Acute Inflammatory Processes Are Induced By Hemolysis and Reversed By Hydroxyurea
Authors: Almeida Camila Bononi , Souza Lucas Eduardo Botelho , Costa Fabio Trindade Maranhão , Werneck Claudio , Covas Dimas T. , Costa Fernando Ferreira , Conran Nicola 
Published: October 2019

Abstract

Background
Plasma free hemoglobin (PFHb) can be elevated by hemolysis in several diseases, including malaria, sepsis and hemolytic anemias such as sickle cell disease (SCD). When PFHb is not neutralized, it can trigger vascular and organ dysfunction, leading to adverse clinical effects. As an example, increased PFHb in SCD is proposed to reduce nitric oxide (NO) bioavailability and induce vascular oxidative stress and inflammation. Vaso-occlusive (VO) processes in SCD may be triggered by the adhesion of leukocytes (WBC), and subsequently red blood cells, to the vessel wall. Hydroxyurea (HU), used as a fetal Hb-inducing drug in SCD, may also exert some of its effects by acting as an NO donor. We induced hemolytic processes in mice to observe WBC recruitment and inflammatory processes; the ability of HU to affect such processes was also investigated.

Methods
Hemolysis was induced in wild-type C57BL/6 mice (2-4 months old) by injecting water (H2O). For intravital microscopy (IVM), cremaster muscles were prepared before or after administration of H2O and/or other agents, and WBC adhesion and extravasation were quantified (7-8 microvenules/mouse). Inflammation status after TNF-α (i.p., 210 min) or H2O (15 min) administration was evaluated by IVIS (Caliper) using a chemiluminescent probe to detect WBC myeloperoxidase. PFHb was measured with Drabkin's solution.

Results
Mice that received H2O presented marked vascular hemolysis within 15 min; levels of PFHb were doubled in H2O mice, compared to mice receiving PBS (2.57±0.43g/dL, 1.08±0.08g/dL Hb, respect., p<0.01, N=3-5) and were similar to PFHb levels observed in a SCD mouse model (data not shown). In contrast, mice that received a TNF-α-inflammatory stimulus showed no alteration in PFHb (1.29±0.43 g/dL, 1.23±0.09 g/dL; PBS and TNF-α, respect., N=3, P>0.05). IVIS demonstrated induction of significant systemic inflammation by H2O, compared to PBS control, particularly in the abdominal area (27.8x105±9.7x105p/s, 6.6x105±1.0x105p/s, respect., P<0.05, N=3), almost reaching levels observed for TNF-α-treated mice (41.0x105±1.7x105p/s, p<0.01 comp. to PBS, N=3). IVM showed that these same TNF-α and H2O stimuli induced WBC adhesion to vessel walls and WBC extravasation (See Table). When an NO donor (DEANO) was given together with H2O, this drug was able to prevent WBC recruitment, reducing both WBC adhesion and extravasation (See Table), similar to effects that were observed when DEANO was given post TNF-α to inflammatory mice (data not shown). Surprisingly, when HU was given following H2O-induced hemolysis, both increased WBC adhesion and extravasation were reversed in a similar manner to that observed for DEANO. Importantly, neither the administration of DEANO nor HU altered H2O-induced elevations in PFHb (p>0.05, data not shown). Finally, using ODQ, a soluble guanylyl cyclase inhibitor, together with HU, the beneficial effects of HU on H2O-induced WBC recruitment were abolished (See Table).

Conclusion
This H2O-mouse model displays substantial hemolysis, culminating in rapid-systemic inflammation and significant alterations in WBC recruitment to the vessel wall, comparable to those of a major inflammatory stimulus. Acute HU administration abolished H2O-induced WBC recruitment in a manner similar to that of an established NO donor. The anti-inflammatory effects of HU in this model were not mediated by the inhibition of hemolysis, rather they were reversed by inhibition of cGMP production. Data indicate that hemolytic events alone may be sufficient to induce major inflammatory events. As WBC recruitment and inflammation are thought to trigger SCD VO processes, results indicate that hemolysis could contribute to this phenomenon. Furthermore, HU may have important NO/cGMP-mediated anti-inflammatory effects, possibly indicating potential for use in other diseases that exhibit hemolysis or inflammation.

Disclosures:
No relevant conflicts of interest to declare.