Lactobacillus rhamnosus L34 and Lactobacillus casei L39 suppress Clostridium difficile-induced IL-8 production by colonic epithelial cells

Abstract

Background: Clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as C. difficile-associated disease (CDAD).With increased severity and failure of treatment in CDAD, new approaches for prevention and treatment, such as the use of probiotics, are needed. Since the pathogenesis of CDAD involves an inflammatory response with a massive influx of neutrophils recruited by interleukin (IL)-8, this study aimed to investigate the probiotic effects of Lactobacillus spp. on the suppression of IL-8 production in response to C. difficile infection.

Results: We screened Lactobacillus conditioned media from 34 infant fecal isolates for the ability to suppress C. difficile-induced IL-8 production from HT-29 cells. Factors produced by two vancomycin-resistant lactobacilli, L. rhamnosus L34 (LR-L34) and L.casei L39 (LC-L39), suppressed the secretion and transcription of IL-8 without inhibiting C. difficile viability or toxin production. Conditioned media from LR-L34 suppressed the activation of phospho-NF-κB with no effect on phospho-c-Jun. However, LC-L39 conditioned media suppressed the activation of both phospho-NF-κB and phospho-c-Jun. Conditioned media from LR-L34 and LC-L39 also decreased the production of C. difficile-induced GM-CSF in HT-29 cells. Immunomodulatory factors present in the conditioned media of both LR-L34 and LC-L39 are heat-stable up to 100°C and > 100 kDa in size.

Conclusions: Our results suggest that L. rhamnosus L34 and L. casei L39 each produce factors capable of modulating inflammation stimulated by C. difficile. These vancomycin-resistant Lactobacillus strains are potential probiotics for treating or preventing CDAD.

Figure 1

Infant feces-derived Lactobacillus spp. produce factors that suppress pro-inflammatory cytokine production by C. difficile-stimulated HT-29 intestinal epithelial cells. LCM from specific strains of human-derived lactobacilli were found to significantly inhibit IL-8 production by HT-29 cells stimulated with C. difficile. Cells were stimulated with C. difficile in the presence of LCM for 24 h. (A) IL-8 production was monitored by ELISA, and (B) GM-CSF production was monitored by a Luminex premixed cytokine assay by Millipore. The results were from three independent experiments in triplicate for figure (A) and one experiment in triplicate for figure (B) and are expressed as the mean ± SEM, **p-value < 0.01 as compared to medium control.

Figure 2

Lactobacillus soluble factors suppress IL-8 transcription in HT-29 cells. IL-8 gene expression was determined in C. difficile-stimulated HT-29 cells after 4 h incubation with medium control or LCM from either LR-L34 or LC-L39. Quantitative real-time PCR was conducted with primers specific to IL-8 and GAPDH transcripts. Gene expression data were normalized to housekeeping gene, GAPDH. Fold change ratios of IL-8 (LCM strain/medium control) from one experiment in triplicate were calculated, and results represent the mean ± SEM, ***p-value < 0.001 as compared to medium control.

Figure 3

Human-derived Lactobacillus spp. suppress activation of C. difficile-induced transcription factors. Concentrations of activated NF-κB (A) and c-Jun (B) were determined by western blot on whole cell lysates of HT-29 cells stimulated with C. difficile with or without medium control or LCM treatment. Concentrations were measured at 15 and 30 min using antibodies corresponding to p-NF-κB p65, NF-κB p65, β-actin, p-c-Jun, and c-Jun. Relative protein concentrations were determined by densitometry, and activated transcription factors were normalized to their non-activated counterpart (p-NF-κB p65 (Ser 536) to NF-κB p65; p-c-Jun to c-Jun). The results were from three independent experiments in duplicate and are expressed as the mean ± SEM, *p-value <0.05 and **p-value <0.01.

Figure 4

IL-8 suppression by LCM after heat treatment and size fractionation. Soluble factors in LCM from LR-L34 and LC-L39 were assayed for heat stability and size prediction. LCM from L. rhamnosus L34 and L. casei L39 was either heated to boiling for various time points (A) or size fractionated by filtration (B) and the effects on IL-8 production by C. difficile-stimulated HT-29 cells was evaluated by ELISA. The results were from three independent experiments in triplicate and are expressed as the mean ± SEM, **p-value <0.01 and ***p-value <0.001.