Intestinal lymphatic transport of Smilax china L. pectic polysaccharide via Peyer's patches and its uptake and transport mechanisms in mononuclear phagocytes.

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.

[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.

Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

Introduction: During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. Methods: High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. Results: The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1ß and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. Discussion: We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

Evaluation of Toll-like Receptor 4 (TLR4) Involvement in Human Atrial Fibrillation: A Computational Study.

In the present study, we have explored the involvement of Toll-like Receptor 4 (TLR4) in atrial fibrillation (AF), by using a meta-analysis of publicly available human transcriptomic data. The meta-analysis revealed 565 upregulated and 267 downregulated differentially expressed genes associated with AF. Pathway enrichment analysis highlighted a significant overrepresentation in immune-related pathways for the upregulated genes. A significant overlap between AF differentially expressed genes and TLR4-modulated genes was also identified, suggesting the potential role of TLR4 in AF-related transcriptional changes. Additionally, the analysis of other Toll-like receptors (TLRs) revealed a significant association with TLR2 and TLR3 in AF-related gene expression patterns. The examination of MYD88 and TICAM1, genes associated with TLR4 signalling pathways, indicated a significant yet nonspecific enrichment of AF differentially expressed genes. In summary, this study offers novel insights into the molecular aspects of AF, suggesting a pathophysiological role of TLR4 and other TLRs. By targeting these specific receptors, new treatments might be designed to better manage AF, offering hope for improved outcomes in affected patients.

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Glutathione-depleting Liposome Adjuvant for Augmenting the Efficacy of a Glutathione Covalent Inhibitor Oridonin for Acute Myeloid Leukemia Therapy.

BACKGROUND: Discrepancies in the utilization of reactive oxygen species (ROS) between cancer cells and their normal counterparts constitute a pivotal juncture for the precise treatment of cancer, delineating a noteworthy trajectory in the field of targeted therapies. This phenomenon is particularly conspicuous in the domain of nano-drug precision treatment. Despite substantial strides in employing nanoparticles to disrupt ROS for cancer therapy, current strategies continue to grapple with challenges pertaining to efficacy and specificity. One of the primary hurdles lies in the elevated levels of intracellular glutathione (GSH). Presently, predominant methods to mitigate intracellular GSH involve inhibiting its synthesis or promoting GSH efflux. However, a conspicuous gap remains in the absence of a strategy capable of directly and efficiently clearing GSH. METHODS: We initially elucidated the chemical mechanism underpinning oridonin, a diminutive pharmacological agent demonstrated to perturb reactive oxygen species, through its covalent interaction with glutathione. Subsequently, we employed the incorporation of maleimide-liposomes, renowned for their capacity to disrupt the ROS delivery system, to ameliorate the drug's water solubility and pharmacokinetics, thereby enhancing its ROS-disruptive efficacy. In a pursuit to further refine the targeting for acute myeloid leukemia (AML), we harnessed the maleic imide and thiol reaction mechanism, facilitating the coupling of Toll-like receptor 2 (TLR2) peptides to the liposomes' surface via maleic imide. This strategic approach offers a novel method for the precise removal of GSH, and its enhancement endeavors are directed towards fortifying the precision and efficacy of the drug's impact on AML targets. RESULTS: We demonstrated that this peptide-liposome-small molecule machinery targets AML and consequently induces cell apoptosis both in vitro and in vivo through three disparate mechanisms: (I) Oridonin, as a Michael acceptor molecule, inhibits GSH function through covalent bonding, triggering an initial imbalance of oxidative stress. (II) Maleimide further induces GSH exhaustion, aggravating redox imbalance as a complementary augment with oridonin. (III) Peptide targets TLR2, enhances the directivity and enrichment of oridonin within AML cells. CONCLUSION: The rationally designed nanocomplex provides a ROS drug enhancement and targeted delivery platform, representing a potential solution by disrupting redox balance for AML therapy.

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Glycyrrhizin attenuates renal inflammation in a mouse Con A-hepatitis model via the IL-25/M2 axis.

Glycyrrhizin (GL) has immunoregulatory effects on various inflammatory diseases including hepatitis and nephritis. However, the mechanisms underlying the anti-inflammatory effect of GL on renal inflammation are not fully understood. Hepatorenal syndrome (HRS) is a functional acute renal impairment that occurs in severe liver disease, and we found that kidney injury also occurs in Con A-induced experimental hepatitis in mice. We previously found that GL can alleviate Con A-induced hepatitis by regulating the expression of IL-25 in the liver. We wanted to investigate whether GL can alleviate Con A-induced nephritis by regulating IL-25. IL-25 regulates inflammation by modulating type 2 immune responses, but the mechanism by which IL-25 affects kidney disease remains unclear. In this study, we found that the administration of GL enhanced the expression of IL-25 in renal tissues; the latter promoted the generation of type 2 macrophages (M2), which inhibited inflammation in the kidney caused by Con A challenge. IL-25 promoted the secretion of the inhibitory cytokine IL-10 by macrophages but inhibited the expression of the inflammatory cytokine IL-1ß by macrophages. Moreover, IL-25 downregulated the Con A-mediated expression of Toll-like receptor (TLR) 4 on macrophages. By comparing the roles of TLR2 and TLR4, we found that TLR4 is required for the immunoregulatory effect of IL-25 on macrophages. Our data revealed that GL has anti-inflammatory effects on Con A-induced kidney injury and that the GL/IL-25/M2 axis participates in the anti-inflammatory process. This study suggested that GL is a potential therapeutic for protecting against acute kidney injury.

Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

A Review: The Significance of Toll-Like Receptors 2 and 4, and NF-κB Signaling in Endothelial Cells during Atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that begins with endothelial activation followed by a series of inflammatory responses, plaque formation, and finally rupture. An early event in endothelial dysfunction is activation of the nuclear factor-κB (NF-κB) signaling axis. Toll-like receptors (TLRs) in endothelial cells (ECs) play an essential role in recognizing pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and lifestyle-associated molecular patterns (LAMPs). Activation of the canonical NF-κB pathway stimulates the expression of cytokines, chemokines, and an array of additional genes which activate and amplify AS-associated inflammatory responses. In this review, we discuss the involvement of TLR2/4 and NF-κB signaling in ECs during AS initiation, as well as regulation of the inflammatory response during AS by noncoding RNAs, especially microRNA (miRNA) and circular RNA (circRNA).

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Radix Sophorae Flavescentis of Sophora flavescens Aiton inhibits LPS-induced macrophage pro-inflammatory response via regulating CFHR2 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Long-term chronic inflammation often leads to chronic diseases. Although Sophora flavescens has been shown to have anti-inflammatory properties, its detailed molecular mechanism is still unknown. AIM OF STUDY: This study investigated the effect of Radix Sophorae Flavescentis on the LPS-induced inflammatory response in macrophages. MATERIALS AND METHODS: LPS was used to induce the peritoneal macrophages to simulate the inflammatory environment in vitro. Different concentrations of Radix Sophorae Flavescentis-containing (medicated) serum were used for intervention. The peritoneal macrophages were identified by using hematoxylin-eosin and immunofluorescence staining. ELISA was used to measure the TNF-α and IL-6 expression to determine the concentration of LPS. ELISA and Western blot (WB) were used to detect the PGE2 and CFHR2 expression in each group, respectively. The lentiviral vector for interference and overexpression of the CFHR2 gene was constructed, packaged, and transfected into LPS-induced macrophages. The transfection efficiency was verified by WB. Then, ELISA was used to detect the TNF-α, PGE2, and IL-6 expression. WB was used to detect the CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression. RESULTS: The primary isolated cells were identified as macrophages. The LPS-treated macrophages exhibited significantly higher expression of PGE2 and CFHR2, and the inflammatory factors TNF-α and IL-6, as well as iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression compared with the control group (P < 0.05). The TNF-α, PGE2, and IL-6 levels, as well as CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression were considerably lower in the LPS-induced+10% medicated-serum group, LPS-induced+20% medicated-serum group, and shCFHR interference group compared with the LPS group (P < 0.05). CONCLUSION: Radix Sophorae Flavescentis might mediate CFHR2 expression and play an important role in inhibiting the LPS-induced pro-inflammatory response of macrophages. Radix Sophorae Flavescentis could be a potential treatment for LPS-induced related inflammatory diseases.

Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2.

Inflammatory bowel disease (IBD) is characterized by recurrent inflammatory reactions in the intestinal mucosa, including ulcerative colitis (UC) and Crohn's disease (CD). The expression of Toll-like receptor 2 (TLR2) has been observed to increase during the progression of IBD. Flavokawain B (FKB), a natural chalcone with potent anti-inflammatory activity, exerts its effects through inhibition of the NF-κB pathway. In this study, we aimed to investigate the effects and mechanisms of FKB targeting TLR2 in IBD. C57BL/6 J mice were treated with 2.5% dextran sulfate sodium (DSS) for 7 days, with administration of FKB or TLR2 inhibitor C29 starting on day 2 to establish the model of IBD. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with the TLR2 agonist Pam3CSK4 to explore the therapeutic effect of FKB and its pharmacological mechanism. Compared with the model group, the FKB-treated group showed significant reductions in colitis-related injuries in the IBD mouse model, including weight gain, increased colon length and reduced inflammation. FKB decreased the formation of TLR2-MyD88 complex by targeting TLR2, leading to suppression of downstream NF-κB signaling pathway. Similar therapeutic effects were observed in the C29-treated group. Additionally, in vitro data suggested that FKB exerted its anti-inflammatory effect by targeting TLR2 and inhibiting Pam3CSK4-induced activation of the NF-κB pathway. The anti-inflammatory effects of FKB were demonstrated through drug affinity responsive target stability assay and cellular thermal shift assay, revealing its binding affinity to TLR2. By inhibiting the activation of the TLR2/NF-κB signaling pathway, FKB effectively prevented DSS-induced IBD and exhibited promising potential as a therapeutic candidate for IBD treatment.

Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis.

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

Immune response to colonization of Candida albicans in mice treated with Cefoperazone.

Candida species are a normal human flora in humans' digestive and reproductive systems, oral cavity, skin, and mucosal surfaces. This study aimed to detect the immunological role of Candida infection by using some immunological markers. The results of levels in serum showed high concentrations of IgA (56.20 ± 12 pg/ml,29.55 ± 4.5 pg/ml respectively) and IgG (12.05 ± 3.218 pg/ml, 3.836 ± 1.23 pg/ml respectively) in mice infected with C. albicans and mice treated with Cefoperazone and infected with Candida with significant differences (P value < 0.05). The results showed high serum levels of IL-17(191.5 ± 42.81 pg/ml) and TLR2(7.651 ± 1.5 pg/ml) in group mice infected with C. albicans compared with negative control and group mice treated with Cefoperazone. Also, high levels of IL-17 (91.33 ± 4.816 pg/ml) and TLR2 (2.630 ± 0.5 pg/ml) in group mice treated with Cefoperazone and infected with Candida compared with negative control and group mice treated with Cefoperazone (P value < 0.05). The results of antibodies and immunological markers in the intestine showed high levels of IgA and IgG in mice infected with C.albicans (55.7 ± 4.9 pg/ml, 18.19 ± 0.63 pg/ml respectively).Also,IgA and IgG in mice treated with Cefoperazone and infected with Candida were high level (43.04 ± 2.1 pg/ml, 2.927 ± 0.2 pg/ml respectively) in mice infected with C. albicans with significant differences (P value < 0.05). The results levels of IL-17 and TLR2 were increased in mice infected with C. albicans (191.5 ± 42.81 pg/ml, 7.651 ± 1.5 pg/ml respectively) and mice treated with Cefoperazone and infected with Candida (91.33 ± 4.816 pg/ml,2.630 ± 0.5 pg/ml respectively) with significant differences (P < 0.05). In conclusion, this study demonstrated that cefoperazone treatment and infection by Candida albicans changed the microbiome components in the gut and finally can change host immune responses. It was observed that elevated levels of the antibodies production (IgA and IgG) and immunological markers (IL-17, and TLR2) in serum and the gut.

Protein Expression of TLR2, TLR4, and TLR9 on Monocytes in TB, HIV, and TB/HIV.

Toll-like receptors (TLRs) have a critical role in recognizing pathogenic patterns and initiating immune responses against TB and HIV. Previously, studies described the gene expression of TLRs in patients with TB and HIV. Here, we demonstrated TLRs protein expressions and their association with clinical status and plasma markers in TB, HIV, and TB/HIV coinfection. The phenotyping of TLR2, TLR4, and TLR9 on CD14+ monocytes and their subsets were determined by multicolor flow cytometry. Host plasma biomarkers and microbial indices were measured using Luminex Multiplex assay and standard of care tools, respectively. TLR2 expression significantly enhanced in TB, slightly increased in HIV but slightly reduced in TB/HIV coinfection compared to apparently health controls (HC). On the other hand, TLR4 expression was significantly increased in TB, HIV, and TB/HIV compared to HC. Expression of TLR4 was equally enhanced on classical and intermediate monocytes while higher TLR2 expression on intermediate than classical monocytes. TLR4 had a positive correlation pattern with plasma biomarkers while TLR2 had an inverse correlation pattern. TLR4 is associated with disease severity while TLR2 is with the immune-competent status of patients. Our findings demonstrated that the pattern of TLR expression is disease as well as monocyte subset specific and distinct factors drive these differences.

Introduction of protein vaccine candidate based on AP65, AP33, and α-actinin proteins against Trichomonas vaginalis parasite: an immunoinformatics design.

BACKGROUND: Trichomonas vaginalis is the most common nonviral sexually transmitted disease (STI) worldwide. Vaccination is generally considered to be one of the most effective methods of preventing infectious diseases. Using AP65, AP33 and α-actinin proteins, this research aims to develop a protein vaccine against Trichomonas vaginalis. METHODS: Based on the B-cell and T-cell epitope prediction servers, the most antigenic epitopes were selected, and with the necessary evaluations, epitope-rich domains of three proteins, AP65, AP33, and α-actinin, were selected and linked. Subsequently, the ability of the vaccine to interact with toll-like receptors 2 and 4 (TLR2 and TLR4) was assessed. The stability of the interactions was also studied by molecular dynamics for a duration of 100 nanoseconds. RESULTS: The designed protein consists of 780 amino acids with a molecular weight of 85247.31 daltons. The results of the interaction of the vaccine candidate with TLR2 and TLR4 of the immune system also showed that there are strong interactions between the vaccine candidate protein with TLR2 (-890.7 kcal mol-1) and TLR4 (-967.3 kcal mol-1). All parameters studied to evaluate the stability of the protein structure and the protein-TLR2 and protein-TLR4 complexes showed that the structure of the vaccine candidate protein is stable alone and in complex with the immune system receptors. Investigation of the ability of the designed protein to induce an immune response using the C-ImmSim web server also showed that the designed protein is capable of stimulating B- and T-cell lymphocytes to produce the necessary cytokines and antibodies against Trichomonas vaginalis. CONCLUSIONS: Overall, our vaccine may have potential protection against Trichomonas vaginalis. However, for experimental in vivo and in vitro studies, it may be a good vaccine candidate.

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.