Emilie Vazeille

The ubiquitin-proteasome and the mitochondria-associated apoptotic pathways are sequentially downregulated during recovery after immobilization-induced muscle atrophy

Immobilization produces morphological, physiological, and biochemical alterations in skeletal muscle leading to muscle atrophy and long periods of recovery. Muscle atrophy during disuse results from an imbalance between protein synthesis and proteolysis but also between apoptosis and regeneration processes. This work aimed to characterize the mechanisms underlying muscle atrophy and recovery following immobilization by studying the regulation of the mitochondria-associated apoptotic and the ubiquitin-proteasome-dependent proteolytic pathways. Animals were subjected to hindlimb immobilization for 4-8 days (I4 to I8) and allowed to recover after cast removal for 10-40 days (R10 to R40). Soleus and gastrocnemius muscles atrophied from I4 to I8 to a greater extent than extensor digitorum longus and tibialis anterior muscles. Gastrocnemius muscle atrophy was first stabilized at R10 before being progressively reduced until R40. Polyubiquitinated proteins accumulated from I4, whereas the increased ubiquitination rates and chymotrypsin-like activity of the proteasome were detectable from I6 to I8. Apoptosome and caspase-3 or -9 activities increased at I6 and I8, respectively. The ubiquitin-proteasome-dependent pathway was normalized early when muscle stops to atrophy (R10). By contrast, the mitochondria-associated apoptotic pathway was first downregulated below basal levels when muscle started to recover at R15 and completely normalized at R20. Myf 5 protein levels decreased from I4 to I8 and were normalized at R10. Altogether, our results suggest a two-stage process in which the ubiquitin-proteasome pathway is rapidly up- and downregulated when muscle atrophies and recovers, respectively, whereas apoptotic processes may be involved in the late stages of atrophy and recovery.

Mannan-binding lectin deficiency results in unusual antibody production and excessive experimental colitis in response to mannose-expressing mild gut pathogens

Background In Crohns disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection. Methods ASCA were measured by ELISA. MBL was assessed by ELISA and quantitative PCR. Wild type and MBL-deficient mice were administered dextran sulfate sodium (DSS) in the presence or absence of viable Candida albicans or adhesive invasive Escherichia coli (AIEC). Mice were infected with C albicans to assess generation of anti-yeast mannan antibodies. Results MBL expression was virtually undetectable in the intestinal mucosa of both healthy controls and patients with CD, irrespective of macroscopic inflammation, indicating that systemic MBL must be responsible for the reduced risk of complicated disease in MBL-competent patients with CD. MBL-deficient mice showed enhanced DSS colitis upon oral challenge with C albicans or AIEC. C albicans could be recovered from the kidneys of colitic/C albicans-fed MBL-deficient, but not wild type mice. Infection with C albicans induced high titres of anti-C albicans mannan IgM and IgG in MBL-deficient mice but only a modest and transient IgM response with no class switch to IgG in wild type mice. Cross-reactive ASCA IgM continuously increased in MBL-deficient mice but rapidly declined after transient induction in wild type mice. In MBL-deficient mice, increased C albicans dissemination correlated with reduced early retention in the circulation. Conclusions These results suggest that systemic MBL helps to prevent excessive inflammation upon access of normally mild pathogens across the damaged intestinal epithelium. Lack of this innate defence promotes antibody responses with cross-reactive potential against common mannan epitopes. These interpretations are compatible with the increased prevalence of ASCA and complicated disease phenotypes in MBL-deficient patients with CD.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation.

AIM To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients. METHODS Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67. RESULTS Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohns disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation. CONCLUSION In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis.

Lack of muscle recovery after immobilization in old rats does not result from a defect in normalization of the ubiquitin-proteasome and the caspase-dependent apoptotic pathways

Immobilization periods increase with age because of decreased mobility and/or because of increased pathological episodes that require bed‐rest. Then, sarcopaenia might be partially explained by an impaired recovery of skeletal muscle mass after a catabolic state due to an imbalance of muscle protein metabolism, apoptosis and cellular regeneration. Mechanisms involved during muscle recovery have been little studied and in elderly they remain almost unknown. We show, in rats, that a short immobilization period during ageing initiated muscle atrophy that was indeed not recovered after 40 days. Immobilization was associated with an activation of both the ubiquitin–proteasome and the mitochondria‐associated apoptotic pathways and the inflammatory and redox processes, and a decrease of cellular regeneration. We show that the lack of muscle recovery during ageing is not due to a defect in proteolysis or apoptosis down‐regulation. These observations lead us to hypothesize that muscle protein synthesis activation after immobilization was altered during ageing.

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease

Objective Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. Design We performed a genetic association study in a large multicentre cohort of patients with Crohns disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. Results Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (pFDR=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. Conclusions Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Role of Meprins to Protect Ileal Mucosa of Crohn's Disease Patients from Colonization by Adherent-Invasive E. coli

Ileal lesions in Crohns disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.

Curcumin treatment prevents increased proteasome and apoptosome activities in rat skeletal muscle during reloading and improves subsequent recovery.

Immobilization is characterized by activation of the ubiquitin (Ub)-proteasome-dependent proteolytic system (UPS) and of the mitochondrial apoptotic pathway. Increased oxidative stress and inflammatory response occur in immobilized skeletal muscles. Curcumin exhibits antioxidant and anti-inflammatory properties, blocked proteasome activation in intact animals, and may favor skeletal muscle regeneration. We therefore measured the effects of curcumin on immobilization-induced muscle atrophy and subsequent recovery. Rats were subjected to hindlimb immobilization for 8 days (I8) and allowed to recover for 10 days (R10). Fifty percent of the rats were injected daily with either curcumin or vehicle. Proteolytic and apoptotic pathways were studied in gastrocnemius muscles. Curcumin treatment prevented the enhanced proteasome chymotrypsin-like activity and the trend toward increased caspase-9-associated apoptosome activity at I8 in immobilized muscles. By contrast, the increase of these two activities was blunted by curcumin at R10. Curcumin did not reduce muscle atrophy at I8 but improved muscle recovery at R10 and the cross-sectional area of muscle fibers of immobilized muscles. Curcumin reduced the increased protein levels of Smac/DIABLO induced by immobilization and enhanced the elevation of X-linked inhibitory apoptotic protein levels at R10. Ub-conjugate levels and caspase-3 activity increased at I8 and were normalized at R10 without being affected by curcumin treatment. Altogether, the data show that curcumin treatment improved recovery during reloading. The effect of curcumin during the atrophic phase on proteasome activities may facilitate the initiation of muscle recovery after reloading. These data also suggest that this compound may favor the initial steps of muscle regeneration.

Antioxidant supplementation had positive effects in old rat muscle, but through better oxidative status in other organs.

OBJECTIVE Aged muscle is characterized by a defect in the ability of leucine to stimulate protein synthesis. We showed previously that antioxidant supplementation improved the anabolic response to leucine of old muscle and reduced inflammation. The aim of the present study was to determine if the positive effects observed in muscle could be related to an improvement of local muscle oxidative status. METHODS Two groups of 20-mo-old male Wistar rats were supplemented or not with rutin, vitamin E, vitamin A, zinc, and selenium during 7 wk. We measured body weight, food intake, oxidative status in muscle and other tissues, gastrocnemius muscle proteolytic activities, and liver glutathione metabolism. RESULTS Antioxidant supplementation had no effect on muscle antioxidant capacity, superoxide dismutase activities, and myofibrillar protein carbonyl content and induced an increase in muscle cathepsin activities. In other tissues, antioxidant supplementation increased liver glutathione (reduced plus oxidized glutathione) content, reduced oxidative damage in the liver and spleen (as measured by γ-keto-aldehyde content), and reduced heart thiobarbituric acid-reactive substances. CONCLUSION Our results showed that the positive effects of antioxidant supplementation observed previously on the anabolic response to leucine of old muscle were not directly related to an improvement of in situ muscle oxidative status. It could result from reduced systemic inflammation/oxidative stress. The dialog between muscle and other organs should be studied more thoroughly, especially during aging.

Postoperative Complications after Ileocecal Resection in Crohn’s Disease: A Prospective Study From the REMIND Group

Objectives:We sought to determine the frequency of and risk factors for early (30-day) postoperative complications after ileocecal resection in a well-characterized, prospective cohort of Crohn’s disease patients.Methods:The REMIND group performed a nationwide study in 9 French university medical centers. Clinical-, biological-, surgical-, and treatment-related data on the 3 months before surgery were collected prospectively. Patients operated on between 1 September 2010 and 30 August 2014 were included.Results:A total of 209 patients were included. The indication for ileocecal resection was stricturing disease in 109 (52%) cases, penetrating complications in 88 (42%), and medication-refractory inflammatory disease in 12 (6%). A two-stage procedure was performed in 33 (16%) patients. There were no postoperative deaths. Forty-three (21%) patients (23% of the patients with a one-stage procedure vs. 9% of those with a two-stage procedure, P=0.28) experienced a total of 54 early postoperative complications after a median time interval of 5 days (interquartile range, 4–12): intra-abdominal septic complications (n=38), extra-intestinal infections (n=10), and hemorrhage (n=6). Eighteen complications (33%) were severe (Dindo–Clavien III–IV). Reoperation was necessary in 14 (7%) patients, and secondary stomy was performed in 8 (4.5%). In a multivariate analysis, corticosteroid treatment in the 4 weeks before surgery was significantly associated with an elevated postoperative complication rate (odds ratio (95% confidence interval)=2.69 (1.15–6.29); P=0.022). Neither preoperative exposure to anti-tumor necrosis factor (TNF) agents (n=93, 44%) nor trough serum anti-TNF levels were significant risk factors for postoperative complications.Conclusions:In this large, nationwide, prospective cohort, postoperative complications were observed after 21% of the ileocecal resections. Corticosteroid treatment in the 4 weeks before surgery was significantly associated with an elevated postoperative complication rate. In contrast, preoperative anti-TNF therapy (regardless of the serum level or the time interval between last administration and surgery) was not associated with an elevated risk of postoperative complications.

Monocyte-derived Macrophages from Crohn’s Disease Patients Are Impaired in the Ability to Control Intracellular Adherent-Invasive Escherichia coli and Exhibit Disordered Cytokine Secretion Profile

BACKGROUND Ileal lesions of Crohns disease [CD] patients are colonised by adherent-invasive Escherichia coli [AIEC] able to survive in macrophage cell lines. We analysed the ability of monocyte-derived macrophages [MDM] from CD patients to control AIEC intracellular replication and the pro-inflammatory cytokine response of the infected-MDM. METHODS Peripheral blood MDM were obtained from 24 CD genotyped for NOD2 and ATG16L1 mutations, 5 ulcerative colitis [UC] patients and 12 healthy controls [HC]. The numbers of intracellular bacteria were determined using gentamicin assay. Cytokine secretion was quantified by ELISA assay. RESULTS We observed that higher levels of bacteria were internalised within MDM from CD patients than MDM from HC or UC patients. MDM from CD patients were unable to restrict AIEC intracellular replication. Infection of MDM from CD patients with AIEC resulted in significantly increased secretion of IL-6 and tumour necrosis factor alpha [TNF α] than did infection with non-pathogenic E. coli. AIEC-infected MDM from CD patients exhibited a disordered cytokines secretion compared with MDM from UC patients and HC. AIEC-infected MDM from patients with quiescent CD released significantly higher amounts of IL-6 and TNF-alpha than those with active disease or those from HC. The level of secreted TNF-alpha was correlated to the number of intracellular AIEC in MDM from CD patients. Treatment of MDM with infliximab did not change the MDM behaviour. CONCLUSIONS MDM from CD patients are unable to restrict intracellular AIEC replication, leading to disordered inflammatory response influenced by disease activity.