P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner

Abstract

Hemolytic diseases are frequently linked to multiorgan failure subsequent to vascular damage. Deciphering the mechanisms leading to organ injury upon hemolytic event could bring out therapeutic approaches. Complement system activation occurs in hemolytic disorders, such as sickle cell disease, but the pathological relevance and the acquisition of a complement-activating phenotype during hemolysis remain unclear. Here we found that intravascular hemolysis, induced by injection of phenylhydrazine, resulted in increased alanine aminotransferase plasma levels and NGAL expression. This liver damage was at least in part complement-dependent, since it was attenuated in complement C3-/- mice and by injection of C5-blocking antibody. We evidenced C3 activation fragments' deposits on liver endothelium in mice with intravascular hemolysis or injected with heme as well as on cultured human endothelial cells (EC) exposed to heme. This process was mediated by TLR4 signaling, as revealed by pharmacological blockade and TLR4 deficiency in mice. Mechanistically, TLR4-dependent surface expression of P-selectin triggered an unconventional mechanism of complement activation by noncovalent anchoring of C3 activation fragments, including the typical fluid-phase C3(H2O), measured by surface plasmon resonance and flow cytometry. P-selectin blockade by an antibody prevented complement deposits and attenuated the liver stress response, measured by NGAL expression, in the hemolytic mice. In conclusion, these results revealed the critical impact of the triad TLR4/P-selectin/complement in the liver damage and its relevance for hemolytic diseases. We anticipate that blockade of TLR4, P-selectin, or the complement system could prevent liver injury in hemolytic diseases like sickle cell disease.

Keywords: P-selectin; TLR-4; complement; endothelium; heme.

Conflict of interest statement

Conflict of interest statement: L.T.R. receives research funding from CSL Behring. The remaining authors declare no conflict of interest.

Figures

Fig. 1.

Pattern of staining for C3 activation fragments in the liver of mice with intravascular hemolysis and their dependence on TLR4 and heme. (A and B) C3 fragments’ deposition on vascular endothelium. (A) Double-staining for vWF (green) and C3b/iC3b (red) of WT and TLR4−/− mice liver frozen sections treated with PBS, heme, or PHZ. Colocalization is in orange. Focus on macrovessels, in particular, central hepatic veins. White arrows point to C3 activation fragments’ deposition along endothelium. (B) Staining quantification, presented as fold change (FC) of the double-positive C3 activation fragments (C3 act fr)/vWF area of each liver section, normalized to the staining of the PBS-injected group of the WT mice. Comparison of WT and TLR4−/− (n ≥ 3) mice. *P < 0.05, **P < 0.005; two-way ANOVA with Tukey’s test for multiple comparisons. (C) Frozen liver sections of WT mice, treated with PHZ ± Hx, were stained for vWF (green) and C3 activation fragments (red).

Fig. 2.

Hemolysis triggers liver injury in a TLR4- and complement-dependent manner. WT, TLR4−/−, and C3−/− mice were injected with PBS, heme, or PHZ. Livers were recovered after 24 h. (A) ALT activity was measured in WT and TLR4−/− mice, presented as fold change (FC), compared with the PBS group (n ≥ 4). (B) ALT activity was measured in WT and C3−/− mice (n ≥ 3). (C) Gene expression of NGAL in livers from WT and TLR4−/− mice, FC (n ≥ 4). (D) Gene expression of NGAL in livers from WT and C3−/− mice, FC (n ≥ 3). (E–G) Efficacy of C5 blockade to prevent liver injury. (E) C5a levels in the plasma of mice, injected with PHZ or pretreated with irrelevant IgG (Irr IgG) or anti–C5-blocking Ab BB5.1 (α-C5). The levels of C5a are compared at day 3 (D-3) and day 1 (D-1) post PHZ injection. The prevention of C5a release indicated that the Ab exerted its blocking effect (n ≥ 4). (F) ALT activity levels (FC) or (G) quantification of the percentage of NGAL positive area in the liver (FC) in the mice, injected with PBS or PHZ, pretreated with Irr IgG or anti-C5. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001; two-way ANOVA with Tukey’s test for multiple comparisons. Values are box plots with median and Min/Max points in A, B, and E–G, and mean ± SEM in C and D.

Fig. 3.

P-selectin is induced secondary to heme-mediated TLR4 activation and serves as an anchoring platform for C3 activation fragments. (A and B) Interactions between C3b/P-selectin and C3(H2O)/P-selectin were studied by surface plasmon resonance by injecting increased concentration of C3b (A) or freeze/thaw native C3 (B) on P-selectin–coated chip. (C and D) HUVEC were treated with 100 µM of heme and exposed to 33% NHS for 30 min at 37 °C. Cells were detached, washed 3× at pH 7.2 or pH 2.5, and stained for C3a (C) or C3 activation fragments (C3 act fr) (D) deposition. (E and F) HUVEC were treated with 100 µM of heme ± 400 nM of TAK-242 ± irrelevant Ab (irr Ig) or anti–P-selectin Ab (αP-sel) for 30 min at 37 °C and exposed to 33% NHS (with 10 mM EGTA and 2 mM MgCl2) for 30 min at 37 °C. Cells were detached and stained for C3 activation fragments’ deposition (n > 5, flow cytometry). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001; two-way ANOVA with Tukey’s test for multiple comparisons. Values are box plots with median and Min/Max points. FC, fold change, compared with basal level.

Fig. 4.

P-selectin blockade prevents complement deposits and liver stress response in mice with intravascular hemolysis. Anti–P-selectin administration prevents complement activation on endothelium and increases of liver injury marker NGAL in hemolytic conditions. WT mice were injected with PBS, heme, or PHZ after first administration of an irrelevant (Irr Ig) or blocking Ab against P-selectin (αP-sel). (A) Gene expression of NGAL in livers. (B and C) Staining quantification (n ≥ 4) for vascular C3 activation fragments’ deposits (C3 act fr) (B) after injection of heme or (C) after induction of hemolysis by PHZ. (D) Examples of double-staining for vWF (green) and C3 activation fragments (red) of liver frozen sections by IF after PHZ injection. Focus on macrovessels, in particular central hepatic veins (Left) and on sinusoidal capillaries (Right). White arrows point to colocalization between C3 activation fragments and vWF (orange). *P < 0.05, ****P < 0.0001; two-way ANOVA with Tukey’s test for multiple comparisons. Values are represented as mean ± SEM in A and box plots with median and Min/Max points in B and C. FC, fold change, compared with PBS-injected mice.