Innate Immune Pattern Recognition Research Articles

Molecular definition of the endogenous Toll-like receptor signalling pathways.

Innate immune pattern recognition receptors, such as the Toll-like receptors (TLRs), are key mediators of the immune response to infection and central to our understanding of health and disease1. After microbial detection, these receptors activate inflammatory signal transduction pathways that involve IκB kinases, mitogen-activated protein kinases, ubiquitin ligases and other adaptor proteins. The mechanisms that connect the proteins in the TLR pathways are poorly defined. To delineate TLR pathway activities, we engineered macrophages to enable microscopy and proteomic analysis of the endogenous myddosome constituent MyD88. We found that myddosomes form transient contacts with activated TLRs and that TLR-free myddosomes are dynamic in size, number and composition over the course of 24 h. Analysis using super-resolution microscopy revealed that, within most myddosomes, MyD88 forms barrel-like structures that function as scaffolds for effector protein recruitment. Proteomic analysis demonstrated that myddosomes contain proteins that act at all stages and regulate all effector responses of the TLR pathways, and genetic analysis defined the epistatic relationship between these effector modules. Myddosome assembly was evident in cells infected with Listeria monocytogenes, but these bacteria evaded myddosome assembly and TLR signalling during cell-to-cell spread. On the basis of these findings, we propose that the entire TLR signalling pathway is executed from within the myddosome.

A mechanistic modelling approach of the host-microbiota interactions to investigate beneficial symbiotic resilience in the human gut.

The health and well-being of a host are deeply influenced by the interactions with its gut microbiota. Contrasted environmental conditions, such as diseases or dietary habits, play a pivotal role in modulating these interactions, impacting microbiota composition and functionality. Such conditions can also lead to transitions from beneficial to detrimental symbiosis, viewed as alternative stable states of the host-microbiota dialogue. This article introduces a novel mathematical model exploring host-microbiota interactions, integrating dynamics of the colonic epithelial crypt, microbial metabolic functions, inflammation sensitivity and colon flows in a transverse section. The model considers metabolic shifts in epithelial cells based on butyrate and hydrogen sulfide concentrations, innate immune pattern recognition receptor activation, microbial oxygen tolerance and the impact of antimicrobial peptides on the microbiota. Using the model, we demonstrated that a high-protein, low-fibre diet exacerbates detrimental interactions and compromises beneficial symbiotic resilience, underscoring a destabilizing effect towards an unhealthy state. Moreover, the proposed model provides essential insights into oxygen levels, fibre and protein breakdown, and basic mechanisms of innate immunity in the colon and offers a crucial understanding of factors influencing the colon environment.

Assessment on malvidin-3-glucoside interaction with TLR4 via multi-spectroscopic analysis and molecular docking

As one innate immune pattern recognition receptor, Toll-like receptor 4 (TLR4) recently has been considered as a critical player in glucolipid metabolism. Blueberries contain high level of anthocyanins, especially malvidin-3-glucoside (Mv-3-glc), which contribute the anti-inflammatory, hypoglycemic, and hypolipidemic effects. It is speculated that Mv-3-glc is able to possess these functions by binding to TLR4. Here, the noncovalent interactions of Mv-3-glc and TLR4 was explored through multi-techniques including fluorescence and ultraviolet–visible (UV–Vis) absorption spectroscopy, as well as molecular docking. The results demonstrated that Mv-3-glc was able to quench TLR4 intrinsic fluorescence effectively. A stable complex was formed spontaneously and the reaction was exothermic. The degree of binding of Mv-3-glc to TLR4 showed a strong dependence on the chemical concentration, temperature, and pH values. The negative signs for enthalpy (ΔH = -69.1 ± 10.8 kJ/mol) and entropy (ΔS = -105.0 ± 12.3 J/mol/K) from the interaction of the Mv-3-glc and TLR4 shows that the major driving forces are the hydrogen bonding and van der Waals' force, which is consistent with the molecular docking results. In addition, molecular docking predicted that the active center with specific amino acid residues, Phe126, Ser127, Leu54, Ile153, and Tyr131 was responsible for the site of Mv-3-glc binding to TLR4/myeloid differentiation protein-2 (MD-2). These findings confirmed that Mv-3-glc could bind to TLR4, which would be beneficial to understand the target therapeutic effects of blueberry anthocyanins on TLR4 in regulating glucolipid metabolism.

Cytosolic DNA sensor AIM2 promotes KRAS-driven lung cancer independent of inflammasomes.

Constitutively active KRAS mutations are among the major drivers of lung cancer, yet the identity of molecular co-operators of oncogenic KRAS in the lung remains ill-defined. The innate immune cytosolic DNA sensor and pattern recognition receptor (PRR) Absent-in-melanoma 2 (AIM2) is best known for its assembly of multiprotein inflammasome complexes and promoting an inflammatory response. Here, we define a role for AIM2, independent of inflammasomes, in KRAS-addicted lung adenocarcinoma (LAC). In genetically defined and experimentally induced (nicotine-derived nitrosamine ketone; NNK) LAC mouse models harboring the KrasG12D driver mutation, AIM2 was highly upregulated compared with other cytosolic DNA sensors and inflammasome-associated PRRs. Genetic ablation of AIM2 in KrasG12D and NNK-induced LAC mouse models significantly reduced tumor growth, coincident with reduced cellular proliferation in the lung. Bone marrow chimeras suggest a requirement for AIM2 in KrasG12D-driven LAC in both hematopoietic (immune) and non-hematopoietic (epithelial) cellular compartments, which is supported by upregulated AIM2 expression in immune and epithelial cells of mutant KRAS lung tissues. Notably, protection against LAC in AIM2-deficient mice is associated with unaltered protein levels of mature Caspase-1 and IL-1β inflammasome effectors. Moreover, genetic ablation of the key inflammasome adapter, ASC, did not suppress KrasG12D-driven LAC. In support of these invivo findings, AIM2, but not mature Caspase-1, was upregulated in human LAC patient tumor biopsies. Collectively, our findings reveal that endogenous AIM2 plays a tumor-promoting role, independent of inflammasomes, in mutant KRAS-addicted LAC, and suggest innate immune DNA sensing may provide an avenue to explore new therapeutic strategies in lung cancer.

Abstract 2691: Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancer cases, and is among the most aggressive and lethal malignancies worldwide. PDAC is driven by genetic alterations in the pancreatic epithelium (e.g. KRAS, TRP53) coupled with a dysregulated innate immune response, the latter leading to an inflammatory tumor microenvironment enriched in innate immune cells (e.g. macrophages). However, immune-based treatment regimens for PDAC patients have primarily focused on immunotherapy with adaptive immune checkpoint inhibitors (e.g. PD-1) which disappointingly have yielded minimal clinical benefit. Thus, there is an unmet clinical need to identity disease-associated innate immune regulators as therapeutic targets in PDAC. Innate immune responses depend on a series of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs) that are expressed in immune and non-immune cells. PRRs are classified into several structurally and functionally conserved subfamilies, including Toll-like receptors (TLRs), Absent in melanoma 2 (AIM2)-like receptors (ALRs), and Nod-like receptors (NLRs). A subset of NLRs and AIM2 are also well documented for their formation of multiprotein complexes called ‘inflammasomes’ - comprising the adaptor ASC and Caspase-1 - which direct the maturation and release of bioactive pro-inflammatory cytokines, IL-18 and IL-1β. However, the role of these critical innate immune regulators in PDAC has been underexplored, and is virtually unknown. By coupling endoscopic ultrasound-guided fine needle aspiration (EUS FNA) to capture tumor biopsies from all stages of PDAC, with whole transcriptome profiling of PDAC patient primary tumors, we reveal specific enrichment of the innate immune TLR2 and ASC inflammasome molecular pathways. Augmented TLR2 and ASC expression was prognostic for low survival, and a “TLR2 activation” signature was also predictive for chemoresistance. Antibody-mediated anti-TLR2 therapy suppressed tumor growth in human PDAC tumor xenografts (patient and cell line) in both immunocompromised and ‘humanized’ mice. With respect to ASC, using the genetically engineered KPC PDAC mouse model, we show that Caspase-1 activation, a read-out for inflammasome activation, is upregulated in pancreatic tumors. Moreover, genetic ablation of ASC in KPC mice markedly suppressed the development of pancreatic tumors, which was associated with significant reductions in levels of both Caspase-1 activation and inflammation. Transcriptome profiling further identified unique downstream molecular networks associated with a range of oncogenic cellular processes. Taken together, our results support innate immune TLR2- and ASC inflammasome-based therapeutic targeting in PDAC, with further clinical utility that ASC inflammasome and TLR2 activation is prognostic, with TLR2 also predictive for chemoresponsiveness. Citation Format: Bassam Kashgari, Louise McLeod, Linden J. Gearing, Daniel Croagh, Brendan J. Jenkins. Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2691.

The Role of the Toll-like Receptor 2 and the cGAS-STING Pathways in Breast Cancer: Friends or Foes?

Breast cancer stands as a primary malignancy among women, ranking second in global cancer-related deaths. Despite treatment advancements, many patients progress to metastatic stages, posing a significant therapeutic challenge. Current therapies primarily target cancer cells, overlooking their intricate interactions with the tumor microenvironment (TME) that fuel progression and treatment resistance. Dysregulated innate immunity in breast cancer triggers chronic inflammation, fostering cancer development and therapy resistance. Innate immune pattern recognition receptors (PRRs) have emerged as crucial regulators of the immune response as well as of several immune-mediated or cancer cell-intrinsic mechanisms that either inhibit or promote tumor progression. In particular, several studies showed that the Toll-like receptor 2 (TLR2) and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways play a central role in breast cancer progression. In this review, we present a comprehensive overview of the role of TLR2 and STING in breast cancer, and we explore the potential to target these PRRs for drug development. This information will significantly impact the scientific discussion on the use of PRR agonists or inhibitors in cancer therapy, opening up new and promising avenues for breast cancer treatment.

The battle between the innate immune cGAS-STING signaling pathway and human herpesvirus infection.

The incidence of human herpesvirus (HHVs) is gradually increasing and has affected a wide range of population. HHVs can result in serious consequences such as tumors, neonatal malformations, sexually transmitted diseases, as well as pose an immense threat to the human health. The cGAS-STING pathway is one of the innate immune pattern-recognition receptors discovered recently. This article discusses the role of the cGAS-STING pathway in human diseases, especially in human herpesvirus infections, as well as highlights how these viruses act on this pathway to evade the host immunity. Moreover, the author provides a comprehensive overview of modulators of the cGAS-STING pathway. By focusing on the small molecule compounds based on the cGAS-STING pathway, novel targets and concepts have been proposed for the development of antiviral drugs and vaccines, while also providing a reference for the investigation of disease models related to the cGAS-STING pathway. HHV is a double-stranded DNA virus that can trigger the activation of intracellular DNA sensor cGAS, after which the host cells initiate a cascade of reactions that culminate in the secretion of type I interferon to restrict the viral replication. Meanwhile, the viral protein can interact with various molecules in the cGAS-STING pathway. Viruses can evade immune surveillance and maintain their replication by inhibiting the enzyme activity of cGAS and reducing the phosphorylation levels of STING, TBK1 and IRF3 and suppressing the interferon gene activation. Activators and inhibitors of the cGAS-STING pathway have yielded numerous promising research findings in vitro and in vivo pertaining to cGAS/STING-related disease models. However, there remains a dearth of small molecule modulators that have been successfully translated into clinical applications, which serves as a hurdle to be overcome in the future.

Liposome-Based Co-Immunotherapy with TLR Agonist and CD47-SIRPα Checkpoint Blockade for Efficient Treatment of Colon Cancer

Antitumor immunity is an essential component of cancer therapy and is primarily mediated by the innate immune response, which plays a critical role in initiating and shaping the adaptive immune response. Emerging evidence has identified innate immune checkpoints and pattern recognition receptors, such as CD47 and Toll-like receptor 7 (TLR7), as promising therapeutic targets for cancer treatment. Based on the fusion protein Fc-CV1, which comprises a high-affinity SIRPα variant (CV1), and the Fc fragment of the human IgG1 antibody, we exploited a preparation which coupled Fc-CV1 to imiquimod (TLR7 agonist)-loaded liposomes (CILPs) to actively target CT26. WT syngeneic colon tumor models. In vitro studies revealed that CILPs exhibited superior sustained release properties and cell uptake efficiency compared to free imiquimod. In vivo assays proved that CILPs exhibited more efficient accumulation in tumors, and a more significant tumor suppression effect than the control groups. This immunotherapy preparation possessed the advantages of low doses and low toxicity. These results demonstrated that a combination of immune checkpoint blockade (ICB) therapy and innate immunity agonists, such as the Fc-CV1 and imiquimod-loaded liposome preparation utilized in this study, could represent a highly effective strategy for tumor therapy.

Detection of Nanoparticles' Ability to Stimulate Toll-Like Receptors Using HEK-Blue Reporter Cell Lines.

Nanoparticles can be used to formulate Toll-like receptor (TLR) agonists as vaccine and immunotherapy adjuvants or contain undesirable contaminants (e.g., endotoxin, CpG DNA, flagellin) with TLR-agonist activity. In both scenarios, the activation of the innate immune pattern recognition receptor leads to the inflammatory response that can be beneficial as in the case with vaccines and immunotherapies or adverse as in the case with contaminants. The protocol described herein utilizes commercially available reporter cell lines expressing individual TLRs, which, upon activation with their cognate agonists, stimulate the cells to produce secreted alkaline phosphatase detectable using a plate reader.

492. Dectin-1 Stimulation Promotes a Distinct Inflammatory Signature in the Setting of Aging and HIV-infection

Abstract Background Dectin-1 is an innate immune pattern recognition receptor that recognizes and binds b-1,3/1,6 glucans on fungal pathogens. Methods We evaluated Dectin-1 function in innate immune cells (Monocytes and Dendritic cells) in a cohort of HIV-positive and HIV-negative young and older adults (n= 81). PBMCs were stimulated with whole glucan particles (1,3/1,6-b-glucan, WGP), a specific Dectin-1 agonist and analyzed via intracellular cytokine staining (ICS) and multicolor flow cytometry of monocytes and dendritic cells. Sorted CD14+ CD16+ monocytes stimulated with WGP and subjected to RNA-seq analysis. Results Stimulation of monocytes with β-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-a, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-a production. This increase in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-a and coagulation response that was already increased at baseline, and a persistent IFN-α and IFN-γ response. HIV-older adults also demonstrated an activated dendritic cell signature and M1 macrophage signature upon stimulation that was not seen in young individuals. Pathways upregulated in all cohorts were the signaling pathways MTORC-1, hypoxia pathway (pathways involved in trained immunity) and the unfolded protein response. Of note MTORC-1 signaling was upregulated at baseline in HIV-Older adults. Conclusion Overall, our study demonstrates that increased age and HIV-infection can affect the function of Dectin-1 and lead to a distinct inflammatory signature. Dectin-1 stimulation in HIV-Older adults led to a more pro-inflammatory phenotype that was demonstrated through both protein and gene expression. Dectin-1 induced inflammation in HIV-infected individuals may be contributing to the pro-inflammatory environment that is seen in the setting of both aging and HIV-infection. Disclosures All Authors: No reported disclosures.

Physiological mechanisms of TLR4 in glucolipid metabolism regulation: Potential use in metabolic syndrome prevention

Over-nourishment or an unbalanced diet has been linked to an increase in the prevalence of metabolic syndrome. An imbalance in glucolipid metabolism is a major cause of metabolic syndrome, which has consequences for human health. Toll-like receptor 4 (TLR4), a member of the innate immune pattern recognition receptor family, is involved in inflammation-related disorders, autoimmune diseases, and tumors. Recent research has shown that TLR4 plays a key role in glucolipid metabolism, which is linked to insulin resistance, intestinal flora, and the development of chronic inflammation. TLR4 activation regulates glucolipid metabolism and contributes to the dynamic relationship between innate immunity and nutrition-related disorders. Further, TLR4 regulates glucolipid metabolism by controlling glycolysis and pyruvate oxidative decarboxylation, interfering with insulin signaling, regulating adipogenic gene expression levels, influencing preadipocyte differentiation and lipid accumulation, and altering the intestinal microbiota and permeability. TLR4 functions may provide new therapeutic applications for the prevention and treatment of metabolic syndrome. The purpose of this review is to enrich mechanistic research of diabetes, atherosclerosis, and other nutrition-related disorders by summarizing the role of TLR4 in the regulation of glucolipid metabolism as well as its physiological mechanisms.

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

Activation and regulation mechanisms of NOD-like receptors based on structural biology.

Innate immunity is a primary defense system against microbial infections. Innate immune pattern recognition receptors (PRRs) play pivotal roles in detection of invading pathogens. When pathogens, such as bacteria and viruses, invade our bodies, their components are recognized by PRRs as pathogen-associated molecular patterns (PAMPs), activating the innate immune system. Cellular components such as DNA and RNA, acting as damage-associated molecular patterns (DAMPs), also activate innate immunity through PRRs under certain conditions. Activation of PRRs triggers inflammatory responses, interferon-mediated antiviral responses, and the activation of acquired immunity. Research on innate immune receptors is progressing rapidly. A variety of these receptors has been identified, and their regulatory mechanisms have been elucidated. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) constitute a major family of intracellular PRRs and are involved in not only combating pathogen invasion but also maintaining normal homeostasis. Some NLRs are known to form multi-protein complexes called inflammasomes, a process that ultimately leads to the production of inflammatory cytokines and induces pyroptosis through the proteolytic cascade. The aberrant activation of NLRs has been found to be associated with autoimmune diseases. Therefore, NLRs are considered targets for drug discovery, such as for antiviral drugs, immunostimulants, antiallergic drugs, and autoimmune disease drugs. This review summarizes our recent understanding of the activation and regulation mechanisms of NLRs, with a particular focus on their structural biology. These include NOD2, neuronal apoptosis inhibitory protein (NAIP)/NLRC4, NLR family pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, and NLRP9. NLRs are involved in a variety of diseases, and their detailed activation mechanisms based on structural biology can aid in developing therapeutic agents in the future.

Novel Scaffolds for Modulation of NOD2 Identified by Pharmacophore-Based Virtual Screening.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an innate immune pattern recognition receptor responsible for the recognition of bacterial peptidoglycan fragments. Given its central role in the formation of innate and adaptive immune responses, NOD2 represents a valuable target for modulation with agonists and antagonists. A major challenge in the discovery of novel small-molecule NOD2 modulators is the lack of a co-crystallized complex with a ligand, which has limited previous progress to ligand-based design approaches and high-throughput screening campaigns. To that end, a hybrid docking and pharmacophore modeling approach was used to identify key interactions between NOD2 ligands and residues in the putative ligand-binding site. Following docking of previously reported NOD2 ligands to a homology model of human NOD2, a structure-based pharmacophore model was created and used to virtually screen a library of commercially available compounds. Two compounds, 1 and 3, identified as hits by the pharmacophore model, exhibited NOD2 antagonist activity and are the first small-molecule NOD2 modulators identified by virtual screening to date. The newly identified NOD2 antagonist scaffolds represent valuable starting points for further optimization.

The Role of Cytokines and Pattern Recognition Receptors in Inflammation Caused by Helicobacter pylori Infection in Gastric Cancer

One of the most common and most critical mortality causes in modern society is gastrointestinal (GI) cancer. Helicobacter pylori is one of the most potent risk factors of gastric cancer. This bacterium can suppress the immune system by various pathogenic factors and mechanisms, lead to chronic inflammatory responses, and play a critical role in the induction of cancer. The first part of this article begins with a description of H. pylori carcinogenicity and in the next part, the roles of innate immunity pattern recognition receptors (PRRs), Toll-like receptors (TLRs), cytokines, and inflammatory regulatory mechanisms in H. pylori infection are discussed. According to our current knowledge, screening and on-time diagnosis of the disease, timely treatment, and immediate eradication of H. pylori are the most critical ways to barricade gastric cancer progression. In the end, it is suggested that, considering the expensive costs of gastric cancer treatment, H. pylori diagnostic screening tests be done at least annually in middle-aged and at-risk people.

Context-dependent functions of pattern recognition receptors in cancer.

The immune system plays a critical role in shaping all facets of cancer, from the early initiation stage through to metastatic disease and resistance to therapy. Our understanding of the importance of the adaptive arm of the immune system in antitumour immunity has led to the implementation of immunotherapy with immune checkpoint inhibitors in numerous cancers, albeit with differing efficacy. By contrast, the clinical utility of innate immunity in cancer has not been exploited, despite dysregulated innate immunity being a feature of at least one-third of all cancers associated with tumour-promoting chronic inflammation. The past two decades have seen innate immune pattern recognition receptors (PRRs) emerge as critical regulators of the immune response to microbial infection and host tissue damage. More recently, it has become apparent that in many cancer types, PRRs play a central role in modulating a vast array of tumour-inhibiting and tumour-promoting cellular responses both in immune cells within the tumour microenvironment and directly in cancer cells. Herein, we provide a comprehensive overview of the fast-evolving field of PRRs in cancer, and discuss the potential to target PRRs for drug development and biomarker discovery in a wide range of oncology settings.

Toll-like receptors change morphine-induced antinociception, tolerance and dependence: Studies using male and female TLR and signalling gene KO mice

Toll-like receptors (TLR) have been proposed as a site of action that alters opioid pharmacodynamics. However, a comprehensive assessment of acute opioid antinociception, tolerance and withdrawal behaviours in genetic null mutant strains with altered innate immune signalling has not been performed. Nor has the impact of genetic deletion of TLR2/4 on high-affinity opioid receptor binding. Here we show that diminished TLR signalling potentiates acute morphine antinociception equally in male and female mice. However, only male TIR8 null mutant mice showed reduced morphine analgesia. Analgesic tolerance was prevented in TLR2 and TLR4 null mutants, but not MyD88 animals. Withdrawal behaviours were only protected in TLR2−/− mice. In silico docking simulations revealed opioid ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. There was no binding of [3H](-)-naloxone or [3H]diprenorphine to TLR4 in the concentrations explored. These data confirm that opioids have high efficacy activity at innate immune pattern recognition binding sites but do not bind to TLR4 and identify critical pathway and sex-specific effects of the complex innate immune signalling contributions to opioid pharmacodynamics. These data further support the behavioural importance of the TLR-opioid interaction but fail to demonstrate direct evidence for high-affinity binding of the TLR4 signalling complex to ligands.

Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Full-length transcriptome sequencing of Heliocidaris crassispina using PacBio single-molecule real-time sequencing

The lack of high-throughput sequencing data makes the research progress of Heliocidaris crassispina slow. Therefore, we used PacBio single-molecule real-time sequencing to generate the first full-length transcriptome. Here, 31,181 isoforms were obtained, with an average length of 2383.20 and a N50 length of 2732 bp. Meanwhile, 764 alternative splicing (AS) events, 5098 long-noncoding RNAs (LncRNAs), 6978 simple sequence repeats (SSRs), and 950 hypothetical transcript factors (TFs) were identified. Moreover, five key innate immune pattern recognition receptors (PRRs), including toll-like receptor (TLR), NACHT domain and leucine-rich repeat (NLR), scavenger receptor cysteine-rich (SRCR), peptidoglycan recognition proteins (PGRP), and gram-negative binding proteins (GNBP), were searched in the transcriptome. In addition, 37 isoforms enriched in KEGG and GO immune systems were also detected. The study provid abundant data support for the current research on H. crassispina.

Novel Evidence That Alternative Activation Pathway of Complement Cascade (ComC) Regulates Optimal Homing and Engraftment of Hematopoietic Stem/Progenitor Cells (HSPCs) in Reactive Oxygen Species (ROS) - Nlrp3 Inflammasome-Dependent Manner

Background. We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required not only for mobilization of hematopoietic stem progenitor cells (HSPCs) but also for their proper homing and engraftment after transplantation to bone marrow (BM) (Leukemia 2012; 26:106-16). The ComC consists of zymogen proteins that become activated in a cascade-mediated manner by the i) classical, ii) mannan-binding lectin (MBL), or iii) alternative pathway. However, it is not clear which of these pathways plays a crucial role in inducing state of sterile inflammation in recipient BM conditioned for transplantation. Interestingly, the alternative pathway of ComC activation, in contrast to the other two pathways, is not triggered by antibodies or specific structures expressed on the surface of invading microorganisms, but is continuously activated and “ticking” by the spontaneous hydrolysis of the third component of the ComC (C3), which is the most abundant complement protein present in blood plasma. This process of C3 hydrolysis is hyperactivated in response to tissue/organ damage and changes the structure of C3 in order to promote binding of factor B (FB) that initiates the amplification process by which more C3b molecules and C3b-Bb convertases are created responsible for activation of the ComC. We also recently demonstrated that homing and engraftment of HSPCs to BM is mediated by activation of innate immunity pattern recognition receptor Nlrp3 inflammasome (Leukemia 2020; 34:1512-1523). On the other hand reactive oxygen species (ROS) are known activators of Nlrp3 inflammasome. Hypothesis. We hypothesized that sensitive and “continuously ticking” in peripheral blood (PB) alternative pathway of ComC activation facilitates homing and engraftment of HSPCs in response to myeloablation triggered sterile inflammation of hematopoietic microenvironment in donor BM. We also hypothesized that this depends on the activation of the Nlrp3 inflammasome in ROS-dependent manner. Materials and Methods. Activation of ComC and Nlrp3 inflammasome in BM of conditioned for hematopoietic transplantation by lethal irradiation mice was evaluated by C5a ELISA assay and immunofluorescence glow assay measuring activation of Nlrp3 inflammasome product that is activated caspase-1, respectively. We also run shotgun proteomic analysis with BM conditioned media and BMMNCs extracts. The release of ROS was measured by a colorimetric assay. To assess the involvement of alternative pathway of ComC activation, we performed homing and engraftment experiments in wild-type (WT) and FB-deficient mice, that have defect in alternative pathway of ComC activation. FB-KO and WT animals were transplanted with WT BMMNC. We also analyzed changes in the BM microenvironment in response to lethal irradiation in WT and FB-KO mice at mRNA and protein level. Results. We demonstrate for a first time that conditioning for transplantation by myeloablative irradiation induces in BM state of sterile inflammation reflected by activation of ComC, the release of reactive oxygen species (ROS), and activation of Nlrp3 inflammasome in ROS-dependent manner. Moreover, as compared to WT animals, all these pathways were significantly inhibited in FB-KO mice. As a result of this FB-KO animals displayed defective homing and engraftment after transplantation of WT BMMNC. This correlated at molecular level by the decreased expression of cell adhesion molecules and group of structural proteins involved in so called “docking structures” necessary for cell migration and homing as well as for Nlrp3 inflammasome complex and caspase 1, 2, 4 and 6. Moreover, FB-KO mice demonstrated lower level of SDF-1 and KL in BM after myeloablative conditioning for transplantation. Conclusions. We provide for a first time an evidence that myeloablative conditioning for transplantation by lethal irradiation activates in ComC-ROS-Nlrp3 inflammasome - dependent manner a state of sterile inflammation in the BM microenvironment, required for optimal homing and engraftment. This data also explains this phenomenon at molecular level and provides an evidence for a crucial involvement of alternative pathway of ComC activation. DisclosuresNo relevant conflicts of interest to declare.