Invasive and Non-invasive Clinical Haemophilus influenzae Type A Isolates Activate Differentiated HL-60 Cells In Vitro

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Courtney L. Ferris
Marina Ulanova


Background: The effective elimination of encapsulated bacteria like Haemophilus influenzae type a (Hia) relies on immune mechanisms such as complement-mediated opsonophagocytosis by neutrophils in coordination with opsonization by anti-capsular antibodies. This study evaluated if Hia could activate the immune response through neutrophils and if these responses differed between encapsulated versus unencapsulated or invasive versus non-invasive strains.

Methods: HL-60-derived neutrophil-like cells (dHL-60), differentiated with 1.25% dimethyl sulfoxide over 9 days, were used in an opsonophagocytosis assay and in vitro infection model to measure Hia’s susceptibility to killing and dHL-60 surface molecule expression, respectively. The impact of strain-specific features on the immune response was investigated using clinical isolates of a dominant North American sequence type (ST)-23, including Hia 11-139 (encapsulated, invasive), 14-61 (encapsulated, non-invasive), 13-0074 (unencapsulated, invasive), as well as a representative ST-4 isolate (Hia 13-240, encapsulated, invasive), and a nontypeable strain (NTHi 375, unencapsulated, non-invasive).

Results: Unencapsulated and non-invasive Hi strains were more susceptible to killing by the innate immune response while the ST-23 invasive strain, Hia 11-139 required serum antibodies for destruction. Flow cytometry analysis showed increased expression of co-stimulatory molecule ICAM-1 and Fc receptors (CD89, CD64) but decreased expression of the Fc receptor CD16, revealing potential mechanisms of neutrophil-mediated defense against Hia that extend to both non-invasive and invasive strains.

Conclusions: Hia clinical isolates with diverse pathogenicity illustrated contrasting susceptibility to killing by immune mechanisms while maintaining the same capacity to activate neutrophil-like cells, further underscoring the need for additional studies on Hia’s pathogenesis.


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1. Slack MPE. A review of the role of Haemophilus influenzae in community-acquired pneumonia. Pneumonia (Nathan). 2015;6:26–43. doi: 10.15172/pneu.2015.6/520. PMID: 31641576; PMCID: PMC5922337

2. Murphy T. Haemophilus Species (Including H. influenzae and Chancroid). In: Mandell G, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 7th ed. Philadelphia, PA: Churchill Livingstone/Elsevier; 2010. p. 2911–2919.

3. Gilsdorf JR. Hib Vaccines: Their Impact on Haemophilus influenzae Type b Disease. J Infect Dis. 2021;224(Suppl 4):S321–S330. doi: 10.1093/infdis/jiaa537. PMCID: PMC8482018

4. Ulanova M, Tsang RSW. Invasive Haemophilus influenzae disease: changing epidemiology and host-parasite interactions in the 21st century. Infect Genet Evol. 2009;9(4):594–605. doi: 10.1016/j.meegid.2009.03.001. PMID: 19460326

5. Tsang RSW, Bruce MG, Lem M, Barreto L, Ulanova M. A review of invasive Haemophilus influenzae disease in the Indigenous populations of North America. Epidemiol Infect. 2014;142(7):1344–1354. doi: 10.1017/S0950268814000405. PMID: 24598220; PMCID: PMC9151223

6. Ulanova M, Tsang RSW. Haemophilus influenzae serotype a as a cause of serious invasive infections. Lancet Infect Dis. 2014;14(1):70–82. doi: 10.1016/S1473-3099(13)70170-1. PMID: 24268829

7. Sadarangani M. Protection Against Invasive Infections in Children Caused by Encapsulated Bacteria. Front Immunol. 2018;9:2674. doi: 10.3389/fimmu.2018.02674. PMID: 30515161; PMCID: PMC6255856

8. Scapini P, Cassatella MA. Social networking of human neutrophils within the immune system. Blood. 2014;124(5):710–719. doi: 10.1182/blood-2014-03-453217. PMID: 24923297

9. Li Y, Wang W, Yang F, Xu Y, Feng C, Zhao Y. The regulatory roles of neutrophils in adaptive immunity. Cell Commun Signal. 2019;17(1):147. doi: 10.1186/s12964-019-0471-y. PMID: 31727175; PMCID: PMC6854633

10. Moxon ER, Kroll JS. The Role of Bacterial Polysaccharide Capsules as Virulence Factors. In: Jann K, Jann B, editors. Bacterial Capsules. Berlin, Heidelberg: Springer; 1990. p. 65–85. PMID: 2404690

11. Cerquetti M, Cardines R, Ciofi degli Atti ML, Giufré M, Bella A, Sofia T, Mastrantonio P, Slack M. Presence of Multiple Copies of the Capsulation b Locus in Invasive Haemophilus influenzae Type b (Hib) Strains Isolated from Children with Hib Conjugate Vaccine Failure. J Infect Dis. 2005;192(5):819–823. doi: 10.1086/432548. PMID: 16088831

12. Kapogiannis BG, Satola S, Keyserling HL, Farley MM. Invasive Infections with Haemophilus influenzae Serotype a Containing an IS1016-bexA Partial Deletion: Possible Association with Virulence. Clin Infect Dis. 2005;41(11):e97–e103. doi: 10.1086/498028. PMID: 16267724

13. Nix EB, Williams K, Cox AD, St Michael F, Romero-Steiner S, Schmidt DS, McCready WG, Ulanova M. Naturally acquired antibodies against Haemophilus influenzae type a in Aboriginal adults, Canada. Emerg Infect Dis. 2015;21(2):273–279. doi: 10.3201/eid2102.140722. PMID: 25626129; PMCID: PMC4313637

14. Ulanova M, Tsang RSW, Nix EB, Kelly L, Shuel M, Lance B, Canadian Immunization Research Network Investigators. Epidemiology of invasive Haemophilus influenzae disease in northwestern Ontario: comparison of invasive and noninvasive H. influenzae clinical isolates. Can J Microbiol. 2023;69(6):219–227. doi: 10.1139/cjm-2022-0208. PMID: 36753721

15. Tsang RSW, Shuel M, Wylie J, Lefebvre B, Hoang L, Law DKS. Population genetics of Haemophilus influenzae serotype a in three Canadian provinces. Can J Microbiol. 2013;59(5):362–364. doi: 10.1139/cjm-2013-0156. PMID: 23647351

16. Mell JC, Sinha S, Balashov S, Viadas C, Grassa CJ, Ehrlich GD, Nislow C, Redfield RJ, Garmendia J. Complete Genome Sequence of Haemophilus influenzae Strain 375 from the Middle Ear of a Pediatric Patient with Otitis Media. Genome Announc. 2014;2(6):e01245-14. doi: 10.1128/genomeA.01245-14. PMID: 25477405; PMCID: PMC4256186

17. Fleck RA, Romero-Steiner S, Nahm MH. Use of HL-60 Cell Line To Measure Opsonic Capacity of Pneumococcal Antibodies. Clin Diagn Lab Immunol. 2005;12(1):19–27. doi: 10.1128/CDLI.12.1.19-27.2005. PMID: 15642980; PMCID: PMC540204

18. Winter LE, Barenkamp SJ. Human antibodies specific for the high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae mediate opsonophagocytic activity. Infect Immun. 2003;71(12):6884–6891. doi: 10.1128/IAI.71.12.6884-6891.2003. PMCID: PMC308909

19. Müller J, Janz S. In vitro cytotoxicity of neutrophil-like human HL-60 cells undergoing an oxidative burst with Escherichia coli reporter strains. Toxicol In Vitro. 1994;8(3):437–440. doi: 10.1016/0887-2333(94)90165-1. PMID: 20692935

20. Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT. Complement System Part I – Molecular Mechanisms of Activation and Regulation. Front Immunol. 2015;6:262. doi: 10.3389/fimmu.2015.00262. PMID: 26082779; PMCID: PMC4451739

21. Taylor CM, Roberts IS. Capsular Polysaccharides and Their Role in Virulence. Contrib Microbiol. 2005;12:55–66. doi: 10.1159/000081689. PMID: 15496776

22. Duell BL, Su YC, Riesbeck K. Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen. FEBS Letters. 2016;590(21):3840–3853. doi: 10.1002/1873-3468.12351. PMID: 27508518

23. Langereis JD, Weiser JN. Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae. mBio. 2014;5(4):e01478-14. doi: 10.1128/mBio.01478-14. PMCID: PMC4120200

24. Choi J, Nix EB, Gaultier GN, Cox AD, McCready W, Ulanova M. Naturally occurring bactericidal antibodies specific for Haemophilus influenzae lipooligosaccharide are present in healthy adult individuals. Vaccine. 2015;33(16):1941–1947. doi: 10.1016/j.vaccine.2015.02.060. PMID: 25738817

25. Nix EB, Choi J, Anthes C, Gaultier GN, Thorgrimson J, Cox AD, Tsang RSW, McCready WG, Boreham D, Ulanova M. Characterization of natural bactericidal antibody against Haemophilus influenzae type a in Canadian First Nations: A Canadian Immunization Research Network (CIRN) Clinical Trials Network (CTN) study. PLoS One. 2018;13(8):e0201282. doi: 10.1371/journal.pone.0201282. PMCID: PMC6093645

26. Urban CF, Lourido S, Zychlinsky A. How do microbes evade neutrophil killing? Cell Microbiol. 2006;8(11):1687–1696. doi: 10.1111/j.1462-5822.2006.00792.x. PMID: 16939535

27. Dudukina E, de Smit L, Verhagen GJA, van de Ende A, Marimón JM, Bajanca-Lavado P, Ardanuy C, Marti S, de Jonge MI, Langereis JD. Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands. Infect Immun. 2020;88(10):e00454-20. doi: 10.1128/IAI.00454-20. PMCID: PMC7504965

28. Futosi K, Fodor S, Mócsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. 2013;17(3):638–650. doi: 10.1016/j.intimp.2013.06.034. PMCID: PMC3827506

29. Bui TM, Wiesolek HL, Sumagin R. ICAM-1: A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis. J Leukoc Biol. 2020;108(3):787–799. doi: 10.1002/JLB.2MR0220-549R. PMCID: PMC7977775

30. Zhong H, Lin H, Pang Q, Zhuang J, Liu X, Li X, Liu J, Tang J. Macrophage ICAM-1 functions as a regulator of phagocytosis in LPS induced endotoxemia. Inflamm Res. 2021;70(2):193–203. doi: 10.1007/s00011-021-01437-2. PMCID: PMC7817350

31. Wehrli M, Cortinas-Elizondo F, Hlushchuk R, Daudel F, Villiger PM, Miescher S, Zuercher AW, Djonov V, Simon HU, Gunten S von. Human IgA Fc Receptor FcαRI (CD89) Triggers Different Forms of Neutrophil Death Depending on the Inflammatory Microenvironment. J Immunol. 2014;193(11):5649–5659. doi: 10.4049/jimmunol.1400028. PMID: 25339672

32. van Gool MMJ, van Egmond M. IgA and FcαRI: Versatile Players in Homeostasis, Infection, and Autoimmunity. Immunotargets Ther. 2021;9:351–372. doi: 10.2147/ITT.S266242. PMCID: PMC7801909

33. Aleyd E, Hout MWM van, Ganzevles SH, Hoeben KA, Everts V, Bakema JE, Egmond M van. IgA Enhances NETosis and Release of Neutrophil Extracellular Traps by Polymorphonuclear Cells via Fcα Receptor I. J Immunol. American Association of Immunologists; 2014;192(5):2374–2383. doi: 10.4049/jimmunol.1300261. PMID: 24493821

34. Paniagua N, Garcia G, Salgado A, Ventura-Ayala L, Navarro MDC, Torres M, Juarez E, Pedraza-Sanchez S. The IgG Fc receptor, CD16, interacts with LPS and is internalized during the in vitro stimulation of human monocytes. Front Immunol. 2015;6. doi: 10.3389/conf.fimmu.2015.05.00372.

35. Wang Y, Jönsson F. Expression, Role, and Regulation of Neutrophil Fcγ Receptors. Front Immunol. 2019;10:1958. doi: 10.3389/fimmu.2019.01958. PMCID: PMC6718464

36. Qureshi SS, Lewis SM, Gant VA, Treacher D, Davis BH, Brown KA. Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol. 2001;125(2):258–265. doi: 10.1046/j.1365-2249.2001.01596.x. PMCID: PMC1906134

37. Sack U. CD64 expression by neutrophil granulocytes. Cytometry B Clin Cytom. 2017;92(3):189–191. doi: 10.1002/cyto.b.21216. PMID: 25522066

38. Blanter M, Gouwy M, Struyf S. Studying Neutrophil Function in vitro: Cell Models and Environmental Factors. J Inflamm Res. 2021;14:141–162. doi: 10.2147/JIR.S284941. PMCID: PMC7829132

39. Tsang RSW, Ulanova M. The changing epidemiology of invasive Haemophilus influenzae disease: Emergence and global presence of serotype a strains that may require a new vaccine for control. Vaccine. 2017;35(33):4270–4275. doi: 10.1016/j.vaccine.2017.06.001. PMID: 28666758

40. Cox AD, Barreto L, Ulanova M, Bruce MG, Tsang R, Conference contributors. Developing a vaccine for Haemophilus influenzae serotype a: Proceedings of a workshop. Can Commun Dis Rep. 2017;43(5):89–95. doi: 10.14745/ccdr.v43i05a02. PMID: 29770071; PMCID: PMC5764720

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