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Immunology and Infection

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Published: March 2nd, 2016

DOI:

10.3791/53964

1Baylor Institute for Immunology Research, Baylor Research Institute

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Inflammation is a fundamental immune mechanism of defense against infectious agents. It promotes pathogen eradication and repair of damaged tissue. It also facilitates the recovery to a normal healthy state1. However, dysregulated inflammation often leads to chronic inflammatory diseases2. Airway inflammation is an initial trigger for different pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis3.

The non-typeable (unencapsulated) Haemophilus influenzae (NTHi) is associated with chronic upper and lower lung inflammatory diseases4,5. It is....

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All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Baylor Research Institute.

1. Culturing Non-typeable Haemophilus influenzae (NTHi) and Preparing the Inoculum

  1. Plate NTHi on a chocolate agar plate and keep the plate upside down in a humidified CO2 incubator overnight at 37 °C and 5% CO2. The following day, culture the bacteria in brain heart infusion broth. Then, add 1 .......

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Intra-tracheal instillation resulted in a markedly increased number of leukocytes in the BALF (Figure 1A, left panel) than installation with saline. The differential count analysis of the leukocytes clearly showed increased neutrophil infiltration (Figure 1, right panel). The FACS analysis of the cells in the BALF further confirmed the increased number of neutrophils (Figure 1B). Histological analysis of H&E-stained sections of the lu.......

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Herein, we describe a unique and minimally invasive procedure to inoculate the lungs of mice with a bacterial lung pathogen. We demonstrate that this procedure can be used to study the function of different genes using mice that are deficient in genes of inflammatory signaling pathways. This procedure can also be used to study the inflammatory responses to viral and fungal lung infections. The advantages of this procedure over other methods such as intranasal or aerosol inhalation are (1) in this procedure, the pathogeni.......

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We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

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Name Company Catalog Number Comments
Chocolate agar plate Fisher Scientific CAS50-99-7
Dextrose Anhydrous Themo Scientific R01300
Heparin Hospira,Inc RL-3010
Deft quick solution Sigma GS500-500ML
Syringe needle 20/26G  BD (REF305115/175)
Iml syringe BD REF 309602
Catheter 20GA BD REF 381433
Dissecting Scissors, straight, 10 cm long kentscientific INS600393
Iris Forceps, serrated, 10cm long kentscientific INS650915
Tweezer #5 Stainless steel, 11cm long kentscientific INS600095
10% Formalin Fisher Scientific CAS 67-56-1
Agarose peqlab 35-1020
5ml polystyrene round-bottom tubes BD REF 352058
1.5 ml Microcentrifuge tubes Light Labs A-7001-R
Reasy Mini  kit  Qiagen 74104
Pellet pestile motor (Tissue homoginizer) Sigma Z359971-1EA
96 well microtiter plates V bottom Thermo 2605
1X PBS Gibco 10010-023
OneComp eBeads eBioscience 01-1111-42
CD45.2-APC eBioscience 17-0454-81 Working dilution 1:100
Ly-6G-eFlor 450 eBioscience 48-5931-82 Working dilution 1:100
BSA HyClone SH30574.03
RBC Lysis Buffer (10X) Biolegend 420301
Live/Dead fixable aqua dead cell stain kit Invitrogen L-34957
EDTA (0.5M) lifetechnologies 15575-020
CD16/CD32 FcBlock BD 553142
Facs tubes polystyrene round bottom tube BD 352052
Formaldehyde Polyscience 4018

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