Title: Intestinal infection results in impaired lung innate immunity to 1 secondary respiratory infection. 2 Subtitle: Lung immunity after gut inflammation. 3 Tetramer and antibody surface-staining of lung single cell suspensions

Pneumonia and diarrhea are among the leading causes of death worldwide, and 27 epidemiological studies have demonstrated that diarrhea is associated with an 28 increased risk of subsequent pneumonia. Our aim was to determine the impact of 29 intestinal infection on innate immune responses in the lung. Using a mouse model of 30 intestinal infection by Salmonella enterica serovar Typhimurium ( S . Typhimurium), we 31 investigated how infection in the gut compartment can modulate immunity in the lungs 32 and impact susceptibility to bacterial ( Klebsiella pneumoniae ) challenge. We found 33 alterations in frequencies of innate immune cells in lungs of intestinally-infected mice 34 compared to uninfected mice. On subsequent challenge with K. pneumoniae we found 35 that mice with prior intestinal infection have higher lung bacterial burden and 36 inflammation, increased neutrophil margination, and neutrophil extracellular traps 37 (NETs), but lower overall numbers of neutrophils, compared to mice without prior 38 intestinal infection. Together, these results suggest that intestinal infection impacts lung 39 innate immune responses, most notably neutrophil characteristics, potentially resulting 40 in increased susceptibility to secondary pneumonia. 41 42 43 46 47 Human studies shown that children with diarrhea are at higher risk of subsequent lung infection. How intestinal infections impact lung immunity is not well known. In the present study, we reveal that bacterial infection of the intestinal mucosa may compromise lung immunity, offering new insights into increased susceptibility to respiratory infections. We found that upon respiratory infection, mice with prior intestinal infection are more moribund and despite having higher bacterial burden, they show reduced numbers of neutrophils in the lung compared to mice without prior intestinal infection. We also found excessive neutrophil extracellular traps formation in the lungs 57 of mice with prior intestinal infection, providing evidence of increased pulmonary tissue damage. Collectively, these data identify a direct link between pulmonary and enteric infection and suggests gut infections impair neutrophils responses in the lungs. infection impacts immunity in the lung is not known. In this study, we examine the impact of intestinal infection on the immune response in the lungs of mice using Salmonella enterica serovar Typhimurium (S. Typhimurium), an established model of intestinal infections. We found that mice infected with S. Typhimurium have increased susceptibility to respiratory Klebsiella pneumoniae infection compared to mice without prior intestinal infection. Prior intestinal infection modulated effector cells of innate immunity in the lung, contributing to respiratory immune dysregulation and a higher K. pneumoniae bacterial burden. This study furthers our understanding of the gut-lung immunological crosstalk, and begins to define the mechanisms of increased susceptibility to secondary pneumonia following diahrreal infections.