Toll-like Receptor Research Articles

DNA and RNA Methylation in Periodontal and Peri-implant Diseases.

Periodontal and peri-implant diseases are primarily biofilm-induced pathologies in susceptible hosts affecting the periodontium and dental implants. Differences in disease susceptibility, severity, and patterns of progression have been attributed to immune regulatory mechanisms such as epigenetics. DNA methylation is an essential epigenetic mechanism governing gene expression that plays pivotal roles in genomic imprinting, chromosomal stability, apoptosis, and aging. Clinical studies have explored DNA methylation inhibitors for cancer treatment and predictive methylation profiles for disease progression. In periodontal health, DNA methylation has emerged as critical, evidenced by clinical studies unraveling its complex interplay with inflammatory genes and its regulatory role in periodontitis contributing to disease severity. Human studies have shown that methylation enzymes associated with gene reactivation (e.g., ten-eleven translocation-2) are elevated in periodontitis compared with gingivitis. Dysregulation of these genes can lead to the production of inflammatory cytokines and an altered initial response to bacteria via the toll-like receptor signaling pathway in periodontal diseases. In addition, in peri-implant diseases, this dysregulation can result in altered DNA methylation levels and enzymatic activity influenced by the properties of the titanium surface. Beyond traditional perspectives, recent evidence highlights the involvement of RNA methylation (e.g., N6-methyladenosine [m6A], N6,2'-0-dimethyladenosine [m6Am]) in periodontitis and peri-implantitis lesions, playing vital roles in the innate immune response, production of inflammatory cytokines, and activation of dendritic cells. Both DNA and RNA methylation can influence the gene expression, virulence, and bacterial behavior of well-known periodontal pathogens such as Porphyromonas gingivalis. Alterations in bacterial methylation patterns result in changes in the metabolism, drug resistance, and gene expression related to survival in the host, thereby promoting tissue degradation and chronic inflammatory responses. In summary, the present state-of-the-art review navigates the evolving landscape of DNA and RNA methylation in periodontal and peri-implant diseases, integrating recent developments and mechanisms to reshape the understanding of epigenetic dynamics in oral health.

Fetal hepatocytes protect the HSPC genome via fetuin-A.

The maintenance of genomic integrity in rapidly proliferating cells is a substantial challenge during embryonic development1-3. Although numerous cell-intrinsic mechanisms have been revealed4-7, little is known about genome-protective effects and influences of developmental tissue microenvironments on tissue-forming cells. Here we show that fetal liver hepatocytes provide protection to haematopoietic stem and progenitor cell (HSPC) genomes. Lineage tracing and depletion in mice demonstrated that delayed hepatocyte development in early fetal livers increased the chromosomal instability of newly colonizing HSPCs. In addition, HSPCs developed tolerance to genotoxins in hepatocyte-conditioned medium, suggesting that hepatocytes protect the HSPC genome in a paracrine manner. Proteomic analyses demonstrated the enrichment of fetuin-A in hepatocyte-conditioned medium but not in early fetal livers. Fetuin-A activates a Toll-like receptor pathway to prevent pathogenic R-loop accumulation in HSPCs undergoing DNA replication and gene transcription in the fetal liver. Numerous haematopoietic regulatory genes frequently involved in leukaemogenic mutations are associated with R-loop-enriched regions. In Fetua-knockout mice, HSPCs showed increased genome instability and susceptibility to malignancy induction. Moreover, low concentrations of fetuin-A correlated with the oncogenesis of childhood leukaemia. Therefore, we uncover a mechanism operating in developmental tissues that offers tissue-forming cell genome protection and is implicated in developmental-related diseases.

Advanced MicroRNA delivery for lung inflammatory therapy: surfactant protein A controls cellular internalisation and degradation of extracellular vesicles

IntroductionAlveolar macrophages (AMs) are the first line of defence against pathogens that initiate an inflammatory response in the lungs and exhibit a strong affinity for surfactant protein A (SP-A). Extracellular...

Targeting acetylated high mobility group box 1 protein (HMGB1) and toll-like receptor (TLR4) interaction to alleviate hypertension and neuroinflammation in fructose-fed rats.

Our previous study reported that fructose intake increased systemic blood pressure and reduced nitric oxide (NO) in the nucleus tractus solitarius (NTS) due to oxidative stress and neuroinflammation. However, it remains unclear how reactive oxygen species (ROS) reduce NO and how this process impacts neuroinflammation in the NTS. This study aimed at investigating the effect of ROS on acetylation of high mobility group box 1 protein (HMGB1) in the NTS of fructose-induced hypertensive rats. Male Wistar-Kyoto (WKY) rats were fed with 10% fructose water to elevate blood pressure. Thereafter, CLI-095 and glycyrrhizic acid (GA) treatments were delivered for up to 2weeks (1mg·12 μL-1·day-1, by intracerebroventricular injection) to reduce the negative effects of toll-like receptor 4 (TLR4) and HMGB1 activation. Two weeks of CLI-095 and GA treatment reduced systemic blood pressure and significantly preserved neuronal and endothelial nitric oxide synthase (nNOS and eNOS) availability against the inflammatory insults of fructose consumption. Both CLI-095 and GA halted the interaction of acetylated HMGB1 and TLR4. Two weeks of CLI-095 and GA treatment markedly reduced NTS inflammation (pro-inflammatory cytokines and microglial activation) and lowered serum norepinephrine levels. Our data reveal novel pharmacological properties for CLI-095 and GA, which improved blood pressure and inflammatory conditions by decreasing the interaction of acetylated HMGB1 with TLR4. These findings challenge the commonly accepted dogma that essential hypertension is specifically mediated by neuroinflammation due to acetylated HMGB1 coupling to TLR4.

Modulation of Innate Immunity via Pattern Recognition Receptors (PRRs) for Antiviral and Antitumor Therapies

Pattern recognition receptors (PRRs) are vital components of the innate immune system, playing a crucial role in detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). This review explores the modulation of innate immunity via PRRs for antiviral and antitumor therapies. PRRs, including toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs), initiate immune responses that enhance the recognition and elimination of pathogens and tumor cells. TLRs, for instance, trigger pro-inflammatory cytokine production and activate dendritic cells, facilitating adaptive immune responses. The therapeutic potential of PRR agonists is significant; they can be used as standalone treatments or in combination with existing antiviral and cancer therapies. PRR agonists enhance vaccine efficacy, promote robust antiviral responses, and improve the recognition of tumor antigens. Additionally, oncolytic viruses can exploit PRR pathways to stimulate innate and adaptive immunity against tumors. However, challenges such as the risk of excessive inflammation and the need for targeted delivery of PRR agonists remain. Future research should focus on optimizing PRR-targeted strategies to harness their full therapeutic potential while minimizing adverse effects. This review highlights the promising role of PRRs in enhancing immune responses and discusses their implications for developing innovative antiviral and antitumor therapies. Keywords: Pattern recognition receptors (PRRs), Innate immunity, Antiviral therapies, Antitumor therapies, Immune modulation

Uncovering the Significance of JNK/AKT Axis in the Autophagic Regulation of Leishmania major Infection.

The role of autophagy in host induced by infection of parasites of the Leishmania genus remains inadequately understood. Leishmania parasites modulate host macrophages to promote its survival by inducing autophagy response in the host cell. In this study, we conducted an investigation of L. major infection, focusing on host autophagy processes where we reconstructed two mathematical models elucidating autophagy induction and inhibition processes and its impact on parasite survival. Our models presented systems modulatory dynamics of the parasite-mediated host autophagy. Our work highlighted the pivotal role of signaling molecules associated with the immune response which included signaling induced by Toll-like receptor (TLR), specifically through regulation of JNK and AKT. Both molecules emerged as key regulators of host autophagy process, highlighting that JNK/AKT signaling axis may be a potential avenue for innovative therapeutic approaches in targeting leishmaniasis. Also, ATG16L complex was identified as a critical determinant in shaping the course of leishmanial infection through formation of autophagosomes. Through invitro analyses in differentiated human monocyte cell line, we observed an increase in nitric oxide synthase (iNOS) concentration upon autophagy inhibition, while autophagy induction resulted in decreased iNOS concentration. This suggested that autophagy induction favors parasite survival in the host, potentially by providing a nutrient source that may be advantageous for the parasite. Inhibition of host autophagy promoted parasite elimination. Hence, our work proposed an avenue for strategically blocking host autophagy which enumerates a targeted approach for combating leishmaniasis.

Epstein-Barr virus and immunity.

The Epstein-Barr virus has infected the vast majority of the world's population. EBV is the most common herpes virus in the human population. In childhood, viral infection is either asymptomatic or can lead to infectious mononucleosis. In a small proportion of latently infected people, especially in immunosuppressed patients, EBV causes lymphoid and epithelial malignancies and a number of autoimmune diseases, including one of the causes of the development of multiple sclerosis. Innate immunity is the first line of antiviral defense, which EBV evades using a number of strategies to achieve successful infection. EBV disrupts the innate immune signaling pathways activated by Toll-like, RIG-I-like, NOD-like and AIM2-like receptors, as well as cyclic GMP-AMP synthetase. EBV also antagonizes interferon production and signaling, including the TBK1-IRF3 and JAK-STAT pathways. Through differential modulation of proviral and antiviral mechanisms of action of caspases and other cellular life cycle regulators at different stages of infection, EBV actively interferes with apoptotic and inflammatory pathways to continue effective infection. By turning the activation of innate immunity to its advantage by triggering a pro-inflammatory reaction and proteolytic cleavage of caspases that exhibit proviral activity, the virus establishes latency or enters the lytic reactivation phase, which contributes to the development of serious life-threatening diseases, including oncogenesis. The outcome of EBV infection is regulated by a subtle interaction between innate, adaptive immunity, and viral replication. In the absence of licensed preventive vaccines, immunocorrection and antiviral therapy are the only possible ways to combat the virus and prevent diseases associated with it. Understanding the mechanisms of action of certain EBV genes involved in the life cycle at different stages of infection will help in the future to find the right approach to the development of preventive and therapeutic drugs against EBV.

Genomic Insights into Host Susceptibility to Periprosthetic Joint Infections: A Comprehensive Literature Review

Periprosthetic joint infection (PJI) is a multifactorial disease, and the risk of contracting infection is determined by the complex interplays between environmental and host-related factors. While research has shown that certain individuals may have a genetic predisposition for PJI, the existing literature is scarce, and the heterogeneity in the assessed genes limits its clinical applicability. Our review on genetic susceptibility for PJI has the following two objectives: (1) Explore the potential risk of developing PJI based on specific genetic polymorphisms or allelic variations; and (2) Characterize the regulatory cascades involved in the risk of developing PJI. This review focused on clinical studies investigating the association between genetic mutations or variations with the development of PJI. The genes investigated in these studies included toll-like receptors and humoral pattern recognition molecules, cytokines, chemokines, mannose-binding lectin (MBL), bone metabolism molecules, and human leukocyte antigen. Among these genes, polymorphisms in IL-1, MBL, vitamin D receptors, HLA-C, and HLA-DQ might have a relevant impact on the development of PJI. The literature surrounding this topic is limited, but emerging transcriptomic and genome-wide association studies hold promise for identifying at-risk genes. This advancement could pave the way for incorporating genetic testing into preoperative risk stratification, enhancing personalized patient care.

Spatial immune heterogeneity in a mouse tumor model after immunotherapy.

Cancer immunotherapy is increasingly used in clinical practice, but its success rate is reduced by tumor escape from the immune system. This may be due to the genetic instability of tumor cells, which allows them to adapt to the immune response and leads to intratumoral immune heterogeneity. The study investigated spatial immune heterogeneity in the tumor microenvironment and its possible drivers in a mouse model of tumors induced by human papillomaviruses (HPV) following immunotherapy. Gene expression was determined by RNA sequencing and mutations by whole exome sequencing. A comparison of different tumor areas revealed heterogeneity in immune cell infiltration, gene expression, and mutation composition. While the mean numbers of mutations with every impact on gene expression or protein function were comparable in treated and control tumors, mutations with high or moderate impact were increased after immunotherapy. The genes mutated in treated tumors were significantly enriched in genes associated with ECM metabolism, degradation, and interactions, HPV infection and carcinogenesis, and immune processes such as antigen processing and presentation, Toll-like receptor signaling, and cytokine production. Gene expression analysis of DNA damage and repair factors revealed that immunotherapy upregulated Apobec1 and Apobec3 genes and downregulated genes related to homologous recombination and translesion synthesis. In conclusion, this study describes the intratumoral immune heterogeneity, that could lead to tumor immune escape, and suggests the potential mechanisms involved.

Deciphering the pharmacological mechanism of Radix astragali for allergic rhinitis through network pharmacology and experimental validation

Radix Astragali (RA) has been recognized for its therapeutic potential in allergic rhinitis (AR), yet its potential pharmacological mechanisms remain elusive. This study systematically investigated the physicochemical properties and biological activities of RA’s phytochemicals, aiming to elucidate their targets and mechanisms in AR treatment. We identified 775 potential targets of RA’s key phytochemicals and intersected these with 29,544 AR-related disease targets, pinpointing 747 shared therapeutic targets. A protein-protein interaction network analysis categorized these targets into five subclusters, with TNF, NFKB1, IKBKB, NFKBIA, and CHUK emerging as central nodes. Enrichment analysis revealed their roles in inflammatory and immune responses, particularly through the NF-κB, TNF, IL-17, Toll-like receptor, and NOD-like receptor signaling pathways. Molecular docking and dynamics simulations confirmed the strong binding affinity and stability of RA’s phytochemicals to these targets. In vivo, RA intervention effectively reversed the expression of key inflammatory markers in an IL-13-induced nasal mucosa inflammation model. Our findings suggest that RA’s multitargeted approach involves the modulation of critical inflammatory pathways, highlighting its therapeutic potential.

IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis.

This study investigated the mechanisms driving the induction and sustained presence of neutrophil extracellular traps (NETs) in the synovial microenvironment of rheumatoid arthritis (RA). Synovial tissue and fluid samples were collected from patients with RA and osteoarthritis (OA), and NET levels and cytokine concentrations were measured using a cytometric bead array and enzyme-linked immunosorbent assay (ELISA). The ability of interleukin-33 (IL-33) to induce NET formation was evaluated using quantitative assays, immunofluorescence staining, live-cell imaging, and electron microscopy. Coincubation experiments of NETs with fibroblast-like synovial cells (FLSs) were conducted, and a modified Transwell migration assay was designed to assess neutrophil migration. The role of IL-33 and NETs in RA progression was further investigated using a collagen antibody-induced arthritis (CAIA) mouse model. The results revealed an increase in NETs and IL-33 levels in the synovial fluid of RA patients, with a significant positive correlation between them. NET formation assays confirmed that IL-33 activates neutrophils to produce NETs and that neutrophils from RA patients exhibit increased responsiveness to IL-33 stimulation. Both in vitro and in vivo evidence has demonstrated that NETs stimulate FLSs to secrete IL-33 and the chemokine CXCL8 via Toll-like receptor 9, promoting further neutrophil recruitment and increasing NET production within the RA synovium. This study reveals a novel positive feedback loop involving NETs and FLSs that is mediated by IL-33 that increases NET accumulation in RA. Targeting IL-33 or NET formation and amplification may offer new therapeutic strategies for managing RA.

Immunoinformatics design of a multi-epitope vaccine for Chlamydia trachomatis major outer membrane proteins

Chlamydia trachomatis (CT) remains a significant infectious cause of blindness and sexually transmitted infections worldwide. The objective and novelty of this study lie in using different serovars of CT to design a broad-spectrum multi-epitope vaccine that might confer immunity against different CT infections. As the major outer membrane protein in CT has good immunodominance properties and high conservation and also determines the several serotypes of CT, it is selected as an antibody target in this study. T-cell and B-cell epitopes from serovars A, B, D, E, L1, and L2 were predicted and combined into a single construct by incorporating adjuvants and linkers to enhance immunogenicity and stability. Physicochemical characterization confirmed the constructed vaccine’s anti-allergic, immunogenicity, and thermostable characteristics, followed by structural modeling to refine its 3D configuration. The 3D model structure of the vaccine was validated through the Ramachandran plot and ProSA z-score. Molecular docking studies of the vaccine demonstrated stable binding with toll-like receptor 3, along with molecular dynamics simulations and binding free energy calculations supporting the complex’s stability. In silico cloning has indicated a high potential for expression in Escherichia coli. Lastly, immune simulations revealed robust activation of B cells, cytotoxic T cells, and antigen-presenting cells, alongside significant production of IgM, IgG antibodies, and balanced Th1/Th2 cytokine response, which is crucial for effective immunity. These results suggest the multi-epitope vaccine could effectively induce comprehensive immune responses against CT, highlighting the need for further in vivo validation to advance this promising candidate toward clinical use.

Cathelicidin: Insights into Its Impact on Metabolic Syndrome and Chronic Inflammation

Background/Objectives: LL-37 is associated with metabolic syndrome (MetS), a constellation of risk factors comprising obesity, insulin resistance (IR), dyslipidemia, and hypertension, which elevates the risk of cardiovascular disease and type 2 diabetes. Methods: In this narrative review, we analyzed the literature focusing on recent developments in the relationship between cathelicidin and various components of MetS to provide a comprehensive overview. Results: Studies have shown that LL-37 is linked to inflammation in adipose tissue (AT) and the development of IR in obesity. Cathelicidin can enhance inflammation by activating pro-inflammatory genes, as well as modulate the inflammatory response. The mechanisms of IR include the activation of complex signaling pathways that induce inflammation and reduce insulin signaling in adipocytes. The activation of Toll-like receptors (TLRs) by cathelicidin stimulates the secretion of pro-inflammatory cytokines, contributing to the disruption of insulin function in adipose cells. Cathelicidin also influences lipid metabolism, with recent research showing a negative relationship between LL-37 levels and HDL cholesterol. Therefore, LL-37 is involved not only in the regulation of inflammation but also in lipid metabolism, potentially aggravating the cardiovascular complications associated with MetS. Conclusions: Cathelicidin plays a crucial role in regulating the balance between inflammatory and anti-inflammatory responses in MetS. Understanding the impact of LL-37 on these mechanisms may unveil novel approaches for addressing MetS and its associated complications.

Designing and immunomolecular analysis of a new broad-spectrum multiepitope vaccine against divergent human papillomavirus types.

Human papillomavirus (HPV), which is transmitted through sexual activity, is the primary cause of cervical cancer and the fourth most common type of cancer in women. In this study, an immunoinformatics approach was employed to predict immunodominant epitopes from a diverse array of antigens with the ultimate objective of designing a potent multiepitope vaccine against multiple HPV types. Immunodominant B cell, cytotoxic T cell (CTL), and helper T cell (HTL) epitopes were predicted using bioinformatics tools These epitopes were subsequently analyzed using various immunoinformatics tools, and those that exhibited high antigenicity, immunogenicity, non-allergenicity, non-toxicity, and excellent conservation were selected. The selected epitopes were linked with appropriate linkers and adjuvants to formulate a broad-spectrum multiepitope vaccine candidate against HPV. The stability of the multiepitope vaccine candidate was confirmed through structural analysis, and docking results indicated a high affinity for Toll-like receptors (TLR2 and TLR4). Molecular dynamics simulations demonstrated a persistent interaction of TLR2 and TLR4 with the multiepitope vaccine candidate. In silico immunological simulations showed that three injections of the multiepitope vaccine candidate resulted in high levels of B- and T-cell immune responses. Moreover, the in silico cloning results indicated that the multiepitope vaccine candidate could be expressed in substantial amounts in E. coli. The results of this study imply that designing a broad-spectrum vaccine against various HPV types using computational methods is plausible; however, experimental validation and safety testing to confirm the findings is essential.

An engineered α1β1 integrin-mediated FcγRI signaling component to control enhanced CAR macrophage activation and phagocytosis

Treatment of solid tumors remains difficult, and therefore there has been increased focus on chimeric antigen receptor macrophages (CAR-M) to challenge solid tumors. However, CAR domain design of of adoptive cell therapy, which leads to differences in antitumor activity and triggered antitumor potential, remains poorly understood for macrophages. We developed an α1β1 integrin-mediated Fc-gamma receptor I (FcγRI) signaling component for CAR-M specific activation and its antitumor potential. We evaluated CAR-M effects with α1β1 integrin-mediated FcγRI signaling (ACT CAR-M) on the activation and antitumor phagocytic response of macrophages in vitro. Subcutaneous tumor model in BALB/c mice and carcinomatosis model in immunodeficient mice were used to test the antitumor effect of ACT CAR-M compared with CD3ζ CAR-M. The α1β1 integrin-mediated FcγRI signaling engagement of CAR-M was associated with enhanced macrophage activation and specific phagocytosis in primary human macrophages, and significantly improved tumor control and survival in multiple cancer models when compared to CD3ζ CAR-M. RNA-sequencing suggested that α1β1 integrin-mediated FcγRI engagement increased antitumor immunity by enhancing pro-inflammatory M1 phenotype-associated pathways, such as Toll-like receptor signaling, tumor necrosis factor signaling, and IL-17 signaling. α1β1 integrin-mediated FcγRI signaling engagement markedly enhanced antitumor effects of CAR-M immunotherapy, which is proposed as an advanced engineering CAR domain material to expand the clinical application of CAR-M.

Evolution and expression of TLR5a and TLR5b in lamprey (Lampetra japonica)

The lamprey serves as a key model organism for studying the origin and evolution of species, embryonic development, and the immune system. The immune system primarily relies on pattern recognition receptors, including Toll-like receptors (TLRs), with Toll-like receptor 5 (TLR5) having a particularly complex evolutionary history. Currently, although TLR5 is being identified in an expanding array of taxonomic groups, a comprehensive study on its evolutionary aspects is yet to be conducted. In this study, we identified Lj-TLR5a and Lj-TLR5b in Lampetra japonica and examined their distribution across various tissues in this species. Furthermore, we conducted preliminary investigations into their immune functions and discovered that, as primitive genes, they are highly sensitive to various pathogens. Upon recognizing flagellar proteins, both Lj-TLR5a and Lj-TLR5b work together; however, these TLRs may function independently in response to other stimuli. Subsequently, we performed comprehensive structural and evolutionary analyses of the TLR5 family, incorporating TLR5 data from various species at different evolutionary stages. Our findings revealed that TLR5a and TLR5b in lamprey are in a relatively primitive evolutionary state. Meanwhile, TLR5L differentiated during the early stages of evolution and exhibits a pseudogenic trend throughout this evolutionary process; notably, this TLR is currently preserved only in certain amphibian and reptile species. In cartilaginous fish, only one type of TLR5 is usually retained, whereas bony fish typically possess both TLR5 and TLR5S. TLR5S has a relatively simple structure, likely arising from repetitive whole-genome events in bony fishes. Among bony fishes, flesh-finned fish were found to retain only one TLR5, which eventually evolved into the TLR5 found in quadrupeds. In summary, this study provides significant insights into the origin and evolution of the TLR5 family by analyzing the evolutionary status and immune functions of Lj-TLR5a and Lj-TLR5b in Japanese lampreys.

Dietary sanguinarine supplementation improves the growth performance and intestinal immunity of broilers

Dietary sanguinarine (SAN) can enhance the growth performance of poultry and livestock, but the regulatory mechanism of the SAN monomer on intestinal homeostasis and how it promotes growth performance has not yet been clarified. In this study, 200 chickens were divided into four groups and fed different doses of SAN (0, 0.225, 0.75, 2.25mg/kg) for transcriptome and microbiota analysis. The data showed that different doses of SAN supplementation increased the feed conversion rate (FCR) of 22 to 42 d old and 1 to 42 d old broilers (P < 0.01), and 0.225 mg/kg SAN reduced the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN) in serum (P < 0.01). Dietary SAN increased the villus height and the villus height/crypt depth (V/C) ratio in the ileum (P < 0.01). The levels of tight junction proteins (zonula occludens-1, occludin and claudin-1) were up-regulated in the ileum and cecum (P < 0.01) and the levels of immunoglobulin (Ig) A, IgM, IgG, interleukin (IL)-4, IL-10 and interferon (IFN)-γ were up-regulated in the serum and ileum (P < 0.01). RNA-seq analysis revealed 385 differentially expressed genes (DEGs) (|log2 fold change| ≥ 1, FDR < 0.05) between the SAN group and CON group. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed 15 pathways mostly associated with the immune system. Additionally, the reverse transcription-PCR results showed that the relative mRNA expression of β-defensin and mucin 2 were up-regulated (P < 0.01) and Toll-like receptor (TLR) 2 and TLR 4 mRNA were down-regulated by SAN (P < 0.01), which was consistent with the transcriptomic analysis. Western blot analysis also showed that SAN reduced the expression of inflammatory proteins such as TLR4, nuclear factor-kappa B and IL-1β in the ileum (P < 0.01). In addition, at the genus level, SAN significantly increased the relative abundance of bacteria (Bacteroides, unclassified_f__Lachnospiraceae, Lactobacillus and Romboutsia) involved in acetate and butyrate production in the cecum, which are associated with enhanced intestinal immune function and maintaining intestinal health. In conclusion, SAN ameliorates the growth performance of broilers, enhances intestinal immune function, regulates the structure of microbiota and maintains intestinal health.

Cooperative tumor inhibition by CpG-oligodeoxynucleotide and cyclic dinucleotide in head and neck cancer involves T helper cytokine and macrophage phenotype reprogramming

Head and neck cancer ranks as the sixth most common cancer worldwide, highlighting the critical need for the development of new therapies to enhance treatment efficacy. The activation of innate immune receptors given their potent immune stimulatory properties aid in the eradication of cancer cells. In this study, we investigated the immune mechanism and anti-tumor function of a Toll-like receptor 9 (TLR9) agonist, CpG-oligodeoxynucleotide-2722 (CpG-2722), in combination with cyclic dinucleotides, which are agonists of stimulator of interferon genes (STING). Our results revealed that CpG-2722 stimulation increased the expression of Th1 pro-inflammatory cytokines. Stimulation by STING agonists exhibited lower expression of Th1 cytokines but higher expression of Th2 cytokines compared to CpG-2722. However, the combination of these two agonists significantly enhanced Th1 cytokines while reducing Th2 cytokines. Moreover, in vivo experiment showed that both CpG-2722 and 2'3'-c-di-AMP suppressed head and neck tumor growth, with their combination proving more effective than the use of these agonists alone. The combined treatment cooperatively promoted the production of Th1 cytokines and type I interferons, while suppressing Th2 cytokines in the tumors as observed in vitro. Additionally, it led to the accumulation of M1 macrophages, dendritic cells, and T cells, shaping a favorable tumor microenvironment for T cell-mediated tumor killing. The anti-tumor activity of the CpG-2722 and 2'3'-c-di-AMP combination depends on the macrophage presence but does not directly activate M1 macrophage polarization, instead working through a reprogrammed cytokine profile. Furthermore, this combination shows a cooperative anti-tumor activity with anti-PD-1 in treating head and neck tumors. Overall, these findings highlight a Th response and macrophage phenotype reprograming involved functional mechanism underlying the cooperative activity of the combination of TLR9 and STING agonists in the immunotherapy of head and neck cancer.

Moxibustion inhibits the macrophage M1 polarization toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa B signaling pathway by regulating T-cell immunoglobulin and mucin-containing protein-3 in rheumatoid arthritis.

To explore whether moxibustion exerts therapeutic effects on rheumatoid arthritis (RA) by regulating the expression of T-cell immunoglobulin and mucin-containing protein-3 (TIM-3) and subsequently modulating the macrophage M1 polarization toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) signaling pathway. We utilized moxibustion treatment in RA rat models using the Zusanli (ST36) and Shenshu (BL23) acupoints. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of the synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Enzyme-linked immunosorbent assay (ELISA) was applied to verify the efficacy of moxibustion in reducing inflammation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of the TIM-3/TLR4-MyD88-NF-κB signaling pathway-related molecules, and Western blot was used to detect the contents of synovial NF-κB. We established the Freund's complete adjuvant (FCA)-induced RA model in rats. The expression level of M1 polarization signaling pathway TLR4-MyD88-NF-κB and the inflammatory factors interleukin-12(IL-12), tumor necrosis factor alpha (TNF-α), and tumor necrosis factor beta (TNF-β) were significantly increased in the RA model. After moxibustion treatment, the expression level of TLR4-MyD88-NF-κB was significantly decreased, and the inflammatory factors IL-12, TNF-α, and TNF-β were decreased, but the expression level was significantly increased in the RA model. When TIM-3 expression was inhibited, the expression level of TLR4-MyD88-NF-κB, and the inflammatory factors IL-12, TNF-α, and TNF-β were not suppressed, even after moxibustion treatment. Moxibustion regulates the key target TIM-3 by acting on the Zusanli (ST36) and Shenshu (BL23) points, thereby inhibiting the M1 polarization of macrophages; that is, it inhibits the TLR4-MyD88-NF-κB signaling pathway, and finally achieves alleviation of pathological changes and anti-inflammatory effects.

Introducing Degradable Cationic Nanogels Carrying TLR9 Stimulating Oligonucleotides.

Cationic, core-crosslinked nanogel particles are prepared from synthetic biodegradable materials. These fully hydrophilic nanogels offer superior customizability compared to common lipid nanoparticles, thereby circumventing intrinsic immune stimulatory properties. Electrostatic loading allows for complexation of nucleic acids including the immune stimulatory Toll-like receptor 9 (TLR9) agonistCpG-ODN (cytidine-phosphate-guanosine oligodeoxynucleotide). Only when complexed inside nanogels, one can control CpG-ODN's biodistribution, prevent systemic toxicity, and increase bioavailability at target locations. Nanogels are prepared from cyclic aliphatic carbonate monomers with reactive pentafluorophenyl (PFP) esters. First, block copolymers are acquired via cationic ring opening polymerization (CROP) onto polyethylene glycol (PEG). Upon self-assembly in ethanol, the micelles' core is cationically core-crosslinked, and the resulting fully hydrophilic particles exhibit sizes of ≈20nm, also upon loading with CpG-ODN. In vitro, they promote intracellular delivery devoid of carrier-related toxicities, while retaining the immune stimulatory properties of the TLR agonist cargo. In vivo, the nanogels reduce systemic liver immune responses and remain near the injection site. Additionally, delivery into cDC1 (conventional dentritic cells type 1) antigen-presenting cells, which are highly relevant for antitumoral T-cell immune responses, is confirmed. Altogether, cationic, core-crosslinked polycarbonate nanogels show promise for nucleic acid delivery, showcasing controlled immunostimulatory cargo delivery and an enhanced hydrolytic biodegradability profile, highly valuable for cancer immunotherapy.