Caspase-4/11 alters circulating immune cells and mediates organ damage in Townes SCD mice Free

Sickle cell disease (SCD) is an inherited red blood cell disorder that is characterized by constant lysis of red blood cells and subsequent chronic inflammation and end-organ damage. Chronic hemolysis and the release of free heme in SCD activates a variety of inflammatory proteins including the pro-inflammatory protein caspase-4. In macrophages, activation of caspase-4 by heme leads to release of the potent inflammatory cytokine IL-1β and pyroptosis, a type of inflammatory cell death.Caspase-4 is also hypothesized to have a role in neutrophil extracellular trap (NET) formation and acute lung injury in SCD. To test the impact of caspase-4 on inflammation and organ damage, we used the well-characterized Townes mouse model of sickle cell disease. In Townes HbSS mice, murine α-globin genes were replaced with the human α-globin gene and the murine β-globin genes were replaced with the human γ-globin gene and the human β-globin gene found in patients with SCD. Like patients with SCD, these mice have severe anemia, heightened inflammation, and signs of chronic organ damage. We used CRISPR/Cas9 to disrupt the gene for caspase-11, the mouse homolog of caspase-4, in the Townes mouse. We then characterized how loss of caspase-11 impacted hematological parameters and markers of organ damage in Townes HbSS mice.

There were no significant differences in red blood cell counts, hemoglobin levels, hematocrit, or reticulocyte counts between HbSS Casp11^+/+and HbSS Casp11^-/-mice, indicating that caspase-11 does not impact the severity of hemolysis in these mice.

Strikingly, we observed significant differences in levels of circulating immune cells between these two groups. At 8 weeks and at 22 weeks, HbSS Casp11^+/+and HbSS Casp11^-/-mice had similar total white blood cell counts (8 weeks 38.2 K/uL vs. 37.0 K/uL, p = 0.716 ; 22 weeks 35.8 K/uL vs. 35.5 K/uL, p = 0.941). However, HbSS Casp11^-/-mice had significantly higher levels of circulating neutrophils (8 weeks 24.6% vs. 30.6%, p= 0.001; 22 weeks 18.4% vs. 24.7% p = 0.023) and lower levels of circulating lymphocytes (8 weeks 71.9% vs. 65.7% p = 0.001; 22 weeks 77.6% vs. 71.0%, p = 0.022). There were no significant differences in platelet levels, monocyte %, eosinophil %, or basophil % between the two groups. Other groups have reported caspase-11 deficient mice have defects in NET formation and chemotaxis. We hypothesize that elevated circulating neutrophil levels in HbSS Casp11^-/-mice are due to deficits in neutrophil function and trafficking, suggesting caspase-11 contributes to heightened inflammation in SCD.

Given the importance of caspase-11 in innate immune function, we expected to observe fewer signs of chronic vaso-occlusion and consequent organ damage in HbSS Casp11^-/-mice. We found that at 22 weeks, livers were significantly lighter in weight in HbSS Casp11^-/-mice (% of total body weight 6.9% vs 6.4%, p = 0.034, n = 35). Based on histopathology, we found significant decreases in the amount of liver ischemia (ischemic foci seen in 5/5 HbSS Casp11^+/+mice and 0/5 HbSS Casp11^-/-mice) and bile duct injury (5/5 HbSS Casp11^+/+mice and 0/5 HbSS Casp11^-/-mice) in caspase-11 null mice. HbSS Casp11^-/-mice also tended to have less reactive hepatocytes (observed in 4/5 HbSS Casp11^+/+ mice and 1/5 HbSS Casp11^-/-mice) and less lobular inflammation (3/5 HbSS Casp11^+/+and 0/5 HbSS Casp11^-/-mice). HbSS Casp11^-/- mice also had significantly lower levels of serum ALT and AST (22 weeks ALT 404.3 U/L vs 190.9 U/L, p = 0.046; AST 259 U/L vs 150 U/L, p = 0.048), suggesting loss of caspase-11 protects mice from liver injury. Spleen histopathology revealed that HbSS Casp11^-/- mice tended to have better retained red pulp/white pulp architecture and lower levels of extramedullary hematopoiesis. This was despite comparable levels of vascular congestion and increased spleen weight in HbSS Casp11^-/-mice (% of total body weight 8 weeks 6.0% vs 6.8% p = 0.031; 22 weeks 6.6% vs 7.8%, p = 0.002).

Overall, we have determined that caspase-11 plays a major role in mediating organ damage in a mouse model of SCD, likely through modulating immune mediators of vaso-occlusion like neutrophils. Thus, the human ortholog of caspase-11, caspase-4, may be a promising therapeutic target to mitigate inflammation and subsequent organ damage in SCD.