Shpigel Lab

Recent Publications

The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence

Hagit Salamon, Einat Nissim-Eliraz, Oded Ardronai, Israel Nissan, and Nahum Y. Shpigel. 2020. “The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence.” Vet Res, 51, 1, Pp. 77. Publisher's Version Abstract
Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts

Einat Nissim-Eliraz, Eilam Nir, Noga Marsiano, Simcha Yagel, and Nahum Y Shpigel. 2020. “NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts.” bioRxiv, Pp. 2020.07.23.212621. Publisher's Version Abstract
The single-epithelial cell layer of the gut mucosa serves as an essential barrier between the host and luminal microflora and plays a major role in innate immunity against invading pathogens. Nuclear factor kB (NF-kB), a central component of the cellular signaling machinery, regulates immune response and inflammation. NF-kB proteins are activated by signaling pathways downstream to microbial recognition receptors and cytokines receptors. Highly regulated NF-kB activity in intestinal epithelial cells (IEC) is essential for normal gut homeostasis; dysregulated activity has been linked to a number of disease states, including inflammatory bowel diseases (IBD) such as Crohns Disease (CD). Our aim was to visualize and quantify spatial and temporal dynamics of NF-kB activity in steady state and inflamed human gut. Lentivirus technology was used to transduce the IEC of human gut xenografts in SCID mice with a NF-kB luminescence reporter system. NF-kB signaling was visualized and quantified using low resolution, intravital imaging of the whole body and high resolution, immunofluorescence microscopic imaging of the tissues. We show that NF-kB is activated in select subset of IEC with low leaky NF-kB activity. These unique inflammatory epithelial cells are clustered in the gut into discrete hotspots of NF-kB activity that are visible in steady state and selectively activated by systemic LPS and human TNFa; or luminal bacteria. The presence of inflammatory hotspots in the normal and inflamed gut might explain the patchy mucosal lesions characterizing CD and thus could have important implications for diagnosis and therapy.Competing Interest StatementThe authors have declared no competing interest.

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. 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.