PPE38 Protein of Mycobacterium tuberculosis Inhibits Macrophage MHC Class I Expression and Dampens CD8+ T Cell Responses

Abstract

Suppression of CD8⁺ T cell activation is a critical mechanism used by Mycobacterium tuberculosis (MTB) to escape protective host immune responses. PPE38 belongs to the unique PPE family of MTB and in our previous study, PPE38 protein was speculated to participate in manipulating macrophage MHC class I pathway. To test this hypothesis, the function of mycobacterial PPE38 protein was assessed here using macrophage and mouse infection models. Decreased amount of MHC class I was observed on the surface of macrophages infected with PPE38-expressing mycobacteria. The transcript of genes encoding MHC class I was also inhibited by PPE38. After infection of C57BL/6 mice with Mycobacterium smegmatis expressing PPE38 (Msmeg-PPE38), decreased number of CD8⁺ T cells was found in spleen, liver, and lungs through immunohistochemical analysis, comparing to the control strain harboring empty vector (Msmeg-V). Consistently, flow cytometry assay showed that fewer effector/memory CD8⁺ T cells (CD44^highCD62L^low) were activated in spleen from Msmeg-PPE38 infected mice. Moreover, Msmeg-PPE38 confers a growth advantage over Msmeg-V in C57BL/6 mice, indicating an effect of PPE38 to favor mycobacterial persistence in vivo. Overall, this study shows a unique biological function of PPE38 protein to facilitate mycobacteria to escape host immunity, and provides hints for TB vaccine development.

Keywords: CD8+ T cells; MHC class I; Mycobacterium; PPE38; macrophages.

Figure 1

PPE38 protein inhibited the MHC class I expression in macrophages. RAW264.7 cells were infected with either M. marinum strains (WT Mm, Tn-MmPPE38, and Comp-MmPPE38, MOI = 1) or M. smegmatis strains (Msmeg-V, Msmeg-PPE38, and Msmeg-PPE68, MOI = 10). The expression of MHC class I was detected by FACS at (A,B) 4 hpi for M. marinum strains or (C,D) 2 hpi for M. smegmatis strains by FACS analysis. (E) Total RNA was isolated from infected RAW264.7 cells. The level of MHC class I was measured by qRT-PCR and expression levels were normalized to corresponding GAPDH levels. Data were generated from four independent experiments. ^*p < 0.05.

Figure 2

PPE38 dampened the expression of MHC class I in peritoneal macrophages. C57BL/6 mice were infected with Msmeg-V and Msmeg-PPE38 (2 × 10⁷), the peritoneal macrophages collected from infected mice were stained with FITC conjugated anti-CD11b mAbs, and PE-conjugated anti-MHC class I mAbs. (A) Expression of MHC class I were analyzed by FACS from 1^*10⁶ cells at 6 hpi (gate R1–R3) and 1 dpi (gate R1′–R3′). Peritoneal macrophages were first gated as CD11b⁺ (gate R3 and R3′), then two subgroup of PMs were observed as CD11b^highMHC class I^high (gate R1 and R1′) and CD11b^lowMHC class I^low (gate R2 and R2′). (B) Histograms and (C) bar graphs show the Mean Fluorescence Intensity of MHC class I of macrophages. (D,E) Isolated mouse peritoneal macrophages from mice pretreated with 3% thioglycollate medium were cultured for 24 h and then infected with Msmeg-V and Msmeg-PPE38 (MOI = 10). The amount of MHC class I was measured via FACS from 1^*10⁵ cells. Bar graphs show the mean ± SD, representing three independent experiments. ^*p < 0.05. ^**p < 0.01.

Figure 3

C57BL/6 mice infected with Msmeg-PPE38 showed reduced distribution of CD8⁺ T cells in infected organs as compared to mice infected with Msmeg-V. The (A) spleen, (B) liver and (C) lungs sections of C57BL/6 mice either treated with PBS or infected with 2 × 10⁷ of Msmeg-V or Msmeg-PPE38 at 6 dpi were stained with anti-CD8 antibody and DAPI. Histograms and bar graphs show the number of CD8⁺ T cells. Arrowheads show CD8⁺ T cell. Photographs of representative sections were shown (× 400 time magnified). CD8+ T cells were counted manually from three different fields of each mice. Bar graphs show the mean ± SD, representing two independent experiments. ^*p < 0.05. ^**p < 0.01. ^***p < 0.001, ^****p < 0.0001.

Figure 4

PPE38 inhibited CD8⁺ T response in infected spleen. Spleen cells derived from three C57BL/6 mice were collected at 6 dpi in each infected group. (A) Representative flow cytometry profiles of CD8⁺ T cells which were gated first as CD3⁺/CD8⁺ cells (B) he expression of cell surface markers of CD8⁺ T cells including CD44 and CD62L was examined by FACS analysis using the respective PE-Cy7 or PerCP-Cy5.5 mAbs. (C) Bar graphs show the mean ± SD percentages of CD44^highCD62L^low CD8⁺ T cells, representing two independent experiments. ^*p < 0.05.

Figure 5

M. smegmatis survival is enhanced in the C57BL/6 mice by expressing PPE38. C57BL/6 mice and BALB/c mice were infected with MS-PPE38 and MS-V (2 × 10⁷), and then the bacteria loads in lungs, liver and spleen were counted at 1, 6, and 9 dpi of C57BL/6 mice (A) and BALB/c mice (B). Data represent mean ± SD of five mice per group for each time point. ^*p < 0.05.