Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden

Fatty acid metabolism in Saccharomyces cerevisiae and effects of fatty acid metabolites on neutrophil function

In the presence of arachidonic acid (AA), Saccharomyces cerevisiae produces prostaglandin E2 (PGE2). S. cerevisiae and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE2 can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE2 on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE2 (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE2 (100 nM). The biochemical pathway utilized by S. cerevisiae to produce PGE2 is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE2 may have on neutrophils. S. cerevisiae does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in S. cerevisiae and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (Pot1, Pox1, Faa1 and Faa2) were upregulated up to 2.3-fold. The results demonstrate that low concentrations of PGE2 enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE 2 may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE2 is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE2 in S. cerevisiae. Experiments testing PGE2 levels in knock-out strains of yeast will identify genes involved in PGE2 production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE 2 in consumables

Immunoregulatory Mechanisms in a Mouse Model of Hookworm Infection

Parasitic worms infect billions of people worldwide. In humans, these infections and inflammation are chronic and debilitating. Therefore, it is imperative to explore host factors that regulate immunity and promote parasite clearance and host protection. In this thesis we discuss two such host factors; resistin like molecules and endocannabinoids.We describe here that during infection with the rodent hookworm Nippostrongylus brasiliensis (Nb), a host molecule named RELM alpha dampens Th2 inflammatory responses. We use bone marrow (BM) chimera technology to show that RELM alpha from BM-derived cells and not non BM cells dampens Th2 immunity and prevents worm clearance. We show that of the BM cells, CD11c+ lung macrophages are the dominant source of RELM alpha. Next, we employ a macrophage-worm co-culture system and found that RELM alpha impairs the ability of macrophages to attach to and kill worms. By conducting gene expression analysis, we show that RELM alpha decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Second, we explored how endocannabinoid signaling influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that function in feeding behavior and metabolism. Following Nb infection, we detected elevated levels of endocannabinoids 2-AG and AEA in the worm infected mouse lung and intestine. To test how endocannabinoids influence helminth infection, we utilized pharmacological inhibitors of cannabinoid receptors. We found inhibition of CB1R resulted in higher worm and eggs burdens which was correlated with decreased amounts of the Th2 cytokine IL-5 in the host. Interestingly, bioinformatic analysis revealed putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. Additionally, we found that all lifecycle stages of Nb produce endocannabinoids. Therefore, we report for the first time that helminth and host-derived endocannabinoids promote host immune responses and reduce parasite burden.Collectively, these studies demonstrate two mechanisms of immunomodulation during helminth infection. In the first mechanism, BM-derived RELM alpha dampens immune responses and inhibits macrophage killing of worms. In the second mechanism, we describe how host and parasite-derived endocannabinoids promote host immunity by upregulating Th2 immunity which drives parasite clearance. In summary, we have identified key regulators of immune responses in helminth infection

Eosinophil diversity in asthma

peer reviewedEosinophils are a type of granulated innate immune cells that have long been implicated in a specific type of asthma, referred to as eosinophilic asthma. Several immunotherapeutics that target and deplete eosinophils or limit their numbers are currently widely used and provide improved disease outcome in severe eosinophilic asthma. Current clinical results provide conclusive evidence of a generally detrimental role of eosinophils in asthma. Yet, a small but growing body of reports suggests that eosinophils may be more diverse than currently appreciated. In this review, we explore pre-clinical and clinical evidence that suggests the existence of eosinophil subsets with potentially distinct functional roles in asthma. We conclude by discussing state-of-the-art strategies for deciphering heterogeneity of this complex cell type, and argue this knowledge could translate into the improved personalized treatment of severe eosinophilic asthma. © 2020 Elsevier Inc

Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (hRETNTg(+)) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of Nippostrongylus brasiliensis (Nb)-infected hRETNTg(+) mice after a fatal LPS dose compared with naive mice or Nb-infected hRETNTg(-) mice. Employing immunoprecipitation assays, hRETNTg(+)Tlr4(-/-) mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis

Hematopoietic cell-derived RELMalpha regulates hookworm immunity through effects on macrophages.

Resistin-like molecule alpha (RELMalpha) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMalpha dampens Th2 inflammatory responses. RELMalpha is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMalpha is unknown. We generated bone marrow (BM) chimeras that were RELMalpha deficient (RELMalpha(-/) (-) ) in BM or non BM cells and infected them with Nb. Non BM RELMalpha(-/-) chimeras had comparable inflammatory responses and parasite burdens to RELMalpha(+/+) mice. In contrast, both RELMalpha(-/-) and BM RELMalpha(-/-) mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c(+) lung macrophages were the dominant BM-derived source of RELMalpha and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMalpha regulates macrophage-mediated Nb killing. Compared to RELMalpha(+) (/+) macrophages, RELMalpha(-/-) macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMalpha partially reversed this phenotype. Gene expression analysis revealed that RELMalpha decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMalpha is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMalpha is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMalpha acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion