Leo R. Fitzpatrick

Efficacy of a novel sphingosine kinase inhibitor in experimental Crohn’s disease

AimActivation of sphingosine kinase (SK) is a key response to many inflammatory processes. The present studies test the hypothesis that an orally available SK inhibitor, ABC294640, would be effective in rodent models of Crohn’s disease.MethodsTrinitrobenzene sulfonic acid (TNBS) was administered rectally to mice and rats. Rats were treated with ABC294640 orally alone or in combination with olsalazine and disease progression was monitored.ResultsFor both rodent species, treatment with ABC294640 attenuated disease progression. Colon samples from the ABC294640-treated animals had improved histology and cytokine parameters when compared with vehicle-treated animals. The expression of SK was similarly increased in TNBS-treated animals and in human colon tissue specimens from inflammatory bowel disease patients relative to normal, control patients.ConclusionsSphingosine kinase may be a critical mediator of colonic damage during intestinal inflammation, and pharmacologic inhibitors of this enzyme may prove useful in the treatment of Crohn’s disease.

MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.

The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the α-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region (P=0.0012–0.04), and one in the 3′-untranslated region (P=2 × 10−7) that displayed associations with UC. Moreover, meprin-α mRNA was decreased in inflamed mucosa of IBD patients. Meprin-α knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-α expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.

4SC-101, a novel immunosuppressive drug, inhibits IL-17 and attenuates colitis in two murine models of inflammatory bowel disease†

Background: Dihydroorotate dehydrogenase (DHODH) is a key enzyme involved in pyrimidine biosynthesis. DHODH is a known target for the treatment of autoimmune diseases. 4SC‐101 is a novel immunosuppressive drug that inhibits DHODH. A goal of our study was to examine the in vitro effects of 4SC‐101 on IL‐17 production by mononuclear cells. In addition, we evaluated the efficacy of 4SC‐101 against acute TNBS (2,4,6‐tritrobenzene sulfonic acid) and chronic dextran sodium sulfate (DSS)‐induced colitis in mice. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy human donors were used to evaluate cellular proliferation and cytokine (IL‐17, TNF‐&agr;) production. The oral effects of 4SC‐101 (100 or 200 mg/kg) were examined following induction of chronic colitis by the administration of 3% DSS (4 cycles) to Balb/c mice. Morphometric and histological indices of colitis were evaluated as indicators of drug efficacy. 4SC‐101 was also administered for 6 days after the intracolonic administration of TNBS (20 mg in 50% ethanol) to female Balb/c mice. The colons were analyzed for overall macroscopic damage, ulceration, total length, distal segment weight, MPO activity, and histological pathology as indicators for the effectiveness of 4SC‐101. Results: In vitro, 4SC‐101 is a potent inhibitor of human DHODH, inhibits lymphocyte proliferation, and uniquely blocks phytohemagglutinin‐stimulated IL‐17 production by lymphocytes. In vivo, oral administration of 4SC‐101 effectively improved both chronic DSS and acute TNBS colitis in mice. In these colitis models the overall efficacy profile of 4SC‐101 was similar to that of dexamethasone. Conclusions: 4SC‐101 is a novel immunosuppressive drug with excellent potential for the treatment of intestinal inflammation. (Inflamm Bowel Dis 2010)

In vitro and in vivo effects of gliotoxin, a fungal metabolite : Efficacy against dextran sodium sulfate-induced colitis in rats

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit cytokine production from macrophage and colonic epithelial cell lines, as well as whether it inhibited nuclear factor-kappa B in these same cell types. Second, we evaluated whether gliotoxin could reduce dextran sodium sulfate-induced colitis in rats. A disease activity index, myeloperoxidase activity, and cytokine levels were evaluated on either day 7 or 21. In both cell lines, gliotoxin dose dependently inhibited cytokine production and nuclear factor-kappa B. On day 21, gliotoxin significantly reduced disease activity (diarrhea and bloody stools) in rats. On day 7, gliotoxin treatment significantly improved various indices of colitis, including colonic cytokine levels. Decreased food consumption and weight gain was evident with a larger dose of gliotoxin. In summary, gliotoxin, a nuclear factor-kappa B inhibitor, effectively reduced dextran sodium sulfate-induced colitis in rats. However, gliotoxin exhibited a narrow therapeutic to toxicity ratio in these rats.

In vitro and in vivo effects of the probiotic Escherichia coli strain M-17: immunomodulation and attenuation of murine colitis

We examined the in vitro and in vivo effects of a probiotic, Escherichia coli strain M-17 (EC-M17), on NF-kappaB signalling, cytokine secretion and efficacy in dextran sulfate sodium (DSS)-induced murine colitis. NF-kappaB signalling was assessed using an NF-kappaB luciferase reporter cell line that was stimulated with TNF-alpha (100 ng/ml). p65 Nuclear binding and cytokine secretion (TNF-alpha, IL-1beta and IL-6) were evaluated using a RAW 264.7 macrophage cell line that was exposed to lipopolysaccharide (LPS; 5 microg/ml). Mice were administered vehicle, EC-M17, metronidazole, or EC-M17 plus metronidazole for 13 d. During the final 6 d, mice also received 2 % DSS. Parameters evaluated included disease activity index (DAI), histology, myeloperoxidase and NF-kappaB p65. EC-M17 dose dependently inhibited TNF-alpha-induced NF-kappaB signalling. At 5 x 109 colony-forming units/ml, EC-M17 inhibited NF-kappaB by >95 %. LPS-induced nuclear p65 binding was significantly inhibited (78 %; P 90 %) the LPS-induced secretion of TNF-alpha, IL-1beta and IL-6. In mice with DSS-induced colitis, EC-M17, metronidazole, and EC-M17 plus metronidazole significantly reduced DAI and colonic histology scores. Both EC-M17 and metronidazole reduced colonic IL-12, IL-6, IL-1beta and interferon-gamma. The combination of EC-M17 plus metronidazole resulted in more substantial cytokine reductions than were found with either treatment alone, and combination therapy significantly (P < 0.05 in both cases) reduced IL-1beta compared with EC-M17 and colonic histology scores compared with metronidazole. Alone, and in combination with metronidazole, EC-M17 improved murine colitis, probably due to an inhibitory effect on NF-kappaB signalling.

Effects of the probiotic formulation VSL#3 on colitis in weanling rats.

Background: Only a few studies have used models of inflammatory bowel disease (IBD) with weanling animals. Previously, the effects of probiotics have not been assessed in such IBD models. The objectives of our study were 2-fold: to establish a suitable model of dextran sulfate sodium (DSS)–induced colitis in weanling rats and to determine the effects of the probiotic formulation VSL#3 on DSS-induced colitis in weanling animals. Materials and Methods: Rats were weaned on postnatal day 21 and administered 2%, 2.5%, or 3% (wt/vol) DSS in drinking water. In subsequent experiments, newly weaned animals were administered vehicle or VSL#3 (0.06, 0.6, or 6 mg) by orogastric gavage. These treatments were given to animals maintained on water (postnatal days 21–28) and then on DSS (postnatal days 28–35). Disease activity indices were determined on a routine basis. On day 35, rats were euthanized. The total colon length was determined. Other parameters of colitis were measured from the distal colon. These parameters included myeloperoxidase (MPO), interleukin (IL)–1β, inhibitory κB-α (IκB-α), and histological assessment of crypt damage and inflammation. Results: DSS 2% was optimal for inducing colitis in weanling rats without significant morbidity. VSL#3 treatments improved various parameters of 2% DSS–induced colitis in weanling rats. The 0.6- and 6-mg doses of VSL#3 were most effective for attenuating this colitis. Conclusions: The probiotic formulation VSL#3 improved DSS-induced colitis in weanling rats. This improvement of colitis involved changes in colonic IκB-α, IL-1β, and MPO, which are suggestive of immune modulation by VSL#3.

Enhanced Intestinal Expression of the Proteasome Subunit Low Molecular Mass Polypeptide 2 in Patients with Inflammatory Bowel Disease

PurposeLow molecular mass polypeptide 2 is an inducible immunoproteasome subunit. The expression of low molecular mass polypeptide 2 has not been examined in the intestine of patients with inflammatory bowel disease. This study was designed to determine whether the intestinal expression of low molecular mass polypeptide 2 was enhanced in a group of patients with inflammatory bowel disease compared with a group of control patients without inflammatory bowel disease. Moreover, we examined the association between low molecular mass polypeptide 2 expression and histologic pathology in these patients.MethodsTwenty-one patients participated in the study. These included six control subjects without inflammatory bowel disease, eight patients with ulcerative colitis, and seven patients with Crohn’s disease. Intestinal low molecular mass polypeptide 2 expression was evaluated by immunohistochemistry, as well as by Western blot. Histology scores (0–40 severity scale) were determined on the same sections of intestine as those used for low molecular mass polypeptide 2 histochemistry.ResultsBy immunohistochemistry, low molecular mass polypeptide 2 expression was significantly enhanced (P < 0.05 vs. control subjects) throughout visibly diseased areas of colon, rectum, and ileum from patients with inflammatory bowel disease. Low molecular mass polypeptide 2 expression also was increased in macroscopically normal intestine from patients with inflammatory bowel disease compared with normal tissue from control subjects. There was a significant correlation (P < 0.0001) between low molecular mass polypeptide 2 expression and histologic pathology in our patients. Western blot results confirmed that low molecular mass polypeptide 2 expression was enhanced in patients with ulcerative colitis (3.1-fold) and in patients with Crohn’s disease (3.5-fold).ConclusionsIntestinal low molecular mass polypeptide 2 expression is significantly increased in inflammatory bowel disease. The association between intestinal low molecular mass polypeptide 2 expression and histologic pathology suggests that this proteasome subunit plays a role in the pathogenesis of inflammatory bowel disease.

Dextran sulfate sodium-induced colitis is associated with enhanced low molecular mass polypeptide 2 (LMP2) expression and is attenuated in LMP2 knockout mice.

Low molecular mass polypeptide 2 (LMP2) is an inducible proteasome subunit. Our goals were to examine LMP2 expression in mice with dextran sulfate sodium (DSS)-induced colitis and to evaluate colitis in LMP2 knockout (LMP2–/–) mice. Mice were given 2.5% DSS in the drinking water. On day 0, 2, 4, or 6 after DSS treatment, LMP2 expression was determined in the distal colon by western blot and immunohistochemistry. Parameters of colitis were measured in LMP2–/– mice or wild-type mice. LMP2 expression was enhanced in the colon of DSS-treated mice at all time points. Symptoms of DSS-induced colitis were always lower in LMP2–/– mice. Normalized histology scores and colonic IL-1ß levels increased over the 6-day study period in wild-type mice. These parameters were significantly reduced in LMP2–/– mice that consumed DSS for 6 days. Enhanced LMP2 expression contributes to the pathogenesis of DSS-induced colitis in mice.

Inhibition of IL-17 as a Pharmacological Approach for IBD

Several experimental approaches have been utilized, in order to critically examine the roles of IL-17 family members in intestinal inflammation. These approaches have included: (1) the use of IL-17A and IL-17F-deficient mice, (2) specific antibodies directed against IL-17, (3) an IL-17 vaccine, (4) methods to block the IL-17 receptor and (5) small-molecule inhibitors of IL-17. Previous studies found somewhat conflicting results in preclinical models of Inflammatory Bowel Disease (IBD), using specific strains of IL-17-deficient mice. This paper will review the preclinical results using various pharmacological approaches [specific IL-17 antibodies, an IL-17 receptor fusion protein, IL-12/IL-23 p40 subunit and IL-17 vaccine approaches, as well as a small molecule inhibitor (Vidofludimus)] to inhibit IL-17 in animal models of IBD. Recent clinical results in patients with IBD will also be discussed for Secukinumab (an IL-17A antibody), Brodalumab (an IL-17 receptor antibody) and two small-molecule drugs (Vidofludimus and Tofacitinib), which inhibit IL-17 as part of their overall pharmacological profiles. This review paper will also discuss some pharmacological lessons learned from the preclinical and clinical studies with anti-IL-17 drugs, as related to drug pharmacodynamics, IL-17 receptor subtypes and other pertinent factors. Finally, future pharmacological approaches of interest will be discussed, such as: (1) Retinoic acid receptor-related orphan nuclear receptor gamma t (Rorγt) antagonists, (2) Retinoic acid receptor alpha (RARα) antagonists, (3) Pim-1 kinase inhibitors and (4) Dual small-molecule inhibitors of NF-κB and STAT3, like synthetic triterpenoids.

NF-κB1 p105 Regulates T Cell Homeostasis and Prevents Chronic Inflammation

Transcription factor NF-κB is regulated by a family of inhibitors, IκBs, as well as the NF-κB1 and NF-κB2 precursor proteins, p105 and p100. Although the different NF-κB inhibitors can all inhibit NF-κB in vitro, their physiological functions are incompletely understood. In this study, we demonstrate that p105 plays an important role in the regulation of T cell homeostasis and prevention of chronic inflammation. Mice lacking p105, but expressing the mature NF-κB1 p50, spontaneously develop intestinal inflammation with features of human inflammatory bowel disease. This inflammatory disorder occurs under specific pathogen-free conditions and critically involves T cells. Consistently, the p105-deficient mice have reduced frequency of naive T cells and increased frequency of memory/effector T cells in the peripheral lymphoid organs. Although p105 is dispensable for the production of immunosuppressive regulatory T cells, p105 deficiency renders CD4 T cells more resistant to Treg-mediated inhibition. We further show that the loss of p105 results in hyperproduction of Th17 subset of inflammatory T cells. Together, these findings suggest a critical role for NF-κB1 p105 in the regulation of T cell homeostasis and differentiation and the control of chronic inflammation.