Human isotype‐dependent inhibitory antibody responses against Mycobacterium tuberculosis

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B‐cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB‐exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti‐MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies

The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell

Macrophage nuclear receptors: Emerging key players in infectious diseases.

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a variety of cells, including macrophages. For decades, NRs have been therapeutic targets because their activity can be pharmacologically modulated by specific ligands and small molecule inhibitors. NRs regulate a variety of processes, including those intersecting metabolic and immune functions, and have been studied in regard to various autoimmune diseases. However, the complex roles of NRs in host response to infection are only recently being investigated. The NRs peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptors (LXRs) have been most studied in the context of infectious diseases; however, recent work has also linked xenobiotic pregnane X receptors (PXRs), vitamin D receptor (VDR), REV-ERBα, the nuclear receptor 4A (NR4A) family, farnesoid X receptors (FXRs), and estrogen-related receptors (ERRs) to macrophage responses to pathogens. Pharmacological inhibition or antagonism of certain NRs can greatly influence overall disease outcome, and NRs that are protective against some diseases can lead to susceptibility to others. Targeting NRs as a novel host-directed treatment approach to infectious diseases appears to be a viable option, considering that these transcription factors play a pivotal role in macrophage lipid metabolism, cholesterol efflux, inflammatory responses, apoptosis, and production of antimicrobial byproducts. In the current review, we discuss recent findings concerning the role of NRs in infectious diseases with an emphasis on PPARγ and LXR, the two most studied. We also highlight newer work on the activity of emerging NRs during infection

Increased phagolysosomal fusion during CREB inhibition requires RIPK3 activity.

A) MDMs were plated on glass coverslips and pretreated with for 60 min with DMSO or CREB inhibitor 666–15 +/- Nec-1, GSK’872, or NSA, then infected with mCherry M.tb H37Rv (red) MOI 10. MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 4–5 donors. B) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are cumulative ± SEM of n = 4–5 donors. One-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

CREB signaling in human macrophages inhibits phagolysosomal fusion.

A) MDMs were plated on glass coverslips and pretreated with DMSO or CREB inhibitor 666–15 for 60 min then infected with mCherry expressing M.tb H37Rv (red) MOI 10. MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 3 donors. B) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are cumulative ± SEM of n = 3 donors. Unpaired t test; *p < 0.05, ***p < 0.01.</p

MLKL is essential for phagolysosomal fusion in the absence of CREB signaling in <i>M</i>.<i>tb</i>-infected human macrophages.

MDMs were plated on glass coverslips and transfected with siRNA targeting MLKL or scrambled control siRNA for 48h. MDMs were then pretreated for 60 min with CREB inhibitor 666–15 and infected with mCherry M.tb H37Rv (red) MOI 10. A) Knockdown of MLKL was verified by WB and percent signal quantified relative to scrambled control. A representative experiment is shown and graphed data are cumulative of n = 3 donors. Unpaired t test. B) MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 3 donors. C) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. Data are shown as fold change of percent colocalization compared to scrambled control for each time point. White arrows indicate colocalization. Data are cumulative ± SEM of n = 3 donors. Unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

CREB inhibition induces activation of the necroptotic signaling pathway in <i>M</i>.<i>tb</i>-infected macrophages, but does not affect cell viability.

MDMs were pretreated with 666–15 or DMSO control for 60 min and subsequently infected with M.tb H37Rv at MOI 2 (A,D) or MOI 10 (B,C). A) Bright field images of the cells were taken at 40x; Data shown are representative of n = 10 donors. B,C) Cell lysates were collected and probed by WB blot for the indicated phosphorylated and total proteins. Densitometry was determined and graphed as fold change ± SEM of phosphorylated protein to total protein; A representative experiment is shown and graphed data are cumulative of n = 4 donors. One-way ANOVA with Tukey’s post-test. D) Membrane integrity and cell viability was determined by Cytotox Glo assay. Data are cumulative ± SEM of n = 3 donors. Two-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001.</p