Abstract
Undernutrition in women and its propagation in their children remains a major global health challenge. Worldwide, one in five children under five years of age manifests undernutrition as impaired linear growth, or stunting. Stunting, defined as having a length-for-age z-score (LAZ) <-2, is associated with poor developmental outcomes, including impaired neurodevelopment and immune responses. Furthermore, stunting and its long-term sequelae persist across generations, contributing to an intergenerational cycle of undernutrition. Existing nutritional interventions fail to substantially improve stunting, highlighting the need to (i) better understand other factors contributing to stunting and (ii) focus clinical efforts towards treating maternal undernutrition. Environmental enteric dysfunction (EED) is a subclinical inflammatory enteropathy of the small intestine proposed to contribute to stunting. EED is diagnosed histologically by blunted intestinal villi, epithelial barrier disruption and an immunoinflammatory infiltrate in the lamina propria. However, the pathophysiology of EED remains poorly defined. Furthermore, the small intestinal microbiota remains understudied due to difficulties in sampling. Evidence that the small intestinal microbiota contributes to EED pathogenesis comes in part from our collaborative work with colleagues at icddr,b (the International Centre for Diarrhoeal Disease Research, Bangladesh) who oversaw the Bangladesh EED (BEED) study. In this study children, aged 12-18 months residing in an urban slum in the Mirpur district of Dhaka who failed a nutritional intervention for their stunting, underwent esophago-gastroduodenoscopy (EGD) to obtain duodenal mucosal biopsies, aspirates of duodenal luminal fluid and plasma samples. A set of 14 bacteria taxa (defined by 16S rRNA amplicon sequencing) were identified in the duodenal microbiota of >80% of children with EED. Moreover, the absolute abundances of these ‘core taxa’ were positively correlated with duodenal mucosal and systemic inflammation in study participants and negatively correlated with linear growth. Our lab showed that introduction of a consortium of bacteria cultured from these duodenal aspirates, including these core taxa, produced small intestinal inflammation in young adult gnotobiotic, providing initial preclinical evidence for a causal role for the duodenal microbiota in a small intestinal enteropathy. Additionally, the BEED study revealed that EED is very prevalent in malnourished (low-BMI) women of reproductive age living in Mirpur. Building upon this work, my thesis aimed to elucidate the mechanisms by which the pattern of intergenerational undernutrition manifests through the transmission of the small intestinal microbiota from mother to child. Here, I present a novel gnotobiotic mouse model of EED involving intergenerational transmission of two different consortia of bacterial strains cultured from duodenal aspirates of children in the BEED study with histopathologic evidence of EED – one that did, and one did not, produce intestinal and systemic inflammation. Histological analyses, protein biomarkers, bulk tissue RNA-Seq and flow cytometry were used to define host pathology in postnatal day 37 (P37) offspring of gnotobiotic female mice colonized with one or the other consortium prior to pregnancy; inflammation was characterized along the length of the intestine, as well as systemically, including in the immune-privileged brain. Single nucleus RNA-Seq (snRNA-Seq) of small intestinal tissue segments further defined alterations in expression of signaling pathways related to intestinal epithelial regeneration, epithelial barrier integrity and immune function, while snRNA-Seq of the cerebral cortex revealed perturbed glial- and endothelial-neuronal intercellular signaling involved in neuronal development, angiogenesis and inflammation. Together, these results provide evidence of a causal role of the small intestinal microbiota in the intestinal and systemic pathology that is manifested in EED. To nominate microbial mediators of the enteropathy, I pursued a series of follow-up gnotobiotic mouse experiments combined with multi-omic analyses. Cohousing young adult mice colonized 9 days earlier with one or the other consortium, revealed the dominance of the inflammatory EED donor-derived consortium compared to the non-inflammatory consortium, reflected by induced inflammation in cohoused mice originally colonized with the non-inflammatory consortium. Metagenome-assembled genomes (MAGs) defined the composition of bacterial communities along the length of the gut; three MAGs were found to be consistently associated with host pathology in both the cohousing and intergenerational transmission models; they represent organisms that are commonly found in the oral microbiota. Genome annotations and microbial RNA-Seq provided context for predicted microbial functions contributing to fitness and pathogenicity of these MAGs. Finally, the addition of cultured bacterial isolates representing these MAGs into mice harboring the non-inflammatory consortium was sufficient to induce features of the enteropathy – most notably in the colon. These findings support the concept that ‘decompartmentalization’, where members of the oral microbiota are established in the intestinal microbiota, is a contributor to EED. In summary, this thesis illustrates an approach for elucidating structure-function relationships in the human small intestinal microbiota - a poorly explored community due to difficulties in obtaining samples. These types of preclinical models should help guide subsequent efforts to better understand the role of the small intestinal microbiota in the pathogenesis of EED, identify candidate therapeutic targets and validate candidate therapeutic agents for treating EED in children (and their mothers).
Committee Chair
Jeffrey Gordon
Committee Members
Adrianus Boon; Ali Ellebedy; Daved Fremont; Deborah Lenschow; Robyn Klein; Sean Whelan
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Molecular Genetics & Genomics)
Document Type
Dissertation
Date of Award
5-2-2025
Language
English (en)
DOI
https://doi.org/10.7936/3kmt-ab14
Recommended Citation
Kao, Clara, "Role of the small intestinal microbiota in the pathogenesis of environmental enteric dysfunction in undernourished children and mothers" (2025). Arts & Sciences Theses and Dissertations. 3544.
The definitive version is available at https://doi.org/10.7936/3kmt-ab14