Rv2626c (rRv2626c) promotes pro-inflammatory cytokine response in RAW 264·7 murine macrophage cells in a dose-dependent manner. RAW 264·7 (3 × 10⁵) macrophages were treated with various concentrations of rRv2626c. The positive control group received a combination of lipopolysaccharide (LPS) and interferon (IFN)-γ. After 48 hr the culture supernatants were harvested and assayed for tumour necrosis factor (TNF)-α (a) and interleukin (IL)-12 (b) levels by enzyme immunoassay (EIA). Data represent the mean ± standard deviation (SD) of three triplicate experiments. Statistical significance was determined using Student’s t-test and the effect was found to be significant at P < 0·001.

Figure 6

Recombinant Rv2626c (rRv2626c) promotes the expression of T helper type 1 (Th-1) cytokines in peripheral blood mononuclear cells (PBMCs) isolated from patients with active tuberculosis (TB) disease. PBMCs (2 × 10⁵) from healthy controls (n = 9) and patients with TB (n = 48) were stimulated with rRv2626c at a concentration of 5 μg/ml for 72 hr. Culture supernatants were collected and the expression of interferon (IFN)-γ (a), tumour necrosis factor (TNF)-α (b) and interleukin (IL)-12 (c) was quantified by enzyme immunoassay (EIA). Statistical significance was determined using Student’s t-test and the effect was found to be significant at P < 0·001.

Figure 7

Recombinant Rv2626c (rRv2626c) increases the expression of costimulatory molecules such as B7-1, B7-2 and CD40 in RAW 264.7 macrophages. RAW 264·7 macrophages were treated with rRv2626c at 3 μg/ml for 24 hr in the presence or absence of interferon (IFN)-γ and lipopolysaccharide (LPS). Cells were washed, incubated with mouse anti-B7-1 (a), mouse anti-B7-2 (b) and mouse anti-CD40 (c) and then incubated with anti-mouse fluorescein isothiocyanate (FITC) conjugate. The control group received isotype immunoglobulin G2a (IgG2a). This experiment is representative of three experiments showing similar results.