Anti-Inflammatory Effects of Lactobacillus Rahmnosus and Bifidobacterium Breve on Cigarette Smoke Activated Human Macrophages

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a major global health problem with cigarette smoke (CS) as the main risk factor for its development. Airway inflammation in COPD involves the increased expression of inflammatory mediators such as CXCL-8 and IL-1β which are important mediators for neutrophil recruitment. Macrophages are an important source of these mediators in COPD. Lactobacillus rhamnosus (L. rhamnosus) and Befidobacterium breve (B. breve) attenuate the development of 'allergic asthma' in animals but their effects in COPD are unknown.

Objective: To determine the anti-inflammatory effects of L. rhamnosus and B. breve on CS and Toll-like receptor (TLR) activation.

Design: We stimulated the human macrophage cell line THP-1 with CS extract in the presence and absence of L. rhamnosus and B. breve and measured the expression and release of inflammatory mediators by RT-qPCR and ELISA respectively. An activity assay and Western blotting were used to examine NF-κB activation.

Results: Both L. rhamnosus and B. breve were efficiently phagocytized by human macrophages. L. rhamnosus and B. breve significantly suppressed the ability of CS to induce the expression of IL-1β, IL-6, IL-10, IL-23, TNFα, CXCL-8 and HMGB1 release (all p<0.05) in human THP-1 macrophages. Similar suppression of TLR4- and TLR9-induced CXCL8 expression was also observed (p<0.05). The effect of L. rhamnosus and B. breve on inflammatory mediator release was associated with the suppression of CS-induced NF-κB activation (p<0.05).

Conclusions: This data indicate that these probiotics may be useful anti-inflammatory agents in CS-associated disease such as COPD.

Fig 1. THP-1 cells efficiently phagocytose bacteria.

THP-1 cells (1x10⁶) were incubated with FITC-labelled L. rhamnosus (LR) or B. breve (BB) at a ratio of 1:10 for 2h at 37°C in the presence or absence of 1.5% cigarette smoke extract (CSE). Cells were washed and the presence of intracellular label detected in 40,000 events by flow cytometry. The uptake of FITC-dextran was used as a positive control (C) and all results are presented as means ± S.E.M. of the uptake compared to this. N = 4 independent experiments, *p<0.05, **p<0.01 versus control.

Fig 2. The effect of L. rahmonusus (LR) and B. breve (BB) on cigarette smoke extract (CSE)-, lipopolysaccharide (LPS)- and CpG-induced CXCL-8 release by THP-1 macrophages.

Cells 1x10⁶/ml were pretreated for 2hrs with 10 bacteria/cell (1), 20 bacteria/cell (2) and 50 bacteria/cell (3) LR and BB in the presence and absence of 1.5% CSE and CXCL-8 release (A) and mRNA expression (B) determined after 16 and 3hrs respectively. Expression of CXCL-8 mRNA is expressed as a ratio of the housekeeping gene GAPDH. Cell viability as assessed by Annexin V expression was not affected by any treatment (C). A similar effect of LR and BB on (D) LPS (1000ng/ml)-and (E) CpG oligonucleotide (3μM)-stimulated CXCL-8 release at 16h was also seen. (F) Cells were preincubated for 2 h with E.coli (at various ratios of bacteria to cells) and then stimulated with 1.5% CSE and CXCL-8 release determined after 16hrs. (G) U937 macrophages were stimulated with LR and BB in the presence and absence of 1.5% CSE and CXCL-8 release determined after 16hrs. Data are presented as mean ± S.E.M. of three independent experiments. *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated CXCL-8.

Fig 3. Effects of L. rhamnosus (LR) and B. breve (BB) on the release of cytokines induced by cigarette smoke extract (CSE).

THP-1 cells (1x10⁶/ml) were preincubated for 2h with LR or BB (both at 10 bacteria/cell) in the presence or absence of 1.5% CSE and the release of IL-23 (A), IL-10 (B), IL-1β (C), TNF-α (D) and IL-6 (E) assessed after 16hrs. Data are presented as mean ± S.E.M. of three independent experiments. *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated mediator release.

Fig 4. Effects of L. rhamnosus (LR) and B. breve (BB) on cigarette smoke extract (CSE)-induced NF-κB activation.

Cells 5x10⁴ /well were preincubated for 2h with LR or BB (both at 10 bacteria/cell) and then incubated for 24h in the presence or absence of 1.5% CSE. SEAP activity was measured in cell culture supernatant (A). *p<0.05 compared to the control and ^#p< 0.05 compared to CSE alone. The effect of RL and BB on NF-κB p65 nuclear import was also assessed by Western blotting with histone H1 (H1) as a nuclear loading control (B). A representative blot of 3 independent experiments is shown (upper panel) with the mean±S.E.M. data presented graphically (lower panel). *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated p65 nuclear import.

Fig 5. Modulation of IL-1β in THP-1 cells by L. rhamnosus (LR) and B. breve (BB) in the presence of cigarette smoke extract (CSE).

A representative immunoblot for IL-1β within whole cell extracts of THP-1 cells (5×10⁶) preincubated for 2h with LR and BB (both at 10 bacteria/cell) in the presence or absence of 1.5% CSE for 6hrs (A). Histone H1 (H1) is used as a housekeeping control. Graphical analysis of at least n = 3 independent experiments is presented (B) as the mean±S.E.M. of the ratio to GAPDH expression. *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated IL-1β expression.

Fig 6. Effects of L. rhamnosus (LR), B. breve (BB) and cigarette smoke extract (CSE) on expression and release of HMGB1.

Cells were preincubated with LR or BB (both at 10 bacteria/cell) for 2h before stimulation with 1.5% CSE for 5h for analysis of intracellular HMGB1 by Western blotting (A) and 16h for detection of HMGB1 release by ELISA (B). A representative blot of the results from 3 independent experiments is shown with histone H1 (H1) as a housekeeping protein. Data in B are presented as the mean±S.E.M. of 3 independent experiments. *p < 0.05; **p < 0.01 versus control and ^#p<0.05, ^##p<0.01 versus CSE-stimulated HMGB1 release.