Faculty Bibliography

Recent developments in organoid technology have enabled the creation of patient-derived intestinal organoids (PDIOs) that recapitulate the structural, functional, genetic, and epigenetic features of their original tissues. However, conventional passage-derived organoids inevitably yield heterogeneous populations in size and number, leading to inconsistent results even under identical conditions. To address this, a standardized approach, referred to here as "single cell-seeded PDIOs," was established. In this method, mature PDIOs were enzymatically dissociated into single cells and seeded at a defined number into individual wells of a 96-well plate. This controlled seeding normalized the size and number of PDIOs. Compared with passage-derived organoids, single cell-seeded PDIOs displayed reduced inter-well variability in organoid numbers and intra-well variability in organoid sizes, which enables the determination of generation efficiency and improves the reproducibility of viability assays. Moreover, this platform is compatible with downstream analysis, including transcriptomic analysis and protein expression profiling. Collectively, this approach may enhance experimental consistency and provide a practical foundation for reproducible PDIO-based studies.

Plasmacytoid dendritic cells (pDCs) mount powerful antiviral type I interferon (IFN-I) responses, yet only a fraction of pDCs produces high levels of IFN-I. Here we report that peripheral pDCs in naive mice comprise three subsets (termed A, B and C) that represent progressive differentiation stages. This heterogeneity was generated by tonic IFN-I signaling elicited in part by the cGAS/STING and TLR9 DNA-sensing pathways. A small 'IFN-I-naive' subset (pDC-A) could give rise to other subsets; it was expanded in STING deficiency or after the IFN-I receptor blockade, but was abolished by exogenous IFN-I. In response to RNA viruses, pDC-A showed increased Bcl2-dependent survival and superior IFN-I responses, but was susceptible to virus infection. Conversely, the majority of pDCs comprised the 'IFN-I-primed' subsets (pDC-B/C) that showed lower IFN-I responses and poor survival, but did not support virus replication. Thus, tonic IFN-I signaling decreases the cytokine-producing capacity and survival of pDCs but increases their virus resistance, facilitating optimal antiviral responses.

Persistent viral infections are associated with long-term health issues and prolonged transmission. How external perturbations after initial exposure affect the duration of infection is unclear. Here we discovered that murine astrovirus, an enteric RNA virus, persists indefinitely when mice remain unperturbed but is cleared rapidly after cage change. In addition to eliminating the external viral reservoir, cage change also induced interferon-stimulated genes in the intestinal epithelium that are necessary for viral clearance. We further identified that displacing infected animals initially caused a temporary period of immune suppression through the stress hormone corticosterone, which was followed by an immune rebound characterized by activation of CD8 T cells responsible for downstream epithelial antiviral responses. Our findings show how viral persistence can be disrupted by preventing re-exposure and activating immunity upon stress recovery, indicating that external factors can be manipulated to shorten the duration of a viral infection.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1^T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1^T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1^T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1^T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

BACKGROUND & AIMS/OBJECTIVE:The role of goblet cells in small intestinal inflammation in Crohn's disease is unknown. Polymorphisms of NOD2 confer risk for Crohn's disease (CD) and associate with small intestinal disease location. We previously showed in mice that Nod2 deficiency leads to overexpansion of Phocaeicola vulgatus in the gut and downstream goblet cell defects, which preceded small intestinal inflammation. In this study, we ask whether goblet cell defects occur in CD patients with NOD2 polymorphisms and investigate in mice how P. vulgatus signals through the intestinal epithelium. METHODS:We performed a retrospective study of patients with CD to assess clinical outcomes and goblet cell histology by NOD2 status. We evaluated the contribution of microbiota and MyD88 signaling in the intestinal epithelium to goblet cell defects in the setting of Nod2 deficiency using genetic mouse models and germ-free mice. RESULTS:mice harboring P. vulgatus. Finally, we show that P. vulgatus requires complex microbiota to exert its effects in Nod2-deficient mice. CONCLUSIONS:Goblet cell defects may be a harbinger of small intestinal inflammation in CD patients, particularly in the postoperative setting. Our findings in mice show that small intestinal goblet cell loss associated with Nod2 mutation is induced by microbiome dysbiosis and epithelial MyD88, in part due to TLR4 signaling.

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

Inbred mice used for biomedical research display an underdeveloped immune system compared with adult humans, which is attributed in part to the artificial laboratory environment. Despite representing a central component of adaptive immunity, the impact of the laboratory environment on the B cell compartment has not been investigated in detail. Here, we performed an in-depth examination of B cells following rewilding, the controlled release of inbred laboratory mice into an outdoor enclosure. In rewilded mice, we observed B cells in circulation with increased signs of maturation, alongside heightened germinal center responses within secondary lymphoid organs. Rewilding also expanded B cells in the gut, which was accompanied by elevated systemic levels of immunoglobulin G (IgG) and IgM antibodies reactive to the microbiota. Our findings indicate that exposing laboratory mice to a more natural environment enhances B cell development to better reflect the immune system of free-living mammals.

UNLABELLED:isolates with low intrinsic virulence. IMPORTANCE/OBJECTIVE:infection.

The relative and synergistic contributions of genetics and environment to interindividual immune response variation remain unclear, despite implications in evolutionary biology and medicine. Here we quantify interactive effects of genotype and environment on immune traits by investigating C57BL/6, 129S1 and PWK/PhJ inbred mice, rewilded in an outdoor enclosure and infected with the parasite Trichuris muris. Whereas cellular composition was shaped by interactions between genotype and environment, cytokine response heterogeneity including IFNγ concentrations was primarily driven by genotype with consequence on worm burden. In addition, we show that other traits, such as expression of CD44, were explained mostly by genetics on T cells, whereas expression of CD44 on B cells was explained more by environment across all strains. Notably, genetic differences under laboratory conditions were decreased following rewilding. These results indicate that nonheritable influences interact with genetic factors to shape immune variation and parasite burden.