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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Abstract

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses. Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Introduction

Haemophilus influenzae (Hi) is a normal commensal bacterium present in the pharynx of most healthy adults. Hi may have a polysaccharide capsule (types A-F, e.g., type B or HiB) or lack a capsule and be nontypeable (NTHi)1. Colonization of the mucosa with this bacterium begins in early childhood, and there is a turnover of different colonizing strains2. This bacterium is also capable of invasion of both the upper and lower respiratory tract; in this context, it may induce activation of the immune response and inflammation3,4. This inflammatory response ma....

Protocol

This work was approved by the human research ethics committee of Monash Health. The protocol follows the guidelines of the human research ethics committee.

1. Antigenic preparation

NOTE: Three different antigenic preparations can be used to assess the immune response to Hi. These are 1) a subcellular component (typically from the bacterial cell wall); 2) killed and inactivated bacteria; and 3) live bacteria. Determine the use of each antigenic preparation prior to the.......

Representative Results

The representative results show how inflammatory immune responses to NTHi can be assessed/quantitated by flow cytometry and confocal microscopy. A key part of the interpretation of the results is the comparison in fluorescence between control and stimulated samples. A number of preliminary experiments are usually required to optimize the staining of samples. How many different colors can be examined simultaneously will depend on the number of channels available on the flow cytometer/confocal microscope. Results are shown.......

Discussion

The methods listed here use fluorescence-based flow cytometry and confocal microscopy techniques that can be used in conjunction to obtain detailed information about the inflammatory lung response to Hi.

Establishing the appropriate antigenic formulation of Hi to be used is critical, and it is advisable to have specific input from a microbiologist in this regard. Live Hi induces a stronger response, while killed Hi preparations and Hi components are more standardized and are easier to store. P.......

Acknowledgements

The authors would like to thank the staff of Clinical Immunology at Monash Health for their assistance with this work.

....

Materials

NameCompanyCatalog NumberComments
Ammonium chlorideSigma Aldrich213330
BrefeldinSigma AldrichB6542
CD28Thermofisher16-0289-81
CD49dThermofisher534048
DAPI prolong goldThermofisherP36931
DHR123Sigma Aldrich109244-58-8
Filcon sterile nylon meshBecton Dickinson340606
Gelatin substrate, EnzchekMolecular probesE12055
MACS mix tube rotaterMiltenyi Biotec130-090-753
MedimachineBecton DickinsonCatalogue number not available
Medicons 50 µmBecton Dickinson340592
PansorbinSigma Aldrich507858
Propidium iodideSigma AldrichP4170
SaponinSigma Aldrich8047152
Superfrost slidesThermofisher11562203

References

  1. Smith-Vaughan, H. C., Sriprakash, K. S., Leach, A. J., Mathews, J. D., Kemp, D. J. Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic. Infection and Immunity. 66, 3403-3409 (1998).
  2. Murphy, T. F.

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