The presence of a variety of TLR ligands on BG likely explains the greater immunostimulatory potential of BG over LPS, without overt inflammation. Additionally, BG treatment enhanced the bactericidal efficacy of second-line drugsDLM and BDQ. of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial Fluorocurarine chloride burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of Fluorocurarine chloride key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, Fluorocurarine chloride this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection. (Mtb) is an intracellular pathogen that is capable of infecting a variety of cell types including epithelial, myeloid and lymphoid cell lineages. This pathogen has evolved numerous strategies to counteract, escape, subvert or delay the host protective immune responses. In innate immune cells, such as macrophages and dendritic cells (DC), Mtb hinders phago-lysosomal fusion (6), limits MHC antigen presentation (7), inhibits apoptosis (8), and dampens the migratory potential of DC (9). At the adaptive immunity level, Mtb-specific CD8 T cells were found to exhibit suppressed cytotoxic activity and proliferative ability due to impaired differentiation (10, 11). Importantly, Mtb also skews the protective Th1-mediated immunity toward Th2 responses by perturbing IFN signaling and inducing high IL-4 levels, which results in reduced iNOS activity, impaired apoptosis of infected cells, increased regulatory T cell numbers and greater iron availability to intracellular Mtb (12, 13). Fluorocurarine chloride Host-directed therapies (HDT) have been increasingly explored LFNG antibody as alternative or adjunct TB treatment that focus on potentiating the host (immune) responses to improve mycobacterial killing (14, 15). Some notable examples include interferon (IFN) or therapy (16C18), antibody-based therapy (19C21), metabolic pathways targeting approaches (22, 23) and therapeutic vaccination with non-pathogenic mycobacteria or Mtb fragments (24C26). Here, we investigated the therapeutic potential of bacterial ghosts (BG) against TB. BG are cytoplasm-free, intact bacterial cell envelopes that are obtained through the conditional expression of plasmid-encoded gene E from the bacteriophage X174 (27). Integration of the 91 amino-acid polypeptide E in the bacterial envelope triggers a fusion process of the inner and outer membranes to form a transmembrane tunnel structure through which the cytoplasmic content is expelled driven by a proton-motive force (28, 29). To date, BG have been made from a variety of pathogens including K12 (30), enterotoxigenic and enterohemaorrhagic (EHEC, ETEC) (31), (32), (33), (34), and (35) for both veterinary and clinical vaccine purposes. BG have also been evaluated as drug delivery (36) and adjuvant (37) systems. Additionally, mucosal routes, including oral, aerosol and intranasal, have been deemed suitable for BG administration (38C41). The presence of various pathogen associated molecular patterns (PAMPs) in Fluorocurarine chloride the cell wall of BGlipopolysaccharide (LPS), peptidoglycan, glycolipids, flagellin, and lipoproteinsmakes them potent activators of innate immune cells, which leads to the production of pro-inflammatory cytokines and bactericidal elements, such as reactive oxygen and nitrogen intermediates (ROIs and RNIs) (37, 42, 43). Furthermore, through their ability to activate DC, BG have also been shown to promote greater pathogen-specific antibody responses (40), increased T lymphocytes recruitment and proliferation with their associated cytokine production (39, 41, 44, 45). In this study, the immunostimulatory properties of BG were assessed in the context of mycobacterial infection and our data demonstrate that BG can enhance mycobacterial killing and improve the efficacy of second-line TB drugs. With greater development to further boost the extent of killing, BG may represent a promising option for HDT. Methods Bacteria, BG, and Mammalian Cell Culture Cultures of BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco?, NJ, USA) at 37C until OD600nm 0.5C0.7 and then stored at ?80C in 7H9 and 25% glycerol (1st Base, Singapore). Mtb H37Rv were cultivated in 50 mL 7H9 rolling culture and processed as described above. Bacterial ghosts (BG) prepared from NM522, as previously described (30), were obtained from Biotech Innovation Research Development & Consulting (BIRD-C, Austria). Bone marrow cells flushed out from femurs and tibia of 7C8 weeks-old female WT or TLR4 KO C57BL/6 mice were differentiated into macrophages (BMMO) over 6C8 days at 37C, under 5% CO2 in complete differentiation medium containing DMEM (Gibco),.
