Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13) — A novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-β1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD).The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])₂]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p < 0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p < 0.0001). TGF-β mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals.In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.     

The JAK2 inhibitor AG490 regulates the Treg/Th17 balance and alleviates DSS-induced intestinal damage in IBD rats

The pathogenesis of inflammatory bowel disease (IBD) remains unclear, and it is currently believed that an imbalance in regulatory T (Treg) cells/T helper 17 cells (Th17 cells) is related to the occurrence and development of IBD. Recently, the JAK2 inhibitor AG490 has been used in animal models such as rheumatoid arthritis and bronchial asthma models and shown to exert immunoregulatory functions that improve disorder in the Treg/Th17 cell balance. This study aimed to evaluate the effect of AG490 on the intestinal inflammatory process in an IBD rat model. A dextran sulfate sodium (DSS)-induced IBD rat model was established, and disease activity index (DAI) scores were calculated. The histopathological damage score was determined by haematoxylin-eosin (H&E) staining. Treg/Th17 cells in the spleen were detected by flow cytometry. The levels of interleukin (IL)-10, IL-6 and IL-17A were detected by enzyme-linked immunosorbent assay (ELISA). AG490 attenuated DSS-induced IBD injury by regulating the Treg/Th17 balance and related cytokine secretion to reduce the DAI and colonic tissue damage. Thus, AG490 may be a new method for effective treatment of IBD.     

Progress in the development of aminopeptidase N (APN/CD13) inhibitors

Aminopeptidase N (APN; CD13) is a member of zinc-containing ectoenzymes family involved in the degradation of neutral or basic amino acids (Ala>Phe>Leu>Gly) from N-terminal of bioactive peptides and amide or arylamide derivatives of amino acids. The expression of APN being up regulated has been implicated in the pathogenesis of a variety of diseases such as cancer, leukemia, diabetic nephropathy, and rheumatoid arthritis. Thus, APN inhibitors (APNIs) are expected to be useful for the treatment of these disorders. This article reviews briefly the structure characteristic and possible function of APN. The proposed biomolecular structures and mechanism of action used in the design of APNIs are thoroughly covered. Major emphasis is on recently published potent, small molecular weight APNIs and their essential structure activity relationship (SAR). Finally, available clinical results of compounds in development are summarized in this review.     

Interleukin-21 (IL-21) controls inflammatory pathways in the gut

In both Crohn's disease (CD) and ulcerative colitis (UC), the major forms of inflammatory bowel diseases (IBD) in humans, the pathologic process consists of an aberrant local immune response to components of the bacterial microflora, due to abnormally strong effector cell activity that is poorly controlled by counter-regulatory mechanisms. There is also evidence that mucosal immune cells actively interact with non-immune cells to promote tissue damage, and that cytokines are essential mediators of this cross-talk. Interleukin-21 (IL-21), the latest member of the common gamma-chain-dependent cytokine family, is a product of activated CD4+ T cells and natural killer T cells. IL-21 is produced in excess in CD tissue, where it helps sustain the ongoing Th1 inflammation. High IL-21 production occurs also in the inflamed colon of most patients with UC, a disease that is not associated with a marked Th1 cell response. This suggests that, in the gut, IL-21 can modulate additional inflammatory pathways other than enhancing Th1 cell immunity. Indeed, IL-21 stimulates the secretion of extracellular matrix degrading enzymes by fibroblasts, and of the T cell chemoattractant, MIP-3 alpha, by epithelial cells. These data collectively indicate that IL-21 is a mediator of the chronic inflammatory response in CD and UC, and suggest that IL-21 may be an emerging therapeutic target in IBD.     

Cytokines in colitis associated cancer: potential drug targets?

In inflammatory bowel disease (IBD), such as UC and CD, the development of colorectal carcinoma can be initiated through chronic inflammation, depending on the duration and severity of the disease. Growing evidence supports a role for various cytokines, released by epithelial and immune cells, in the pathogenesis of colitis associated cancer (CAC). For instance, TNF-alpha has been recently shown to promote tumor development in experimental colitis. Due to its role in the pathogenesis of IBD, TNF-alpha blockade has become one of the cornerstones of IBD therapy. Thus, anti-TNF-alpha strategies could also provide effective anti-tumor therapies. TGF-beta has been shown to attenuate an anti-tumor immune-response through the induction of regulatory T cells in spontaneous and inflammation associated cancer. However, IL-6 signaling promotes tumor growth in experimental CAC, and this signaling is inhibited by TGF-beta. Members of the IL-12 family, such as IL-12, IL-23, and IL-27, have been implicated in pathogenesis of colitis and IL-23 seems to be involved in inflammation associated carcinogenesis. However, a role for those cytokines in CAC remains to be shown. Whereas the above-mentioned cytokines promote tumor growth, others provide an anti tumor effect. IL-10 deficient mice develop colitis and CAC a few weeks after birth. Besides its immunosuppressive properties, IL-10 also has anti angiogenic and both tumor promoting and -inhibiting properties, which could be responsible for these observations. Since most of the above mentioned cytokines are involved in both, inflammation and carcinogenesis, it is difficult to account their contribution to the individual steps during pathogenesis of CAC. However, as already shown in IBD, cytokines may also provide promising therapeutic targets in CAC.     

Peanut shell extract inhibits the development of dextran sulfate sodium (DSS)-induced colitis

Inflammatory bowel diseases (IBD) induce inflammation in the colon and small intestine. IBD include ulcerative colitis and Crohn's disease, with such common symptoms as severe diarrhea, fever, and blood in the stool. In the current study, we explored the ability of peanut shell extract (PSE) to alleviate IBD in an experimental colonic inflammation model. Colitis was induced by orally administered dextran sulfate sodium (DSS) in mice. Peanut shell extract was prepared using a method of aqueous ethanol. DSS treatment reduced the colon length and mouse body weight, and aggravated disease condition compared with untreated control mice. Oral administration of 400 mg/kg PSE alleviated colon shortening, body weight loss, DAI, and colon injury score in DSS-induced colitis. These physiological improvements were validated by reduced levels of proinflammatory cytokines and infiltrating macrophage accumulation in the inflamed colon in the PSE administered group. These observations suggest that PSE may be developed as an alternative natural extract for the prevention or treatment of IBD.     

Effect of recombinant serine protease from adult stage of Trichinella spiralis on TNBS-induced experimental colitis in mice

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic autoimmune disease. At present, worms and their products has been shown to have protective effects on immune-mediated diseases. Therefore, we aimed to investigate the effect of the recombination Trichinella spiralis (T. spiralis, Ts) adult serine protease-like protein rTs-ADSp-7 on a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD mouse model. Colitis was induced by intrarectal administration of a TNBS solution. The disease activity index (DAI), which included weight loss, diarrhoea, and bloody stool, was measured. Colon segments were stained with haematoxylin and eosin (H.E.) for histopathological score. Cytokine release in the serum was analysed by meso scale discovery (MSD). Cytokine release in the colon was detected by ELISA. Splenocytes were separated, and the cytokine profiles of Th1 (IFN-γ), Th2 (IL-4), Th17 (IL-17A) and Treg cells were analysed by flow cytometry. Our result showed that rTs-ADSp-7 reduced the clinical disease activity of TNBS-induced colitis in mice. In addition, we found that rTs-ADSp-7 reduced the production of Th1- and Th17-related cytokines while upregulating the expression of Th2- and Treg-related cytokines in TNBS-induced colitis mice. rTs-ADSp-7 also increased the population of Th2 and Treg cells in TNBS-induced colitis mice. rTs-ADSp-7 alleviated the severity of TNBS-induced colitis while balancing the CD4⁺ T cell immune response. rTs-ADSp-7 has therapeutic potential for colitis treatment and can be used as a helminth-derived protein therapy for CD or other Th1 immunity-mediated diseases.     

Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH ／ 3T3 fibroblasts

AIM: To investigate the regulatory mechanism of transforming growth factor-betaon activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS: The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-β1…     

Recent advance in aminopeptidase N (APN/CD13) inhibitor research

APN is an important zinc dependent metallo-exopeptidase, which can degrade the extracellular matrix, and plays an important role in tumor invasion, metastasis and angiogenesis. it has been considered as a suitable target for anti-cancer drug design. Recently, research of structure-based APN inhibitors is becoming an interesting field, and many APN inhibitors have been reported. Here we review a series of APN inhibitors and the recent progress in this field.     

Therapeutic potential of chloroquine in a murine model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammation of the gastrointestinal tract which is mainly caused by dysregulated gut immune response to commensal flora. Very limited treatment options with marginal efficacy are available along with surgery which has high risk of reoccurrence. As both innate and adaptive immune responses have been found altered in IBD, a good therapeutic strategy could be to restrict both of them under chronic inflammatory conditions. Effect of chloroquine on TLR9 signaling is well reported, while there are limited studies on non-endosomal TLRs as well as T cell responses. Hence, we studied its effect on other TLRs as well as T cell response along with testing it as a potential therapeutics in IBD using murine preclinical colitis model. Chloroquine significantly suppressed the TLR2 as well as TLR9 signaling in both in vitro as well as in vivo experimental settings, while it had no effect on TLR4 pathway. It also suppressed the T cell cytokine and proliferative responses. In, DSS-induced murine colitis model, chloroquine administration, significantly improved body weight loss, colon length shortening, tissue damage and inflammatory cell infiltration. Based on our findings in preclinical murine model of IBD, chloroquine has the potential to be considered as a therapeutic option in clinics through inhibition of diverse TLR and T cell responses.     

Maresin 1 alleviates dextran sulfate sodium-induced ulcerative colitis by regulating NRF2 and TLR4/NF-kB signaling pathway

ObjectiveUlcerative colitis (UC) is one of the most common gastrointestinal diseases, characterized as a chronic, relapsing inflammation that causes damage to the colonic mucosa. Maresin 1 (MaR1), a specialized proresolving mediator, has powerful anti-inflammatory activity that prevents the occurrence of various inflammatory diseases. The aim of this study was to explore the role and potential mechanism of MaR1 in DSS-induced ulcerative colitis.MethodsIn the present study, we established dextran sulfate sodium (DSS)-induced ulcerative colitis rat model in vivo. Rats with colitis received tail vein injection of MaR1, with or without intraperitoneal injection of ML385. The changes of body weight, colon length, disease activity index (DAI), colonic histopathology, inflammatory cytokines, the activity of myeloperoxidase (MPO) and reactive oxygen species (ROS), and infiltration of macrophages expressing F4/80 were analyzed for the evaluation of colitis severity. In addition, protein expressions were detected using western blot.ResultsMaR1 significantly reduced inflammatory cytokines production, and restored body weight, DAI and colonic histopathology. Besides, MaR1 improved the expression of tight junction (TJ) proteins and reduced the infiltration of neutrophil and macrophages, as well as a decreased activity of MPO and ROS. Meanwhile, MaR1 activated Nrf2 signaling and decreased toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) activation. Furthermore, ML385, an inhibitor of Nrf2, significantly reversed the protective effect of MaR1.ConclusionMaR1 play a protective role in DSS-induced colitis by activating Nrf2 signaling and inactivating Nrf2-mediated TLR4/NF-κB signaling pathway, which mediate proinflammatory mediators and intestinal TJ proteins in rats, providing novel insights into the therapeutic strategy of colitis.     

Therapeutic potential of carbon monoxide (CO) for intestinal inflammation

The pathogenesis of inflammatory bowel disease (IBD) is complicated and even several therapeutic strategies have been developed, they are not adequate for achieving mucosal remission in all IBD patients. Several reports have described the role of carbon monoxide (CO) in protection against chronic intestinal inflammation. CO has recently emerged as a potent immunomodulatory entity, anti-inflammatory agent, and homeostasis of physiological condition. CO reduces lipopolysaccharide-induced proinflammatory cytokines in macrophages via the effect of MAPK pathways. Interleukin-6, one of the important cytokines in the pathogenesis of IBD is also regulated by CO. Epithelial cell restitution is reported to be important factor to control IBD and CO has been reported to enhance colonic epithelial restitution through FGF15/19 expression in colonic myofibroblasts. CO also reduced mucosal damage and inflammation in several experimental animal colitis models such as interleukin-10(-/-) mouse model, TCRα(-/-) mouse model, dextran sodium sulfate colitis model, and trinitrobennzen sulfonic acid colitis model. Taken together, CO has anti-inflammatory and enhancement of restitution examined in vitro model and in vivo experimental colitis model. These results indicate that CO may have a potential to be one of the therapeutic strategies in IBD patients.     

Inhibition of glycolipid biosynthesis by N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin protects against the inflammatory response in hapten-induced colitis

Since glycolipid biosynthesis is potentially involved in immunological and inflammatory responses, we tested the effect of a novel inhibitor of intracellular glycolipid biosynthesis N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) in two hapten-induced colitis models: trinitrobenzene sulphonic acid (TNBS)- and oxazolone (4-ethoxymethylene-2phenyl-2oxazoline-5-one; Oxa)-induced colitis. AMP-DNM was given either by intraperitoneal injection or orally via the diet. Mice treated with AMP-DNM had less severe colitis and a more rapid weight recovery, less edema and less wall thickness. Cellular infiltration, goblet cell loss and myeloperoxidase (MPO) activity were reduced in colons of AMP-DNM-treated animals. Intralesional IFN-gamma and IL-18 production were lower in mice of the AMP-DNM-treated groups. Furthermore, AMP-DNM treatment reduced the serum anti-TNBS and anti-Oxa antibody levels. Our findings show that the glycolipid biosynthesis inhibitor AMP-DNM has a strong anti-inflammatory and immune suppressive activity on both TNBS- and Oxa-induced colitis. The data also provide evidence that glycolipid biosynthesis is involved in the inflammatory cascade in these inflammatory bowel disease (IBD) models.     

IL-33 induces both regulatory B cells and regulatory T cells in dextran sulfate sodium-induced colitis

Interleukin (IL)-33 is a member of the IL-1 family. Serum levels of IL-33 are increased in inflammatory bowel diseases (IBD), suggesting that IL-33 is involved in the pathogenesis of IBD, although its role is not clear. In this study, we investigated the role of IL-33 in the regulation of T-helper (Th) cell and B cell responses in mesenteric lymph nodes (MLN) in mice with dextran sulfate sodium (DSS)-induced colitis. Here, we showed that IL-33-treated mice were susceptible to DSS-induced colitis as compared with PBS-treated mice. The production of spontaneous inflammatory cytokines production by macrophages or dendritic cells (DC) in MLN significantly increased, and the responses of Th2, regulatory T cells (Treg) and regulatory B cells (Breg) were markedly upregulated, while Th1 responses were significantly downregulated in MLN of IL-33-treated mice with DSS-induced colitis. Our results demonstrate that IL-33 contributes to the pathogenesis of DSS-induced colitis in mice by promoting Th2 responses, but suppressing Th1 responses, in MLN. Moreover, IL-33 treatment increased Breg and Treg responses in MLN in mice with DSS-induced colitis. Therefore, modulation of IL-33/ST2 signaling is implicated as a novel biological therapy for inflammatory diseases associated with Th1 responses.     

Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10(-/-) mice by attenuating the activation of T cells and promoting their apoptosis

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptor induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10^−/− mice. JWH-133 effectively attenuated the overall clinical score, and reversed colitis-associated pathogenesis and decrease in body weight in IL-10^−/− mice. After JWH-133 treatment, the percentage of CD4⁺ T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells declined in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) of mice with chronic colitis. JWH-133 was also effective in ameliorating dextran sodium sulfate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodo-pravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, and suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD.     

Effect of recombinant Trichinella spiralis cysteine proteinase inhibitor on TNBS-induced experimental inflammatory bowel disease in mice

Inflammatory bowel disease (IBD) is a chronic autoimmune disease with a high recurrence rate. Ulcerative colitis (UC) and Crohn's disease (CD) are two types of IBD. At present, parasite-derived cysteine protease inhibitors have received extensive attention from researchers, and experiments have confirmed that these protease have an effect on certain autoimmune diseases. So we conducted experiments to investigate the effect of Trichinella spiralis cysteine protease inhibitors on TNBS-induced mouse CD models. In this experiment, 72 male BALB/c mice aged 6–8 weeks were randomly divided into two groups: prevention group and therapy group. The mice were sacrificed and harvested on the 7th day after the model was established to measure the changes of various indicators of colitis.The comparison of the TsCystatin + TNBS group with the PBS + TNBS group showed that the DAI score, MPO activity, and colonic macroscopic and microscopic damage significantly reduced, IFN-γ significantly decreased, IL-4 expression increased, and NF-κB expression decreased. The percentage of CD4+CD25+Foxp3+ Treg and CD8+CD28− Treg in spleen, and the proportion of CD4+/CD8+ Treg cells decreased. In the therapy group, we found no significant difference between the TNBS+PBS group and TNBS + TsCystatin group.Treatment with TsCystatin exerted a good intervention effect on the TNBS-induced mouse CD model. TsCystatin possibly induced a Th2-type immune response in the body, which balanced the Th1-type immune response induced by TNBS administration, thereby relieving colitis.     

TGF-beta 1 differentially regulates IL-2 expression and [3H]-thymidine incorporation in CD3 epsilon mAb- and CD28 mAb-activated splenocytes and thymocytes

Transforming growth factor-beta(1) (TGF-beta(1)) is a critical bifunctional regulator of inflammatory responses. Evidence strongly suggests that these regulatory consequences are, at least in part, a result of profound pleiotropic effects on T lymphocyte effector function. The mechanisms underlying the contradictory biological effects of TGF-beta(1) remain ambiguous. The objective of the present studies was to test the hypothesis that the concentration of TGF-beta(1) and the temporal relationship between activation of the T cell receptor (TCR) and the TGF-beta receptor regulate the effect of TGF-beta(1) on T lymphocyte activation and proliferation. Toward this end, we have quantified the concentration- and time-dependent effect of TGF-beta(1) on interleukin-2 (IL-2) protein secretion as an index of T lymphocyte activation and [3H]-thymidine incorporation as an index of cell proliferation in primary splenocytes and thymocytes. Our results suggest that TGF-beta(1) stimulates IL-2 production at low concentrations (0.1-1 pg/ml) and conversely inhibits IL-2 production at high concentrations (1-10 ng/ml) in CD3epsilon monoclonal antibody (mAb)+/-CD28 mAb-activated splenocytes. Additionally, concentrations of TGF-beta(1) that stimulate IL-2 production in CD3epsilon mAb+CD28 mAb-activated splenocytes concominantly inhibit splenocyte proliferation under similar conditions. Furthermore, we provide evidence suggesting that the effects of TGF-beta(1) on T lymphocytes are dependent upon the temporal relationship between activation of the TCR and the TGF-beta receptor. A time-dependent loss of a stimulatory effect and a concomitant gain of an inhibitory response by TGF-beta(1) on IL-2 production in response to CD3epsilon and CD28 mAbs is observed when TGF-beta(1) is added following T lymphocyte activation. In summary, these data unequivocally demonstrate that the orchestration of paradoxical effects of TGF-beta(1) on T-lymphocyte function is dependent upon the concentration of TGF-beta(1) and the temporal relationship between activation of signaling through the TCR and the TGF-beta receptor. Future mechanistic studies addressing the putative role that these factors play in modulating the effects of TGF-beta(1) on T lymphocyte activity will undoubtedly provide valuable insight towards the pharmacological intervention of inflammatory responses.