Mitochondrial Dysfunction in CD4+ T Effector Memory RA+ Cells.

Human ageing is accompanied by poor responses to infection and decreased vaccine efficacy. While the causes of this can be attributed to defects in the immune system that increase with age, it is unknown whether mitochondrial dysfunction may also contribute to these phenomena. This study aims to assess mitochondrial dysfunction in CD4+ terminal effector memory T cells re-expressing CD45RA (TEMRA) cells and other CD4+ memory T cell subtypes, which are increased in number in the elderly population, with respect to how their metabolic responses to stimulation are altered compared to CD4+ naïve T cells. In this study, we show that CD4+ TEMRA cells exhibit altered mitochondrial dynamics compared to CD4+ naïve cells and CD4+ central and effector memory cells, with a 25% reduction in OPA1 expression. CD4+ TEMRA and memory cells show increased upregulation of Glucose transporter 1 following stimulation and higher levels of mitochondrial mass compared to CD4+ naïve T cells. Additionally, TEMRA cells exhibit a decrease in mitochondrial membrane potential compared to other CD4+ memory cell subsets by up to 50%. By comparing young to aged individuals, more significant mitochondria mass and lower membrane potential were observed in CD4+ TEMRA of young individuals. In conclusion, we suggest that CD4+ TEMRA cells may be impaired with respect to their metabolic response to stimulation, possibly contributing to impaired responses to infection and vaccination.

3D cyclorama for digital unrolling and visualisation of deformed tubes.

Colonic crypts are tubular glands that multiply through a symmetric branching process called crypt fission. During the early stages of colorectal cancer, the normal fission process is disturbed, leading to asymmetrical branching or budding. The challenging shapes of the budding crypts make it difficult to prepare paraffin sections for conventional histology, resulting in colonic cross sections with crypts that are only partially visible. To study crypt budding in situ and in three dimensions (3D), we employ X-ray micro-computed tomography to image intact colons, and a new method we developed (3D cyclorama) to digitally unroll them. Here, we present, verify and validate our '3D cyclorama' method that digitally unrolls deformed tubes of non-uniform thickness. It employs principles from electrostatics to reform the tube into a series of onion-like surfaces, which are mapped onto planar panoramic views. This enables the study of features extending over several layers of the tube's depth, demonstrated here by two case studies: (i) microvilli in the human placenta and (ii) 3D-printed adhesive films for drug delivery. Our 3D cyclorama method can provide novel insights into a wide spectrum of applications where digital unrolling or flattening is necessary, including long bones, teeth roots and ancient scrolls.

Staphylococcus aureus internalization in mast cells in nasal polyps: Characterization of interactions and potential mechanisms.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps is a common chronic condition. The exact cause of nasal polyps remains unknown. Recently, we made the novel observation of intracellular localization of Staphylococcus aureus within mast cells in nasal polyps. OBJECTIVE: This follow-up study aimed to further characterize interactions between S aureus and mast cells in this setting and elucidate potential internalization mechanisms with particular emphasis on the role of staphylococcal enterotoxin B (SEB). METHODS: A prospective study was performed using an explant tissue model with ex vivo inferior turbinate mucosa obtained from patients with chronic rhinosinusitis with nasal polyps (n = 7) and patients without CRS (n = 5). Immunohistochemistry was used to characterize S aureus uptake into mast cells and investigate the effects of SEB on this process. An in vitro cell-culture model was used to investigate mast cell-S aureus interactions by using a combination of fluorescent in situ hybridization, confocal laser scanning microscopy, scanning electron microscopy, transmission electron microscopy, and proliferation assays. RESULTS: S aureus was captured by extracellular traps and entered mast cells through phagocytosis. Proliferating intracellular S aureus led to the expansion and eventual rupture of mast cells, resulting in release of viable S aureus into the extracellular space. The presence of SEB appeared to promote internalization of S aureus into mast cells. CONCLUSION: This study provides new insights into the interactions between S aureus and mast cells, including the internalization process, and demonstrates a prominent role for SEB in promoting uptake of the bacteria into these cells.

Relationships Between Ion Channels, Mitochondrial Functions and Inflammation in Human Aging.

Aging is often associated with a loss of function. We believe aging to be more an adaptation to the various, and often continuous, stressors encountered during life in order to maintain overall functionality of the systems. The maladaptation of a system during aging may increase the susceptibility to diseases. There are basic cellular functions that may influence and/or are influenced by aging. Mitochondrial function is amongst these. Their presence in almost all cell types makes of these valuable targets for interventions to slow down or even reserve signs of aging. In this review, the role of mitochondria and essential physiological regulators of mitochondria and cellular functions, ion channels, will be discussed in the context of human aging. The origins of inflamm-aging, associated with poor clinical outcomes, will be linked to mitochondria and ion channel biology.

Adaptive NKG2C+CD57+ Natural Killer Cell and Tim-3 Expression During Viral Infections.

Repetitive stimulation by persistent pathogens such as human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV) induces the differentiation of natural killer (NK) cells. This maturation pathway is characterized by the acquisition of phenotypic markers, CD2, CD57, and NKG2C, and effector functions-a process regulated by Tim-3 and orchestrated by a complex network of transcriptional factors, involving T-bet, Eomes, Zeb2, promyelocytic leukemia zinc finger protein, and Foxo3. Here, we show that persistent immune activation during chronic viral co-infections (HCMV, hepatitis C virus, and HIV) interferes with the functional phenotype of NK cells by modulating the Tim-3 pathway; a decrease in Tim-3 expression combined with the acquisition of inhibitory receptors skewed NK cells toward an exhausted and cytotoxic phenotype in an inflammatory environment during chronic HIV infection. A better understanding of the mechanisms underlying NK cell differentiation could aid the identification of new immunological targets for checkpoint blockade therapies in a manner that is relevant to chronic infection and cancer.

The application of silver nano-particles on developing potential treatment for chronic rhinosinusitis: Antibacterial action and cytotoxicity effect on human nasal epithelial cell model.

Chronic rhinosinusitis (CRS) has raised attentions both in many countries due to its high relapsing rate and the resistance of certain treatments especially antibiotics application on both acute and chronic bacterial rhinosinusitis. The aim of this research is stressing on developing an effective alternative treatment for treating CRS and reducing the use of antibiotics to avoid further resistance forming. The antibacterial functions of silver nano-particles (AgNPs) are well known according to previous reports and studies. However, for developing a suitable treatment for further clinical application, a variety of AgNPs cell cytotoxicity experiments and AgNPs antibacterial properties experiments were examined in vitro in this study. For imitating the clinical condition of CRS, the human nasal epithelial cell line (RPMI2650) has chosen as experimental model. Moreover, Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) were selected for antibacterial function experiments. The analytical results demonstrated that 5ppm of AgNPs not only maintains >80% of cell activity to RPMI2650, but also possesses >80% of antibacterial function to S. aureus and 100% of antibacterial function to E. coli. Therefore, 5ppm of AgNPs might be considered as a promising antibacterial agent for treating CRS.

Hot topics in reactive oxygen therapy: Antimicrobial and immunological mechanisms, safety and clinical applications.

Reactive oxygen species (ROS), when combined with various delivery mechanisms, has the potential to become a powerful novel therapeutic agent against difficult-to-treat infections, especially those involving biofilm. It is important in the context of the global antibiotic resistance crisis. ROS is rapidly active in vitro against all Gram-positive and Gram-negative bacteria tested. ROS also has antifungal and antiviral properties. ROS prevents the formation of biofilms caused by a range of bacterial species in wounds and respiratory epithelium. ROS has been successfully used in infection prevention, eradication of multiresistant organisms, prevention of surgical site infection, and intravascular line care. This antimicrobial mechanism has great potential for the control of bioburden and biofilm at many sites, thus providing an alternative to systemic antibiotics on epithelial/mucosal surfaces, for wound and cavity infection, chronic respiratory infections and possibly recurrent urinary infections as well as local delivery to deeper structures and prosthetic devices. Its simplicity and stability lend itself to use in developing economies as well.

CD57 in human natural killer cells and T-lymphocytes.

The CD57 antigen (alternatively HNK-1, LEU-7, or L2) is routinely used to identify terminally differentiated 'senescent' cells with reduced proliferative capacity and altered functional properties. In this article, we review current understanding of the attributes of CD57-expressing T-cells and NK cells in both health and disease and discuss how this marker can inform researchers about their likely functions in human blood and tissues in vivo. While CD57 expression on human lymphocytes indicates an inability to proliferate, these cells also display high cytotoxic potential, and CD57(pos) NK cells exhibit both memory-like features and potent effector functions. Accordingly, frequencies of CD57-expressing cells in blood and tissues have been correlated with clinical prognosis in chronic infections or various cancers and with human aging. Functional modulation of senescent CD57(pos) T-cells and mature CD57(pos) NK cells may therefore represent innovative strategies for protection against human immunological aging and/or various chronic diseases.

Abnormal thymic stromal lymphopoietin expression in the duodenal mucosa of patients with coeliac disease.

OBJECTIVE: The short isoform of thymic stromal lymphopoietin (TSLP), a cytokine constitutively expressed by epithelial cells, is crucial in preserving immune tolerance in the gut. TSLP deficiency has been implicated in sustaining intestinal damage in Crohn's disease. We explored mucosal TSLP expression and function in refractory and uncomplicated coeliac disease (CD), a T-cell-mediated enteropathy induced by gluten in genetically susceptible individuals. DESIGN: TSLP isoforms-long and short-and receptors-TSLPR and interleukin (IL)-7Rα-were assessed by immunofluorescence, immunoblotting and qRT-PCR in the duodenum of untreated, treated, potential and refractory patients with CD. The ability of the serine protease furin or CD biopsy supernatants to cleave TSLP was evaluated by immunoblotting. The production of interferon (IFN)-γ and IL-8 by untreated CD biopsies cultured ex vivo with TSLP isoforms was also assessed. RESULTS: Mucosal TSLP, but not TSLPR and IL-7Rα, was reduced in untreated CD and refractory CD in comparison to treated CD, potential CD and controls. Transcripts of both TSLP isoforms were decreased in active CD mucosa. Furin, which was overexpressed in active CD biopsies, was able to cleave TSLP in vitro. Accordingly, refractory and untreated CD supernatants showed higher TSLP-degrading capacity in comparison to treated CD and control supernatants. In our ex vivo model, both TSLP isoforms significantly downregulated IFN-γ and IL-8 production by untreated CD biopsies. CONCLUSIONS: Reduced mucosal TSLP expression may contribute to intestinal damage in refractory and untreated CD. Further studies are needed to verify whether restoring TSLP might be therapeutically useful especially in refractory patients with CD.

Compartmentalization of immunosenescence: a deeper look at the mucosa.

Developments in medical care and living conditions led to an astonishing increase in life-span perspective and subsequently a rise in the old population. This can be seen as a success for public health policies but it also challenges society to adapt, in order to cope with the potentially overwhelming cost for the healthcare system. A fast-growing number of older people lose their ability to live independently because of diseases and disabilities, frailty or cognitive impairment. Many require long-term care, including home-based nursing, communities and hospital-based care. Immunosenescence, an age-related deterioration in immune functions, is considered a major contributory factor for the higher prevalence and severity of infectious diseases and the poor efficacy of vaccination in the elderly. When compared with systemic immunosenescence, alterations in the mucosal immune system with age are less well understood. For this reason, this area deserves more extensive and intensive research and support. In this article, we provide an overview of age-associated changes occurring in systemic immunity and discuss the distinct features of mucosal immunosenescence.

Influence of matrix metalloproteinase-12 on fibrinogen level.

In vitro studies have shown that matrix metalloproteinase-12 (MMP12) can degrade fibrinogen, a clotting factor whose level predicts risk of advanced atherosclerosis and myocardial infarction. In this study, we found that mean plasma fibrinogen level was approximately 10-fold higher in MMP12 knockout mice than wildtype mice (p=0.0006). Differential allelic expression analysis of human MMP12 gene polymorphism rs17368582 in human vascular tissues showed an allele-specific effect on MMP12 expression, with one allele (T) having 1.6 fold higher expression level than the other allele (C) (p=0.0006). In a population cohort, we found that individuals homozygous for the MMP12 low expression allele had higher plasma fibrinogen levels (2.95 mg/mL compared with 2.61 mg/mL in other individuals, p=0.029) and increased risk of advanced atherosclerosis [odds ratio 6.3 (95% CI 1.9-20.8), p=0.003] and myocardial infarction [hazard ratio 5.6 (95% CI 1.7-18.3), p=0.005]. In summary, our study in mouse and humans provides in vivo evidence of an effect of MMP12 on fibrinogen level.

Histone deacetylase inhibitors and their potential role in inflammatory bowel diseases.

IBDs (inflammatory bowel diseases) are lifelong manifestations that significantly impair the quality of life of those who suffer from them. Although many therapies are now available, including immunomodulatory drugs such as Infliximab which have efficacy in IBD, not all patients respond and some patients generate autoantibodies against these drugs. Hence the search for novel treatments is ongoing. HDACs (histone deacetylases) are responsible for condensation of chromatin in the nucleus of cells and inhibition of gene transcription and are often dysregulated during cancer. HDAC inhibitors allow normal gene transcription to be restored and provide attractive therapeutic options, as they have been shown to be anti-inflammatory and anti-proliferative in cancer. Indeed, two HDAC inhibitors have been recently approved for the treatment of cutaneous T-cell lymphoma in the U.S.A. Recent research using animal models has shown that HDAC inhibitors may have a beneficial effect in colitis by boosting levels of Foxp3+ (forkhead box P3+) T-regulatory cells that dampen inflammation. In the present paper, we outline the background to IBD, HDACs and their inhibitors as well as discussing their current use in models of IBD.

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli.

Ileal lesions in Crohn's 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 ß. 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 ß. 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.

Stromelysin-1 and macrophage metalloelastase expression in the intestinal mucosa of Crohn's disease patients treated with infliximab.

BACKGROUND AND AIMS: The mechanism by which anti-tumor necrosis factor (TNF)-alpha therapy promotes rapid closure of fistulas and mucosal wound healing in Crohn's disease (CD) remains unclear. An ex-vivo model of gut T-cell mediated injury indicated that TNF-alpha blockade prevents tissue damage concomitant with matrix metalloproteinase (MMP) inhibition. We, therefore, hypothesized that the chimeric anti-TNF-alpha antibody infliximab facilitates wound healing in CD by downregulating tissue degrading MMPs. We focused on MMP-3 (stromelysin-1) and MMP-12 (macrophage metalloelastase) as these two enzymes have been linked to connective tissue destruction in CD. METHODS: Endoscopic biopsies were taken from 10 CD patients immediately before and after 10 weeks of treatment with infliximab. Before treatment, biopsies were taken from macroscopically inflamed areas, and after treatment were collected from the same locations as before treatment. The degree of mucosal damage was assessed by using a histological scoring system. MMP transcripts were detected by in-situ hybridization on paraffin sections. MMP proteins were determined by immunoblotting on mucosal homogenates. RESULTS: Six out of 10 patients had a clinical response to infliximab. MMP-3 and MMP-12 transcripts and proteins, which were highly expressed in CD inflamed mucosa, decreased after treatment in those patients who responded to infliximab. MMP-3 and MMP-12 downregulation was accompanied by a concomitant improvement of the histologic score. No change in MMP expression was found in nonresponders. CONCLUSION: The downregulation of tissue degrading MMPs in CD mucosa may explain the wound repair capacity of infliximab in healing fistulas and ulcers.

Matrix metalloproteinase-3 production by gut IgG plasma cells in chronic inflammatory bowel disease.

BACKGROUND: In both ulcerative colitis (UC) and Crohn's disease (CD) there is a marked increase in mucosal IgG plasma cells (PC), although their precise role is not well established. In this study we isolated gut PCs from patients with IBD and normal controls and analyzed cytokine production, matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 production, and PC longevity ex vivo. METHODS: Lamina propria mononuclear cells (LPMCs) were isolated from patients with CD (n = 19), UC (n = 27), and normal controls (n = 42). PCs were further selected by immunomagnetic isolation using CD138 microbeads. Cytokine, MMP-3, and TIMP-1 expression was investigated by Taqman polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), Western blotting, and confocal microscopy. PC lifespan in vitro was studied by ELISpot analysis. RESULTS: PCs from both controls and IBD patients contained high levels of transcripts for TGFbeta, whereas they did not contain significant transcripts for IL-4, IL-5, IL-10, IFNgamma, TNF, or IL-12p40. PCs from patients with CD and UC expressed significantly higher levels of MMP-3 protein and transcripts than controls (P < 0.0001). The vast majority of MMP-3-expressing PCs were IgG+ve. In culture, IgA PCs from both IBD patients and controls persisted for only a few days, but IgG PCs from IBD patients persisted for at least 3 weeks. CONCLUSIONS: We have demonstrated that IgG PCs from patients with IBD express large amounts of MMP-3 and that they appear to be long-lived. These results identify a new pathway by which IgG PCs may damage the gut.

Functional modulation of Crohn's disease myofibroblasts by anti-tumor necrosis factor antibodies.

BACKGROUND & AIMS: Infliximab induces immune cell apoptosis by outside-to-inside signaling through transmembrane tumor necrosis factor-alpha (mTNF). However, in inflamed gut, myofibroblasts also produce TNF-alpha, and the affects of anti-TNF antibodies on these structural cells are unknown. We investigated the action of infliximab on apoptosis, the production of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP)-1, and migration of Crohn's disease (CD) myofibroblasts. METHODS: Colonic myofibroblasts were isolated from patients with active CD and controls. mTNF was evaluated by Western blotting and flow cytometry. Infliximab-treated myofibroblasts were analyzed for apoptosis by Annexin V staining and caspase-3. TIMP-1 and MMPs were measured by Western blotting, and fibroblast migration was assessed by using an in vitro wound-healing scratch assay. RESULTS: CD myofibroblasts showed higher mTNF expression than control myofibroblasts. Infliximab had no effect on CD myofibroblast apoptosis, caspase-3 activation, and production of MMP-3 and MMP-12. However, infliximab induced a significant dose-dependent increase in TIMP-1 production, which was inhibited by the p38 mitogen-activated protein kinase inhibitor SB 203580. The anti-TNF agents adalimumab, etanercept, and p55 TNF-receptor-human IgG fusion protein also increased TIMP-1 production. The migration of CD myofibroblasts was enhanced significantly by infliximab and recombinant human TIMP-1, and infliximab-induced migration was inhibited by anti-TIMP-1 neutralizing antibody. Infliximab also decreased CD myofibroblast collagen production. CONCLUSIONS: Our findings show a novel therapeutic pathway for anti-TNF therapies in enhancing TIMP-1 production and myofibroblast migration, which may reduce MMP activity and facilitate the wound healing.

Role of macrophage metalloelastase in gut inflammation.

Matrix metalloproteinases (MMPs) are involved in a number of physiological and pathologic processes including the inflammation found in IBD. We have shown that MMP-3 is upregulated in Crohn's disease and in ulcerative colitis. This study shows a potential role for MMP-12 in these idiopathic diseases.