Sepsis Induced Red Cell Dysfunction (SiRD): Physiology and Mechanisms

Abstract BACKGROUND: In sepsis, many red blood cell (RBC) defects are described: ↑ oxygen (O2) affinity, ↓ deformability, ↑ aggregation & adhesion, and dysregulated RBC-based vasoregulation. We suggest these comprise a unique class of organ (RBC) failure, which we term sepsis-induced RBC dysfunction (SiRD). We propose SiRD arises from RBC metabolic failure and depowered antioxidant systems - resulting in impaired RBC physiology that disables O2 transport. We employed a murine cecal ligation and puncture (CLP) sepsis model to characterize SiRD features and explore mechanisms of SiRD initiation. METHODS: Mice underwent sham (Gcont), mild (Gmild: D1 mortality 10%), or severe CLP (Gsev: D1 mortality 50%); N=4-8/group. D1 RBCs were collected. SiRD phenotype was characterized by analyzing: (1) O2 affinity (p50, Bohr effect), (2) deformability (LORRCA - elongation index, EI), (3) osmotic fragility (LORRCA - ΔOsm resilience), (4) aggregation (LORRCA - syllectogram) and (5) vasoregulation (organ chamber bioassay - RBC hypoxic vasodilaton). Mechanistic explanation for the SiRD phenotype was explored by evaluating: (6) Ca+ accumulation (flow cytometry), (7) caspase3 activation (flow cytometry), (8) cdB3 proteolysis (western blot), (9) RBC metabolomic analysis (13C1,2,3-glucose based MS, +/- exogenous oxidant stress), (10) nitric oxide content (photolysis:chemiluminescence - total RBC NO, FeNO, and SNO content), and (11) eryptosis:phosphatidylserine (PS) exposure (flow cytometry). SiRD physiologic impact was evaluated by (12) quantifying tissue O2 delivery impairment in vivo (HIF-1a bioluminescent mouse, IVIS). RESULTS: RBCs from septic mice demonstrated the following physiologic Δs: (1) ↑ O2 affinity (L-shifted HbO2 dissociation) at 3 pHs (7.2, 7.4, and 7.6, with ↓ Bohr effect), with ↑ p50 (Gsev vs Gcont) (pH7.2: 47.8±1.2* vs. 53.3±0.9 Torr; pH7.4: 37.2±1.1** vs. 42.9±0.7 Torr; and pH7.6: 29.0±0.6*** vs. 34.2±0.8 Torr); (2) ↓ RBC deformability - with similar EImax, shear stress (SS)1/2 was ↑ in Gsev vs Gcont (0.92 vs. 0.65 Pa); (3) ↑ osmotic fragility - min & max tolerated osmolality (Omin & Omax): Omin in Gcont, Gmild, and Gsev were 166.1±8.9, 158.8±4.5, and 159.1±2.8 mOsm/kg; and Omax were 336.0±21.5, 315.3±9.1, and 314.0±8.5 mOsm/kg, respectively; (4) ↑ aggregation - syllectogram backscatter intensity (Isc): in Gsev, Isc ↓ from 32.5±5.0 to 12.5±2.8 Au; but in Gcont, Isc only changed from 31.1±3.4 to 23.6±4.6 Au; (5) hypoxic vasodilation response ↓ in Gsev RBCs vs Gcont (8.9±4.2% vs 10.2±4.0%*, respectively). Exploration of potential mechanisms for SiRD revealed: (6) ↑ RBC Ca+ (Fluo3-based flow cytometry): ↑ mean florescence intensity (MFI) with sepsis severity (Gcont: 61.3±11.6; Gmild: 73.4±14.6, and Gsev: 105±36.0); (7) MIF of active caspase3 probe (CaspGlow-DEVD-FMK) did not change following CLP; however, after incubation with 0.1mM H2O2, MIF for Gsev ↑ significantly (Gcont: 549.3±28.8 vs. Gsev: 3123±782.2*); (8) ↑ cdB3 proteolysis was observed, Gsev > Gmild; (9) principal component and pathway analysis of metabolomic data identified sepsis severity-based constraints in the hexose monophosphate shunt pathway & linked antioxidant systems (NADPH and GSH); (10) RBC SNOHb & membrane SNO ↓ in Gsev vs Gcont (0.08 ± 0.04* & 0.04 ± 0.02* vs 0.22 ± 0.08 & 0.17 ± 0.07 SNOHb (SNO:Hb molar ratio) and membrane SNO mol/mg protein, respectively; (11) ↑ %age of eryptotic RBCs after CLP (annexin V-based flow cytometry): Gsev annexin V positive sub-population was 5.3±2.9%**, compared to Gcont 1.3±0.6% - following oxidative stress (0.1mM H2O2), annexin V positive sub- population in Gcont ↑ further to 11.5±4.1%; whilst Gsev, ↑ to 31.0±9.2%***. (12) O2 delivery impairment was quantified by imaging (IVIS) HIF-bioluminescent mice (FVB/Gt(ROSA)26Sor tm2(HIF1A/luc)kael): D1 normalized luciferase activity in Gsev was ↑ 3.4±0.8* fold vs baseline; a similar difference was observed after washed RBC exchange from Gsev → Gcont mice. (NB: * P CONCLUSIONS: In sepsis, RBCs undergo major biochemical & physiologic alterations that impair O2 delivery, which we term SiRD. We propose SiRD arises from acquired RBC metabolic constraints, oxidative RBC injuries and impaired physiology, comprising a distinct form of organ (RBC) failure that may influence sepsis outcome. This well characterized model will allow further characterization, mechanistic study, and therapeutic exploration. Disclosures Doctor: KaloCyte, Inc.: Equity Ownership, Research Funding; Washington University in St. Louis: Patents & Royalties; NIH: Research Funding; DoD: Research Funding; Biogen: Consultancy; Children's Discovery Institute: Research Funding.