Mucosal Immunology

המעבדה לאימונולוגיה של מערכת העיכול
Head of the lab:
Prof. Iris Dotan, M.D
Phone: 03-9377237, 03-9377238
email: irisdo@clalit.org.il,
Lab Manager:
Dr. Keren Rabinowitz, PhD
Phone: 03-9376626, 052-8657705
email: k.rabinowitz@gmail.com
Research team:
  • עמיתת מחקר: ד"ר שרית כהן קדר
  • עוזרת מחקר: אפרת שחם ברדה
  • עוזרת מחקר: דניאלה קייזר
  • עוזרת מחקר: שושנה שוורץ
Students:
  • Ph.D. student: Kawsar Kaboub
  • Ph.D. student: Vadim (Dani) Dubinsky (In collaboration with Prof. Uri Gophna, TAU)
  • Ph.D. student: Hanan Abu-Taha
Research Areas:

Our lab focuses on Translational Research in Gastroenterology. We apply skills in Mucosal Immunology to relevant clinical questions, derived from tight collaboration  and interaction with the Division of Gastroenterology, Rabin Medical Center (https://hospitals.clalit.co.il/rabin/he/departments-and-clinics/gastroenterology/Pages/gastroenterology.aspx(. Our primary field of interest is studying immune homeostatic responses in the intestinal mucosa. How does the intestinal epithelium respond to commensal flora (bacteria, fungi) and consequently activates the intestinal immune system. We identified genetic, molecular, microbial, serologic, immune, nutritional and environmental factors associated with intestinal inflammation. Specifically, we focus on inflammatory pathways developing in patients undergoing total proctocolectomy and ileal pouch anal anastomosis reconstruction (pouch surgery) and those with early inflammatory bowel disease. We aim to expand our mechanistic understanding of intestinal responses to commensal flora, nutritional choices and biological therapies. We use our biomaterial repository (collected in a longitudinal and multidisciplinary approach) in order to gain meaningful and clinically relevant information regarding inflammatory pathways and ways to modify/manipulate them so that IBD can be prevented, better treated, and even cured.

·     Biomarker-based multidisciplinary team (Bio-MDT) approach to personalized microbial-targeted treatment of pouchitis and Crohn's disease

 

·     
Commensal fungi and their cell wall β-glucans direct differential response in human intestinal epithelial cells, suggesting a mechanism for mucosal tolerance.

Heat-killed C. albicans interferes with zymosan induced cytokine secretion. SW480 (A,B) and HT-29 (C) cells were stimulated with zymosan,heat killed C. albicans (HKCA), or both, for 20 hours. Cytokine concentration in the supernatants was assessed. *p<0.05; **p<0.01 vs. zymosan.

C. albicans induced Syk phosphorylation in the normal human colonic epithelium. Human colonic mucosal explants were stimulated ex-vivo with heat-killed C. albicans. Frozen sections were stained with antibodies against phospho-Syk (red) or the epithelial marker EpCAM (green) as well as nuclear counterstain with DAPI (blue).

Heat-killed C. albicans interferes with zymosan induced cytokine secretion. SW480 (A,B) and HT-29 (C) cells were stimulated with zymosan, heat killed C. albicans (HKCA), or both, for 20 hours. Cytokine concentration in the supernatants was assessed. *p<0.05; **p<0.01 vs. zymosan.

C. albicans induced Syk phosphorylation in the normal human colonic epithelium. Human colonic mucosal explants were stimulated ex-vivo with heat-killed C. albicans. Frozen sections were stained with antibodies against phospho-Syk (red) or the epithelial marker EpCAM (green) as well as nuclear counterstain with DAPI (blue).

·   Human intestinal epithelial cells respond to commensal fungi by autophagy and LC3-associated phagocytosis

Active autophagy may be monitored in human intestinal mucosal sections. Paraffin embedded mucosal sections from human ileum and colon of the same individual were stained with Ab against LC3 and GABARAP (yellow and pink pseudo-colors respectively) as well as with the epithelial marker EpCAM. Magnification x20 (LC3 stained images: zoom x2)

Active autophagy may be monitored in human intestinal mucosal sections. Paraffin embedded mucosal sections from human ileum and colon of the same individual were stained with Ab against LC3 and GABARAP (yellow and pink pseudo-colors respectively) as well as with the epithelial marker EpCAM. Magnification x20 (LC3 stained images: zoom x2)

·   The JAK inhibitor-tofacitinib inhibits signaling pathways ex-vivoand has functional implications in human intestinal mucosa.

Tofacitinib prevented IL-13-induced decrease in TEER and increase Claudin2 levels. Polarized T84 cells  were treated with Tofa at the indicated concentrations apically for 1 hour prior to basolateral administration of IL-13 (100ng/ml) for 24 hours. Cells permeability was assessed by TEER measurements, expressed as a percentage of initial values (A). CLDN2 mRNA levels was determined by quantitative real-time PCR (B). p-STAT6 and CLDN2 protein expression were assessed by Western Blot
Tofacitinib prevented IL-13-induced decrease in TEER and increase Claudin2 levels. Polarized T84 cells were treated with Tofa at the indicated concentrations apically for 1 hour prior to basolateral administration of IL-13 (100ng/ml) for 24 hours. Cells permeability was assessed by TEER measurements, expressed as a percentage of initial values (A). CLDN2 mRNA levels was determined by quantitative real-time PCR (B). p-STAT6 and CLDN2 protein expression were assessed by Western Blot

·  

Mucus production can be analyzed in human intestinal mucosal sections. Paraffin embedded mucosal sections from human ileum were stained with Ab against MUC2 and EpCAM (green and red pseudo-colors respectively). Magnification x20.

Mucosal barrier alterations in inflammatory bowel diseases (IBD), specifically mucin characteristics, modifying factors and specific markers in the mucosa of patients with pouches.

 

Mucus production can be analyzed in human intestinal mucosal sections. Paraffin embedded mucosal sections from human ileum were stained with Ab against MUC2 and EpCAM (green and red pseudo-colors respectively). Magnification x20.

 

 

·        Predominantly antibiotic-resistant intestinal microbiome persists in patients with pouchitis who respond to antibiotic therapy

Inferred fluoroquinolones (FQ) resistance of the microbiome in patients over time. The analysis is based on point mutations in gyrA alleles for each bacterial genus, summarized to the order level; "R” signifies that all members carry at least one gyrA mutation (a resistant allele), while "S” signifies that all members carry the sensitive allele; orders marked in white were below detection. Relative abundance of gyrA variants during: (A) Ciprofloxacin and metronidazole treatment (C+M); (B) no antibiotic treatment. Green boxes indicate samples taken during a pause in antibiotic treatment of over 30 days. The plot includes 23 patients (113 longitudinal samples). (C) The total FQ-resistant fraction (at least 1 mutation) of the microbiome across all samples (assembled metagenomes, n=215) in Abx+ and Abx- groups; *P=1.4x10-13, Mann-Whitney test.
 Inferred fluoroquinolones (FQ) resistance of the microbiome in patients over time. The analysis is based on point mutations in gyrA alleles for each bacterial genus, summarized to the order level; "R” signifies that all members carry at least one gyrA mutation (a resistant allele), while "S” signifies that all members carry the sensitive allele; orders marked in white were below detection. Relative abundance of gyrA variants during: (A) Ciprofloxacin and metronidazole treatment (C+M); (B) no antibiotic treatment. Green boxes indicate samples taken during a pause in antibiotic treatment of over 30 days. The plot includes 23 patients (113 longitudinal samples). (C) The total FQ-resistant fraction (at least 1 mutation) of the microbiome across all samples (assembled metagenomes, n=215) in Abx+ and Abx- groups; *P=1.4x10-13, Mann-Whitney test.

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