Pathogen-associated Molecular Patterns Research Articles

Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effect of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune related-genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65×108 CFU/mL) were evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV) group, recombinant plasmid pEGFP-Gal9 treatment (Gal9) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O.macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O.macrolepis infected with A. hydrophila in Gal9 group at 24 h significantly lower than that in EV group (P < 0.05), and the survival rate has increased from 15% to 35%. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play important regulatory in glycolysis / gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.

In Vitro Analysis of Tandem Peptides from Human CD5 and CD6 Scavenger Receptors as Potential Anti-Cryptococcal Agents.

Cryptococcus neoformans is included in the World Health Organization fungal priority pathogen list, complied to expedite improved research and public-health interventions. The limited number of available antifungal drugs, their associated toxicity, and the emergence of drug-resistant strains make the development of new therapeutic strategies mandatory. Pattern-recognition receptors (PRRs) from the host's innate immune system constitute a potential source of new antimicrobial agents. CD5 and CD6 are lymphoid members of the ancient scavenger receptor cysteine-rich superfamily (SRCR-SF) which bind pathogen-associated molecular patterns (PAMPs) of fungal and bacterial origin. Evidence supports the concept that such binding maps to 11-mer sequences present in each of their three SRCR extracellular domains. Herein, we have designed synthetic peptides containing tandems of such 11-mer sequences (namely CD5-T and CD6-T) and analyzed their C. neoformans-binding properties in vitro. Our results show both inhibitory effects on fungal growth and an ability to impact capsule formation and titanization, two critical virulence factors of C. neoformans involved in immune evasion. These effects hold promise for CD5-T and CD6-T peptides as single or adjuvant therapeutic agents against cryptococcosis.

Overexpression of plant chitin receptors in wheat confers broad‐spectrum resistance to fungal diseases

SUMMARYWheat (Triticum aestivum L.) is a globally staple crop vulnerable to various fungal diseases, significantly impacting its yield. Plant cell surface receptors play a crucial role in recognizing pathogen‐associated molecular patterns (PAMPs) and activating PAMP‐triggered immunity, boosting resistance against a wide range of plant diseases. Although the role of plant chitin receptor CERK1 in immune recognition and defense has been established in Arabidopsis and rice, its function and potential agricultural applications in enhancing resistance to crop diseases remain largely unexplored. Here, we identify and characterize TaCERK1 in Triticeae crop wheat, uncovering its involvement in chitin recognition, immune regulation, and resistance to fungal diseases. By a comparative analysis of CERK1 homologs in Arabidopsis and monocot crops, we demonstrate that AtCERK1 in Arabidopsis elicits the most robust immune response. Moreover, we show that overexpressing TaCERK1 and AtCERK1 in wheat confers resistance to multiple fungal diseases, including Fusarium head blight, stripe rust, and powdery mildew. Notably, transgenic wheat lines with moderately expressed AtCERK1 display superior disease resistance and heightened immune responses without adversely affecting growth and yield, compared to TaCERK1 overexpression transgenics. Our findings highlight the significance of plant chitin receptors across diverse plant species and suggest potential strategies for bolstering crop resistance against broad‐spectrum diseases in agricultural production through the utilization of plant immune receptors.

Features of the expression of genes of antimicrobial peptides and the microbiome at the level of the mucous membrane of the upper respiratory tract during aging

Today, the proportion of elderly and senile people is steadily growing throughout the world. The most important factors in the first line of immune defense of the mucous membranes of the upper respiratory tract are β-defensins, which are a group of secretory proteins with antimicrobial activity. The aim of this study was to study the expression of genes for antimicrobial peptides β-defensins and the composition of the microbiome of the mucous membranes of the upper respiratory tract in elderly people and long-livers with various aging phenotypes.The main study group included 67 centenarians and 49 elderly people, who were further divided into two subgroups depending on the course of aging (pathological and successful aging). Nucleic acids were isolated from nasopharyngeal scrapings and the expression levels of the DEFB1 and DEFB4 genes were determined using real-time polymerase chain reaction. The composition of the microbiota in nasopharyngeal swabs was determined by MALDI-TOF mass spectrometry.In analyzing the expression of the DEFB1 gene in elderly people and centenarians with successful and pathological aging phenotypes, no difference was revealed between the groups. Expression of the DEFB4 gene was increased in centenarians with pathological aging compared to centenarians with successful aging and in the elderly group. Excessive production of antimicrobial peptides is dual in nature; on the one hand, they provide the first line of defense against microorganisms, and on the other, they are cytotoxic to their own cells. An increase in the expression of the DEFB4 gene during aging may be due to an increase in the number of pathogen-associated molecular patterns, which can be one’s own microbiota and/or components of microbial metabolism. Analysis of the microbiota composition showed an increase in biodiversity in individuals with a successful aging phenotype compared to a pathological phenotype. Particular attention is paid to Staphylococcus spp., the species composition of which depends on the aging phenotype. In the pathological aging group, the frequency of St. aureus colonization is significantly higher than in the successful aging group.Thus, overexpression of the DEFB4 gene and changes in the composition of the microbiota of the mucous membranes of the upper respiratory tract may be one of the mechanisms explaining the increased susceptibility to infections in various aging phenotypes.

Rapid assembly of mixed thiols for toll-like receptor-based electrochemical pathogen sensing.

Herein, we describe a rapid and facile fabrication of electrochemical sensors utilizing two different toll-like receptor (TLR) proteins as biorecognition elements to detect bacterial pathogen associated molecular patterns (PAMPs). Using potential-assisted self-assembly, binary mixtures of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MCH), or MUA and an in-house synthesized zwitterionic sulfobetaine thiol (DPS) were assembled on a gold working electrode within 5 minutes, which is >200 times shorter than other TLR sensors' preparation time. Electrochemical methods and X-ray photoelectron microscopy were used to characterize the SAM layers. SAMs composed of the betaine terminated thiol exhibited superior resistance to nonspecific interactions, and were used to develop the TLR sensors. Biosensors containing two individually immobilized TLRs (TLR4 and TLR9) were fabricated on separate MUA-DPS SAM modified Au electrodes (MUA-DPS/Au) and tested for their response towards their respective PAMPs. The changes to electron transfer resistance in EIS of the TLR4/MUA-DPS/Au sensor showed a detection limit of 4 ng mL-1 for E. coli 0157:H7 endotoxin (lipopolysaccharide, LPS) and a dynamic range of up to 1000 ng mL-1. The TLR4-based sensor showed negligible response when tested with LPS spiked human plasma samples, showing no interference from the plasma matrix. The TLR9/MUA-DPS/Au sensor responded linearly up to 350 μg mL-1 bacterial DNA, with a detection limit of 7 μg mL-1. The rapid assembly of the TLR sensors, excellent antifouling properties of the mixed SAM assembly, small size and ease of operation of EIS hold great promise for the development of a portable and automated broad-spectrum pathogen detection and classification tool.

A Mitogen-Activated Protein Kinase Pathway Is Required for Bacillus amyloliquefaciens PMB05 to Enhance Disease Resistance to Bacterial Soft Rot in Arabidopsis thaliana.

When a plant is infected by a pathogen, endogenous immune responses are initiated. When the initiation of these defense responses is induced by a pathogen-associated molecular pattern (PAMP) of a pathogen, it is called PAMP-triggered immunity (PTI). Previous studies have shown that Bacillus amyloliquefaciens PMB05 can enhance PTI signals and improve disease control of bacterial soft rot and wilt in Arabidopsis thaliana. In the context of controlling bacterial wilt disease, the involvement of a mitogen-activated protein kinase (MAPK) signaling pathway has been established. Nevertheless, it remains unclear whether this pathway is also required for B. amyloliquefaciens PMB05 in controlling bacterial soft rot. In this study, A. thaliana ecotype Columbia (Col-0) and its mutants on a MAPK pathway-related pathway were used as a model and established that the ability of B. amyloliquefaciens PMB05 to control soft rot requires the participation of the MAPK pathway. Moreover, the enhancement of disease resistance by PMB05 is highly correlated with the activation of reactive oxygen species generation and stomata closure, rather than callose deposition. The spray inoculation method was used to illustrate that PMB05 can enhance stomatal closure, thereby restricting invasion by the soft rot bacterium. This control mechanism has also been demonstrated to require the activation of the MAPK pathway. This study demonstrates that B. amyloliquefaciens PMB05 can accelerate stomata closure via the activation of the MAPK pathway during PTI, thereby reducing pathogen invasion and achieving disease resistance against bacterial soft rot.

Abstract B061: Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy

Abstract Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) has a dismal 5-year overall survival (OS) rate of 3%. Current standard of care provides only modest anti-cancer impact. Preclinical models show that combinations of myeloid agonists can induce strong anti- tumor activity in PDAC tumors resistant to immunotherapy, including immune checkpoint blockade (anti-CTLA4, anti-PD1). This study combines odetiglucan, a beta glucan pathogen- associated molecular pattern (PAMP), with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody, in the maintenance setting after cytotoxic chemotherapy induction. Preliminary findings are presented as the study was closed early for financial reasons. Methods: Patients with mPDAC who had not progressed after 4-8 months of 1L chemotherapy were enrolled and received weekly odetiglucan (4 mg/kg) plus every 3-week dosing of CDX-1140 (1.5 mg/kg). The primary endpoints were safety, MTD and RP2D, with secondary endpoints including DOR, PFS, ORR, and OS. Tissue and blood samples were collected at baseline and during treatment to evaluate immune pharmacodynamic responses. Results: A total of 5 patients received treatment on study including 4 patients who were efficacy evaluable and 1 patient who withdrew due to adverse event (arthralgia) after beginning treatment. Treatment-related emergent adverse events were seen in all patients with most common AEs including grade 1-2 arthralgia, chills, and fatigue. Grade 3 AEs included arthralgia (n=1), fatigue (n=1), and leukopenia (n=2). The ORR included 1 PR, 1 SD, and 2 PD. Two patients remained on treatment for &amp;gt;100 days. The pharmacodynamic profile was consistent with the mode of action of odetiglucan and CDX-1140, including a reduction in peripheral Dectin-1 expressing monocytes and transient decreases in peripheral B cells. Patients with disease control (PR or SD, n=2) compared to patients with progression (PD, n=2) showed expansion of peripheral Ki67+ CD8+ T cells following treatment. Analysis of a paired tumor biopsy from 1 patient with PD confirmed penetration of odetiglucan into a liver metastasis with expansion of CD11b+ macrophages and a reduction of cytokeratin+ tumor content, but without significant changes in T cell infiltration. Conclusions: The combination of odetiglucan and CDX-1140 was feasible and safe when administered as maintenance therapy in patients with mPDAC whose disease had not progressed on 1L chemotherapy. Treatment produced encouraging activity with changes in immunological correlates associated with disease control. These early results support further investigation combining odetiglucan with a CD40 agonist in mPDAC. Citation Format: Mark H O'Hara, Max Wattenberg, Ignacio Garrido-Laguna, Eileen M O'Reilly, Michael Yellin, Tibor Keler, Michele Gargano, Nick Niles, Jeremy Drees, Nandita Bose, Gregory L Beatty. Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B061.

Abstract PR-10: Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy

Abstract Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) has a dismal 5-year overall survival (OS) rate of 3%. Current standard of care provides only modest anti-cancer impact. Preclinical models show that combinations of myeloid agonists can induce strong anti-tumor activity in PDAC tumors resistant to immunotherapy, including immune checkpoint blockade (anti-CTLA4, anti-PD1). This study combines odetiglucan, a beta glucan pathogen-associated molecular pattern (PAMP), with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody, in the maintenance setting after cytotoxic chemotherapy induction. Preliminary findings are presented as the study was closed early for financial reasons. Methods: Patients with mPDAC who had not progressed after 4-8 months of 1L chemotherapy were enrolled and received weekly odetiglucan (4 mg/kg) plus every 3-week dosing of CDX-1140 (1.5 mg/kg). The primary endpoints were safety, MTD and RP2D, with secondary endpoints including DOR, PFS, ORR, and OS. Tissue and blood samples were collected at baseline and during treatment to evaluate immune pharmacodynamic responses. Results: A total of 5 patients received treatment on study including 4 patients who were efficacy evaluable and 1 patient who withdrew due to adverse event (arthralgia) after beginning treatment. Treatment-related emergent adverse events were seen in all patients with most common AEs including grade 1-2 arthralgia, chills, and fatigue. Grade 3 AEs included arthralgia (n=1), fatigue (n=1), and leukopenia (n=2). The ORR included 1 PR, 1 SD, and 2 PD. Two patients remained on treatment for &amp;gt;100 days. The pharmacodynamic profile was consistent with the mode of action of odetiglucan and CDX-1140, including a reduction in peripheral Dectin-1 expressing monocytes and transient decreases in peripheral B cells. Patients with disease control (PR or SD, n=2) compared to patients with progression (PD, n=2) showed expansion of peripheral Ki67+ CD8+ T cells following treatment. Analysis of a paired tumor biopsy from 1 patient with PD confirmed penetration of odetiglucan into a liver metastasis with expansion of CD11b+ macrophages and a reduction of cytokeratin+ tumor content, but without significant changes in T cell infiltration. Conclusions: The combination of odetiglucan and CDX-1140 was feasible and safe when administered as maintenance therapy in patients with mPDAC whose disease had not progressed on 1L chemotherapy. Treatment produced encouraging activity with changes in immunological correlates associated with disease control. These early results support further investigation combining odetiglucan with a CD40 agonist in mPDAC. Citation Format: Mark H O'Hara, Max Wattenberg, Ignacio Garrido-Laguna, Eileen M O'Reilly, Michael Yellin, Tibor Keler, Michele Gargano, Nick Niles, Jeremy Drees, Nandita Bose, Gregory L Beatty. Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-10.

Lipoteichoic Acid from Heyndrickxia coagulans HOM5301 Modulates the Immune Response of RAW 264.7 Macrophages.

Heyndrickxia coagulans (formerly Bacillus coagulans) has been increasingly utilized as an immunomodulatory probiotics. Oral administration of H. coagulans HOM5301 significantly boosted both innate and adaptive immunity in mice, particularly by increasing the phagocytic capacity of monocytes/macrophages. Lipoteichoic acid (LTA), a major microbe-associated molecular pattern (MAMP) in Gram-positive bacteria, exhibits differential immunomodulatory effects due to its structural heterogeneity. We extracted, purified, and characterized LTA from H. coagulans HOM5301. The results showed that HOM5301 LTA consists of a glycerophosphate backbone. Its molecular weight is in the range of 10-16 kDa. HOM5301 LTA induced greater productions of nitric oxide, TNFα, and IL-6 in RAW 264.7 macrophages compared to Staphylococcus aureus LTA. Comparative transcriptome and proteome analyses identified the differentially expressed genes and proteins triggered by HOM5301 LTA. KEGG analyses revealed that HOM5301 LTA transcriptionally and translationally activated macrophages through two immune-related pathways: cytokine-cytokine receptor interaction and phagosome formation. Protein-protein interaction network analysis indicated that the pro-inflammatory response elicited by HOM5301 LTA was TLR2-dependent, possibly requiring the coreceptor CD14, and is mediated via the MAPK and NF-kappaB pathways. Our results demonstrate that LTA is an important MAMP of H. coagulans HOM5301 that boosts immune responses, suggesting that HOM5301 LTA may be a promising immunoadjuvant.

ABHD8 antagonizes inflammation by facilitating chaperone-mediated autophagy-mediated degradation of NLRP3

ABSTRACT The NLRP3 inflammasome is a multiprotein complex that plays a vital role in the innate immune system in response to microbial infections and endogenous danger signals. Aberrant activation of the NLRP3 inflammasome is implicated in a spectrum of inflammatory and autoimmune diseases, emphasizing the necessity for precise regulation of the NLRP3 inflammasome to maintain immune homeostasis. The protein level of NLRP3 is a limiting step for inflammasome activation, which must be tightly controlled to avoid detrimental consequences. Here, we demonstrate that ABHD8, a member of the α/β-hydrolase domain-containing (ABHD) family, interacts with NLRP3 and promotes its degradation through the chaperone-mediated autophagy (CMA) pathway. ABHD8 acts as a scaffold to recruit palmitoyltransferase ZDHHC12 to NLRP3 for its palmitoylation as well as subsequent CMA-mediated degradation. Notably, ABHD8 deficiency results in the stabilization of NLRP3 protein and promotes NLRP3 inflammasome activation. We further confirm that ABHD8 overexpression ameliorates LPS- or alum-triggered NLRP3 inflammasome activation in vivo. Interestingly, the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs the ABHD8-NLRP3 association, resulting in an elevation in NLRP3 protein level and excessive inflammasome activation. These findings demonstrate that ABHD8 May represent a potential therapeutic target in conditions associated with NLRP3 inflammasome dysregulation. Abbreviations: 3-MA: 3-methyladenine; ABHD: α/β-hydrolase domain-containing; BMDMs: Bone marrow-derived macrophages; CFZ: carfilzomib; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DAMPs: danger/damage-associated molecular patterns; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; NH4Cl: ammonium chloride; NLRP3: NLR family pyrin domain containing 3; PAMPs: pathogen-associated molecular patterns; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Innate immune sensing of cell death in disease and therapeutics.

Innate immunity, cell death and inflammation underpin many aspects of health and disease. Upon sensing pathogens, pathogen-associated molecular patterns or damage-associated molecular patterns, the innate immune system activates lytic, inflammatory cell death, such as pyroptosis and PANoptosis. These genetically defined, regulated cell death pathways not only contribute to the host defence against infectious disease, but also promote pathological manifestations leading to cancer and inflammatory diseases. Our understanding of the underlying mechanisms has grown rapidly in recent years. However, how dying cells, cell corpses and their liberated cytokines, chemokines and inflammatory signalling molecules are further sensed by innate immune cells, and their contribution to further amplify inflammation, trigger antigen presentation and activate adaptive immunity, is less clear. Here, we discuss how pattern-recognition and PANoptosome sensors in innate immune cells recognize and respond to cell-death signatures. We also highlight molecular targets of the innate immune response for potential therapeutic development.

Single von Willebrand factor C-domain protein-2 confers immune defense against bacterial infections in the silkworm, Bombyx mori

Single-domain von Willebrand factor type C proteins (SVWCs), primarily found in arthropods, responds to infections caused by various pathogens. Three SVWCs have been identified in the silkworm and BmSVWC2 might play a crucial role in the immune system. However, the regulatory mechanism of BmSVWC2 remains largely unknown. This study aimed to investigate the biochemical functions of BmSVWC2 in the immune system of B. mori comprehensively. Phylogenetic analysis revealed that BmSVWC1, BmSVWC3, and BmSVWC2 were distributed in diverse groups, suggesting distinct biochemical functions. The mRNA and protein levels of BmSVWC2 increased significantly in response to bacterial infection. BmSVWC2 exhibited clear binding activity to the polysaccharide pathogen-associated molecular patterns of bacteria and fungi, enhancing bacterial clearance in vivo but not in vitro. RNA-sequencing assays of the fat body and hemocytes showed that numerous immune genes were markedly up-regulated with higher level of BmSVWC2, primarily affecting recognition, signaling, and response production of the Toll and immune deficiency (IMD) signaling pathways. This led to the production of various antimicrobial peptides and significant antibacterial activities in the hemolymph. BmSVWC2 up-regulated phagocytosis-related genes in the fat body and hemocytes, and phagocytosis assays confirmed that BmSVWC2 improved the phagocytic ability of hemocytes against bacteria. Additionally, BmSVWC2 induced the expression of nitric oxide synthetase (NOS) in the fat body, and bioassays confirmed that BmSVWC2 increased NOS activity in the fat body and hemolymph, resulting in nitric oxide accumulation. However, BmSVWC2 did not affect phenoloxidase activity, despite it caused differential expression of a few serine proteases and serine protease inhibitors. Co-immunoprecipitation and mass spectrometry assays showed that BmSVWC2 interacted with 30 K proteins, such as 30 K protein 2, 30 K pBmHPC-19, 30 K 19G1-like, 30 K protein 8, 30 K protein 7, 30 K pBmHPC-23, and low molecular mass lipoprotein 4-like. Our study provides a comprehensive characterization of BmSVWC2 and elucidates the mechanism underlying its regulation of immune responses activation.

NOD1 and NOD2: Essential Monitoring Partners in the Innate Immune System.

Nucleotide-binding oligomerization domain containing 1 (NOD1) and NOD2 are pivotal cytoplasmic pattern-recognition receptors (PRRs) that exhibit remarkable evolutionary conservation. They possess the ability to discern specific peptidoglycan (PGN) motifs, thereby orchestrating innate immunity and contributing significantly to immune homeostasis maintenance. The comprehensive understanding of both the structure and function of NOD1 and NOD2 has been extensively elucidated. These receptors proficiently recognize an array of damage-associated molecular patterns (DAMPs) as well as pathogen-associated molecular patterns (PAMPs), subsequently mediating inflammatory responses and autophagy. In recent years, emerging evidence has highlighted the crucial roles played by NOD1 and NOD2 in regulating infectious diseases, metabolic disorders, cancer, and autoimmune conditions, among others. Perturbation in either their loss or excessive activation can detrimentally impact immune homeostasis. This review offers a comprehensive overview of the structural characteristics, subcellular localization, activation mechanisms, and significant roles of NOD1 and NOD2 in innate immunity and related disease.

Identifying RNA Sensors in Antiviral Innate Immunity.

The innate immune system plays a pivotal role in pathogen recognition and the initiation of innate immune responses through its Pathogen Recognition Receptors (PRRs), which detect Pathogen-Associated Molecular Patterns (PAMPs). Nucleic acids, including RNA and DNA, are recognized as particularly significant PAMPs, especially in the context of viral pathogens. During RNA virus infections, specific sequences in the viral RNA mark it as non-self, enabling host recognition through interactions with RNA sensors, thereby triggering innate immunity. Given that some of the most lethal viruses are RNA viruses, they pose a severe threat to human and animal health. Therefore, understanding the immunobiology of RNA PRRs is crucial for controlling pathogen infections, particularly RNA virus infections. In this chapter, we will introduce a "pull-down" method for identifying RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensors.

In Vitro and In Vivo Evaluation of Virus-Induced Innate Immunity in Mouse.

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-β) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.

Identifying a Gene Deficiency in the Antiviral Innate Immune Signaling Pathway.

Innate immunity is an important defense barrier for the human body. After viral pathogen-associated molecular patterns (PAMPs) are detected by host-pathogen recognition receptors (PRRs), the associated signaling pathways trigger the activation of the interferon (IFN) regulatory factor (IRF) family members and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). However, any gene defects among the signaling adaptors will compromise innate immune efficiency. Therefore, investigating genetic defects in the antiviral innate immune signaling pathway is important. We summarize the commonly used research methods related to antiviral immune gene defects and outline the relevant research protocols, which will help investigators study antiviral innate immunity.

C-ter100 peptide derived from Vibrio vEP-45 protease acts as a pathogen-associated molecular pattern to induce inflammation and innate immunity.

The bacterium Vibrio vulnificus causes fatal septicemia in humans. Previously, we reported that an extracellular metalloprotease, vEP-45, secreted by V. vulnificus, undergoes self-proteolysis to generate a 34 kDa protease (vEP-34) by losing its C-terminal domain to produce the C-ter100 peptide. Moreover, we revealed that vEP-45 and vEP-34 proteases induce blood coagulation and activate the kallikrein/kinin system. However, the role of the C-ter100 peptide fragment released from vEP-45 in inducing inflammation is still unclear. Here, we elucidate, for the first time, the effects of C-ter100 on inducing inflammation and activating host innate immunity. Our results showed that C-ter100 could activate NF-κB by binding to the receptor TLR4, thereby promoting the secretion of inflammatory cytokines and molecules, such as TNF-α and nitric oxide (NO). Furthermore, C-ter100 could prime and activate the NLRP3 inflammasome (NLRP3, ASC, and caspase 1), causing IL-1β secretion. In mice, C-ter100 induced the recruitment of immune cells, such as neutrophils and monocytes, along with histamine release into the plasma. Furthermore, the inflammatory response induced by C-ter100 could be effectively neutralized by an anti-C-ter100 monoclonal antibody (C-ter100Mab). These results demonstrate that C-ter100 can be a pathogen-associated molecular pattern (PAMP) that activates an innate immune response during Vibrio infection and could be a target for the development of antibiotics.

Comparative Analysis of Transcriptomes Reveals Pathways and Verifies Candidate Genes for Clubroot Resistance in Brassica oleracea.

Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both resistant (R) and susceptible (S) B. oleracea plants infected by P. brassicae. Then the comparative analysis was carried out between the R and S samples at different time points during the infection stages to reveal clubroot resistance related pathways and candidate genes. Compared with 0 days after inoculation, a total of 4991 differential expressed genes were detected from the S pool, while only 2133 were found from the R pool. Gene function enrichment analysis found that the effector-triggered immunity played a major role in the R pool, while the pathogen-associated molecular pattern triggered immune response was stronger in the S pool. Simultaneously, candidate genes were identified through weighted gene co-expression network analysis, with Bol010786 (CNGC13) and Bol017921 (SD2-5) showing potential for conferring resistance to clubroot. The findings of this research provide valuable insights into the molecular mechanisms underlying clubroot resistance and present new avenues for further research aimed at enhancing the clubroot resistance of B. oleracea through breeding.

Identification and expression analysis of NOD-like receptor gene family in response to Aeromonas hydrophila and poly I:C challenge in Chinese soft-shelled turtle (Pelodiscus sinensis)

Nucleotide-binding domain (NOD)-like receptors (NLRs), a type of pattern recognition receptor (PRR), specifically recognize pathogen-associated molecular patterns and damage-associated molecular patterns, playing crucial roles in the immune system. Herein, the composition, expression pattern, and co-expressed pathways of NLRs in Pelodiscus sinensis (PsNLRs) were analyzed. Eight PsNLRs, located on seven chromosomes, were first identified from the genome. Phylogenetic analysis revealed that PsNLRs were most closely related to the NLRs of the western painted turtle and the green sea turtle, and could be clustered into four subfamilies. Besides, the expression profile of PsNLRs exhibited tissue-specificity after infection with Aeromonas hydrophila or poly I:C. Nonetheless, PsCIITA and PsNLRP3.2 displayed infection-specific expression patterns, whereas other PsNLRs showed similar expression patterns during A. hydrophila and poly I:C infections. Weighted gene co-expression network analysis found that six PsNLRs were generated in the turquoise module, in which several immune-related signaling pathways were enriched, including “NOD-like receptor signaling pathway”, “NF-κB signaling pathway”, “PI3K-Akt signaling pathway”, “Chemokine signaling pathway”, “Antigen processing and presentation”, and ”Toll-like receptor signaling pathway”. Additionally, three immune-related genes, NF-κB1, TRAF3, and PIK3CG, were identified as hub genes. These results indicated that PsNLRs may play key roles in regulating immune responses in P. sinensis. Overall, an in-depth study of the NLR gene family in P. sinensis would yield insights into developing strategies to combat bacterial and viral infections.

Toll-like receptor 21 in Labeo rohita recognizes double-stranded RNA and lipopolysaccharides by engaging the critical motifs in the LRR domain and gets activated against bacterial assaults

TLR (Toll-like receptor)-21 is a non-mammalian TLR and exhibits a unique function within the innate immune systems of fishes, birds, and amphibians. Despite its important role as PRR (pattern recognition receptor), research on TLR21 in many fish species, as well as in rohu (Labeo rohita), remains relatively limited. This article describes the molecular cloning of LrTLR21 (TLR21 in L. rohita), 3D (3-dimensional) modelling of its LRR (leucine-rich repeat)-regions, prediction of LRR18 to LRR20 as the LPS (lipopolysaccharide)-binding site, and LRR1 to LRR5 and LRR21 to LRR23 as the poly I:C (polyinosinic-polycytidylic acid) binding sites. It also describes the response of LrTLR21 in response to Aeromonas hydrophila and Edwardsiella tarda infections and PAMPs (pathogen-associated molecular patterns) (LPS and poly I:C)-stimulations. The ORF (open reading frame) of the LrTLR21 comprises 2955 nucleotides, encoding 984 aa (amino acid) residues with molecular weight and isoelectric point of 113.791 kDa and ∼8.79, respectively. Domain analysis of the deduced LrTLR21 displayed the existence of a signal peptide (residues 1–24), 26 LRR regions (residues 61–685), a TM (transmembrane) domain (residues 736–758), and a TIR (Toll/interleukin-1 receptor) domain (residues 790–937). The 3D model of LrTLR21-LRR regions has parallel β-sheets and few α-helices. Phylogenetically, LrTLR21 is closely related to the Onychostoma macrolepis and Carassius gibelio TLR21, and during ontogenesis, it is expressed in most of the developmental stages. In rohu fingerlings, it is consistently expressed in all examined tissues viz., skin, liver, heart, blood, eye, muscle, kidney, intestine, brain, gill, and spleen. Upon exposure to E. tarda and A. hydrophila infections, as well as LPS and poly I:C stimulations, the expression of LrTLR21 is significantly (p < 0.05) increased in the blood, kidney, liver, and gill. In the RBCs (red blood cells), PBLs (peripheral blood leukocytes), and kidney macrophages, LrTLR21 is also significantly induced following in-vivo and in-vitro LPS and poly I: C-stimulations. These findings on LrTLR21 are the first ones showing its structural insights and PAMPs binding motifs and its key role in recognizing pathogens and their PAMPs in RBCs, PBLs, and macrophages.