Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

Summary

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

Abstract

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.

To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Introduction

The human intestine is colonized with a large consortium of microorganisms that forms the microbiota, who has developed a mutually beneficial relationship with the host during the evolution. In this relationship, the host provides a secure niche for the microbiota, whereas the microbiota provides vitamins, nutrient digestion and protection from pathogens to the host, where the microbiota resides¹. When this beneficial relationship between the host and the microbiota is disturbed, diseases can develop, such as inflammatory bowel disease (IBD). IBD is a multifactorial chronic inflammatory disease of the intestine caused by genetic and environment....

Protocol

Mice were bred and kept under specific pathogen-free (SPF) conditions in the animal facility of Department of Biomedicine, University of Basel (Basel, Switzerland). All mouse experiments were performed in accordance with the Swiss Federal and Cantonal regulations (animal protocol number 2816 [Canton of Basel-Stadt]).

1. Preparation of LPS Solution

1. Open the stock of LPS purified from Escherichia coli 0111:B4 under sterile conditions and reconstitute it in water to the concentration of 5 mg/mL.
2. Dilute the LPS stock with the sterile phosphate buffered saline (PBS) to the working concentration of 0.2 µg/µ....

Results

To mimic the consequences for the host after the entrance of bacteria or bacterial-derived products that occurs after intestinal barrier breach, LPS was injected into C57Bl/6 mice in a sublethal dose (2 µg/g body weight). Every single mouse was monitored and scored for the occurrence of endotoxemia with parameters listed in score sheet that includes, the appearance of the mice, the activity of the animals, the condition of eyes, and the respiration rate and quality (Table 1

Discussion

This protocol mimics immunological processes that occur after the invasion by microbial-derived products. Critical steps within the protocol are the selection of the mouse line, the hygiene status of the mice, the dose of LPS, the monitoring of the animals for the occurrence of endotoxemia, and the time point of experiment termination. Most importantly, the genetic background of the mouse strain has to be considered. Different mouse strains have different susceptibility to LPS. For example, the C3H/HeJ and C57BL/10ScCr m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DreamTaq Green PCR Master Mix (2x)	Thermo Fisher Scientific, Waltham, MA, USA	K1081
High Capacity cDNA Reverse Transcription Kit,	Applied Biosystems, Foster City, CA, USA	4368813
RNase-Free DNase Set,	Qiagen, Hilden, Germany	79254
LPS Escherichia coli O111:B4	Invivogen, San Diego, CA, USA.	tlrl-eblps
Omnican 50 Single-use insulin syringe	B. Braun Melsungen, Melsungen, Germany	9151125
Bioanalyzer 2100	Agilent Technologie, Santa Clara, USA	not applicable
Centrifuge 5430	Eppendorf, Hamburg, Germany	not applicable
Centrifuge Mikro 220R	Hettich, Kirchlengern, Germany	not applicable
Dissection tools	Aesculap, Tuttlingen, Germany	not applicable
Fast-Prep-24 5G Sample Preparation System	M.P. Biomedicals, Santa Ana, CA, USA	not applicable
NanoDrop ND-1000	NanoDrop Products, Wilmington, DE, USA	not applicable
TRI Reagent	Zymo Research, Irvine, CA, USA	R2050-1