Dual RNA Seq of Salmonella-positive and Salmonella-negative small intestinal epithelial cells using the neonatal infection model

The intestinal epithelium is the first line of defence against invasive enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal small intestine. Instead, here we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighbouring intestinal epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid co-cultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis and identified the critical involvement of the 'eat-me' signals and adaptors phosphatidylserine and C1q as well as the 'eat-me' receptors integrin-v (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighbouring epithelial cells may represent a previously unrecognised mechanism of neonatal antimicrobial host defense to maintain barrier integrity. C57BL/6 mice will be infected on day 1 after birth with 100 CFU S. Tm. carrying a FPV-mCherry plasmid (pmCherry, kindly provided by Leigh Knodler, NIH, Hamilton, USA). Enterocytes will be isolated from total small intestinal tissues using a previously established EDTA-based protocol at d4p.i. (Lotz et al., 2006). Alexa Fluor® conjugated EpCam and CD45 antibodies in combination with flow cytometric (FACS) sorting will be used to further enrich enterocytes and remove immune cells. EpCam+ CD45- enterocytes (Ep+) will then be pooled from several animals of an infected litter and individually gated and sorted according to their intracellular Salmonella status (Ep+Sal+ = pmCherry+ or Ep+Sal- = pmCherry-). In parallel, EpCam+ CD45- enterocytes (Ep+) will be sorted from non-infected age-matched (5 day old) neonates using a similar protocol and allow defining the gate for Salmonella negative enterocytes. RNA will be isolated from the four independent FACS sorting experiments using the QIAGEN RNeasy Micro or RNeasy Midi Kit for Ep+Sal+ population or Ep+Sal- population and Ep+ population from non-infected neonates, respectively. cDNA libraries were generated according to published protocol (Westermann et al., 2016) and gene expression analysis was performed using DESeq2 as described in Westermann et al. paper (Westermann et al., 2016)