Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection

Abstract

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamycin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.

Ecotin protectsSalmonellaTyphimurium against the microbicidal activity of host proteases

“…Recruitment of PMNs into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to STm infection and it has been shown that intraluminal PMNs protect the cecal epithelium from STm induced loss of integrity [32]. Here we found that Ecotin was dispensable for PMN recruitment and bacteria amount inside the luminal PMNs were similar upon WT or eco STm infection.…”

The microbiota conditions a gut milieu that selects for wild-type Salmonella Typhimurium virulence

“…Earlier work using genetically barcoded S . Typhimurium populations had shown that the pathogen population structure in the cecum lumen is established by bacterial growth, influx, efflux, and death and that the Salmonella population structures of the feces resemble those in the cecum [ 46 – 49 ]. Thus, by analyzing the feces, we could assess the clonal composition of the Salmonella population within the host (that is the cecum lumen) and obtain information about the pathogen population that can be transmitted to new hosts.…”

“…Moreover, in streptomycin pretreated mice, gut inflammation induced by wild-type S . Typhimurium elicits such a pronounced anti-microbial defense that even gut-luminal loads of the inflammation-adapted pathogen transiently decline by 10 to 10,000-fold at day 2 of the infection [ 48 , 49 ] before regrowing to carrying capacity (≈10 9 CFU/g in cecum content or feces). Based on this previous knowledge and our data presented above, we hypothesized that wild-type S .…”

“…Typhimurium gut infection has a pronounced effect on the gut-luminal bacterial community between days 1 and 2 p.i. [ 48 , 49 ], which starts to recover after day 2. However, neither the introduction of individual strains nor the supplementation of propionate or butyrate in the drinking water of mice (as examples for prominent microbiota fermentation products) prevented the selection for S .Tm* hilD ( S15B Fig ).…”

“…Typhimurium in streptomycin pretreated mice not only prevents gut microbiota regrowth, but also it has particularly strong bactericidal effects at days 1 to 2 p.i. [ 48 , 49 ], which are strong enough to reduce the gut-luminal loads of the inflammation-adapted pathogen by 10 to 10,000-fold at day 2 of the infection [ 48 , 49 ], before it can regrow to carrying capacity (≈10 9 CFU/g in cecum content or feces). The co-housing experiment in Fig 5 provides further evidence that microbiota can (re)-establish in the infected gut as early as day 4 p.i.…”