D609 Suppresses Antituberculosis Response by Regulating Dendritic Cells Antigen Presentation

Abstract

Objective: To elucidate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in the antituberculosis (anti-TB) immune response mediated by dendritic cells (DCs).

Methods: In vivo, C57BL/6J mice infected with the Mycobacterium tuberculosis strain H37Rv. Before infection, the mice were pretreated with the PC-PLC inhibitor D609. Bacillary loads in lung and spleen tissues were quantified through colony-forming unit (CFU) assays. Hematoxylin and eosin (H&E) staining was performed to assess inflammatory infiltration and tissue damage. Levels of inflammatory mediators in peripheral venous blood were quantified using enzyme-linked immunosorbent assays (ELISAs). Flow cytometry was employed to determine the proportions of conventional DCs (cDCs) and their subsets, cDC1 and cDC2, within lung, spleen, and lymph node tissues. In vitro, mouse bone marrow-derived dendritic cells (BMDCs) pretreated with D609. The expression levels of chemokines and pro-inflammatory cytokines were assessed via quantitative polymerase chain reaction (qPCR) and ELISA. BMDCs were loaded with H37Rv expressing red fluorescent protein (RFP-H37Rv) or DQ-OVA, and flow cytometry was utilized to analyze the impact of D609 on antigen phagocytosis and processing. Furthermore, flow cytometry was employed to evaluate the effect of D609 pretreatment on the expression levels of costimulatory molecules on BMDCs. The capacity of D609-treated BMDCs to activate and proliferate T cells, as well as to induce interferon-gamma (IFN-γ) secretion, was assessed through a DC-T cell coculture system.

Results: In vivo analysis revealed that mice pretreated with D609 exhibited a marked increase in tissue bacterial load, enhanced inflammatory infiltration, and a reduction in pro-inflammatory mediator expression in peripheral venous blood. There was a notable decrease in the number of cDCs in lung and lymph node tissues, with a pronounced reduction in cDC1 in the lungs and cDC2 in the lymph nodes. In vitro studies demonstrated that D609 pretreated BMDCs displayed a significant decline in inflammatory mediator production, antigen phagocytosis, and antigen processing capabilities, potentially due to altered expression of costimulatory molecules. Coculture experiments indicated that D609 pretreated BMDCs showed a substantial reduction in their ability to stimulate T cell activation, proliferation, and IFN-γ secretion.

Conclusion: Our findings suggest that PC-PLC plays a critical role in the functionality of DCs, including the production of chemokines and pro-inflammatory cytokines, migration to lymph nodes, and antigen presentation to T cells, which collectively contribute to T cell activation and effective clearance of Mycobacterium tuberculosis. Further investigation into the regulatory mechanisms of PC-PLC in DCs may uncover novel therapeutic targets for the development of advanced anti-TB treatments.

Keywords: D609; Mycobacterium tuberculosis; antigen presentation; dendritic cells.

Figure 1

In vivo immune response of D609 treated mice infected with H37Rv. (A) In vivo H37Rv load in the lung and spleen of D609 (200 ng) treated mice at 2 and 5 weeks post H37Rv infection (n = 5). (B) The results of H&E staining and tissue score of the lung of D609 treated mice (n = 5，five randomly selected fields of view for tissue score statistics). (C) H&E staining of the spleen tissues of D609 treated mice. The amount of splenic MGCs was quantified (n = 5, 30 randomly selected fields of view for statistics). Yellow arrows indicate MGCs. (D) ELISA assays of cytokine expression in the lung of mice 2 weeks post H37Rv infection (n = 5). A two‐way ANOVA with Šidák's post hoc test (A) and an unpaired t‐test (B–D) were used for statistical analysis. Data are presented as mean ± SD and are representative of at least three experiments with similar observations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; ELISA, enzyme‐linked immunosorbent assay; H&E, hematoxylin and eosin; MGCs, multinucleated giant cells; SD, standard deviation.

Figure 2

D609 modulation of dendritic cell subtype abundance in vivo. (A) Impact of D609 (200 ng) on dendritic cell abundance in the pulmonary, splenic, and inguinal lymph nodes (LN) microenvironment of mice 2 weeks post H37Rv infection. Quantification of conventional dendritic cells (cDCs) using flow cytometry analysis with CD11c and MHC‐II markers (n = 5). (B) Flow cytometry analysis of cDC1, cDC2 ratios using CD8 and CD11b markers within the cDC population (n = 5). A two‐way ANOVA with Šidák's post hoc test (A, B) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least three experiments with similar observations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; SD, standard deviation.

Figure 3

D609 modulates the immune function of BMDCs. (A, B) The effect of D609 on cell viability. The effect of D609 on cell activity was detected by Annexin V/PI (A) and CCK‐8 (B) (n = 5). The concentrations of D609 were 10, 20, 30, and 40 μg/mL, respectively. (C) Quantitative real‐time PCR analysis of Il12‐p35, IL‐10, and Ccr7 in D609‐treated BMDCs infected with H37Rv (MOI = 2, n = 5). (D) ELISA analysis of IL‐12p70 and IL‐10 in D609‐treated BMDCs infected with H37Rv (MOI = 2, n = 5). (E) Flow cytometry analysis of red fluorescence‐positive BMDCs treated with D609, followed by infection with H37Rv‐RFP (MOI = 10, n = 5). (F) Flow cytometry analysis of green fluorescence‐positive BMDCs treated with D609, followed by incubation with DQ‐OVA (n = 5). (G) Flow cytometry analysis of CD80, CD86, CD40, and MHC‐II expression on the surface of H37Rv‐infected BMDCs at MOI = 2 at 24 hpi (n = 5). Statistical analysis was conducted employing a one‐way analysis of variance (ANOVA) test (B), a two‐way ANOVA test with Šidák's post hoc analysis (C, D, F, G), and an unpaired t‐test (E). Data are presented as mean ± SD and are representative of at least three experiments with similar observations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. BMDCs, bone marrow‐derived dendritic cells; CCK‐8, Cell Counting Kit‐8; ELISA, enzyme‐linked immunosorbent assay; SD, standard deviation.

Figure 4

D609 regulates BMDCs‐mediated T‐cell immunity. (A, B) Flow cytometry analysis was performed on the mean fluorescence intensity of activation markers CD25 and CD69 of OT‐II naïve CD4⁺ T cells cocultured with BMDCs loaded with OVA or OVA_323‐339, CD25 MFI (A), CD69 MFI (B), (n = 3). (C) Flow cytometry analysis of T proliferation of CFSE‐stained OT‐II naïve CD4⁺ T cells cocultured with BMDCs loaded with OVA or OVA_323‐339, (n = 5). (D) Flow cytometry analysis of CD4⁺IFN‐γ⁺ T differentiation of OT‐II naïve CD4⁺ T cells cocultured with BMDCs loaded with OVA or OVA_323‐339, (n = 5). A two‐way ANOVA with Šidák's post hoc test (A–D) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least three experiments with similar observations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ANOVA, analysis of variance; BMDCs, bone marrow‐derived dendritic cells; SD, standard deviation.