Unravelling mechanisms of bacterial recognition by Acanthamoeba: insights into microbial ecology and immune responses.

Acanthamoeba, are ubiquitous eukaryotic microorganisms, that play a pivotal role in recognizing and engulfing various microbes during predation, offering insights into microbial dynamics and immune responses. An intriguing observation lies in the apparent preference of Acanthamoeba for Gram-negative over Gram-positive bacteria, suggesting potential differences in the recognition and response mechanisms to bacterial prey. Here, we comprehensively review pattern recognition receptors (PRRs) and microbe associated molecular patterns (MAMPs) that influence Acanthamoeba interactions with bacteria. We analyze the molecular mechanisms underlying these interactions, and the key finding of this review is that Acanthamoeba exhibits an affinity for bacterial cell surface appendages that are decorated with carbohydrates. Notably, this parallels warm-blooded immune cells, underscoring a conserved evolutionary strategy in microbial recognition. This review aims to serve as a foundation for exploring PRRs and MAMPs. These insights enhance our understanding of ecological and evolutionary dynamics in microbial interactions and shed light on fundamental principles governing immune responses. Leveraging Acanthamoeba as a model organism, provides a bridge between ecological interactions and immunology, offering valuable perspectives for future research.

The implication of dendritic cells in lung diseases: Immunological role of toll-like receptor 4.

The immune responses play a profound role in the progression of lung lesions in both infectious and non-infectious diseases. Dendritic cells, as the "frontline" immune cells responsible for antigen presentation, set up a bridge between innate and adaptive immunity in the course of these diseases. Among the receptors equipped in dendritic cells, Toll-like receptors are a group of specialized receptors as one type of pattern recognition receptors, capable of sensing environmental signals including invading pathogens and self-antigens. Toll-like receptor 4, a pivotal member of the Toll-like receptor family, was formerly recognized as a receptor sensitive to the outer membrane component lipopolysaccharide derived from Gram-negative bacteria, triggering the subsequent response. Moreover, its other essential roles in immune responses have drawn significant attention in the past decade. A better understanding of the implication of Toll-like receptor 4 in dendritic cells could contribute to the management of pulmonary diseases including pneumonia, pulmonary tuberculosis, asthma, acute lung injury, and lung cancer.

Monocytic Phagocytes in the Immunopathogenesis of Cytokine Storm Syndromes.

Cytokine storm syndromes (CSSs) are caused by a dysregulated host immune response to an inciting systemic inflammatory trigger. This maladaptive and harmful immune response culminates in collateral damage to host tissues resulting in life-threatening multisystem organ failure. Knowledge of the various immune cells that contribute to CSS pathogenesis has improved dramatically in the past decade. Monocytes, dendritic cells, and macrophages, collective known as monocytic phagocytes, are well-positioned within the immune system hierarchy to make key contributions to the initiation, propagation, and amplification of the hyperinflammatory response in CSS. The plasticity of monocytic phagocytes also makes them prime candidates for mediating immunoregulatory and tissue-healing functions in patients who recover from cytokine storm-mediated immunopathology. Therefore, approaches to manipulate the myriad functions of monocytic phagocytes may improve the clinical outcome of CSS.

Dual transcriptomic analysis reveals early induced Castanea defense-related genes and Phytophthora cinnamomi effectors.

Phytophthora cinnamomi Rands devastates forest species worldwide, causing significant ecological and economic impacts. The European chestnut (Castanea sativa) is susceptible to this hemibiotrophic oomycete, whereas the Asian chestnuts (Castanea crenata and Castanea mollissima) are resistant and have been successfully used as resistance donors in breeding programs. The molecular mechanisms underlying the different disease outcomes among chestnut species are a key foundation for developing science-based control strategies. However, these are still poorly understood. Dual RNA sequencing was performed in C. sativa and C. crenata roots inoculated with P. cinnamomi. The studied time points represent the pathogen's hemibiotrophic lifestyle previously described at the cellular level. Phytophthora cinnamomi expressed several genes related to pathogenicity in both chestnut species, such as cell wall-degrading enzymes, host nutrient uptake transporters, and effectors. However, the expression of effectors related to the modulation of host programmed cell death (elicitins and NLPs) and sporulation-related genes was higher in the susceptible chestnut. After pathogen inoculation, 1,556 and 488 genes were differentially expressed by C. crenata and C. sativa, respectively. The most significant transcriptional changes occur at 2 h after inoculation (hai) in C. sativa and 48 hai in C. crenata. Nevertheless, C. crenata induced more defense-related genes, indicating that the resistant response to P. cinnamomi is controlled by multiple loci, including several pattern recognition receptors, genes involved in the phenylpropanoid, salicylic acid and ethylene/jasmonic acid pathways, and antifungal genes. Importantly, these results validate previously observed cellular responses for C. crenata. Collectively, this study provides a comprehensive time-resolved description of the chestnut-P. cinnamomi dynamic, revealing new insights into susceptible and resistant host responses and important pathogen strategies involved in disease development.

Microglia and Systemic Immunity.

Microglia are specialized immune cells that reside in the central nervous system (CNS) and play a crucial role in maintaining the homeostasis of the brain microenvironment. While traditionally regarded as a part of the innate immune system, recent research has highlighted their role in adaptive immunity. The CNS is no longer considered an immune-privileged organ, and increasing evidence suggests bidirectional communication between the immune system and the CNS. Microglia are sensitive to systemic immune signals and can respond to systemic inflammation by producing various inflammatory cytokines and chemokines. This response is mediated by activating pattern recognition receptors (PRRs), which recognize pathogen- and danger-associated molecular patterns in the systemic circulation. The microglial response to systemic inflammation has been implicated in several neurological conditions, including depression, anxiety, and cognitive impairment. Understanding the complex interplay between microglia and systemic immunity is crucial for developing therapeutic interventions to modulate immune responses in the CNS.

Screening and Characterization of Immunobiotic Lactic Acid Bacteria with Porcine Immunoassay Systems.

Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research has allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models, utilizing transfectant cells expressing PRRs along with an established porcine intestinal epitheliocyte (PIE) cell line, have proven to be valuable tools for immunobiotic selection and for gaining insights into the molecular mechanisms responsible for their beneficial effects.

The Molecular Signature Related to Local Inflammatory and Immune Response in Canine Cutaneous Hypersensitivity Reactions: A Preliminary Study.

Cutaneous hypersensitivity reactions (CHRs) are complex inflammatory skin disorders that affect humans and dogs. This study examined the inflammatory and immune responses leading to skin damage, inflammation, and irritation by investigating gene expression through quantitative PCR (qPCR) and protein localization through the immunohistochemistry (IHC) of specific receptors and molecules involved in CHRs. Formalin-fixed paraffin-embedded (FFPE) samples from canine CHR skin (n = 20) and healthy dog skin (n = 3) were analyzed for expression levels of eight genes, including members of the pattern recognition receptor (PRR) family, CD209 and CLEC4G, the Regakine-1-like chemokine, and acute phase proteins (APPs), LBP-like and Hp-like genes. Additionally, we examined the local involvement of IL-6, Janus Kinase 1 (JAK1), and the signal transducer activator of transcription 3 (STAT3) in the CHR cases. The study demonstrated statistically significant increases in the expression levels of CD209, Hp-like (p < 0.01), LBP-like, Regakine-1-like, and CLEC4G (p < 0.05) genes in CHRs compared to healthy controls. Conversely, IL-6, JAK1, and STAT3 showed no significant difference between the two groups (p > 0.05). Protein analysis revealed JAK1 and STAT3 expression in CHR hyperplastic epithelial cells, dermal fibroblasts, and endothelial cells of small capillaries, indicating a possible involvement in the JAK/STAT pathway in local inflammatory response regulation. Our findings suggest that the skin plays a role in the development of CHRs.

Molecular and functional in vivo characterisation of murine Dectin-1 isoforms.

Dectin-1 is a C-type lectin-receptor involved in sensing fungi by innate immune cells. Encoded by the Clec7a gene, Dectin-1 exists in two major splice isoforms, Dectin-1a and 1b, which differ in the presence of a membrane-proximal stalk domain. As reported previously, this domain determines degradative routes for Dectin-1a and 1b leading to the generation of a stable N-terminal fragment exclusively from Dectin-1a. Here, we narrow down the responsible part of the stalk and demonstrate the stabilisation of the Dectin-1a N-terminal fragment in tetraspanin-enriched microdomains. C57BL/6 and BALB/c mice show divergent Dectin-1 isoform expression patterns, which are caused by a single nucleotide polymorphism in exon 3 of the Clec7a gene, leading to a non-sense Dectin-1a mRNA in C57BL/6 mice. Using backcrossing, we generated mice with the C57BL/6 Clec7a allele on a BALB/c background and compared these to the parental strains. Expression of the C57BL/6 allele leads to the exclusive presence of the Dectin-1b protein. Furthermore, it was associated with higher Dectin-1 mRNA expression, but less Dectin-1 at the cell surface according to flow cytometry. In neutrophils, this altered ROS production induced by Dectin-1 model ligands, while cellular responses in macrophages and dendritic cells were not significantly influenced by the Dectin-1 isoform pattern.

Increased viral tolerance mediates by antiviral RNA interference in bat cells.

Bats harbor highly virulent viruses that can infect other mammals, including humans, posing questions about their immune tolerance mechanisms. Bat cells employ multiple strategies to limit virus replication and virus-induced immunopathology, but the coexistence of bats and fatal viruses remains poorly understood. Here, we investigate the antiviral RNA interference pathway in bat cells and discover that they have an enhanced antiviral RNAi response, producing canonical viral small interfering RNAs upon Sindbis virus infection that are missing in human cells. Disruption of Dicer function results in increased viral load for three different RNA viruses in bat cells, indicating an interferon-independent antiviral pathway. Furthermore, our findings reveal the simultaneous engagement of Dicer and pattern-recognition receptors, such as retinoic acid-inducible gene I, with double-stranded RNA, suggesting that Dicer attenuates the interferon response initiation in bat cells. These insights advance our comprehension of the distinctive strategies bats employ to coexist with viruses.

Identification of Nonsynonymous SNPs in Immune-Related Genes Associated with Pneumonia Severity in Pigs.

We previously showed that several polymorphisms in genes encoding pattern recognition receptors that cause amino acid substitutions alter pathogen recognition ability and disease susceptibility in pigs. In this study, we expanded our analysis to a wide range of immune-related genes and investigated polymorphism distribution and its influence on pneumonia in multiple commercial pig populations. Among the polymorphisms in 42 genes causing 634 amino acid substitutions extracted from the swine genome database, 80 in 24 genes were found to have a minor allele frequency of at least 10% in Japanese breeding stock pigs via targeted resequencing. Of these, 62 single nucleotide polymorphisms (SNPs) in 23 genes were successfully genotyped in 862 pigs belonging to four populations with data on pneumonia severity. Association analysis using a generalized linear mixed model revealed that 12 SNPs in nine genes were associated with pneumonia severity. In particular, SNPs in the cellular receptor for immunoglobulin G FCGR2B and the intracellular nucleic acid sensors IFI16 and LRRFIP1 were found to be associated with mycoplasmal pneumonia of swine or porcine pleuropneumonia in multiple populations and may therefore have wide applications in the improvement of disease resistance in pigs. Functional analyses at the cellular and animal levels are required to clarify the mechanisms underlying the effects of these SNPs on disease susceptibility.

Harnessing cGAS-STING axis for therapeutic benefits in systemic lupus erythematosus.

The cyclic GMP-AMP synthase (cGAS), a prominent intracellular DNA sensor in mammalian cells, controls the innate immune response and the stimulator of interferon genes (STING)-mediated synthesis of pro-inflammatory cytokines, such as type-I interferon (IFN-I). For decades, IFN-I has been hypothesized to be essential in the development of systemic lupus erythematosus (SLE), a chronic multisystem autoimmunity characterized by immune complex (IC) deposition in small vessels. Recent findings revealed that the activation of the cGAS-STING pathway by self-DNA would propagate the autoimmune responses via upregulating IFN-I production in SLE. In this review, we aimed to provide a comprehensive outlook of the role of the cGAS-STING pathway in SLE pathobiology, as well as, a better understanding of current therapeutic opportunities targeting this axis.

Novel ApeC-containing protein mediates the recognition and internalization of Vibrio splendidus in Apostichopus japonicus.

Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

Zebrafish use conserved CLR and TLR signaling pathways to respond to fungal PAMPs in zymosan.

Pattern recognition receptors (PRRs) such as C-type lectin receptors (CLRs) and Toll-like receptors (TLRs) are used by hosts to recognize pathogen-associated molecular patterns (PAMPs) in microorganisms and to initiate innate immune responses. While PRRs exist across invertebrate and vertebrate species, the functional homology of many of these receptors is still unclear. In this study, we investigate the innate immune response of zebrafish larvae to zymosan, a ß-glucan-containing particle derived from fungal cell walls. Macrophages and neutrophils robustly respond to zymosan and are required for zymosan-induced activation of the NF-κB transcription factor. Full activation of NF-κB in response to zymosan depends on Card9/Syk and Myd88, conserved CLR and TLR adaptor proteins, respectively. Two putative CLRs, Clec4c and Sclra, are both required for maximal sensing of zymosan and NF-κB activation. Altogether, we identify conserved PRRs and PRR signaling pathways in larval zebrafish that promote recognition of fungal PAMPs. These results inform modeling of human fungal infections in zebrafish and increase our knowledge of the evolution and conservation of PRR pathways in vertebrates.

Exploring the innate immune system of Urechis unicinctus: Insights from full-length transcriptome analysis.

The Echiura worm Urechis unicinctus refers to a common benthic invertebrate found in the intertidal zone of Huanghai as well as Bohai Bay. U. unicinctus is known to contain various physiologically active substances, making it highly valuable in terms of its edibility, medicinal properties, and economic potential. Nonetheless, the limited study on the immune system of U. unicinctus poses difficulties for its aquaculture and artificial reproduction. Marine invertebrates, including shellfish and U. unicinctus, are thought to primarily depend on their innate immune system for disease protection, owing to the severalinnate immune molecules they possess. Herein, we employed PacBio single-molecule real-time (SMRT) sequencing technology to perform the full-length transcriptome analysis of U. unicinctus individuals under five different conditions (room temperature (RT), low temperature (LT), high temperature (HT), without water (DRY), ultraviolet irradiation (UV)). Concequently, we identified 59,371 unigenes that had a 2,779 bp average length, 2,613 long non-coding RNAs (lncRNAs), 59,190 coding sequences (CDSs), 35,166 simple sequence repeats (SSRs), and 1,733 transcription factors (TFs), successfully annotating 90.58 % (53,778) of the unigenes. Subsequently, key factors associated with immune-related processes, such as non-self-recognition, cellular immune defenses, and humoral immune defenses, were searched. Our study also identified pattern recognition receptors (PRRs) that included 17 peptidoglycan recognition proteins (PGRPs), 13 Gram-negative binding proteins (GNBPs), 18 scavenger receptors (SRs), 74 toll-like receptors (TLRs), and 89 C-type lectins (CLTs). Altogether, the high-quality transcriptome obtained data will offer valuable insights for further investigations into U. unicinctus innate immune response, laying the foundation for subsequent molecular biology studies and aquaculture.

Host Factors Modulate Virus-Induced IFN Production via Pattern Recognition Receptors.

Innate immunity is the first line of defense in the human body, and it plays an important role in defending against viral infection. Viruses are identified by different pattern-recognition receptors (PRRs) that activate the mitochondrial antiviral signaling protein (MAVS) or transmembrane protein 173 (STING), which trigger multiple signaling cascades that cause nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) to produce inflammatory factors and interferons (IFNs). PRRs play a pivotal role as the first step in pathogen induction of interferon production. Interferon elicits antiviral activity by inducing the transcription of hundreds of IFN-stimulated genes (ISGs) via the janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway. An increasing number of studies have shown that environmental, pathogen and host factors regulate the IFN signaling pathway. Here, we summarize the mechanisms of host factor modulation in IFN production via pattern recognition receptors. These regulatory mechanisms maintain interferon levels in a normal state and clear viruses without inducing autoimmune disease.

Harnessing paraprobiotics and postbiotics for enhanced immune function in Asian seabass (Lates calcarifer): Insights into pattern recognition receptor signaling.

The Asian seabass, Lates calcarifer, is a key species in Asian aquaculture due to its nutritional value and adaptability. However, disease outbreaks, particularly viral and bacterial infections, pose significant challenges to its production. Immunostimulants offer promising solutions but raise safety concerns. Paraprobiotics and postbiotics (CPP) emerge as safer alternatives, exerting health benefits without live microorganisms. This study investigated the potential of probiotic paraprobiotic and postbiotic supplements derived from Bacillus subtilis to enhance the immune response and antioxidant capacity of Asian seabass and improve their resistance to Streptococcus iniae infection. Analysis of antioxidant activity and lipid peroxidation revealed significant improvements in fish supplemented with CPP, indicating their effectiveness in mitigating oxidative stress. Immunological assays demonstrated enhanced growth performance and serum immunity, including increased alternative complement activity, immunoglobulin levels, and phagocytic activity, in supplemented fish. Furthermore, upregulated expression of proinflammatory cytokines (TNF-α, IL-6, IL-1ß) and pattern recognition receptors (NLRC3, TLR22, MDA5) in immune tissues. Fish supplemented with CPP exhibited higher resistance and survival rates against S. iniae infection challenge compared to control groups. The study elucidates the mechanisms underlying the immunomodulatory effects of CPP, shedding light on their potential applications in aquaculture.

Characterization of DmToll and DmToll7 homologue in Litopenaeus vannamei based on structure analysis.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that recognize invading pathogens and activate downstream signaling pathways. The number of 10 Tolls is found in Litopenaeus vannamei but have not yet been identified as the corresponding Toll homologue of model animal. In this study, we predicted the three-dimensional (3D) structures of 10 LvTolls (LvToll1-10) with AlphaFold2 program. The per-residue local distance difference test (pLDDT) scores of LvTolls showed the predicted structure of LvTolls had high accuracy (pLDDT>70). By structural analysis, 3D structures of LvToll2 and LvToll3 had high similarity with Drosophila melanogaster Toll and Toll7, respectively. 3D structure of LvToll7 and LvToll10 were not similar to that of other LvTolls. Moreover, we also predicted that LvSpätzle4 had high structural similarity to DmSpätzle. There were 9 potential hydrogen bonds in LvToll2-LvSpätzle4 complex. Importantly, co-immunoprecipitation assay showed that LvToll2 could bind with LvSpätzle4. Collectively, this study provides new insight for researching invertebrate immunity by identifying the protein of model animal homologue.

Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4-specific LPS Mimetics with Picomolar Potency.

Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, Toll-like Receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing ß,ß-diglucosamine, which was investigated by NMR, we developed two sets of lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1'-glycosylation towards fully orthogonally protected nonreducing GlcNß(1↔1')ßGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

Aging is associated with an insufficient early inflammatory response of lung endothelial cells in SARS-CoV-2 infection.

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.