Pathogen-associated Molecular Pattern Molecules Research Articles

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. Invitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Neutralizing Staphylococcus aureus PAMPs that Trigger Cytokine Release from THP-1 Monocytes.

Innate immunity has considerable specificity and can discriminate between individual species of microbes. In this regard, pathogens are "seen" as dangerous to the host and elicit an inflammatory response capable of destroying the microbes. This immune discrimination is achieved by toll-like receptors on host cells recognizing pathogens, such as Staphylococcus aureus, and microbe-specific pathogen-associated molecular pattern (PAMP) molecules, such as lipoteichoic acid (LTA). PAMPs impede wound healing by lengthening the inflammatory phase of healing and contributing to the development of chronic wounds. Preventing PAMPs from triggering the release of inflammatory cytokines will counteract the dysregulation of inflammation. Here, we use ELISA to evaluate the use of cationic molecules branched polyethylenimine (BPEI), PEGylated BPEI (PEG-BPEI), and polymyxin-B to neutralize anionic LTA and lower levels of TNF-α cytokine release from human THP-1 monocytes in a concentration-dependent manner. Additional data collected with qPCR shows that BPEI and PEG-BPEI reduce the expression profile of the TNF-α gene. Similar effects are observed for the neutralization of whole-cell S. aureus bacteria. In vitro cytotoxicity data demonstrate that PEGylation lowers the toxicity of PEG-BPEI (IC50 = 2661 μm) compared to BPEI (IC50 = 853 μM) and that both compounds are orders of magnitude less toxic than the cationic antibiotic polymyxin-B (IC50 = 79 μM). Additionally, the LTA neutralization ability of polymyxin-B is less effective than BPEI or PEG-BPEI. These properties of BPEI and PEG-BPEI expand their utility beyond disabling antibiotic resistance mechanisms and disrupting S. aureus biofilms, providing additional justification for developing these agents as wound healing therapeutics. The multiple mechanisms of action for BPEI and PEG-BPEI are superior to current wound treatment strategies that have a single modality.

Importance of the Frequency at Using Immunomodulatory Therapies with Evidence Based and Standardized Plant Biomodulators

Many years ago, a great deal of research was carried out to improve the suppressed activity of innate immune system in cancer patients using various immunomodulators originated from microorganisms or plants. However, more than 30 years ago these investigations were stopped since their repeated applications often led to a tolerance which could not be explained at the level of science at that time and in spite of a great amount of data, the curative role of innate system was poorly understood. Today, growing evidence suggests that the adaptive cytotoxic T cells have rather prophylactic effects working more proficiently in the early phases of tumor development, whereas the innate cellular system acts more curatively in parallel with disease progression. It’s now also known, that tumor-induced dysregulation of the innate immune system leads to a decreased function of type-1 effector cells and a predominance of type-2 cells, promoting the development of tumors. In this review article a number of results originating from old publications are discussed and presented again in order to interpret them according to our current knowledges which appear to be helpful for more correct application of standardized and evidence based immunomodulators (SEIM) from plant or microbes. Conclusions: 1.) Since the chemistry is not able to produce appropriate structures of Pathogen Associated Molecular Pattern (PAMP) molecules, we need PAMP containing SEIM from plants and microbes which can activate the type-1 phagocytic cells by binding their Pattern Recognition / Toll Like Receptors (RCC/TLR) on their cell membrane. 2.) PAMP molecules have a direct effect only on RCC/TLR molecules of phagocytes and only thereafter with a minimum time delay of 24 hours are the regulatory cascade mechanisms activated which can increase the function of the for the curative tumor defense important MHC-I unrestricted effectors (such as NK, γδT and type-1 NKT cells). 3.) A permanent activation of phagocytic cells can result in a decrease in MHC-1 unrestricted immune function, which may explain the tolerance observed often during continuous SEIM treatments. 4.) Since short-term activations of type-1 phagocytic cells causes more antitumor benefit, on the base of kinetic investigations a necessity to insert of 72h therapy-free intervals during SEIM therapy must be taken into consideration.

Impact of house dust mite in vitiligo skin: environmental contribution to increased cutaneous immunity and melanocyte detachment.

Vitiligo is an autoimmune skin disorder characterized by loss of melanocytes. Protease-mediated disruption of junctions between keratinocytes and/or keratinocyte intrinsic dysfunction may directly contribute to melanocyte loss. House dust mite (HDM), an environmental allergen with potent protease activity, contributes to respiratory and gut disease but also to atopic dermatitis and rosacea. To verify if HDM can contribute to melanocyte detachment in vitiligo and if so, by which mechanism(s). Using primary human keratinocytes, human skin biopsies from healthy donors and patients with vitiligo, and 3D reconstructed human epidermis, we studied the effect of HDM on cutaneous immunity, tight and adherent junction expression and melanocyte detachment. HDM increased keratinocyte production of vitiligo-associated cytokines and chemokines and increased expression of toll-like receptor (TLR)-4. This was associated with increased in situ matrix-metalloproteinase (MMP)-9 activity, reduced cutaneous expression of adherent protein E-cadherin, increased soluble E-cadherin in culture supernatant and significantly increased number of suprabasal melanocytes in the skin. This effect was dose-dependent and driven by cysteine protease Der p1 and MMP-9. Selective MMP-9 inhibitor, Ab142180, restored E-cadherin expression and inhibited HDM-induced melanocyte detachment. Keratinocytes from patients with vitiligo were more sensitive to HDM-induced changes than healthy keratinocytes. All results were confirmed in a 3D model of healthy skin and in human skin biopsies. Our results highlight that environmental mite may act as an external source of pathogen-associated molecular pattern molecules in vitiligo and topical MMP-9 inhibitors may be useful therapeutic targets. Whether HDM contributes to the onset of flares in vitiligo remains to be tested in carefully controlled trials.

"Open Sesame" to the complexity of pattern recognition receptors of myeloid-derived suppressor cells in cancer.

Pattern recognition receptors are primitive sensors that arouse a preconfigured immune response to broad stimuli, including nonself pathogen-associated and autologous damage-associated molecular pattern molecules. These receptors are mainly expressed by innate myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Recent investigations have revealed new insights into these receptors as key players not only in triggering inflammation processes against pathogen invasion but also in mediating immune suppression in specific pathological states, including cancer. Myeloid-derived suppressor cells are preferentially expanded in many pathological conditions. This heterogeneous cell population includes immunosuppressive myeloid cells that are thought to be associated with poor prognosis and impaired response to immune therapies in various cancers. Identification of pattern recognition receptors and their ligands increases the understanding of immune-activating and immune-suppressive myeloid cell functions and sheds light on myeloid-derived suppressor cell differences from cognate granulocytes and monocytes in healthy conditions. This review summarizes the different expression, ligand recognition, signaling pathways, and cancer relations and identifies Toll-like receptors as potential new targets on myeloid-derived suppressor cells in cancer, which might help us to decipher the instruction codes for reverting suppressive myeloid cells toward an antitumor phenotype.

Effect of Holstein genotype on ex-vivo cytokine response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during the periparturient period

Effects of Holstein genotype on innate immune response were assessed with ex-vivo lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation of whole blood from unselected (UH, n = 10) and contemporary (CH, n = 11) Holsteins that differ in production by more than 4,500 kg/lactation. Blood was collected at −14, 7, 28, and 49 days in milk (DIM), mixed with a pathogen-associated molecular pattern (PAMP) molecule (0.01 or 1.0 µg LPS or 10 or 100 µg LTA per mL blood) and incubated (4 h, 37 °C). Plasma cytokines were quantified by ELISA, log10-transformed and analyzed by repeated measures with DIM as the repeated effect. Cytokine responses increased with PAMP dose and decreased as DIM increased. There was a genotype by LPS dose interaction for IL-1β as response to the low dose was greater in UH but did not differ between genotypes for the high dose. The IL-1β response was greater while the IL-6 response to LTA tended to be greater in UH than in CH cows. The more negative energy balance of CH cows did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of CH cows to LPS and to LTA.

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.

Role of Microbiota-Derived Metabolites in Alcoholic and Non-Alcoholic Fatty Liver Diseases.

Chronic liver disease encompasses diseases that have various causes, such as alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Gut microbiota dysregulation plays a key role in the pathogenesis of ALD and NAFLD through the gut–liver axis. The gut microbiota consists of various microorganisms that play a role in maintaining the homeostasis of the host and release a wide number of metabolites, including short-chain fatty acids (SCFAs), peptides, and hormones, continually shaping the host’s immunity and metabolism. The integrity of the intestinal mucosal and vascular barriers is crucial to protect liver cells from exposure to harmful metabolites and pathogen-associated molecular pattern molecules. Dysbiosis and increased intestinal permeability may allow the liver to be exposed to abundant harmful metabolites that promote liver inflammation and fibrosis. In this review, we introduce the metabolites and components derived from the gut microbiota and discuss their pathologic effect in the liver alongside recent advances in molecular-based therapeutics and novel mechanistic findings associated with the gut–liver axis in ALD and NAFLD.

PDI family thioredoxin from disk abalone (Haliotis discus discus): Responses to stimulants (PAMPs, bacteria, and viral) and functional characterization

Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress, compared to the pcDNA3.1(+) transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress.

Post-Translational Modification of HMGB1 Disulfide Bonds in Stimulating and Inhibiting Inflammation.

High mobility group box 1 protein (HMGB1), a highly conserved nuclear DNA-binding protein, is a “damage-associated molecular pattern” molecule (DAMP) implicated in both stimulating and inhibiting innate immunity. As reviewed here, HMGB1 is an oxidation-reduction sensitive DAMP bearing three cysteines, and the post-translational modification of these residues establishes its proinflammatory and anti-inflammatory activities by binding to different extracellular cell surface receptors. The redox-sensitive signaling mechanisms of HMGB1 also occupy an important niche in innate immunity because HMGB1 may carry other DAMPs and pathogen-associated molecular pattern molecules (PAMPs). HMGB1 with DAMP/PAMP cofactors bind to the receptor for advanced glycation end products (RAGE) which internalizes the HMGB1 complexes by endocytosis for incorporation in lysosomal compartments. Intra-lysosomal HMGB1 disrupts lysosomal membranes thereby releasing the HMGB1-transported molecules to stimulate cytosolic sensors that mediate inflammation. This HMGB1-DAMP/PAMP cofactor pathway slowed the development of HMGB1-binding antagonists for diagnostic or therapeutic use. However, recent discoveries that HMGB1 released from neurons mediates inflammation via the TLR4 receptor system, and that cancer cells express fully oxidized HMGB1 as an immunosuppressive mechanism, offer new paths to targeting HMGB1 for inflammation, pain, and cancer.

Self-extracellular RNA promotes pro-inflammatory response of astrocytes to exogenous and endogenous danger signals

ObjectiveAstrocytes participate in the local innate immune response of the central nervous system. In response to stress such as ischemia, activated cells release endogenous factors known as damage-associated molecular patterns (DAMPs). Self-extracellular RNA (eRNA) is such a ubiquitous alarm signal. However, it is unclear whether eRNA is involved in the early acute phase of cerebral ischemia and is sufficient to sensitize astrocytes towards a DAMP or PAMP (pathogen-associated molecular pattern) reaction.MethodsPro-inflammatory activation upon eRNA stimulation was characterized in primary murine astrocyte cultures. In vivo, an experimental stroke model was used to localize and quantify eRNA in murine brain sections. Using primary cortical neurons and the mouse hippocampal neuronal cell line HT-22, neuronal RNA release upon stress conditions related to cerebral hypoxia/ischemia was analyzed.ResultsWhile low-dose eRNA alone did not promote pro-inflammatory activation of astrocytes in culture, it strongly enhanced the expression of pro-inflammatory cytokines in the presence of either Pam2CSK4, a synthetic PAMP molecule that mimics bacterial infection, or high mobility group box 1 (HMGB1), a prominent DAMP. Synergism of eRNA/Pam2CSK4 and eRNA/HMGB1 was prevented by blockage of the astroglial toll-like receptor (TLR)-2. Inhibition of NF-κB- and mitogen-activated protein kinase-dependent signaling pathways hampered eRNA/Pam2CSK4-mediated pro-inflammatory activation of astrocytes. In vivo, the amount of non-nuclear, presumably extracellular ribosomal RNA in close proximity to neurons significantly accumulated across the infarct core and peri-infarct areas that was accompanied by transcriptional up-regulation of various pro-inflammatory factors. Accordingly, the exposure of neurons to hypoxic/ischemic stress in vitro resulted in the release of eRNA, partly mediated by active cellular processes dependent on the cytosolic calcium level.ConclusionThe DAMP signal eRNA can sensitize astrocytes as active players in cerebral innate immunity towards exogenous and endogenous activators of inflammation (PAMPs and DAMPs) in a synergistic manner via TLR2-NF-κB-dependent signaling mechanisms. These findings provide new insights into the pathogenesis of ischemic stroke and other inflammatory neurological disorders. Further studies will clarify whether administration of RNase in vivo may serve as an effective treatment for inflammatory brain pathologies.

Effect of polymyxin B on ex vivo tumor necrosis factor-alpha responsiveness of blood leukocytes in Danish Holstein Friesian cows

Whole blood stimulation assay (WBA) with killed gram-positive and gram-negative udder pathogens were used to investigate the interference of the endotoxin-binding antibiotic polymyxin B (PMB) on the ex vivo TNF-α response. Blood samples were collected from first to third lactating dairy cows in their early lactation (<50 days in milk, n = 32) period. The WBA was stimulated with both inactivated bacteria (e.g., dead Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis), at a concentration of 2.5 × 106/mL; and pathogen-associated molecular pattern molecules, namely E. coli LPS (10 μg/mL), and S. aureus peptidoglycan (PG, 10 μg/mL). The PMB was added at a concentration of 0, 12.5, 25, 50, 100, and 200 μg/mL to each stimulant, respectively. All bacteria stimulants resulted in an increased TNF-α response compared to the negative control. The PMB affected the TNF-α responses of gram-positive (except S. dysgalactaie), gram-negative bacteria; and bacterial cell wall components at a PMB concentration of 25–50 μg/mL. The LPS and E. coli had similar TNF-α response but PG had a lower TNF-α response than gram-positive bacteria. The doses of PMB (≥ 25 μg/mL) should be used with caution when using different types of pathogens or should be avoided in ex vivo TNF-α studies.

Molecular Characteristics of RAGE and Advances in Small-Molecule Inhibitors.

Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily. RAGE binds and mediates cellular responses to a range of DAMPs (damage-associated molecular pattern molecules), such as AGEs, HMGB1, and S100/calgranulins, and as an innate immune sensor, can recognize microbial PAMPs (pathogen-associated molecular pattern molecules), including bacterial LPS, bacterial DNA, and viral and parasitic proteins. RAGE and its ligands stimulate the activations of diverse pathways, such as p38MAPK, ERK1/2, Cdc42/Rac, and JNK, and trigger cascades of diverse signaling events that are involved in a wide spectrum of diseases, including diabetes mellitus, inflammatory, vascular and neurodegenerative diseases, atherothrombosis, and cancer. Thus, the targeted inhibition of RAGE or its ligands is considered an important strategy for the treatment of cancer and chronic inflammatory diseases.

Perioperative alterations in the intestinal microbiota and functional changes mediate innate immune activation after small bowel transplantation

AimSmall bowel transplantation (SBT) is the only therapy for end-stage short bowel syndrome. However, complicated pathological changes and an increased risk of postoperative infections in the perioperative period are major obstacles to patient survival, but the associated mechanisms remain unclear. MethodsTo explore perioperative alterations in the intestinal microbiota and their functional changes after SBT, 16S rRNA sequencing of ileostomy effluents and plasma analysis were performed pre-SBT and periodically post-SBT. ResultsThe results suggested that the presence of Proteobacteria accelerated bacterial motility and chemotaxis during the first week in post-SBT recipients. Altered gut microbiota impaired intestinal barrier integrity and upregulated 16S rDNA, pathogen-associated molecular pattern (PAMP) and pattern-recognition molecule (PRM) levels in peripheral circulation. Importantly, the levels of neutrophils, monocytes, cytotoxic T lymphocytes, and natural killer cells and the expression of proinflammatory cytokines were increased in the peripheral blood and had potential roles in activating innate immune-mediated inflammatory injury after SBT. ConclusionTogether, our results suggest that altered microbiota and functional changes are probably related to innate immune-mediated inflammatory injury and graft survival after SBT, suggesting that the monitoring and regulation of intestinal microbiota are necessary for SBT patients.

Liposome engraftment and antigen combination potentiate the immune response towards conserved epitopes of the malaria vaccine candidate MSP2

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored surface antigen on merozoites of the malaria parasite Plasmodium falciparum. It consists of highly conserved N- and C-terminal domains, and a central polymorphic region that allows all MSP2 alleles to be categorized into the 3D7 or FC27 family. Previously it has been shown that epitope accessibility differs between lipid-bound and lipid-free MSP2, suggesting that lipid interactions modulate the conformation and antigenicity in a way that may better mimic native MSP2 on the merozoite surface. Therefore, we have immunised mice with MSP2 engrafted onto liposomes using a C-terminal tether that mimics the native GPI anchor. To improve the immunogenicity of the formulated antigen, liposomes were supplemented with Pathogen Associated Molecular Pattern molecules, specifically agonists of the Toll-like receptor 4 (TLR4) or TLR2. Induced antibodies were directed mostly towards conserved epitopes, predominantly in the conserved C-terminal region of MSP2. We also found that immunisation with a combination of 3D7 and FC27 MSP2 enhanced antibody responses to conserved epitopes, and that the overall responses of mice immunised with MSP2-engrafted liposomes were comparable in magnitude to those of mice immunised with MSP2 formulated in Montanide ISA720. The antibodies elicited in mice by immunising with MSP2-engrafted liposomes recognised the native form of parasite MSP2 on western blots and were found to be cross-reactive with isolated 3D7 and FC27 merozoites when investigated by ELISA. The liposome-tethered MSP2 induced higher titres of complement-fixing antibodies to 3D7 and FC27 MSP2 than did MSP2 formulated in Montanide ISA720. Our results indicate that liposomal formulation represents a viable strategy for eliciting a strong immune response that favours conserved epitopes in MSP2 and thus a strain-transcendent immune response.

Pathogen-associated selection on innate immunity genes (TLR4, TLR7) in a neotropical rodent in landscapes differing in anthropogenic disturbance

Toll-like receptors (TLRs) form part of the innate immune system and can recognize structurally conserved pathogen-associated molecular pattern (PAMP) molecules. Their functional importance in the resistance to pathogens has been documented in laboratory experimental settings and in humans. TLR diversity, however, has been rarely investigated in wildlife species. How the genetic diversity of TLRs is associated with various pathogens and how it is shaped by habitat disturbance are understudied. Therefore, we investigated the role of genetic diversity in the functionally important parts of TLR4 and TLR7 genes in resistance towards gastrointestinal nematodes and Hepacivirus infection. We chose a generalist study species, the rodent Proechimys semispinosus, because it is highly abundant in three Panamanian landscapes that differ in their degree of anthropogenic modification. We detected only two TLR7 haplotypes that differed by one synonymous single-nucleotide polymorphism (SNP) position. The TLR4 variability was higher, and we detected four TLR4 haplotypes that differed at one synonymous SNP and at three amino acid positions within the leucine-rich repeat region. Only TLR4 haplotypes had different frequencies in each landscape. Using generalized linear models, we found evidence that nematode loads and virus prevalence were influenced by both specific TLR4 haplotypes and landscape. Here, the variable “landscape” served as a surrogate for the important influential ecological factors distinguishing landscapes in our study, i.e. species diversity and host population density. Individuals carrying the common TLR4_Ht1 haplotype were less intensely infected by the most abundant strongyle nematode. Individuals carrying the rare TLR4_Ht3 haplotype were all Hepacivirus-positive, where those carrying the rare haplotype TLR4_Ht4 were less often infected by Hepacivirus than individuals with other haplotypes. Our study highlights the role of TLR diversity in pathogen resistance and the importance of considering immune genetic as well as ecological factors in order to understand the effects of anthropogenic changes on wildlife health.

ACOD1 in immunometabolism and disease.

Immunometabolism plays a fundamental role in health and diseases and involves multiple genes and signals. Aconitate decarboxylase 1 (ACOD1; also known as IRG1) is emerging as a regulator of immunometabolism in inflammation and infection. Upregulation of ACOD1 expression occurs in activated immune cells (e.g., macrophages and monocytes) in response to pathogen infection (e.g., bacteria and viruses), pathogen-associated molecular pattern molecules (e.g., LPS), cytokines (e.g., TNF and IFNs), and damage-associated molecular patterns (e.g., monosodium urate). Mechanistically, several immune receptors (e.g., TLRs and IFNAR), adapter proteins (e.g., MYD88), ubiquitin ligases (e.g., A20), and transcription factors (e.g., NF-κB, IRFs, and STATs) form complex signal transduction networks to control ACOD1 expression in a context-dependent manner. Functionally, ACOD1 mediates itaconate production, oxidative stress, and antigen processing and plays dual roles in immunity and diseases. On the one hand, activation of the ACOD1 pathway may limit pathogen infection and promote embryo implantation. On the other hand, abnormal ACOD1 expression can lead to tumor progression, neurodegenerative disease, and immune paralysis. Further understanding of the function and regulation of ACOD1 is important for the application of ACOD1-based therapeutic strategies in disease.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Abstract B159: Investigating the neuroimmune interaction between nociceptive neurons and dendritic cells

Abstract Nociceptive somatosensory neurons (nociceptors) have recently been established as controllers of immune responses in the context of infections, allergic airway inflammation, and imiquimod (IMQ)-induced psoriasiform skin inflammation. In most of these models, myeloid cells including dendritic cells (DCs) were shown to be a target of the actions of nociceptors. DCs form a bridge between the innate and adaptive immune system and it is mostly through their actions that the adaptive immune system gets activated. Notably, DCs were previously found to engage in a direct physical interaction with nociceptors as well as to be affected by soluble neuropeptides produced by the latter. Conflicting results have, however, been reported with regards to the function and the requirement of neuropeptides for the nociceptor:DC interaction under in vivo settings, suggesting a degree of context-dependency, while the requirement for physical interaction of the two cell types has not been tested. Inherently, in vivo approaches are not readily amenable for dissection of the molecular underpinnings of the interaction, and involvement of other, accessory cells can be difficult to exclude. Consequently, we have established an in-vitro nociceptor:DC co-culture system, which, compared to in vivo settings, allows us to dissect nociceptor:DC interactions in a less complex, reductionist setting.Using the in vitro co-culture setup, we show that dendritic cells and nociceptors engage in tight physical interactions, as well as that nociceptors produce chemo-attractant signals, which act directly on DCs. Further, we demonstrate that nociceptors enhance production of IL-6 and IL12-p40 cytokines by dendritic cells in response to IMQ, a TLR7 agonist. Strikingly, this effect was only apparent when DCs and nociceptors were allowed direct contact, suggesting a contact or proximity-based mechanism of interaction. In addition to IMQ, we identify other pathogen-associated molecular pattern (PAMP) molecules, which elicit a cytokine response that can be enhanced in the presence of nociceptors. Next, using live cell imaging, we showed that, as described previously, treatment of nociceptors with capsaicin, a TRPV-1 channel agonist, results in calcium flux and membrane depolarization of the neuronal cells. Strikingly, similar changes can be observed in DCs interacting with the neurons, while DCs alone do not exhibit any such behavior when treated with capsaicin. To further substantiate our findings, we utilized channelrhodopsin-expressing neurons, which can be activated by blue light without any other perturbations of the system. Importantly, light-induced activation of such neurons was sufficient to drive a calcium flux in the interacting DCs.Finally, to gain an unbiased view of the effects that nociceptors have on DCs, we performed RNA sequencing of DCs after co-culture. Strikingly, we observed profound changes in gene expression across DC subsets when DCs were cultured with neurons without any further stimulation as well as when they were treated with IMQ in the co-culture.In summary, we have developed an in vitro system that allows us to investigate the interactions between nociceptors and DCs. Using this system, we have confirmed that nociceptors directly communicate with DCs, we addressed the requirement for physical interactions of the two cell types and we have begun to shed light on the molecular underpinnings of these interactions. Citation Format: Pavel Hanc, Siyi Huang, Ulrich H. von Andrian. Investigating the neuroimmune interaction between nociceptive neurons and dendritic cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B159.

Tough and Immunosuppressive Titanium-Infiltrated Exoskeleton Matrices for Long-Term Endoskeleton Repair.

Although biodegradable membranes are essential for effective bone repair, severe loss of mechanical stability because of rapid biodegradation, soft tissue invasion, and excessive immune response remain intrinsically problematic. Inspired by the exoskeleton-reinforcing strategy found in nature, we have produced a Ti-infiltrated chitin nanofibrous membrane. The membrane employs vapor-phase infiltration of metals, which often occurs during metal oxide atomic layer deposition (ALD) on organic substrates. This metal infiltration manifests anomalous mechanical improvement and stable integration with chitin without cytotoxicity and immunogenicity. The membrane exhibits both impressive toughness (∼13.3 MJ·m-3) and high tensile strength (∼55.6 MPa), properties that are often mutually exclusive. More importantly, the membrane demonstrates notably enhanced resistance to biodegradation, remaining intact over the course of 12 weeks. It exhibits excellent osteointegrative performance and suppresses the immune response to pathogen-associated molecular pattern molecules indicated by IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor expression. We believe the excellent chemico-biological properties achieved with ALD treatment can provide insight for synergistic utilization of the polymers and ALD in medical applications.