ABSTRACT
Helminths are distinct from microbial pathogens in terms of size and complexity, and are likely the evolutionary driving force for type 2 immunity. CD4+ helper T cells can both coordinate worm clearance and prevent immunopathology, but issues of T cell antigen specificity in the context of helminth-induced Th2 and T regulatory cell (Treg) responses have not been addressed. Herein, a novel transgenic line of the gastrointestinal nematode Strongyloides ratti was generated that expresses the immunodominant CD4+ T cell epitope 2W1S as a fusion protein with green fluorescent protein (GFP) and FLAG peptide in order to track and study helminth-specific CD4+ T cells. C57BL/6 mice infected with this stable transgenic line (termed Hulk) underwent a dose-dependent expansion of activated CD44+CD11a+ 2W1S-specific CD4+ T cells, preferentially in the lung parenchyma. Transcriptional profiling of 2W1S-specific CD4+ T cells isolated from mice infected with either Hulk or the enteric bacterial pathogen Salmonella expressing 2W1S revealed that pathogen context exerted a dominant influence over CD4+ T cell phenotype. Interestingly, Hulk-elicited 2W1S-specific CD4+ T cells exhibited both Th2 and Treg phenotypes and expressed high levels of the EGFR ligand amphiregulin, which differed greatly from the phenotype of 2W1S-specific CD4+ T cells elicited by 2W1S-expressing Salmonella. While immunization with 2W1S peptide did not enhance clearance of Hulk infection, immunization did increase total amphiregulin production as well as the number of amphiregulin-expressing CD3+ cells in the lung following Hulk infection. Altogether, this new model system reveals that helminth-specific CD4+ T cells can adopt effector as well as immunosuppressive and wound reparative phenotypes. This report establishes a new resource for studying the nature and function of helminth-specific T cells.
Dr. Douglas' Dissertation Defense Powerpoint Presentation:
Reference: Douglas, Bonnie B, "Antigen Specific CD4+ T Cell Responses against a Gastrointestinal Nematode" (2021). Dissertations available from ProQuest. AAI28770912.
Read More: https://repository.upenn.edu/dissertations/AAI28770912
ABSTRACT
The gastrointestinal (GI) epithelium is a wide array of cell lineages that form a dynamic barrier to the external environment that regulates nutrient absorption, immunity and a physical barrier against organisms entering the intestinal lamina propria. However, the specific proteins expressed by epithelia to maintain GI function during homeostasis and inflammation and to promote tissue repair following injury are incompletely understood. This body of work demonstrates that one member of the Leucine rich repeat Ig domain containing nogo interacting protein family (LINGO) called LINGO3 serves a critical and previously unappreciated role in maintaining the intestinal barrier under homeostasis and in promoting tissue regeneration following acute tissue injury in the GI tract. Under steady-state conditions in co-housed animals, Lingo3 gene deficient (Lingo3 KO) mice have a marked dysregulation in the architecture of the adherens junction complex between small and large intestinal epithelial cells, which correlated with increased intestinal permeability and serum endotoxin levels. This mucosal barrier defect was also associated with a significant increase in Type 1 cytokines and pro-inflammatory myeloid lineage antigen presenting cells. Interestingly, Lingo3 deficiency did not confer resistance or susceptibility to the enteropathogenic bacterium Citrobacter rodentium. However, when Lingo3 KO mice were exposed to the dextran sodium sulfate (DSS) model of colitis, they developed significantly worse disease than co-housed wild-type (WT) controls as defined by overall morbidity, clinical score, reduced colon length, and histological changes, particularly during the recovery phase of DSS. Curiously, this basal defect and DSS recovery phase defect was remarkably similar to the phenotype of mice genetically deficient in Trefoil factor 2 (Tff2), a goblet cell-derived cytokine known to promote tissue repair. This dissertation also demonstrates that treatment of DSS-treated WT mice with a long-acting agonist of TFF2 (TFF2-Fc) promotes faster tissue recovery than Lingo3 KO mice. This enhanced tissue recovery mediated by TFF2-Fc was associated with a greater number of intestinal epithelial cells showing phosphorylation of ERK, which is a key transcription factor in the MAPK pathway that promotes cellular proliferation. To directly test whether LINGO3 and TFF2 functioned to drive epithelial cell regeneration distinct from their impact upon inflammation, we generated small and large intestinal organoids. Results show that both Lingo3 deficient and Tff2 deficient organoids had defective growth kinetics and impaired architectural structure. Strikingly, both Lingo3 deficient and Tff2 deficient small intestinal organoids had decreased mRNA transcript levels of the intestinal stem cell marker, Lgr5, implying that defects in stem cell abundance and/or function. In conclusion, the body of work comprising this dissertation demonstrates that LINGO3 serves a previously unappreciated role in mucosal epithelial cell barrier function under homeostasis and during tissue repair following colitic injury. This role partially functions through the reparative cytokine TFF2, which collectively warrants further investigation into how this putative TFF2-LINGO3 axis regulates enterocyte and intestinal stem cell function during health and disease.
Reference: Zullo, Kelly, "Leucine Rich Repeat Ig-Domain Containing Nogo Interacting Protein 3 (lingo3) Is A Novel Regulator Of Mucosal Homeostasis" (2019). Publicly Accessible Penn Dissertations. 3704.
Read More: https://repository.upenn.edu/dissertations/AAI28770912
