Project description:Hyperglycemia is a hallmark in prediabetes and type 2 diabetes mellitus (T2DM) which increases risk of micro and macrovascular complications such as diabetic retinopathy, diabetic nephropathy (microvascular complications), and peripheral vascular disease, cerebrovascular disease and cardiovascular diseases (macrovascular complications). Endothelial cells are affected in both cases. In this study, we investigated the miRNA expression changes in HUVECs during different glucose treatment (5mM, 10mM, 25mM and 40mM glucose) at various time intervals (6, 12, 24 and 48hrs). The results of miRNA microarray showed there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico prediction showed that the following pathways: Regulation of actin cytoskeleton, PI3K-Akt signaling pathway, Apoptosis, Neurotrophin signaling pathway, and Insulin signaling pathway, were dysregulated during hyperglycemia. Majority of the pathways are related to apoptosis. 10 miRNAs (miR-26a-5p, -26b-5p, 29b-3p, 29c-3p, 125b-1-3p, -130b-3p, - 140-5p, -221-3p, -192-5p, and -320a,) showed increased expression with increasing concentration of glucose treatment. miR-26a-5p, -29b-3p, - 140-5p, -221-3p, and -192-5p are involves in endothelial apoptosis. Our study revealed miRNAs (miR-29b-3p and – 192-5p) with known mRNA targets (BCL2 and MCL1) showed expression pattern inversely correlating with their respective target mRNAs. Therefore these miRNAs could involve in the endothelial dysfunction due to hyperglycemia.
Project description:Cystic Fibrosis Related Diabetes (CFRD), the main co-morbidity in Cystic Fibrosis (CF), is associated with higher rates of lung function decline. We hypothesize that airway epithelial barrier function is impaired in CF and is further exacerbated under hyperglycemia, worsening pulmonary outcomes. Using 16HBE cells as a model cell line, we studied the effects of hyperglycemia in airway epithelial barrier function. Results show increased paracellular dye flux in CF cells in response to insulin treatment under hyperglycemia, suggesting impaired barrier integrity. Gene expression experiments identified Claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells. Further investigation into CLDN4 protein localization by confocal microscopy showed that CLDN4 was tightly localized at tight junctions in WT cells and localization did not change under hyperglycemia. ln contrast, CLDN4 was less well-localized in CF cells at normal glucose and localization was worsened in CF cells conditioned to hyperglycemia. Treatment with highly effective modulator compounds (ETI) reversed this trend, and CFTR rescue by ETI in CF cells was not affected by insulin treatment or hyperglycemia. Bulk RNA sequencing showed differences in transcriptional responses in CF compared to WT cells under normal or high glucose, highlighting PTPRG as a promising target for further investigation.
Project description:Using DropSeq single cell RNA sequencing, we report that neuronal derived-IL-18 is required for goblet cell expression of intestinal antimicrobial protein expression Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the anti-microbial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA-hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron derived IL-18 signaling controls tissue wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Project description:Using RNA sequencing, we report that neuron derived-IL-18 is required for intestinal antimicrobial protein expression Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the anti-microbial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA-hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron derived IL-18 signaling controls tissue wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Project description:Environmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study utilized a novel microarray method to interrogate the host transcriptome in feces in Malawian children with EED. Our data showed that the children studied had a range of %L values, consistent a spectrum of EED from normal to severe. We identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B-cells, and mediators that dampen cellular responses to hormones. EED associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial and parasitic microbes and enhanced phagocytosis. Several mucins, regulatory factors and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than normal children. In conclusion, EED represents the focused activation of elements of the immune system and is associated with widespread intestinal barrier disruption. The differentially expressed transcripts may be explored as potential biomarkers. In 259 children, EED was measured by lactulose permeability (%L) in the small intestine. After isolating low copy numbers of mRNA, the transcriptome was reliably and reproducibly profiled. mRNA copy number was correlated with %L using analyses of covariance. The transcripts identified were mapped to biological pathways and processes.
Project description:In diabetes, the kidney contributes to the development of diabetic hyperglycemia by increasing glucose reabsorption from the primary urine and by upregulating gluconeogenesis in the proximal tubule. However, these two processes are also controlled by the circadian clock, a mechanism that synchronizes a large number of specific renal functions with environmental daily cycles. Here, we investigated the (patho)physiological role of intrinsic renal tubule circadian clocks in the diabetic kidney. We demonstrate that diabetic mice devoid of the circadian transcriptional regulator BMAL1 in the renal tubule exhibit additional enhancement of renal gluconeogenesis, exacerbated hyperglycemia, increased glucosuria, polyuria and renal hypertrophy. Collectively, our results suggest that diabetic hyperglycemia can be worsened by dysfunction or misalignment of intrinsic renal circadian clocks.