Potential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homologue in Crohn's Disease Fibrosis.

BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of Crohn's disease [CD]. It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. METHODS: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures [13 zones collected in five patients]. Proteins of interests were validated by immunohistochemistry [IHC] in ileal and colonic samples of stricturing CD [n = 44], pure inflammatory CD [n = 29], and control [n = 40] subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. RESULTS: Proteomic study revealed an endoplasmic reticulum [ER] stress proteins increase in the epithelium of CD ileal fibrotic strictures, including anterior gradient protein 2 homologue [AGR2] and binding-immunoglobulin protein [BiP]. This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. CONCLUSIONS: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.

Solute carrier family 12 member 2 as a proteomic and histological biomarker of dysplasia and neoplasia in ulcerative colitis.

BACKGROUND AND AIMS: Ulcerative colitis (UC) patients have a greater risk of developing colorectal cancer through inflammation-dysplasia-carcinoma sequence of transformation. The histopathological diagnosis of dysplasia is therefore of critical clinical relevance, but dysplasia may be difficult to distinguish from inflammatory changes. METHODS: A proteomic pilot study on 5 UC colorectal dysplastic patients highlighted proteins differentially distributed between paired dysplastic, inflammatory and normal tissues. The best candidate marker was selected and immunohistochemistry confirmation was performed on AOM/DSS mouse model lesions, 37 UC dysplasia, 14 UC cancers, 23 longstanding UC, 35 sporadic conventional adenomas, 57 sporadic serrated lesions and 82 sporadic colorectal cancers. RESULTS: Differential proteomics found 11 proteins significantly more abundant in dysplasia compared to inflammation, including Solute carrier family 12 member 2 (SLC12A2) which was confidently identified with 8 specific peptides and was below the limit of quantitation in both inflammatory and normal colon. SLC12A2 immunohistochemical analysis confirmed the discrimination of preneoplastic and neoplastic lesions from inflammatory lesions in mice, UC and in sporadic contexts. A specific SLC12A2 staining pattern termed "loss of gradient" reached 89% sensitivity, 95% specificity and 92% accuracy for UC-dysplasia diagnosis together with an inter-observer agreement of 95.24% (multirater κfree of 0.90; IC95%: 0.78 - 1.00). Such discrimination could not be obtained by Ki67 staining. This specific pattern was also associated with sporadic colorectal adenomas and cancers. CONCLUSIONS: We found a specific SLC12A2 immunohistochemical staining pattern in precancerous and cancerous colonic UC-lesions which could be helpful for diagnosing dysplasia and cancer in UC and non-UC patients.

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages.

BACKGROUND: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient's survival. METHODS: We analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohistochemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages. RESULTS: Out of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed different expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues. CONCLUSION: We highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers.

Comparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work.

Selective glucocorticoid receptor modulator compound A, in contrast to prednisolone, does not induce leptin or the leptin receptor in human osteoarthritis synovial fibroblasts.

OBJECTIVE: Glucocorticoids are powerful anti-inflammatory compounds that also induce the expression of leptin and leptin receptor (Ob-R) in synovial fibroblasts through TGF-ßsignalling and Smad1/5 phosphorylation. Compound A (CpdA), a selective glucocorticoid receptor agonist, reduces inflammation in murine arthritis models and does not induce diabetes or osteoporosis, thus offering an improved risk:benefit ratio in comparison with glucocorticoids. Due to the detrimental role of leptin in OA pathogenesis, we sought to determine whether CpdA also induced leptin and Ob-R protein expression as observed with prednisolone. METHODS: Human synovial fibroblasts and chondrocytes were isolated from the synovium and cartilage of OA patients after joint surgery. The cells were treated with prednisolone, TGF-ß1, TNF-α and/or CpdA. Levels of leptin, IL-6, IL-8, MMP-1 and MMP-3 were measured by ELISA and expression levels of Ob-R phospho-Smad1/5, phospho-Smad2, α-tubulin and glyceraldehyde 3-phosphate dehydrogenase were analysed by western blotting. RESULTS: CpdA, unlike prednisolone, did not induce leptin secretion or Ob-R protein expression in OA synovial fibroblasts. Moreover, CpdA decreased endogenous Ob-R expression and down-regulated prednisolone-induced leptin secretion and Ob-R expression. Mechanistically, CpdA, unlike prednisolone, did not induce Smad1/5 phosphorylation. CpdA, similarly to prednisolone, down-regulated endogenous and TNF-α-induced IL-6, IL-8, MMP-1 and MMP-3 protein secretion. The dissociative effect of CpdA was confirmed using chondrocytes with no induction of leptin secretion, but with a significant decrease in IL-6, IL-8, MMP-1 and MMP-3 protein secretion. CONCLUSION: CpdA, unlike prednisolone, did not induce leptin or Ob-R in human OA synovial fibroblasts, thereby demonstrating an improved risk:benefit ratio.

Matrix metalloproteinase-19 deficiency promotes tenascin-C accumulation and allergen-induced airway inflammation.

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing the recruitment and clearance of inflammatory cells and modifying the biological activity of many peptide mediators by cleavage. MMP-19 is newly described, and it preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The present study sought to assess the expression of MMP-19 in a murine asthma model, and to address the biological effects of MMP-19 deficiency in mice. Allergen-exposed, wild-type mice displayed increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs, as detected by immunohistochemistry. After an allergen challenge of MMP-19 knockout (MMP-19(-/-)) mice, exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue, along with increased airway responsiveness to methacholine. A shift toward increased T helper-2 lymphocyte (Th2)-driven inflammation in MMP-19(-/-) mice was demonstrated by (1) increased numbers of cells expressing the IL-33 receptor T(1)/ST(2) in lung parenchyma, (2) increased IgG(1) levels in serum, and (3) higher levels of IL-13 and eotaxin-1 in lung extracts. Tenascin-C was found to accumulate in peribronchial areas of MMP-19(-/-) after allergen challenges, as assessed by Western blot and immunohistochemistry analyses. We conclude that MMP-19 is a new mediator in asthma, preventing tenascin-C accumulation and directly or indirectly controlling Th2-driven airway eosinophilia and airway hyperreactivity. Our data suggest that MMP-19 may act on Th2 inflammation homeostasis by preventing the accumulation of tenascin protein.

Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice.

The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.

Role of A disintegrin and metalloprotease-12 in neutrophil recruitment induced by airway epithelium.

Among proteases, metalloproteases are implicated in tissue remodeling, as shown in numerous diseases including allergy. ADAMs (A Disintegrin And Metalloprotease) metalloproteases are implicated in physiologic processes such as cytokine and growth factor shedding, cell migration, adhesion, or repulsion. Our aim was to measure ADAM-12 expression in airway epithelium and to define its role during the allergic response. To raise this question, we analyzed the ADAM-12 expression ex vivo after allergen exposure in patients with allergic rhinitis and in vitro in cultured primary human airway epithelial cells (AEC). Clones of BEAS-2B cells transfected with the full-length form of ADAM-12 were generated to study the consequences of ADAM-12 up-regulation on AEC function. After allergen challenge, a strong increase of ADAM-12 expression was observed in airway epithelium from patients with allergic rhinitis but not from control subjects. In contrast with the other HB-epidermal growth factor sheddases, ADAM-10 and -17, TNF-alpha in vitro increased the expression of ADAM-12 by AEC, an effect amplified by IL-4 and IL-13. Up-regulation of ADAM-12 in AEC increased the expression of alpha3 and alpha4 integrins and to the modulation of cell migration on fibronectin but not on collagen. Moreover, overexpression of ADAM-12 in BEAS-2B enhanced the secretion of CXCL1 and CXCL8 and their capacity to recruit neutrophils. CD47 was strongly decreased by ADAM-12 overexpression, a process associated with a reduced adhesion of neutrophils. These effects were mainly dependent on epidermal growth factor receptor activation. In summary, ADAM-12 is produced during allergic reaction by AEC and might increase neutrophil recruitment within airway mucosa.

ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo.

ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motifs), the first described member of the ADAMTS family, is differentially expressed in various tumors. However, its exact role in tumor development and progression is still unclear. The aim of this study was to investigate the effects of ADAMTS-1 transfection in a bronchial epithelial tumor cell line (BZR) and its potential to modulate tumor development. ADAMTS-1 overexpression did not affect in vitro cell properties such as (a) proliferation in two-dimensional culture, (b) proliferation in three-dimensional culture, (c) anchorage-independent growth in soft agar, (d) cell migration and invasion in modified Boyden chamber assay, (e) angiogenesis in the aortic ring assay, and (f) cell apoptosis. In contrast, ADAMTS-1 stable transfection in BZR cells accelerated the in vivo tumor growth after s.c. injection into severe combined immunodeficient mice. It also promoted a stromal reaction characterized by myofibroblast infiltration and excessive matrix deposition. These features are, however, not observed in tumors derived from cells overexpressing a catalytically inactive mutant of ADAMTS-1. Conditioned media from ADAMTS-1-overexpressing cells display a potent chemotactic activity toward fibroblasts. ADAMTS-1 overexpression in tumors was associated with increased production of matrix metalloproteinase-13, fibronectin, transforming growth factor beta (TGF-beta), and interleukin-1beta (IL-1beta). Neutralizing antibodies against TGF-beta and IL-1beta blocked the chemotactic effect of medium conditioned by ADAMTS-1-expressing cells on fibroblasts, showing the contribution of these factors in ADAMTS-1-induced stromal reaction. In conclusion, we propose a new paradigm for catalytically active ADAMTS-1 contribution to tumor development, which consists of the recruitment of fibroblasts involved in tumor growth and tumor-associated stroma remodeling.