Immunophenotype and metabolism are linked in peripheral blood neutrophils from patients with kidney cancer

The aim of the present study was to analyze the relationships between expression of activation and adhesion receptors on peripheral blood neutrophils, and intracellular activity of some neutrophil enzymes in patients with kidney cancer (KC). Patients and methods: the KC patients (n = 72) (T3N0M0, clear-cell type) were examined prior to surgical treatment at the Krasnoyarsk Regional Oncology Center. The diagnosis was verified histologically for all KC patients. The phenotype of blood neutrophils was studied using flow cytometry. The surface receptor expression levels of the neutrophils were evaluated by mean fluorescence intensity. NAD and NADP-dependent dehydrogenases activities in purified peripheral blood neutrophils were measured by bioluminescent method. Results: we have found that the phenotypic alterations in circulating KC patients’ neutrophils appeared along with inhibition of main intracellular metabolic processes and were closely linked with them. The features of the phenotypic imbalance in the neutrophils from KC patients were associated with a decrease in blood cells expressing adhesive (CD11b and CD62L) and functional (CD64 and HLA-DR) receptors. Moreover, the patient’s neutrophils expressed CD11b, CD16 and HLA-DR on their cell surface more intensively, than neutrophilic leukocytes from control group. These phenotypic changes in KC patients’ blood neutrophils occurred in parallel with pronounced decrease in immature cells numbers. The metabolic changes of neutrophil cytoplasmic compartment in KC patients were determined by a decrease in Glu6PDH activity (a key and initializing enzyme of the pentose phosphate cycle) and NADH-LDH (anaerobic glycolysis). Mitochondrial metabolism in neutrophils of KC patients was characterized by multidirectional changes in the activity of NAD- and NADP-dependent glutamate dehydrogenases (decreased activity of NAD-dependent and increased activity of NADP-dependent) and a decrease in NADH-MDH activity. The established features in mitochondrial enzymes activities suggest some disturbances of NAD-dependent processes that could lead to down-regulation of aerobic energy processes. We guess that the decreased activity of plastic and energy processes in blood neutrophils of KC patients could affect the receptor expression levels. By means of correlation analysis, we have found that the relationships in KC patients were determined by negative effects of NADHGDH and NADH-LDH activities upon expression of activation and adhesion receptors in blood neutrophils. Of these enzymes, only glutathione reductase activity in neutrophils from KC patients was positively linked with the CD23 and HLA-DR expression. Thus, an increase in activity of energy processes (e.g., coupling the tricarboxylic acid cycle to amino acid metabolism) in blood neutrophils from the patients with kidney cancer could stimulate expression levels of activation and adhesion receptors and potentially increase antitumor activity of neutrophils.

1. Bao Y., Ledderose C., Seier T., Graf A.F., Brix B., Chong E., Junger W.G. Mitochondria regulate neutrophil activation by generating ATP for autocrine purinergic signaling. J. Biol. Chem., 2014, Vol. 289, no. 39, pp. 26794-26803.

2. Bednarska K., Klink M., Wilczyński J.R., Szyłło K., Malinowski A., Sułowska Z., Nowak M. Heterogeneity of the Mac-1 expression on peripheral blood neutrophils in patients with different types of epithelial ovarian cancer. Immunobiology, 2016, Vol. 221, no. 2, pp. 323-332.

3. Brandau S., Dumitru C.A., Lang S. Protumor and antitumor functions of neutrophil granulocytes. Semin. Immunopathol., 2013, Vol. 35, no. 2, pp. 163-176.

4. da Silva K.D., Caldeira P.C., Alves A.M., Vasconcelos A.C.U., Gomes A.P.N., de Aguiar M.C.F., Tarquinio S.B.C. High CD3(+) lymphocytes, low CD66b(+) neutrophils, and scarce tumor budding in the invasive front of lip squamous cell carcinomas. Arch. Oral. Biol., 2019, Vol. 104, pp. 46-51.

5. Dahlgren C., Gabl M., Holdfeldt A., Winther M., Forsman H. Basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria. Biochem. Pharmacol., 2016, Vol. 114, pp. 22-39.

6. Delebarre M., Dessein R., Lagrée M., Mazingue F., Sudour-Bonnange H., Martinot A., Dubos F. Differential risk of severe infection in febrile neutropenia among children with blood cancer or solid tumor. J. Infect., 2019, Vol. 79, no. 2, pp. 95-100.

7. Fan H.J., Tan Z.B., Wu Y.T., Feng X.R., Bi Y.M., Xie L.P., Zhang W.T., Ming Z., Liu B., Zhou Y.C. The role of ginsenoside Rb1, a potential natural glutathione reductase agonist, in preventing oxidative stress-induced apoptosis of H9C2 cells. J. Ginseng. Res., 2020, Vol. 44, no. 2, pp. 258-266.

8. Gatti A., Ceriani C., De Paschale M., Magnani C., Villa M., Viganò P., Clerici P., Brando B. Quantification of neutrophil and monocyte CD64 expression: a predictive biomarker for active tuberculosis. Int. J. Tuberc. Lung Dis., 2020, Vol. 24, no. 2, pp. 196-201.

9. Giese M.A., Hind L.E., Huttenlocher A. Neutrophil plasticity in the tumor microenvironment. Blood, 2019, Vol. 133, no. 20, pp. 2159-2167.

10. Goto K., Matsuyama R., Suwa Y., Arisaka S., Kadokura T., Sato M., Mori R., Kumamoto T., Taguri M., Endo I. The maximum chemiluminescence intensity predicts severe neutropenia in gemcitabine-treated patients with pancreatic or biliary tract cancer. Cancer Chemother. Pharmacol., 2018, Vol. 82, no. 6, pp. 953-960.

11. Granot Z. Neutrophils as a therapeutic target in cancer. Front. Immunol., 2019, Vol. 10, 1710. doi: 10.3389/fimmu.2019.01710.

12. Kelm M., Lehoux S., Azcutia V., Cummings R.D., Nusrat A., Parkos C.A., Brazil J.C. Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18. FASEB J., 2020, Vol. 34, no. 2, pp. 2326-2343.

13. Kudryavtsev I.V., Subbotovskaya A.I. Application of six-color flow cytometric analysis for immune profile monitoring. Medical Immunology (Russia), 2015, Vol. 17, no. 1, pp. 19-26. doi: 10.15789/1563-0625-2015-1-19-26.

14. Kumar S., Dikshit M. Metabolic insight of neutrophils in health and disease. Front. Immunol., 2019, Vol. 10, 2099. doi: 10.3389/fimmu.2019.02099.

15. Kurtasova L.M., Savchenko A.A., Shkapova E.A. Clinical aspects of functional disorders of neutrophilic granulocytes in oncopathology. Novosibirsk: Nauka, 2009. 183 p.

16. Lokwani R., Wark P.A., Baines K.J., Fricker M., Barker D., Simpson J.L. Blood Neutrophils In COPD But Not Asthma Exhibit A Primed Phenotype With Downregulated CD62L Expression. Int. J. Chron. Obstruct. Pulmon. Dis., 2019, Vol. 14, pp. 2517-2525.

17. Maecker H., McCoy P., Nussenblatt R. Standardizing immunophenotyping for the human immunology project. Nat. Rev. Immunol., 2012, Vol. 12, pp. 191-200.

18. Mahmoodpoor A., Paknezhad S., Shadvar K., Hamishehkar H., Movassaghpour A.A., Sanaie S., Ghamari A.A., Soleimanpour H. Flow cytometry of CD64, HLA-DR, CD25, and TLRs for diagnosis and prognosis of sepsis in critically ill patients admitted to the intensive care unit: a review article. Anesth. Pain. Med., 2018, Vol. 8, no. 6, e83128. doi: 10.5812/aapm.83128.

19. Matlung H.L., Babes L., Zhao X.W., van Houdt M., Treffers L.W., van Rees D.J., Franke K., Schornagel K., Verkuijlen P., Janssen H., Halonen P., Lieftink C., Beijersbergen R.L., Leusen J.H.W., Boelens J.J., Kuhnle I., van der Werff Ten Bosch J., Seeger K., Rutella S., Pagliara D., Matozaki T., Suzuki E., Menke-van der Houven van Oordt C.W., van Bruggen R., Roos D., van Lier R.A.W., Kuijpers T.W., Kubes P., van den Berg T.K. Neutrophils Kill antibodyopsonized cancer cells by trogoptosis. Cell Rep., 2018, Vol. 23, no. 13, pp. 3946-3959.

20. Mishalian I., Granot Z., Fridlender Z.G. The diversity of circulating neutrophils in cancer. Immunobiology, 2017, Vol. 222, Iss. 1, pp. 82-88.

21. Pan Z., Zhang L., Liu C., Huang X., Shen S., Lin X., Shi C. Cisplatin or carboplatin? Neutrophil to lymphocyte ratio may serve as a useful factor in small cell lung cancer therapy selection. Oncol. Lett., 2019, Vol. 18, no. 2, pp. 1513-1520.

22. Pirozzolo G., Gisbertz S.S., Castoro C., van Berge Henegouwen M.I., Scarpa M. Neutrophil-to-lymphocyte ratio as prognostic marker in esophageal cancer: a systematic review and meta-analysis. J. Thorac. Dis., 2019, Vol. 11, no. 7, pp. 3136-3145.

23. Rice C.M., Davies L.C., Subleski J.J., Maio N., Gonzalez-Cotto M., Andrews C., Patel N.L., Palmieri E.M., Weiss J.M., Lee J.M., Annunziata C.M., Rouault T.A., Durum S.K., McVicar D.W. Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression. Nat. Commun., 2018, Vol. 9, no. 1, 5099. doi: 10.1038/s41467-018-07505-2.

24. Richer B.C., Salei N., Laskay T., Seeger K. Changes in neutrophil metabolism upon activation and aging. Inflammation, 2018, Vol. 41, no. 2, pp. 710-721.

25. Savchenko A.A. Evaluation of NAD(P)-dependent dehydrogenase activities in neutrophilic granulocytes by the bioluminescent method. Bulletin of Experimental Biology and Medicine (Russia), 2015, Vol. 159, no. 5, pp. 692-695.

26. Savchenko A.A., Zdzitovetskii D.E., Borisov A.G., Luzan N.A. Chemiluminescent and enzyme activity of neutrophils in patients with widespread purulent peritonitis depending on the outcome of disease. Annals of the Russian Academy of Medical Sciences, 2014, Vol. 69, no. 5-6, pp. 23-28.

27. Savchenko А.А., Borisov A.G., Cherdancev D.V., Pervova O.V., Kudryavtsev I.V., Gvozdev I.I., Moshev A.V. Features of the phenotype and NAD(P)-dependent dehydrogenases activity in neutrophil by patients with widespread purulent peritonitis in prognosis for sepsis development. Russian Journal of Infection and Immunity, 2018, Vol. 8, no. 3, pp. 369-376.

28. Shkapova E.A., Kurtasova L.M., Savchenko A.A. Lucigenin- and luminol-dependent chemiluminescence of blood neutrophils in patients with renal cancer. Bulletin of Experimental Biology and Medicine, 2010, Vol. 149, no. 2, pp. 239-241.

29. Sica A., Guarneri V., Gennari A. Myelopoiesis, metabolism and therapy: a crucial crossroads in cancer progression. Cell Stress, Vol. 3, no. 9, pp. 284-294.

30. Sumida K., Wakita D., Narita Y., Masuko K., Terada S., Watanabe K., Satoh T., Kitamura H., Nishimura T. Anti-IL-6 receptor mAb eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T-cell responses. Eur. J. Immunol., 2012, Vol. 42, no. 8, pp. 2060-2072.

31. Tan C., Gu J., Chen H., Li T., Deng H., Liu K., Liu M., Tan S., Xiao Z., Zhang H., Xiao X. Inhibition of aerobic glycolysis promotes neutrophil to influx to the infectious site via CXCR2 in sepsis. Shock, 2020, Vol. 53, no. 1, pp. 114-123.

32. Thwe P.M., Ortiz D.A., Wankewicz A.L., Hornak J.P., Williams-Bouyer N., Ren P. Closing the Brief case: recurrent chromobacterium violaceum bloodstream infection in a glucose-6-phosphate dehydrogenase (G6PD)- deficient patient with a severe neutrophil defect. J. Clin. Microbiol., 2020, Vol. 58, no. 2, pii: e00314-19. doi: 10.1128/JCM.00314-19.

33. Veglia F., Perego M., Gabrilovich D. Myeloid-derived suppressor cells coming of age. Nat. Immunol., 2018, Vol. 19, no. 2, pp. 108-119.

34. Veglia F., Tyurin V.A., Blasi M., De Leo A., Kossenkov A.V., Donthireddy L., To T.K.J., Schug Z., Basu S., Wang F., Ricciotti E., DiRusso C., Murphy M.E., Vonderheide R.H., Lieberman P.M., Mulligan C., Nam B., Hockstein N., Masters G., Guarino M., Lin C., Nefedova Y., Black P., Kagan V.E., Gabrilovich D.I. Fatty acid transport protein 2 reprograms neutrophils in cancer. Nature, 2019, Vol. 569, no. 7754, pp. 73-78.

35. Won W.J., Deshane J.S., Leavenworth J.W., Oliva C.R., Griguer C.E. Metabolic and functional reprogramming of myeloid-derived suppressor cells and their therapeutic control in glioblastoma. Cell Stress, 2019, Vol. 3, no. 2, pp. 47-65.

36. Wu L., Saxena S., Awaji M., Singh R.K. Tumor-associated neutrophils in cancer: Going Pro. Cancers (Basel), 2019, Vol. 11, no. 4, E564. doi: 10.3390/cancers11040564.

37. Zeindler J., Angehrn F., Droeser R., Däster S., Piscuoglio S., Ng C.K.Y., Kilic E., Mechera R., Meili S., Isaak A., Weber W.P., Muenst S., Soysal S.D. Infiltration by myeloperoxidase-positive neutrophils is an independent prognostic factor in breast cancer. Breast Cancer Res. Treat., 2019, Vol. 177, no. 3, pp. 581-589.