TLR4 Receptor Research Articles

Coinfection with porcine circovirus type 2 and Glaesserella parasuis serotype 4 enhances pathogenicity in piglets

Coinfection of Porcine circovirus type 2 (PCV2) and Glaesserella parasuis type 4 (GPS4) is widespread clinically, resulting in high morbidity and mortality, however, interactions between the two pathogens during coinfection and the coinfection pathogenesis are poorly understood. In this study, a piglet model coinfected with PCV2 and GPS4 was established; coinfection of the piglets' group showed more obvious symptoms, such as high fever and emaciation, and more severe histological lesions appeared in various organs. Importantly, piglets in the coinfection group produced lower levels of PCV2 and GPS4 antibodies, and showed high levels of inflammatory cytokines, TLR2, and TLR4, while the levels of CD4, CD8, MHC II, costimulatory molecules, and IL-12p40 were decreased. In addition, a model of macrophage 3D4/21 cells coinfection with PCV2 and GPS4 was established, coinfected cells exhibited increased expression of the cytokines IL-6, IL-8, TNF-α, IL-1β, and the receptors TLR2, TLR4, while decreased MHC II. We further demonstrate that cytokine production is associated with the activation of NF-κB and NLRP3 inflammasome signaling pathways, and TLR4 is also involved. Altogether, our findings suggest that coinfection with PCV2 and GPS4 exacerbates the inflammatory response, resulting in severe tissue damage, and probably impaired macrophage antigen presentation and T cell activation, resulting in immune dysregulation, aggravating host infection.

Influence of X chromosome in sex-biased autoimmune diseases

Females have better ability to resolve infections, compared to males, but also, a greater susceptibility to develop autoimmunity. Besides the initial interest on the contribution of sex-steroid hormone signaling, the role of genetic factors linked to X chromosome has recently focused much attention. In human and mouse, the number of X chromosomes, rather than sex-steroid hormones, have been found associated with higher risk or susceptibility to develop autoimmunity, particularly rheumatic diseases, such as SLE, Sjögren's syndrome or Scleroderma. For all of these diseases, the Toll-like receptor TLR7 and TLR8, encoded on the same locus in the human Xp, have been demonstrated to be causal in disease development through gene dosage effect or gain of function mutations. During embryonic development in female mammals, one X chromosome is stochastically inactivated to balance X-linked gene expression between males and females, a process known as X chromosome inactivation (XCI). Nevertheless, some genes including immune related genes can escape XCI to variable degree and penetrance, resulting in a bi-allelic expression in some immune cells, such as TLR7. Because tight regulation of TLR expression is necessary for a healthy, self-tolerant immune environment, XCI escape has been proposed as a mechanism contributing to this sexual dimorphism. In this review, we will summarize general mechanisms of XCI, and describe the known escapee's genes in immune cells, the cellular diversity created by such mechanisms and its potential implication in autoimmune diseases, with a particular focus on the X-linked genes and immune cell populations involved in SLE. Whether dysregulated expression of X-linked genes could contribute to the enhanced susceptibility of females to develop such diseases remains to be proven. Shedding lights onto the X-linked genetic mechanisms contributing to modulation of immune cell functions will undoubtedly provide new insights into the intricate mechanisms underlying sex differences in immunity and autoimmunity.

Inflammation Regulation by Bacterial Molecular Patterns.

Stimulation of innate immunity by bacterial molecular patterns can induce an enhanced cellular immune response to pathogens that are associated with innate immune memory shaped by epigenetic changes. Immunological memory can be expressed in the acceleration/intensification of inflammation, as well as in the exact opposite-to maintain tolerance and non-response to a repeated stimulus. Tolerance is one of the central concepts of immunity and is ensured by the consistency of all parts of the immune response. The severe consequences of inflammation force researchers to study in detail all stages of the downstream pathways that are activated after exposure to a stimulus, while the formation of non-response to a pro-inflammatory stimulus has not yet received a detailed description. Elucidation of the mechanism of tolerance is an urgent task for the prevention and treatment of inflammatory diseases. The aim of this investigation was to study the dynamic changes in the gene expression of A20 and ATF3, the inflammation suppressors, against the background of the expression of the genes of the innate immunity receptors TLR4 and NOD2 and the pro-inflammatory cytokine TNF-α under the influence of TLR4 and NOD2 agonists, lipopolysaccharide (LPS) and glucosaminylmuramyl dipeptide (GMDP). The mechanism of inflammation regulation by bioregulators of bacterial origin-LPS and GMDP-was evaluated in vitro in human peripheral blood mononuclear cells and in vivo after i.p. administration of LPS and GMDP to mice. Gene expression was assessed by RT-PCR. Innate immune receptors and the pro-inflammatory cytokine TNF-α were found to develop early in response to LPS and GMDP, both in vitro and in vivo. Genes of cytosolic proteins controlling inflammation (A20 and ATF3) were expressed later. Prior exposure of the innate immune system to LPS and muramyl peptides may modulate host defense against acute inflammation. As a result of the study, new data were obtained on dynamic changes in deubiquitinase A20 and the transcription factor ATF3, which are involved in the limitation and suppression of inflammatory reactions caused by fragments of bacterial cell walls-LPS and GMDP. Thus, bioregulators of bacterial origin LPS and GMDP, along with pro-inflammatory factors, activate the expression of genes that suppress inflammation, which should be considered when analyzing data from studies of the pro-inflammatory properties of LPS and GMDP and when developing drugs based on them.

Devising a multi epitope vaccine toward the dengue virus using the computational method in Bangladesh

Dengue is one of the major mosquito-borne diseases that still threaten humans and kill countless people. It is caused by a positive-stranded RNA virus that included the family of Flaviviridae. Dengue fever is an exquisite feverish viral disease dispatched by Aedes mosquitoes' sting, posing any one of the four dengue viral serotypes. This virus transmits through a vertical transmission way using a full unique system. Unfortunately, there is no still effective developed vaccine to eradicate this disease. Computational methods were used in this work to build and we suggest a multi-epitope vaccination for the dengue virus in Asia. This work employed different immunoinformatics and bioinformatics databases to predict powerful epitopes on the dengue virus envelope protein utilizing in silico techniques. We identified a total of 14 epitopes from the target envelope protein by evaluating their ability to induce both innate and acquired immunity via T and B lymphocyte-mediated reactions. Epitope conservation was taken into account because Dengue virus is an RNA virus, and all selected epitopes were 100% conserved. Antigenicity of the final multi-epitope vaccine component was 0.7055. Disulfide engineering was conducted at an area of high mobility to improve vaccine protein stability. In addition, codon adaptation and in silico cloning were used to guarantee that the planned subunit vaccine in E. coli was expressed at a greater level. Lastly, a molecular docking and simulation analysis was carried out for the vaccination protein and the TLR-4 receptor in order to assess the binding free energy and stability of the combination for this reason, the suggested in silico vaccine has to be tested for safety and immunogenicity in order to guarantee an active immunity against the Dengue virus.

Immunoinformatics design of multi-epitope vaccine using surface cell antigen OmpB and heat shock protein GroEL against rickettsioses

Rickettsioses, caused by intracellular bacteria of the rickettsia genus, pose a global health threat, particularly affecting underserved communities. Delays in diagnosis and treatment due to limited healthcare access result in severe cases and fatalities. To address this, a multi-epitope vaccine targeting OmpB and GroEL proteins in epidemic and endemic typhus rickettsia was developed using immunoinformatics techniques. T-cell and B-cell epitopes were predicted from OmpB and GroEL proteins, resulting in 5 CTL, 4 HTL, and 4 B-cell epitopes. These epitopes were linked appropriately and combined with suitable adjuvants to construct the vaccine. The vaccine's physicochemical properties and tertiary structure were validated within acceptable ranges. Molecular docking analysis demonstrated favorable binding with TLR4 receptors and MHC molecules, further confirmed by molecular dynamics simulation. Immune simulation analysis predicted T-cell, IFN, and IL-2 responses upon vaccine administration. The vaccine sequence was then optimized and cloned into a plasmid vector pET-23a(+) at the Hind and AlwNI restriction sites. In this in silico experiment, a multi-epitope vaccine derived from OmpB and GroEL proteins is presented, the first validation steps of which have been completed. Subject to successful in vivo studies, this vaccine could prove to be a promising therapeutic strategy against rickettsiosis and address a critical public health challenge.

Nicotinamide mononucleotides alleviated neurological impairment via anti-neuroinflammation in traumatic brain injury.

Traumatic brain injury (TBI) is one of the main factors of death and disability in adults with a high incidence worldwide. Nervous system injury, as the most common and serious secondary injury after TBI, determines the prognosis of TBI patients. NAD+ has been confirmed to have neuroprotective effects in neurodegenerative diseases, but its role in TBI remains to be explored. In our study, nicotinamide mononucleotides (NMN), a direct precursor of NAD+, was used to explore the specific role of NAD+ in rats with TBI. Our results showed that NMN administration markedly attenuated histological damages, neuronal death, brain edema, and improved neurological and cognitive deficits in TBI rats. Moreover, NMN treatment significantly suppressed activated astrocytes and microglia after TBI, and further inhibited the expressions of inflammatory factor. Besides, RNA sequencing was used to access the differently expressed genes (DEGs) and their enriched (Kyoto Encyclopedia of Genes and Genomes) KEGG pathways between Sham, TBI, and TBI+NMN. We found that 1589 genes were significantly changed in TBI and 792 genes were reversed by NMN administration. For example, inflammatory factor CCL2, toll like receptors TLR2 and TLR4, proinflammatory cytokines IL-6, IL-11 and IL1rn which were activated after TBI and were decreased by NMN treatment. GO analysis also demonstrated that inflammatory response was the most significant biological process reversed by NMN treatment. Moreover, the reversed DEGs were typically enriched in NF-Kappa B signaling pathway, Jak-STAT signaling pathway and TNF signaling pathway. Taken together, our data showed that NMN alleviated neurological impairment via anti-neuroinflammation in traumatic brain injury and the mechanisms may involve TLR2/4-NF-κB signaling.

Human lactoferrin modulates gene expression of the cytokine IL4 and the receptor TLR4 in the rat spleen under stress and upon the lipopolysaccharide administration

ABSTRACT Lactoferrin is a multifunctional glycoprotein of the transferrin family with a molecular mass of about 80 kDa. We studied the effect of human lactoferrin on stress- and lipopolysaccharide-induced changes in blood corticosterone levels, as well as on the gene expression of the cytokine IL4 and the pattern-recognition receptor TLR4 in rat splenocytes. Stress in rats was modeled by swimming in cold water (1-4°C) for 2 min. Lactoferrin and lipopolysaccharide (LPS) were administered intraperitoneally before the stress exposure. Corticosterone level in the plasma was determined by enzyme immunoassay, and changes in gene expression were assessed by real-time polymerase chain reaction (PCR) with reverse transcription. We showed that preventive intraperitoneal administration of lactoferrin reduced the stress and LPS-induced increase in the gene expression of both IL4 and TLR4 in rat splenocytes but did not change the concentration of corticosterone in the blood.

The study of NF-κB transcription factor activation by Pseudomonas aeruginosa recombinant proteins in eukaryotic cell culture.

The transcription factor NF-κB is a key factor in the activation of immune responses; it is in turn activated by pattern recognition receptors, such as TLR and NLR receptors. The search for ligands activating innate immunity receptors is an important scientific problem due to the possibility of their use as adjuvants and immunomodulators. In this study the effect of recombinant Pseudomonas aeruginosa OprF proteins and a toxoid (a deletion atoxic form of exotoxin A) on the activation of TLR4, TLR9, NOD1, and NOD2 receptors has been investigated. The study was carried out using free and co-adsorbed on Al(OH)₃ P. aeruginosa proteins and eukaryotic cells encoding these receptors and having NF-κB-dependent reporter genes. The enzymes encoded by the reported genes are able to cleave the substrate with the formation of a colored product, the concentration of which indicates the degree of receptor activation. It was found that free and adsorbed forms of the toxoid were able to activate the TLR4 surface receptor for lipopolysaccharide. OprF and the toxoid activated the intracellular NOD1 receptor, but only in the free form. This may be due to the fact that the cell lines used were not able to phagocytize aluminum hydroxide particles with protein adsorbed on them.

Post-synthetic benzylation of the mRNA 5' cap via enzymatic cascade reactions.

mRNAs are emerging modalities for vaccination and protein replacement therapy. Increasing the amount of protein produced by stabilizing the transcript or enhancing translation without eliciting a strong immune response are major steps towards overcoming the present limitations and improving their therapeutic potential. The 5' cap is a hallmark of mRNAs and non-natural modifications can alter the properties of the entire transcript selectively. Here, we developed a versatile enzymatic cascade for regioselective benzylation of various biomolecules and applied it for post-synthetic modification of mRNA at the 5' cap to demonstrate its potential. Starting from six synthetic methionine analogues bearing (hetero-)benzyl groups, S-adenosyl-l-methionine analogues are formed and utilized for N7G-cap modification of mRNAs. This post-synthetic enzymatic modification exclusively modifies mRNAs at the terminal N7G, producing mRNAs with functional 5' caps. It avoids the wrong orientation of the 5' cap-a problem in common co-transcriptional capping. In the case of the 4-chlorobenzyl group, protein production was increased to 139% during in vitro translation and to 128-150% in four different cell lines. This 5' cap modification did not activate cytosolic pathogen recognition receptors TLR3, TLR7 or TLR8 significantly more than control mRNAs, underlining its potential to contribute to the development of future mRNA therapeutics.

Immunomodulatory activity of polysaccharide from Trametes gibbosa (Pers.) Fr (Basidiomycota, Fungi) mediated by TLR4 signaling pathway

Background: Trametes species possess remarkable immunomodulatory and anticancer effects which are mainly related to the activation of innate immune receptors by their polysaccharide constituents. In this study, we investigate the effect of Trametes gibbosa (Pers.) Fr. polysaccharide fraction (TGP) on activation of TLR-4 receptor and subsequent release of IL-8 in HEK-Blue™ hTLR4 cells.Materials and Methods: The polysaccharide fraction was purified using ethanol precipitation and dialysis methods. The total sugar content and monosaccharide composition were analyzed by phenol–sulfuric acid and chromatographic methods. FT-IR spectroscopy was also performed for structure characterization of the polysaccharide. The activation of TLR4 was determined by measuring the secreted embryonic alkaline phosphatase in the culture media.Results: The results indicated that the total sugar content of TGP was about 90%, which glucose was the major constituents. FT-IR analysis showed the characteristic bands of polysaccharides. TGP was able to activate the TLR-4 signaling pathway in a dose-dependent manner. Moreover, the significant increase of IL-8 was observed in cells treating with TGP. The HEK-Blue Null2™ reporter cells lacking TLR4, did not respond to LPS and TGP.Conclusion: The results suggest that TLR4 signaling cascade serve as targets for immunomodulatory activity of T. gibbosa which could address the anticancer properties of Trametes species.

Prognostic value of TLR4 receptors in cerebrospinal fluid for formation of cerebral vasospasm and delayed cerebral ischemia

Prognostic value of TLR4 receptors in cerebrospinal fluid for formation of cerebral vasospasm and delayed cerebral ischemia

Insight into TLR4 receptor inhibitory activity via QSAR for the treatment of Mycoplasma pneumonia disease.

Mycoplasma pneumoniae (MP) is one of the most common pathogenic organisms causing upper and lower respiratory tract infections, lung injury, and even death in young children. Toll-like receptors (TLRs) play an important role in innate immunity by allowing the host to recognize pathogens invading the body. Previous studies demonstrated that TLR4 is a potential therapeutic target for the treatment of MP pneumonia. Therefore, the present study aimed to screen biologically active ingredients that target the TLR4 receptor pathway. We first used molecular docking to screen out the active compounds inhibiting the TLR4 pathway, and then used regression and classification machine learning algorithms to establish a quantitative structure-activity relationship (QSAR) model to predict the biological activity of the screened compounds. A total of 78 molecules were used in QSAR modelling, which were retrieved from the ChEMBL database. The QSAR models had acceptable correlation coefficients of R 2 on the training and testing dataset in the range of 0.96 to 0.91 and 0.93 to 0.76, respectively. The multiclass classification models showed accuracy on training and testing data within ranges of 1.0 to 0.70, 0.96 to 0.63, and log loss ranges from 0.27 to 8.63, respectively. In addition, molecular descriptors and fingerprints have been studied as structural elements involved in increased and decreased inhibitory activities. These results provide a quantitative analysis of QSAR and classification models applicable for high-throughput screening, as well as insights into the mechanisms of inhibition of TLR4 antagonists.

Study on the mechanism of Phellinus igniarius total flavonoids in reducing uric acid and protecting uric acid renal injury in vitro

BackgroundUric acid nephropathy (UN) is a complication of hyperuricemia (HUA), which has a great impact on people's lives. Here, we evaluated the therapeutic potential of total flavonoids of Phellinus igniarius (TFPI) in vivo and studied the anti UN effect of TFPI in vitro. MethodsHyperuricemia was induced by intraperitoneal injection of potassium oxonate in ICR mice. After intervention with TFPI, we evaluated the levels of serum uric acid (UA) and creatinine (CR), and the contents of xanthine oxidase (XOD) and adenosine deaminase (ADA) in liver. To explore the effect and molecular mechanism of TFPI on UN, we treated HK-2 cells with monosodium urate (MSU) to study the effect of TFPI on apoptosis and inflammation. In addition, to explore the mechanism of TFPI on uric acid transport we evaluated the relationship between uric acid transporter ABCG2 and inflammatory signaling pathway TLR4-NLRP3. ResultsIn the model mice, TFPI significantly decreased the levels of UA and Cr, which may be related to the inhibition of XOD enzyme activity. In HK-2 cells, the response of TFPI to MSU can effectively inhibit apoptosis and activation of TLR4-NLRP3 signaling pathway and promote the expression of ABCG2. ConclusionsTFPI can significantly inhibit the release of inflammatory factors and promote the expression of ABCG2 by targeting TLR4 receptor and NLRP3 inflammasome. And targeted inhibition of XOD enzyme activity to reduce uric acid level and inhibit the development of UN.

AW-NI-2: A HIGH-FIBRE DIET ATTENUATES HYPERTENSION BY IMPROVING GUT BARRIER INTEGRITY VIA SCFA-RECEPTOR SIGNALLING

Objective: The gut microbiome has been recently discovered to play a role in blood pressure (BP) regulation. Short-chain fatty acids (SCFAs), produced from the fermentation of fibre by the gut microbiota, are protective in experimental hypertension models. However, whether the protective effects of fibre are dependent on SCFA-receptor (GPR41, GPR43) signalling is not known. This is relevant as hypertensive patients have a deficiency in GPR43. We aimed to determine whether SCFA-receptor signalling is relevant in hypertension, whether the protective effects of a high-fibre diet depend on SCFA-receptor signalling, and the underlying mechanisms involved. Design and Methods: We induced hypertension using angiotensin II (Ang-II, 0.5 mg/kg/day) in wild-type and the novel GPR41/43 double knockout (DKO) male mice (n = 9–15). We fed wild-type and DKO mice high-fibre (n = 6–7) or standard diets (n = 9–15). Another cohort received TLR4 antagonist treatment (n = 6–7). We measured BP by tail-cuff, performed Mason trichrome staining for fibrosis in the heart and kidney, and immunofluorescence staining for zonulin-1, a key tight junction protein to determine gut permeability, in the colon. Results: When fed a standard diet and challenged with Ang-II, DKO mice had significantly higher BP, mortality, cardiorenal hypertrophy and fibrosis, and lower zonulin-1 in the colon compared to wild-type mice (all P < 0.05). A high-fibre diet partially protected DKO mice, resulting in significantly lower BP, mortality, cardiorenal hypertrophy, and fibrosis (all P < 0.05). Mice fed high-fibre had significantly higher zonulin-1 expression in the colon (P < 0.05), meaning that a high-fibre diet improved gut barrier integrity. This suggests that the protective effects of high-fibre partially depend on GPR41/GPR43 signalling. Hypertensive wild-type and DKO mice had significantly higher plasma TLR4 activation suggesting its ligand, microbial lipopolysaccharides (LPS), is in the circulation (P < 0.05). TLR4 activation leads to inflammation and subsequent tissue damage that could be responsible for the phenotype observed. Thus, we treated hypertensive DKO and wild-type mice with a TLR4 antagonist (i.p., injection of 50 μg/kg/body weight twice weekly). The severity of DKO hypertensive mice was rescued to the level of WT when treated with the TLR4 antagonist - no difference from wild-type mice in terms of BP, mortality, organ weights, and fibrosis. Conclusions: We revealed for the first time that a high-fibre diet attenuates hypertension by improving gut barrier integrity via the SCFA-receptors GPR41 and GPR43. This leads to lower translocation of harmful microbial substances such as LPS from the gut and activation of its receptor TLR4 to induce inflammatory responses that promote hypertension.

Trypanosoma brucei Lipophosphoglycan Activates Host Immune Responses via the TLR-mediated p38 MAP Kinase and NF-κB Pathways

Objective: This study was aimed at investigating the immunoregulatory effects of trypanosomal lipophosphoglycan (LPG) anchored to trypanosome membranes, including the formation of neutrophil extracellular traps (NETs) and neutrophil cytokine release after parasite infection. The interaction of cell surface TLR receptors with LPG, which signals cellular responses during Trypanosma brucei infection, was systematically investigated. Methods: The cytokine expression profile in neutrophils after exposure to T. brucei LPG, and the involvement of TLR2, TLR4, p38 MAP kinase, and NF-κB in NET formation were studied with molecular immunological approaches including quantitative PCR, western blotting and immunofluorescence. Results: T. brucei-derived LPG induced phosphorylation of p38 MAP kinase and NF-κB, thereby stimulating neutrophil secretion of IL-1β, IL-8, and TNF-α. The blockade of Toll-like receptor 2/4 and specific inhibitors of MyD88, p38 MAP kinase, and NF-κB decreased cytokine release and the phosphorylation of both kinases. Furthermore, the exposure of neutrophils containing LPG to IL-1β and LPG-induced cell supernatants promoted the release of NETs. Conclusion: Our findings suggest that T. brucei LPG activates neutrophil IL-1β secretion via the TLR-mediated p38 MAP kinase and NF-κB pathways, thereby promoting the formation of LPG-stimulated NETs.

Neutrophil Extracellular Traps Delay Diabetic Wound Healing by Inducing Endothelial-to-Mesenchymal Transition via the Hippo pathway.

Diabetic foot ulcers (DFUs) are among the most frequent complications of diabetes with significant morbidity and mortality. Diabetes can trigger neutrophils to undergo histone citrullination by protein arginine deiminase 4 (encoded by Padi4 in mice) and release neutrophil extracellular traps (NETs). The specific mechanism of NETs-mediated wound healing impairment in diabetes remains unknown. In this study, we show neutrophils are more susceptible to NETosis in diabetic wound environments. Via in vitro experiments and in vivo models of wound healing using wide-type and Padi4 -/- mice, we demonstrate NETs can induce the activation of PAK2 via the membrane receptor TLR-9. Then PAK2 phosphorylates the intracellular protein Merlin/NF2 to inhibit the Hippo-YAP pathway. YAP binds to transcription factor SMAD2 and translocates from the cytoplasm into the nucleus to promote endothelial-to-mesenchymal transition (EndMT), which ultimately impedes angiogenesis and delays wound healing. Suppression of the Merlin/YAP/SMAD2 pathway can attenuate NET-induced EndMT. Inhibition of NETosis accelerates wound healing by reducing EndMT and promoting angiogenesis. Cumulatively, these data suggest NETosis delays diabetic wound healing by inducing EndMT via the Hippo-YAP pathway. Increased understanding of the molecular mechanism that regulates NETosis and EndMT will be of considerable value for providing cellular targets amenable to therapeutic intervention for DFUs.

Synthesis and immunopharmacological evaluation of novel TLR7 agonistic triazole tethered imidazoquinolines.

Toll-like receptors-7 and -8 are expressed abundantly on antigen-presenting cells, and their agonists make potential adjuvant candidates for the development of new efficacious vaccines. In view of the importance of new efficacious imidazoquinoline based adjuvants, herein we have synthesized a focused library of a new class of imidazoquinolines retaining the N-isobutyl substitution of an imidazole moiety as in imiquimod and introduced a 1,2,3-triazolyl moiety upon alkyl substitution at the imidazolemethyne carbon employing triazolyl click chemistry. All the novel analogues were characterized using various spectroscopic techniques and the target specificity of these molecules was determined using HEK TLR7/8 transfected cell lines. TLR7/8 activity and also the molecular docking results correlated primarily to the position of the substituent for aromatic groups and also to the chain length in alkyl substitutions. The immunomodulatory properties of these analogues were evaluated using murine DC activation and also with hPBMC activation markers, cytokines which revealed that these analogues after modification were able to target the TLR7 receptors and also had a pro-inflammatory immune response.

Inhibitory effects of Ephedra alte on IL-6, hybrid TLR4, TNF-α, IL-1β, and extracted TLR4 receptors: in silico molecular docking

Inflammation is a physiological reaction of the immune system required to remove the presence of pathogenic germs. Many herbal-derived extracts and phytoconstituents show anti-inflammatory effects. Among these natural phytoconstituents is Ephedra alte (E. alte), which shows pepsin enzyme inhibitory, antibacterial, and antioxidant activities. In this work, molecular docking study is conducted on five major human anti-inflammatory cytokines receptors (IL-6, hybrid TLR4, TNF-α, IL-1β, and extracted TLR4) to explore the molecular recognition process and complex ligand-receptor interactions of E. alte phytoconstituents. Human TLR4 receptor has been computationally extracted, for the first time, from the hybrid TLR4 human and VLRB inshore hagfish. Among E. alte phytoconstituents, only β-Sitosterol and Androstan-3-one have better LBE (Lowest Binding Energy) scores with inhibition constant (Ki) values than those of other tested compounds. The β-Sitosterol and Androstan-3-one results indicate that these compounds could be efficient inhibitors of inflammation and reduce the oxidative stress by interfering with the activity of the five studied proteins.

Microglial subpopulations with distinct transcriptome signatures vary across brain regions in the resting mouse brain

Microglia are crucial for tissue homeostasis and its disturbance. However, microglial heterogeneity and its relationship with microglial activation in physiological conditions remain elusive. Using single-cell RNA sequencing, we identified microglial subpopulations with distinct transcriptome signatures in the resting brain. The distribution of two major, continuous subpopulations varied across brain regions, especially between cerebral cortices and the hypothalamus. Lipopolysaccharide and chronic social defeat stress, both of which involve the innate immune receptor TLR4, upregulate the marker genes of selective microglial subpopulations. These findings suggest that microglial subpopulations contribute to the heterogeneity of microglial transcriptome and responsiveness within and across brain regions.

The Effect of CD300A Receptor on Caspase-1 Activity in the Context of Cell Death and on Its Activators Nlrp3 and Asc in Sertoli Cells

The phagocytosis of spermatozoa cytoplasm by Sertoli cells is a key maturation event during spermiation and takes place in a blood-testis barrier maintained immune-privileged environment that is essential for the prevention of the sperm self-antigens from presentation. We have recently discovered Nlrp3 inflammasome to be functional in Sertoli cells upon TLR4 and NOD receptors challenge in ATP danger presence, resulting in release of pro-inflammatory cytokines and cell death that could potentially result in breaking of the selftolerance. In this context we have investigated, the considered inhibitory, phagocytic receptor CD300a that is able to detect eventually sperm cell surface apoptotic signals. We found CD300a to be essential for Nlrp3 inflammasome activation via CD300a-dependent Asc expression and both caspase-1 and caspase-3 activation, since its gene silencing abrogated these events. At the same time CD300a demonstrated bi-directional regulatory abilities, supressing Gasdermin D inflammasome effector pro- and activated forms. CD300a had a restrictive effect on cell population level reducing caspase positive cells in strong caspase-1 and 3, and weak caspase-3 expressing dead cells, suggesting key regulatory role in Sertoli cell faith. CD300a should be further explored as its potential perturbation could play a role in putative self-antigen-toleranceperturbation and thus contribute to male infertility.