JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Immunology and Infection

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Published: June 29th, 2021



1Monash Lung and Sleep, Monash Medical Centre, 2Monash University Department of Medicine, Monash Medical Centre, 3Flow Cytometry Science Technology Platform, Francis Crick Institute, 4Clinical Immunology, Monash Medical Centre, 5Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Ammonium chloride Sigma Aldrich 213330
Brefeldin Sigma Aldrich B6542
CD28 Thermofisher 16-0289-81
CD49d Thermofisher 534048
DAPI prolong gold Thermofisher P36931
DHR123 Sigma Aldrich 109244-58-8
Filcon sterile nylon mesh Becton Dickinson 340606
Gelatin substrate, Enzchek Molecular probes E12055
MACS mix tube rotater Miltenyi Biotec 130-090-753
Medimachine Becton Dickinson Catalogue number not available
Medicons 50 µm Becton Dickinson 340592
Pansorbin Sigma Aldrich 507858
Propidium iodide Sigma Aldrich P4170
Saponin Sigma Aldrich 8047152
Superfrost slides Thermofisher 11562203

  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. Haemophilus and Moxarella infections. Harrisons Principles of Internal Medicine. 152, (2018).
  3. King, P. T., Sharma, R. The lung immune response to nontypeable haemophilus influenzae (lung immunity to NTHi). Journal of Immunology Research. , 706376 (2015).
  4. Ahearn, C. P., Gallo, M. C., Murphy, T. F. Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease. Pathogens and Disease. 75, 9 (2017).
  5. Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science. 303, 1532-1535 (2004).
  6. Brinkmann, V., Zychlinsky, A. Neutrophil extracellular traps: is immunity the second function of chromatin. Journal of Cell Biology. 198, 773-783 (2012).
  7. Jorch, S. K., Kubes, P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nature Medicine. 23, 279-287 (2017).
  8. Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. J. Extracellular traps and macrophages: new roles for the versatile phagocyte. Journal of Leukocyte Biology. 97, 1023-1035 (2015).
  9. Cheng, O. Z., Palaniyar, N. NET balancing: a problem in inflammatory lung diseases. Frontiers in Immunology. 4, 1 (2013).
  10. Jacobs, D. M., Ochs-Balcom, H. M., Zhao, J., Murphy, T. F., Sethi, S. Lower airway bacterial colonization patterns and species-specific interactions in chronic obstructive pulmonary disease. Journal of Clinical Microbiology. 56, (2018).
  11. Barenkamp, S. J., Munson, R. S., Granoff, D. M. Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. The Journal of Infectious Diseases. 143, 668-676 (1981).
  12. Barenkamp, S. J. Outer membrane proteins and lipopolysaccharides of nontypeable Haemophilus influenzae. The Journal of Infectious Diseases. 165, 181-184 (1992).
  13. Johnston, J. W. Laboratory growth and maintenance of Haemophilus influenzae. Current Protocols in Microbiology. , (2010).
  14. King, P. T., et al. Adaptive immunity to nontypeable Haemophilus influenzae. American Journal of Respiratory and Critical Care Medicine. 167, 587-592 (2003).
  15. Coleman, H. N., Daines, D. A., Jarisch, J., Smith, A. L. Chemically defined media for growth of Haemophilus influenzae strains. Journal of Clinical Microbiology. 41, 4408-4410 (2003).
  16. King, P. T., Ngui, J., Gunawardena, D., Holmes, P. W., Farmer, M. W., Holdsworth, S. R. Systemic humoral immunity to non-typeable Haemophilus influenzae. Clinical & Experimental Immunology. 153, 376-384 (2008).
  17. King, P. T., et al. Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression. PLoS One. 10, 0120371 (2015).
  18. Aaron, S. D., et al. Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine. 163, 349-355 (2001).
  19. King, P. T., et al. Lung T-cell responses to nontypeable Haemophilus influenzae in patients with chronic obstructive pulmonary disease. The Journal of Allergy and Clinical Immunology. 131, 1314-1321 (2013).
  20. Tsujikawa, T., et al. Robust cell detection and segmentation for image cytometry reveal th17 cell heterogeneity. Cytometry A. 95, 389-398 (2019).
  21. Sharma, R., O'Sullivan, K. M., Holdsworth, S. R., Bardin, P. G., King, P. T. Visualizing macrophage extracellular traps using confocal microscopy. Journal of Visualized Experiments: JoVE. (128), e56459 (2017).
  22. Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K., Ren, Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiology. 15, 36 (2015).
  23. Ueckert, J. E., Nebe von-Caron, G., Bos, A. P., ter Steeg, P. F. Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Letters in Applied Microbiology. 25, 295-299 (1997).
  24. Essilfie, A. T., et al. Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma. Thorax. 67, 588-599 (2012).
  25. Huvenne, W., et al. Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice. Respiratory Research. 12, 69 (2011).
  26. Radhakrishna, N., Farmer, M., Steinfort, D. P., King, P. A Comparison of Techniques for Optimal Performance of Bronchoalveolar Lavage. Journal of Bronchology & Interventional Pulmonology. 22, 300-305 (2015).
  27. Quatromoni, J. G., Singhal, S., Bhojnagarwala, P., Hancock, W. W., Albelda, S. M., Eruslanov, E. An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. Journal of Leukocyte Biology. 97, 201-209 (2015).
  28. Tighe, R. M., et al. Improving the quality and reproducibility of flow cytometry in the lung. An official American thoracic society workshop report. American Journal of Respiratory and Critical Care Medicine. 61, 150-161 (2019).
  29. Yu, Y. R., et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PLoS One. 11, 0150606 (2016).
  30. Duan, M., et al. Distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease. Journal of Immunology. 189, 946-955 (2012).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved