Stability in metabolic phenotypes and inferred metagenome profiles before the onset of colitis-induced inflammation.

Inflammatory bowel disease (IBD) is associated with altered microbiota composition and metabolism, but it is unclear whether these changes precede inflammation or are the result of it since current studies have mainly focused on changes after the onset of disease. We previously showed differences in mucus gut microbiota composition preceded colitis-induced inflammation and stool microbial differences only became apparent at colitis onset. In the present study, we aimed to investigate whether microbial dysbiosis was associated with differences in both predicted microbial gene content and endogenous metabolite profiles. We examined the functional potential of mucus and stool microbial communities in the mdr1a -/- mouse model of colitis and littermate controls using PICRUSt on 16S rRNA sequencing data. Our findings indicate that despite changes in microbial composition, microbial functional pathways were stable before and during the development of mucosal inflammation. LC-MS-based metabolic phenotyping (metabotyping) in urine samples confirmed that metabolite profiles in mdr1a -/- mice were remarkably unaffected by development of intestinal inflammation and there were no differences in previously published metabolic markers of IBD. Metabolic profiles did, however, discriminate the colitis-prone mdr1a -/- genotype from controls. Our results indicate resilience of the metabolic network irrespective of inflammation. Importantly as metabolites differentiated genotype, genotype-differentiating metabolites could potentially predict IBD risk.

Arginase-1-expressing macrophages are dispensable for resistance to infection with the gastrointestinal helminth Trichuris muris.

Alternatively activated macrophages (AAMs) have key roles in the immune response to a variety of gastrointestinal helminths such as Heligmosomoides bakeri and Nippostrongylus brasiliensis. In addition, AAMs have been implicated in the resolution of infection-induced pathology in Schistosoma mansoni infection. AAMs exert their activity in part via the enzyme arginase-1 (Arg1), which hydrolyses L-arginine into urea and ornithine, and can supply precursor substrate for proline and polyamine production. Trichuris muris is a worm that resides in the large intestine with resistance being characterized by a Th2 T-cell response, which drives alternatively activated macrophage production in the local environment of the infection. To investigate the role of AAMs in T. muris infection, we used independent genetic and pharmacologic models of arginase deficiency. In acute infection and Th2-dominated immunity, arginase-deficient models expelled worms normally. Macrophage-Arg1-deficient mice showed cytokine and antibody levels comparable to wild-type animals in acute and chronic infection. We also found no role for AAMs and Arg1 in infection-induced pathology in the response to T. muris in either chronic (Th1 dominated) or acute (Th2 dominated) infections. Our data demonstrate that, unlike other gastrointestinal helminths, Arg1 expression in AAMs is not essential for resistance to T. muris in effective resolution of helminth-induced inflammation.

Increased natural killer cell activity in patients with an abdominal aortic aneurysm.

BACKGROUND: Natural killer (NK) cells have an emerging role in the development of chronic disease and in the direction and maintenance of inflammatory responses. Abdominal aortic aneurysms (AAA) is a chronic inflammatory disorder of unknown aetiology. The aim was to investigate whether NK cells showed altered function in patients with an AAA. METHODS: The presence, phenotype and function of peripheral blood and tissue NK cells from patients with an AAA, peripheral vascular disease (PVD) and healthy age-sex-matched controls were assessed before and after surgery. RESULTS: Patients with an AAA had significantly higher (P < 0.010) percentages of peripheral blood NK cells (mean (95 per cent c.i.) 23.8 (2.6) per cent) than patients with PVD (17.4 (2.9) per cent) and control subjects (16.2 (2.8) per cent). The NK cells from patients with an AAA had increased cytotoxicity on a per cell basis towards both an NK-sensitive target cell line and human aortic smooth muscle cells. Increased NK cell proportions (22.7 (3.5) per cent) and cytotoxic activity, together with higher C-reactive protein values, persisted after successful AAA repair. CONCLUSION: These data support the hypothesis that increased NK cytotoxicity could be a contributing factor in the generation or potentiation of inflammation in patients with an AAA.

Colonic epithelial cell mediated suppression of CD4 T cell activation.

BACKGROUND AND AIMS: As the first point of contact with enteric antigens, intestinal epithelial cells (IEC) may be key in regulating mucosal immune responses. We determined therefore if murine colonic epithelial cells (CEC) have tolerogenic or activating effects on CD4 T cells. METHODS: Using a novel CEC, macrophages, and CD4 T cell coculture system, mitogen and antigen specific responses of naïve and antigen primed CD4 T cells were assessed. RESULTS: Although a proportion of CEC express the costimulatory molecules B7.1, B7.2, CD40, and CD54, they were unable to promote mitogen or antigen driven activation of CD4 T cells, even in the presence of exogenous costimulatory signals. CD4 T cells cocultured with CEC were CD25lo and CD45RBlo and remained in the G1 phase of the cell cycle. CEC were also able to prevent CD4 T cell activation by professional antigen presenting cells. CEC mediated suppression of T cell activation was cell contact dependent and transforming growth factor beta independent. CONCLUSIONS: These observations suggest that CEC contribute to the maintenance of T cell tolerance in the gut by preventing inappropriate activation of CD4 T cells.

Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

Transforming growth factor-beta promotes 'death by neglect' in post-activated human T cells.

Transforming growth factor-beta (TGF-beta) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound-healing, we studied the effects of TGF-beta on mitogen-stimulated peripheral blood cells. TGF-beta added at the initiation of mitogenesis did not significantly alter T-cell activation, proliferation, CD45 isoform switching, or activation-induced cell death. By contrast, TGF-beta added 72 hr post-activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF-beta had no effect on mitogen-induced up-regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas-sensitive Jurkat cell line by activated T cells. Furthermore, TGF-beta had no direct effect on levels of mRNA for members of the bcl family (bcl-X, bfl-1, bik, bak, bax, bcl-2 and mcl-1). These findings suggest that TGF-beta does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin-2, which can 'rescue' lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro-apoptotic effects of TGF-beta on post-activated T cells, thus demonstrating that TGF-beta increases the cytokine-dependence of T cells for survival. We propose a novel role for TGF-beta in the suppression of inflammation by promoting the elimination of post-activated T cells once the initiating stimulus has been resolved.

Dynamic modeling and optimal fed-batch feeding strategies for a two-phase partitioning bioreactor.

A dynamic model for the degradation of phenol in a two-phase partitioning bioreactor has been developed based on mechanistic balances around the bioreactor. The key process characteristics including substrate transfer between the organic and aqueous phases, substrate inhibition, oxygen limitation, and cell entrainment were incorporated into the model. The model predictions were validated against existing experimental data obtained for a 2-L bioreactor, and good correlation was observed for the time frames of the simulations, as well as for trends in cell and substrate concentrations. Optimal fed-batch, phenol feeding strategies were then developed based on two approaches: (1) maximization of phenol consumption in a fixed time interval and (2) consumption of a fixed amount of phenol in minimal time. The optimal feeding policies, determined using the Iterative Dynamic Programming algorithm, provided substantial improvements in the amount of phenol consumed when compared to a typical experimental heuristic approach. For example, 45.73 g of phenol was predicted to be consumed in 50 h (not including lag phase) using the optimal feeding profile compared to 10.26 g of phenol consumed in the simulated experimental approach. Oxygen limitation was predicted to be a recurring operational challenge in the partitioning bioreactor, and had a strong impact on the optimization results.

Inhibition of T cell activation by normal human biliary epithelial cells.

BACKGROUND/AIMS: Human intrahepatic biliary epithelial cells can express immune recognition elements and are targets for immune attack in several liver pathologies. The aim of this study was to investigate the ability of biliary epithelial cells to act as accessory cells for T cell activation in normal and inflammatory conditions. METHODS: Normal biliary epithelial cells were cocultured with allogeneic unstimulated and mitogen- or antigen-stimulated peripheral blood lymphocytes. T cell responses were assessed by flow cytometry. RESULTS: Biliary epithelial cells did not induce allostimulation in resting T cells and inhibited T cell activation in response to either phytohaemagglutinin, mitogenic anti-CD3 antibody or recall antigen, irrespective of the presence of accessory cells. Biliary epithelial cells did not affect T cell viability, promote or inhibit activation-induced apoptosis nor modulate expression of CD95/Fas. In presence of biliary epithelial cells, stimulated T cells failed to develop an antigen-committed (CD45R0hi) phenotype and were unresponsive to subsequent CD3 ligation. However, T cells underwent normal activation in the presence of biliary epithelial cells which had been pre-treated with Interferon gamma or TGFbeta, cytokines implicated in liver disease. CONCLUSIONS: In normal liver, biliary epithelial cells inhibit rather than promote T cell activation, but their anergising effects may be overcome in response to trauma.

Expression of CD44 on bile ducts in primary sclerosing cholangitis and primary biliary cirrhosis.

AIM: To examine expression of CD44, a transmembrane glycoprotein involved in lymphocyte homing and activation, in inflammatory liver diseases. METHODS: Formalin fixed, paraffin embedded tissues were obtained from normal, uninvolved liver from patients undergoing partial hepatectomy for metastatic carcinoma (9) and transplant hepatectomy specimens from patients with primary biliary cirrhosis (12), primary sclerosing cholangitis (8), autoimmune hepatitis (3), hepatitis C (3), and secondary sclerosing cholangitis (1). Expression of CD44 (using antibodies to three core epitopes), HLA-DR, and lymphocyte phenotypic markers was studied by immunohistochemistry. RESULTS: CD44 expression was not detected in either hepatocytes or biliary epithelial cells in normal livers. In sections from all 27 transplant hepatectomy specimens, CD44 was positive in bile duct epithelial cells but not in hepatocytes. The proportion of CD44+ ducts was much higher in biliary disease than in chronic hepatitis. By contrast, expression of HLA-DR was detected in a relatively small percentage of bile ducts. Activated, memory phenotype CD4+ T lymphocytes were increased in the parenchyma of all diseased livers and an infiltrate of activated CD8+ cells within the biliary epithelium was evident in inflammatory biliary disease. CONCLUSIONS: CD44 appears to play an important role in the development of autoimmune biliary disease by promoting lymphoepithelial interactions, whereas HLA-DR may be involved in the subsequent progression of these conditions.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

BACKGROUND/AIMS: Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. METHODS: Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. RESULTS: All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. CONCLUSIONS: These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production.

Examination of a wide range of inbred mice of diverse genetic backgrounds and major histocompatibility complex haplotypes revealed a dramatic difference in the susceptibilities of males and females to Toxoplasma gondii infection. Female mice were found to be more susceptible to acute infection, as determined by higher mortality levels, than male mice, while those female mice surviving to have chronic infections harbored more cysts in their brains than did surviving males. This phenomenon was therefore investigated in greater depth immunologically in the BALB/K mouse, a strain showing moderate susceptibility to infection with T. gondii. Plasma tumor necrosis factor alpha (TNF-alpha) levels were elevated in both male and female BALB/K mice on days 8 and 10 postinfection, but not thereafter, with males producing significantly higher levels than females. However, it was not until day 12 postinfection that the first deaths occurred, and these were among female mice, indicating that TNF-alpha production was not responsible for mortality. In vitro examination of T. gondii-specific T-cell proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice than in their female counterparts. This difference was most apparent in splenocyte cultures initiated at day 15 postinfection, where complete suppression of proliferation was noted in the splenocytes from female mice but not from male mice. Analysis of tissue culture supernatants from these cultures revealed distinct differences in the kinetics of production as well as the quantities of gamma interferon (IFN-gamma) and interleukin 10 (IL-10) produced. Spleen cells from male mice produced higher levels of IFN-gamma in the early stages of infection than those from female mice. IFN-gamma levels were highest in the supernatants from male splenocyte cultures initiated at day 15 postinfection. Similar levels of IFN-gamma were not obtained from the supernatants of female splenocyte cultures until day 22 postinfection. IL-10 production, on the other hand, peaked at maximal levels in the cell cultures from both sexes initiated at day 22 postinfection. These results suggest that, in male mice, a rapid response to infection with high levels of TNF-alpha and IFN-gamma helps to control parasite multiplication, after which IL-10 production may be important in down regulating these potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms of T-cell proliferation and IFN-gamma production compared with their male counterparts may account for their poor survival rates and higher cyst burdens.