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

Abstract

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.

Introduction

The ectopeptidases Dipeptidylpeptidase IV (DPIV/CD26, EC 3.4.14.5) and Aminopeptidase N (APN/CD13, EC 3.4.11.2) are involved in the control of immune cell activation and therefore emerged as new drug targets for the treatment of diseases associated with an imbalanced T cell response.

DPIV is a 220–240 kDa type II transmembrane protein, expressed as a highly glycosylated homodimer. On immune cells its enzymatic activity is linked to the T cell activation antigens TA-1 in humans and THAM-1 in mice, clustered as CD26. While resting T cells carry low to medium amounts of DPIV at their surface, expression is strongly up-regulated upon T cell activation [1], [2], [3], [4]. DPIV belongs to a group of proteins, which is summarized under the term DASH (DPIV activity and structural homologues) and is characterized by a similar substrate specificity and overlapping inhibitor profile [5]. DPIV cleaves off dipeptides from the N-terminus of polypeptides, if proline is in the penultimate position. Based on its unique enzymatic activity DPIV is faced with a multiple substrate scenario, which involves several chemokines, (e.g. SDF-α, RANTES), cytokines, peptide hormones (glucose regulation) as well as neuropeptides (e.g. substance P, neuropeptide Y) [6].

Aminopeptidase N, identical with the myeloid linage antigen CD13, is a zinc-dependent metalloprotease with a molecular mass of 150 kDa. The proteolytically active homodimer is expressed as type II transmembrane protein. Since resting T lymphocytes lack immunochemically detectable APN expression, an elevated APN gene transcription and surface expression of APN was reported on activated T cells derived from local sites of inflammation and following TCR engagement or mitogenic stimulation [7], [8]. Recent experimental work provided evidence that particularly CD4+CD25+ regulatory T cells express significant amounts of APN [9].

APN contributes to the proteolysis of neuropeptides, angiotensins, cytokines and immunomodulatory peptides and is involved in the degradation of extracellular matrix proteins and in antigen processing. This enzyme is catalyzing the hydrolysis of neutral amino acids from the N-terminus of oligo-peptides, but stops hydrolysis if proline is in second position of the N-terminal sequence [7], [8]. Thereby it is putatively generating DPIV-susceptible substrates.

Due to their similar expression pattern, subcellular localization and their unique substrate specificities, both peptidases are believed to co-operate in the limited proteolysis of biological active proteins and thereby to modulate cellular communication. Targeting DPIV or APN on activated T lymphocytes from mouse and humans by specific synthetic inhibitors was found to result in a suppression of cell proliferation and a reduced synthesis of proinflammatory cytokines without affecting cellular viability [10], [11], [12], [13], [14]. Most interestingly, both, the inhibition of DPIV or APN, leads to an increasing transcription, synthesis and secretion of the most potent immunosuppressive cytokine TGFβ1, suggesting that both enzymes plays a key role in limiting and terminating immune activation [11], [12], [13], [14]. Moreover, APN inhibitors were recently found to be capable of preserving and promoting the suppressive activity of CD4+CD25^bright T regulatory cells [15].

The simultaneous incubation of activated human PBMC and T cells in vitro with specific synthetic inhibitors was found to elicit additive suppressing effects on DNA synthesis and proinflammatory cytokine production, particularly at low inhibitor concentrations [16]. Therefore, both peptidases represent a promising target complex for the treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). Based on the increasing knowledge on the co-function of DPIV and APN in regulating growth and activity of lymphocytes as well as on the described synergistic effect of inhibition of both enzymes in vitro, a novel therapeutic strategy was proposed, which was termed Peptidase-targeted Immunoregulation (PETIR™). The aim is the restoration and maintenance of immune balance by limiting T effector cells activation and by the induction of powerful endogenous immunosuppressive mechanisms, such as TGF-β and regulatory T cells in parallel.

The present study evaluated the efficacy of DPIV and APN inhibitors alone compared to the effect of a combined inhibition of both peptidases in a mouse model of inflammatory bowel disease.