Circulating Heparan Sulfate Fragments Attenuate Histone-Induced Lung Injury Independently of Histone Binding

Abstract

Extracellular histones are cationic damage-associated molecular pattern molecules capable of directly inducing cellular injury via charge-mediated interactions with plasma membranes. Accordingly, histones released into the plasma during critical illness are known to contribute to the onset and propagation of lung injury. Vascular injury (with consequent degradation of the endothelial glycocalyx) simultaneously releases anionic heparan sulfate fragments (hexa- to octasaccharides in size) into the plasma. It is unknown whether this endogenous release of heparan sulfate fragments modulates charge-dependent histone cytotoxicity, or if exogenous heparan sulfate fragments could therapeutically attenuate histone-induced lung injury. Using isothermic calorimetry, we found that extracellular histones only bind to heparan sulfate fragments ≥ 10 saccharides in size, suggesting that glycocalyx-derived heparan sulfate hexa/octasaccharides are incapable of intercepting/neutralizing circulating histones. However, we found that even heparan sulfate fragments incapable of histone binding (e.g., tetrasaccharides) attenuated histone-induced lung injury in vivo, suggesting a direct, size-independent protective effect of heparan sulfate. We found that histones had no effect on human neutrophils ex vivo but exerted toll-like receptor-independent cytotoxicity on human pulmonary microvascular endothelial cells in vitro. This cytotoxicity could be prevented by either the addition of negatively charged (i.e., highly sulfated) heparan sulfate tetrasaccharides (incapable of binding histones) or decasaccharides (capable of binding histones). Taken together, our findings suggest that heparan sulfate oligosaccharides may directly exert pulmonary endothelial-protective effects that attenuate histone-mediated lung injury.

Figure 1. Size-dependence of HS binding to CTH

(a,b) Representative isotherms of calf thymus histone (CTH, 68.2 µM) binding to highly-sulfated HS oligosaccharides (400 µM). No binding was noted with HS tetrasaccharides (dp4, a); similarly negative isotherms were noted with dp6 and dp8 oligosaccharides. In contrast, fragments ≥ 10 saccharides (dp10, b) in size demonstrated both endothermic (downsloping) and exothermic (upsloping) binding reactions. (c) Isothermic calorimetry quantification of number of binding sites (N) and binding affinity of CTH-HS reactions.

Figure 2. Binding of highly-sulfated HS decasaccharides to different histone subtypes

(a-c) Representative isotherms of histone H1 (a), H3 (b), and H4 (c, all 20 µM) binding to highly-sulfated dp10 HS (400 µM). Histone H1-HS binding demonstrated an endothermic (downsloping) reaction; in contrast, H3 and H4 binding to HS was exothermic (upsloping). (d) Isothermic calorimetry quantification of number of binding sites (N) and binding affinity of histone-dp10 HS reactions.

Figure 3. Sulfation-dependence of dp10 HS binding to CTH

a Heparan sulfate, a linear polymer of repeating glucosamine-hexuronic (either glucuronic or iduronic) acid disaccharides, can be sulfated at the 2-O position of iduronic acid ("2S") or the N- or 6-O position of glucosamine ("NS" or "6S"). b-e Representative isotherms of CTH binding to fully sulfated dp10 HS (modeled by heparin, b), N-desulfated dp10 HS (c), 2-O-desulfated dp10 HS (d), and 6-O-desulfated dp10 HS (e, all 20 µM) binding to highly-sulfated dp10 HS (400 µM). f Isothermic calorimetry quantification of number of binding sites (N) and binding affinity of CTH-dp10 HS reactions.

Figure 4. HS oligosaccharides attenuate CTH-induced acute lung injury

Intravenous calf thymus histones (CTH, 25 mg/kg) induce lung injury at 24 h, as quantified by increased lung edema (wet/dry ratio, a), alveolar permeability (bronchoalveolar lavage (BAL) protein, b), and alveolar inflammation (BAL leukocyte/neutrophil infiltration (c,d)). These quantitative measures are consistent with qualitative injury apparent on lung histology (e). These indices of injury were attenuated by highly-sulfated HS fragments (dp4 or dp10 heparin oligosaccharides, 12 µg intravenously injected every 6 h). n = 3 - 4 per group. * P < 0.05 compared to saline/saline control; † P < 0.05 compared to CTH/saline.

Figure 5. CTH do not activate primed or unprimed human neutrophils ex vivo

Neutrophils were isolated from the plasma of healthy humans and treated ex vivo with or without 1 h antecedent LPS 100 ng/ml pretreatment) with fMLP (100 nM, positive control), superoxide dismutase (20 units/ml, negative control), and/or calf thymus histones (CTH, 20 or 200 µg/ml). Superoxide production was measured every 8 min for 4 hours (a); quantification was performed at 40 min (b). n = 3 - 4 biological replicates (each corresponding to a distinct neutrophil collection); * P < 0.05 compared to all other groups.

Figure 6. HS oligosaccharides prevent CTH-induced endothelial cytotoxicity

(a) CTH induce endothelial cell death (as quantified by LDH release from HPMEC-ST1.6R endothelial cells) in a dose-dependent fashion. (b) CTH-induced endothelial (HPMEC-ST1.6R) cytotoxicity is minimally TLR-4 dependent, as demonstrated by only modest protection provided by high-dose TAK-242 (a TLR4 inhibitor). In contrast, HS oligosaccharides (of all sizes) protected against CTH-induced cytotoxicity in HPMEC-ST1.6R cells (c) and primary human lung microvascular endothelial cells (d). (e) The HPMEC-ST1.6R-protective effects of dp4 HS was dependent on N-, 2-O, and 6-O sulfation, while the protective effects of dp10 HS only required N-sulfation. (f) The protective effects of dp4 and dp10 HS were additive to (and therefore likely independent of) high-dose TLR4 inhibition. n > 3 independent replicates for all groups. * P < 0.05 compared to control group.

Figure 7. HS oligosaccharides prevent low-dose CTH-induced endothelial activation

In contrast to high-dose CTH-induced cytotoxicity, lower doses of CTH induce endothelial cell activation, as quantified in HPMEC-ST1.6R cells by TNFα expression (a) and in primary human lung microvascular endothelial cells by loss of cell-membrane VE-Cadherin (b). (c) Low-dose CTH activation of HPMEC-ST1.6R endothelial cells is TLR-4 dependent, as demonstrated by suppression of TNFα expression by TAK-242. This protective effect is similar to (and not augmented by) dp4 or dp10 HS, suggesting that TAK-242 and HS oligosaccharides inhibit endothelial activation by a shared pathway. (d) Highly-sulfated dp4 and dp10 HS oligosaccharides did not impact LPS-induced TNF-α expression in HPMEC-ST1.6R cells, suggesting that these oligosaccharides do not function as nonspecific TLR4 inhibitors. (e) Low-dose CTH exposure does not activate MLKL, a mediator of necroptosis. MLKL activation is not influenced by HS oligosaccharides. n > 3 independent replicates for all groups. * P < 0.05 compared to control group.