Diarrheal diseases remain a leading cause of deaths in children (~1.2 million/year) worldwide. In addition to its significant mortality and morbidity i developing countries, even in western countries, diarrhea associated with IBD and infection by food-borne pathogens or hospital acquired infections remains a major healthcare challenge. Therefore, better and novel treatment modalities are needed. In this regard, studies during our previous funding cycle provided pioneering in vitro and in vivo evidence for a potential probiotic Lactobacillus acidophilus (LA) based approach for the amelioration of diarrhea. We demonstrated that the mechanism of LA-mediated antidiarrheal effects involved stimulation of intestinal NaCl absorption, involving upregulation of the activities of NHE3 and DRA (the key intestinal apical membrane Na+/H+ and Cl-/HCO3- exchangers, respectively). Most importantly, the bacteria-free culture supernatant (CS) of LA was equally effective in stimulating NaCl absorption, defining a novel therapeutic modality to utilize bacteria-derived bioactive molecules, rather than live bacteria, for treating diarrheal disorders. Our studies further indicated that LA r LA- CS-induced enhancement of Cl-/HCO3- exchange activity involved increased levels of DRA on the apical membrane as well as its increased expression via transcriptional mechanisms. However, detailed cellular and molecular events governing LA- or LA-CS-induced alterations in DRA trafficking and regulation of DRA expression at the transcriptional level under normal physiology and under disease states are not known. Therefore, based on our published reports and strong preliminary data presented here we hypothesized that bacteria-free culture supernatant of L. acidophilus stimulates intestinal epithelial Cl-/HCO3- exchange activity via multiple mechanisms involving alterations of both intracellular trafficking of DRA as well as DRA expression. We also hypothesize that LA or LA-CS can combat the repression of DRA in models of IBD. We will test these hypotheses by systematic set of approaches utilizing both in vitro and in vivo models and state-of-the-art imaging and advanced molecular biology techniques. Studies proposed in Specific Aim 1 will investigate the mechanisms of LA-CS induced modulation of DRA trafficking in Caco2 cell monolayers and elucidate the role of lipid rafts, cytoskeletal and PDZ proteins;
Specific Aim 2 will focus on elucidating mechanisms of LA-CS-induced stimulation of DRA transcription in Caco2 cells;and studies in Specific Aim 3 will examine the effects of short-term as well as long-term LA treatment on DRA function and membrane targeting mechanisms in in-vivo mouse models including IBD models. These studies will not only enhance our understanding of the mechanisms underlying proabsorptive/antidiarrheal role of probiotic LA but will also highlight the potential of the secreed bioactive molecules in LA-CS in combating the IBD associated diarrhea.

Public Health Relevance

Diarrheal diseases still result in ~1.2 million deaths/year worldwide in children under the age of five. In this regard, probiotics are beneficial bacteria which have been shown to be useful in clinical trials in treating diarrheal diseases. The studies proposed in this application will determine molecular and cellular mechanisms responsible for antidiarrheal effects of a probiotic bacteria Lactobacillus acidophilus. Understanding of the mechanisms of its beneficial effects and the bacteria derived molecules responsible for them may assist in designing novel and superior treatment strategies in for diarrheal diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK081858-05
Application #
8576042
Study Section
Special Emphasis Panel (ZRG1-DKUS-L (03))
Program Officer
Grey, Michael J
Project Start
2008-07-01
Project End
2018-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$274,050
Indirect Cost
$56,550
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Ma, Ke; Malhotra, Pooja; Soni, Vinay et al. (2014) Overactivation of intestinal SREBP2 in mice increases serum cholesterol. PLoS One 9:e84221
Xie, Ping; Joladarashi, Darukeshwara; Dudeja, Pradeep et al. (2014) Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology. Am J Physiol Renal Physiol 306:F1260-74
Malhotra, Pooja; Soni, Vinay; Kumar, Anoop et al. (2014) Epigenetic modulation of intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) gene expression by DNA methylation. J Biol Chem 289:23132-40
Singh, Varsha; Kumar, Anoop; Raheja, Geetu et al. (2014) Lactobacillus acidophilus attenuates downregulation of DRA function and expression in inflammatory models. Am J Physiol Gastrointest Liver Physiol 307:G623-31
Anbazhagan, Arivarasu N; Priyamvada, Shubha; Kumar, Anoop et al. (2014) Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 306:G123-31
Borthakur, Alip; Bhattacharyya, Sumit; Kumar, Anoop et al. (2013) Lactobacillus acidophilus alleviates platelet-activating factor-induced inflammatory responses in human intestinal epithelial cells. PLoS One 8:e75664
Malhotra, Pooja; Boddy, Craig S; Soni, Vinay et al. (2013) D-Glucose modulates intestinal Niemann-Pick C1-like 1 (NPC1L1) gene expression via transcriptional regulation. Am J Physiol Gastrointest Liver Physiol 304:G203-10
Saksena, Seema; Priyamvada, Shubha; Kumar, Anoop et al. (2013) Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 304:G615-22
Gill, Ravinder K; Kumar, Anoop; Malhotra, Pooja et al. (2013) Regulation of intestinal serotonin transporter expression via epigenetic mechanisms: role of HDAC2. Am J Physiol Cell Physiol 304:C334-41
Singla, Amika; Kumar, Anoop; Priyamvada, Shubha et al. (2012) LPA stimulates intestinal DRA gene transcription via LPA2 receptor, PI3K/AKT, and c-Fos-dependent pathway. Am J Physiol Gastrointest Liver Physiol 302:G618-27

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