Associations of Toll-like Receptor Gene Polymorphisms with NETosis Activity as Prognostic Criteria for the Severity of Pneumonia

Karnaushkina M.A., Guryev A.S., Mironov K.O., Dunaeva E.A., Korchagin V.I., Bobkova O.Yu., Vasilyeva I.S., Kassina D.V., Litvinova M.M.

Key words: gene polymorphism; TLR1; TLR2; TLR4; neutrophilic extracellular traps; pneumonia.

2021, volume 13, issue 3, page 47.

DOI: https://doi.org/10.17691/stm2021.13.3.06

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The aim of the study was to determine the molecular genetic prognostic criteria for the severity of the course pneumonia based on the analysis of the association of genetic polymorphism in toll-like receptors with the severity of NETosis.

Materials and Methods. The study included 38 patients with the main diagnosis of community-acquired pneumonia with a severe course. All the patients underwent standard clinical laboratory examinations, computed tomography of the thoracic organs, microbiological examination of blood and tracheobronchial aspirate. The level of neutrophilic extracellular traps (NETs) in blood smears was determined on the 1^st–2^nd and 5^th–7^th days of hospitalization. Genotyping of rs5743551 (TLR1), rs5743708 (TLR2), and rs4986790 (TLR4) polymorphic loci was performed by pyrosequencing.

Results. The level of NETs on the 1^st day of admission was statistically significantly lower in heterozygous and homozygous carriers of rs4986790 (TLR4) polymorphism (AG and GG genotypes) compared with patients with the wild-type genotype (AA genotype) (p<0.05). When comparing the number of NETs with genotypes for rs5743708 (TLR2) and rs5743551 (TLR1) polymorphisms, no statistically significant correlation was found (p>0.05). The study of the NET level in dynamics demonstrated a decrease in the NETosis activity of neutrophils during the first week of hospitalization (p<0.05). The presence of the G allele in the patient’s genotype for rs5743551 (TLR1) polymorphism increases the risk of a poor outcome of the disease (p<0.0001) (OR=20.3; 95% CI (4.3–135.0)).

Conclusion. The obtained data suggest that level of NETs is a marker of the activity of neutrophils which are closely related to the studied genetic polymorphisms, and affects the prognosis of the pneumonia outcome.

Dandachi D., Rodriguez-Barradas M.C. Viral pneumonia: etiologies and treatment. J Investig Med 2018; 66(6): 957–965, https://doi.org/10.1136/jim-2018-000712.
Bilichenko T.N., Bystritskaya E.V., Chuchalin A.G., Belevsky A.S., Batyn S.Z. Mortality from respiratory diseases in 2014–2015 and ways to reduce it. Pulmonologiya 2016; 26(4): 389–397, https://doi.org/10.18093/0869-0189-2016-26-4-389-397.
Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y., Weiss D.S., Weinrauch Y., Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science 2004; 303(5663): 1532–1535, https://doi.org/10.1126/science.1092385.
Fuchs T.A., Abed U., Goosmann C., Hurwitz R., Schulze I., Wahn V., Weinrauch Y., Brinkmann V., Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007; 176(2): 231–241, https://doi.org/10.1083/jcb.200606027.
Sousa-Rocha D., Thomaz-Tobias M., Diniz L.F., Souza P.S., Pinge-Filho P., Toledo K.A. Trypanosoma cruzi and its soluble antigens induce NET release by stimulating toll-like receptors. PLoS One 2015; 10(10): e0139569, https://doi.org/10.1371/journal.pone.0139569.
Yipp B.G., Petri B., Salina D., Jenne C.N., Scott B.N., Zbytnuik L.D., Pittman K., Asaduzzaman M., Wu K., Meijndert H.C., Malawista S.E., de Boisfleury Chevance A., Zhang K., Conly J., Kubes P. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 2012; 18(9): 1386–1393, https://doi.org/10.1038/nm.2847.
Yipp B.G., Kubes P. NETosis: how vital is it? Blood 2013; 122(16): 2784–2794, https://doi.org/10.1182/blood-2013-04-457671.
Rijkers G.T., Holzer L., Dusselier T. Genetics in community-acquired pneumonia. Curr Opin Pulm Med 2019; 25(3): 323–329, https://doi.org/10.1097/mcp.0000000000000580.
Pilsczek F.H., Salina D., Poon K.K., Fahey C., Yipp B.G., Sibley C.D., Robbins S.M., Green F.H., Surette M.G., Sugai M., Bowden M.G., Hussain M., Zhang K., Kubes P. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 2010; 185(12): 7413–7425, https://doi.org/10.4049/jimmunol.1000675.
Siebert J.N., Hamann L., Verolet C.M., Gameiro C., Grillet S., Siegrist C.A., Posfay-Barbe K.M. Toll-interleukin 1 receptor domain-containing adaptor protein 180L single-nucleotide polymorphism is associated with susceptibility to recurrent pneumococcal lower respiratory tract infections in children. Front Immunol 2018; 9: 1780, https://doi.org/10.3389/fimmu.2018.01780.
Wurfel M.M., Gordon A.C., Holden T.D., Radella F., Strout J., Kajikawa O., Ruzinski J.T., Rona G., Black R.A., Stratton S., Jarvik G.P., Hajjar A.M., Nickerson D.A., Rieder M., Sevransky J., Maloney J.P., Moss M., Martin G., Shanholtz C., Garcia J.G., Gao L., Brower R., Barnes K.C., Walley K.R., Russell J.A., Martin T.R. Toll-like receptor 1 polymorphisms affect innate immune responses and outcomes in sepsis. Am J Respir Crit Care Med 2008; 178(7): 710–720, https://doi.org/10.1164/rccm.200803-462oc.
Ministry of Health of the Russian Federation. Vnebol’nichnaya pnevmoniya u vzroslykh: klinicheskie rekomendatsii [Community-acquired pneumonia in adults: clinical guidelines]. Moscow; 2019; 97 р.
Fine M.J., Auble T.E., Yealy D.M., Hanusa B.H., Weissfeld L.A., Singer D.E., Coley C.M., Marrie T.J., Kapoor W.N. A prediction rule to identify low-risk patients with community-acquired pneumonia. N Engl J Med 1997; 336(4): 243–250, https://doi.org/10.1056/nejm199701233360402.
Mukhopadhyay A., Tai B.C., See K.C., Ng W.Y., Lim T.K., Onsiong S., Ee S., Chua M.J., Lee P.R., Loh M.L., Phua J. Risk factors for hospital and long-term mortality of critically ill elderly patients admitted to an intensive care unit. Biomed Res Int 2014; 2014: 960575, https://doi.org/10.1155/2014/960575.
Gur’ev A.S., Mosal’skaya D.V., Volkov A.Yu. Method for determining the relative amount of etiologically transformed phagocytes. Patent RU 2712179. 2019.
Mironov K.O., Dunaeva E.A., Dribnokhodova O.P., Shipulin G.A. Experience in using genetic analysis systems based on pyrosequencing technology. Spravocnik zaveduusego KDL 201-6; 5: 33–42.
Kloek A.T., Brouwer M.C., van de Beek D. Host genetic variability and pneumococcal disease: a systematic review and meta-analysis. BMC Med Genomics 2019; 12(1): 130, https://doi.org/10.1186/s12920-019-0572-x.
Kumpf O., Giamarellos-Bourboulis E.J., Koch A., Hamann L., Mouktaroudi M., Oh D.Y., Latz E., Lorenz E., Schwartz D.A., Ferwerda B., Routsi C., Skalioti C., Kullberg B.J., van der Meer J.W., Schlag P.M., Netea M.G., Zacharowski K., Schumann R.R. Influence of genetic variations in TLR4 and TIRAP/Mal on the course of sepsis and pneumonia and cytokine release: an observational study in three cohorts. Crit Care 2010; 14(3): R103, https://doi.org/10.1186/cc9047.
Agnese D.M., Calvano J.E., Hahm S.J., Coyle S.M., Corbett S.A., Calvano S.E., Lowry S.F. Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections. J Infect Dis 2002; 186(10): 1522–1525, https://doi.org/10.1086/344893.
Lorenz E., Mira J.P., Frees K.L., Schwartz D.A. Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock. Arch Intern Med 2002; 162(9): 1028–1032, https://doi.org/10.1001/archinte.162.9.1028.
Rahman S., Gadjeva M. Does NETosis contribute to the bacterial pathoadaptation in cystic fibrosis? Front Immunol 2014; 5: 378, https://doi.org/10.3389/fimmu.2014.00378.
Beiter K., Wartha F., Albiger B., Normark S., Zychlinsky A., Henriques-Normark B. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006; 16(4): 401–407, https://doi.org/10.1016/j.cub.2006.01.056.
Buchanan J.T., Simpson A.J., Aziz R.K., Liu G.Y., Kristian S.A., Kotb M., Feramisco J., Nizet V. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 2006; 16(4): 396–400, https://doi.org/10.1016/j.cub.2005.12.039.
Thammavongsa V., Missiakas D.M., Schneewind O. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science 2013; 342(6160): 863–866, https://doi.org/10.1126/science.1242255.
van Strijp J.A., Rooijakkers S.H. Entrapment exploited. Trends Microbiol 2014; 22(2): 55–57, https://doi.org/10.1016/j.tim.2013.12.010.
Berends E.T.M., Horswill A.R., Haste N.M., Monestier M., Nizet V., von Köckritz-Blickwede M. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2010; 2(6): 576–586, https://doi.org/10.1159/000319909.
Gur’ev A., Mosalskaia D., Lopatin A., Volkov A. Prognostic value of cellular markers in sepsis: extracellular DNA traps and platelet count relation. Berlin, 32nd Annual Congress of the European Society of Intensive Care Medicine ESICM LIVES 2019. Intensive Care Med Exp 2019; 000809: 237–238, https://doi.org/10.1186/s40635-019-0265-y.
Hoogerwerf J.J., de Vos A.F., Bresser P., van der Zee J.S., Pater J.M., de Boer A., Tanck M., Lundell D.L., Her-Jenh C., Draing C., von Aulock S., van der Poll T. Lung inflammation induced by lipoteichoic acid or lipopolysaccharide in humans. Am J Respir Crit Care Med 2008; 178(1): 34–41, https://doi.org/10.1164/rccm.200708-1261oc.
Skerrett S.J., Braff M.H., Liggitt H.D., Rubens C.E. Toll-like receptor 2 has a prominent but nonessential role in innate immunity to Staphylococcus aureus pneumonia. Physiol Rep 2017; 5(21): e13491, https://doi.org/10.14814/phy2.13491.

Karnaushkina M.A., Guryev A.S., Mironov K.O., Dunaeva E.A., Korchagin V.I., Bobkova O.Yu., Vasilyeva I.S., Kassina D.V., Litvinova M.M. Associations of Toll-like Receptor Gene Polymorphisms with NETosis Activity as Prognostic Criteria for the Severity of Pneumonia. Sovremennye tehnologii v medicine 2021; 13(3): 47, https://doi.org/10.17691/stm2021.13.3.06