Human Intestinal Endothelial Cells Research Articles

Direct therapeutic effect of sulfadoxine-pyrimethamine on nutritional deficiency-induced enteric dysfunction in a human Intestine Chip

Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth. Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs). Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips. Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials. The Bill and Melinda Gates Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the HDDC Organoid Core of the P30 DK034854.

Analysis of the interferon-γ-induced secretome of intestinal endothelial cells: putative impact on epithelial barrier dysfunction in IBD.

The development of inflammatory bowel diseases (IBD) involves the breakdown of two barriers: the epithelial barrier and the gut-vascular barrier (GVB). The destabilization of each barrier can promote initiation and progression of the disease. Interestingly, first evidence is available that both barriers are communicating through secreted factors that may accordingly serve as targets for therapeutic modulation of barrier functions. Interferon (IFN)-γ is among the major pathogenesis factors in IBD and can severely impair both barriers. In order to identify factors transmitting signals from the GVB to the epithelial cell barrier, we analyzed the secretome of IFN-γ-treated human intestinal endothelial cells (HIEC). To this goal, HIEC were isolated in high purity from normal colon tissues. HIEC were either untreated or stimulated with IFN-γ (10U/mL). After 48h, conditioned media (CM) were harvested and subjected to comparative hyper reaction monitoring mass spectrometry (HRM™ MS). In total, 1,084 human proteins were detected in the HIEC-CM. Among these, 43 proteins were present in significantly different concentrations between the CM of IFN-γ- and control-stimulated HIEC. Several of these proteins were also differentially expressed in various murine colitis models as compared to healthy animals supporting the relevance of these proteins secreted by inflammatory activated HIEC in the inter-barrier communication in IBD. The angiocrine pathogenic impact of these differentially secreted HIEC proteins on the epithelial cell barrier and their perspectives as targets to treat IBD by modulation of trans-barrier communication is discussed in detail.

NKG2D ligand expression in Crohn's disease and NKG2D-dependent stimulation of CD8+ T cell migration

Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1–6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohn's disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4–6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8+ T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56+ T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8+ T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D+ lymphocytes into the inflamed CD intestine.

P-196 YI Human Intestinal Mesenchymal Cells Exhibit a Pro-Fibrogenic Phenotype in Response to Adipose Tissue Derived-Mediators—Linking Fat to Intestinal Fibrosis

Mesenteric adipose tissue, mainly consisting of preadipocytes and adipocytes, is increasingly considered an active player in the pathogenesis of inflammatory bowel disease (IBD). Adipocyte hyperplasia and creeping fat in Crohn's disease (CD) are associated with stricture formation, which is caused by excessive extracellular matrix (ECM) deposition by myofibroblasts and muscle cells. We therefore investigated whether mesenteric adipocyte/preadipocyte-derived mediators induce a pro-fibrogenic response by human intestinal myofibroblasts (HIF) and muscle cells (HIMC) and the mechanisms involved. HIF and HIMC derived from IBD and control tissues were exposed to 6 different adipokines (leptin, chemerin, resistin, visfatin, vaspin, nesfatin), free fatty acids (FFA) of different length (palmitate, stearate, oleate, decanoate, butyrate) as well as TNF, IL-1β, TGF-β1, bFGF, or their combinations, and cell proliferation was assessed by 3H-thymidine incorporation and cell counting. Modulation of αSMA was investigated by immunoblot; fibronectin (FN), collagen I (Col 1) and IL-6 content in the supernatants was measured by ELISA, RIA or immunoblot; and migration by Boyden chamber assay. p38MAPK, PKC, PI3K, NFκB, TLR2, TLR4 or MyD88 signaling was blocked by specific inhibitors or siRNA, respectively. Primary human intestinal endothelial cells, preadipocytes and adipocytes as well as the intestinal epithelial cell line CaCo2 served as controls. bFGF and long chain, but not short chain, FFA increased proliferation of HIF and HIMC 2.2 and 3.4-fold, respectively (P < 0.001). When mediators were combined a striking synergistic effect of bFGF and long-chain FFA was observed, with a dramatic 12-fold increase in HIF and HIMC proliferation. Synergy was dependent on p38MAPK, PKC and PI3K, but not MyD88, TLR2, TLR4, NFκB or CD36. Supernatants of human intestinal adipocytes, but not preadipocytes increased proliferation of HIMC. The effect of palmitate, the most abundant long-chain FFA, on mesenchymal cell proliferation was specific, since it failed to induce proliferation in intestinal epithelial, endothelial cells as well as preadipocytes and adipocytes. TGF-β1, bFGF and TNF induced a marked increase in FN secretion, while TGF-β1, bFGF and IL-1β enhanced the production of Col-1 (P < 0.05–0.001) by HIF. The HIF inflammatory response, as measured by IL-6, was increased by IL-1β, TNF and TGF-β1 (P < 0.05), and TGF-β1 upregulated αSMA expression. HIF- and HIMC-conditioned medium induced HIF and HIMC migration with further increase after exposure to TGF-β1 and bFGF (P < 0.01). Adipokines and FFA failed to increase matrix production, IL-6 secretion, αSMA expression or cell migration. The above results were largely independent of the IBD or control origin of HIF or HIMC. Mesenteric adipocyte/preadipocyte-derived mediators induce a differential and selective fibrogenic response by HIF and HIMC. bFGF and long-chain, but not short chain, FFA synergistically increased HIF and HIMC proliferation in a p38MAPK-, PKC- and PI3K-dependent, but NFκB-, MyD88-, TLR2-, TLR4- and CD36-independent manner. Adipocyte-, but not preadipocyte-conditioned medium enhanced HIMC proliferation. TGF-β1, bFGF, TNF and IL-1β, but not adipokines or FFA increased ECM secretion, IL-6 production, αSMA expression and cell migration. These results indicate that fat-derived mediators actively contribute to intestinal fibrosis.

Investigating the role of proinflammatory CD16+ monocytes in the pathogenesis of inflammatory bowel disease

Infiltrating monocytes and macrophages contribute to the initiation and perpetuation of mucosal inflammation characteristic for human inflammatory bowel disease (IBD). Peripheral blood monocytes expressing the low-affinity Fcgamma receptor CD16 have been identified previously as a major proinflammatory cell population, based on their unique cytokine secretion profile. However, the contribution of these cells to the pathogenesis of inflammatory bowel disease remains to be elucidated. Thus, in this study we investigated whether the peripheral CD16(+) monocyte count correlates with common IBD disease parameters, and whether these cells infiltrate the intestinal mucosa under inflammatory conditions. We observed that CD16(+) peripheral blood monocytes are increased significantly in active Crohn's disease, particularly in patients with high Crohn's disease activity index and colonic involvement. Furthermore, we found that CD16(+) cells are a major contributor to the inflammatory infiltrate in Crohn's disease mucosa, although their spontaneous migration through primary human intestinal endothelial cells is limited. Our data suggest that lamina propria, but not peripheral blood, CD16(+) monocytes are a crucial proinflammatory cell population in IBD, and a potential target for anti-inflammatory therapy.

Hyaluronan (HA) deposition precedes and promotes leukocyte recruitment in intestinal inflammation.

Increased hyaluronan (HA) deposition is a common feature of inflamed tissues, including inflammatory bowel disease (IBD)-involved intestines. However, whether HA accumulation promotes or is the result of intestinal inflammation is unknown. Using the mouse dextran sulfate sodium (DSS)-induced experimental model of colitis, we investigated changes in HA deposition in the colon over time in conjunction with evolving pathological changes of tissue architecture. Profound changes in colon HA deposition occurred within 3-7 days of oral DSS administration and, more important, they preceded the inflammatory infiltrate. Interestingly, HA deposition within blood vessels of the colon is observed as early as 3 days during the course of colitis induction, well before any significant inflammatory infiltrate. HA deposition is also observed in blood vessels of inflamed human colon of IBD patients. We determined that human intestinal endothelial cells generate HA in response to proinflammatory stimuli by demonstrating a TNF-alpha-induced increase in hyaluronan synthase-3 mRNA expression and the accumulation of HA cable-like structures that are adhesive for leukocytes. Additionally, IBD mucosal endothelial cells produce higher levels of cell surface HA in response to TNF-alpha than non-IBD control cells. Therefore, HA deposition is an early event in inflamed gut tissue, preceding and likely promoting leukocyte infiltration.

Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

Human intestinal microvascular endothelial cells express Toll-like receptor 5: a binding partner for bacterial flagellin.

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

Butyrate modulates gene and protein expression in human intestinal endothelial cells

We hypothesized that sodium butyrate, a product of enteric bacterial fermentation, modulates gene expression in gut microvascular endothelium which plays a central role in mucosal immunity. We examined sodium butyrate’s effect on LPS-induced gene and protein expression in primary cultures of human intestinal microvascular endothelial cells. cDNA array analysis revealed that sodium butyrate augmented ICAM-1 mRNA expression, while it inhibited IL-6 and COX-2 expression in response to LPS stimulation. These results were confirmed at the protein level. Prostaglandin E2 production by LPS was also strongly inhibited by butyrate. The pattern of altered gene expression by butyrate was reproduced by the histone deacetylase inhibitor tricostatin A, suggesting that the regulatory mechanism of butyrate on HIMEC gene expression involves histone deacetylase inhibition. IκBα degradation and NF-κB activation were unaffected by butyrate. In addition to effects on epithelium, sodium butyrate modulates the innate mucosal immune response towards LPS through effects on microvascular endothelial function.

Major histocompatibility complex class II–dependent antigen presentation by human intestinal endothelial cells

Background & Aims: In the normal gut, human intestinal microvascular endothelial cells (HIMECs) express major histocompatibility complex (MHC) class II molecules. Enhanced expression is found in chronic inflammation. We examined the cytokine regulation of MHC class II molecules and the associated invariant chain (Ii) in HIMECs and investigated whether such cells can process and present a complex protein antigen to T cells. Methods: Enzyme-linked immunosorbent assay, flow cytometry, immunoelectron microscopy, as well as T-cell activation assay with HIMECs and HLA-DR–restricted T-cell clones were employed. Results: In unstimulated HIMEC monolayers, HLA-DR, -DP, and -DQ and Ii were undetectable at the protein level, but interferon gamma (IFN-γ) (100 U/mL) induced expression that peaked for DR after 2–3 days, for DP after 4–6 days, for DQ after 10–12 days, and for Ii after 2–3 days. Tumor necrosis factor α had no effect alone but enhanced class II expression in combination with IFN-γ, most notably for DQ and DP. HLA-DR3–restricted and Mycobacterium tuberculosis heat shock 65-kilodalton–specific T-cell clones were activated to produce IFN-γ in response to relevant antigen presented by IFN-γ–treated HIMECs. This response was inhibited by blocking monoclonal antibody to HLA-DR and by chloroquine when compared to professional antigen-presenting cells, HIMECs activated T-cell clones quite efficiently. Conclusions: These data suggest that microvascular endothelial cells can present complex protein antigens in the human gut. GASTROENTEROLOGY 1998;114:649-656