Abstract
Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-α (TNFα). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1 h, followed by a wash. Media containing gentamicin was added and collected at 6 h post-treatment. Compared to CTL, 10 8 ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization ( P < 0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFα mRNA expression by IPEC-J2 cells treated apically with 10 8 ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6 h ( P < 0.05). However, BL increased IL8 mRNA at 1.5 h ( P < 0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST ( P < 0.05) and BL (1.5 and 3.0 h; P < 0.05), but not LR or SC. Only ST increased TNFα mRNA relative to CTL ( P < 0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5 h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL ( P < 0.0001), reduced by LR ( P < 0.05), and LR + ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST ( P < 0.0001), but blunted basolaterally in BL + ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.
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