pf-3512676 and Tuberculosis

Intracellular bacterium, Mycobacterium tuberculosis (M. tb), infects specifically macrophages as host cells. IRAK-M, a member of IRAK family, is a negative regulator in TLR signaling and specifically expresses in monocytes and macrophages. The role of IRAK-M in intracellular growth of M. tb and macrophage polarization was explored, for deeply understanding the pathogenesis of M. tb, the significance of IRAK-M to innate immunity and pathogen-host interaction.. IRAK-M expression was detected in M. tb infected macrophages and in human lung tissue of pulmonary tuberculosis with immunofluorescence staining, Western blot and immunohistochemistry. IRAK-M knock-down and over-expressing cell strains were constructed and intracellular survival of M. tb was investigated by acid-fast staining and colony forming units. Molecular markers of M1-type (pSTAT1 and iNOS) and M2-type (pSTAT6 and Arg-1) macrophages were detected using Western blot in IRAK-M knockdown U937 cells infected with M. tb H37Rv. U937 cells were stimulated with immunostimulant CpG7909 into M1 status and then infected with M. tb H37Rv. Expression of IRAK-M, IRAK-4 and iNOS was detected with immunofluorescence staining and Western blot, to evaluate the effect of IRAK-M to CpG directed M1-type polarization of macrophages during M. tb infection. Molecules related with macrophage's bactericidal ability such as Hif-1 and phosphorylated ERK1/2 were detected with immunohistochemistry and Western blot.. IRAK-M increased in M. tb infected macrophage cells and also in human lung tissue of pulmonary tuberculosis. IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. During M. tb infection, IRAK-M knockdown induced M1-type, while inhibited M2-type polarization of macrophage. M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which was reversed by IRAK-M knockdown in U937 cells. IRAK-M affected Hif-1 and MAPK signaling cascade during M. tb infection.. Conclusively, IRAK-M might alter the polarity of macrophages, to facilitate intracellular survival of M. tb and affect Th1-type immunity of the host, which is helpful to understanding the pathogenesis of M. tb.

Topics: Bacterial Load; Cells, Cultured; Gene Expression; Gene Knockdown Techniques; Host-Pathogen Interactions; Humans; Hydrolases; Immunity, Innate; Immunohistochemistry; Interleukin-1 Receptor-Associated Kinases; Jurkat Cells; Lentivirus; Lung; Macrophages; MAP Kinase Signaling System; Mycobacterium tuberculosis; Nitric Oxide Synthase Type II; Oligodeoxyribonucleotides; Signal Transduction; Tuberculosis; Tuberculosis, Pulmonary; U937 Cells

This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis.. ELISA was used to detect specific antibody and IFN-γ expression in sera; ELISPOT, to detect IFN-γ expression in splenocytes; MTT assay and FACS, to detect T-lymphocytes proliferation in spleens; and RT-PCR, to detect cytokines expression in lungs of mice after immunization. Bacterial load and histopathological lesions in lungs or spleens of mice challenged with Myco. tuberculosis H37Rv strain were analysed. Compared with incomplete Freund's adjuvant, CpG7909 induced more potent production of Pe685a-specific IgG2a/IgG1 antibody and higher expression of IFN-γ in sera, stimulated more generation of antigen-specific IFN-γ-secreting splenocytes, enhanced frequencies of CD3(+) CD4(+) and CD3(+) CD8(+) T-lymphocytes in spleen and increased transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung. However, lower bacterial load in lung and less severe lung pathology were not observed in CpG7909 group mice.. CpG7909 is able to enhance immunological effects of Pe685a subunit vaccine, but does not confer significant protective efficacy against Myco. tuberculosis infection.. CpG7909 as an adjuvant of subunit vaccine against Myco. tuberculosis is worthy of further investigation.

Topics: Acyltransferases; Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antibody Formation; Antigens, Bacterial; Bacterial Load; Bacterial Proteins; Female; Freund's Adjuvant; Immunoglobulin G; Interferon-gamma; Interleukin-6; Lipids; Lung; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; Oligodeoxyribonucleotides; Recombinant Proteins; Spleen; T-Lymphocytes; Toll-Like Receptor 9; Tuberculosis; Tuberculosis Vaccines; Tumor Necrosis Factor-alpha; Vaccines, Subunit