Effects of Acute Luminal Perfusion of Lactobacillus rhamnosus GG on Mouse Small Intestinal Mucosa
Authors: Veltri John Gerardo , Yang Bowen , Pearce Sarah , Occi Jim , Husain Seema , Gao Nan , Soteropoulos Patricia , Connell Nancy , Ferraris Ronaldo 
Published: June 2021

Lactobacillus rhamnosus GG (LGG) is a Gram positive facultative anaerobic lactic acid bacterium that has been associated with the treatment and prevention of intestinal inflammatory disorders. Proposed mechanisms, including increased barrier function, exclusion of pathogenic species, and modulation of cytokine production, were obtained mainly from experimental approaches relying on in vivo LGG incorporation into feed and drinking water where LGG effects may be confounded by trillions of resident bacteria as well as on in vitro LGG incubation of isolated cells and immortalized cell lines that may not fully replicate in vivo LGG‐mucosa interactions. Thus, we perfused LGG at 37°C into the chyme‐free intestinal lumen of anesthetized nongerm‐free mice, to determine acute mucosal responses when LGG are the sole bacteria challenging the gut.Commercial grade lyophilized LGG was reconstituted and determined to be viable via confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining, as well as growth curve analysis. OD600 absorbance values were obtained and used to calculate a generation time of 2.27 hours, further showing the viability of LGG prior to perfusion. Mice were placed under general anesthesia and surgery was performed to directly perfuse LGG into the proximal small intestine via catheters (15 mL/h over 4 h). The inflowing LGG perfusate had 3.3×10^8 CFU/mL in Krebs Ringer Buffer with inulin (7 mg/mL), well above the recommended 10^7 CFU/mL probiotic concentration shown to induce mucosal responses. Control perfusate contained only inulin.Mucosal mRNA expression of cytokines IL6 and IL2 as well as mucin Muc 2 increased with LGG perfusion. Multiplex ELISA analysis of whole tissue proteins showed LGG‐induced increases in cytokines IFN‐γ, IL10, IL2, IL4, IL5, and IL6 (P<0.05). RNA from mucosal scrapes were then submitted for Next Generation Sequencing to determine LGG‐induced changes in the mucosal transcriptome. Analysis of sequencing data determined that LGG perfusion (n = 4) altered the mRNA levels of 441 genes (fold change>1.5, P<0.01). Chodl (endocytosis of bacteria), Cyp1a1 (expression depends on TLR2), and Tmprss15 (serine protease in host defense) expression increased by > 7‐fold while Defa3 (a defensin), Plb1 and Pla2g2 (phospholipases), CCR6 (dendritic and T cells migration and recruitment) and Lyz (lysozyme) were each downregulated by > 5‐fold. Ingenuity Pathway Analysis (IPA) was performed to identify canonical pathways affected by LGG. The most significant were the LPS/IL‐1 Mediated Inhibition of RXR Function, Glutathione‐mediated Detoxification, and Aryl Hydrocarbon Receptor Signaling (P<0.01). IPA also revealed upstream regulators Cftr, mir‐21, Tp53, Tcf3, CDKN1A, POR and TGFb1 to have the greatest predicted activation (z‐score >2.7, P<0.01), and upstream regulators Rabl6, Foxm1, Myc, S100a6 and NFkb1 to have the greatest predicted inhibition (z‐score <‐2.4, P<0.01). Most of these LGG‐affected genes, pathways and transcription factors are involved in bacterial recognition, host defense, host metabolism and inflammation.Our initial findings indicate strong modulation of the gut mucosa of mice perfused solely with LGG, and parallel those of previous probiotic research relying on feeding and in vitro techniques. Thus, intestinal perfusion is an excellent method for use in studies seeking to understand acute, singular effects of probiotic and prebiotic supplements on the intestinal mucosa.Support or Funding InformationSupported by NSF Grant No. IOS‐1121049 and 1456673 (RPF), NIH Grant No. R01‐DK‐102934 (NG)