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 environmental factors that occur in two major forms, Crohn's disease (CD) and ulcerative colitis (UC). Despite similarities between the two IBD forms, they are characterized by certain differences in the location and nature of inflammatory modifications. CD is a relapsing transmural inflammatory disorder that can potentially extend to any part of the gastrointestinal tract, while UC is non-transmural and is restricted to the colon. Furthermore, mutations in Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a pattern recognition receptor (PRR) that recognizes muramyl dipeptide (MDP), a component of the cell wall of most Gram-positive and – negative bacteria, is associated with CD². Furthermore, Escherichia coli (E. coli), Listeria and Streptococci and their products were all found within macrophages in CD patients that have entered the host after a barrier breach³. When bacteria or their products enter the host during the development of CD, the immune system develops a response leading to the production of circulating anti-bacterial antibodies⁴. Maybe, the most convincing evidence for the role of the microbiota in the pathogenesis of IBD stems from mouse models. When animals are treated with antibiotics, or when mice are kept in germ-free (GF) conditions, the severity of the disease is reduced in most colitis models, such as in IL-10-/- mice that do not develop colitis in GF facilities^5,6. Furthermore, colitis also disturbs the composition of the microbiota, which is characterized by an imbalanced composition and reduced richness called dysbiosis⁷. The consequence of IBD can be an increased intestinal permeability that can lead to the entrance of microbes and microbial-derived products into the host.

In animals, the application of Dextran Sodium Sulfate (DSS) induces an intestinal epithelial breach leading to an increased permeability of the epithelial barrier⁸. Portal LPS concentrations are elevated in animals with DSS colitis⁹. Interestingly, animals lacking the C type lectin receptor specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) are protected from DSS colitis and LPS-induced endotoxemia¹⁰. To further disseminate into the host, bacteria or bacteria derived products have to pass the vascular barrier¹¹, the peritoneal cavity, in which the small and large intestine is located, the mesenteric lymph nodes and/or the liver¹². To reduce the complexity of this system, a defined bacterial-derived compound was used. LPS, which causes endotoxemia after intraperitoneal (i.p.) or intravenous (i.v.) injection¹³ was injected into mice, to study the expression of the interleukins Il6 and Ilb and the cytokine Tnfa in response to LPS.

LPS is a pathogen-associated molecular pattern (PAMP) expressed as a cell-wall component of Gram-negative bacteria, that consists of lipid A (the main PAMP in the structure of LPS), a core oligosaccharide and an O side chain¹⁴. Toll-like receptor 4 (TLR4) expressed by dendritic cells, macrophages, and epithelial cells recognizes LPS¹⁵, that requires co-receptors for appropriate binding. The acute phase protein LPS-binding protein (LBP) binds LPS to form a complex that transfers LPS to the cluster of differentiation 14 (CD14), a glycosylphosphatidylinositol-anchored membrane protein. CD14 further shuttles LPS to Lymphocyte antigen 96 or also known as MD-2, which is associated with the extracellular domain of TLR4. The binding of LPS to MD-2 facilitates the dimerization of TLR4/MD-2 to induce conformational changes to recruit intracellular adaptor molecules to activate the downstream signaling pathway¹⁴, which includes the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway and the TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent pathway¹⁶. The recognition of LPS by TLR4 then activates the NF-κB pathway and induces the expression of proinflammatory cytokines, such as TNFα, IL-6, and IL-1β¹⁷.

In particular, when LPS is injected into animals, the concentration of LPS given to the animals, the genetic background of the animal and the diet has to be considered. High concentrations of LPS leads to a septic shock, characterized by hypotension and multiple organ failures, and finally to death¹⁸. Mice are less sensitive to LPS compared to humans, where LPS concentrations between 2-4 ng/kg body weight (BW) are able to induce a cytokine storm¹⁹. For mice, the lethal dose (LD50), which induces death in half of the mice ranges from 10-25 mg/kg BW²⁰ depending on the mouse strain used. For the commonly used mouse strains, C57Bl/6 and BALB/c, the lethal dose 50% (LD50) is 10 mg/kg BW. In contrast, the strains C3H/HeJ and C57BL/10ScCr are protected from LPS induced endotoxemia, which is due to mutations in Tlr4²¹. Consequently, Tlr4-deficient mice are hyporesponsive to injections with LPS²². Other genetically modified mouse lines, such as PARP1-/- mice²³ are resistant to LPS-induced toxic shock.

The mouse model described here uses a sublethal dose of LPS administered systemically to mimic the consequences of LPS dissemination after a barrier breach of the body`s surfaces. The chosen LPS concentration (2 mg/kg BW) did not induce mortality in C56Bl/6 mice, but the induced release of pro-inflammatory cytokines.

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 Open the stock of LPS purified from Escherichia coli 0111:B4 under sterile conditions and reconstitute it in water to the conce…

Representative 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

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