Na+-taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice

Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+-Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 μm in DDC-fed mice, 432±280 to 762±288 μm in Atp8b1-G308V mice and from 522±130 to 3625±378 μm in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by ∼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018)

New perspective on dextran sodium sulfate colitis: antigen-specific T cell development during intestinal inflammation.

CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens

Th17 cells are detected in the spleen after colitis resolution.

<p>IFNγ and IL-17A producing CD4+ T cells were detected in the spleens and mLNs, 14 days after the start of DSS using intracellular cytokine staining. A) Percentages depicted are the populations of cytokine expressing CD4+ cells within the total CD4+ population. Bars indicate the mean, N = 8 mice per group. ** P < 0.01. B) Representative FACS contour dot plots for spleen and mLN showing intracellular staining of IL-17A and IFNγ within the gated CD4+ T cell population. Percentages within CD4+ T cell population are shown.</p

NTCP deficiency in mice protects against obesity and hepatosteatosis

Bile acids play a major role in the regulation of lipid and energy metabolism. Here we propose the hepatic bile acid uptake transporter Na+ taurocholate cotransporting polypeptide (NTCP) as a target to prolong postprandial bile acid elevations in plasma. Reducing hepatic clearance of bile acids from plasma by genetic deletion of NTCP moderately increased plasma bile acid levels, reduced diet-induced obesity, attenuated hepatic steatosis, and lowered plasma cholesterol levels. NTCP and G protein–coupled bile acid receptor–double KO (TGR5–double KO) mice were equally protected against diet-induced obesity as NTCP–single KO mice. NTCP-KO mice displayed decreased intestinal fat absorption and a trend toward higher fecal energy output. Furthermore, NTCP deficiency was associated with an increased uncoupled respiration in brown adipose tissue, leading to increased energy expenditure. We conclude that targeting NTCP-mediated bile acid uptake can be a novel approach to treat obesity and obesity-related hepatosteatosis by simultaneously dampening intestinal fat absorption and increasing energy expenditure

OVA-directed, CD4+ Foxp3-T cells are detected only in mice treated with DSS and OVA.

<p>Both mLN and spleen cell suspensions were prepared 14 days after the start of the DSS and OVA treatment for all groups. Cells were stimulated with OVA, and CD69 expression was measured using flow cytometry. A) Representative FACS dot plots of the CD69 expression in CD4+ Foxp3- (Tconv) and CD4+ Foxp3+ (Treg) T cells in the spleen. Percentages shown are the % CD69+ cells in the specific gated Tconv or Treg populations. B) The mean percentage CD69 expression was calculated for Tconv and Treg in the spleen cells of each group. C) Representative FACS plots of the CD69 expression in Tconv and Treg cells in the mLN. D) The mean percentage CD69 expression was calculated for Tconv and Treg cells in the mLN. Results are expressed as mean + SEM, N = 6-9 per group. *** P < 0.001; **** P < 0.0001.</p

During colitis, T cells accumulate in the inflamed regions of the colon.

<p>Mice were treated with DSS for 6 days and sacrificed on day 7. The mice displayed signs of colitis including (A) an increased Disease Activity Index (DAI) and (B) shortened colons. C) Immunohistochemical staining of CD3+ cells in colons obtained from both control (left panes) and DSS-treated (right panes) mice. Top panes are 200x (bar: 5 µm) and bottom panes are 400x magnification (bar: 1 µm). Increased CD3+ cells are observed in inflamed colons. D) Naïve (CD4+ CD62L+ CD44-), central memory (T_CM, CD4+ CD62L+ CD44+) and effector memory (T_EM, CD4+ CD62L-CD44+) T cells were measured in the mLNs and colon mononuclear cell suspensions using flow cytometry. Results are expressed as mean + SEM, N = 4-6 mice per group. *** P < 0.001; **** P < 0.0001.</p

Oral antigens are presented in the draining lymph nodes of both healthy and DSS treated mice.

<p>A) OVA presentation in the gastrointestinal tract was visualized by the proliferation of adoptively transferred, CFSE labeled, OTII T cells. Representative FACS dot plots displaying OTII T cell proliferation within the mLNs of both healthy and DSS-treated mice after oral gavage of saline, “no antigen” or oral gavage with OVA, “ovalbumin”. The loss of CFSE intensity is an indication of dividing T cells. B) Percent proliferated OTII cells found within isolated mLNs. C) Percent proliferated OTII cells in the non-local, axillary lymph nodes. Results for (B) and (C) are expressed as mean + SEM, N = 4 mice per group, pooled from two independent experiments. * P < 0.05; ** P < 0.01.</p

DSS and OVA treated mice have the same clinical phenotype as mice treated with DSS alone.

<p>Mice were treated with both DSS and OVA for 6 days, and several clinical parameters were measured during disease progression and after sacrifice. A) Percent weight gain relative to starting weight was measured in each individual mouse for 14 days. B) DAI was calculated on day 6 and day 13 for all groups as described in the materials and methods. N = 10-15 mice before day 7, N = 5-10 after day 7, mice are pooled from two independent experiments. Mesenteric LNs C) and spleens D) were obtained from mice sacrificed on day 14. Cell suspensions were prepared and total cells counted in each organ. Samples (N = 5) were pooled from two independent experiments. E) SAA was measured in the serum from mice sacrificed on day 14. Samples (N = 5) were pooled from two independent experiments. F) Several colons collected from mice on day 14 were examined and scored for histological damage parameters as described in the materials and methods (N = 3, from a single experiment). G) Representative photos are shown for colonic sections from control, DSS-treated, OVA treated controls and OVA and DSS-treated mice. Bar is 5 µm. All graphical results are expressed as mean + SEM in the bar graphs. * P < 0.05; ** P < 0.01.</p

DSS and OVA treated mice have aspecific T cell responses similar to mice treated with DSS alone.

<p>Mice were either treated with DSS alone or with DSS and OVA. Spleens and mLNs were removed, stimulated and examined by flow cytometry. A) Percentages of CD4+ T cells were determined within cultures after stimulation with anti-CD3 for 48 hours. N = 4-9 mice per group. B) The ratios of the percentages of Foxp3- and Foxp3+ cells (Foxp3-/Foxp3+) were determined within the CD4+ T cell population. N = 4-9 mice per group. Using intracellular cytokine staining, percentages of IL-17A+ (C) and IFNγ+ cells (D) within the CD4+ cell population were determined after stimulation. N = 3 mice per group. All graphical results are expressed as mean + SEM in the bar graphs.</p