IFNλ signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection.

Interferon lambda (IFN-λ) plays diverse roles in bacterial infections. Previously we showed that IFN-λ is induced in the lungs of B. pertussis-infected adult mice and exacerbates inflammation. Here, we report that mice lacking the IFN-λ receptor (IFNLR1) specifically on neutrophils (MRP8creIFNLR1fl/fl mice) exhibit reduced lung bacterial load and inflammation compared to WT mice during B. pertussis infection. In B. pertussis-infected wild type (WT) mice, lung type I and III IFN responses were higher than in MRP8creIFNLR1fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of IFN-γ-producing neutrophils in the lungs of MRP8creIFNLR1fl/fl mice compared to WT mice. IFNLR1-/- neutrophils incubated with B. pertussis exhibited higher killing compared to WT neutrophils. Treatment of WT neutrophils with IFN-λ further decreased their bacterial killing capacity and treatment of WT mice with IFN-λ increased lung bacterial loads. Contributing to the differential killing, we found that IFNLR1-/- neutrophils exhibit higher levels of reactive oxygen species, myeloperoxidase [1], matrix metalloproteinase-9 (MMP9) activity, neutrophil extracellular traps (NETs), and IFN-γ secretion than WT neutrophils, and inhibiting NADPH oxidase inhibited bacterial killing in IFNLR1-/- neutrophils. B. pertussis induced IFN-λ secretion and IFNLR1 gene expression in mouse and human neutrophils and this was dependent on the bacterial virulence protein pertussis toxin (PTX). PTX enhanced bacterial loads in WT but not in MRP8creIFNLR1fl/fl or IFNLR1-/- mice. Thus, PTX disrupts neutrophil function by enhancing type III IFN signaling, which prevents neutrophils from effectively clearing B. pertussis during infection, leading to higher bacterial loads and exacerbation of lung inflammation.

Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice.

Many respiratory infections are selectively injurious to infants, yet the etiology of age-associated susceptibility is unknown. One such bacterial pathogen is Bordetella pertussis. In adult mice, innate interferon γ (IFN-γ) is produced by natural killer (NK) cells and restricts infection to the respiratory tract. In contrast, infant pertussis resembles disease in NK cell- and IFN-γ-deficient adult mice that experience disseminated lethal infection. We hypothesized that infants exhibit age-associated deficits in NK cell frequency, maturation, and responsiveness to B. pertussis, associated with low IFN-γ levels. To delineate mechanisms behind age-dependent susceptibility, we compared infant and adult mouse models of infection. Infection in infant mice resulted in impaired upregulation of IFN-γ and substantial bacterial dissemination. B. pertussis-infected infant mice displayed fewer pulmonary NK cells than adult mice. Furthermore, the NK cells in the infant mouse lungs had an immature phenotype, and the infant lung showed no upregulation of the IFN-γ-inducing cytokine IL-12p70. Adoptive transfer of adult NK cells into infants, or treatment with exogenous IFN-γ, significantly reduced bacterial dissemination. These data indicate that the lack of NK cell-produced IFN-γ significantly contributes to infant fulminant pertussis and could be the basis for other pathogen-induced, age-dependent respiratory diseases.

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease.

The enteric nervous system (ENS), a collection of neural cells contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the neural crest and remains largely unchanged thereafter. Here, we show that the lineage composition of maturing ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal dysmotility which is associated with reduction in neural crest-derived neuronal markers and concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS.

COUNTEN, an AI-Driven Tool for Rapid and Objective Structural Analyses of the Enteric Nervous System.

The enteric nervous system (ENS) consists of an interconnected meshwork of neurons and glia residing within the wall of the gastrointestinal (GI) tract. While healthy GI function is associated with healthy ENS structure, defined by the normal distribution of neurons within ganglia of the ENS, a comprehensive understanding of normal neuronal distribution and ganglionic organization in the ENS is lacking. Current methodologies for manual enumeration of neurons parse only limited tissue regions and are prone to error, subjective bias, and peer-to-peer discordance. There is accordingly a need for robust, and objective tools that can capture and quantify enteric neurons within multiple ganglia over large areas of tissue. Here, we report on the development of an AI-driven tool, COUNTEN (COUNTing Enteric Neurons), which is capable of accurately identifying and enumerating immunolabeled enteric neurons, and objectively clustering them into ganglia. We tested and found that COUNTEN matches trained humans in its accuracy while taking a fraction of the time to complete the analyses. Finally, we use COUNTEN's accuracy and speed to identify and cluster thousands of ileal myenteric neurons into hundreds of ganglia to compute metrics that help define the normal structure of the ileal myenteric plexus. To facilitate reproducible, robust, and objective measures of ENS structure across mouse models, experiments, and institutions, COUNTEN is freely and openly available to all researchers.