Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages

Abstract

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the β-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

FIGURE 1.

TOLLIP interrupts time-dependent expression of TLRs on MΦ. A, cells were cultured with (black line) or without (shaded) HlyA and analyzed by flow cytometry for the expression of TLR1, -2, -4, and -6 at time points shown between 2–48 h. B, cell lysate was prepared after 10 h of incubation of MΦ with and without HlyA, electrophoresed, and immunoblotted for detection of TOLLIP. The blot was reprobed with anti-β-actin Ab to ensure equal loading of protein in both lanes. C, MΦ of C57BL/6 mice were cultured in absence and presence of HlyA or with anti-TLR4 Ab plus HlyA and analyzed for expression of TLR2 and -6. Up-regulation of TLR2 was also analyzed on MΦ of C3H/HeJ mice treated with or without HlyA. The data given were obtained in one of three representative experiments. *, p < 0.05.

FIGURE 2.

TLR4-dependent, HlyA-induced expression of MyD88 and NF-κB. A, total RNA was extracted from the cells cultured with or without HlyA and subjected to RT-PCR using the corresponding primers of MyD88 and GAPDH. Ethidium bromide-stained PCR products were photographed, and then images were digitized and analyzed. PCR products were quantified and expressed as ratio of the product to GAPDH band density. B, MΦ were cultured with HlyA, and cell lysate at each time point was normalized for the protein content and immunoprecipitated (IP) with anti-TLR4 Ab or anti-TLR2 Ab. The precipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-MyD88 Ab to assess the association of MyD88 with either of the two TLRs. The data given were obtained in one of three representative experiments. C, cells were cultured in absence (thin black line), presence (thick black line) of HlyA and anti-TLR4 Ab plus HlyA (thin gray line), incubated in Hepes-Triton buffer (pH 7.4) to obtain pure nuclei and analyzed for NF-κB translocation. The shaded profile indicates isotype-matched control. The bar diagram represents mean fluorescence intensity (MFI) of the cells as mean ± S.E. of three independent experiments. *, p < 0.05.

FIGURE 3.

HlyA-induced time-dependent up-regulation of activation, co-stimulatory and MHC class II (I-Ab) molecules are controlled by TLR4 and 2. A and B, cells were cultured with (black line) and without (shaded) HlyA for CD80–CD86, CD40, and MHC class II (I-Ab) expression at the indicated time points. C, cells were cultured with (gray line) and without (shaded) HlyA or with anti-TLR4 Ab plus HlyA (black line) for the expression of CD86 and CD40 at 2 h. The bar diagram represents mean fluorescence intensity (MFI) of the cells as mean ± S.E. **, p < 0.005. D, cells were cultured with (gray line) and without (shaded) HlyA or with Abs as indicated plus HlyA (black line) for expression of CD40 at 24 h. **, p < 0.005. E, C3H/HeJ MΦ were cultured with (black line) and without (shaded) HlyA for CD86 and CD40 at the indicated time points. F, C3H/HeJ MΦ were cultured with (black line) and without (shaded) HlyA or anti-TLR2 Ab plus HlyA (gray line) for CD40 at 24 h. The bar diagram represents mean fluorescence intensity of untreated (open), treated (solid), and anti-TLR2 Ab plus HlyA treated (hatched) cells as mean ± S.E. *, p < 0.05. G, cell lysate was prepared of MΦ treated with and without HlyA, which was immunoblotted for Bcl-x_L and reprobed with anti-β-actin Ab to ensure equal loading of protein.

FIGURE 4.

Quantification of cytokines by ELISA. MΦ obtained from C57BL/6 and C3H/HeJ mice were cultured in absence and presence of HlyA or with anti-TLR4 Ab/anti-TLR2 Ab plus HlyA. The supernatants were assayed for TNF-α after 24 h and IL-10/IL-12 p70 after 48 h of culture. Data represent mean ± S.E. of three independent experiments, each done in triplicate. *, p < 0.05; **, p < 0.005.

FIGURE 5.

Jacalin- and VCC⁵⁰-induced expression of TLRs, co-stimulatory and activation molecules. MΦ obtained from C3H/HeJ and C57BL/6 mice were cultured with (black line) and without (shaded) jacalin or VCC⁵⁰. Up-regulation of TLR4, TLR2, CD86, and CD40 were analyzed by flow cytometry.

FIGURE 6.

HlyA-induced apoptosis of PerC MΦ. A, the cells were cultured for 3 and 24 h with and without HlyA. Annexin V and 7-amino actinomycin D (7-AAD) positive apoptotic cells were detected by flow cytometry. The data represent percentage of annexin V/7-amino actinomycin D-stained untreated and HlyA-treated MΦ. The number in each quadrant indicates the cells in percent. The bar diagrams show the percentage of CD11b⁺ apoptotic cells as the mean ± S.E. of three independent experiments. The percentage of apoptotic MΦ of C57BL/6 mice treated with anti-TLR4 Ab plus HlyA and that of C3H/HeJ mice are shown at 3 h. **, p < 0.005. B, the cells were cultured for 3 h in the absence (shaded) and presence (black line) of HlyA and analyzed by flow cytometry for the intracellular expression of caspase-8 and caspase-9. Similarly, the cells after culture were analyzed for active caspase-3. The cells in percent under the marker boundary were considered to be positive for caspase-3. Lysate was prepared from HlyA-treated and -untreated cells, electrophoresed, and analyzed by Western blotting for the presence of caspase-6 and caspase-7.

FIGURE 7.

Diagrammatic representation of V. cholerae HlyA-induced apoptosis and activation of MΦ.