Differentiation of macrophages in the presence of anaphylatoxin C3a: effect on efferocytosis

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Abstract

BACKGROUND: Macrophages are unique professional phagocytes involved in both innate and adaptive immune responses. This is made possible by their functional plasticity—the ability to acquire different phenotypes. Switching between subtypes during interactions with other immune system components is referred to as polarization. Anaphylatoxin C3a has been shown to influence macrophage polarization. Depending on the polarization state, macrophages exhibit different phagocytic activities. Given the contradictory evidence regarding C3a’s effects on macrophages, determining its impact on phagocytosis in general and on efferocytosis in particular is of special interest.

AIM: The work aimed to investigate the effect of anaphylatoxin C3a on efferocytosis by M0 and M2 macrophages.

METHODS: The experiments were conducted using human peripheral blood mononuclear cells. Macrophages were differentiated in the presence of C3a, and M2 polarization was induced with interleukin-4. Efferocytosis was assessed using confocal microscopy, whereas phagocytosis was evaluated via flow cytometry. Real-time reverse transcription polymerase chain reaction was used to measure expression of the efferocytosis receptor genes mertk, axl, and tyro3.

RESULTS: M2 macrophages demonstrated greater efferocytic capacity compared to M0 macrophages. Anaphylatoxin C3a did not affect the phagocytosis of dead Escherichia coli bacteria but inhibited the phagocytosis of apoptotic cells in both unpolarized M0 and alternatively activated M2 macrophages. It was also shown that C3a reduces the expression of the Tyro3 receptor gene in both M0 and M2 macrophages.

CONCLUSION: Anaphylatoxin C3a exerts a suppressive effect on efferocytosis, likely through modulation of apoptotic cell recognition processes.

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About the authors

Aleksandra A. Dmitrieva

Institute of Experimental Medicine

Author for correspondence.
Email: aleksandra-2001@mail.ru
ORCID iD: 0000-0003-2680-4069
SPIN-code: 3009-2698
Russian Federation, Saint Petersburg

Anna A. Ivanova

Institute of Experimental Medicine

Email: anna.ivantcova@gmail.com
ORCID iD: 0000-0002-8673-9628
SPIN-code: 5306-1995
Russian Federation, Saint Petersburg

Alexandra V. Burnusuz

Institute of Experimental Medicine

Email: alexandraburnusuz@gmail.com
ORCID iD: 0000-0002-2281-5548
Russian Federation, Saint Petersburg

Sergey V. Orlov

Institute of Experimental Medicine

Email: serge@iem.spb.ru
ORCID iD: 0000-0002-3134-1989
SPIN-code: 1690-8110

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

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2. Fig. 1. Verification of macrophage polarization: a, measurement of CD206 expression levels by flow cytometry. Number of replicates: n = 3. The gray peak corresponds to cells stained with nonspecific fluorescently labeled antibodies (isotype control). The background fluorescence is indicated by a cut-off line. Dashed line, M0 macrophages (median 9.5; first and third quartiles 7.3 and 12.4, respectively); solid line, M2 macrophages (median 13.9; first and third quartiles 9.4 and 19.9, respectively); *p < 0.05 (Mann–Whitney test); b, IL-1Ra levels in the culture supernatants of M0 and M2 macrophages measured by enzyme-linked immunosorbent assay. Box plots represent medians and interquartile ranges; *p < 0.05 (Mann–Whitney test); c, mRNA levels of cd206, il1ra, and pparγ in macrophages. Box plots represent medians and interquartile ranges; *p < 0.05 (Mann–Whitney test).

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3. Fig. 2. Efferocytosis of cells by macrophages. Macrophages (M0 and M2) were differentiated and polarized in the presence of C3a (10 nM): a, confocal microscopy; macrophages were stained with DAPI, and apoptotic Jurkat cells were labeled with CFDA-SE. The image was obtained by merging signals from three channels (DIC, CFSE, DAPI). Arrows indicate macrophages that had engulfed apoptotic cells, whereas crossed-out arrows indicate events that could not be identified as efferocytosis; b, quantitative assessment of efferocytosis, with the y-axis showing the efferocytic index: the ratio of the number of internalized apoptotic cells to the total number of macrophages in the field of view (100 fields of view for each data point), multiplied by 100%. Box plots represent the medians and interquartile ranges of the analyzed data. *p < 0.05 (Kruskal–Wallis test).

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4. Fig. 3. Phagocytosis of E. coli by macrophages. Number of replicates: n = 3. The vertical line indicates the autofluorescence threshold of macrophages. Blue: macrophages that phagocytosed E. coli–FITC (median 235; first and third quartiles 229 and 243, respectively); black line: macrophages differentiated in the presence of C3a (10 nM) and that phagocytosed E. coli–FITC (median 271; first and third quartiles 262 and 275, respectively). *p < 0.05 (Mann–Whitney test).

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5. Fig. 4. mRNA levels of TAM receptor genes mertk, axl, and tyro3 in macrophages. Box plots represent the medians and interquartile ranges. *p < 0.05 (Kruskal–Wallis test).

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