Catharina Alam

Keratins in health and disease

The cytoprotective keratins (K) compose the intermediate filaments of epithelial cells and their inherited and spontaneous mutations give rise to keratinopathies. For example, mutations in K1/K5/K10/K14 cause epidermal skin diseases whereas simple epithelial K8/K18/K19 variants predispose to development of several liver disorders. Due to their abundance, tissue- and context-specific expression, keratins constitute excellent diagnostic markers of both neoplastic and non-neoplastic diseases. During injury and in disease, keratin expression levels, cellular localization or posttranslational modifications are altered. Accumulating evidence suggests that these changes modulate multiple processes including cell migration, tumor growth/metastasis and development of infections. Therefore, our understanding of keratins is shifting from diagnostic markers to active disease modifiers.

Effects of a germ-free environment on gut immune regulation and diabetes progression in non-obese diabetic (NOD) mice

Aims/hypothesisMicrobial factors influence the development of diabetes in NOD mice. Studies in germ-free animals have revealed important roles of microbiota in the regulation of Th17 and forkhead box P3 (FOXP3)+ T regulatory (Treg) activation in the intestine. However, the effects of intestinal microbiota in immune regulation and diabetes development in NOD mice are still poorly understood.MethodsA colony of germ-free NOD mice was established to evaluate the effects of intestinal microbiota on regulatory immunity in the gut, and on the development of insulitis and diabetes in NOD mice.ResultsDiabetes developed in roughly equal numbers in germ-free and specific pathogen-free NOD mice. Insulitis was accentuated in germ-free NOD mice; yet insulin preservation was unaltered. Germ-free NOD mice showed increased levels of Il17 (also known as Il17a) mRNA in the colon, and of Th17 and Th1 cells in the mesenteric and pancreatic lymph nodes, while Foxp3 mRNA and FOXP3+ Tregs were reduced. In the islet infiltrates, FOXP3+CD4+ T cells were slightly increased in germ-free mice. B cells appeared less activated in the peritoneum and were less abundant in islet infiltrates.Conclusions/interpretationThese results indicate that lack of intestinal microbiota promotes an imbalance between Th1, Th17 and Treg differentiation in the intestine. This imbalance is associated with accelerated insulitis, but intact recruitment of FOXP3+ Tregs into islets, suggesting: (1) a microbial dependence of local induction of Treg in the gut and draining lymph nodes; but (2) a potentially compensatory function of naturally occurring Tregs in the islets, which may help control diabetogenic T cells.

Inflammatory tendencies and overproduction of IL-17 in the colon of young NOD mice are counteracted with diet change.

OBJECTIVE Dietary factors influence diabetes development in the NOD mouse. Diet affects the composition of microbiota in the distal intestine, which may subsequently influence intestinal immune homeostasis. However, the specific effects of antidiabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear. RESEARCH DESIGN AND METHODS Gut microbiota of NOD mice fed a conventional diet or ProSobee formula were compared using gas chromatography. Colonic lamina propria immune cells were characterized in terms of activation markers, cytokine mRNA and Th17 and Foxp3+ T-cell numbers, using real-time PCR and flow cytometry. Activation of diabetogenic CD4 T-cells by purified B-cells was assessed in both groups. Immune tolerance to autologous commensal bacteria was evaluated in vitro using thymidine-incorporation tests. RESULTS Young NOD mice showed a disturbed tolerance to autologous commensal bacteria. Increased numbers of activated CD4 T-cells and (CD11b+CD11c+) dendritic cells and elevated levels of Th17 cells and IL23 mRNA were moreover observed in colon lamina propria. These phenomena were abolished when mice were fed an antidiabetogenic diet. The antidiabetogenic diet also altered the expression levels of costimulatory molecules and the capacity of peritoneal B-cells to induce insulin-specific CD4 T-cell proliferation. CONCLUSIONS Young NOD mice show signs of subclinical colitis, but the symptoms are alleviated by a diet change to an antidiabetogenic diet. Disrupted immune tolerance in the distal intestine may influence peritoneal cell pools and B-cell–mediated activation of diabetogenic T-cells.

Ly6C supports preferential homing of central memory CD8(+) T cells into lymph nodes

Ly6C is a murine cell‐surface antigen expressed by plasma cells, subsets of myeloid cells and many T cells, including memory T cells. We previously documented that Ly6C crosslinking induces LFA‐1 clustering on naïve CD8+ T cells. Here, we show that in vitro and in vivo differentiation of naïve CD8+ T cells into central (Tcm) but not effector (Tem) memory T cells enhances Ly6C expression, and its crosslinking induces strong LFA‐1 clustering on Tcm. Blocking Ly6C function inhibits in vivo Tcm homing to LNs as efficiently as blocking L‐selectin but it does not potentiate the inhibition provided by blocking either L‐selectin or LFA‐1 function. Thus, Ly6C, L‐selectin and LFA‐1 all appear to be part of a common homing pathway. In vitro, Ly6C crosslinking enhances Tcm adherence to ICAM‐1 in the presence of CCL21. In summary, Tcm homing involves Ly6C, in addition to L‐selectin and LFA‐1, and appears to potentiate firm adhesion of Tcm to ICAM‐1 in synergy with a chemokine. We propose that Ly6C augments Tcm compartmentalization into LNs during their homing.

Keratin 8 modulates β-cell stress responses and normoglycaemia.

Summary Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on &bgr;-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8+/+) and K8 knockout (K8−/−) mice. Islet &bgr;-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8+/+ and K8−/− mice. K8 and K18 were the predominant keratins in islet &bgr;-cells and K8−/− mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8−/− &bgr;-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8−/− mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8−/− mice. &bgr;-cell keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8+/+ mice and in diabetic NOD mice, suggesting a role for keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for keratins in &bgr;-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.

Enhanced trafficking to the pancreatic lymph nodes and auto-antigen presentation capacity distinguishes peritoneal B lymphocytes in non-obese diabetic mice

Aims/hypothesisNOD.Igµnull mice lacking mature B cells are highly resistant to diabetes and display poor CD4 T cell responses to autoantigens. Nevertheless, the degree to which different B cell subsets contribute to diabetes in NOD mice remains unresolved. Due to their role in the recognition of microbial and autoantigens, peritoneal B cell characteristics were examined in NOD mice to see if they differ developmentally, phenotypically or functionally in aspects relevant to diabetogenesis.MethodsThe population dynamics, activation state, migratory behaviour and antigen presentation function were investigated in NOD peritoneal B cells.ResultsNOD peritoneal B cells were found to express abnormally high levels of co-stimulatory molecules (CD40, CD86 and CD69). In contrast, the expression of l-selectin and integrin α4β1 was markedly reduced in NOD mice compared with BALB/c and C57BL/6 mice. The number of B cells in the peritoneum was lower in NOD than in control mice throughout development; migration of B cells from the peritoneum to the pancreatic lymph nodes in NOD mice was enhanced tenfold. NOD B cells showed no chemotactic response to sphingosine-1-phosphate, which normally acts to retain B cells in the peritoneum. Peritoneal B cells of NOD mice also presented insulin autoantigen to CD4 T cells, inducing T cell proliferation.Conclusions/interpretationNOD peritoneal B cells are hyperactivated, migrate to the pancreatic lymph nodes and are capable of driving insulin-specific CD4 T cell activation. These characteristics could make them important for inducing or amplifying T cell responses against islet-antigens.

Bacterial cellulose–kaolin nanocomposites for application as biomedical wound healing materials

This short communication provides preliminary experimental details on the structure–property relationships of novel biomedical kaolin–bacterial cellulose nanocomposites. Bacterial cellulose is an effective binding agent for kaolin particles forming reticulated structures at kaolin–cellulose interfaces and entanglements when the cellulose fraction is sufficiently high. The mechanical performance of these materials hence improves with an increased fraction of bacterial cellulose, though this also causes the rate of blood clotting to decrease. These composites have combined potential as both short-term (kaolin) and long-term (bacterial cellulose) wound healing materials.

The Amount of Keratins Matters for Stress Protection of the Colonic Epithelium

Keratins (K) are important for epithelial stress protection as evidenced by keratin mutations predisposing to human liver diseases and possibly inflammatory bowel diseases. A role for K8 in the colon is supported by the ulcerative colitis-phenotype with epithelial hyperproliferation and abnormal ion transport in K8-knockout (K8−/−) mice. The heterozygote knockout (K8+/−) colon appears normal but displays a partial ion transport-defect. Characterizing the colonic phenotype we show that K8+/− colon expresses ~50% less keratins compared to K8 wild type (K8+/+) but de novo K7 expression is observed in the top-most cells of the K8+/− and K8−/− crypts. The K8+/− colonic crypts are significantly longer due to increased epithelial hyperproliferation, but display no defects in apoptosis or inflammation in contrast to K8−/−. When exposed to colitis using the dextran sulphate sodium-model, K8+/− mice showed higher disease sensitivity and delayed recovery compared to K8+/+ littermates. Therefore, the K8+/− mild colonic phenotype correlates with decreased keratin levels and increased sensitivity to experimental colitis, suggesting that a sufficient amount of keratin is needed for efficient stress protection in the colonic epithelia.

In Vivo Imaging of Reactive Oxygen and Nitrogen Species in Murine Colitis

Background:Traditional techniques analyzing mouse colitis are invasive, laborious, or indirect. Development of in vivo imaging techniques for specific colitis processes would be useful for monitoring disease progression and/or treatment effectiveness. The aim was to evaluate the applicability of the chemiluminescent probe L-012, which detects reactive oxygen and nitrogen species, for in vivo colitis imaging. Methods:Two genetic colitis mouse models were used; K8 knockout (K8−/−) mice, which develop early colitis and the nonobese diabetic mice, which develop a transient subclinical colitis. Dextran sulphate sodium was used as a chemical colitis model. Mice were anesthetized, injected intraperitoneally with L-012, imaged, and quantified for chemiluminescent signal in the abdominal region using an IVIS camera system. Results:K8−/− and nonobese diabetic mice showed increased L-012-mediated chemiluminescence from the abdominal region compared with control mice. L-012 signals correlated with the colitis phenotype assessed by histology and myeloperoxidase staining. Although L-012 chemiluminescence enabled detection of dextran sulphate sodium–induced colitis at an earlier time point compared with traditional methods, large mouse-to-mouse variations were noted. In situ and ex vivo L-012 imaging as well as [18F]FDG-PET imaging of K8−/− mice confirmed that the in vivo signals originated from the distal colon. L-012 in vivo imaging showed a wide variation in reactive oxygen and nitrogen species in young mice, irrespective of K8 genotype. In aging mice L-012 signals were consistently higher in K8−/− as compared to K8+/+ mice. Conclusions:In vivo imaging using L-012 is a useful, simple, and cost-effective tool to study the level and longitudinal progression of genetic and possibly chemical murine colitis.

Peritoneal cavity is a route for gut-derived microbial signals to promote autoimmunity in non-obese diabetic mice.

Macrophages play a crucial role in innate immune reactions, and peritoneal macrophages (PMs) guard the sterility of this compartment mainly against microbial threat from the gut. Type 1 diabetes (T1D) is an autoimmune disease in which gut microbiota and gut immune system appear to contribute to disease pathogenesis. We have recently reported elevated free radical production and increased permeability of gut epithelium in non‐obese diabetic (NOD) mice. Impaired barrier function could lead to bacterial leakage to the peritoneal cavity. To explore the consequences of impaired gut barrier function on extra‐intestinal immune regulation, we characterized peritoneal lavage cells from young newly weaned NOD mice. We detected a rapid increase in the number of macrophages 1–2 weeks after weaning in NOD mice compared to C57BL/6 and BALB/c mice. Interestingly, this increase in macrophages was abrogated in NOD mice that were fed an antidiabetogenic diet (ProSobee), which improves gut barrier function. Macrophages in young (5‐week‐old) NOD mice displayed a poor TNF‐α cytokine response to LPS stimulation and high expression of interleukin‐1receptor‐associated kinase‐M (IRAK‐M), indicating prior in vivo exposure to TLR‐4 ligand(s). Furthermore, injection of LPS intraperitoneally increased T cell CD69 expression in pancreatic lymph node (PaLN), suggestive of T cell activation. Leakage of bacterial components such as endotoxins into the peritoneal cavity may contribute to auto‐reactive T cell activation in the PaLN.