Hemolysis Controls Pro-Inflammatory Monocyte Priming Via Induction of and Interaction with Circadian Clock Protein Rev-Erbα in Sickle Cell Disease

Background:  Patients with SCD tolerate a systemic pro-inflammatory vascular milieu created by chronic ischemia/reperfusion injury and profound erythrocyte hemolysis.   In addition to this chronic low level inflammation, exposure to relatively innocuous, sub-clinical inflammatory stimuli appears to ignite an exaggerated, potentially fatal inflammatory response in patients.  The etiology of this inflammatory hyper-reactivity is not well understood.   There is ample evidence that, in steady state, a cadre of inflammatory cells, especially monocytes, exhibit a primed phenotype.  Such priming, or propensity to activate, likely contributes to baseline inflammation, and is requisite for the inflated inflammatory response.  Monocytes are quite unique amongst the leukocytes in that their inflammatory potential, including Il-6 release, is governed by the mammalian circadian clock.    A role for the rhythmic oscillation of clock proteins as a controller of inflammation in SCD has never been demonstrated.  However, a binding partner for heme, the nuclear receptor rev-erbα, is implicated as a regulator of clock controlled genes.

Objective:  To test the hypothesis that hemolysis, via heme-induced perturbation of the clock protein Rev-erbα, forms the basis for an enhanced inflammatory response in the monocyte.

Methods:    Intraperitoneal low dose lipopolysaccharide (LPS) was used to elicit an inflammatory response in the Townes mouse model of SCD.  Plasma from the mice was acquired 6 hours after LPS injection.  Analysis of 25 cytokines was accomplished using luminex methods.  Monocytes were modeled in vitro using THP-1 cells.  Simultaneous analysis of 84 induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. Inflammatory cytokine levels in cell supernatants were determined via ELISA. 

Results:  We challenged the mice with low dose LPS (<10ng).   Interrogation of the inflammatory cytokines in these mice revealed no change in any cytokine tested in the AA mice, but 20 out of 25 inflammatory cytokines were upregulated in mice with the SS genotype.  The monocyte-based cytokines were clearly target of LPS activation in the SS mice. TNF-α and Il-1β were both upregulated 20 fold and 80 fold respectively in the SS mice.  KC levels (the murine equivalent of Il-8) levels were increased 80 fold in the SS mice treated with LPS. Il-6 levels, however, were the most pronounced with a 40,000 fold increase over PBS injected SS mice.   We then evaluated the role of hemolysis on monocyte inflammatory potential in vitro.  Sustained monocyte exposure to physiological levels of heme in SCD alone could induce a low level of inflammatory gene expression and Il-6 release.  However, sustained exposure to heme dramatically increased Il-6 release from the monocyte in response to LPS.  Expression of the Il-6 gene was also increased, but the peak gene expression was time delayed compared to  LPS treatment alone.  In fact, we noted this phase shifting of inflammatory gene expression in the heme primed cells.  LPS induced the release of significantly more TNF-α and Il-1β into the culture media in the presence of heme – consistent with the notion of heme setting a hyperactive threshold in response to LPS.  We also noted that heme induced expression of the clock gene rev-erbα, and that antagonizing the activity of rev-erbα ablated the enhanced inflammatory response induced by LPS in the heme primed cells. 

 Conclusion:  These data provide evidence that hemolysis may play an important role in the hyper-inflammatory monocyte response via heme- induced dysregulation of the circadian clock.  These novel observations provide entirely new avenues of anti-inflammatory therapy in SCD.