A synbiotic mixture of Bifidobacterium breve M16-V, oligosaccharides and pectin, enhances Short Chain Fatty Acid production and improves lung health in a preclinical model for pulmonary neutrophilia

Abstract

Introduction: Pulmonary neutrophilia is a hallmark of numerous airway diseases including Chronic Obstructive Pulmonary Disease (COPD), Neutrophilic asthma, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS) and COVID-19. The aim of the current study was to investigate the effect of dietary interventions on lung health in context of pulmonary neutrophilia.

Methods: Male BALB/cByJ mice received 7 intra-nasal doses of either a vehicle or lipopolysaccharides (LPS). To study the effect of nutritional interventions they received 16 intra-gastric doses of either a vehicle (PBS) or the following supplements (1) probiotic Bifidobacterium breve (B. breve) M16-V; (2) a prebiotic fiber mixture of short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides, and low-viscosity pectin in a 9:1:2 ratio (scGOS/lcFOS/lvPectin); and (3) A synbiotic combination B. breve M16-V and scGOS/lcFOS/lvPectin. Parameters for lung health included lung function, lung morphology and lung inflammation. Parameters for systemic immunomodulation included levels of fecal short chain fatty acids and regulatory T cells.

Results: The synbiotic supplement protected against the LPS induced decline in lung function (35% improved lung resistance at baseline p = 0.0002 and 25% at peak challenge, p = 0.0002), provided a significant relief from pulmonary neutrophilia (40.7% less neutrophils, p < 0.01) and improved the pulmonary neutrophil-to-lymphocyte ratio (NLR) by 55.3% (p = 0.0033). Supplements did not impact lung morphology in this specific experiment. LPS applied to the upper airways induced less fecal SCFAs production compared to mice that received PBS. The production of acetic acid between day -5 and day 16 was increased in all unchallenged mice (PBS-PBS p = 0.0003; PBS-Pro p < 0.0001; PBS-Pre, p = 0.0045; PBS-Syn, p = 0.0005) which upon LPS challenge was only observed in mice that received the synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin (p = 0.0003). A moderate correlation was found for butyric acid and lung function parameters and a weak correlation was found between acetic acid, butyric acid and propionic acid concentrations and NLR.

Conclusion: This study suggests bidirectional gut lung cross-talk in a mouse model for pulmonary neutrophilia. Neutrophilic lung inflammation coexisted with attenuated levels of fecal SCFA. The beneficial effects of the synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin on lung health associated with enhanced levels of SCFAs.

Keywords: Short Chain Fatty Acids; acetate; butyrate; gut-lung axis; neutrophil to lymphocyte ratio; nutraceuticals; pulmonary neutrophilia; synbiotics.

Figure 1

The timeline (above) depicts the experimental design for the LPS induced mouse model for pulmonary neutrophilia. The timeline shows the days on which the airways were challenged with either LPS or PBS control (dark grey circles) and the days on which the intragastric prophylactic supplements were provided (solid light grey circles). The arrows indicate the 4 timepoints at which feces was collected for the analysis of Short Chain Fatty Acids (SCFA). Lung function, lung morphology and lung inflammation was analyzed at day 16 (red circle). The graph (below) depicts body weight which was analyzed throughout the experiment as key indicator for mouse well-being. Stunted growth as a result of the first LPS challenge remained within acceptable levels of discomfort reaching a max of 3.5%, the day after the first LPS challenge (n.s.).

Figure 2

(A) Basal tidal volume was not impacted by intranasal LPS challenge. Lung resistance was negatively impacted by the intranasal LPS challenge at baseline and at increasing doses of Metacholine (indicated by ****p < 0.0001). The synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin significantly dampened the negative effect of LPS on lung resistance by 34.9% at baseline and 25% at peak challenge (indicated by ***p < 0.001 throughout the challenge). (B) The linear regression graph presents the pattern that step-wise methacholine challenge induces on the tidal Volume. A significant change (indicated by ^p = 0.0093) was seen between the effect of methacholine on tidal volume of the LPS-placebo and PBS-placebo. A significant treatment effect on tidal volume was not observed. (C) Simple linear regression analysis confirmed a strong significant change between the lung resistance dose–response curves of the PBS-placebo and LPS-placebo groups (indicated by ^^^^p < 0.0001). A treatment effect of synbiotics was also observed within the LPS challenged groups (indicated by ***p = 0.001). The lineair regression analysis furthermore pointed toward indications for a beneficial treatment effect of Probiotics, p < 0.0001 and Prebiotics p = 0.0042 on altering the effect of methacholine compared to mice that received a placebo supplement.

Figure 3

(A) Representative micrographs of H&E-stained sections of lung parenchyma of 3 different mice per group are shown. All LPS challenged groups showed areas of inflammation and remodeling of the lung parenchyma which was absent in mice that received i.n. PBS. The LPS effect includes cellular infiltrates intraluminal-, peribronchial-, and perivascular; thickening of bronchial-, and alveolar walls; and detachments of mucosal epithelium. (B) Representative micrographs of H&E-stained sections of the distal alveolar airspaces of 3 different mice per group are shown. The images depict larger open spaces in the LPS challenged groups which can be a signals for alveolar wall breakdown. (C) The graph depicts the calculated Lineair mean intercept (Lm score) as a measure of the disruption of alveolar walls. Lm was lowest in the PBS-PBS control group. The unpaired t test with Welch’s correction provided a p-value of 0.0469 for the difference between PBS-PBS and LPS-PBS. Of all tested treatments, the synbiotic group showed the lowest Lm-Score, which was however not significantly different from the LPS-PBS group.

Figure 4

(A) The total amount of cells in BALF were significantly increased in mice that were challenged with LPS compared to PBS (indicated by ****p < 0.0001). The synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin significantly dampened the negative effect of LPS on total influx of cells by 28.6% compared to placebo (indicated by *p < 0.01). (B) Representative micrographs of Diff-Quik stained BAL cells showing the morphological differences between Macrophages (M), Lymphocytes (L), Neutrophils (N). Challenged mice showed high numbers of neutrophils which appear as cells with a typical multi-lobed nucleus and neutral-stained cytoplasmic granules. Red Blood Cells (RBC) were observed but not quantified. (C) LPS induced a significant increase in macrophages, neutrophils and lymphocytes compared to mice that received i.n. PBS (indicated by ****p < 0.0001). The synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin significantly reduced the numbers of neutrophils (indicated by **p < 0.01). (D) LPS induced a significant increase in the BALF Neutrophil to Lymphocyte Ratio (NLR) (indicated by #p < 0.0001, analyzed by t test). The synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin provided a significantly lower NLR compared to placebo (indicated by #p < 0.01). (E) BALF Cytokine analysis showed that LPS induced a 55.3% increase in MIP1a (indicated by #p = 0.0451) and a 49.6% increase in KC (indicated by #p = 0.0081). The synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin significantly dampened MIP1a (indicated by #p < 0.05) but not KC.

Figure 5

The graphs depicts the analysis of the numbers of systemic Tregs in thoracic lympnodes, mesenteric lympnodes and spleen tissue. The highest numbers of Tregs were counted in thoracic lympnodes and spleen of the mice that received the synbiotic formula of B Breve M16-V and GOS:FOS:lvPectin 9:1:2. Prebiotic supplementation with GOS:FOS:lvPectin 9:1:2 had a high count in mesenteric lymphnodes of unchallenged mice but not in challenged mice. This preliminary screening did not result in any significant results but a cautiously optimistic alignment with other data warrants further investigation.

Figure 6

The graphs depict the analysis of acetic acid, butyric acid and propionic acid in fecal samples on 4 time points. Unchallenged mice (striped bars) showed a significant increase in the production of acetic acid over time (**p < 0.01; ***p < 0.001; ****p < 0.0001). In the LPS challenged mice the increase was only visible in mice that received the synbiotic mixture of B. breve M16-V and GOS:FOS:lvPectin (***p < 0.001). Mice that received this synbiotic mixture also had a significant increase in levels of butyric acid and propionic acid on day 16. The probiotic B. breve M16-V on itself only induced a significant increase in butyric acid in unchallenged mice. The prebiotic supplement GOS:FOS:lvPectin upregulated butyric acid in challenged mice but not acetic acid nor propionic acid.

Figure 7

(A) Graph depicts Pearson correlations analysis of overlapping lung health and SCFA data points. A moderate correlation is observed between levels of butyric acid and improved lung function parameters. Levels of acetic acid were weakly correlated to improved lung resistance and tidal volume and propionic acid was moderately correlated to tidal volume and weakly to lung resistance. (B) Levels of acetic acid, butyric acid were moderately correlated to improvement of the BALF Neutrophil to Lymphocyte Ratio (NLR). To label the strength of the Pearson associations, the r values between, 0–0.3 (or −0.3) are considered as weak, 0.3–0.7 (or −0.7) as moderate, 0.7–1.0 (or −1) as a strong positive (or negative) correlations (85).