Maria Rosaria Fiorentino, PhD
Mechanisms of the host-microbial interaction in the human gastrointestinal tract
Maria Rosaria Fiorentino, PhD, Lecturer on Pediatrics, Harvard Medical School, Molecular Biologist, MassGeneral Hospital for Children
The intestinal epithelium is the largest mucosal surface providing an interface between the external environment and the internal host milieu.
The gut mucosa represents the “battlefield” where friends (i.e., nutrients and enteric microflora) and foes (i.e., pathogenic microorganisms and their toxins) need to be selectively recognized to reach an ideal balance between tolerance and immune response to non-self antigens. In normal homeostasis, the gastrointestinal epithelial layer forms a tight, but selective barrier: microbes and most antigens are held at bay, but nutrients from the essential to the trivial are absorbed efficiently. What is becoming increasingly clear is that defects in epithelial permeability are associated with a large number of local and even systemic disorders. A common feature of enteric infections is their ability to increase epithelial permeability, an effect that initiates and promotes the host immune response.
My research focuses mainly on understanding the mechanisms of the host-microbial interaction in the human gastrointestinal tract, in order to identify the strategies used by pathogens to attack their hosts and the specific mechanisms hosts put in place to defend themselves. In the last four years I have been interested on defining the functional and immune response of gastro-intestinal epithelial cells to bacterial infections leading to pathologies such as gastric cancer, typhoid fever and dysenteries. By the development and application of new methodologies, I have been studying in vitro and ex vivo the interplay between enteric bacterial human pathogens and the host in order to shed light on clinical condition in which this interplay may lead to disease status. I am working with Helicobacter pylori, Salmonella enterica serovar Typhi and Shigella flexneri and dysenteriae 1 (each primarily affecting a distinct major segment of the GI tract, i.e., the stomach, ileum and colon, respectively) and some typhoid and dysenteriae vaccine candidates utilizing human cell co-culture systems, novel ex-vivo animal models, and human intestinal biopsy explants obtained during routine endoscopic procedures. Besides infecting cell monolayers and recently also 3D organoids, I also infect ex-vivo mice intestinal tissues stripped of the muscularis and mounted on snapwells or Ussing Chambers. These techniques give me the unique opportunity to evaluate the effects of bacteria infection exclusively on epithelial cells, allowing an in-depth analysis of epithelial barrier dysfunctions.
Figure 1. Tight-junction organization is disrupted following exposure to wild type Shigella but not to vaccine candidates . A, D, G. Uninfected monolayer immunostained for ZO-1, Claudin-1 or Occludin, respectively. B, C. Caco2 cells infected with wild-type S. flexneri 2a (B) or CVD 1208S (C) at 106 CFU, stained for ZO-1. E, F. Caco-2 monolayers infected with wild-type S. flexneri 2a (E) or CVD 1208S (F) at 106 CFU and stained for Claudin-1. H, I. Monolayers infected with wild-type S. flexneri 2a (H) or CVD 1208S (I) at 106 CFU and stained for Occludin. J, K, L. Caco-2 cells treated with S. dysenteriae 1 filtered supernatant (J), live wild-type (K) or attenuated CVD 1256 (L) S. dysenteriae 1 strains applied at 105 CFU and stained for ZO-1. Nuclei are stained with DAPI (blu). Bar 25 µm.
Epithelial/endothelial barrier dysfunctions are associated to a wide array of diseases. This is not surprising, considering that our immune system and our physiology are based on barriers. Because of my interest in understanding how loss of barrier function leads to a disease state, I am also involved in a project aimed to evaluate the state of the blood-brain-barrier (BBB) in Schizophrenia and Autism Spectrum Disorders (ASDs). Dysfunction of the BBB has been associated with numerous inflammatory neurologic disorders, such as stroke, epilepsy, Parkinson’s and Alzheimer’s disease. Schizophrenia and ASDs are complex conditions that stem from the classic genes-environment interaction paradigm. There are no defined mechanisms explaining how environmental triggers can lead to these conditions. One hypothesis based on the gut-brain axis connection suggests that inappropriate antigens trafficking through an impaired intestinal barrier followed by passage of these antigens through a permissive blood-brain barrier can be part of the chain of events leading to these diseases. However, while evidence of a permeable gut barrier in ASDs and schizophrenia is increasingly reported, no information is available concerning a similar breach for the blood-brain barrier (BBB). My efforts are aimed to understand whether the “leaky” gut-blood-brain-barrier notion might be implicated in the pathogenesis of a subgroup of these two neuropsychiatric disorders.
Figure 2. Occludin is hyperphosphorylated on threonine and translocates into the cytoplasm, in Caco2 monolayers infected with S. Typhi and leading vaccine candidates CVD908-htrA and CVD909. We analyzed the solubility of occludin in the form of X-100 Triton soluble (S) and insoluble (I) fractions as an indication of its association with tight-junctions. A. and D. Western blot of protein samples blotted with anti-occludin (upper panel), anti-phosphothreonine (middle panel) and anti-actin (lower panel) as loading control, 4 and 22 hours following exposure to bacteria, respectively. B ,C and D,E. Quantification of the data shown in A. Data have been normalized to the actin loading control.
My third interest lies in uncovering the molecular mechanisms by which gluten ingestion causes the complete disruption of the intestinal epithelial barrier in people affected by celiac disease. We know that gliadin (a protein of gluten) can cross the epithelial barrier and activate the secretion of zonulin, leading to increased intestinal permeability. My specific interest is to identify the molecular mechanism by which zonulin affects tight junctions, altering intestinal permeability.
Ultimately, the outcome of my research may lead to innovative preventive and/or therapeutic approaches for the treatment of devastating diseases, including autoimmune, chronic inflammatory and tumor diseases, currently orphan of any effective treatment.
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