Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin.

Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α-helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF-hand protein family, a coiled-coil dimer-forming region and a carboxyl-terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c-heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c-HMM co-produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin-activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of -18.44 kcal/mol, inhibits the motor function of Myo5c with a half-maximal concentration of 280 nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin-producing ß-cells located within the pancreatic islets of Langerhans.

Potentiation of Lipotoxicity in Human EndoC-ßH1 ß-Cells by Glucose is Dependent on the Structure of Free Fatty Acids.

SCOPE: Lipotoxicity is a significant element in the development of type 2 diabetes mellitus (T2DM). Since pro-diabetic nutritional patterns are associated with hyperglycemia as well as hyperlipidemia, the study analyzes the effects of combining these lipid and carbohydrate components with a special focus on the structural fatty acid properties such as increasing chain length (C16-C20) and degree of saturation with regard to the role of glucolipotoxicity in human EndoC-ßH1 ß-cells. METHODS AND RESULTS: ß-cell death induced by saturated FFAs is potentiated by high concentrations of glucose in a chain length-dependent manner starting with stearic acid (C18:0), whereas toxicity remains unchanged in the case of monounsaturated FFAs. Interference with FFA desaturation by overexpression and inhibition of stearoyl-CoA-desaturase, which catalyzes the rate-limiting step in the conversion of long-chain saturated into corresponding monounsaturated FFAs, does not affect the potentiating effect of glucose, but FFA desaturation reduces lipotoxicity and plays an important role in the formation of lipid droplets. Crucial elements underlying glucolipotoxicity are ER stress induction and cardiolipin peroxidation in the mitochondria. CONCLUSION: In the context of nutrition, the data emphasize the importance of the lipid component in glucolipotoxicity related to the development of ß-cell dysfunction and death in the manifestation of T2DM.

Advanced Glycation End-Products (AGEs) of Lysine and Effects of Anti-TCR/Anti-TNF-α Antibody-Based Therapy in the LEW.1AR1-iddm Rat, an Animal Model of Human Type 1 Diabetes.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1-iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the Nε-monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites Nε-carboxymethyl lysine (CML), furosine and Nε-monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine (p = 0.0289) and free CML (p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine (p = 0.0076) and CML (p = 0.0270). In the spleen, group differences were found for proteinic furosine (p = 0.0114) and free furosine (p = 0.0368), as well as for proteinic CML (p = 0.0502) and proteinic MML (p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen (r = -0.8271, p < 0.0001) compared to pancreas (r = 0.5805, p = 0.0073) and kidney (r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups.

New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells.

Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells.

The importance of aquaporin-8 for cytokine-mediated toxicity in rat insulin-producing cells.

Aquaporin-8 (AQP8) is a peroxiporin, a transmembrane water and hydrogen peroxide (H2O2) transport protein expressed in the mitochondrial and plasma membranes of pancreatic ß-cells. AQP8 protein expression is low under physiological conditions, but it increases after cytokine exposure both, in vitro and in vivo, possibly related to a NF-κB consensus sequence in the promoter. AQP8 knockdown (KD) insulin-producing RINm5F cells are particularly susceptible to cytokine-mediated oxidative stress. Cytokine (a mixture of IL-1ß, TNF-α, and IFN-γ) treated AQP8 KD cells exhibited pronounced sensitivity to reactive oxygen and nitrogen species (ROS and RNS), resulting in a significant loss of ß-cell viability due to enhanced toxicity of the increased concentrations of H2O2 and hydroxyl radicals (●OH) in mitochondria of AQP8 KD cells. This viability loss went along with increased caspase activities, reduced nitrite concentration (representative of nitric oxide (NO●) accumulation) and increased lipid peroxidation. The explanation for the increased toxicity of the proinflammatory cytokines in AQP8 KD cells resides in the fact that efflux of the H2O2 generated during oxidative stress in the ß-cell mitochondria is hampered through the loss of the peroxiporin channels in the mitochondrial membranes of the AQP8 KD cells. The increased proinflammatory cytokine toxicity due to loss of AQP8 expression in the KD ß-cell mitochondria is thus the result of increased rates of apoptosis. This decreased cell viability is caused by increased levels of oxidative stress along with a ferroptosis-mediated cell death component due to decreased NO● generation.

MCPIP1 is a novel link between diabetogenic conditions and impaired insulin secretory capacity.

During diabetes development insulin production and glucose-stimulated insulin secretion (GSIS) are defective due to inflammation-related, yet not fully understood mechanisms. MCPIP1 (monocyte chemotactic protein-induced protein-1) is a strong regulator of inflammation, and acts predominantly as a specific RNase. The impact of MCPIP1 on insulin secretory capacity is unknown. We show that the expression of the ZC3H12A gene, which encodes MCPIP1, was induced by T1DM- and by T2DM-simulating conditions, with a stronger effect of cytokines. The number of MCPIP1-positive pancreatic islet-cells, including beta-cells, was significantly higher in diabetic compared to nondiabetic individuals. In the 3'UTR regions of mRNAs coding for Pdx1 (pancreatic and duodenal homeobox 1), FoxO1 (forkhead box protein O1), and of a novel regulator of insulin handling, Grp94 (glucose-regulated protein 94), MCPIP1-target structures were detected. Overexpression of the wild type MCPIP1wt, but not of the mutant MCPIP1D141N (lacking the RNase activity), decreased the expression of genes involved in insulin production and GSIS. Additionally INS1-E-MCPIP1wt cells exhibited a higher Ire1 (inositol-requiring enzyme 1) expression. MCPIP1wt overexpression blunted GSIS and glucose-mediated calcium influx with no deleterious effects on glucose uptake or glucokinase activity. We identify MCPIP1 as a new common link between diabetogenic conditions and beta-cell failure. MCPIP1 may serve as an interesting target for novel beta-cell protective approaches.

The central role of glutathione peroxidase 4 in the regulation of ferroptosis and its implications for pro-inflammatory cytokine-mediated beta-cell death.

Pro-inflammatory cytokines are crucial mediators of beta-cell destruction in type 1 diabetes mellitus (T1DM). The involvement of ferroptosis as a form of oxidative non-apoptotic cell death in T1DM pathogenesis has not been elucidated so far. Moreover, the role of glutathione peroxidase 4 (GPx4) as an antioxidative enzyme and a major regulator of ferroptosis remains elusive. Assessment of GPx4 expression in different pancreatic islet cell types revealed a predominant expression in beta-cells. Silencing of GPx4 by RNA interference and exposure to tert-butyl hydroperoxide (tert-BHP) caused ferroptosis in rat pancreatic beta-cells as evidenced by non-apoptotic cell death in association with increased lipid peroxidation, disturbed ATP synthesis, reduced GSH content, and GPx4 degradation. GPx4 overexpression as well as the ferroptosis inhibitor ferrostatin-1 effectively attenuated beta-cell death induced by tert-BHP. Notably, beta-cell toxic cytokines did not induce ferroptosis although beta-cells underwent cell death. Inhibition of iNOS by Nω-nitro-L-arginine however led to a massive lipid peroxidation upon exposure to pro-inflammatory cytokines. Hence, nitric oxide produced during pro-inflammatory cytokine action prevents the induction of ferroptosis, thereby favouring apoptosis as a primary cell death mechanism. The extraordinarily high abundance of the phospholipid hydroperoxidase GPx4 in beta-cells in contrast to the very low expression in other islet cell types points to a susceptibility of beta-cells to the accumulation of toxic lipid peroxides. Overall, these data strongly suggest that GPx4 is indispensable for beta-cell function under physiological conditions. On the other hand, our results exclude an involvement of ferroptosis as an alternative beta-cell death mode under pro-inflammatory cytokine attack.

Translation of curative therapy concepts with T cell and cytokine antibody combinations for type 1 diabetes reversal in the IDDM rat.

Proinflammatory cytokines released from the pancreatic islet immune cell infiltrate in type 1 diabetes (T1D) cause insulinopenia as a result of severe beta cell loss due to apoptosis. Diabetes prevention strategies targeting different cytokines with antibodies in combination with a T cell antibody, anti-TCR, have been assessed for therapy success in the LEW.1AR1-iddm (IDDM) rat, an animal model of human T1D. Immediately after diabetes manifestation, antibody combination therapies were initiated over 5 days with anti-TNF-α (tumour necrosis factor), anti-IL-1ß (interleukin), or anti-IFN-γ (interferon) together with anti-TCR for the reversal of the diabetic metabolic state in the IDDM rat. Anti-TCR alone showed only a very limited therapy success with respect to a reduction of immune cell infiltration and beta cell mass regeneration. Anti-TCR combinations with anti-IL-1ß or anti-IFN-γ were also not able to abolish the increased beta cell apoptosis rate and the activated immune cell infiltrate leading to a permanent beta cell loss. In contrast, all anti-TCR combinations with anti-TNF-α provided sustained therapy success over 60 to 360 days. The triple combination of anti-TCR with anti-TNF-α plus anti-IL-1ß was most effective in regaining sustained normoglycaemia with an intact islet structure in a completely infiltration-free pancreas and with a normal beta cell mass. Besides the triple combination, the double antibody combination of anti-TCR with anti-TNF-α proved to be the most suited therapy for reversal of the T1D metabolic state due to effective beta cell regeneration in an infiltration free pancreas. KEY MESSAGES: Anti-TCR is a cornerstone in combination therapy for autoimmune diabetes reversal. The combination of anti-TCR with anti-TNF-α was most effective in reversing islet immune cell infiltration. Anti-TCR combined with anti-IL-1ß was not effective in this respect. The combination of anti-TCR with anti-TNF-α showed a sustained effect over 1 year.

Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.

Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein­coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10-12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in ß-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.

Rat Models of Human Type 1 Diabetes.

Rat models of human type 1 diabetes have been shown to be of great importance for the elucidation of the mechanisms underlying the development of autoimmune diabetes. The three major well-established spontaneous rat models are the BioBreeding (BB) diabetes-prone rat, the Komeda diabetes-prone (KDP) rat, and the IDDM (LEW.1AR1-iddm) rat. Their distinctive features are described with special reference to their pathology, immunology, and genetics and compared with the situation in patients with type 1 diabetes mellitus. For all three established rat models, a distinctive genetic mutation has been identified that is responsible for the manifestation of the diabetic syndrome in these rat strains.

Remission of autoimmune diabetes by anti-TCR combination therapies with anti-IL-17A or/and anti-IL-6 in the IDDM rat model of type 1 diabetes.

BACKGROUND: The cytokine IL-17 is a key player in autoimmune processes, while the cytokine IL-6 is responsible for the chronification of inflammation. However, their roles in type 1 diabetes development are still unknown. METHODS: Therefore, therapies for 5 days with anti-IL-17A or anti-IL-6 in combination with a T cell-specific antibody, anti-TCR, or in a triple combination were initiated immediately after disease manifestation to reverse the diabetic metabolic state in the LEW.1AR1-iddm (IDDM) rat, a model of human type 1 diabetes. RESULTS: Monotherapies with anti-IL-6 or anti-IL-17 showed no sustained anti-diabetic effects. Only the combination therapy of anti-TCR with anti-IL-6 or anti-IL-17 at starting blood glucose concentrations up to 12 mmol/l restored normoglycaemia. The triple antibody combination therapy was effective even up to very high initial blood glucose concentrations (17 mmol/l). The ß cell mass was raised to values of around 6 mg corresponding to those of normoglycaemic controls. In parallel, the apoptosis rate of ß cells was reduced and the proliferation rate increased as well as the islet immune cell infiltrate was strongly reduced in double and abolished in triple combination therapies. CONCLUSIONS: The anti-TCR combination therapy with anti-IL-17 preferentially raised the ß cell mass as a result of ß cell proliferation while anti-IL-6 strongly reduced ß cell apoptosis and the islet immune cell infiltrate with a modest increase of the ß cell mass only. The triple combination therapy achieved both goals in a complimentary anti-autoimmune and anti-inflammatory action resulting in sustained normoglycaemia with normalized serum C-peptide concentrations.

Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a LADA Rat Model Compared With Type 1 Diabetes.

Approximately 10% of patients with type 2 diabetes suffer from latent autoimmune diabetes in adults (LADA). This study provides a systematic assessment of the pathology of the endocrine pancreas of patients with LADA and for comparison in a first rat model mimicking the characteristics of patients with LADA. Islets in human and rat pancreases were analyzed by immunohistochemistry for immune cell infiltrate composition, by in situ RT-PCR and quantitative real-time PCR of laser microdissected islets for gene expression of proinflammatory cytokines, the proliferation marker proliferating cell nuclear antigen (PCNA), the anti-inflammatory cytokine interleukin (IL) 10, and the apoptosis markers caspase 3 and TUNEL as well as insulin. Human and rat LADA pancreases showed differences in areas of the pancreas with respect to immune cell infiltration and a changed ratio between the number of macrophages and CD8 T cells toward macrophages in the islet infiltrate. Gene expression analyses revealed a changed ratio due to an increase of IL-1ß and a decrease of tumor necrosis factor-α. IL-10, PCNA, and insulin expression were increased in the LADA situation, whereas caspase 3 gene expression was reduced. The analyses into the underlying pathology in human as well as rat LADA pancreases provided identical results, allowing the conclusion that LADA is a milder form of autoimmune diabetes in patients of an advanced age.

Asymmetric dimethylation and citrullination in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, and effects of anti-TCR/anti-TNF-α antibody-based therapy.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.

MCPIP1 regulates the sensitivity of pancreatic beta-cells to cytokine toxicity.

The autoimmune-mediated beta-cell death in type 1 diabetes (T1DM) is associated with local inflammation (insulitis). We examined the role of MCPIP1 (monocyte chemotactic protein-induced protein 1), a novel cytokine-induced antiinflammatory protein, in this process. Basal MCPIP1 expression was lower in rat vs. human islets and beta-cells. Proinflammatory cytokines stimulated MCPIP1 expression in rat and human islets and in insulin-secreting cells. Moderate overexpression of MCPIP1 protected insulin-secreting INS1E cells against cytokine toxicity by a mechanism dependent on the presence of the PIN/DUB domain in MCPIP1. It also reduced cytokine-induced Chop and C/ebpß expression and maintained MCL-1 expression. The shRNA-mediated suppression of MCPIP1 led to the potentiation of cytokine-mediated NFκB activation and cytokine toxicity in human EndoC-ßH1 beta-cells. MCPIP1 expression was very high in infiltrated beta-cells before and after diabetes manifestation in the LEW.1AR1-iddm rat model of human T1DM. The extremely high expression of MCPIP1 in clonal beta-cells was associated with a failure of the regulatory feedback-loop mechanism, ER stress induction and high cytokine toxicity. In conclusion, our data indicate that the expression level of MCPIP1 affects the susceptibility of insulin-secreting cells to cytokines and regulates the mechanism of beta-cell death in T1DM.

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation.

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.

ß-Cell DNA Damage Response Promotes Islet Inflammation in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic ß-cells are destroyed by islet-infiltrating T cells. Although a role for ß-cell defects has been suspected, ß-cell abnormalities are difficult to demonstrate. We show a ß-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The ß-cell DDR is more frequent in islets infiltrated by CD45+ immune cells, suggesting a link to islet inflammation. The ß-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1ß and Cxcl10. ß-Cell-specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that ß-cell DDR is an early event in T1D, possibly contributing to autoimmunity.

Changes in immune cell frequencies in primary and secondary lymphatic organs of LEW.1AR1-iddm rats, a model of human type 1 diabetes compared to other MHC congenic LEW inbred strains.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes, which arose through a spontaneous mutation in the Dock8 gene within the MHC congenic background strain LEW.1AR1. This mutation not only mediates diabetes development but also leads to a variable T cell frequency in peripheral blood. In this study, the immune cell frequencies of primary and secondary lymphatic organs of LEW.1AR1-iddm rats were analysed at days 40 and 60 and compared to other MHC congenic LEW rat strains. In LEW.1AR1-iddm rats, the secondary lymphatic organs such as lymph nodes and spleen showed a reduced, around 15% in comparison to all other strains, but very variable T cell frequency, mirroring the fluctuating T cell content in blood. On the other hand, the frequency of B cells was increased by 10% in the lymph nodes and by 5% in the spleen. Thus, the decreasing number of T cells in blood could not be caused by an increase of T cells in secondary lymphatic organs. The frequency of single- or double-positive T cells in the thymus was unaffected. The T cell frequencies in the other analysed strains were more stable and mostly higher in all secondary lymphatic organs. Obviously, the Dock8 mutation leads to variabilities of T cell frequencies in blood as well as in secondary lymphatic organs. In conclusion, the Dock8 mutation was responsible for changed immune cell frequencies in different compartments and together with the RT1B/Du haplotype causing immune imbalances and development of autoimmune diabetes.

Restoration of mucosal integrity and epithelial transport function by concomitant anti-TNFα treatment in chronic DSS-induced colitis.

Impaired salt and water absorption is a hallmark of diarrhea in IBD. In the present study, the therapeutic effect of continuous anti-TNFα treatment on the progression of inflammation and colonic transport dysfunction during chronic dextran sulfate sodium (DSS)-induced colitis was investigated. Chronic colitis was induced by three DSS exposure cycles. Mice received TNFα monoclonal antibody treatment twice weekly after the end of the first 5-day DSS drinking period. Mice developed chronic DSS-induced colitis characterized by a typical immune cell infiltration composed of CD3+ T cells and CD68+ macrophages, both expressing high levels of the pro-inflammatory cytokines IL-1ß and TNFα, a loss of NHE3 and PDZK1 in the brush border region of the absorptive enterocyte and a decrease of colonic fluid absorption in vivo, measured by colonic single pass perfusion. Concomitant anti-TNFα treatment resulted in a significant reduction of mucosal immune cell infiltration and expression of the pro-inflammatory cytokines IL-1ß and TNFα. It also resulted in a normalization of NHE3-mediated fluid absorption and a restoration of NHE3 and PDZK1 location in the apical and subapical region of the enterocytes. Here, we show for the first time that in this chemically induced murine colitis model, anti-TNFα treatment significantly decreased inflammatory activity, improved mucosal integrity and restored transport function despite an ongoing inflammatory insult. Anti-TNFα treatment may therefore be beneficial in patients with IBD even in spite of an absence of complete mucosal healing. KEY MESSAGES: Chronic DSS treatment caused a loss of NHE3 and PDZK1 in the brush border region of the absorptive enterocyte and decreases colonic fluid absorption. In DSS-induced colitis, anti-TNFα treatment reduced mucosal immune cell infiltration and expression of the pro-inflammatory cytokines IL-1ß and TNFα. In DSS-induced colitis, anti-TNFα treatment normalized NHE3-mediated fluid absorption and restored NHE3 and PDZK1 location in the enterocytes. In DSS-induced colitis, anti-TNFα treatment decreased inflammatory activity, improved mucosal integrity, and restored transport function.

Enhanced activation of interleukin-10, heme oxygenase-1, and AKT in C5aR2-deficient mice is associated with protection from ischemia reperfusion injury-induced inflammation and fibrosis.

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.