Incorporation of galactose in the lipooligosaccharide of non-typeable Haemophilus influenzae

    Langereis, J.D. | St. Radboud Universiteit Nijmegen | 11 augustus 2014 | 7.1.14.046CO

International Symposium Neutrophil 2014 - Incorporation of galactose in the lipooligosaccharide of non-typeable Haemophilus influenzae prevents IgM binding, complement activation and phagocytosis by neutrophils.

International Symposium Neutrophil 2014 in Montreal, Canada, which was held from May 31st through June 3rd was the second bi-annual symposium (first symposium was in 2012) focused on the role of neutrophillic granulocytes (Neutrophils) in health and disease. This year, over 300 experts on neutrophil research attended this meeting. During this symposium, 35 25 minute talks and 6 15 minute “trainee” talks were presented.
This symposium included 6 sessions:
I) Neutrophil functional responses,
II) Regulatory mechanisms,
III) Neutrophils in inflammation and immunity,
IV) Poster session with 170 posters,
V) Pathogen-neutrophil interaction and
VI) Neutrophils as therapeutic target.
Especially neutrophil extracellular trap (NET) formation was a topic addressed extensively during this symposium. In addition, other new functions for neutrophils in immune regulation and pathogenesis were presented. 

Neutrophils in health and disease

Neutrophillic granulocytes are the largest white blood cell population in the blood. Neutrophils are a crucial component of the innate immune system that control bacterial and fungal infection through various killing mechanisms (1). These mechanisms include 1) a powerful oxidative burst, 2) the release of proteolytic enzymes stored in granules, 3) phagocytic capacity and 4) the formation of NETs (1).
Although neutrophils are professional phagocytes, pathogens have developed mechanisms that prevent phagocytosis or extracellular killing by neutrophils. In addition, influx and activation of neutrophils are also often associated with tissue damage and, therefore, contribute to tissue pathology in patients with acute or chronic diseases.


Non-typeable Haemophilus influenzae and neutrophil-mediated killing

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human restricted pathogen. NTHi is one of the most frequently isolated bacterium colonizing the lower airways of patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchiectasis or asthma (2-5). The immune response to NTHi includes secretion of cytokines and chemotactic compounds by the respiratory epithelium and resident immune cells, which attracts various types of immune cell, including large numbers of neutrophils, to the site of inflammation (3, 6). Neutrophils posses multiple anti-microbial functions contributing to the clearance of bacterial pathogens (7). Even though neutrophils are specialized killers, these cells are often not able to clear NTHi infections from the lungs, resulting in bacterial colonization for extended periods of time (2).

During the poster session (Session IV), I presented my work with the title: Incorporation of galactose in the lipooligosaccharide of non-typeable Haemophilus influenzae prevents IgM binding, complement activation and phagocytosis by neutrophils.

Bacteria need to be opsonized by antibodies and/or complement factors such as C3b and C5b to be efficiently phagocytosed by neutrophils (8). Unencapsulated bacteria, like NTHi, are particularly susceptible to complement-mediated killing and subsequent phagocytosis by neutrophils due to the lack of a protective surface structure. In response to this threat, NTHi has developed multiple strategies to evade the bactericidal activity of the human complement system. For instance, NTHi binds complement control proteins C4b binding protein (C4BP) and factor H (9-11). In addition, variation and modifications in its lipooligosaccharide (LOS) composition has been shown to prevent complement activation (12-15). 

We used transposon sequencing (Tn-seq) technology to identify genes required for NTHi to survive in the presence of human neutrophils. We identified an important role for phase-variable incorporation of oligosaccharides into the LOS of NTHi, shielding an epitope that is recognized by naturally-acquired IgM, thereby preventing complement activation, opsonization and killing by human neutrophils.

Other host-pathogen interaction data presented

Various oral and poster presentations covered host-pathogen interactions during this symposium. Especially session V, Pathogen-neutrophil interaction, was of interest, although it covered mostly parasite infections. Prof. Akira Takashima showed convincing data that neutrophils could differentiate into a hybrid leukocyte population exhibiting dual functionality of professional phagocyte, the general action of neutrophils, as well as an antigen presenting phenotype for which more data is available in literature (16-18).

A large number of posters in session IV covered the inability of NETs to directly kill pathogens such as Mycobacterium tuberculosis (Filio-Rodriguez et. al.), Candida albicans (Rapala-Kozik et. al.) and Staphylococcus aureus (Lefrancais et. al.). Interestingly, Lefrancais et. al. showed that formation of NETs in a mouse model for S. aureus lung infections was associated with increased lung permeability and increased death. Therefore, formation of NETs in the lungs appears to be increase lung damage. Since NTHi was shown to induce NETs in vitro (19), this phenomenon might also contribute to disease pathology due to long-term colonization of the lungs with NTHi.

Multiple mechanisms used by a variety of pathogens were found to impair neutrophil function. For instance, Neisseria gonorrhoeae prevented ROS production by eliminating expression of opacity-associated proteins (Smirnov et. al.), Pseudomonas aeruginosa inhibited granule fusion and ROS production, which was dependent on the type III secretion system (Vareechon et. al.), Yersinia YopH interfered with neutrophil signal transduction pathways (Mecsas et. al.) and Streptococus pneumonia evaded neutrophil responses through hydrolysis of platelet-activating factor (PAF) (Hergott et. al.). 

Conclusions

The neutrophil field is expanding because novel functions, such as antigen presentation and immune regulation, are identified for this innate immune cell. However, the “classical” anti-microbial functions are shown to be beneficial, as well as detrimental in development of various acute and chronic diseases. Therefore, increasing bactericidal activity of neutrophils without increasing tissue damage might contribute to eliminating long-term bacterial colonization of the lungs of patients with COPD and other chronic lung diseases and thereby preventing further disease progression.


References
1. Nathan C. 2006. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6:173-182.
2. Murphy TF, Brauer AL, Schiffmacher AT, Sethi S. 2004. Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease. American journal of respiratory and critical care medicine 170:266-272.
3. Hill AT, Campbell EJ, Hill SL, Bayley DL, Stockley RA. 2000. Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis. The American journal of medicine 109:288-295.
4. Roman F, Canton R, Perez-Vazquez M, Baquero F, Campos J. 2004. Dynamics of long-term colonization of respiratory tract by Haemophilus influenzae in cystic fibrosis patients shows a marked increase in hypermutable strains. Journal of clinical microbiology 42:1450-1459.
5. Zhang Q, Illing R, Hui CK, Downey K, Carr D, Stearn M, Alshafi K, Menzies-Gow A, Zhong N, Fan Chung K. 2012. Bacteria in sputum of stable severe asthma and increased airway wall thickness. Respiratory research 13:35.
6. Zhang M, Li Q, Zhang XY, Ding X, Zhu D, Zhou X. 2010. Relevance of lower airway bacterial colonization, airway inflammation, and pulmonary function in the stable stage of chronic obstructive pulmonary disease. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 29:1487-1493.
7. Kolaczkowska E, Kubes P. 2013. Neutrophil recruitment and function in health and inflammation. Nature reviews. Immunology 13:159-175.
8. Langereis JD. 2013. Neutrophil integrin affinity regulation in adhesion, migration, and bacterial clearance. Cell adhesion & migration 7:476-481.
9. Hallstrom T, Jarva H, Riesbeck K, Blom AM. 2007. Interaction with C4b-binding protein contributes to nontypeable Haemophilus influenzae serum resistance. Journal of immunology 178:6359-6366.
10. Hallstrom T, Zipfel PF, Blom AM, Lauer N, Forsgren A, Riesbeck K. 2008. Haemophilus influenzae interacts with the human complement inhibitor factor H. Journal of immunology 181:537-545.
11. Rosadini CV, Ram S, Akerley BJ. 2014. Outer Membrane Protein P5 Is Required for Resistance of Nontypeable Haemophilus influenzae to Both the Classical and Alternative Complement Pathways. Infection and immunity 82:640-649.
12. Langereis JD, Stol K, Schweda EK, Twelkmeyer B, Bootsma HJ, de Vries SP, Burghout P, Diavatopoulos DA, Hermans PW. 2012. Modified lipooligosaccharide structure protects nontypeable Haemophilus influenzae from IgM-mediated complement killing in experimental otitis media. mBio 3:e00079-00012.
13. Ho DK, Ram S, Nelson KL, Bonthuis PJ, Smith AL. 2007. lgtC expression modulates resistance to C4b deposition on an invasive nontypeable Haemophilus influenzae. Journal of immunology 178:1002-1012.
14. Clark SE, Snow J, Li J, Zola TA, Weiser JN. 2012. Phosphorylcholine allows for evasion of bactericidal antibody by Haemophilus influenzae. PLoS pathogens 8:e1002521.
15. Virji M, Weiser JN, Lindberg AA, Moxon ER. 1990. Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells. Microbial pathogenesis 9:441-450.
16. Pliyev BK, Sumarokov AB, Buriachkovskaia LI, Menshikov M. 2011. Extracellular acidosis promotes neutrophil transdifferentiation to MHC class II-expressing cells. Cellular immunology 271:214-218.
17. Ostanin DV, Kurmaeva E, Furr K, Bao R, Hoffman J, Berney S, Grisham MB. 2012. Acquisition of antigen-presenting functions by neutrophils isolated from mice with chronic colitis. Journal of immunology 188:1491-1502.
18. Matsushima H, Geng S, Lu R, Okamoto T, Yao Y, Mayuzumi N, Kotol PF, Chojnacki BJ, Miyazaki T, Gallo RL, Takashima A. 2013. Neutrophil differentiation into a unique hybrid population exhibiting dual phenotype and functionality of neutrophils and dendritic cells. Blood 121:1677-1689.
19. Juneau RA, Pang B, Weimer KE, Armbruster CE, Swords WE. 2011. Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps. Infection and immunity 79:431-438.

 

Keyword: neutrophillic granulocytes

Terug naar het overzicht

Gratis nieuwsbrief

Blijf op de hoogte van het laatste nieuws over subsidies, zorg en onderzoek naar astma, COPD en andere longziekten.