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. Author manuscript; available in PMC: 2024 Aug 8.
Published in final edited form as: Immunity. 2023 Jul 26;56(8):1809–1824.e10. doi: 10.1016/j.immuni.2023.06.026

Figure 2. Complement factor H controls cell-autonomous C3 activation in monocyte-derived macrophages and delays the resolution of inflammation.

Figure 2.

(A) C3 and C5 mRNA and protein levels of monocyte-derived macrophages (Mo-Macs) from thioglycolate-injected mice measured by qRT-PCR and ELISA. (B) C3 mean fluorescence unit (MFI) of peritoneal Ly6Chi and Ly6Clo monocytes of thioglycolate-injected mice determined by intracellular flow cytometry. (C) Intracellular C3 levels normalized to cellular protein content in cell lysates of sorted circulating Ly6Chi monocytes and resident peritoneal macrophages (Res-Macs) from unchallenged mice as well as Ly6Chi monocytes and Mo-Macs from thioglycolate-injected mice, quantified by ELISA. (D) Intracellular C3 levels of peritoneal Ly6Chi monocytes of thioglycolate-injected Cfh+/+ and Cfh−/− mice quantified by ELISA. Representative histograms show C3 levels within peritoneal Ly6Chi monocytes assessed by intracellular flow cytometry. (E-G) Characterization of Mo-Macs in the peritoneal lavage fluid of thioglycolate-injected Cfh+/+ vs Cfh−/− mice. (E) Intracellular C3 levels of adherent Mo-Macs by ELISA. (F) Bar graphs and representative cytometry plots show surface and intracellular C3 staining of Mo-Macs measured by flow cytometry. (G) C3a/C3 ratio, a measure of complement activation, as judged by total intracellular C3a and C3 levels in lysates of adherent Mo-Macs quantified by ELISA. (H-L) Characterization of the resolution of inflammation in the peritoneal lavage fluid of Cfh+/+ vs Cfh−/− mice 2, 24, 72 and 168 hours after thioglycolate injection. Absolute numbers of (H) Mo-Macs and (I) neutrophils, quantified by flow cytometry. (J) Resolution curves and (K) the corresponding resolution interval calculated by the resolution indices extrapolated from the curves are shown. (L) Absolute numbers of early (AnnV+7-AAD−) and late (AnnV+7-AAD+) apoptotic cells, assessed by flow cytometry. Each symbol represents individual mice. Data are representative of at least three independent experiments. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).