Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

Abstract

Background: Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γ responsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30-100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation.

Methodology/principal findings: Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ) were treated with exosomes +/- IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibility complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infected cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection.

Conclusions: Our study suggests that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanism by which M.tb may exert its suppression of a host immune response beyond the infected cell.

Figure 1. Exosomes isolated from M.tb infected cells inhibit IFN-γ induced surface expression of MHC class II and CD64 on BMMØ.

Exosomes were isolated from uninfected RAW264.7 cells (Un exo) or RAW264.7 cells infected with M.tb H37Rv (Rvexo). BMMØ were treated with exosomes for 18 hours or left untreated (RC) followed by +/− IFN-γ stimulation for an additional 18 hours. Cells were stained with either PE conjugated anti-MHC class II or anti-CD64 and analyzed by flow cytometry. Shown are the number of cells stained with MHC class II (A) or CD64 (B) and representative FACS plots for each treatment; MHC-II (C) and CD64 (D). Isotype control antibody was used to define background staining. Results are representative of three individual experiments plus standard deviations; asterisk (*) indicates a p value ≤0.05 between +/− IFN-γ treatments.

Figure 2. Exosome-mediated inhibition of MHC-II and CD64 expression is partially dependent on exosomes containing mycobacterial lipoproteins.

Exosomes were isolated from RAW264.7macrophages infected with wild-type or LspA-deficient M.tb. BMMØ were treated with the exosomes or left untreated (RC) for 18 hours and then incubated for an additional 18 hours +/− IFN- γ. Cells were stained with PE-conjugated anti-MHC class II or anti-CD64 antibody and analyzed by flow cytometry. Isotype control antibodies were used to define background staining. Shown is the mean fluorescence intensity for each sample with isotype control values subtracted from each value for MHC-II (A) and CD64 (B). Also shown are the representative FACS plots for MHC-II (C) and CD64 (D) expression. Results are representative of two independent experiments plus standard deviation and p value <0.05 between +/− IFN-γ treatments are indicated by asterisk (*).

Figure 3. Theinhibition of MHC class II and CD64 by Rvexosomesis dependent on macrophage expression of TLR2.

Untreated or exosome-treated BMMØ isolated from TLR2-deficient mice were stimulated +/− IFN-γ. Macrophages were harvested, stained with PE-conjugated anti-MHC class II or anti-CD64 antibody and analyzed by flow cytometry. Shown are the mean fluorescence intensity values for MHC-II (A) and CD64 (B) expression. Results are representative of two independent experiments plus standard deviation and p value <0.05 between +/− IFN-γ treatments are indicated by asterisk (*).

Figure 4. Exosomes from M.tb-infected cells do not block IFN-γ induced STAT1 phosphorylation but do inhibit IFN-γ induced expression of CIITA.

BMMØ were treated +/− exosomes isolated from RAW264.7 macrophagesas described for figure 1 followed by a 30 minute incubation with IFN-γ. Cells were lysed and analyzed by Western blot for p-STAT1 (Tyr701) (A). The p44/42 MAP Kinase antibody was used as a loading control as described previously (17). BMMØ were treated with exosomes and stimulated +/− IFN-γ for 18 hours. Cells were harvested for qRT-PCR using specific primers for target gene (CIITA) and reference gene (GAPDH). Shown is the relative mRNA expression compared to untreated cells for CIITA normalized to GAPDH (B). Results are representative of two separate experiments plus standard deviation and p value <0.05 shown by asterisk (*).

Figure 5. Exosomes isolated from M.tb-infected cells inhibit a subset of IFN-γ inducible genes which partially overlaps with those inhibited by an M.tb infection.

BMMØ were treated with exosomes isolated from RAW264.7 cells (uninfected/infected)or were infected with M.tb followed by incubation +/− IFN-γ. RNA was isolated, converted to double-stranded cDNA, labeled with Cy3 and hybridized to Musmusculus 4×72 whole genome array. Genes up-regulated or down-regulated by each treatment were identified on the basis of ≥2 fold change in gene expression and a p value ≤0.05 as defined through three independent microarray experiments. Results are represented as Venn diagrams showing the total number of genes identified as well as the number of genes which overlap between treatment groups (A and B). Genes induced by IFN-γ were further analyzed for suppression by exosome pre-treatment or H37Rv infection. Similar analysis was performed on genes suppressed by IFN-γ whose expression was “rescued” by exosome pre-treatment or H37Rv infection. Results are depicted as pie charts showing the number of IFN-γ-induced genes not inhibited by any treatment, those inhibited by treatment or infection and those common to both groups (C). Similarly, results are shown for genes rescued by exosomes or H37Rv infection (D).