TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Exploring the S Protein of SARS-CoV-2 to Design a Novel Multi-Epitope Vaccine against COVID-19 Based on Immunoinformatics Approaches.

BACKGROUND: Developing a novel COVID-19 multi-epitope vaccine (CoVMEV) is essential to containing the SARS-CoV-2 pandemic. METHODS: The virus's immunodominant B and T cell epitopes from the S protein were found and joined to create the CoVMEV. Bioinformatics techniques were used to investigate the secondary and tertiary structures, as well as the physical and chemical properties of CoVMEV. RESULTS: CoVMEV exhibited high antigenicity and immunogenicity scores, together with good water solubility and stability. Toll-like receptor 2 (TLR2) and toll-like receptor4 (TLR4), which are critical in triggering immunological responses, were also strongly favoured by CoVMEV. Molecular dynamics simulation and immune stimulation studies revealed that CoVMEV effectively activated T and B lymphocytes, and increased the number of active CD8+ T cells than similar vaccines. CONCLUSION: CoVMEV holds promise as a potential vaccine candidate for COVID-19, given its robust immunogenicity, stability, antigenicity, and capacity to stimulate a strong immune response. This study presents a significant design concept for the development of peptidyl vaccines targeting SARS-CoV-2. Further investigation and clinical trials will be crucial in assessing the efficacy and safety of CoVMEV as a potential vaccine for COVID-19.

Design of multi-epitope chimeric vaccine against Monkeypox virus and SARS-CoV-2: A vaccinomics perspective.

The current era we experience is full with pandemic infectious agents that no longer threatens the major local source but the whole globe. Almost the most emerging infectious agents are severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), followed by monkeypox virus (MPXV). Since no approved antiviral drugs nor licensed active vaccines are yet available, we aimed to utilize immunoinformatics approach to design chimeric vaccine against the two mentioned viruses. This is the first study to deal with design divalent vaccine against SARS-CoV-2 and MPXV. ORF8, E and M proteins from Omicron SARS-CoV-2 and gp182 from MPXV were used as the protein precursor from which multi-epitopes (inducing B-cell, helper T cells, cytotoxic T cells and interferon-É£) chimeric vaccine was contrived. The structure of the vaccine construct was predicted, validated, and docked to toll-like receptor-2 (TLR-2). Moreover, its sequence was also used to examine the immune simulation profile and was then inserted into the pET-28a plasmid for in silico cloning. The vaccine construct was probable antigen (0.543) and safe (non-allergen) with strong binding energy to TLR-2 (-1169.8 kcal/mol) and found to have significant immune simulation profile. In conclusion, the designed chimeric vaccine was potent and safe against SARS-CoV-2 and MPXV, which deserves further consideration.

[Research progress on Toll-like receptor 2 (TLR2) with Mycobacterium tuberculosis infection].

Toll-like receptor 2 (TLR2) is a pattern recognition receptor expressed on the surface of leukocytes. Various ligands can activate or inhibit TLR2, therefore regulating the inflammation and apoptosis of immune cells. Mycobacterium tuberculosis (MTB) typically parasitizes macrophages. Further, after infecting the body, MTB can interact with TLR2 on the surface of various immune cells, including macrophages, leading to the release of cytokines that can affect the state and proliferation of MTB in the body. Additional research is needed to understand the polymorphism of TLR2 at the molecular level. Current studies indicate that the majority of TLR2 polymorphisms are not associated with susceptibility to MTB infection. This review provides an overview of the researches related to TLR2 and its ligands, the immune regulation activities of TLR2 following MTB infection, and the association of TLR2 polymorphism with susceptibility to MTB.

Design of a novel multiepitope vaccine with CTLA-4 extracellular domain against Mycoplasma pneumoniae: A vaccine-immunoinformatics approach.

BACKGROUND: Community-acquired pneumonia often stems from the macrolide-resistant strain of Mycoplasma pneumoniae, yet no effective vaccine exists against it. METHODS: This study proposes a vaccine-immunoinformatics strategy for Mycoplasma pneumoniae and other pathogenic microbes. Specifically, dominant B and T cell epitopes of the Mycoplasma pneumoniae P30 adhesion protein were identified through immunoinformatics method. The vaccine sequence was then constructed by coupling with CTLA-4 extracellular region, a novel molecular adjuvant for antigen-presenting cells. Subsequently, the vaccine's physicochemical properties, antigenicity, and allergenicity were verified. Molecular dynamics modeling was employed to confirm interaction with TLR-2, TLR-4, B7-1, and B7-2. Finally, the vaccine underwent in silico cloning for expression. RESULTS: The vaccine exhibited both antigenicity and non-allergenicity. Molecular dynamics simulation, post-docking with TLR-2, TLR-4, B7-1, and B7-2, demonstrated stable interaction between the vaccine and these molecules. In silico cloning confirmed effective expression of the vaccine gene in insect baculovirus vectors. CONCLUSION: This vaccine-immunoinformatics approach holds promise for the development of vaccines against Mycoplasma pneumoniae and other pathogenic non-viral and non-bacterial microbes.

Dominant B cell-T cell epitopes instigated robust immune response in-silico against Scrub Typhus.

Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

Characterization and expression profiles of toll-like receptor genes (TLR2 and TLR5) in immune tissues of hybrid yellow catfish under bacterial infection.

The yellow catfish (Pelteobagrus fulvidraco) is one of the most economically important freshwater species in Asia. However, pathogenic bacterial infections often cause high rates of mortality and economic losses in practical aquaculture. Previous studies in mammals have shown that Toll-like receptor 2 (TLR2) and Toll-like receptor 5 (TLR5) are involved in the recognition of cell wall components such as lipopolysaccharides and flagella of various bacteria, thereby acting as key regulators in the innate immunity response. However, TLR2 and TLR5 in yellow catfish have not been characterized. In the present study, TLR2 and TLR5 were examined through comparative genomic approaches. The gene structure, collinearity, protein spatial structure, and phylogenetic relationships were compared with those in multiple representative vertebrates. Meanwhile, quantitative real-time PCR was conducted to explore transcriptional changes in TLR2 and TLR5 in immune tissues after infection with exogenous A. hydrophila and E. tarda. The results demonstrated the presence of TLR2 and TLR5 in yellow catfish. However, a systematic analysis showed that TLR2 was not associated with the arrangement of diverse neighboring genes. The expression of hybrid yellow catfish TLR2 transcripts in multiple tissues (including liver, spleen, kidney, and intestine) was significantly up-regulated after infection with A. hydrophila and E. tarda, suggesting that hybrid yellow catfish TLR2 and TLR5 may participate in the immune process. Taken together, the results indicate that TLR2 and TLR5 are conserved in terms of evolution and possess significant antibacterial activity as well as regulatory properties in immune-related tissues and thus play key roles in host defense against pathogen invasion.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

In silico design of a novel multi-epitope vaccine against HCV infection through immunoinformatics approaches.

Infection with the hepatitis C virus (HCV) is one of the causes of liver cancer, which is the world's sixth most prevalent and third most lethal cancer. The current treatments do not prevent reinfection; because they are expensive, their usage is limited to developed nations. Therefore, a prophylactic vaccine is essential to control this virus. Hence, in this study, an immunoinformatics method was applied to design a multi-epitope vaccine against HCV. The best B- and T-cell epitopes from conserved regions of the E2 protein of seven HCV genotypes were joined with the appropriate linkers to design a multi-epitope vaccine. In addition, cholera enterotoxin subunit B (CtxB) was included as an adjuvant in the vaccine construct. This study is the first to present this epitopes-adjuvant combination. The vaccine had acceptable physicochemical characteristics. The vaccine's 3D structure was predicted and validated. The vaccine's binding stability with Toll-like receptor 2 (TLR2) and TLR4 was confirmed using molecular docking and molecular dynamics (MD) simulation. The immune simulation revealed the vaccine's efficacy by increasing the population of B and T cells in response to vaccination. In silico expression in Escherichia coli (E. coli) was also successful.

Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae.

Streptococcus pneumoniae, a common colonizer of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy participants of controlled human infection studies. We hypothesized that surface expression of pneumococcal lipoproteins, recognized by the innate immune receptor TLR2, mediates epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signaling. Loss of lgt also led to the concomitant decrease in interferon signaling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in ~10% of global pneumococcal isolates. While palA was enriched in the carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonization is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonized by pneumococci without becoming unwell. It is therefore important to better understand how the colonization process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonization and disease.

TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

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.

TLR2 is non-redundant in the population and subpopulation responses to Mycobacterium tuberculosis in macrophages and in vivo.

Tuberculosis (TB), caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is a global health threat. Targeting host pathways that modulate protective or harmful components of inflammation has been proposed as a therapeutic strategy that could aid sterilization or mitigate TB-associated permanent tissue damage. In purified form, many Mtb components can activate innate immune pathways. However, knowledge of the pathways that contribute most to the observed response to live Mtb is incomplete, limiting the possibility of precise intervention. We took a systematic, unbiased approach to define the pathways that drive the earliest immune response to Mtb. Using a macrophage model of infection, we compared the bulk transcriptional response to infection with the response to a panel of Mtb-derived putative innate immune ligands. We identified two axes of response: an NF-kB-dependent response similarly elicited by all Mtb pathogen-associated molecular patterns (PAMPs) and a type I interferon axis unique to cells infected with live Mtb. Consistent with growing literature data pointing to TLR2 as a dominant Mtb-associated PAMP, the TLR2 ligand PIM6 most closely approximated the NF-kB-dependent response to the intact bacterium. Quantitatively, the macrophage response to Mtb was slower and weaker than the response to purified PIM6. On a subpopulation level, the TLR2-dependent response was heterogeneously induced, with only a subset of infected cells expressing key inflammatory genes known to contribute to the control of infection. Despite potential redundancies in Mtb ligand/innate immune receptor interactions during in vivo infection, loss of the TLR2/PIM6 interaction impacted the cellular composition of both the innate and adaptive compartments. IMPORTANCE Tuberculosis (TB) is a leading cause of death globally. Drug resistance is outpacing new antibiotic discovery, and even after successful treatment, individuals are often left with permanent lung damage from the negative consequences of inflammation. Targeting host inflammatory pathways has been proposed as an approach that could either improve sterilization or improve post-treatment lung health. However, our understanding of the inflammatory pathways triggered by Mycobacterium tuberculosis (Mtb) in infected cells and lungs is incomplete, in part because of the complex array of potential molecular interactions between bacterium and host. Here, we take an unbiased approach to identify the pathways most central to the host response to Mtb. We examine how individual pathways are triggered differently by purified Mtb products or infection with the live bacterium and consider how these pathways inform the emergence of subpopulation responses in cell culture and in infected mice. Understanding how individual interactions and immune pathways contribute to inflammation in TB opens the door to the possibility of developing precise therapeutic interventions.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge.

African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling.

Tongue sole creatine kinases function as DAMP and activate antimicrobial immunity via TLR2.

Creatine kinase (CK) is an enzyme that regulates adenosine triphosphate (ATP) metabolism to maintain energy homeostasis. Although CK has been reported to be involved in pathogen infection, the immune function of CK remains elusive. In this study, we identified two muscle-type CK from the teleost tongue sole Cynoglossus semilaevis (designated CsCKM-1 and CsCKM-2). Bacterial infection modulated CsCKM-1/2 expression in tongue sole tissues and induced the release of CsCKM-1/2 into serum. Recombinant CsCKM-1/2 (rCsCKM-1/2) exhibited robust kinase activity and bound to bacterial pathogens and pathogen-associated molecular patterns. rCsCKM-1/2 also bound to tongue sole peripheral blood leukocytes (PBLs) and promoted PBLs to uptake bacterial pathogens, inhibit bacterial proliferation, and express proinflammatory cytokines. When co-expressed in HEK293T cells, CsCKM-1/2 were found to interact with the leucine rich domain of toll-like receptor 2 (TLR2). The presence of TLR2 antagonist significantly reduced CsCKM-1/2-induced immune response and antibacterial effect. Taken together, these results indicated that tongue sole creatine kinases function as damage-associated molecular pattern (DAMP) molecules and play an important role in antimicrobial immunity via TLR2.

Gene expression analysis of toll like receptor 2 and 4, Dectin-1, Osteopontin and inflammatory cytokines in human dental pulp ex-vivo.

BACKGROUND: Toll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation. OBJECTIVE: The aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls. METHODS: A total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05. RESULTS: Gene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis. CONCLUSION: Dectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship.

Decreased Interleukin-1 Family Cytokine Production in Patients with Nontuberculous Mycobacterial Lung Disease.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the Mycobacterium avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin-1ß (IL-1ß), IL-18, IL-1α, and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1ß production by macrophages infected with MAC. Toll-like receptor 2 (TLR2) and TWIK2 (a two-pore domain K+ channel) were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all four cytokines, whereas a two-pore domain K+ channel inhibitor decreased the production of IL-1ß, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. A reduced monocyte ratio might contribute to the attenuated production of IL-1 family cytokines by PBMCs of NTMPD patients in response to MAC stimulations. Collectively, our findings suggest that the attenuated IL-1 response may increase susceptibility to NTM pulmonary infection through multiple factors, including impaired expression of the TLR2 and TWIK2 and reduced monocyte ratio. IMPORTANCE Upon MAC stimulation, the production of IL-1 family cytokines and IL-10 by PBMCs of NTMPD patients was attenuated compared with that of healthy household contacts. Upon MAC stimulation, the expression of TLR2 and TWIK2 (one of the two-pore domain K+ channels) was attenuated in PBMCs of NTMPD patients compared with that of healthy household contacts. The production of IL-1 family cytokines by MAC-stimulated PBMCs and MAC-infected monocytes of healthy donors was reduced by a TLR2 inhibitor and two-pore domain K+ channel inhibitor. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. Collectively, our data suggest that defects in the expression of TLR2 and TWIK2 in human PBMCs or monocytes and reduced monocyte ratio are involved in the reduced production of IL-1 family cytokines, and it may increase susceptibility to NTM pulmonary infection.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

Akkermansia muciniphila phospholipid induces homeostatic immune responses.

Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.