Platelet hyaluronidase-2 regulates the early stages of inflammatory disease in colitis.

Platelets are specialized cells essential for hemostasis that also function as crucial effectors capable of mediating inflammatory and immune responses. These sentinels continually survey their environment and discriminate between homeostatic and danger signals such as modified components of the extracellular matrix. The glycosaminoglycan hyaluronan (HA) is a major extracellular matrix component that coats the vascular lumen and, under normal conditions, restricts access of inflammatory cells. In response to tissue damage, the endothelial HA matrix enhances leukocyte recruitment and regulates the early stages of the inflammatory response. We have shown that platelets can degrade HA from the surface of activated endothelial cells via the enzyme hyaluronidase-2 (HYAL2) and that HYAL2 is deficient in platelets isolated from patients with inflammatory bowel disease (IBD). Platelets are known to be involved in the pathogenesis of several chronic disease states, including IBD, but they have been largely overlooked in the context of intestinal inflammation. We therefore wanted to define the mechanism by which platelet HYAL2 regulates the inflammatory response during colitis. In this study, we provide evidence that HA catabolism is disrupted in human intestinal microvascular endothelial cells isolated from patients with IBD. Furthermore, mice deficient in HYAL2 are more susceptible to an acute model of colitis, and this increased susceptibility is abrogated by transfusion of HYAL2-competent platelets. Finally, we show that platelets, via HYAL2-dependent degradation of endothelial HA, regulate the early stages of inflammation in colitis by limiting leukocyte extravasation.

Hyaluronan 35 kDa enhances epithelial barrier function and protects against the development of murine necrotizing enterocolitis.

BACKGROUND: Disruption of tight junctions (TJs) predisposes to bacterial translocation, intestinal inflammation, and necrotizing enterocolitis (NEC). Previously, studies showed that hyaluronan (HA), a glycosaminoglycan in human milk, maintains intestinal permeability, enhances intestinal immunity, and reduces intestinal infections. In this study, we investigated the effects of HA 35 kDa on a NEC-like murine model. METHODS: Pups were divided into Sham, NEC, NEC+HA 35, and HA 35. Severity of intestinal injury was compared using a modified macroscopic gut scoring and histologic injury grading. The effect of HA 35 on intestinal permeability was determined by measuring FITC dextran and bacterial translocation. RNA and protein expression of TJ proteins (claudin-2, -3, -4, occludin, and ZO-1) were compared between the groups. RESULTS: Pups in the NEC+HA 35 group had increased survival and lower intestinal injury compared to untreated NEC. In addition, HA 35 reduced intestinal permeability, bacterial translocation, and proinflammatory cytokine release. Ileal expression of claudin-2, -3, -4, occludin, and ZO-1 was upregulated in NEC+HA 35 and HA 35 compared to untreated NEC and shams. CONCLUSION: These findings suggest that HA 35 protects against NEC partly by upregulating intestinal TJs and enhancing intestinal barrier function.

Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

BACKGROUND: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. RESULTS: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. CONCLUSIONS: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

MicroRNA 181b-3p and its target importin α5 regulate toll-like receptor 4 signaling in Kupffer cells and liver injury in mice in response to ethanol.

Increased inflammatory signaling by Kupffer cells contributes to alcoholic liver disease (ALD). Here we investigated the impact of small, specific-sized hyaluronic acid of 35 kD (HA35) on ethanol-induced sensitization of Kupffer cells, as well as ethanol-induced liver injury in mice. Unbiased analysis of microRNA (miRNA) expression in Kupffer cells identified miRNAs regulated by both ethanol and HA35. Toll-like receptor 4 (TLR4)-mediated signaling was assessed in primary cultures of Kupffer cells from ethanol- and pair-fed rats after treatment with HA35. Female C57BL6/J mice were fed ethanol or pair-fed control diets and treated or not with HA35. TLR4 signaling was increased in Kupffer cells by ethanol; this sensitization was normalized by ex vivo treatment with HA35. Next generation sequencing of Kupffer cell miRNA identified miRNA 181b-3p (miR181b-3p) as sensitive to both ethanol and HA35. Importin α5, a protein involved in p65 translocation to the nucleus, was identified as a target of miR181b-3p; importin α5 protein was increased in Kupffer cells from ethanol-fed rats, but decreased by HA35 treatment. Overexpression of miR181b-3p decreased importin α5 expression and normalized lipopolysaccharide-stimulated tumor necrosis factor α expression in Kupffer cells from ethanol-fed rats. In a mouse model of ALD, ethanol feeding decreased miR181b-3p in liver and increased expression of importin α5 in nonparenchymal cells. Treatment with HA35 normalized these changes and also protected mice from ethanol-induced liver and intestinal injury. CONCLUSION: miR181b-3p is dynamically regulated in Kupffer cells and mouse liver in response to ethanol and treatment with HA35. miR181b-3p modulates expression of importin α5 and sensitivity of TLR4-mediated signaling. This study identifies a miR181b-3p-importin α5 axis in regulating inflammatory signaling pathways in hepatic macrophages. (Hepatology 2017;66:602-615).

Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix.

Dynamic alterations of the extracellular matrix in response to injury directly modulate inflammation and consequently the promotion and resolution of disease. During inflammation, hyaluronan (HA) is increased at sites of inflammation where it may be covalently modified with the heavy chains (HC) of inter-α-trypsin inhibitor. Deposition of this unique, pathological form of HA (HC-HA) leads to the formation of cable-like structures that promote adhesion of leukocytes. Naive mononuclear leukocytes bind specifically to inflammation-associated HA matrices but do not adhere to HA constitutively expressed under homeostatic conditions. In this study, we have directly investigated a role for the blood-coagulation protease thrombin in regulating the adhesion of monocytic cells to smooth muscle cells producing an inflammatory matrix. Our data demonstrate that the proteolytic activity of thrombin negatively regulates the adhesion of monocytes to an inflammatory HC-HA complex. This effect is independent of protease-activated receptor activation but requires proteolytic activity toward a novel substrate. Components of HC-HA complexes were predicted to contain conserved thrombin-susceptible cleavage sites based on sequence analysis, and heavy chain 1 (HC1) was confirmed to be a substrate of thrombin. Thrombin treatment is sufficient to cleave HC1 associated with either cell-surface HA or serum inter-α-trypsin inhibitor. Furthermore, thrombin treatment of the inflammatory matrix leads to dissolution of HC-HA cable structures and abolishes leukocyte adhesion. These data establish a novel mechanism whereby thrombin cleavage of HC1 regulates the adhesive properties of an inflammatory HA matrix.

Hyaluronan Depolymerization by Megakaryocyte Hyaluronidase-2 Is Required for Thrombopoiesis.

Hyaluronan is the predominant glycosaminoglycan component of the extracellular matrix with an emerging role in hematopoiesis. Modulation of hyaluronan polymer size is responsible for its control over cellular functions, and the balance of hyaluronan synthesis and degradation determines its molecular size. Although two active somatic hyaluronidases are expressed in mammals, only deficiency in hyaluronidase-2 (Hyal-2) results in thrombocytopenia of unknown mechanism. Our results reveal that Hyal-2 knockout mice accumulate hyaluronan within their bone marrow and within megakaryocytes, the cells responsible for platelet generation. Proplatelet formation by Hyal-2 knockout megakaryocytes was disrupted because of abnormal formation of the demarcation membrane system, which was dilated and poorly developed. Importantly, peptide-mediated delivery of exogenous hyaluronidase rescued deficient proplatelet formation in murine and human megakaryocytes lacking Hyal-2. Together, our data uncover a previously unsuspected mechanism of how hyaluronan and Hyal-2 control platelet generation.

Platelet hyaluronidase-2: an enzyme that translocates to the surface upon activation to function in extracellular matrix degradation.

Following injury, platelets rapidly interact with the exposed extracellular matrix (ECM) of the vessel wall and the surrounding tissues. Hyaluronan (HA) is a major glycosaminoglycan component of the ECM and plays a significant role in regulating inflammation. We have recently reported that human platelets degrade HA from the surfaces of activated endothelial cells into fragments capable of inducing immune responses by monocytes. We also showed that human platelets contain the enzyme hyaluronidase-2 (HYAL2), one of two major hyaluronidases that digest HA in somatic tissues. The deposition of HA increases in inflamed tissues in several inflammatory diseases, including inflammatory bowel disease (IBD). We therefore wanted to define the mechanism by which platelets degrade HA in the inflamed tissues. In this study, we show that human platelets degrade the proinflammatory matrix HA through the activity of HYAL2 and that platelet activation causes the immediate translocation of HYAL2 from a distinct population of α-granules to platelet surfaces where it exerts its catalytic activity. Finally, we show that patients with IBD have lower platelet HYAL2 levels and activity than healthy controls.

The extracellular matrix in IBD: a dynamic mediator of inflammation.

PURPOSE OF REVIEW: The extracellular matrix (ECM) is a frequently overlooked component of the pathogenesis of inflammatory bowel disease (IBD). However, the functional and clinically significant interactions between immune as well as nonimmune cells with the ECM have important implications for disease pathogenesis. In this review, we discuss how the ECM participates in process associated with IBD that involves diverse cell types of the intestine. RECENT FINDINGS: Remodeling of the ECM is a consistent feature of IBD, and studies have implicated key ECM molecules in IBD pathogenesis. While the majority of prior studies have focused on ECM degradation by proteases, more recent studies have uncovered additional degrading enzymes, identified fragments of ECM components as potential biomarkers, and revealed that ECM synthesis is increased in IBD. These new studies support the notion that the ECM, rather than acting as a passive element, is an active participant in promoting inflammation. SUMMARY: New studies have offered exciting clues about the function of the ECM during IBD pathogenesis. The balance of ECM synthesis and turnover is altered in IBD, and the molecules involved exhibit discreet biological functions that regulate inflammation on the basis of specific cell type and matrix molecule.

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

BACKGROUND & AIMS: Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS: Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS: CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells. CONCLUSIONS: CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

The epithelial danger signal IL-1α is a potent activator of fibroblasts and reactivator of intestinal inflammation.

Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC-released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α-positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1ß. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α-mediated IEC-fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD.

Determination of hyaluronan molecular mass distribution in human breast milk.

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M⩽20 kDa, M∼20 to 60 kDa, and M∼60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M⩾110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.

Human milk hyaluronan enhances innate defense of the intestinal epithelium.

Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human ß-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human ß-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hß D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease.

OBJECTIVE: Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model. DESIGN: Experimental colitis was induced in ceruloplasmin null (Cp(-/-)) and wild-type (WT) mice by dextran sulphate sodium administration. The role of ceruloplasmin was further evaluated by transplantation of WT macrophages into Cp(-/-) mice. RESULTS: Cp(-/-) mice rapidly lost weight and were moribund by day 14, while WT mice survived at least 30 days. Colon culture supernatants from Cp(-/-) mice exhibited elevated levels of TNFα, KC and MCP-1, indicative of increased inflammation and neutrophil and macrophage infiltration. Elevated leucocytes and severe histopathology were observed in Cp(-/-) mice. Elevated protein carbonyl content was detected in colons from Cp(-/-) mice suggesting ceruloplasmin antioxidant activity might contribute to its protective function. Unexpectedly, intraperitoneal administration of human ceruloplasmin into Cp(-/-) mice did not afford protection. Bone marrow transplantation from WT mice or injection of isolated peripheral blood monocytes markedly reduced severity of colitis and morbidity in Cp(-/-) mice. CONCLUSION: Macrophage-derived ceruloplasmin contributes importantly to protection against inflammation and tissue injury in acute and chronic experimental colitis. The findings suggest that defects in ceruloplasmin expression or processing may influence the onset or progression of inflammatory bowel disease in patients.

Specific-sized hyaluronan fragments promote expression of human ß-defensin 2 in intestinal epithelium.

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human ß-defensin 2 (HßD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HßD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HßD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HßD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HßD2 protein.

Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein.

Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ~150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20-150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ~150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases.

Increased levels of survivin, via association with heat shock protein 90, in mucosal T cells from patients with Crohn's disease.

BACKGROUND & AIMS: Defective apoptosis of lamina propria T cells (LPTs) is involved in the pathogenesis of Crohn's disease. Survivin, a member of the inhibitors of apoptosis family, prevents cell death and regulates cell division. Survivin has been studied extensively in cancer, but little is known about its role in Crohn's disease. METHODS: LPTs were isolated from mucosal samples of patients with Crohn's disease or ulcerative colitis and healthy individuals (controls). LPTs were activated with interleukin-2 or via CD3, CD2, and CD28 signaling, and cultured at 42°C to induce heat shock. Survivin expression was assessed by immunohistochemistry, confocal microscopy, and immunoblotting; survivin levels were reduced by RNA interference. Cell viability, apoptosis, and proliferation were measured by trypan blue exclusion, annexin-V/7-Aminoactinomycin D staining, and uptake of [3]thymidine, respectively. RESULTS: LPTs from patients with Crohn's disease had higher levels of survivin than LPTs from patients with ulcerative colitis or controls. RNA knockdown of survivin in LPTs inhibited their proliferation and promoted apoptosis. Levels of survivin were low in LPTs from patients with ulcerative colitis and controls as a result of ubiquitin-mediated proteasome degradation. In LPTs from patients with Crohn's disease, survivin bound to the heat shock protein (HSP)90, and therefore was resistant to proteasome degradation. Incubating LPTs with 17-N-allylamino-17-demethoxygeldanamycin, an inhibitor of HSP90, reduced levels of survivin and induced apoptosis. CONCLUSIONS: Levels of survivin are increased in LPTs from patients with Crohn's disease (compared with ulcerative colitis and controls) because survivin interacts with HSP90 and prevents proteasome degradation. This allows LPTs to avoid apoptosis. Strategies to restore apoptosis to these cells might be developed to treat patients with Crohn's disease.

35kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.

Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35 kDa have multiple protective effects in the gut and are well-tolerated in humans. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3ß and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.

The protein kinase R modifies gut physiology to limit colitis.

Here we investigate the function of the innate immune molecule protein kinase R (PKR) in intestinal inflammation. To model a colitogenic role of PKR, we determine the physiological response to dextran sulfate sodium (DSS) of wild-type and two transgenic mice strains mutated to express either a kinase-dead PKR or to ablate expression of the kinase. These experiments recognize kinase-dependent and -independent protection from DSS-induced weight loss and inflammation, against a kinase-dependent increase in the susceptibility to DSS-induced injury. We propose these effects arise through PKR-dependent alteration of gut physiology, evidenced as altered goblet cell function and changes to the gut microbiota at homeostasis that suppresses inflammasome activity by controlling autophagy. These findings establish that PKR functions as both a protein kinase and a signaling molecule in instituting immune homeostasis in the gut.

Inflammation-induced endothelial-to-mesenchymal transition: a novel mechanism of intestinal fibrosis.

In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-ß1, IL-1ß, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-ß1, IL-1ß and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.