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Shigella infection is facilitated by interaction of human enteric α-defensin 5 with colonic epithelial receptor P2Y11

ABSTRACT: Human enteric α-defensin 5 (HD5) acts as a “double-edged sword” in host immunity due to its prominent role in promoting pathogen infections, yet the mechanistic basis of its infection-enhancing operation remains unclarified. Here we show that HD5 induces abundant filopodial extensions in epithelial cells which capture Shigella, a major human enteroinvasive pathogen that exploits filopodia for invasion, at high efficiency, suggesting a novel human-specific mechanism of HD5-augmented bacterial invasion. Using multi-omics screening and both gut-on-chip and in vivo models, we identify the HD5 receptor as P2Y11, a purinergic receptor distributed apically on the luminal surface of human colonic epithelium, which to our knowledge is the first HD5 receptor for a specific cellular function. cAMP-PKA signaling was identified to be the main pathway mediating the cytoskeleton-regulating activity of HD5, whose critical residues involved were also determined. In illuminating this mechanism of Shigella invasion, our findings raise the possibility of new intervention strategies against HD5-augmented infections.

ORGANISM(S): Homo Sapiens

SUBMITTER: Dan Xu

PROVIDER: PXD057873 | iProX | Thu Nov 14 00:00:00 GMT 2024

REPOSITORIES: iProX

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Project description:Besides its well-documented antimicrobial functions, human enteric α-defensins 5 (HD5) has been recently found to act as a "double-edged sword" in intestinal defense by enhancing Shigella infection through inducing filopodial-like extensions via activating P2Y11 receptor in human colonic epithelial cells. Nonetheless, the physiological implications of HD5-P2Y11 interaction, beyond its involvement in Shigella infection, have not been comprehensively elaborated. Here, by using primary human colonic epithelial cells, human colonoid organoids, and a gut-on-a-chip model, we showed that HD5 plays an essential roles in establishing and restoring the human intestinal barrier by promoting cell adhesion in suspension or detached cells. Mechanistic investigation demonstrates that although P2Y11 still serves as the responsible receptor mediating the the barrier-protecting effect of HD5, a cell state-dependent alternative downstream signaling cascade was uncovered. In well-adhered cells, HD5-P2Y11 signaling preferentially engages the cAMP-PKA pathway to induce exploratory protrusions like filopodia. Conversely, in suspension cells or detached cells where adhesion is compromised, the signaling shifts priority to the FAK pathway to rapidly reinforce cell adhesion. This dynamic switching allows HD5 to precisely orchestrate epithelial barrier maintenance and repair. This pathway involves the phosphorylation and activation of FAK at key tyrosine residues (Tyr397, Tyr576, Tyr925), which in turn activates the small GTPase Rac1. This HD5-P2Y11-FAK-Rac1 axis enhances barrier function by promoting the formation and stabilization of focal adhesions, accelerating cell attachment, and facilitating epithelial migration for efficient repair. By delineating this novel pathway and its context-dependent regulation, the study fills the knowledge gap to understand the paradoxical roles of HD5 in human intestinal physiology and more importantly, provides a new perspective to understand the physiological significance of this human-specific defensin, extending its role beyond direct antimicrobial action to a key regulator of cytoskeletal dynamics and intestinal homeostasis.

Project description:BACKGROUND Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS With ~4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glia reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity. CONCLUSIONS Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

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Shigella infection is facilitated by interaction of human enteric α-defensin 5 with colonic epithelial receptor P2Y11

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