ELP1, the Gene Mutated in Familial Dysautonomia, Is Required for Normal Enteric Nervous System Development and Maintenance and for Gut Epithelium Homeostasis.

Familial dysautonomia (FD) is a rare sensory and autonomic neuropathy that results from a mutation in the ELP1 gene. Virtually all patients report gastrointestinal (GI) dysfunction and we have recently shown that FD patients have a dysbiotic gut microbiome and altered metabolome. These findings were recapitulated in an FD mouse model and moreover, the FD mice had reduced intestinal motility, as did patients. To understand the cellular basis for impaired GI function in FD, the enteric nervous system (ENS; both female and male mice) from FD mouse models was analyzed during embryonic development and adulthood. We show here that not only is Elp1 required for the normal formation of the ENS, but it is also required in adulthood for the regulation of both neuronal and non-neuronal cells and for target innervation in both the mucosa and in intestinal smooth muscle. In particular, CGRP innervation was significantly reduced as was the number of dopaminergic neurons. Examination of an FD patient's gastric biopsy also revealed reduced and disoriented axons in the mucosa. Finally, using an FD mouse model in which Elp1 was deleted exclusively from neurons, we found significant changes to the colon epithelium including reduced E-cadherin expression, perturbed mucus layer organization, and infiltration of bacteria into the mucosa. The fact that deletion of Elp1 exclusively in neurons is sufficient to alter the intestinal epithelium and perturb the intestinal epithelial barrier highlights a critical role for neurons in regulating GI epithelium homeostasis.

Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.

Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of ß-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug (SNAI2) and Snail (SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of ß1 integrin and cellular differentiation state.

Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor.

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-Dependent Repression of Claudin-2.

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein-coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A(-/-) mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A(-/-) colons showed significantly more TNF-α(+) and Ly6C(hi)MHCII(-) proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A(-/-) mice. G2A(-/-) mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4(+) lymphocytes were recruited to inflamed G2A(-/-) colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A(-/-) mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α(+) monocyte and enhanced their maturation from Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues.

Intestinal Epithelial Ecto-5'-Nucleotidase (CD73) Regulates Intestinal Colonization and Infection by Nontyphoidal Salmonella.

Ecto-5'-nucleotidase (CD73) is expressed abundantly on the apical surface of intestinal epithelial cells (IECs) and functions as the terminal enzyme in the generation of extracellular adenosine. Previous work demonstrated that adenosine signaling in IECs results in a number of tissue-protective effects during inflammation; however, a rationale for its apical expression has been lacking. We hypothesized that the highly polarized expression of CD73 is indicative of an important role for extracellular adenosine as a mediator of host-microbe interactions. We show that adenosine harbors bacteriostatic activity against Salmonella enterica serovar Typhimurium that is not shared by the related purine metabolite 5'-AMP, inosine, or hypoxanthine. Analysis of Salmonella colonization in IEC-specific CD73 knockout mice (CD73f/fVillinCre ) revealed a nearly 10-fold increase in colonization compared to that in controls. Despite the increased luminal colonization by Salmonella, CD73f/fVillinCre mice were protected against Salmonella colitis and showed reduced Salmonella burdens in viscera, suggesting that adenosine promotes dissemination. The knockdown of CD73 expression in cultured IECs resulted in dramatic defects in intraepithelial localization and replication as well as defective transepithelial translocation by Salmonella In conclusion, we define a novel antimicrobial activity of adenosine in the gastrointestinal tract and unveil an important role for adenosine as a regulator of host-microbe interactions. These findings have broad implications for the development of new therapeutic agents for infectious disease.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

HIF1A reduces acute lung injury by optimizing carbohydrate metabolism in the alveolar epithelium.

BACKGROUND: While acute lung injury (ALI) contributes significantly to critical illness, it resolves spontaneously in many instances. The majority of patients experiencing ALI require mechanical ventilation. Therefore, we hypothesized that mechanical ventilation and concomitant stretch-exposure of pulmonary epithelia could activate endogenous pathways important in lung protection. METHODS AND FINDINGS: To examine transcriptional responses during ALI, we exposed pulmonary epithelia to cyclic mechanical stretch conditions--an in vitro model resembling mechanical ventilation. A genome-wide screen revealed a transcriptional response similar to hypoxia signaling. Surprisingly, we found that stabilization of hypoxia-inducible factor 1A (HIF1A) during stretch conditions in vitro or during ventilator-induced ALI in vivo occurs under normoxic conditions. Extension of these findings identified a functional role for stretch-induced inhibition of succinate dehydrogenase (SDH) in mediating normoxic HIF1A stabilization, concomitant increases in glycolytic capacity, and improved tricarboxylic acid (TCA) cycle function. Pharmacologic studies with HIF activator or inhibitor treatment implicated HIF1A-stabilization in attenuating pulmonary edema and lung inflammation during ALI in vivo. Systematic deletion of HIF1A in the lungs, endothelia, myeloid cells, or pulmonary epithelia linked these findings to alveolar-epithelial HIF1A. In vivo analysis of ¹³C-glucose metabolites utilizing liquid-chromatography tandem mass-spectrometry demonstrated that increases in glycolytic capacity, improvement of mitochondrial respiration, and concomitant attenuation of lung inflammation during ALI were specific for alveolar-epithelial expressed HIF1A. CONCLUSIONS: These studies reveal a surprising role for HIF1A in lung protection during ALI, where normoxic HIF1A stabilization and HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation.

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis.

Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine-creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2-dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.

Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.

Increased susceptibility of natural killer T-cell-deficient mice to acetaminophen-induced liver injury.

UNLABELLED: Acetaminophen (APAP) overdose causes severe, fulminant liver injury. The underlying mechanism of APAP-induced liver injury (AILI), studied by a murine model, displays similar characteristics of injury as those observed in patients. Previous studies suggest that aside from APAP-induced direct damage to hepatocytes, the hepatic innate immune system is activated and may contribute to the overall pathogenesis of AILI. The current study employed the use of two murine natural killer (NK) cells with T-cell receptor (NKT) cell knockout models (CD1d(-/-) and Jα18(-/-) ) to elucidate the specific role of NKT cells in AILI. Compared to wild-type (WT) mice, NKT cell-deficient mice were more susceptible to AILI, as indicated by higher serum alanine transaminase levels and mortality. Increased levels of cytochrome P450 2E1 (CYP2E1) protein expression and activities, which resulted in increased APAP protein adduct formation, were observed in livers of APAP-treated NKT cell-deficient mice, compared to WT mice. Compared to WT mice, starvation of NKT cell-deficient mice induced a higher increase of ketone bodies, which up-regulate CYP2E1 through protein stabilization. CONCLUSION: Our data revealed a novel role of NKT cells in regulating responses to starvation-induced metabolic stress. Elevated ketone body production in NKT cell-deficient mice resulted in increased CYP2E1-mediated APAP biotransformation and susceptibility to AILI.

A simplified protocol for deriving sterile, infectious murine Heligmosomoides polygyrus bakeri larvae.

Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae.

An endogenously anti-inflammatory role for methylation in mucosal inflammation identified through metabolite profiling.

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.

IFN-γ attenuates hypoxia-inducible factor (HIF) activity in intestinal epithelial cells through transcriptional repression of HIF-1ß.

Numerous studies have revealed that hypoxia and inflammation occur coincidentally in mucosal disorders, such as inflammatory bowel disease. During inflammation, epithelial-expressed hypoxia-inducible factor (HIF) serves an endogenously protective function. In this study, we sought to explore how mucosal immune responses influence HIF-dependent end points. Guided by a screen of relevant inflammatory mediators, we identified IFN-γ as a potent repressor of HIF-dependent transcription in human intestinal epithelial cells. Analysis of HIF levels revealed that HIF-1ß, but not HIF-1α, is selectively repressed by IFN-γ in a JAK-dependent manner. Cloning and functional analysis of the HIF-1ß promoter identified a prominent region for IFN-γ-dependent repression. Further studies revealed that colonic IFN-γ and HIF-1ß levels were inversely correlated in a murine colitis model. Taken together, these studies demonstrated that intestinal epithelial HIF is attenuated by IFN-γ through transcriptional repression of HIF-1ß. These observations are relevant to the pathophysiology of colitis (i.e., that loss of HIF signaling during active inflammation may exacerbate disease pathogenesis).

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification.

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-kappaB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.

Gut microbiome dysbiosis drives metabolic dysfunction in Familial dysautonomia.

Familial dysautonomia (FD) is a rare genetic neurologic disorder caused by impaired neuronal development and progressive degeneration of both the peripheral and central nervous systems. FD is monogenic, with >99.4% of patients sharing an identical point mutation in the elongator acetyltransferase complex subunit 1 (ELP1) gene, providing a relatively simple genetic background in which to identify modifiable factors that influence pathology. Gastrointestinal symptoms and metabolic deficits are common among FD patients, which supports the hypothesis that the gut microbiome and metabolome are altered and dysfunctional compared to healthy individuals. Here we show significant differences in gut microbiome composition (16 S rRNA gene sequencing of stool samples) and NMR-based stool and serum metabolomes between a cohort of FD patients (~14% of patients worldwide) and their cohabitating, healthy relatives. We show that key observations in human subjects are recapitulated in a neuron-specific Elp1-deficient mouse model, and that cohousing mutant and littermate control mice ameliorates gut microbiome dysbiosis, improves deficits in gut transit, and reduces disease severity. Our results provide evidence that neurologic deficits in FD alter the structure and function of the gut microbiome, which shifts overall host metabolism to perpetuate further neurodegeneration.

Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism.

Cell-free hemoglobin (Hb) exposure may be a pathogenic mediator in the development of pulmonary arterial hypertension (PAH), and when combined with chronic hypoxia the potential for exacerbation of PAH and vascular remodeling is likely more pronounced. We hypothesized that Hb may contribute to hypoxia-driven PAH collectively as a prooxidant, inflammatory, and nitric oxide (NO) scavenger. Using programmable micropump technology, we exposed male Sprague-Dawley rats housed under room air or hypoxia to 12 or 30 mg per day Hb for 3, 5, and 7 wk. Blood pressure, cardiac output, right ventricular hypertrophy, and indexes of pulmonary vascular remodeling were evaluated. Additionally, markers of oxidative stress, NO bioavailability and inflammation were determined. Hb increased pulmonary arterial (PA) pressure, pulmonary vessel wall stiffening, and right heart hypertrophy with temporal and dose dependence in both room air and hypoxic cohorts. Hb induced a modest increase in plasma oxidative stress markers (malondialdehyde and 4-hydroxynonenal), no change in NO bioavailability, and increased lung ICAM protein expression. Treatment with the antioxidant Tempol attenuated Hb-induced pulmonary arterial wall thickening, but not PA pressures or ICAM expression. Chronic exposure to low plasma Hb concentrations (range = 3-10 µM) lasting up to 7 wk in rodents induces pulmonary vascular disease via inflammation and to a lesser extent by Hb-mediated oxidation. Tempol demonstrated a modest effect on the attenuation of Hb-induced pulmonary vascular disease. NO bioavailability was found to be of minimal importance in this model.