ABSTRACT
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.
Subject(s)
Betacoronavirus/physiology , COVID-19 Vaccines/immunology , Coronavirus Infections/immunology , Severe acute respiratory syndrome-related coronavirus/physiology , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Conserved Sequence/genetics , Evolution, Molecular , Humans , Immunization , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccine DevelopmentABSTRACT
The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.
Subject(s)
Kupffer Cells/physiology , Listeria monocytogenes/immunology , Listeriosis/immunology , Liver/immunology , Macrophages/immunology , Neutrophils/immunology , Peritoneum/microbiology , Animals , Cell Communication , Cell Self Renewal , Host-Pathogen Interactions , Humans , Immunity, Innate , Kupffer Cells/microbiology , Liver/microbiology , Liver/pathology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Neutrophil Infiltration , Peritoneum/pathologyABSTRACT
Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.
Subject(s)
COVID-19 , Pulmonary Disease, Chronic Obstructive , Serpins , Cytokines , Epithelial Cells , Humans , Peptide Hydrolases , Pulmonary Disease, Chronic Obstructive/drug therapy , SARS-CoV-2 , Sequence Analysis, RNA , Serpins/pharmacology , Serpins/therapeutic useABSTRACT
Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, in vitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1ß, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1ß expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in in vitro studies.
Subject(s)
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculosis , Animals , Cytokines , Macrophages , Mice , Mice, Inbred C57BLABSTRACT
Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.
Subject(s)
Macrophages/metabolism , Monocytes/metabolism , Polysaccharides/metabolism , Chromatography, Liquid , Glycomics , Glycopeptides/analysis , Glycosylation , Humans , Polysaccharides/chemistry , Tandem Mass SpectrometryABSTRACT
Paratuberculosis and bovine tuberculosis are two mycobacterial diseases of ruminants which have a considerable impact on livestock health, welfare, and production. These are chronic "iceberg" diseases which take years to manifest and in which many subclinical cases remain undetected. Suggested biomarkers to detect infected or diseased animals are numerous and include cytokines, peptides, and expression of specific genes; however, these do not provide a strong correlation to disease. Despite these advances, disease detection still relies heavily on dated methods such as detection of pathogen shedding, skin tests, or serology. Here we review the evidence for suitable biomarkers and their mechanisms of action, with a focus on identifying animals that are resilient to disease. A better understanding of these factors will help establish new strategies to control the spread of these diseases.
Subject(s)
Biomarkers/analysis , Disease Resistance , Immunologic Factors/analysis , Mycobacterium Infections/veterinary , Ruminants , Animals , Mycobacterium Infections/geneticsABSTRACT
Damaging inflammation is a hallmark of Mycobacterium tuberculosis infection, and understanding how this is regulated is important for the development of new therapies to limit excessive inflammation. The E3 ubiquitin ligase, Roquin, is involved in immune regulation; however, its role in immunity to M. tuberculosis is unknown. To address this, we infected mice with a point mutation in Roquin1/Rc3h1 (sanroque). Aerosol-infected sanroque mice showed enhanced control of M. tuberculosis infection associated with delayed bacterial dissemination and upregulated TNF production in the lungs after 2 wk. However, this early control of infection was not maintained, and by 8 wk postinfection sanroque mice demonstrated an increased bacterial burden and dysregulated inflammation in the lungs. As the inflammation in the lungs of the sanroque mice could have been influenced by emerging autoimmune conditions that are characteristic of the mice aging, the function of Roquin was examined in immune cell subsets in the absence of autoimmune complications. M. bovis bacillus Calmette-Guérin-primed sanroque T cells transferred into Rag1-/- mice provided equivalent protection in the spleen and liver. Interestingly, the transfer of mycobacteria-specific (P25 CD4+ TCR transgenic) wild-type spleen cells into sanroqueRag1-/- mice actually led to enhanced protection with reduced bacterial load, decreased chemokine expression, and reduced inflammation in the lungs compared with transfers into Rag1-/- mice expressing intact Roquin. These studies suggest that modulation of Roquin in myeloid cells may reduce both inflammation and bacterial growth during the chronic phase of M. tuberculosis infection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Inflammation/immunology , Lung/immunology , Mycobacterium tuberculosis/physiology , Myeloid Cells/immunology , Tuberculosis/immunology , Ubiquitin-Protein Ligases/metabolism , Animals , Bacterial Load , CD4-Positive T-Lymphocytes/microbiology , CD4-Positive T-Lymphocytes/transplantation , Cells, Cultured , Lung/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/microbiology , Transplantation Chimera , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
The cytokine Tumor Necrosis Factor Superfamily member 14, TNFSF14 (or LIGHT), is a controversial player in numerous diseases. We investigated the role of endogenously expressed TNFSF14 in diet-induced obesity in vivo. Firstly, we studied the effects of Tnfsf14 ablation on the development of obesity, glucose intolerance, insulin resistance, steatosis, tissue inflammation, and mitochondrial respiration in the liver. Secondly, we examined the role of TNFSF14 expression in hematopoietic cells on obesity and insulin sensitivity. Male Tnfsf14 knockout (KO) and wild type mice were fed chow or high fat diet (HFD) for 12 weeks and were assessed for weight gain, glucose intolerance, insulin resistance, hepatosteatosis, mitochondrial dysfunction, and cytokine expression. Wild-type mice were also reconstituted with bone marrow cells from Tnfsf14 knockout mice and were fed chow or HFD for 12 weeks. These mice were examined for weight gain and insulin resistance. HFD fed mice had elevated circulating levels of serum TNFSF14. Liver and white adipose tissue are potential sources of this elevated TNFSF14. Tnfsf14 deficient mice displayed increased obesity, glucose intolerance, insulin resistance, hepatosteatosis, and mitochondrial dysfunction compared to control mice on a HFD. Hepatic cytokine profiling pointed to a potential novel role of decreased IL-6 in the metabolic disturbances in obesogenic Tnfsf14 knockout mice. Bone marrow cells from Tnfsf14 deficient mice appeared to promote diet-induced obesity, insulin resistance and reduced FGF21 levels in white adipose tissue and liver. Our novel data suggest that Tnfsf14 ablation exacerbates parameters of the metabolic syndrome under high fat feeding conditions and provides evidence to support the development of TNFSF14 agonists as potential therapeutics in diet-induced obesity.
Subject(s)
Fibroblast Growth Factors/metabolism , Insulin/metabolism , Interleukin-6/metabolism , Liver/physiology , Metabolic Diseases/immunology , Obesity/immunology , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Adipose Tissue/metabolism , Animals , Diet, High-Fat , Disease Models, Animal , Humans , Insulin Resistance/genetics , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Tumor Necrosis Factor Ligand Superfamily Member 14/geneticsABSTRACT
Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases, e.g., ß-galactosidase, ß-hexosaminidases and α-/ß-mannosidases, was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.
Subject(s)
Cell-Derived Microparticles/metabolism , Glycoproteins/genetics , Host-Pathogen Interactions , Macrophages/metabolism , Mycobacterium tuberculosis/growth & development , Proteome/genetics , Carbohydrate Sequence , Cell Line , Cell-Derived Microparticles/chemistry , Cell-Derived Microparticles/microbiology , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Gene Expression Regulation , Glycoproteins/metabolism , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Humans , Lectins/genetics , Lectins/metabolism , Lysosomes/chemistry , Lysosomes/metabolism , Macrophages/chemistry , Macrophages/microbiology , Mannose/chemistry , Mannose/metabolism , Mycobacterium tuberculosis/pathogenicity , Proteome/metabolism , Sialic Acids/chemistry , Sialic Acids/metabolism , Signal TransductionABSTRACT
We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.
Subject(s)
Macrophages/immunology , Macrophages/metabolism , Salmonella typhimurium/physiology , Salmonella/physiology , Toll-Like Receptors/metabolism , Zinc/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cells, Cultured , Copper , Cytoplasmic Vesicles/chemistry , Cytoplasmic Vesicles/metabolism , Gene Expression Regulation, Bacterial , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Toll-Like Receptors/geneticsABSTRACT
Microparticles (MPs) are small membranous particles (100-1000 nm) released under normal steady-state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb-infected (TBinf-MP) and uninfected human THP-1 monocytic cells. MP proteins were analyzed by GeLC-MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf-MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP-1 cells to TBinf-MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.
Subject(s)
Cytokines/metabolism , Interferon Type I/metabolism , Macrophages/metabolism , Macrophages/microbiology , Mycobacterium tuberculosis/pathogenicity , Ubiquitins/metabolism , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line/microbiology , Cell-Derived Microparticles/metabolism , Cytokines/genetics , Gene Expression Regulation/drug effects , Host-Pathogen Interactions , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Monocytes/metabolism , Monocytes/microbiology , Proteins/genetics , Proteins/metabolism , RNA-Binding Proteins , Tandem Mass Spectrometry , Tuberculosis/metabolism , Ubiquitins/geneticsABSTRACT
Tuberculosis (TB) remains a major public health issue. New tests to aid diagnoses and monitor the response to therapy are urgently required. There is growing interest in the use of microRNA (miRNA) profiles as diagnostic, prognostic or predictive markers in a range of clinical and infectious diseases, including Mycobacterium tuberculosis infection, however, challenges exist to accurately normalise miRNA levels in cohorts. This study examined the appropriateness of 12 miRs and RNU6B to normalise circulating plasma miRNA levels in individuals with active TB from 2 different geographical and ethnic regions. Twelve miRs (let-7, miR-16, miR-22, miR-26, miR-93, miR-103, miR-191, miR-192, miR-221, miR-423, miR-425 and miR-451) and RNU6B were selected based on their reported production by lung cells, expression in blood and previous use as a reference miRNA. Expression levels were analysed in the plasma of newly diagnosed TB patients from Australia and China compared with individuals with latent TB infection and healthy volunteers. Analysis with both geNorm and NormFinder software identified miR-93 as the most suitable reference miR in both cohorts, either when analysed separately or collectively. Interestingly, there were large variations in the expression levels of some miRs, in particular miR-192 and let-7, between the two cohorts, independent of disease status. These data identify miR-93 is a suitable reference miR for normalizing miRNA levels in TB patients, and highlight how environmental, and possibly ethnic, factors influence miRNA expression levels, demonstrating the necessity of assessing the suitability of reference miRs within the study population.
Subject(s)
MicroRNAs/blood , Tuberculosis/blood , Tuberculosis/genetics , Adolescent , Adult , Aged , Australia , Case-Control Studies , Cohort Studies , Female , Gene Expression Regulation , Genetic Association Studies , Humans , Male , MicroRNAs/genetics , Middle Aged , Reference Standards , Software , Young AdultABSTRACT
Mycobacterium tuberculosis infection is characterized by a strong inflammatory response whereby a few infected macrophages within the granuloma induce sustained cellular accumulation. The mechanisms coordinating this response are poorly characterized. We hypothesized that microparticles (MPs), which are submicron, plasma membrane-derived vesicles released by cells under both physiological and pathological conditions, are involved in this process. Aerosol infection of mice with M. tuberculosis increased CD45(+) MPs in the blood after 4 wk of infection, and in vitro infection of human and murine macrophages with mycobacteria enhanced MP release. MPs derived from mycobacteria-infected macrophages were proinflammatory, and when injected into uninfected mice they induced significant neutrophil, macrophage, and dendritic cell recruitment to the injection site. When incubated with naive macrophages, these MPs enhanced proinflammatory cytokine and chemokine release, and they aided in the disruption of the integrity of a respiratory epithelial cell monolayer, providing a mechanism for the egress of cells to the site of M. tuberculosis infection in the lung. In addition, MPs colocalized with the endocytic recycling marker Rab11a within macrophages, and this association increased when the MPs were isolated from mycobacteria-infected cells. M. tuberculosis-derived MPs also carried mycobacterial Ag and were able to activate M. tuberculosis-specific CD4(+) T cells in vivo and in vitro in a dendritic cell-dependent manner. Collectively, these data identify an unrecognized role for MPs in host response against M. tuberculosis by promoting inflammation, intercellular communication, and cell migration.
Subject(s)
Cell-Derived Microparticles/immunology , Macrophages/immunology , Macrophages/microbiology , Mycobacterium tuberculosis/immunology , Tuberculosis/immunology , Animals , Antigens, Bacterial/immunology , Biological Transport , CD4-Positive T-Lymphocytes/immunology , Cell Communication/immunology , Cell Line , Cell Movement/immunology , Cell-Derived Microparticles/metabolism , Chemokines/biosynthesis , Chemokines/immunology , Cytokines/biosynthesis , Cytokines/immunology , Dendritic Cells/immunology , Dendritic Cells/microbiology , Endosomes/metabolism , Humans , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Lymphocyte Activation/immunology , Macrophages/metabolism , Mice , Mice, Transgenic , Tuberculosis/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
Non-Tuberculous mycobacteria (NTM) are opportunistic environmental bacteria. Globally, NTM incidence is increasing and modeling suggests that, without new interventions, numbers will continue to rise. Effective treatments for NTM infections remain suboptimal. Standard therapy for Mycobacterium avium complex, the most commonly isolated NTM, requires a 3-drug regime taken for approximately 18 months, with rates of culture conversion reported between 45 and 70%, and high rates of relapse or reinfection at up to 60%. New therapeutic options for NTM treatment are urgently required. A survey of ongoing clinical trials for new NTM therapy listed on ClinicalTrials.Gov using the terms 'Mycobacterium avium', 'Mycobacterium abscessus', 'Mycobacterium intracellulare', 'Non tuberculous Mycobacteria' and 'Nontuberculous Mycobacteria' and a selection criterion of interventional studies using antibiotics demonstrates that most trials involve dose and combination therapy of the guideline based therapy or including one or more of; Amikacin, Clofazimine, Azithromycin and the anti-TB drugs Bedaquiline and Linezolid. The propensity of NTMs to form biofilms, their unique cell wall and expression of both acquired and intrinsic resistance, are all hampering the development of new anti-NTM therapy. Increased investment in developing targeted treatments, specifically for NTM infections is urgently required.
ABSTRACT
ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1ß and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease.
Subject(s)
Bacterial Infections/metabolism , Parasitic Diseases/metabolism , Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Animals , Apoptosis , Bacteria/immunology , Bacterial Infections/immunology , Disease Susceptibility , Humans , Inflammasomes/metabolism , Interleukin-18/biosynthesis , Interleukin-1beta/biosynthesis , Ion Channels/metabolism , Macrophages/metabolism , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Parasites/immunology , Parasitic Diseases/immunology , Phospholipase D/biosynthesis , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTßR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTßR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4(+) T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.
Subject(s)
Immunity, Cellular , Leishmania donovani/pathogenicity , Leishmaniasis, Visceral/pathology , Receptors, Tumor Necrosis Factor, Member 14/metabolism , T-Lymphocytes/immunology , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Cell Proliferation/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-12/biosynthesis , Interleukin-23/biosynthesis , Leishmania donovani/immunology , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Liver/parasitology , Liver/pathology , Lymphotoxin beta Receptor/immunology , Lymphotoxin beta Receptor/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Tumor Necrosis Factor, Member 14/immunology , Signal Transduction , T-Lymphocytes/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 14/geneticsABSTRACT
Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X(7) receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X(7) also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X(7)-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X(7)-deficient peritoneal macrophages. The surface expression of P2X(7) on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X(7) was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X(7) receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.
Subject(s)
Adenosine Triphosphate/immunology , Apoptosis/immunology , Macrophages/immunology , Phagocytosis/immunology , Receptors, Purinergic P2X7/immunology , Adenosine Triphosphate/metabolism , Animals , Cell Separation , Extracellular Fluid/chemistry , Flow Cytometry , Humans , Ligands , Macrophages/cytology , Macrophages/metabolism , Mice , Microscopy, Confocal , Peptides/immunology , Protein Binding , Receptors, Purinergic P2X7/chemistry , Receptors, Purinergic P2X7/metabolism , Receptors, Scavenger/immunology , Receptors, Scavenger/metabolismABSTRACT
Phagocytosis of nonopsonized bacteria is central to innate immunity, but its regulation is less defined. We show that overexpression of the P2X(7) receptor greatly augments the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X(7) expression by siRNA significantly reduces the phagocytic ability of human monocytic cells. An intact P2X(7)-nonmuscle myosin complex is required for phagocytosis of nonopsonized beads because activation of P2X(7) receptors by adenosine triphosphate (ATP), which dissociates myosin IIA from the P2X(7) complex, inhibits this phagocytic pathway. Fresh human monocytes rapidly phagocytosed live and heat-killed Staphylococcus aureus and Escherichia coli in the absence of serum, but the uptake was reduced by prior incubation with ATP, or P2X(7) monoclonal antibody, or recombinant P2X(7) extracellular domain. Injection of beads or bacteria into the peritoneal cavity of mice resulted in their brisk phagocytosis by macrophages, but injection of ATP before particles markedly decreased this uptake. These data demonstrate a novel pathway of phagocytosis of nonopsonized particles and bacteria, which operate in vivo and require an intact P2X(7)-nonmuscle myosin IIA membrane complex. The inhibitory effect of ATP on particle uptake by the macrophage is regulated by the P2X(7) receptor and defines this phagocytic pathway.
Subject(s)
Adenosine Triphosphate/immunology , Escherichia coli/immunology , Macrophages, Peritoneal/immunology , Nonmuscle Myosin Type IIA/immunology , Phagocytosis/immunology , Receptors, Purinergic P2/immunology , Staphylococcus aureus/immunology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Cell Line , Humans , Immunity, Innate/drug effects , Immunity, Innate/physiology , Macrophages, Peritoneal/metabolism , Mice , Nonmuscle Myosin Type IIA/metabolism , Phagocytosis/drug effects , Protein Structure, Tertiary/physiology , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7ABSTRACT
The TNF superfamily member, LIGHT, contributes to optimal T-cell activation in vitro through co-stimulation of dendritic cell cytokine production; however, its role in T-cell-mediated control of intracellular bacterial infections is unknown. Protective immunity against Listeria monocytogenes and Mycobacterium tuberculosis infection requires both antigen-specific CD4(+) and CD8(+) T cells. Using LIGHT-deficient mice we determined that LIGHT was necessary for optimal re-stimulation of anti-listerial CD8(+) T cells in vitro. By contrast, LIGHT(-/-) mice infected with L. monocytogenes generated equivalent T-cell responses and controlled the infection as effectively as normal C57BL/6 mice. Following M. tuberculosis infection, LIGHT(-/-) mice showed a significant increase in bacterial replication in the lungs at 4 weeks, but by 6 weeks had controlled the infection. Analysis of T-cell responses in vivo revealed that LIGHT was dispensable for the activation of primary T-cell responses and the production of IL-12 and IFN-gamma. In addition, LIGHT was not required for the induction of memory T-cell responses to anti-mycobacterial DNA or BCG vaccines and for subsequent protection against tuberculosis challenge. Therefore, LIGHT contributes to the optimal co-stimulation of anti-listerial CD8(+) T-cell responses in vitro and to the early control of M. tuberculosis infection; however, other mechanisms compensate for LIGHT deficiency in the control of these pathogens in vivo.
Subject(s)
Mycobacterium Infections/immunology , Tumor Necrosis Factor Ligand Superfamily Member 14/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Listeria monocytogenes/immunology , Listeria monocytogenes/isolation & purification , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/isolation & purification , Tumor Necrosis Factor Ligand Superfamily Member 14/deficiencyABSTRACT
microRNA are small non-coding RNA molecules which inhibit gene expression by binding mRNA, preventing its translation. As important regulators of gene expression, there is increasing interest in microRNAs as potential diagnostic biomarkers and therapeutic targets. Studies investigating the role of one of the miRNA-miR-652-3p-detail diverse roles for this miRNA in normal cell homoeostasis and disease states, including cancers, cardiovascular disease, mental health, and central nervous system diseases. Here, we review recent literature surrounding miR-652-3p, discussing its known target genes and their relevance to disease progression. These studies demonstrate that miR-652-3p targets LLGL1 and ZEB1 to modulate cell polarity mechanisms, with impacts on cancer metastasis and asymmetric cell division. Inhibition of the NOTCH ligand JAG1 by miR-652-3p can have diverse effects on angiogenesis and immune cell regulation. Investigation of miR-652-3p and other dysregulated miRNAs identified a number of pathways potentially regulated by miR-652-3p. This review demonstrates that miR-652-3p has great promise as a diagnostic or therapeutic target due to its activity across multiple cellular systems.