Evaluation of Leptospira Phagocytosis by Macrophages of C3 Deficient Mice in the Complement System

Author(s)

Tiago Francisco da Silva

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-06-16

Examining board members

Lourdes Isaac; Lorena Bavia; Niels Olsen Saraiva Câmara; Lucile Maria Floeter Winter

• Advisor: Lourdes Isaac

Abstract

Leptospirosis is an important zoonosis for public health, caused by the spirochete bacteria of the genus Leptospira . The innate immune response against these bacteria depends largely on phagocytosis and activation of the Complement System. This system is composed by several proteins that are either soluble or present on the cell surface, participating in both innate and adaptive immunity. C3 is the most abundant protein in this system and its importance is especially confirmed in C3 deficient patients, as they are very highly susceptible to severe and recurrent infections. Several studies have shown that the C3 protein plays a fundamental role in the control of infections, responsible for generating opsonins which facilitate microorganism killing, including leptospires. However, the role of C3 in leukocyte differentiation and intracellular response has not been investigated so far. To better understand the importance of C3 for the development of macrophages, this study aimed to compare several important cell parameters for phagocytosis of leptospires on macrophages of C57Bl/6 (wild) and C57Bl/6 C3-/- mice (C3 deficient). Initially, the cell populations in the peritoneal exsudate were assessed by flow cytometry. Higher number of resident macrophages (F4/80high) and lower number of macrophages differentiated from monocytes (F4/80low) were observed in C3 deficient mice, when compared to wild mice, suggesting that monocyte recruitment to the inflammatory site maybe is impaired. The cytoskeleton plays a crucial role in phagocytosis and is important for migration and formation of cell evaginations and invaginations which allow particle internalization. Considering that, the presence and distribution of -tubulin and F-actin in peritoneal macrophages and bone marrow derived macrophages was analyzed. The absence of C3 significantly interfered in macrophage morphology and size, since they stained less with anti--tubulin and reduced cell size (area), evidenced by the use of phalloidin (reacts with F-actin). Then, the presence of the following cell surface molecules was quantified by flow cytometry: CD11a /CD18, CD11b, CD11c, CD64, CD197, CD206, CD282, CD284, CD36, CD80, MHC II and CD21/CD35. The presence of the following receptors CD282 (TLR2), CD64 (FcR1) and CD36 (scavenger receptor) was approximately 20% less in bone marrow differentiated macrophages from C3 deficient mice, when compared to macrophages from wild mouse strain. However, the presence of these same receptors was similar in peritoneal macrophages from both groups of mice. On the other hand, the presence of the Complement 4 receptor (CR4; CD11c / CD18) was six times higher in peritoneal macrophages and differentiated from the bone marrow of B6.C3-/- We also evaluated the phagocytosis of Zimosan and E. coli K12, which was higher in B6.C3-/- macrophages, which may be linked to the cell morphology changes and expression of certain membrane receptors. No significant difference in the in vitro phagocytosis of leptospires by murine macrophages was observed. However, it is still necessary to improve the experimental conditions. Surprisingly, the in vivo phagocytosis of L. interrogans by peritoneal macrophages was higher in C3 deficient mice. Finally, we measured ROS and MAPK activation in macrophages, after 12-O-tetradecanoylforbol-13-acetate (TPA) treatment. Both were less intense in B6.C3-/- macrophages compared to B6.C3+/+ macrophages, confirming the importance of C3 for cell signaling which remains to be further investigated. Taken together, our results suggest that C3 may influence morphology and macrophage functions during ontogeny of C57Black/6 mice, which is of great importance for innate and acquired immune responses. (AU)

• FAPESP's process: 19/01435-7 - Evaluation of leptospira phagocytosis by peritoneal macrophages from C3 deficient mice
• Grantee: Tiago Francisco da Silva
• Support Opportunities: Scholarships in Brazil - Master