TLR4 Receptor Research Articles

Immunogenic multi-epitope-based vaccine development to combat cyclosporiasis of immunocompromised patients applying computational biology method.

Cyclospora cayetanensis infections, also known as cyclosporiasis, persist to be the prevalent emerging protozoan parasite and an opportunist that causes digestive illness in immunocompromised individuals. In contrast, this causal agent can affect people of all ages, with children and foreigners being the most susceptible populations. For most immunocompetent patients, the disease is self-limiting; in extreme circumstances, this illness can manifest as severe or persistent diarrhea as well as colonize on secondary digestive organs leading to death. According to recent reports, worldwide 3.55% of people are infected by this pathogen, with Asia and Africa being more prevalent. For the treatment, trimethoprim-sulfamethoxazole is the only licensed drug and does not appear to work as well in some patient populations. Therefore, the much more effective strategy to avoid this illness is immunization through the vaccine. This present study uses immunoinformatics for identifying a computational multi-epitope-based peptide vaccine candidate for Cyclospora cayetanensis. Following the review of the literature, a highly efficient, secure, and vaccine complex based on multi-epitopes was designed by utilizing the identified proteins. These selected proteins were then used to predict non-toxic and antigenic HTL-epitopes, B-cell-epitopes, and CTL-epitopes. Ultimately, both a few linkers and an adjuvant were combined to create a vaccine candidate with superior immunological epitopes. Then, to establish the vaccine-TLR complex binding constancy, the TLR receptor and vaccine candidates were placed into the FireDock, PatchDock, and ClusPro servers for molecular docking and iMODS server for molecular-dynamic simulation. Finally, this selected vaccine construct was cloned into Escherichia coli strain-K12; thus, the constructed vaccines against Cyclospora cayetanensiscould improve the host immune response and can be produced experimentally.

Mkp-1 deficiency strengthens immune defense against fungal infection

Abstract Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1−/− mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1−/− mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1−/− mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1−/− bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1−/− BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens. *The studies were supported by NIH grants (AI113930 and AI124029 to Y.L., and AI121196 and AI123253 to J.Z.).

Spinal adenosine 2A receptor inhibition restores phrenic long-term facilitation during mild systemic inflammation

Inflammation undermines several forms of neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by moderate acute intermittent hypoxia (mAIH; 3, 5-min episodes, 5 min intervals; arterial PO2 ~ 40–50 mmHg). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist lipopolysaccharide (LPS; 100 mg/kg, i.p.) abolishes mAIH-induced pLTF by a mechanism that requires spinal p38 MAP kinase and protein phosphatase activity. Neuroinflammation activates microglia and other cells in the CNS, elevating extracellular levels of adenosine—a purine nucleoside known to suppress mAIH-induced pLTF. We hypothesized basal adenosine levels in ventral spinal C 3 -C 5 segments are increased in adult male Sprague-Dawley rats 24 hours post-LPS administration, thereby undermining mAIH-induced pLTF via adenosine 2A receptor (A 2A ) activation. Thus, we predicted that cervical intrathecal administration of the A 2A receptor inhibitor MSX-3 (10 μM, 12 μL) would rescue mAIH-induced pLTF in rats pre-treated with LPS. As predicted, LPS significantly increased basal ventral C 3 -C 5 adenosine levels (p=0.010 vs controls; n=7 per group). In control rats (saline injection, i.p.), MSX-3 enhanced pLTF versus vehicle controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; p=0.002; each n=6). As expected, mAIH-induced pLTF was abolished in LPS-treated rats (4 ± 6% baseline; n=6). MSX-3 restored mAIH-induced pLTF to levels equivalent to MSX-3 treated control rats (120 ± 14%; p<0.001; n=6; versus LPS controls with MSX-3: p=0.539). Thus, mild LPS-induced inflammation abolishes mAIH-induced pLTF by a mechanism associated with increased spinal adenosine levels and A 2A receptor activation. As we move mAIH forward as a therapeutic modality to improve breathing and non-respiratory movements in people with spinal cord injury or ALS, pretreatment with A 2A receptor inhibitors may offset the undermining effects of systemic inflammation associated with these severe neuromuscular disorders. NIH HL147554, HL148030, T32HL134621 (ABM) and the UF McKnight Brain Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 101: Crosstalk Of Pluripotency And Innate Immunity In Smooth Muscle Cell Phenotypic Transitions

Objectives: Smooth muscle cell ( SMC ) phenotypic transitions play a critical role in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms responsible for these transitions are not well understood. We previously found that the embryonic stem cell/pluripotency factor OCT4 is reactivated in SMC in human and mouse atherosclerotic plaques and plays an atheroprotective role by regulating SMC investment into the protective fibrous cap. Loss of OCT4 in SMC is associated with SMC de-differentiation toward a macrophage-like phenotypic state resulting in increased lipid accumulation and phagocytosis. However, the endogenous molecular mechanism responsible for atheroprotective SMC-OCT4-dependent effects remains unknown. Methods and Results: Herein, we performed combinatory bioinformatics analyses on the in vivo and in vitro wild-type and SMC- Oct4 knockout mouse samples and found that loss of Oct4 in SMC resulted in up-regulation of the innate immune receptor TLR4 in numerous experimental conditions. Notably, the TLR4 promoter does not have any putative OCT4 binding sites. First - Using publicly available OCT4 ChIPseq datasets and long non-coding ( lnc )RNA databases, we identified several lncRNAs, including IGF2AS, MALAT1, PLATR10, BACE1, and SRA based on the OCT4 occupancy at promoter and enhancer regions of lncRNA, which in turn, directly target TLR4. To confirm the OCT4-dependent activation of the identified lncRNAs in SMC, qRT-PCR was performed in mouse aortic SMC transfected with Oct4 -blocking siRNA. We found that the Oct4 knockdown in SMC led to significant decreases in all five selected lncRNAs and increases in TLR4 and its downstream gene, TRIF. Second - Intriguingly, we also demonstrated that TLR4 signaling, in turn, promotes hydroxymethylation of the OCT4 promoter and reactivates OCT4 in SMC. Third - Using a Tlr4 -reporter and SMC-lineage tracing mouse model, we also demonstrated that medial differentiated SMC from healthy arteries, but not phenotypically modulated lesion SMC, express TLR4. Conclusions: Together, these results indicate a role of TLR4-signaling in SMC both upstream and downstream of OCT4, suggesting that pluripotency/innate immunity crosstalk plays a critical role in SMC phenotypic transitions.

Staphylococcus aureus increases Prostaglandin E2 secretion in cow neutrophils by activating TLR2, TLR4, and NLRP3 inflammasome signaling pathways.

In clinical settings, dairy cows are often attacked by pathogenic bacteria after delivery, especially Staphylococcus aureus (S. aureus). Neutrophils have long been regarded as essential for host defense against S. aureus. Prostaglandin E2 (PGE2) can additionally be used as an inflammatory mediator in pathological conditions to promote the repair of inflammatory injuries. However, whether S. aureus can promote the accumulation of PGE2 after the infection of neutrophils in cows and its mechanism remain unclear. Lipoprotein is an important immune bioactive ingredient of S. aureus. In this study, the changes in neutrophils were monitored in dairy cows infected with wild-type S. aureus (SA113) and an S. aureus lipoprotein-deficient strain (Δlgt); meanwhile, we established whether pattern recognition receptors mediate this process and whether S. aureus lipoproteins are necessary for causing the release of PGE2 from cow neutrophils. The results showed that Δlgt was less effective than SA113 in inducing the production of IL-1β, IL-6, IL-8, IL-10, and PGE2 within neutrophils; furthermore, TLR2, TLR4, and NLRP3 receptors were found to mediate the inducible effect of lipoprotein on the above inflammation mediators and cytokines, which depended on MAPK and Caspase-1 signaling pathways. In addition, TLR2, TLR4, and NLRP3 inhibitors significantly inhibited PGE2 and cytokine secretion, and PGE2 was involved in the interaction of S. aureus and neutrophils in dairy cows, which could be regulated by TLR2, TLR4, and NLRP3 receptors. We also found that S. aureus was more likely to be killed by neutrophils when it lacked lipoprotein and TLR2, TLR4, and NLRP3 were involved, but PGE2 seemed to have no effect. Taken together, these results suggest that lipoprotein is a crucial component of S. aureus in inducing cytokine secretion by neutrophils as well as killing within neutrophils, which could be accomplished by the accumulation of PGE2 by activating MAPK and the Caspase-1 signaling pathways through TLR2, TLR4, and NLRP3 receptors. These results will contribute to a better understanding of the interaction between S. aureus and host immune cells in dairy cows.

Chitin oligomers promote lymphoid innate and adaptive immune cell activation.

Chitin is a highly abundant N-acetylglucosamine polysaccharide that has been linked to immune responses in the context of fungal infections and allergic asthma, especially to T helper 2 immune responses. Unfortunately, due to the frequent use of crude chitin preparations of unknown purity and degree of polymerization, there is still great uncertainty about how chitin activates different parts of the human immune system. We recently identified chitin oligomers of 6 N-acetylglucosamine units as the smallest immunologically active chitin motif and the innate immune receptor TLR2 as a primary chitin sensor on human and murine myeloid cells, but the response of further immune cells (e.g. lymphoid cells) to oligomeric chitin has not been investigated. Our analysis of primary human immune cells now shows that chitin oligomers activate immune responses of both innate and adaptive lymphocytes: notably, chitin oligomers activated natural killer cells but not B lymphocytes. Moreover, chitin oligomers induced maturation of dendritic cells and enabled potent CD8+ T-cell recall responses. Our results suggest that chitin oligomers not only trigger immediate innate responses in a limited range of myeloid cells but also exert critical activities across the entire human immune system. This highlights chitin oligomer immune activation as an interesting and broadly applicable potential target for both adjuvant development and therapeutic interference in chitin-mediated pathologies.

924 Involvement of keratinocyte TLR2 receptor during fungal infection of the skin

924 Involvement of keratinocyte TLR2 receptor during fungal infection of the skin

Abstract LB028: Reprogramming of tumor-infiltrating immune cells using a tumor-associated macrophage-targeted TLR7 agonist

Abstract Myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are two of the more immunosuppressive cell types in a tumor microenvironment (TME). Although TAMs and MDSCs can be converted from tumor-supporting to tumor-suppressing phenotypes by treatment with proinflammatory immune stimulants (e.g. TLR7 agonists) in vitro, such strategies have proven to be too toxic in vivo because of their systemic activation of immune cells. Therefore, for such immune stimulants to be used to reprogram the immune system in solid tumors, they must be targeted specifically to the immune cells in the TME. One strategy for targeting TAMs/MDSCs is to exploit a receptor that is expressed solely on these cell types. Because folate receptor beta (FRβ) is expressed exclusively on myeloid cells and since FRβ acquires its ability to bind folic acid (FA) only in inflamed tissues, we have previously exploited FA to target attached TLR7 agonists (TLR7a) to FRβ+ TAMs/MDSCs in solid tumors. While significant repolarization of TAMs and MDSCs was observed in this previous study, premature systemic release of the attached TLR7a caused unwanted toxicity. However, since FRβ was later found to reside in the same endosome as TLR7, we hypothesized that constructing a more stable FA-TLR7a conjugate with a non-cleavable linker should not only prevent premature TLR7a release, but also deposit the FA-TLR7a into an endosome containing a TLR7 receptor, thereby enabling selective activation of TAMs and MDSCs in the TME. The data summarized here confirm this hypothesis. Thus, free TLR7-1A was found to stimulate massive production of IL-6, TNF-α and IFNγ upon addition to human peripheral blood in vitro or injected into live mice, suggesting cells in both species respond aggressively to TLR7-1A. However, performance of the same studies with FA-targeted TLR7-1A stimulated barely or nondetectable levels of the inflammatory cytokines, confirming that FA-TLR7-1A is unable to enter and activate cells that lack a folate receptor (FRβ). More importantly, intravenous administration of FA-TLR7-1A into mice bearing an orthotopic 4T1 breast tumor totally blocked metastases and induced tumor regression without causing detectable toxicity. Flow cytometric analyses of the residual tumor tissue further demonstrated that FA-TLR7-1A increased M1/M2 macrophage ratios and elevated infiltration of CD8+ T cells into the tumor masses. Taken together, these data demonstrate that a targeted TLR7 agonist can rejuvenate immune cells in a solid tumor without systemically activating the immune system. Citation Format: John Victor Napoleon, Fenghua Zhang, Rami A. Alfar, Taylor A. Wolt, Philip S. Low. Reprogramming of tumor-infiltrating immune cells using a tumor-associated macrophage-targeted TLR7 agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB028.

Histological Properties of Placentas in Acute Herpes Simplex Virus Infection Affecting Genitals

Acute herpes simplex virus infection in pregnant women results in intrauterine infection of the fetus in 45–50% of cases, thus being the cause of septic complications and severe somatic and neurological disorders in the newborn.The aim of the study was to investigate the placentas of new mothers with newly diagnosed herpes simplex virus type 2 infection (HSV-2) using histological and immunochemical methods.Material and methods. This histological study included 10 placentas of new mothers after operative delivery by caesarean section with newly diagnosed HSV-2 infection. The comparison group (control) consisted of 10 placentas of women with a physiological pregnancy. Histological sections of the placenta were stained with hematoxylin and eosin, Giemsa's solution and Picro-Mallory staining. Identification of the pro- and antiinflammatory phenotype of macrophages (CD68, CD163), subpopulations of CD4 and CD8 lymphocytes, TLR4 receptor expression was performed by immunohistochemical method using specific antibodies. Quantification of the cell population and TLR4 receptors was performed morphometrically. The data obtained were statistically analysed using MS Excel (2016) and SPSS Statistics 17.0.Results. Morphological manifestations of acute herpes simplex virus infection were combined inflammatory and compensatory in nature in the third trimester of pregnancy. The histological picture of the fetal part of the placenta demonstrated a decreased number of M2 (antiinflammatory) macrophages and an increased number of M1 (pro-inflammatory) macrophages. The imbalance between CD163+ and CD68+ profiles of placental macrophages with the numerical predominance of the latter evidenced a pronounced cellular immune response; this, in turn, was supported by intense TLR4 immunopositive staining of the fetal part of the placenta. There was perivillous fibrin deposition of varying degrees, villus agglutination, and necrosis of trophoblast cells associated with a relatively small population of CD4+ and CD8+. Villitis of viral etiology was characterized by destructive infiltration of maternal CD8+ T-lymphocytes penetrating the chorionic villi, combined with activated macrophages in the villi of the fetal part of the placenta. Modifications in the proportion of immune cells during HSV-2 infection supported cytotoxic and autoimmune reactions in the placenta in response to HSV-2 introduction.Conclusion. The results obtained evidence the development of a pronounced immunopathological process in the tissues of the fetal part of the placenta, which results in the decreased protective and compensatory properties of the placenta and increases risk of perinatal complications.

Abstract 6370: Cell-based luminescent reporter bioassays for immunotherapies targeting macrophage effector functions

Abstract Macrophages play a key role in the elimination of cancer via phagocytosis and presentation of tumor antigens to T cells. Antibody-dependent cellular phagocytosis (ADCP), mediated by macrophages and other myeloid cells, is an important mechanism-of-action (MoA) for antibody-based cancer immunotherapies. Therapeutic strategies that enhance ADCP and other macrophage effector functions can augment direct tumor destruction and enable anti-tumor immunity. ADCP is initiated by binding of antibody Fc domains to Fc gamma receptors (FcgRs), and FcgR crosslinking results in signal transduction that drives inflammatory cytokine production and phagocytosis. Simultaneously, macrophage effector functions are regulated by immune checkpoints. Blockade of these immune checkpoints (e.g., SIRPa, ILT4), or agonists of activation pathways (e.g., TLR receptors) can enhance macrophage effector functions. Current assays to measure macrophage function are tedious, low throughput, and highly variable. Here, we present a suite of cell-based luminescent reporter bioassays to measure macrophage effector functions: 1) A monocytic cell background was used to measure ADCP via endogenous expression of multiple FcgRs and integration of a FcgR-activation dependent luminescent reporter. 2) Direct detection of cellular phagocytosis can be measured using the HiBiT split luciferase system. 3) Modulation of macrophage-expressed immune checkpoints can be determined using cell-based reporter bioassays for SIRPa/CD47 and ILT4/HLA-G. 4) Activation of TLRs 1, 2, 4, 5 and 6 can be measured using a TLR reporter bioassay. These bioassays are robust, easy-to-use, and are pre-qualified according to ICH guidelines. Together, these novel reporter bioassays provide a robust, high-throughput toolbox to facilitate discovery and development of immunotherapies targeting macrophage effector functions. Citation Format: Jonathan Mitchell, Julia Gilden, Jim Hartnett, Pete Stecha, Gopal B. Krishnan, Frank Fan, Mei Cong, Jamison Grailer. Cell-based luminescent reporter bioassays for immunotherapies targeting macrophage effector functions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6370.

Abstract 2498: Melanoma cell -derived extracellular vesicles - immunomodulators of the tumor microenvironment

Abstract The role of extracellular vesicles (EVs) in tumor-stroma communication has been under extensive investigation, and their contribution to inflammation and immunosuppression, angiogenesis and metastasis has been demonstrated. However, the exact molecular mechanisms and the EV cargos responsible for mediating these effects are still partly unknown. In tumor microenvironment, macrophages are one of the most abundant cell types and they are composed of several distinct populations that share features of both pro-inflammatory (M1) and immunosuppressive (M2) macrophages. In this study, we investigated the effects of melanoma cell secreted EVs on macrophage polarization. In several cancers hyaluronan synthesis is elevated and overexpression of its producing enzyme, hyaluronan synthase 3 (HAS3), induces EV shedding from the plasma membrane. These HAS3 induced EVs contain a hyaluronan coat around the vesicles. To elucidate the effects of HAS3-induced EVs on target cells, THP1 monocytes were treated with EVs derived from EGFP-HAS3 overexpression MV3 melanoma cells, or from parental MV3 cells, and the expression of cytokines and other factors related in cancer -induced immune modulation was studied. EVs were isolated with ultracentrifugation and characterized with Nanoparticle tracking analysis (NanoSight NS300) and transmission electron microscopy. THP1 cells were activated with PMA for 48 h and thereafter cells were treated with either control EVs from MV3 cells or hyaluronan coated EVs from HAS3 overexpressing cells for 4-24 h. The expression of pro-inflammatory and immunosuppressive markers was studied with qPCR, and the effects on cell morphology with confocal microscopy. The size distribution of EVs derived from MV3 and HAS3-MV3 cells was relatively similar, being approximately 100-200 nm in range, while the particle concentration (/ml) was higher in samples derived from HAS3-MV3 cells. The expression of proinflammatory cytokines and chemokines, like IL-1β and IL-8, and immunosuppressive factors like TSG-6 and PD-L1 was elevated in macrophages treated with MV3 melanoma cell secreted EVs. The effect on expression of the studied factors was more obvious when cells were treated with hyaluronan coated EVs (HAS3-MV3 EVs). This indicates that either the hyaluronan coat around the EVs or their cargo is different compared to control EVs, which causes the effects. Inhibiting TLR4 receptor decreased the cytokine expression induced by HAS3-MV3 EVs, suggesting TLR4-mediated activation after HAS3-MV3 EV exposure. Our results suggest that melanoma cell derived EVs induce mixed M1/M2 polarization of THP1 monocytes. Citation Format: Sanna Marika Pasonen-Seppänen, Piia Takabe, Kirsi Kainulainen, Kaisa Mäki-Mantila, Riku Korkiamäki, Silvia Lopez Borrego, Taija Hukkanen, Niina Aaltonen, Kirsi Rilla. Melanoma cell -derived extracellular vesicles - immunomodulators of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2498.

Reduced IL-8 Secretion by NOD-like and Toll-like Receptors in Blood Cells from COVID-19 Patients

Severe inflammatory responses are associated with the misbalance of innate and adaptive immunity. TLRs, NLRs, and cytokine receptors play an important role in pathogen sensing and intracellular control, which remains unclear in COVID-19. This study aimed to evaluate IL-8 production in blood cells from COVID-19 patients in a two-week follow-up evaluation. Blood samples were taken at admission (t1) and after 14 days of hospitalization (t2). The functionality of TLR2, TLR4, TLR7/8, TLR9, NOD1, and NOD2 innate receptors and IL-12 and IFN-γ cytokine receptors was evaluated by whole blood stimulation with specific synthetic receptor agonists through the quantification of IL-8, TNF-α, or IFN-γ. At admission, ligand-dependent IL-8 secretion was 6.4, 13, and 2.5 times lower for TLR2, TLR4, and endosomal TLR7/8 receptors, respectively, in patients than in healthy controls. Additionally, IL-12 receptor-induced IFN-γ secretion was lower in COVID-19 patients than in healthy subjects. We evaluated the same parameters after 14 days and observed significantly higher responses for TLR2, TLR4, TLR7/8, TLR9, and NOD1, NOD2, and IFN-γ receptors. In conclusion, the low secretion of IL-8 through stimulation with agonists of TLR2, TLR4, TLR7/8, TLR9, and NOD2 at t1 suggests their possible contribution to immunosuppression following hyperinflammation in COVID-19 disease.

Immunological Study of Orthodontically Treated Patients Recovering from COVID-19 in Babylon Province, Iraq

Background and Objectives: This study aimed to examine the levels of the toll-like receptors TLR4 and TLR2 in the blood and saliva of patients with coronavirus disease-2019 (COVID-19) receiving orthodontic care in Babylon Province. Materials and Methods: Sixty serum and saliva samples were collected and divided into three groups of 20 patients: patients with COVID-19 who did not receive orthodontic treatment (group 1); patients with COVID-19 who received orthodontic treatment (group 2); and patients with COVID-19 who received orthodontic treatment and recovered (group 3). Thirty samples of serum and saliva from healthy outpatient clinics comprised the control group. Enzyme-linked immunosorbent assay was used to determine the result. Results: TLR4 levels in saliva increased in all experimental groups (1–3) as compared with the control group. However, only COVID-19 patients and orthodontic patients in group 2 had elevated blood levels of TLR4, whereas the levels were reduced in the other two groups (1 and 3). All experimental groups showed an increase in TLR2 levels in saliva relative to the control group. In the first and second experimental groups, serum TLR2 concentrations increased dramatically, whereas they declined in the third group. There was no correlation between TLR4 and TLR2 in either group of patients. The concentrations of TLR4 and TLR2 in saliva were higher in the experimental groups than in the control group.

The roles of toll-like receptor 4, CD33, CD68, CD69, or CD147/EMMPRIN for monocyte activation by the DAMP S100A8/S100A9.

The S100A8/A9 heterocomplex is an abundant damage-associated molecular pattern and mainly expressed by monocytes, inflammatory activated keratinocytes and neutrophilic granulocytes. The heterocomplex as well as the heterotetramer are involved in a variety of diseases and tumorous processes. However, their detailed mode of action and especially which receptors are involved hereby remains to be fully revealed. Several cell surface receptors are reported to interact with S100A8 and/or S100A9, the best studied being the pattern recognition receptor TLR4. RAGE, CD33, CD68, CD69, and CD147, all of them are involved as receptors in various inflammatory processes, are also among these putative binding partners for S100A8 and S100A9. Interactions between S100 proteins and these receptors described so far come from a wide variety of cell culture systems but their biological relevance in vivo for the inflammatory response of myeloid immune cells is not yet clear. In this study, we compared the effect of CRISPR/Cas9 mediated targeted deletion of CD33, CD68, CD69, and CD147 in ER-Hoxb8 monocytes on S100A8 or S100A9 induced cytokine release with TLR4 knockout monocytes. Whereas deletion of TLR4 abolished the S100-induced inflammatory response in monocyte stimulation experiments with both S100A8 and S100A9, knockouts of CD33, CD68, CD69, or CD147 revealed no effect on the cytokine response in monocytes. Thus, TLR4 is the dominant receptor for S100-triggered inflammatory activation of monocytes.

Effects of rhamnogalacturonan-I type polysaccharide purified from Curcuma longa on immunostimulatory and intracellular signaling pathway mechanisms of macrophages

In previous study, potent anti-metastatic activities of turmeric-derived pectic polysaccharide (TP) via immune enhancing effects of intravenous and oral administration were evaluated. The present study was designed to elucidate intracellular signaling pathway for macrophage activation by the rhamnogalacturonan-I type polysaccharide (TPE-I) purified from TP. TPE-I increased the production of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO) by RAW264.7 cells. In addition, immunoblotting analysis indicated that TPE-I dose-dependently phosphorylated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways. Subsequently, TPE-I induced the translocation of activated NF-κB into the nuclei, which was observed by confocal laser scanning microscopy. In the specific inhibitor experiments, cytokines and chemokine secretions were found to be suppressed by BAY11-7082, JNK inhibitor II, and SB20358. Furthermore, the experiments with neutralizing antibodies showed that pattern recognition receptors, such as dectin-1, toll-like receptor (TLR)2, TLR4, cluster of differentiation (CD)14, complement receptors (CR)3, mannose receptor (MR), and scavenger receptor (SR) were involved in cytokine and chemokine secretions by TPE-I in RAW264.7 cells. Consequently, these results suggest that TPE-I phosphorylated the MAPK and NF-κB signaling pathways via mainly the dectin-1, TLR2, TLR4, CD14, CR3, MR, and SR receptors to macrophage, and subsequently induced the secretion of IL-6, TNF-α and NO.

Construction and validation of a multi-epitope in silico vaccine model for lymphatic filariasis by targeting Brugia malayi: a reverse vaccinology approach

BackgroundLymphatic filariasis (LF), often referred to as elephantiasis, has been identified as one of the 17 neglected tropical diseases by the World Health Organization. Currently, there are no vaccines available to treat this infection in humans. Therefore, with the objective of devising a novel preventive measure, we exploited an immunoinformatics approach to design a multi-epitope-based subunit vaccine for LF, that can elicit a variety of immune responses within the host.In this study, different B cell, TC cell, and TH cell-binding epitopes were screened from the antigenic proteins of Brugia malayi and they were passed through several immunological filters to determine the optimal epitopes.ResultsAs a result, 15 CD8+, 3 CD4+, and 3 B cell epitopes were found to be prominent, antigenic, non-toxic, immunogenic and non-allergenic. The presence of conformational B cell epitopes and cytokine-inducing epitopes confirmed the humoral and cell-mediated immune response that would be triggered by the constructed vaccine model.Following that, the selected epitopes and TLR-4-specific adjuvant were ligated by appropriate peptide linkers to finalize the vaccine construct. Protein–protein docking of the vaccine structure with the TLR4 receptor predicted strong binding affinity and hence putatively confirms its ability to elicit an immune response. Further, the efficiency of the vaccine candidate to provide a long-lasting protective immunity was assessed by in silico immune simulation. The reverse translated vaccine sequence was also virtually cloned in the pET28a (+) plasmid after the optimization of the gene sequence.ConclusionSo taken together, by monitoring the overall in silico assessment, we hypothesize that our engineered peptide vaccine could be a viable prophylactic approach in the development of vaccines against the threat of human lymphatic filariasis.Graphical

Cannabidiol prevents chemotherapy-induced neuropathic pain by modulating spinal TLR4 via endocannabinoid system activation

This study aimed to investigate the effect of cannabidiol (CBD) on type 4 Toll-like receptors (TLR4), glial cells and pro-inflammatory cytokines during the neuropathic pain induced by the chemotherapy agent paclitaxel (PTX), as well as the involvement of the endocannabinoid system in this process. Male C57BL6 mice were subjected to PTX-induced neuropathic pain. To evaluate the involvement of the TLR4, glial cells and cannabinoid CB2 receptor, specific inhibitors or antagonists were intrathecally administered. The western blotting and immunofluorescence assay was performed to evaluate the spinal expression of TLR4, microglia, astrocytes and cannabinoid CB2 receptor. The levels of spinal pro-inflammatory cytokines and endocannabinoids were determined by enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry analysis, respectively. CBD prevented PTX-induced neuropathic pain, and the cannabinoid CB2 receptor antagonist AM630 reversed this effect. In addition, CBD treatment inhibited the spinal expression of TLR4 and Iba1 in mice with neuropathic pain. CBD also increased spinal levels of endocannabinoids anandamide and 2-arachidonoylglycerol, and reduced levels of cytokines in mice with neuropathic pain. CBD was efficient in preventing PTX-induced neuropathic pain, and this effect may involve inhibition of the TLR4 on microglia spinal with activation of the endocannabinoid system.

CEMIP-mediated hyaluronan metabolism facilitates SCLC metastasis by activating TLR2/c-Src/ERK1/2 axis

Small-cell lung cancer (SCLC) is a highly metastatic and recalcitrant malignancy. Metastasis is the major cause of death in patients with SCLC but its mechanism remains poorly understood. An imbalance of hyaluronan catabolism in the extracellular matrix accelerates malignant progression in solid cancers due to the accumulation of low-molecular-weight HA. We previously found that CEMIP, a novel hyaluronidase, may act as a metastatic trigger in SCLC. In the present study, we found that both CEMIP and HA levels were higher in SCLC tissues than in paracancerous tissues from patient specimens and in vivo orthotopic models. Additionally, high expression of CEMIP was associated with lymphatic metastasis in patients with SCLC, and in vitro results showed that CEMIP expression was elevated in SCLC cells relative to human bronchial epithelial cells. Mechanistically, CEMIP facilitates the breakdown of HA and accumulation of LMW-HA. LMW-HA activates its receptor TLR2, and subsequently recruits c-Src to activate ERK1/2 signalling, thereby promoting F-actin rearrangement as well as migration and invasion of SCLC cells. In addition, the in vivo results verified that depletion of CEMIP attenuated HA levels and the expressions of TLR2, c-Src, and phosphorylation of ERK1/2, as well as liver and brain metastasis in SCLC xenografts. Furthermore, the application of the actin filament inhibitor latrunculin A significantly inhibited the liver and brain metastasis of SCLC in vivo. Collectively, our findings reveal the critical role of CEMIP-mediated HA degradation in SCLC metastasis and suggest its translational potential as an attractive target and a novel strategy for SCLC therapy.

Therapeutic effect of Sargassum swartzii against urban particulate matter–induced lung inflammation via toll-like receptor-mediated NF-κB pathway inhibition

The continuous upsurge of airborne particulate matter (PM) is considered a major threat to human health all around worldwide, especially in East Asian countries. PM-induced lung inflammation contributes to acute lung injury (ALI), a critical condition with a high mortality rate. The current study aims at exploring the potential underlying mechanism of sulfated polysaccharides (SPSS) isolated from brown seaweed Sargassum swartzii, located in Hikkaduwa, Sri Lanka, as an anti-inflammatory therapeutic agent against PM-induced lung inflammation. Results revealed that PM stimulation induced nitric oxide (NO) and prostaglandin E2 (PGE2) production as well as pro-inflammatory cytokines secretion including tumor necrosis factor α (TNF-α), and interleukins, IL-1β and IL-6 from MH-S cells. Dose-dependent treatment of SPSS down-regulated these indicators via reducing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) like inflammatory mediators. Moreover, the results showed that initiation of inflammatory response is through identification of PM matter by cell membrane toll-like receptors, TLR2 and TLR4 as well as endosomal TLR7 and TLR3 receptors modulated by NF-κB signaling pathways. Interestingly, these effects were reversed with SPSS in PM-stimulated MH-S cells. Altogether, our findings suggest the potential therapeutic value of SPSS in treating ALI through inflammation suppression via inactivation of TLR/NF-κB signaling pathway.

Epitopes screening and vaccine molecular design of SADS-CoV based on immunoinformatics.

The regional outbreak of the Swine acute diarrhea syndrome coronavirus (SADS-CoV) has seriously threatened the swine industry. There is an urgent need to discover safe and effective vaccines to contain them quickly. The coronavirus spike protein mediates virus entry into host cells, one of the most important antigenic determinants and a potential vaccine target. Therefore, this study aims to conduct a predictive analysis of the epitope of S protein B cells and T cells (MHC class I and class II) by immunoinformatics methods by screening and identifying protective antigenic epitopes that induce major neutralized antibodies and activate immune responses to construct epitope vaccines. The study explored primary, secondary, and tertiary structures, disulfide bonds, protein docking, immune response simulation, and seamless cloning of epitope vaccines. The results show that the spike protein dominant epitope of the screening has a high conservativeness and coverage of IFN-γ, IL-4-positive Th epitope, and CTL epitope. The constructed epitope vaccine interacts stably with TLR-3 receptors, and the immune response simulation shows good immunogenicity, which could effectively activate humoral and cellular immunity. After codon optimization, it was highly likely to be efficiently and stably expressed in the Escherichia coli K12 expression system. Therefore, the constructed epitope vaccine will provide a new theoretical basis for the design of SADS-CoV antiviral drugs and related research on coronaviruses such as SARS-CoV-2.