A New Approach to Analyze the State of the Complement System in Patients with COVID-19. Pilot Study

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Дәйексөз келтіру

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Аннотация

One of the risk factors for infection caused by SARS-CoV-2 is hyperactivation of the complement system, which can lead to activation and damage to the endothelium, thrombosis and, in some severe cases, the development of multiple organ failure. Using quantitative immunohistochemistry, we studied the opsonization of human umbilical vein endothelial cells (HUVEC) by complement factors C3/C3b when exposed to blood plasma from patients with a confirmed diagnosis of COVID-19 in order to evaluate the possibility of using this approach to study the state of the complement system in COVID-19 ex vivo. For these purposes, FITC-labeled antibodies specific to the C3c domain of factors C3/C3b were used. The integral intensity of the recorded fluorescence and the number of C3/C3b-immunopositive structures were chosen as parameters for assessing the binding of C3/C3b to cells. We have shown that when HUVEC are incubated with plasma from patients, the total number of C3/C3b-immunopositive structures recorded on the membrane of these cells is 5.8 ± 2.8 times (mean ± SD, p < 0.001) higher than when exposed to plasma from healthy people. In this case, the integrated fluorescence intensity increased by 6.3 ± 3.2 times (mean ± SD, p ≤ 0.0001) compared to the control. The area of immunopositive structures recorded after exposure to plasma cells from healthy donors and patients with COVID-19 and selected for analysis ranged from 2.2 to 70 μm². Immunopositive particles with an area of 2.2–10.9 μm² after incubation with plasma of COVID-19 patients had a more elongated shape compared to controls. The average number of particles per cell was 0.49 ± 0.06 (mean ± SD, n = 6) in the control, and 2.4 ± 0.4 (mean ± SD, n = 13, p < 0.001) during incubation with patient plasma. Analysis of particle area distribution showed that the most pronounced differences in the number of C3/C3b-immunopositive structures compared to the control were observed among large particles. Thus, we showed an increase in the level of opsonization of endothelial cells by complement factors C3/C3b in the presence of plasma from COVID-19 patients in comparison with control plasma. The increase is due to an increase in the number of C3/C3b-immunopositive structures, mostly large ones. We assume that the proposed approach will allow us to study the role of the complement system in damage to vascular endothelial cells in patients with COVID-19 using an ex vivo model, as well as to evaluate the level of complement activation in the plasma of patients and the effectiveness of their treatment.

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Авторлар туралы

P. Avdonin

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

Хат алмасуға жауапты Автор.
Email: ppavdonin@gmail.com
Ресей, Moscow, 119334

L. Komleva

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

Email: ppavdonin@gmail.com
Ресей, Moscow, 119334

M. Blinova

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

Email: ppavdonin@gmail.com
Ресей, Moscow, 119334

Е. Ivanova

Moscow City Clinical Hospital 52

Email: ppavdonin@gmail.com
Ресей, Moscow, 123182

O. Kotenko

Moscow City Clinical Hospital 52

Email: ppavdonin@gmail.com
Ресей, Moscow, 123182

N. Frolova

Moscow City Clinical Hospital 52

Email: ppavdonin@gmail.com
Ресей, Moscow, 123182

E. Stolyarevich

Moscow City Clinical Hospital 52

Email: ppavdonin@gmail.com
Ресей, Moscow, 123182

E. Rybakova

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

Email: ppavdonin@gmail.com
Ресей, Moscow, 119334

P. Avdonin

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

Email: ppavdonin@gmail.com
Ресей, Moscow, 119334

Әдебиет тізімі

  1. Cugno M., Meroni P.L., Gualtierotti R., Griffini S., Grovetti E., Torri A., Lonati P., Grossi C., Borghi M.O., Novembrino C., Boscolo M., Uceda Renteria S.C., Valenti L., Lamorte G., Manunta M., Prati D., Pesenti A., Blasi F., Costantino G., Gori A., Bandera A., Tedesco F., Peyvandi F. 2021. Complement activation and endothelial perturbation parallel COVID-19 severity and activity. J. Autoimmun. 116, 102560. doi: 10.1016/j.jaut.2020.102560
  2. Noris M., Benigni A., Remuzzi G. 2020. The case of complement activation in COVID-19 multiorgan impact. Kidney Int. 98, 314–322. doi: 10.1016/j.kint.2020.05.013
  3. Cugno M., Gualtierotti R., Possenti I., Testa S., Tel F., Griffini S., Grovetti E., Tedeschi S., Salardi S., Cresseri D., Messa P., Ardissino G. 2014. Complement functional tests for monitoring eculizumab treatment in patients with atypical hemolytic uremic syndrome. J. Thromb. Haemost. 12, 1440–1448. doi: 10.1111/jth.12615
  4. Zelek W.M., Harrison R.A. 2023. Complement and COVID-19: Three years on, what we know, what we don’t know, and what we ought to know. Immunobiology. 228, 152393. doi: 10.1016/j.imbio.2023.152393
  5. Afzali B., Noris M., Lambrecht B.N., Kemper C. 2022. The state of complement in COVID-19. Nat. Rev. Immunol. 22, 77–84. doi: 10.1038/s41577–021–00665–1
  6. Avdonin P.P., Markitantova Yu. V., Rybakova E.Yu., Goncharov N.V., Avdonin P.V. 2023. Activation of complement factor C3/ C3b deposition on the endothelial cell surface by histamine as one of the causes of endothelium damage in COVID-19. Biochem. (Mosc.) Suppl. Ser. A Membr. Cell Biol. 17 (Suppl.1), S51–S58. doi: 10.1134/S1990747823070012
  7. Goncharov N.V., Sakharov I., Danilov S.M., Sakandelidze O.G. 1987. [Use of collagenase from the hepatopancreas of the Kamchatka crab for isolating and culturing endothelial cells of the large vessels in man]. Bull. Eksp. Biol. Med. 104, 376–378.
  8. Galbusera M., Noris M., Gastoldi S., Bresin E., Mele C., Breno M., Cuccarolo P., Alberti M., Valoti E., Piras R., Donadelli R., Vivarelli M., Murer L., Pecoraro C., Ferrari E., Perna A., Benigni A., Portalupi V., Remuzzi G. 2019. An ex vivo test of complement activation on endothelium for individualized eculizumab therapy in hemolytic uremic syndrome. Am.J. Kidney Dis. 74, 56–72. doi: 10.1053/j.ajkd.2018.11.012
  9. Noris M., Galbusera M., Gastoldi S., Macor P., Banterla F., Bresin E., Tripodo C., Bettoni S., Donadelli R., Valoti E., Tedesco F., Amore A., Coppo R., Ruggenenti P., Gotti E., Remuzzi G. 2014. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood. 124, 1715–1726. doi: 10.1182/blood-2014–02–558296
  10. Naß J., Terglane J, Gerke V. 2021. Weibel palade bodies: Unique secretory organelles of endothelial cells that control blood vessel homeostasis. Front. Cell Dev. Biol., 9, 813995. doi: 10.3389/fcell.2021.813995
  11. Del Conde I., Cruz M.A., Zhang H., Lopez J.A., Afshar-Kharghan V. 2005. Platelet activation leads to activation and propagation of the complement system. J. Exp. Med. 201, 871–879. doi: 10.1084/jem.20041497
  12. Cleator J.H., Zhu W.Q., Vaughan D.E., Hamm H.E. 2006. Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP. Blood. 107, 2736–2744. doi: 10.1182/blood-2004–07–2698
  13. Whiss P.A., Andersson R.G., Srinivas U. 1998. Kinetics of platelet P-selectin mobilization: Concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO donor SNAP. Cell Adhes. Commun. 6, 289–300. doi: 10.3109/15419069809010788
  14. Burns A.R., Bowden R.A., Abe Y., Walker D.C., Simon S.I., Entman M.L., Smith C.W. 1999. P-selectin mediates neutrophil adhesion to endothelial cell borders. J. Leukoc. Biol. 65, 299–306. doi: 10.1002/jlb.65.3.299
  15. Repka-Ramirez M.S. 2003. New concepts of histamine receptors and actions. Curr. Allergy Asthma Rep. 3, 227–231. doi: 10.1007/s11882–003–0044–3
  16. Foreman K.E., Vaporciyan A.A., Bonish B.K., Jones M.L., Johnson K.J., Glovsky M.M., Eddy S.M., Ward P.A. 1994. C5a-induced expression of P-selectin in endothelial cells. J. Clin. Invest. 94, 1147–1155. doi: 10.1172/JCI117430
  17. Khew-Goodall Y., Butcher C.M., Litwin M.S., Newlands S., Korpelainen E.I., Noack L.M., Berndt M.C., Lopez A.F., Gamble J.R., Vadas M.A. 1996. Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3. Blood. 87, 1432–1438.
  18. Conti P., Caraffa A., Tete G., Gallenga C.E., Ross R., Kritas S.K., Frydas I., Younes A., Di Emidio P., Ronconi G. 2020. Mast cells activated by SARS-CoV-2 release histamine which increases IL-1 levels causing cytokine storm and inflammatory reaction in COVID-19. J. Biol. Regul. Homeost. Agents. 34, 1629–1632. doi: 10.23812/20–2EDIT
  19. Conway E.M., Mackman N., Warren R.Q., Wolberg A.S., Mosnier L.O., Campbell R.A., Gralinski L.E., Rondina M.T., van de Veerdonk F.L., Hoffmeister K.M., Griffin J.H., Nugent D., Moon K., Morrissey J.H. 2022. Understanding COVID-19-associated coagulopathy. Nat. Rev. Immunol. 22, 639–649. doi: 10.1038/s41577–022–00762–9
  20. Campello E., Bulato C., Spiezia L., Boscolo A., Poletto F., Cola M., Gavasso S., Simion C., Radu C.M., Cattelan A., Tiberio I., Vettor R., Navalesi P., Simioni P. 2021. Thrombin generation in patients with COVID-19 with and without thromboprophylaxis. Clin. Chem. Lab. Med. 59, 1323–1330. doi: 10.1515/cclm-2021–0108
  21. Chang Y., Bai M., You Q. 2022. Associations between serum interleukins (IL-1beta, IL-2, IL-4, IL-6, IL-8, and IL-10) and disease severity of COVID-19: A systematic review and meta-analysis. Biomed. Res. Int. 2022, 2755246. doi: 10.1155/2022/2755246
  22. Benard A., Jacobsen A., Brunner M., Krautz C., Klosch B., Swierzy I., Naschberger E., Podolska M.J., Kouhestani D., David P., Birkholz T., Castellanos I., Trufa D., Sirbu H., Vetter M., Kremer A.E., Hildner K., Hecker A., Edinger F., Tenbusch M., Muhl-Zurbes P., Steinkasserer A., Richter E., Streeck H., Berger M.M., Brenner T., Weigand M.A., Swirski F.K., Schett G., Grutzmann R., Weber G.F. 2021. Interleukin-3 is a predictive marker for severity and outcome during SARS-CoV-2 infections. Nat. Commun. 12, 1112. doi: 10.1038/s41467–021–21310–4
  23. Del Valle D.M., Kim-Schulze S., Huang H.H., Beckmann N.D., Nirenberg S., Wang B., Lavin Y., Swartz T.H., Madduri D., Stock A., Marron T.U., Xie H., Patel M., Tuballes K., Van Oekelen O., Rahman A., Kovatch P., Aberg J.A., Schadt E., Jagannath S., Mazumdar M., Charney A.W., Firpo-Betancourt A., Mendu D.R., Jhang J., Reich D., Sigel K., Cordon-Cardo C., Feldmann M., Parekh S., Merad M., Gnjatic S. 2020. An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat. Med. 26, 1636–1643. doi: 10.1038/s41591–020–1051–9
  24. Huang C., Wang Y., Li X., Ren L., Zhao J., Hu Y., Zhang L., Fan G., Xu J., Gu X., Cheng Z., Yu T., Xia J., Wei Y., Wu W., Xie X., Yin W., Li H., Liu M., Xiao Y., Gao H., Guo L., Xie J., Wang G., Jiang R., Gao Z., Jin Q., Wang J., Cao B. 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 395, 497–506. doi: 10.1016/S0140–6736(20)30183–5
  25. Bettoni S., Galbusera M., Gastoldi S., Donadelli R., Tentori C., Sparta G., Bresin E., Mele C., Alberti M., Tortajada A., Yebenes H., Remuzzi G., Noris M. 2017. Interaction between Multimeric von Willebrand Factor and Complement: A fresh look to the pathophysiology of microvascular thrombosis. J. Immunol. 199, 1021–1040. doi: 10.4049/jimmunol.1601121
  26. Favaloro E.J., Henry B.M., Lippi G. 2021. Increased VWF and decreased ADAMTS-13 in COVID-19: Creating a milieu for (micro)thrombosis. Semin. Thromb. Hemost. 47, 400–418. doi: 10.1055/s-0041–1727282
  27. McConnell M.J., Kawaguchi N., Kondo R., Sonzogni A., Licini L., Valle C., Bonaffini P.A., Sironi S., Alessio M.G., Previtali G., Seghezzi M., Zhang X., Lee A., Pine A.B., Chun H.J., Zhang X., Fernandez-Hernando C., Qing H., Wang A., Price C., Sun Z., Utsumi T., Hwa J., Strazzabosco M., Iwakiri Y. 2021. Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy. J. Hepatol. 75, 647–658 doi: 10.1016/j.jhep.2021.04.050
  28. Bernardo A., Ball C., Nolasco L., Moake J.F., Dong J.F. 2004. Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultralarge von Willebrand factor multimers under flow. Blood. 104, 100–106. doi: 10.1182/blood-2004–01–0107 2004–01–0107
  29. Wagner D.D. 1990. Cell biology of von Willebrand factor. Annu. Rev. Cell Biol. 6, 217–246. doi: 10.1146/annurev.cb.06.110190.001245
  30. Aiello S., Gastoldi S., Galbusera M., Ruggenenti P., Portalupi V., Rota S., Rubis N., Liguori L., Conti S., Tironi M., Gamba S., Santarsiero D., Benigni A., Remuzzi G., Noris M. 2022. C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19. Blood Adv. 6, 866–881. doi: 10.1182/bloodadvances.2021005246
  31. Miteva K.T., Pedicini L., Wilson L.A., Jayasinghe I., Slip R.G., Marszalek K., Gaunt H.J., Bartoli F., Deivasigamani S., Sobradillo D., Beech D.J., McKeown L. 2019. Rab46 integrates Ca2+ and histamine signaling to regulate selective cargo release from Weibel-Palade bodies. J. Cell Biol. 218, 2232–2246. doi: 10.1083/jcb.201810118
  32. Law S.K., Levine R.P. 1977. Interaction between the third complement protein and cell surface macromolecules. Proc. Natl. Acad. Sci. USA. 74, 2701–2705. doi: 10.1073/pnas.74.7.2701
  33. Martin M., Blom A.M. 2016. Complement in removal of the dead – balancing inflammation. Immunol. Rev. 274, 218–232. doi: 10.1111/imr.12462

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2. Fig 1. C3/C3b binding by HUVEC cells under the influence of blood plasma of healthy donors and COVID-19 patients. a - Cell staining with antibodies to C3/C3b and Hoechst 33258 nuclear dye after incubation with blood plasma of healthy donors; b - Cell staining with antibodies to C3/C3b and Hoechst 33258 nuclear dye after incubation with blood plasma of COVID-1 patients; c - Number of nuclei per gender after incubation of cells with blood plasma of healthy donors and COVID-19 patients (p ≥ 0. 8); d - average number of C3/ C3b-immunopositive particles per nucleus in HUVEC cell culture after incubation with blood plasma of healthy donors and COVID-19 patients (***p < 0.001).

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3. Fig. 2. Distribution of C3/C3b-immunopositive structures by number and area in HUVEC cell culture after incubation with plasma of healthy donors and COVID-19 patients. N is the average number of immunopositive structures of a given size range in the scanned field of view obtained from 12 independent random frames (p ≤ 0.001).

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4. Fig. 3. Differences in the shape of C3/C3b-immunopositive structures recorded after incubation of HUVEC cells with plasma of healthy donors and COVID-19 patients. dFmax is the maximum diameter of the Feret object, dFmin is the minimum diameter of the Feret object. The shape was evaluated by the ratio dFmax/dFmin. The higher the ratio, the more elongated shape the particles have. Immunopositive particles with an area of 2.2-10.9 μm2 had a more elongated shape after incubation with plasma from COVID-19 patients compared to controls (*p < 0.05, **p < 0.01).

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