Lactobacillus isolates from healthy volunteers exert immunomodulatory effects on activated peripheral blood mononuclear cells

Abstract

As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immuno-modulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti-CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases.

Keywords: Th1; Th2; cytokine; immuno-modulation; lactobacilli; probiotics.

Fig. 1. Detection of C44 and C48 by RAPD fingerprints.

PCR fingerprinting Lactobacillus strains (with random primer) to cluster identical isolates and the standard strains are shown. Lane 1 and 5: Marker; lane 2 and lane 3: C44; lane 4: LMG9433; lane 6: C48; lane 7: LMG7955. LMG9433 is a standard train of L. acidophilus and LMG7955 is a standard strain of L. paracasie.

Fig. 2. Inhibition of pathogen growth by lactobacilli-free culture medium.

Pathogens were cultured in neutralized and non-neutralized supernatants of Lactobacillus strains C44 or C48 for 5 and 24 hours, respectively. Viable count was performed and changes in percentage of growth were determined. A and B: pathogens cultured in supernatant of C44 for 5 and 24 hours, respectively; C and D: pathogens cultured in supernatant of C48 for 5 and 24 hours, respectively. Pathogens were: C2 (E. coli), C5 (S. typhimurium), C11 (E. faecalis), C14 (K. pneumoniae), C25 (S. flexneri) and QC8 (P. aeruginosa). Values are mean±SD for n = 3.

Fig. 3. IL-10 production from PBMCs stimulated with lactobacilli in different concentrations with or without antibiotics.

IL-10 secretion of PBMCs from 3 donors stimulated with viable C44 and C48 at 10⁶ and 10⁷ CFU/mL in the presence or absence of antibiotics.

Fig. 4. Profiles of cytokines produced by PBMCs induced by different stimuli from bacteria.

Cytokine response after 24 hours of culture with various stimuli of bacteria are shown in A. Ratios of IL-10/IL-12p70 are shown in B. C11 (E. faecalis) and C30 ( B. Bifidum) are controls of opportunistic pathogens and probiotics (v:viable, h-k: heat-killed and D: DNA). Values are mean ± SD for n=3, *P < 0.05, **P < 0.01.

Fig. 5. Profiles of cytokines from anti-CD3 activated PBMCs induced by different stimuli from bacteria.

TCF-β (E), IL-10 (B), IL-6 (A), IL-8, IFN-γ (D) and IL-12p70 (C) response after 24 hours of culture, and IL-4 (F), IL-5 (G), IL-13 (H) response after 72 hours of culture with various stimuli together with anti-CD3 antibody are shown in (A). Ratios of IL-10/IL-12p70 are shown in Fig. I. C11 (E. faecalis) and C30 (B. Bifidum) are controls of opportunistic pathogens and probiotics (viable, heat-killed and DNA of bacteria). Values are mean±SD for n=3. Significance levels are indicated and denoted as *P < 0.05, **P < 0.01.