Bacterial Pathogen-associated Molecular Patterns Research Articles

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

In the Acute Phase of Trypanosoma cruzi Infection, Liver Lymphoid and Myeloid Cells Display an Ambiguous Phenotype Combining Pro- and Anti-Inflammatory Markers.

Multiple cell populations, cellular biochemical pathways, and the autonomic nervous system contribute to maintaining the immunological tolerance in the liver. This tolerance is coherent because the organ is exposed to high levels of bacterial pathogen-associated molecular pattern (PAMP) molecules from the intestinal microbiota, such as lipopolysaccharide endotoxin (LPS). In the case of Trypanosoma cruzi infection, although there is a dramatic acute immune response in the liver, we observed intrahepatic cell populations combining pro- and anti-inflammatory markers. There was loss of fully mature Kupffer cells and an increase in other myeloid cells, which are likely to include monocytes. Among dendritic cells (DCs), the cDC1 population expanded relative to the others, and these cells lost both some macrophage markers (F4/80) and immunosuppressive cytokines (IL-10, TGF-β1). In parallel, a massive T cell response occured with loss of naïve cells and increase in several post-activation subsets. However, these activated T cells expressed both markers programmed cell death protein (PD-1) and cytokines consistent with immunosuppressive function (IL-10, TGF-β1). NK and NK-T cells broadly followed the pattern of T cell activation, while TCR-γδ cells appeared to be bystanders. While no data were obtained concerning IL-2, several cell populations also synthesized IFN-γ and TNF-α, which has been linked to host defense but also to tissue injury. It therefore appears that T. cruzi exerts control over liver immunity, causing T cell activation via cDC1 but subverting multiple populations of T cells into immunosuppressive pathways. In this way, T. cruzi engages a mechanism of hepatic T cell tolerance that is familiar from liver allograft tolerance, in which activation and proliferation are followed by T cell inactivation.

Antimicrobial peptide expression in the cockroach gut during enterobacterial infection is specific and influenced by type III secretion.

ABSTRACTOmnivorous synanthropic cockroaches, such as the German cockroach (Blattella germanica), are reservoirs and vectors of enteric bacterial pathogens. A lifestyle conducive to frequent encounters with high loads of diverse bacteria may have led to the evolution of unique innate immune systems in these insects. The innate immune response of insects relies largely on generalized mechanisms to sense and eliminate foreign microbes. However, analyses of the genomes of common synanthropic cockroaches previously revealed a repertoire of pathogen associated molecular pattern (PAMP) receptors and antimicrobial peptides (AMPs) that is significantly expanded relative to most holometabolous insect models and vectors, supporting the intriguing possibility that cockroaches may encode enhanced recognition within their immune system and may possess an enhanced capacity to fine tune innate immune responses. Investigating how cockroaches respond to infection with enterobacteria provides the opportunity to expand our fundamental knowledge of the regulation of insect innate immunity in a context that is biologically and medically relevant. German cockroaches can harbor both Salmonella enterica serovar Typhimurium and Escherichia coli in their gut without experiencing pathogenesis. The former colonizes the gut and replicates while the latter persists only transiently. We hypothesized that differences in the innate immune response may contribute to or result from the difference in infection dynamics between the two enterobacteria. To test this hypothesis, we used qRT-PCR to analyze expression of five genes encoding representative AMPs (Attacins, Blattellicin, Defensins) in the gut of German cockroaches 1 and 24 h after ingestion of live or heat-killed enterobacteria. We found that robust AMP expression was induced in response to ingestion of a live wild-type strain of S. Typhimurium, but not in response to live E. coli, heat-killed S. Typhimurium, or a live mutant strain of S. Typhimurium lacking type III secretion systems. These results indicate that the cockroach immune system does not respond to stimulation with high levels of ingested bacterial PAMPs such as peptidoglycan. Rather, AMP expression in the gut appears to be induced by active bacterial colonization involving type III secretion. We speculate that this form of regulation may have evolved to prevent over activation of the immune system from frequent ingestion of innocuous, non-colonizing, or non-viable bacteria. While additional work is needed to delineate the molecular mechanisms underlying our observations, our findings provide significant novel insight into the immunological adaptation of cockroaches to life in septic environments as well as the factors that regulate bacterial pathogen transmission by these insects.

Structural Characterization of LpqH (19 kDa surface antigen), an Immunomodulatory Lipoprotein of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, is predominantly a disease of the lungs acquired by inhaling mycobacteria from infected individuals via airborne droplets. As soon as Mycobacterium reaches the lungs, its first encounter happens to be with the resident alveolar macrophages which immediately can phagocytose, but cannot kill the pathogen. Thus, the pathogen‐laden macrophages now disseminate to other organs, aiding in the spread of infection in an undetected manner. Certain mycobacterial surface motifs known as pathogen‐associated molecular patterns (PAMPs), recognize specific pattern recognition receptors (PRRs) present on the host cell surface, thereby mediating these early interactions of the mycobacteria with macrophages. A major group of these bacterial PAMPs includes mycobacterial lipoproteins, of which, the 19 kDa surface antigen LpqH, have a critical role in shaping the host immunological response in tuberculosis. The protein pleiotropically modulates the host immune response by interacting with Toll‐Like Receptors (TLRs) such as TLR2 homodimers and TLR1/TLR2, TLR2/TLR4, and TLR2/TLR6 heterodimers. On one hand, it acts as an apoptosis‐inducing factor for macrophages whereas on the other, it shows that prolonged exposure of LpqH to TLRs eventually favours immune evasion, thereby allowing the dissemination of the pathogen. Despite the essential role of LpqH in tuberculosis,till date, the detailed structure‐function relation of this protein remains to be elucidated. We have determined the high‐resolution crystal structure of Mycobacterium tuberculosisLpqH at a resolution of 1.26Å and refined to Rwork and Rfree of 14.29% and 17.79%, respectively. It has been observed that the structural architecture of LpqH is indeed unique and has never been previously described. Furthermore, sequence identity between LpqH and other well‐known mycobacterial lipoproteins is very low, which further implies that the protein is indeed unique and its structural characterization is vital to the overall understanding of its pathogenesis in case of active tuberculosis.

Pulmonary Endotoxin is Associated with Reflux, Inflammation, and Adverse Outcomes in Lung Transplant Recipients

Purpose Gastroesophageal reflux disease (GERD) and aspiration are risk factors for poor long-term outcomes in lung transplant recipients. However, the mechanisms underlying this relationship are poorly understood. We have previously shown that bile acids and bacterial density in the lung allograft are associated with GERD. As such, we hypothesized that products of the gut microbiome, including pathogen associated molecular patterns (PAMPs), are aspirated into the airways of lung transplant recipients resulting in lung injury. Herein, we measure endotoxin, a bacterial PAMP, in large airway bronchial washes (LABW) of lung transplant recipients and assess the association with GERD, aspiration, inflammation, and allograft survival. Methods Endotoxin was measured using the Limulus amebocyte lysate assay in LABW obtained 2-7 months post-transplant in 61 lung transplant recipients. Proximal and total reflux episodes were measured using 24h pH-impendence testing. Bile acids were measured using mass spectrometry and soluble markers of inflammation were measured with multiplex assay. Results LABW endotoxin was higher in samples that had a positive bacterial culture (p=0.08). Among samples that did not have a positive bacterial culture (n=49), endotoxin concentration was associated with proximal reflux episodes (r=0.312; p=0.02), total reflux episodes (r=0.321; p=0.03), total bile acid concentration (r=0.260; p=0.04), and inflammatory mediators IL-6, IL-8, IL-1ß, and CCL5 (Figure A). Among all 61 lung transplant recipients, those with LABW endotoxin levels in the highest tertile were at increased risk of chronic lung allograft dysfunction (CLAD) [HR: 3.35 (1.43-7.84); p=0.003] (Figure B) and death/retransplant [HR: 4.24 (1.38-13.01); p=0.01]. Conclusion In the absence of overt bacterial infection, endotoxin concentration in LABW was associated with reflux, aspiration, and inflammation. Aspiration of PAMPs may explain why reflux is a risk factor for CLAD. Gastroesophageal reflux disease (GERD) and aspiration are risk factors for poor long-term outcomes in lung transplant recipients. However, the mechanisms underlying this relationship are poorly understood. We have previously shown that bile acids and bacterial density in the lung allograft are associated with GERD. As such, we hypothesized that products of the gut microbiome, including pathogen associated molecular patterns (PAMPs), are aspirated into the airways of lung transplant recipients resulting in lung injury. Herein, we measure endotoxin, a bacterial PAMP, in large airway bronchial washes (LABW) of lung transplant recipients and assess the association with GERD, aspiration, inflammation, and allograft survival. Endotoxin was measured using the Limulus amebocyte lysate assay in LABW obtained 2-7 months post-transplant in 61 lung transplant recipients. Proximal and total reflux episodes were measured using 24h pH-impendence testing. Bile acids were measured using mass spectrometry and soluble markers of inflammation were measured with multiplex assay. LABW endotoxin was higher in samples that had a positive bacterial culture (p=0.08). Among samples that did not have a positive bacterial culture (n=49), endotoxin concentration was associated with proximal reflux episodes (r=0.312; p=0.02), total reflux episodes (r=0.321; p=0.03), total bile acid concentration (r=0.260; p=0.04), and inflammatory mediators IL-6, IL-8, IL-1ß, and CCL5 (Figure A). Among all 61 lung transplant recipients, those with LABW endotoxin levels in the highest tertile were at increased risk of chronic lung allograft dysfunction (CLAD) [HR: 3.35 (1.43-7.84); p=0.003] (Figure B) and death/retransplant [HR: 4.24 (1.38-13.01); p=0.01]. In the absence of overt bacterial infection, endotoxin concentration in LABW was associated with reflux, aspiration, and inflammation. Aspiration of PAMPs may explain why reflux is a risk factor for CLAD.

Perspective on the relationship between microbiota dysbiosis and neuroinflammation: probiotics as a treatment option.

Perspective on the relationship between microbiota dysbiosis and neuroinflammation: probiotics as a treatment option.

The barley HvSTP13GR mutant triggers resistance against biotrophic fungi.

High‐yielding and stress‐resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, which possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analysed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased on bacterial and fungal pathogen‐associated molecular pattern (PAMP) application, suggesting a PAMP‐triggered signalling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is probably targeted by transcription factors downstream of PAMP‐triggered immunity pathways. We confirmed that the nonfunctional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus in barley. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host–pathogen interaction, is a promising approach to achieve broad and durable resistance in plants.

The Effect of TLR Agonists and Myokines on Secretory Activity of Adipogenically Differentiated MSC Cultures.

We studied the effect of bacterial pathogen-associated molecular patterns and myokines on the secretion of adipokines by mesenchymal stem cells (MSC) and products of their adipogenic differentiation. The secretion of adiponectin, adipsin, leptin, and insulin by adipogenically differentiated cell cultures was quantitatively determined using multiplex ELISA. MSC obtained from the stromal vascular fraction of human subcutaneous adipose tissue were shown to secrete a known adipokine adipsin. The ability of white adipocytes to secrete significant amounts of insulin (in vitro) has been shown for the first time. Control cultures of white adipocytes secreted much higher levels of adiponectin, leptin, and insulin when compared to other adipocytes cultures. On the other hand, beige and brown adipocyte cultures secreted more adipsin than white adipocyte cultures. The influence of myokine β-aminoisobutyric acid on the secretion of adipsin in MSC, white, beige, and brown adipocytes was also studied.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system—the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1β and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1β, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49–0.61) and between neutrophils and cytokine responses to R848 (0.38–0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Leaf and tuber treatments with PAMPs trigger organ-specific responses in potato

Organ-specificity of plant defence is an under-represented axis of research in plant biology. However, recent publications point out the importance of this concept in plant–pathogen interactions. Indeed, assuming the resistance status of a whole plant against a given pathogen following studies conducted on a single organ might lead to erroneous conclusions. This study aims at understanding the extent of potato (Solanum tuberosum) organ-specific defence mechanisms after leaf and tuber treatments with pathogen-associated molecular patterns (PAMPs). Our results show that potato leaves and tubers activate contrasting defence responses, in terms of production of reactive oxygen species (ROS) and activation of defence-related genes, following treatments with the Phytophthora PAMP Pep-25 or the bacterial PAMP flg22. Interestingly, flg22 induced ROS production in both leaves and tubers, indicating that this peptide is recognized in these organs. By contrast, ROS production was induced in the leaf but not in the tuber after Pep-25 treatment. Analysis of changes in expression of defence-related genes showed that at least six genes exhibited organ-specific expression patterns. In addition to those early responses, inhibition of potato seedling growth was also found to affect both the aboveground and belowground organs following treatment with Pep-25 and with flg22. Taken together, these experiments will help further the investigation of potato defence against Phytophthora spp.

The Effect of LPS and Flagellin on the Process of Lipolysis in Mesenchymal Stromal Cells during Adipogenic Differentiation.

We analyzed the effects of bacterial pathogen-associated molecular patterns (LPS and flagellin) and adrenergic agonist isoproterenol on the content of total and phosphorylated (Ser552) hormone-sensitive lipase in mesenchymal stromal cells and cell products of their adipogenic differentiation. The expression of hormone-sensitive lipase and an increase in the content of its activated phosphorylated form were demonstrated by Western blotting in cells of all three lines of adipogenic differentiation. Under the influence of flagellin, the content of total and phosphorylated forms of hormone-sensitive lipase increased in brown adipocytes, while LPS induced a decrease in the content of total hormone-sensitive lipase in white adipocytes. We hypothesize that bacterial pathogen-associated molecular patterns can activate lipolysis under pathological conditions associated with slow remodeling of the adipose tissue.

Structural analysis of toll-like receptor 18 from soiny mullet (Liza haematocheila): Giving insights on the ligand binding mechanism of fish specific TLRs

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel β-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel β-sheet (βA-βD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18.

Multiplexed Plasma Immune Mediator Signatures Can Differentiate Sepsis From NonInfective SIRS: American Surgical Association 2020 Annual Meeting Paper.

Sepsis and sterile both release "danger signals' that induce the systemic inflammatory response syndrome (SIRS). So differentiating infection from SIRS can be challenging. Precision diagnostic assays could limit unnecessary antibiotic use, improving outcomes. After surveying human leukocyte cytokine production responses to sterile damage-associated molecular patterns (DAMPs), bacterial pathogen-associated molecular patterns, and bacteria we created a multiplex assay for 31 cytokines. We then studied plasma from patients with bacteremia, septic shock, "severe sepsis," or trauma (ISS ≥15 with circulating DAMPs) as well as controls. Infections were adjudicated based on post-hospitalization review. Plasma was studied in infection and injury using univariate and multivariate means to determine how such multiplex assays could best distinguish infective from noninfective SIRS. Infected patients had high plasma interleukin (IL)-6, IL-1α, and triggering receptor expressed on myeloid cells-1 (TREM-1) compared to controls [false discovery rates (FDR) <0.01, <0.01, <0.0001]. Conversely, injury suppressed many mediators including MDC (FDR <0.0001), TREM-1 (FDR <0.001), IP-10 (FDR <0.01), MCP-3 (FDR <0.05), FLT3L (FDR <0.05), Tweak, (FDR <0.05), GRO-α (FDR <0.05), and ENA-78 (FDR <0.05). In univariate studies, analyte overlap between clinical groups prevented clinical relevance. Multivariate models discriminated injury and infection much better, with the 2-group random-forest model classifying 11/11 injury and 28/29 infection patients correctly in out-of-bag validation. Circulating cytokines in traumatic SIRS differ markedly from those in health or sepsis. Variability limits the accuracy of single-mediator assays but machine learning based on multiplexed plasma assays revealed distinct patterns in sepsis- and injury-related SIRS. Defining biomarker release patterns that distinguish specific SIRS populations might allow decreased antibiotic use in those clinical situations. Large prospective studies are needed to validate and operationalize this approach.

Inositol-requiring enzyme 1 (IRE1) plays for AvrRpt2-triggered immunity and RIN4 cleavage in Arabidopsis under endoplasmic reticulum (ER) stress

Many stresses induce the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, a phenomenon known as ER stress. In response to ER stress, cells initiate a protective response, known as unfolded protein response (UPR), to maintain cellular homeostasis. The UPR sensor, inositol-requiring enzyme 1 (IRE1), catalyzes the cytoplasmic splicing of bZIP transcription factor-encoding mRNAs to activate the UPR signaling pathway. Recently, we reported that pretreatment of Arabidopsis thaliana plants with tunicamycin, an ER stress inducer, increased their susceptibility to bacterial pathogens; on the other hand, IRE1 deficient mutants were susceptible to Pseudomonas syringae pv. maculicola (Psm) and failed to induce salicylic acid (SA)-mediated systemic acquired resistance. However, the functional relationship of IRE1 with the pathogen and TM treatment remains unknown. In the present study, we showed that bacterial pathogen-associated molecular patterns (PAMPs) induced IRE1 expression; however, PAMP-triggered immunity (PTI) response such as callose deposition, PR1 protein accumulation, or Pst DC3000 hrcC growth was not altered in ire1 mutants. We observed that IRE1 enhanced plant immunity against the bacterial pathogen P. syringae pv. tomato DC3000 (Pst DC3000) under ER stress. Moreover, TM-pretreated ire1 mutants were more susceptible to the avirulent strain Pst DC3000 (AvrRpt2) and showed greater cell death than wild-type plants during effector-triggered immunity (ETI). Additionally, Pst DC3000 (AvrRpt2)-mediated RIN4 degradation was reduced in ire1 mutants under TM-induced ER stress. Collectively, our results reveal that IRE1 plays a pivotal role in the immune signaling pathway to activate plant immunity against virulent and avirulent bacterial strains under ER stress.

Changes in H3K27ac at Gene Regulatory Regions in Porcine Alveolar Macrophages Following LPS or PolyIC Exposure.

Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks (H3K4me3, H3K4me1, H3K27ac and H3K27me3), we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C).

ADP-heptose: a bacterial PAMP detected by the host sensor ALPK1.

The innate immune response constitutes the first line of defense against pathogens. It involves the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), the production of inflammatory cytokines and the recruitment of immune cells to infection sites. Recently, ADP-heptose, a soluble intermediate of the lipopolysaccharide biosynthetic pathway in Gram-negative bacteria, has been identified by several research groups as a PAMP. Here, we recapitulate the evidence that led to this identification and discuss the controversy over the immunogenic properties of heptose 1,7-bisphosphate (HBP), another bacterial heptose previously defined as an activator of innate immunity. Then, we describe the mechanism of ADP-heptose sensing by alpha-protein kinase 1 (ALPK1) and its downstream signaling pathway that involves the proteins TIFA and TRAF6 and induces the activation of NF-κB and the secretion of inflammatory cytokines. Finally, we discuss possible delivery mechanisms of ADP-heptose in cells during infection, and propose new lines of thinking to further explore the roles of the ADP-heptose/ALPK1/TIFA axis in infections and its potential implication in the control of intestinal homeostasis.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis.

Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.

1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts.

The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18h before stimulation with lipopolysaccharide (LPSμg/ml), lipoteichoic acid (LTAμg/ml) or polyinosinic:polycytidylic acid (poly I:C-20μg/ml) PAMPs for 6h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, β-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.

Genome-wide DNA hypomethylation shapes nematode pattern-triggered immunity in plants.

Summary A role for DNA hypomethylation has recently been suggested in the interaction between bacteria and plants; it is unclear whether this phenomenon reflects a conserved response.Treatment of plants of monocot rice and dicot tomato with nematode‐associated molecular patterns from different nematode species or bacterial pathogen‐associated molecular pattern flg22 revealed global DNA hypomethylation. A similar hypomethylation response was observed during early gall induction by Meloidogyne graminicola in rice. Evidence for the causal impact of hypomethylation on immunity was revealed by a significantly reduced plant susceptibility upon treatment with DNA methylation inhibitor 5‐azacytidine.Whole‐genome bisulphite sequencing of young galls revealed massive hypomethylation in the CHH context, while not for CG or CHG nucleotide contexts. Further, CHH hypomethylated regions were predominantly associated with gene promoter regions, which was not correlated with activated gene expression at the same time point but, rather, was correlated with a delayed transcriptional gene activation. Finally, the relevance of CHH hypomethylation in plant defence was confirmed in rice mutants of the RNA‐directed DNA methylation pathway and DECREASED DNA METHYLATION 1.We demonstrated that DNA hypomethylation is associated with reduced susceptibility in rice towards root‐parasitic nematodes and is likely to be part of the basal pattern‐triggered immunity response in plants.