Intraepithelial neutrophils in mammary, urinary and gall bladder infections.

Neutrophil mobilization is a crucial response to protect the host against invading microorganisms. Neutrophil recruitment and removal have to be tightly regulated to prevent uncontrolled inflammation and excessive release of their toxic content causing tissue damage and subsequent organ dysfunctions. We show here the presence of live and apoptotic neutrophils in the cytoplasm of inflamed mammary, urinary and gall bladder epithelial cells following infection with E. coli and Salmonella bacteria. The entry process commenced with adherence of transmigrated neutrophils to the apical membrane of inflamed epithelial cells. Next, nuclear rearrangement and elongation associated with extensive actin polymerization enabled neutrophils to crawl and invaginate the apical membrane into cytoplasmic double membrane compartments. Scission of the invaginated cell membrane from the entry point and loss of these surrounding membranes released intracellular neutrophils into the cytoplasm where they undergone apoptotic death. The co-occurrence of this observation with bacterial invasion and formation of intracellular bacterial communities (IBCs) might link entry of infected neutrophils to the formation of IBCs and chronic carriage in E. coli mastitis and cystitis and Salmonella cholecystitis.

Pam3CSK4/TLR2 signaling elicits neutrophil recruitment and restricts invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine mastitis model.

Mastitis-inflammation of the mammary gland is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria, and Escherichia coli is frequently implicated. Intramammary challenge with bacterial LPS is sufficient to elicit the disease. However, using toll-like receptor (TLR) 4-deficient mice, we previously found that mammary pathogenic E. coli is still able to elicit neutrophil recruitment, indicating the presence of bacterial virulence factors other than LPS. To date, no specific virulence factors have been identified in E. coli isolates associated with mastitis, and other microbe-associated molecular patterns (MAMPs), such as bacterial lipoproteins, are prime candidates. The synthetic analog of bacterial lipopeptides, Pam3CSK4, is recognized by TLR2 and mimics the proinflammatory properties of triacylated lipoproteins of Gram-negative bacteria. The aim of the present work was to determine the role of bacterial lipoproteins recognized by TLR2 on mammary cells as virulence factors in the mammary gland. Using the murine mastitis model, we previously showed that following intramammary LPS challenge, neutrophil recruitment is strictly dependent on alveolar macrophages. Thus, the role of alveolar macrophages in the response to intramammary bacterial lipoprotein challenge was also studied. Here, Pam3CSK4 infusion induced mastitis in wild-type mice, but not in TLR2-deficient mice. The wild-type phenotype was not restored by adoptive transfer of TLR2-expressing macrophages into the alveolar milk space of TLR2-deficient mice, indicating that cells other than alveolar macrophages are essential for Pam3CSK4/TLR2 signaling. In contrast to the Pam3CSK4 treatment, infection with E. coli P4 resulted in inflammation, even in the absence of TLR2 signaling, indicating that lipoproteins are sufficient, but not essential virulence factors in the pathogenesis of the intact bacteria. However, in the absence of TLR2, the infecting E. coli P4 invaded the alveolar epithelial cells and formed intracellular bacterial communities, indicating that intact lipoprotein/TLR2 signaling is essential to restricting bacterial invasion.