Shpigel Lab

Recent Publications

Intraepithelial neutrophils in mammary, urinary and gall bladder infections

Dvir Mintz, Hagit Salamon, Michal Mintz, Ilan Rosenshine, and Nahum Y Shpigel. 2019. “Intraepithelial neutrophils in mammary, urinary and gall bladder infections.” Vet Res, 50, 1, Pp. 56. Abstract
Neutrophil mobilization is a crucial response to protect the host against invading microorganisms. Neutrophil recruitment and removal have to be tightly regulated to prevent uncontrolled inflammation and excessive release of their toxic content causing tissue damage and subsequent organ dysfunctions. We show here the presence of live and apoptotic neutrophils in the cytoplasm of inflamed mammary, urinary and gall bladder epithelial cells following infection with E. coli and Salmonella bacteria. The entry process commenced with adherence of transmigrated neutrophils to the apical membrane of inflamed epithelial cells. Next, nuclear rearrangement and elongation associated with extensive actin polymerization enabled neutrophils to crawl and invaginate the apical membrane into cytoplasmic double membrane compartments. Scission of the invaginated cell membrane from the entry point and loss of these surrounding membranes released intracellular neutrophils into the cytoplasm where they undergone apoptotic death. The co-occurrence of this observation with bacterial invasion and formation of intracellular bacterial communities (IBCs) might link entry of infected neutrophils to the formation of IBCs and chronic carriage in E. coli mastitis and cystitis and Salmonella cholecystitis.

Correction of Defective T-regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor Alpha.

Rimma Goldberg, Cristiano Scotta, Dianne Cooper, Einat Nissim-Eliraz, Eilam Nir, Scott Tasker, Peter M Irving, Jeremy Sanderson, Paul Lavender, Fowzia Ibrahim, Jonathan Corcoran, Toby Prevost, Nahum Y Shpigel, Federica Marelli-Berg, Giovanna Lombardi, and Graham M Lord. 2019. “Correction of Defective T-regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor Alpha.” Gastroenterology. Abstract
BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4β7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor alpha (RARA) and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. The pathway that regulates expression of integrin subunit alpha is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4β7, as well as CD62L and FOXP3, compared to cells incubated with rapamycin or rapamycin and ATRA. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labelled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4β7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. clinicaltrials.gov no: NCT03185000.

Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system

N. Nagy, N. Marsiano, R. S. Bruckner, M. Scharl, M. J. Gutnick, S. Yagel, E. Arciero, A. M. Goldstein, and N. Y. Shpigel. 2018. “Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system.” Neurogastroenterol MotilNeurogastroenterol Motil, 30. Abstract
BACKGROUND: Current efforts to develop stem cell therapy as a novel treatment for neurointestinal diseases are limited by the unavailability of a model system to study cell transplantation in the human intestine. We propose that xenograft models support enteric nervous system (ENS) development in the fetal human intestine when transplanted into mice subcutaneously or intra-abdominally. METHODS: Fetal human small and large intestine were grafted onto the small intestinal mesentery and into the subcutaneous tissue of immunodeficient mice for up to 4 months. Intestinal cytoarchitecture and ENS development were studied using immunohistochemistry. KEY RESULTS: In both abdominal and subcutaneous grafts, the intestine developed normally with formation of mature epithelial and mesenchymal layers. The ENS was patterned in two ganglionated plexuses containing enteric neurons and glia, including cholinergic and nitrergic neuronal subtypes. c-Kit-immunoreactive interstitial cells of Cajal were present in the gut wall. CONCLUSIONS & INFERENCES: Abdominal xenografts represent a novel model that supports the growth and development of fetal human intestine. This in vivo approach will be a useful method to study maturation of the ENS, the pathophysiology of neurointestinal diseases, and the long-term survival and functional differentiation of neuronal stem cells for the treatment of enteric neuropathies.

Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5-10 days post-partum

R. Sicsic, T. Goshen, R. Dutta, N. Kedem-Vaanunu, V. Kaplan-Shabtai, Z. Pasternak, Y. Gottlieb, N. Y. Shpigel, and T. Raz. 2018. “Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5-10 days post-partum.” Vet ResVet Res, 49, Pp. 77. Abstract
Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n = 35) were sampled immediately following clinical classification as healthy (n = 21), suffering from metritis (n = 13) or septic metritis (n = 1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8 +/- 9.3%), Firmicutes (27.9 +/- 8.4%) and Bacteroidetes (19.7 +/- 7.2%), while Bacteroidetes (60.3 +/- 10.3%), Fusobacteria (13.4 +/- 5.9%) and Firmicutes (10.5 +/- 3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7 +/- 7.2% vs. 60.3 +/- 10.3%), Fusobacteria (7.5 +/- 5.2% vs. 13.4 +/- 5.9%) and Proteobacteria (31.8 +/- 9.3% vs. 7.3 +/- 5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.

Transplantation of Human Intestine into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas.

Ramona S Bruckner, Einat Nissim-Eliraz, Noga Marsiano, Eilam Nir, Hadar Shemesh, Martin Leutenegger, Claudia Gottier, Silvia Lang, Marianne R Spalinger, Sebastian Leibl, Gerhard Rogler, Simcha Yagel, Michael Scharl, and Nahum Y Shpigel. 2018. “Transplantation of Human Intestine into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas.” J Crohns Colitis. Abstract
Background and Aims: Enteric fistulas represent a severe and medically challenging co-morbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. Methods: Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analyzed the resident immune cells and inflammatory response elicited by systemic LPS in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyze fully-developed xenografts that spontaneously developed enterocutaneous fistulas. Results: Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1β, IL-6, TNF-α, A20 and IkBα was significantly elevated in response to LPS with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4 + lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. Conclusions: Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

T3SS-dependent microvascular thrombosis and ischemic enteritis in human gut xenografts infected with enteropathogenic Escherichia coli

E. Nissim-Eliraz, E. Nir, I. Shoval, N. Marsiano, I. Nissan, H. Shemesh, N. Nagy, A. M. Goldstein, M. Gutnick, I. Rosenshine, S. Yagel, and N. Y. Shpigel. 2017. “T3SS-dependent microvascular thrombosis and ischemic enteritis in human gut xenografts infected with enteropathogenic Escherichia coli.” Infect ImmunInfect Immun. Abstract
Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS) causing the hallmark lesions of attaching and effacing (AE) and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in approximately 50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.
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