Establishment of A Mouse Sepsis-Induced Immunosuppression Model By Intranasal Instillation of Bacteria

Summary

Here, we present a protocol for establishing a mouse sepsis immunosuppression model via intranasal instillation of Klebsiella pneumoniae and validated the model through various indicators.

Abstract

Sepsis is a life-threatening organ failure caused by the host's inappropriate response to infection. Pneumonia is the primary cause of sepsis. Animal models are important tools for studying the pathogenesis of sepsis and evaluating new treatment strategies. Previous animal models of sepsis lacked natural infection pathways, making it difficult to accurately reflect the clinical infection process.

This study introduced a mouse sepsis immunosuppression model established by intranasal instillation of Klebsiella pneumoniae (KP) to simulate the natural respiratory infection pathway. After modeling with different concentrations of KP, the inflammation of the model was evaluated at 12 h for cytokine interleukin-6 (IL-6) levels, and organ damage was evaluated at 24 h for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and blood urea nitrogen (BUN) levels. The survival rate of the model was analyzed after 7 days. The percentage of lymphocytes and white blood cells, the proportion of major histocompatibility complex class II (MHC-II) expression in monocytes, and the mean fluorescence intensity (MFI) of IL-10 and TGF-β1 in CD4⁺ T cells were observed to evaluate the immune suppression in mice. After 7 days of modeling, a secondary infection was performed by intranasal instillation of Pseudomonas aeruginosa (PA). The bacterial load in bronchoalveolar lavage fluid (BALF) 24 h after secondary infection and the mortality rate within 14 days were assessed to further evaluate the model's immune suppression.

Nasal instillation of high-concentration Klebsiella pneumoniae can effectively induce an immune suppression. Its purpose is to simulate sepsis induced by a natural respiratory infection pathway and to provide a standard model for sepsis research in animals.

Introduction

Sepsis is a life-threatening organ failure resulting from the host's inappropriate response to infection^1,2,3. It is one of the leading causes of death in intensive care units (ICUs). Globally, there are 48.9 million sepsis cases annually, with 11 million associated deaths⁴. Despite recent advancements in anti-infective therapy and organ support technologies, the pathogenesis of sepsis remains incompletely understood, and specific treatment options are lacking. As such, sepsis has become a key focus and challenge in clinical medical research⁴.

Animal models play an irreplaceable role in basic and translational medical research, serving as essential tools for investigating sepsis pathogenesis and evaluating novel therapeutic strategies. By simulating the pathophysiological processes of human sepsis, these models not only facilitate exploration of key mechanisms such as immune disorders and organ dysfunction but also provide an experimental platform for drug screening and the development of intervention strategies. Among various animal models, mice are the most commonly used in sepsis research due to their well-defined genetic background, extensive research on their immune system, and ease of manipulation.

Currently, several sepsis animal models based on different paradigms are available, including endotoxin models, bacterial infusion models, cecal ligation and puncture (CLP) models, and colon ascendens stent peritonitis (CASP) models⁵. However, these models lack natural infection routes, making it challenging to truly reflect the clinical infection process. Studies have reported that pneumonia is the primary cause of sepsis in the United States⁶. Therefore, establishing a mouse sepsis model that mimics natural infection routes, exhibits immunosuppressive features, and is easy to operate with high reproducibility is of great significance for in-depth research into the immune mechanisms of sepsis and for evaluating potential intervention strategies.

Klebsiella pneumoniae (KP) accounts for 3% to 8% of all hospital-acquired bacterial infections^7,8,9. This bacterium can enter the body through various portals, including the respiratory, gastrointestinal, and urinary tracts, as well as skin, and infections caused by KP have a poor prognosis, especially in immunocompromised patients^7,10. Given that pneumonia is the leading cause of sepsis and KP is a common respiratory pathogen associated with poor outcomes, KP is an ideal candidate for establishing a clinically relevant sepsis model.

Although a variety of animal models of KP infection, including the intranasal method, have been established previously¹¹, only a few studies have focused on optimizing the model to induce sepsis-associated immunosuppression or defining the optimal KP concentration for model establishment. This gap limits the utility of existing KP models for studying sepsis-induced immune dysfunction.

To address this need, we established a sepsis-induced immunosuppression model in mice via intranasal instillation of KP. Compared to existing models, this model simulates the natural pathways of respiratory infections. The model was further validated to provide a method for subsequent research.

Protocol

All experimental procedures described in this article were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (SCXK(PLA)20120031).

1. Experimental animals

1. Use C57BL/6 mice, male, aged 8–10 weeks, weighing 23–28 g (Animal License No.: SCXK (Xiang) 2019-0004). House the mice in a specific pathogen-free (SPF) environment with free access to food and water. Maintain the housing conditions as follows: room temperature of (24 ± 2ºC), humidity of 50%–55%, and a 12 h/12 h light-dark cycle.

2. Bacterial preparation

1. Use a suitable strain. Here, use Klebsiella pneumoniae American Type Culture Collection (ATCC) strain 43816.
2. Prepare an appropriate culture medium (Luria-Bertani [LB] broth).
3. Prepare bacteria for instillation.
   1. Pick a single colony and inoculate it into 5 mL of LB broth, then incubate overnight (12 h) in a shaker at 37 °C and 220 rpm.
   2. Transfer the overnight bacterial culture to fresh LB broth and culture for 3–4 h until reaching the high-density growth stage (optical density at 600 nm [OD₆₀₀] ≈ 0.8).
   3. After diluting the bacterial solution, take 100 µL and add it to the bottom of the culture dish containing solid culture medium. Place it in a 37 °C incubator for 12 h, count the number of colonies, and obtain the concentration of KP at 2 × 10⁹ CFU/mL, i.e., OD₆₀₀ ≈ 0.8.
   4. Adjust the concentration of the bacteria using 0.9% NaCl solution. Prepare three bacterial suspensions with concentrations of 1 × 10⁸ CFU/mL, 1 × 10⁶ CFU/mL, and 1 × 10⁴ CFU/mL for subsequent use.

3. Intranasal instillation infection (Figure 1)

1. Anesthetize the mouse via intraperitoneal injection of 2% sodium pentobarbital at a dose of 2 µL/g (Figure 1A,B).
2. When the mouse exhibits reduced consciousness but retains the swallowing reflex, aspirate 50 µL of the bacterial suspension (or 0.9% NaCl solution as a control) using a micropipette.
3. Gently hold the mouse's neck with the thumb placed on the front of the neck, and gently grasp the mouse's body with the rest of the fingers. Tilt the mouse's head backward (Figure 1C).
4. Slowly instill the bacterial suspension into the mouse's nostrils using a pipette (approximately 5–10 µL per drop). Instill one drop into one nostril at a time (Figure 1D).
5. After observing that the mouse has inhaled the suspension, quickly lift the mouse upward and then slowly lower it to promote the inhalation of the suspension into the bronchi (Figure 1E,F).
6. Switch to the other nostril and repeat the procedure. Drip alternately into both nostrils, 5-10 µL each time, until a total of 50 µL are inhaled.
7. If the mouse coughs, sighs, or cannot inhale the bacterial solution during the operation, allow the mouse to rest and observe. After the situation stabilizes, continue the step of inhaling to ensure that the mouse inhales all the bacterial solution (or 0.9% NaCl).
8. Fix the mouse in a supine position on a mouse board tilted at 60° until consciousness is restored. Provide supplemental heat to facilitate anesthetic recovery; monitor animals continuously to prevent overheating. After the mouse recover from anesthesia, put them back  return it to its cage with optimal temperature conditions, sufficient water, and food.

4. Model evaluation

NOTE: Four groups of mouse models were established via the aforementioned intranasal instillation method, namely the control group (group A, instilled with PBS), the 1 × 10⁴ KP concentration group (group B), the 1 × 10⁶ KP concentration group (group C), and the 1 × 10⁸ KP concentration group (group D). The model was evaluated using the following methods:

1. Evaluate the 12-h inflammatory indicators.
   1. At 12 h after intranasal instillation of KP, collect blood (≥ 300 µL) via cardiac puncture (n = 5 per group). Separate the serum by centrifugation (1000 × g,10 min) and use it for the detection of cytokine interleukin-6 (IL-6).
2. 24-h organ damage assessment
   1. At 24 h after intranasal instillation of KP, collect blood (≥300 µL) via cardiac puncture (n = 5 per group),use the serum for biochemical indicator detection. Among these indicators, use alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to assess liver damage, creatine kinase (CK) for muscular damage, and blood urea nitrogen (BUN) for kidney damage.
3. Symptom assessment
   1. Monitor the body weight of the mouse daily, and observe hair condition, activity level, and other parameters.
4. 7-day cytokines and immune cells
   1. At 7 days post-modeling (n = 10 [A/B], n = 15 [C], n = 40 [D]), collect blood via cardiac puncture for flow cytometry analysis to assess the percentages of lymphocytes, the proportion of major histocompatibility complex class II (MHC‑II) expression in monocytes, the mean fluorescence intensity MFI) of IL-10 and TGF-β1 in CD4⁺ T cells.
   2. Tissue processing
      1. Mix blood samples (100 µL) with red blood cell lysis buffer (300 µL) at a volume ratio of 1:3. After 15 min of erythrocyte lysis at room temperature, centrifuge the mixture at 400 × g for 5 min, and discard the supernatant.
      2. Resuspend the pellet in 200 µL of red blood cell lysis buffer (volume ratio = 1:2) and incubate for 10 min for secondary lysis.
      3. Following another centrifugation at 400 × g for 5 min, remove the supernatant, and resuspend the pellet in 100 µL of cell stain buffer.
      4. Add 1 µL of each flow cytometry antibody to the samples. Briefly, add CD45 for lymphocyte staining; CD45, CD116, Ly6G, Ly6C, and MHC-II antibodies for MHC‑II detection; CD45, CD3, CD4, CD8, and CD25 antibodies for the measurement of IL‑10 and TGF‑β1. The dilution ratio for each antibody listed above is 1:100.
      5. Incubate all samples at 4 °C for 30 min in the dark. Thereafter, add 1 mL of cell stain buffer, then centrifuge at 400 × g for 5 min; discard the supernatant.
      6. For the detection of lymphocytes and MHC-II, resuspend in 400 µL of cell stain buffer prior to flow cytometry analysis.
      7. Subsequently, perform procedures for the detection of IL‑10 and TGF‑β1 following steps 4.4.2.8–4.4.2.12:
      8. Add 1 mL of fixation buffer and incubate the samples at 4ºC in the dark for 60 min.
      9. After adding 1 mL of perm buffer, centrifuge the mixture at 400 × g for 5 min, and discard the supernatant.
      10. Resuspend the cell pellet in 100 µL of perm buffer, followed by the addition of 1 µL each of Foxp3, IL-10, and TGF-β1 antibodies. Incubate the samples at 4 °C for 30 min in the dark.
      11. Next, add 1 mL of perm buffer, and centrifuge at 400 × g for 5 min before removing the supernatant.
      12. Finally, resuspend the cells in 400 µL of cell stain buffer and detect by flow cytometry.
   3. Gating strategy
      1. To detect lymphocytes, exclude debris and doublets on the FSC-A/FSC-H plot. Identify total leukocytes as CD45⁺ events. Gate the lymphocytes based on low FSC/SSC within the CD45⁺ population.
      2. To detect MHC-II, first gate leukocytes on FSC-A/SSC-A to exclude debris, followed by selection of CD45⁺ cells. Identify myeloid cells as CD11b⁺, with neutrophils excluded by Ly6G negativity. Define classical monocytes as Ly6G⁻ Ly6Chi cells, and assess MHC-II expression within this population.
      3. To detect IL-10 and TGF-β1, exclude debris and doublets by FSC-A/SSC-A and FSC-A/FSC-H. Identify total T cells as CD45⁺CD3⁺ events. Within the CD4⁺CD8⁻ population, identify Tregs as CD25⁺FOXP3⁺ cells. Quantify TGF-β⁺ cells in the Treg subset, and assess IL-10 expression across all CD4⁺ T cells. Set all positive gates using FMO controls.
5. Bacterial count in bronchoalveolar lavage fluid (BALF) at 24 h after secondary infection
   1. On day 7 after model establishment (n = 5 [A/B], n = 10 [C], n = 40 [D]), subject the mouse to intranasal instillation of 50 µL of Pseudomonas aeruginosa (PA) (at a concentration of 6.8 × 10¹⁰ CFU/mL) using the aforementioned intranasal instillation method.
   2. At 24 h after secondary infection with PA, collect 1.5 mL of bronchoalveolar lavage fluid (BALF). Serially dilute the BALF, plate it onto LB liquid medium, and incubate it in a constant-temperature incubator at 37 °C for 24 h before colony counting.
   3. Follow these steps for BALF collection.
      1. Deeply anesthetize (2.5 µL/g 2% sodium pentobarbital) or euthanize (7.5 µL/g 2% sodium pentobarbital) the mouse humanely and place it in a supine position with the neck hyperextended.
      2. Make a midline cervical incision to expose the trachea. Insert a blunt 24-G catheter into the trachea to just beyond the carina and secure it with a suture.
      3. Instill 0.5 mL sterile, ice-cold PBS into the lung via the tracheal cannula. Gently aspirate 3–5 times, recover the fluid, and repeat this lavage cycle 3 times for a total volume of approximately 1.5 mL.
6. Survival analysis
   1. Use a separate cohort of mice for survival analysis (n = 10 [A/B/C], n = 40 [D]).
   2. Record the survival status of the mice within 7 days after infection.
   3. After secondary infection with PA, observe the mortality rate of the mice from day 7 to day 14.
   4. After observation, deeply anesthetize the mice with isoflurane (5%); anesthetic depth was confirmed by the loss of righting and toe-pinch reflexes, regular respiration, and muscle relaxation prior to euthanasia by intraperitoneal injection of an overdose of pentobarbital sodium (150 mg/kg), with death confirmed by cervical dislocation.
7. Statistical analysis
   1. Perform normality tests and homogeneity of variance tests on the quantitative data.
   2. For comparisons between groups, use one-way analysis of variance (ANOVA) if the data are normally distributed and exhibit homogeneous variance; otherwise, use nonparametric tests.
   3. Perform survival analysis using the Kaplan-Meier method. Use the mixed-effects models to test differences in body weight changes between groups at different time points. Consider a p-value ≤ 0.05 statistically significant.

Results

At 12 h after intranasal instillation, the serum IL-6 level in the 1 × 10⁸ KP group was significantly increased (p < 0.0001, n = 5) (Figure 2A). At 24 h after intranasal instillation, the serum levels of ALT (p < 0.01, n = 5) (Figure 2B), AST (p < 0.01, n = 5) (Figure 2C), CK (p < 0.01, n = 5) (Figure 2D), and BUN (p < 0.001, n = 5) (Figure 2E

Discussion

Sepsis is a complex clinical syndrome, and the characteristic immunosuppressive state during its late stage is a key factor leading to patient death^12,13. Therefore, preclinical models that accurately simulate the pathophysiology of sepsis-induced immunosuppression are crucial for in-depth exploration of its mechanisms and the development of new therapies. In this study, we successfully established and validated a mouse sepsis model induced by intranasal instilla...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biochemical incubator	YSEI	SHH-150L	Bacterial incubation
C57BL/6J mice	Vital River Laboratories	8-10 weeks, male
CD116b	BioLegend	101257	Flow Antibodies (Anti-Mouse)
CD25	BD bioscience	740447	Flow Antibodies (Anti-Mouse)
CD3	BioLegend	100203	Flow Antibodies (Anti-Mouse)
CD4	BioLegend	100544	Flow Antibodies (Anti-Mouse)
CD45	BioLegend	103116	Flow Antibodies (Anti-Mouse)
CD8	BioLegend	100752	Flow Antibodies (Anti-Mouse)
Cell stain buffer	BioLegend	420201
Constant temperature shaker	Honour	HNY-100B	Bacterial cultivation
Flow cytometer	Beckman Coulter	CytoFLEX	Flow cytometry analysis
High-speed centrifuge	Beckman Coulter	AIIEGRA
IDEXX catalyst one	IDEXX	Catalyst One	Biochemical analysis
IL-10	BioLegend	505026	Flow Antibodies (Anti-Mouse)
Insulin cyringe	WEGO	1mL	Blood collection
Klebsiella pneumoniae ATCC 43816	biobw.org	bio-81659
LB Broth (powder)	Solarbio	L1010	LB liquid culture medium
LB Broth (powder)	Solarbio	L1015	LB agar plate
LY6C	BioLegend	128010	Flow Antibodies (Anti-Mouse)
Ly6G	BioLegend	127606	Flow Antibodies (Anti-Mouse)
MHC-II	BioLegend	107622	Flow Antibodies (Anti-Mouse)
Micropipette	Eppendorf	P100
Red blood cell lysis buffer	Solarbio	R1010
Spectrophotometer	Eppendorf	Eppendorf6131	Optical density measurement
Statistical software	GraphPad Software	Graphpad Prism10.0
Sterile and enzyme-free pipette tips	Biosharp	200μL
TF diluent buffer	BD Pharmingen	51-90008101
TF fix/perm buffer	BD Pharmingen	51-90008100
TF perm/wash buffer	BD Pharmingen	51-90008102
TGF-β1	BioLegend	141406	Flow Antibodies (Anti-Mouse)
Varioskan LUX multimodemicroplate reader	Thermo Scientific	Varioskan LUX