Paneth cell disruption-induced necrotizing enterocolitis requires live bacteria and occurs independent of TLR4 signaling

Abstract

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in premature infants. Both human surgical specimens and animal models suggest a potential involvement of Paneth cells in NEC pathogenesis. Paneth cells play critical roles in epithelial homeostasis, innate immunity and host-microbial interactions. Yet, the complex interplay between Paneth cell disruption, epithelial barrier dysfunction and microbial-driven inflammation remains unclear in the immature intestine. In this study, mucosal intestinal injury consistent with human NEC was induced in postnatal day 14-16 (P14-P16) mice by disrupting Paneth cells, followed by gavage with Klebsiella pneumonia. Mucosal injury was determined by histology, serum cytokine levels and epithelial barrier dysfunction. Toll-like receptor 4 (TLR4) activation was examined using protein expression, gene expression, and TLR4−/− mice. Finally, the role of bacteria was evaluated using heat-killed bacteria, conditioned media, Bacillus cereus and cecal slurries. We found that live bacteria were required to induce injury; however, TLR4 activation was not required. NEC induced by Paneth cell disruption results in altered localization of tight junction proteins and subsequent loss of barrier function. Prior research has shown a requirement for TLR4 activation to induce NEC-like damage. However, many infants develop NEC in the absence of Gram-negative rod bacteremia, raising the possibility that alternative pathways to intestinal injury exist. In this study, we show a previously unknown mechanism for the development of intestinal injury equivalent to that seen in human NEC and that is not dependent on TLR4 pathways. These data are congruent with the new hypothesis that NEC may be the consequence of several disease processes ending in a final common inflammatory pathway.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine

“…Treatment with diphtheria toxin induces apoptosis of any cells possessing DTR while sparing all other cells. In this model, treatment with diphtheria toxin followed by Klebsiella pneumoniae exposure also produces intestinal injury that is equivalent to human NEC (14,119). These data provide further evidence that it is a disruption of, and not an absence of Paneth cells that contributes to development of NEC-like injury in the immature small intestine.…”

“…Interestingly, when Paneth cells are disrupted in neonatal rats followed by enteral exposure to E. coli, there is not only an increase in bacterial translocation, but also a development of NEC-like injury to the small intestinal tract (105). In adapting this model to mice, our laboratory and others have shown that selective ablation of Paneth cells followed by enteric gavage of Klebsiella pneumoniae in 14-days old mice results in grossly necrotic intestines (89,(117)(118)(119), an increase in serum inflammatory markers (119), and alterations in the microbiome (14) that are consistent with human NEC. The use of 2-weeks old mice in this model is potentially advantageous as well as they possess a gene expression profile of epithelial cell genes that matches the expression profile seen in preterm human infants during the window when they are most susceptible to develop NEC (18,67).…”

“…One critique of this methodology is that dithizone is not specific to Paneth cells but instead is a general chelator of heavy metals. To help resolve this issue, we developed the PC-DTR mouse (14,119,120). The PC-DTR mouse has a human diphtheria toxin receptor (DTR) inserted into mouse Paneth cells targeting the cryptdin-2 promotor (65).…”

Lipopolysaccharide‐induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood

“…Samples were normalized for the amount of protein obtained and maternal serum, placental homogenates, and amniotic fluid samples were quantified for cytokine levels (IL-1β, IL-6, IL-10, KC-GRO TNF, IL-2, IL-4, IL-5, IL-12, and INFγ) using a Meso Scale Discovery technology as previously described. 16 Non-significant cytokines differences are not shown.…”

Single-Immunoglobulin Interleukin-1-Related Receptor regulates vulnerability to TLR4-mediated necrotizing enterocolitis in a mouse model

“…Although we used a well-accepted model of NEC, the relevance of SIGIRR to Paneth cell ablation (19) and trinitrobenzene sulfonic-acid models (20) of experimental NEC need to be investigated, as these models probe different pathogenic mechanisms underlying NEC.…”