DataSheet_1_TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.docx

IntroductionThe incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D.MethodsTo fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study.ResultsWe found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice.DiscussionIn summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.</p

Characteristics of Control Corneas.

<p>Characteristics of Control Corneas.</p

Expression of SFRP Family Proteins in Human Keratoconus Corneas

<div><p>We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.</p></div

Surveyed rivers in distribution areas of Scaly-sided Merganser.

<p>From left to right are Li River, Yuan River, Xiu River, Fu River, Xin River, Rao River.</p

Fig 5 -

NAD+ (a) and NADH (b) concentrations, and the ratio of NADH/NAD+ (c) in skeletal muscle after the intervention. The error bars represent SD. * p < 0.05. NC represents control mice, sedentary in normoxic environment; DC, diabetic control mice, sedentary in normoxic environment; DE, diabetic mice, 4 weeks of exercise in normoxic environment; DH, diabetic mice, sedentary for 4 weeks in hypoxic environment; and DHE, diabetic mice, 4 weeks of exercise in hypoxic environment; DE1, diabetic mice, one hour exposure to exercise in normoxic environment; DH1, diabetic mice, one hour exposure to exercise in hypoxic environment; and DHE1, diabetic mice, one hour exposure to exercise in hypoxic environment. N = 7 in each group.</p

Characteristics of KC Patients.

a<p>Grade 4 KC: Severe; VA &gt;6/7.5 with contact lens correction; severe corneal thinning and Munson’s sign.</p>b<p>DALK: Deep anterior lamellar keratoplasty.</p>c<p>Ectasia developed post-LASIK.</p

SFRP Immunolabelling: Semi-Quantitative Grading^a.

a<p>Semi-quantitative grading = Intensity+% immunolabelling (minimum = 0 and maximum = 6):</p><p><i>Intensity of Immunoreactivity.</i></p><p>0 = no staining.</p><p>0.5 = very weak.</p><p>1 = weak.</p><p>2 = moderate.</p><p>3 = strong.</p><p><i>% Immunolabelling.</i></p><p>0 = 0%.</p><p>1 = 1% to 10%.</p><p>2 = 11% to 50%.</p><p>3 = &gt;50%.</p>b<p>Significance.</p>c<p>SFRP1, 2 and 3 expressed in keratocytes only; SFRP5 expressed in stroma including matrix and cells.</p>d<p>Mann-Whitney test, p&lt;0.05 significant; NS - not significant.</p>e<p>ND - not detected.</p>f<p>KC full thickness graft n = 7/15; deep anterior lamellar keratoplasty (DALK) n = 8/15.</p

Fasting blood glucose (FBG) in skeletal muscle after the intervention.

The error bars represent SD. * p < 0.05. NC represents control mice, sedentary in normoxic environment; DC, diabetic control mice, sedentary in normoxic environment; DE, diabetic mice, 4 weeks of exercise in normoxic environment; DH, diabetic mice, sedentary for 4 weeks in hypoxic environment; and DHE, diabetic mice, 4 weeks of exercise in hypoxic environment; DE1, diabetic mice, one hour exposure to exercise in normoxic environment; DH1, diabetic mice, one hour exposure to exercise in hypoxic environment; and DHE1, diabetic mice, one hour exposure to exercise in hypoxic environment. N = 7 in each group.</p

Graphs summarising semi-quantitative grading for SFRP immunolabelling of KC and control specimens.

<p>The graphs show the range of grading for SFRP1 to 5 immunostaining in the basal epithelium adjacent to the cone region, and a similar region in control corneas. SFRP1 to 4 immunostaining is significantly increased compared to controls (p&lt;0.05, Mann-Whitney test).</p

The data used in statistical analysis of the study.

The data used in statistical analysis of the study.</p