Elafin and its precursor trappin‐2 are known for their contribution to the physiological mucosal shield against luminal microbes. Such a contribution seems to be particularly relevant in the gut, ...where the exposure of host tissues to heavy loads of microbes is constant and contributes to mucosa‐associated pathologies. The expression of trappin‐2/elafin has been shown to be differentially regulated in diseases associated with gut inflammation. Accumulating evidence has demonstrated the protective effects of trappin‐2/elafin in gut intestinal disorders associated with acute or chronic inflammation, or with gluten sensitization disorders. The protective effects of trappin‐2/elafin in the gut are discussed in terms of their pleiotropic modes of action: acting as protease inhibitors, transglutaminase substrates, antimicrobial peptides or as a regulator of pro‐inflammatory transcription factors. Further, the question of the therapeutic potential of trappin‐2/elafin delivery at the intestinal mucosa surface is raised. Whether trappin‐2/elafin mucosal delivery should be considered to ensure intestinal tissue repair is also discussed.
The multidomain serine protease inhibitor lymphoepithelial Kazal-type related inhibitor (LEKTI) represents a key regulator of the proteolytic events occurring during epidermal barrier formation and ...hair development, as attested by the severe autosomal recessive ichthyosiform skin condition Netherton syndrome (NS) caused by mutations in its encoding gene, serine protease inhibitor Kazal-type 5 (SPINK5). Synthesized as a proprotein, LEKTI is rapidly cleaved intracellularly, thus generating a number of potentially bioactive fragments that are secreted. Here, we show that SPINK5 generates three classes of transcripts encoding three different LEKTI isoforms, which differ in their C-terminal portion. In addition to the previously described 15 domain isoform, SPINK5 encodes a shorter LEKTI isoform composed of only the first 13 domains, as well as a longer isoform carrying a 30-amino-acid residue insertion between the 13th and 14th inhibitory domains. We demonstrate that variable amounts of SPINK5 alternative transcripts are detected in all SPINK5 transcriptionally active tissues. Finally, we show that in differentiated cultured human keratinocytes all SPINK5 alternative transcripts are translated into protein and that the LEKTI precursors generate distinct secreted C-terminal proteolytic fragments from a similar cleavage site. Since several data indicate a biological role for the pro-LEKTI-cleaved polypeptides, we hypothesize that the alternative processing of the SPINK5 pre-messenger RNA represents an additional mechanism to further increase the structural and functional diversity of the LEKTI bioactive fragments.
Abnormal gut barrier function is the basis of gut inflammatory disease. It is known that house dust mite (HDM) aero-allergens induce inflammation in respiratory mucosa. We have recently reported ...allergen from Dermatophagoides pteronyssinus (Der p1) to be present in rodent gut.
To examine whether Der p1 is present in human gut and to assess its effect on gut barrier function and inflammation.
Colonic biopsies, gut fluid, serum and stool were collected from healthy adults during endoscopy. Der p1 was measured by ELISA. Effect of HDM was assessed on gut permeability, tight-junction and mucin expression, and cytokine production, in presence or absence of cysteine protease inhibitors or serine protease inhibitors. In vivo effect of HDM was examined in mice given oral HDM or protease-neutralised HDM. Role of HDM in low-grade inflammation was studied in patients with IBS.
HDM Der p1 was detected in the human gut. In colonic biopsies from healthy patients, HDM increased epithelial permeability (p<0.001), reduced expression of tight-junction proteins and mucus barrier. These effects were associated with increased tumour necrosis factor (TNF)-α and interleukin (IL)-10 production and were abolished by cysteine-protease inhibitor (p<0.01). HDM effects did not require Th2 immunity. Results were confirmed in vivo in mice. In patients with IBS, HDM further deteriorated gut barrier function, induced TNF-α but failed to induce IL-10 secretion (p<0.001).
HDM, a ubiquitous environmental factor, is present in the human gut where it directly affects gut function through its proteolytic activity. HDM may be an important trigger of gut dysfunction and warrants further investigation.
While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease ...(IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.
Successful prevention of food allergy requires the identification of the factors adversely affecting the capacity to develop oral tolerance to food antigen in early life.
This study sought to ...determine whether oral exposure to Dermatophagoides pteronyssinus through breast milk affects gut mucosal immunity with long-term effects on IgE-mediated food allergy susceptibility.
Gut immunity was explored in 2-week-old mice breast-fed by mothers exposed to D pteronyssinus, protease-inactivated D pteronyssinus, or to PBS during lactation. We further analyzed oral tolerance to a bystander food allergen, ovalbumin (OVA). In a proof-of-concept study, Der p 1 and OVA levels were determined in 100 human breast milk samples and the association with prevalence of IgE-mediated egg allergy at 1 year was assessed.
Increased permeability, IL-33 levels, type 2 innate lymphoid cell activation, and Th2 cell differentiation were found in gut mucosa of mice nursed by mothers exposed to D pteronyssinus compared with PBS. This pro-Th2 gut mucosal environment inhibited the induction of antigen-specific FoxP3 regulatory T cells and the prevention of food allergy by OVA exposure through breast milk. In contrast, protease-inactivated D pteronyssinus had no effect on offspring gut mucosal immunity. Based on the presence of Der p 1 and/or OVA in human breast milk, we identified groups of lactating mothers, which mirror the ones found in mice to be responsible for different egg allergy risk.
This study highlights an unpredicted potential risk factor for the development of food allergy, that is, D pteronyssinus allergens in breast milk, which disrupt gut immune homeostasis and prevents oral tolerance induction to bystander food antigen through their protease activity.
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Background and Purpose
Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to ...epithelial cells and that chymotrypsin signals to them via protease‐activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells.
Experimental Approach
The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N‐terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC–MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids.
Key Results
We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin‐10 (IL‐10) up‐regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin.
Conclusion and Implications
Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.
The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is ...controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficent mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.--Yamasaki, K., Schauber, J., Coda, A., Lin, H., Dorschner, R. A., Schechter, N. M., Bonnart, C., Descargues, P., Hovnanian, A., Gallo, R. L. Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin.
The basal transcription/repair factor II H (TFIIH), found mutated in cancer-prone or premature aging diseases, plays a still unclear role in RNA polymerase I transcription. Furthermore, the impact of ...this function on TFIIH-related diseases, such as trichothiodystrophy (TTD), remains to be explored. Here, we studied the involvement of TFIIH during the whole process of ribosome biogenesis, from RNAP1 transcription to maturation steps of the ribosomal RNAs. Our results show that TFIIH is recruited to the ribosomal DNA in an active transcription-dependent manner and functions in RNAP1 transcription elongation through ATP hydrolysis of the XPB subunit. Remarkably, we found a TFIIH allele-specific effect, affecting RNAP1 transcription and/or the pre-rRNA maturation process. Interestingly, this effect was observed in mutant TFIIH-TTD cells and also in the brains of TFIIH-TTD mice. Our findings provide evidence that defective ribosome synthesis represents a new faulty mechanism involved in the pathophysiology of TFIIH-related diseases.
Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor ...cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3β (GSK3β) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3β activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3β nonactive form, strengthening the role of PAR2 in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.
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