Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates inflammatory response and homeostasis of spleen and colon in experimental model of Pseudomonas aeruginosa pneumonia

Summary

Background & aims

Recent clinical trials and in vivo models demonstrate probiotic administration can reduce occurrence and improve outcome of pneumonia and sepsis, both major clinical challenges worldwide. Potential probiotic benefits include maintenance of gut epithelial barrier homeostasis and prevention of downstream organ dysfunction due to systemic inflammation. However, mechanism(s) of probiotic-mediated protection against pneumonia remain poorly understood. This study evaluated potential mechanistic targets in the maintenance of gut barrier homeostasis following Lactobacillus rhamnosus GG (LGG) treatment in a mouse model of pneumonia.

Methods

Studies were performed in 6–8 week old FVB/N mice treated (o.g.) with or without LGG (10⁹ CFU/ml) and intratracheally injected with Pseudomonas aeruginosa or saline. At 4, 12, and 24 h post-bacterial treatment spleen and colonic tissue were collected for analysis.

Results

Pneumonia significantly increased intestinal permeability and gut claudin-2. LGG significantly attenuated increased gut permeability and claudin-2 following pneumonia back to sham control levels. As mucin expression is key to gut barrier homeostasis we demonstrate that LGG can enhance goblet cell expression and mucin barrier formation versus control pneumonia animals. Further as Muc2 is a key gut mucin, we show LGG corrected deficient Muc2 expression post-pneumonia. Apoptosis increased in both colon and spleen post-pneumonia, and this increase was significantly attenuated by LGG. Concomitantly, LGG corrected pneumonia-mediated loss of cell proliferation in colon and significantly enhanced cell proliferation in spleen. Finally, LGG significantly reduced pro-inflammatory cytokine gene expression in colon and spleen post-pneumonia.

Conclusions

These data demonstrate LGG can maintain intestinal barrier homeostasis by enhancing gut mucin expression/barrier formation, reducing apoptosis, and improving cell proliferation. This was accompanied by reduced pro-inflammatory cytokine expression in the gut and in a downstream organ (spleen). These may serve as potential mechanistic targets to explain LGG's protection against pneumonia in the clinical and in vivo setting.

Introduction

Infection and pneumonia remain a major cause of morbidity and mortality despite advances in supportive care and antimicrobial therapy [1]. Recently, a multi-national intensive care unit (ICU) survey of over 14,000 patients in more then 1200 ICUs found 51% of patients were considered infected on the day of the survey and 71% were receiving antibiotics. In this study, 64% percent of infections were of respiratory origin and the hospital mortality rate of infected patients was greater than double that of uninfected patients (33% vs. 15%) [1]. Furthermore, antibiotics used to treat these infections lead to loss of commensal gastrointestinal (GI) microbiota and potentially to overgrowth of pathogens (dysbiosis) that may also be resistant to standard antimicrobial agents [2], [3]. The intestine is thought to be the “motor” of systemic inflammatory response syndrome regardless of the location of the initial infection [4]. The effect of alterations in the gut microbiota and gut barrier homeostasis are thought to be transmitted to and propagated by downstream organs, such as the spleen where large immune cell populations are harbored [5]. Alterations in intestinal homeostasis and gut microbiota in sepsis results in increased inflammatory cytokine production [6], gut barrier dysfunction [7], and increased cellular apoptosis [8] all of which may contribute to multiple organ failure (MOF).

A promising intervention to maintain gut integrity and prevent pathologic alterations in the gut microbiota or “dysbiosis” is the use of probiotic bacteria [9]. Probiotic use in ICU has recently been supported by several clinical trials and meta-analysis work [10], [11], [12] showing significant reduction of infections, and specifically ventilator-associated pneumonia (VAP) [13]. Although probiotics show promise as effective treatments in a range of clinical conditions, the specific mechanisms of benefit are complex and not fully described [14]. Based on the results from in vivo and in vitro studies in other models, it appears that probiotics may reduce intestinal epithelial cell apoptosis [15], improve intestinal integrity [16], and prevent bacterial translocation [17], reduce overgrowth of pathogenic bacteria, and potentially suppress inflammatory cytokine production [18], [19].

As stated, some of the most promising data for probiotic use is in prevention and improved outcome from pneumonia in clinical critical care as well as in vivo pneumonia models [10], [20]. Interestingly, recent meta-analysis data indicates that the clinical benefit of probiotics on pneumonia may be most significant in prevention of pneumonia from Pseudomonas aeruginosa [11]. In support of this, our laboratory has also recently shown that Lactobacillus rhamnosus GG (LGG) (a commonly used clinical probiotic) can reduce mortality and bacteremia from experimental P. aeruginosa pneumonia [21]. However, as stated, the mechanisms of probiotic-mediated protection against pneumonia, and sepsis in general, are poorly understood. Given this data, and given that P. aeruginosa pneumonia is one of the most common gram-negative bacteria causing pneumonia in critically ill patients with a high fatality rate [22] we chose to further investigate potential mechanistic targets of LGG in improving outcome from experimental P. aeruginosa pneumonia.