Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the most ancient diseases recorded. In cases of bone TB, it significantly disrupts bone homeostasis, though the precise mechanisms are poorly understood and effective treatment targets are scarce. Our study investigated the role of Rv1509 in the pathogenesis of bone TB. We found that Rv1509 enhances the differentiation of bone marrow macrophages (BMMs) into osteoclasts by activating the TLR2 pathway, which stimulates the production of IL-6 and TNF-α. This, in turn, indirectly inhibits osteoblast differentiation and mineralization. Additionally, Rv1509 directly impairs osteoblast function and enhances the secretion of RANKL via TLR2 signaling, creating a detrimental RANKL/OPG imbalance that promotes osteoclast differentiation and bone degradation. Notably, the injection of Rv1509 into mouse skulls led to extensive bone damage, highlighting its significant role as a virulence factor in the pathogenesis of bone TB.

The widespread chronic enteritis known as Paratuberculosis (PTB) or Johne's disease (JD) is caused by Mycobacterium avium subspecies paratuberculosis (MAP), posing a significant threat to global public health. Given the challenges associated with PTB or JD, the development and application of vaccines are potentially important for disease control. The aim of this study was to design a multi-epitope vaccine against MAP. A total of 198 MAP genomes were analyzed using pan-genome and reverse vaccinology approaches. B-cell and T-cell epitope analysis was performed on the selected promising cross-protective antigens followed by selection of epitopes with high antigenicity, no allergenicity, and no toxicity for the design of the vaccine. The designed vaccine was evaluated through molecular dynamics simulations, molecular docking, and immunological simulations. The results revealed the identification of five promising cross-protective antigens. In total, 10 B-cell epitopes, 10 HTL epitopes, and 9 CTL epitopes were selected for the design of the vaccine. Both the vaccine candidate and the vaccine-TLR4 complex demonstrated considerable stability in molecular dynamics simulations. Molecular docking studies confirmed that the vaccine candidate successfully interacted with TLR4. Immunological simulations showed an increase in both B-cell and T-cell populations after vaccination. Additionally, the vaccine candidate exhibited a codon adaptability index of 1.0 and a GC content of 53.64%, indicating strong potential for successful expression in Escherichia coli. This research developed a multi-epitope vaccine targeting MAP through pan-genomes and reverse vaccinology methods, offering innovative strategies for creating effective vaccines against MAP.

Mycobacterium tuberculosis (MTB) ESX-1, a type VII secretion system, is a key virulence determinant contributing to MTB's survival within lung mononuclear phagocytes (MNPs), but its effect on MNP recruitment and differentiation remains unknown. Here, using multiple single-cell RNA sequencing techniques, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces a transcriptional signature of immune evasion in lung macrophages and BMDM in an ESX-1-dependent manner. Spatial transcriptomics revealed an up-regulation of permissive features within MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 facilitates the recruitment and differentiation of MNPs, which MTB can infect and manipulate for survival. Our dataset across various models and methods could contribute to the broader understanding of recruited cell heterogeneity during MTB lung infection.

Toll-like receptors (TLRs) are crucial components of innate immunity. A specific form of genetic variation in TLR genes may increase the chance of developing leukemia. The present investigation conducted a comprehensive meta-analysis to examine the correlation between three TLR polymorphisms, namely TLR2 (rs3804099), TLR4 (rs4986790), and TLR9 (rs187084), within the leukemia risk group. An in-depth literature search was performed using Web of Science, PubMed, and Google Scholar to identify noteworthy research published in these scientific databases from 2012 to 2024. Research articles were evaluated according to rigorous inclusion criteria, and data was compiled for meta-analysis using Microsoft Excel (Ver. 2013), MedCalc (Ver. 19.3), and RevMan software (Ver. 5.3). Finally, 11 qualified studies were selected for the ongoing investigation, encompassing a combined total of 1315 leukemia cases and 1340 controls. Using a dominant genotype model, the meta-analysis found that the TLR2 (rs3804099) and TLR9 (rs187084) polymorphisms were strongly linked to higher risk of leukemia, with ORs of 2.042 (95% CI: 1.35-3.08, p = 0.001) and 1.38 (95% CI: 1.14-1.67, p = 0.001) respectively. Notably, the TLR4 (rs4986790) polymorphism did not exhibited any substantial correlation with the incidence of leukemia. The results indicate that variations in TLR2 and TLR9 genes could be considered a novel genetic biomarker for the leukemia development, highlighting their potential use in risk assessment and targeted therapies. This emphasizes the possibility of using these variations in evaluating risk and developing targeted remedies. However, greater research capacities are required to research into the fundamental mechanisms and authenticate these trends in other populations.

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical 'GxSxG' motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

Mycobacterium avium subsp. paratuberculosis (MAP) primarily invades ruminants' small intestine via the Peyer's patches in the ileum and jejunum. Despite ongoing efforts to develop effective MAP vaccines, the effects of live-attenuated vaccines on mucosal immunity remain poorly understood. Previous studies indicate that the BacA oral vaccine confers localized protection against MAP in the ileum and ileocecal valve of calves, but not in the jejunum. This protection correlates with heightened levels of peripheral blood immune cells exhibiting pro-inflammatory and memory traits. This study aimed to evaluate immune responses induced by oral BacA vaccination in the ileum and jejunum Peyer's patches, comparing protection at both sites through mucosal immune cell profiling and RNA-seq transcriptome analyses. It represents the first exploration of mucosal immune responses in Peyer's patches following oral MAP vaccination. Oral BacA immunization increased CD4 + IFNγ+ and CD4 + TNFα+ cell frequencies, along with the T effector memory to T central memory cell ratio, in the ileum and jejunum of BacA-vaccinated animals challenged with wildtype MAP, compared to the infection control group challenged solely with wildtype MAP. Immune cells isolated from the ileum of vaccinated-challenged animals exhibited significant upregulation in IFNγ, IP-10, TNFα, IL-2, IL-15, and IL-17 expression upon restimulation compared to the uninfected control group, whereas minimal differences were observed in the jejunum under similar conditions. RNA-seq data further indicated a more robust host response in the ileum across all experimental groups. Gene ontology analyses revealed genes associated with increased phagocytic and apoptotic activities in the vaccinated-challenged group. Overall, the BacA oral vaccine's effectiveness appears to vary primarily due to differences in antigen-specific gene expression between the ileum and jejunum, with the ileum showing a more robust host response. Understanding these effects on young calves' mucosal immunity and how live vaccines modulate immune responses is crucial for advancing mucosal vaccine development against MAP.

Advances in mass spectrometry accelerates the characterization of HLA ligandome, necessitating the development of efficient methods for immunopeptidomics analysis and (neo)antigen prediction. We develop ImmuneApp, an interpretable deep learning framework trained on extensive HLA ligand datasets, which improves the prediction of HLA-I epitopes, prioritizes neoepitopes, and enhances immunopeptidomics deconvolution. ImmuneApp extracts informative embeddings and identifies key residues for pHLA binding. We also present a more accurate model-based deconvolution approach and systematically analyzed 216 multi-allelic immunopeptidomics samples, identifying 835,551 ligands restricted to over 100 HLA-I alleles. Our investigation reveals the effectiveness of the composite model, denoted as ImmuneApp-MA, which integrates mono- and multi-allelic data to enhance predictive performance. Leveraging ImmuneApp-MA as a pre-trained model, we built ImmuneApp-Neo, an immunogenicity predictor that outperforms existing methods for prioritizing immunogenic neoepitope. ImmuneApp demonstrates its utility across various immunopeptidomics datasets, which will promote the discovery of novel neoantigens and the development of new immunotherapies.

The BCG vaccine, Bacille Calmette Guerin, holds the distinction of being the most widely administered vaccine. Remarkably, a century has passed since its discovery; however, puzzlingly, questions persist regarding the effectiveness of the immune response it triggers. After years of diligent observation, it has been deduced that BCG imparts immunity primarily to a specific age group, namely children. This prompts a significant query: the rationale behind BCG's limited efficacy against TB in particular age groups and populations remains elusive. Beyond vaccinations, drug therapy has emerged as an alternative route for TB prevention. Nonetheless, this approach faces challenges in the contemporary landscape, marked by the emergence of new instances of MDR-TB and XDR-TB, compounded by the financial burden of treatment. It's noteworthy that BCG remains the sole WHO-approved vaccine for TB. This comprehensive review delves into several aspects, encompassing the immune response during infection, the shortcomings of BCG in conferring immunity, and the various factors contributing to its limitations. Within this discourse, we explore potential explanations for the observed deficiencies of the BCG vaccine and consider how these insights could catalyze the development of future vaccines. The current landscape of novel vaccine development for TB is illuminated, including a spotlight on the latest vaccine candidates.

Mycobacterium tuberculosis (M. tb) is a significant intracellular pathogen responsible for numerous infectious disease-related deaths worldwide. It uses ESX-1 T7SS to damage phagosomes and to enter the cytosol of host cells after phagocytosis. During infection, M. tb and host mitochondria release dsDNA, which activates the CGAS-STING1 pathway. This pathway leads to the production of type I interferons and proinflammatory cytokines and activates autophagy, which targets and degrades bacteria within autophagosomes. However, the role of type I IFNs in immunity against M. tb is controversial. While previous research has suggested a protective role, recent findings from cgas-sting1 knockout mouse studies have contradicted this. Additionally, a study using knockout mice and non-human primate models uncovered a new mechanism by which neutrophils recruited to lung infections form neutrophil extracellular traps. Activating plasmacytoid dendritic cells causes them to produce type I IFNs, which interfere with the function of interstitial macrophages and increase the likelihood of tuberculosis. Notably, M. tb uses its virulence proteins to disrupt the CGAS-STING1 signaling pathway leading to enhanced pathogenesis. Investigating the CGAS-STING1 pathway can help develop new ways to fight tuberculosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

The Mycobacterial growth inhibition assay (MGIA) is an ex-vivo assay used to measure the overall functional immune response elicited by infection or vaccination. In tuberculosis (TB) vaccine development, MGIA is a potentially important tool for preclinical evaluation of early-stage vaccine candidates to complement existing assays, and to potentially reduce the need for lengthy and costly pathogenic Mycobacterium tuberculosis (Mtb) animal challenge experiments. The conventional method of MGIA in mice entails directly infecting mixed cell cultures, most commonly splenocytes, from immunised mice with mycobacteria. However, this direct infection of mixed cell populations may yield unreliable results and lacks sufficient sensitivity to discriminate well between different vaccines due to the low number of mycobacteria-permissive cells. Here, we modified the assay by inclusion of mycobacteria-infected congenic murine macrophage cell lines as the target cells, and by measuring the total number of killed cells rather than the relative reduction between different groups. Thus, using splenocytes from Mycobacterium bovis BCG immunised mice, and J774 and MH-S (BALB/c background) or BL/6-M (C57Bl/6 background) macrophage cell lines, we demonstrated that the modified assay resulted in at least 26-fold greater mycobacterial killing per set quantity of splenocytes as compared to the conventional method. This increased sensitivity of measuring mycobacterial killing was confirmed using both the standard culture forming unit (CFU) assay and luminescence readings of luciferase-tagged virulent and avirulent mycobacteria. We propose that the modified MGIA can be used as a highly calibrated tool for quantitating the killing capacity of immune cells in preclinical evaluation of vaccine candidates for TB.

The host immune responses play a pivotal role in the establishment of long-term memory responses, which effectively aids in infection clearance. However, the prevailing anti-tuberculosis therapy, while aiming to combat tuberculosis (TB), also debilitates innate and adaptive immune components of the host. In this study, we explored how the front-line anti-TB drugs impact the host immune cells by modulating multiple signaling pathways and subsequently leading to disease relapse. Administration of these drugs led to a reduction in innate immune activation and also the cytokines required to trigger protective T cell responses. Moreover, these drugs led to activation-induced cell death in the mycobacterial-specific T cell leading to a reduced killing capacity. Furthermore, these drugs stalled the T cell differentiation into memory subsets by modulating the activation of STAT3, STAT4, FOXO1, and NFκB transcription factors and hampering the Th1 and Th17-mediated long-term host protective memory responses. These findings suggest the urgent need to augment directly observed treatment, short-course (DOTS) therapy with immunomodulatory agents to mitigate the adverse effects linked to the treatment.IMPORTANCEAs a central component of TB eradication initiatives, directly observed treatment, short-course (DOTS) therapy imparts immune-dampening effects during the course of treatment. This approach undermines the host immune system by delaying the activation process and lowering the immune response. In our investigation, we have unveiled the impact of DOTS on specific immune cell populations. Notably, the signaling pathways involving STAT3 and STAT4 critical for memory responses and NFκβ associated with pro-inflammation were substantially declined due to the therapy. Consequently, these drugs exhibit limited effectiveness in preventing recurrence of the disease. These observations highlight the imperative integration of immunomodulators to manage TB infection.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active β-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

CD4+ T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4+ T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4+ T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4+ T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c+ cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII+ CD11c+ antigen-presenting cells is key to the CD4+ T cell/NK cell protection axis. By contrast, CD8+ T cell control of AEC1 infection appeared to operate independently.

CD4+ T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4+ T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c+ dendritic cells. This mechanism of CD4+ T cell control releases the need for the direct engagement of CD4+ T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens.

Since the discovery of extracellular vesicles (EVs) in mycobacterial species 15 years back, we have learned that this phenomenon is conserved in the Mycobacterium genus and has critical roles in bacterial physiology and host-pathogen interactions. Mycobacterium tuberculosis (Mtb), the tuberculosis (TB) causative agent, produces EVs both in vitro and in vivo including a diverse set of biomolecules with demonstrated immunomodulatory effects. Moreover, Mtb EVs (MEVs) have been shown to possess vaccine properties and carry biomarkers with diagnostic capacity. Although information on MEV biogenesis relative to other bacterial species is scarce, recent studies have shed light on how MEVs originate and are released to the extracellular space. In this minireview, we discuss past and new information about the vesiculogenesis phenomenon in Mtb, including biogenesis, MEV cargo, aspects in the context of host-pathogen interactions, and applications that could help to develop effective tools to tackle the disease.

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

Mycobacterium tuberculosis (MTB) infects and replicates in lung mononuclear phagocytes (MNPs) with astounding ability to evade elimination. ESX-1, a type VII secretion system, acts as a virulence determinant that contributes to MTB's ability to survive within MNPs, but its effect on MNP recruitment and/or differentiation remains unknown. Here, using single-cell RNA sequencing, we studied the role of ESX-1 in MNP heterogeneity and response in mice and murine bone marrow-derived macrophages (BMDM). We found that ESX-1 is required for MTB to recruit diverse MNP subsets with high MTB burden. Further, MTB induces an anti-inflammatory signature in MNPs and BMDM in an ESX-1 dependent manner. Similarly, spatial transcriptomics revealed an upregulation of anti-inflammatory signals in MTB lesions, where monocyte-derived macrophages concentrate near MTB-infected cells. Together, our findings suggest that MTB ESX-1 mediates the recruitment and differentiation of anti-inflammatory MNPs, which MTB can infect and manipulate for survival.

Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.

The caspase recruitment domain-containing protein 9 (CARD9) is an intracellular adaptor protein that is abundantly expressed in cells of the myeloid lineage, such as neutrophils, macrophages, and dendritic cells. CARD9 plays a critical role in host immunity against infections caused by fungi, bacteria, and viruses. A CARD9 deficiency impairs the production of inflammatory cytokines and chemokines as well as migration and infiltration, thereby increasing susceptibility to infections. However, CARD9 signaling varies depending on the pathogen causing the infection. Furthermore, different studies have reported altered CARD9-mediated signaling even with the same pathogen. Therefore, this review focuses on and elucidates the current literature on varied CARD9 signaling in response to various infectious stimuli in humans and experimental mice models.

Intercellular communication among microbes plays an important role in disease exacerbation. Recent advances have described small vesicles, termed as "extracellular vesicles" (EVs), previously disregarded as "cellular dust" to be vital in the intracellular and intercellular communication in host-microbe interactions. These signals have been known to initiate host damage and transfer of a variety of cargo including proteins, lipid particles, DNA, mRNA, and miRNAs. Microbial EVs, referred to generally as "membrane vesicles" (MVs), play a key role in disease exacerbation suggesting their importance in pathogenicity. Host EVs help coordinate antimicrobial responses and prime the immune cells for pathogen attack. Hence EVs with their central role in microbe-host communication, may serve as important diagnostic biomarkers of microbial pathogenesis. In this review, we summarize current research regarding the roles of EVs as markers of microbial pathogenesis with specific focus on their interaction with host immune defence and their potential as diagnostic biomarkers in disease conditions.

Extracellular vesicles (EVs) originating from bacteria function critical roles in bacterial biologic physiology and host-pathogen interactions. Mycobacterium tuberculosis (M. tuberculosis) produces EVs both in vitro and in vivo, with membrane-bound nanoparticles facilitating the transmission of biological molecules including lipids, proteins, nucleic acids and glycolipids, while interacting remotely with the host. Although studies of EVs in mycobacterial infections is still in its infancy, it has already revealed an entirely new aspect of M. tuberculosis-host interactions that may have implications for tuberculosis (TB) pathogenesis. In this review, we discuss the significant functions of M. tuberculosis EVs in elucidating the mechanisms underlying vesicle biogenesis and modulating cellular immune responses, as well as the recent advances and challenges in the development of novel preventive and therapeutic or diagnostic strategies against TB.

Control of Tuberculosis (TB) infection is mainly the result of productive teamwork between T-cell populations and antigen presenting cells (APCs). However, APCs activation at the site of initiating cellular immune response during BCG early infection is not completely understood.

In this study, we injected C57BL/6 mice in intravenous (i.v) or subcutaneous (s.c) route, then splenic or inguinal lymph node (LN) DCs and MΦs were sorted, and mycobacteria uptake, cytokine production, antigen presentation activity, and cell phenotype were investigated and compared, respectively.

Ag85A-specific T-cell immune response began at 6 days post BCG infection, when BCG was delivered in s.c route, Th17 immune response could be induced in inguinal LN. BCG could induce high level of activation phenotype in inguinal LN MΦs, while the MHC II presentation of mycobacteria-derived peptides by DCs was more efficient than MΦs.

The results showed that BCG immunized route can decide the main tissue of T-cell immune response. Compared with s.c injected route, APCs undergo more rapid cell activation in spleen post BCG i.v infection.

Bovine Johne's disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a common member of the microbial flora in healthy individuals. However, problems may arise when GBS-colonized mothers become pregnant. GBS may be transferred from a colonized mother to her newborn or developing fetus, which may result in complications such as miscarriage, pre-term birth, meningitis, pneumonia, or sepsis. Macrophages play an especially important role in the fetal and newborn response to GBS due to the limited development of the adaptive immune system early in life. The goal of this study was to expand what is currently known about how GBS manipulates macrophage cell signaling to evade the immune system and cause disease. To this end, we investigated whether the PI3K-Akt pathway was involved in several key aspects of the macrophage response to GBS. We explored whether certain GBS strains, such as sequence type (ST)-17 strains, rely on this pathway for the more rapid macrophage uptake they induce compared to other GBS strains. Our findings suggest that this pathway is, indeed, important for macrophage uptake of GBS. Consistent with these findings, we used immunofluorescence microscopy to demonstrate that more virulent strains of GBS induce more actin projections in macrophages than less virulent strains. Additionally, we explored whether PI3K-Akt signaling impacted the ability of GBS to survive within macrophages after phagocytosis and whether this pathway influenced the survival rate of macrophages themselves following GBS infection. The PI3K-Akt pathway was found to promote the survival of both macrophages and intracellular GBS following infection. We also observed that inhibition of the PI3K-Akt pathway significantly reduced GBS-mediated activation of NFκB, which is a key regulator of cell survival and inflammatory responses. Overall, these insights into strain-dependent GBS-mediated manipulation of the PI3K-Akt pathway and its downstream targets in infected macrophages may provide new insights for the development of diagnostic and therapeutic tools to combat severe GBS disease.

Mycobacterium tuberculosis is one of the major invasive intracellular pathogens causing most deaths by a single infectious agent. The interaction between host immune cells and this pathogen is the focal point of the disease, Tuberculosis. Host immune cells not only mount the protective action against this pathogen but also serve as the primary niche for growth. Thus, recognition of this pathogen by host immune cells and following signaling cascades are key dictators of the disease state. Immune cells, mainly belonging to myeloid cell lineage, recognize a wide variety of Mycobacterium tuberculosis ligands ranging from carbohydrate and lipids to proteins to nucleic acids by different membrane-bound and soluble pattern recognition receptors. Simultaneous interaction between different host receptors and pathogen ligands leads to immune-inflammatory response as well as contributes to virulence. This review summarizes the contribution of pattern recognition receptors of host immune cells in recognizing Mycobacterium tuberculosis and subsequent initiation of signaling pathways to provide the molecular insight of the specific Mtb ligands interacting with specific PRR, key adaptor molecules of the downstream signaling pathways and the resultant effector functions which will aid in identifying novel drug targets, and developing novel drugs and adjuvants.

In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication.

We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system.

Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1β, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFβ, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated.

Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

HIV and TB are the cause of significant worldwide mortality and pose a grave danger to the global public health. TB is the leading cause of death in HIV-infected persons, with one in four deaths attributable to TB. While the majority of healthy individuals infected with M. tuberculosis (Mtb) are able to control the infection, co-infection with HIV increases the risk of TB infection progressing to TB disease by over 20-fold. While antiretroviral therapy (ART), the cornerstone of HIV care, decreases the incidence of TB in HIV-uninfected people, this remains 4- to 7-fold higher after ART in HIV-co-infected individuals in TB-endemic settings, regardless of the duration of therapy. Thus, the immune control of Mtb infection in Mtb/HIV-co-infected individuals is not fully restored by ART. We do not fully understand the reasons why Mtb/HIV-co-infected individuals maintain a high susceptibility to the reactivation of LTBI, despite an effective viral control by ART. A deep understanding of the molecular mechanisms that govern HIV-induced reactivation of TB is essential to develop improved treatments and vaccines for the Mtb/HIV-co-infected population. We discuss potential strategies for the mitigation of the observed chronic immune activation in combination with both anti-TB and anti-retroviral approaches.

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Mycobacterium avium (M. avium) represents a species of concern, because of its ability to modulate the host's innate immune response, and therefore influence trajectory of adaptative immunity. Since eradicative response against mycobacteria, and M. tuberculosis/M. avium, relies on peptides actively presented on a Major Histocompatibility complex-II (MHC-II) context, we assessed paradoxical stimulation of Dendritic Cell resulting on immature immunophenotype characterized by membrane minor increase of MHC-II and CD40 despite of high expression of the pro-inflammatory tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in supernatants. Identification of M. avium leucine rich peptides forming short α-helices shutting down Type 1T helper (Th1), contribute to the understanding of immune evasion of an increasingly prevalent pathogen, and may provide a basis for future immunotherapy to infectious and non-infectious disease.

Being among the top 10 causes of adult deaths, tuberculosis (TB) disease is considered a major global public health concern to address. The human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb), is an extremely competent and well-versed pathogen that promotes pathogenesis by evading the host immune systems through numerous tactics. Investigations revealed that Mtb could evade the host defense mechanisms by reconfiguring the host gene transcription and causing epigenetic changes. Although results indicate the link between epigenetics and disease manifestation in other bacterial infections, little is known regarding the kinetics of the epigenetic alterations in mycobacterial infection. This literature review discusses the studies in Mtb-induced epigenetic alterations inside the host and its contribution in the host immune evasion strategies. It also discusses how the Mtb-induced alterations could be used as 'epibiomarkers' to diagnose TB. Additionally, this review also discusses therapeutic interventions to be enhanced through remodification by 'epidrugs'.

Toll-like receptors (TLRs) may be involved in the natural history of human papillomavirus (HPV) infection. In our study, we aimed to investigate the association of TLR4 (rs10116253, rs1927911, rs10759931) and TLR9 (rs187084, rs352140) gene polymorphisms with cervical persistent high-risk HPV (HR-HPV) infection, as well as multiple HR-HPV infections.

A total of 269 study subjects were enrolled and grouped by retrospectively analyzing the HR-HPV testing results and other clinical data of 2647 gynecological outpatients from Jingzhou Hospital Affiliated to Yangtze University. We conducted a case-control study to compare the role of TLR4/TLR9 gene polymorphisms between HR-HPV transient and persistent infections, as well as between HR-HPV single and multiple infections. HR-HPV genotypes were detected using Real-time polymerase chain reaction (RT-PCR). PCR-restriction fragment length polymorphism (PCR-RFLP) was used to determine TLR4 and TLR9 gene polymorphisms. Analyses of the different outcome variables (HR-HPV infection status and time for HR-HPV clearance) with respect to TLR4/TLR9 polymorphisms were carried out. Logistic regression analysis was used to determine the association of TLR4/TLR9 genotypes and alleles with HR-HPV infection status. The Kaplan-Meier method with the log-rank test was used to analyze the relationship between TLR4/TLR9 genotypes and the time for HR-HPV clearance.

The mutant genotypes of TLR9 rs187084 and rs352140 were associated with persistent (rs187084: CT and CT+CC; rs352140: CT and CT+TT) and multiple (rs187084: CT and CT+CC; rs352140: CT+TT) (all P < 0.05) HR-HPV infection. However, no association was found between TLR4 polymorphisms and HR-HPV infection status. Kaplan-Meier time to HR-HPV clearance analysis demonstrated that women carrying rs187084 and rs352140 mutant genotypes take longer duration to clear HR-HPV infection compared with wild-type genotype carriers (P1 = 0.012; P2 = 0.031).

Our results suggested that TLR9 polymorphisms, but not TLR4, were associated with cervical persistent and multiple HR-HPV infections, which could be useful as a potential predictor of HR-HPV infection status.

Mycobacterium tuberculosis has thrived in parallel with humans for millennia, and despite our efforts, M. tuberculosis continues to plague us, currently infecting a third of the world's population. The success of M. tuberculosis has recently been attributed, in part, to the PE-PPE family; a unique collection of 168 proteins fundamentally involved in the pathogenesis of M. tuberculosis. The PE-PPE family proteins have been at the forefront of intense research efforts since their discovery in 1998 and whilst our knowledge and understanding has significantly advanced over the last two decades, many important questions remain to be elucidated. This review consolidates and examines the vast body of existing literature regarding the PE-PPE family proteins, with respect to the latest developments in elucidating their evolution, structure, subcellular localisation, function, and immunogenicity. This review also highlights significant inconsistencies and contradictions within the field. Additionally, possible explanations for these knowledge gaps are explored. Lastly, this review poses many important questions, which need to be addressed to complete our understanding of the PE-PPE family, as well as highlighting the challenges associated with studying this enigmatic family of proteins. Further research into the PE-PPE family, together with technological advancements in genomics and proteomics, will undoubtedly improve our understanding of the pathogenesis of M. tuberculosis, as well as identify key targets/candidates for the development of novel drugs, diagnostics, and vaccines.

Mycobacterium tuberculosis is one of the most successful pathogens affecting humans, being the main cause of tuberculosis. It accounts for most infectious agent-related deaths worldwide; it has been estimated that a third of the world's population are bacillus carriers. This pathogen's evolutionary adaptation is mainly due to its ability to block a host's immune system by preventing it using an effective immune response in cases of active tuberculosis. Peptide-based synthetic vaccines represent an alternative for counteracting tuberculosis; however, although peptide antigens can be identified, they are not recognised by a host's immune system. An approach using dendritic cells as immunomodulating agents for increasing synthetic peptides' antigenic capacity has thus been advanced. Dendritic cells obtained from IL to 4- and GM-CSF-treated peripheral blood mononuclear cells were pulsed with synthetic Mtb protein peptides which have been reported as participating in mycobacteria-host interactions; their amino acid sequences were modified to improve MHC-II coupling and thus increase their recognition by a host's immune system. pMHC-II/TCR interaction triggered a lymphocyte response which controlled Mtb intracellular growth in infected macrophages. This work has been aimed at contributing to understanding dendritic cells' role in Mycobacterium tuberculosis protein peptide antigen presentation, thereby increasing individuals' immune response as a means of controlling the disease.