The tripeptide KdPT protects from intestinal inflammation and maintains intestinal barrier function

Abstract

Treatment options for inflammatory bowel disease (IBD) are incompletely helpful, and surgery is often needed. One promising class of future therapeutic agents for IBD is melanocortin-related peptides, which exhibit potent immunomodulatory effects. We investigated KdPT, a tripeptide derivative of the C-terminus of α-melanocyte-stimulating hormone, as an anti-inflammatory small molecule in vivo and in vitro. Intestinal inflammation was studied after oral administration of dextran sodium sulfate and in IL-10 gene-deficient mice. The effects of KdPT on key colonic epithelial cell functions were studied in vitro and in vivo by evaluating proliferation, wound healing, transepithelial resistance, and expression of tight junction proteins. Melanin assays were performed to determine the melanotropic effects of KdPT. KdPT-treated animals showed markedly reduced severity of inflammation in both colitis models. In colonic epithelial cells, KdPT increased proliferation, accelerated closure of wounds, and improved transepithelial electrical resistance after stimulation with interferon-γ/tumor necrosis factor-α. Moreover, treatment with KdPT also prevented the loss of tight junction protein expression and improved barrier function in vivo. KdPT acted independently of IL-1 receptor type I in vivo and did not affect melanogenesis in vitro. KdPT is capable of attenuating the course of experimental colitis in different models and maintains epithelial cell function. Furthermore, KdPT does not induce pigmentation, emphasizing the potential of this small molecule for the future treatment of IBD.

Figure 1

KdPT ameliorates DSS-induced colitis. C57BL/6 WT mice received 3% DSS in their drinking water for 5 days, and inflammation was monitored by daily measurement of individual weights. A: From day 2 onward, one group was treated with 10 μg of KdPT i.p. daily, and control animals received an equivalent volume of PBS i.p. Data are mean ± SE; n = 5 for each group. *P < 0.05. B: Representative histologic images of control mice and KdPT-treated animals 13 days after induction of colitis. C: Histologic scores of colonic tissue according to the method of Dieleman et al of control mice and KdPT-treated animals 13 days after induction of colitis. Data are mean ± SE; n = 5 for each group. *P < 0.003.

Figure 2

Effect of KdPT at various concentrations after i.p. administration and after oral and rectal application in DSS-induced colitis. C57BL/6 mice received various amounts of KdPT by daily i.p. injections starting 2 days after induction of DSS colitis. Inflammation was monitored by individual body weight. A: Overall weight loss was significantly less intense in mice receiving 5 and 25 μg of KdPT daily, whereas lower concentrations were not effective. Data are mean ± SE; n = 5 for each group. *P < 0.05. B: Histologic scores of colonic tissue according to the method of Dieleman et al in control mice and animals treated with different doses of KdPT i.p. Note that KdPT doses of <5 μg of KdPT per day did not ameliorate DSS-induced colitis. Data are mean ± SE; n = 5 to 7 for each group. *P < 0.05. C: Impact of oral administration of KdPT on DSS-induced colitis. C57BL/6 mice received 10 or 100 μg of KdPT by oral gavage 2 days after starting DSS treatment. Note that only 100 μg was effective. Data are mean ± SE; n = 5 for each group. *P < 0.05. D: Rectal application of KdPT (10 μg) improves DSS-induced colitis. Data are mean ± SE; n = 5 for each group. *P < 0.05.

Figure 3

KdPT attenuates colitis in nonsteroidal anti-inflammatory drug–treated IL-10–deficient mice. A: IL-10–deficient mice orally received 100 μg of KdPT per day beginning on day 0 after administration of piroxicam until the end of the experiment on day 31, and control animals received PBS. Note the progressive weight loss in PBS-treated animals compared with KdPT-treated mice. Data are mean ± SE; n = 7 mice. *P < 0.05. B: Histologic scores of colonic tissue in PBS- and KdPT-treated animals after 31 days. Data are mean ± SE; n = 7 per group. *P < 0.01.

Figure 4

Induction of cell proliferation after stimulation with KdPT in DSS-treated and IL-10^−/− mice. Immunohistochemical analysis of the cell proliferation marker Ki-67 in colonic tissue of PBS-treated mice and KdPT-treated animals 13 days after induction of DSS colitis (A) and in IL-10^−/− mice 31 days after piroxicam treatment (B). Data are mean ± SE; n = 5 for each group. *P < 0.05.

Figure 5

Induction of cell proliferation after stimulation with KdPT in vitro. A: MTS activity in Caco-2 cells after stimulation with KdPT. Cells were stimulated with KdPT at the doses indicated for 24 hours. The number of viable Caco-2 cells after treatment with 10⁻⁷ and 10⁻⁹ mol/L KdPT was significantly increased compared with cells incubated with medium alone (mean ± SE relative increase: 1.43 ± 0.06 and 1.64 ± 0.19, respectively) Data are mean ± SE; n = 6, *P < 0.05. B: Increased wound healing in Caco-2 monolayers after stimulation with KdPT. A defined wound was set, and cells were stimulated with medium or medium plus KdPT at various concentrations. Migration of Caco-2 cells after in vitro wounding and KdPT treatment as determined by cell counting in a standardized viewing field. Data are mean ± SE. Independent experiments (n = 5) were performed in quadruplicate. *P < 0.05.

Figure 6

KdPT increases TER in colonic epithelial cells. A: TER in epithelial cells (T84) decreases after stimulation with IFN-γ/TNF-α. After 48 hours, mean ± SE TER was 70.4% ± 7%, and co-stimulation with KdPT significantly increased TER (110% ± 10%). Data are mean ± SE; n = 6. *P = 0.01, 48 hours; **P = 0.04, 72 hours. B: KdPT prevents increased colonic epithelial permeability in acute DSS-induced colitis. To assess the permeability of the colonic epithelium, the colon was perfused with Evans blue in vivo for 15 minutes, and its uptake into the mucosa was quantified spectrophotometrically. Compared with controls (mean ± SE extinction/gram colon tissue: 0.39 ± 0.07), there was a massively increased uptake of Evans blue in the DSS-treated group (mean ± SE extinction/gram colon tissue: 1.11 ± 0.13), indicating a disrupted epithelial barrier. Concomitant treatment with KdPT completely prevented this increase in permeability (mean ± SE: 0.69 ± 0.09). Values are given as mean ± SE; n = 5 per group. *P = 0.026, healthy control versus DSS-treated; **P = 0.48, DSS-treated versus KdPT-treated.

Figure 7

KdPT maintains tight junction protein localization in colonic epithelium in situ. The tight junction proteins ZO-1, occludin (Occ), claudin-1 (Cl-1), Cl-3, and Cl-5 were stained after DSS challenge and in IL-10–deficient mice. In control animals, ZO-1, Occ, Cl-1, and Cl-3 are mostly localized at the apical membrane and are displayed by an intense apical fluorescence band; Cl-5 was additionally expressed at the basolateral membrane. In IL-10 gene–deficient mice (day 31 after the start of KdPT application) and after treatment with DSS (day 10 after the start of DSS application), the apical staining of ZO-1, Occ, Cl-1, Cl-3, and Cl-5 is strongly reduced, whereas in KdPT-treated animals, the junction proteins stain markedly longer. Images are representative of five animals in each group. Arrowheads represent the positive membrane staining of indicated tight junction proteins.

Figure 8

KdPT ameliorates DSS-induced colitis in IL-1R type I–deficient mice. A: IL-1R type I–deficient mice received 3% DSS in their drinking water for 5 days, and inflammation was monitored by daily measurement of individual weights. From day 2 after the start of DSS application, one group was treated with 100 μg of KdPT p.o. daily, and control animals received an equivalent volume of PBS i.p. Data are mean ± SE; n = 7 for each group. *P < 0.05. B: Histologic scores of colonic tissue according to the method of Dieleman et al of control mice and KdPT-treated animals 9 days after induction of colitis. Data are mean ± SE; n = 7 for each group. *P < 0.05.

Figure 9

Saturable uptake of KdPT is dependent on pH and energy. Uptake or transport of KdPT by intestinal epithelial cells was measured in vitro in Caco-2 cells. With increased proton gradient, uptake of KdPT was significantly enhanced approximately threefold (P < 0.05) (A), whereas a temperature of 4°C almost completely abolished its transport (B). C: Saturable uptake of KdPT by Caco-2 cells was shown by increasing the concentration of KdPT up to 500 μmol/L. Data are mean ± SE. All the experiments were performed in triplicate.

Figure 10

PepT1 mediated transport of KdPT in vitro. A: Caco-2 cells expressing the oligopeptide transporter PepT1 effectively transport the PepT1 substrate Gly-Sar, whereas HT-29 cells lack PepT1 expression. In contrast, the transport of radiolabeled KdPT was significantly less effective in Caco-2 cells compared with Gly-Sar, whereas HT-29 cells did not exhibit a significant difference. B: Uptake of Gly-Sar increased from day 4 to day 17, whereas uptake of KdPT significantly dropped. C: Competition assays showed that Gly-Sar uptake was only partially inhibited by high concentrations of KdPT. D: Conversely, excessive amounts of various PepT1 substrates induced a decrease in KdPT uptake in Caco-2 cells. Data are mean ± SE. All the experiments were performed in triplicate.

Figure 11

Intestinal uptake of KdPT in vivo. Whereas absorption of KdPT was maximal in the colon (A, left), PepT1-specific Gly-Sar was more effectively absorbed in the small intestinal tract and followed the PepT1 gradient of protein expression (A, right). PepT1 competitors (cp) totally blocked Gly-Sar uptake, whereas KdPT uptake was unaffected (A, hatched bars). DSS treatment did not modify Gly-Sar uptake (B) but induced significantly increased KdPT absorption in the inflamed colon (C). duo, duodenum; jeju, jejunum.

Figure 12

KdPT does not alter melanin synthesis. Normal human melanocytes were exposed to various doses of KdPT for 5 days. Bovine pituitary extracts (BPE) (containing natural melanocortins) were used as positive control. Melanin amounts in the lysed cells were determined photometrically using a synthetic melanin standard. Melanin measurements were performed in triplicate and represent mean ± SD. Data depict one of three individual experiments with identical results.