The Role Of NF-κB In The Activation Of Human iNKT Cells In Sickle Cell Disease Patients and In Vitro

Sickle cell disease (SCD) is the most common inherited hematologic01 disorder in the United States. It is a chronic degenerative disease that is punctuated by periodic painful vaso-occlusive crises (pVOC). When sickle hemoglobin polymerizes it causes erythrocytes to become rigid and sticky, which in turn provokes vaso-occlusion due to multi-cellular aggregates. Vaso-occlusion causes the clinical complications of SCD, which include acute chest syndrome, strokes, increase risk of infection, organ damage and decreased life expectancy. We have shown that invariant natural killer T (iNKT) cells are pivotal in causing inflammation and injury in sickle cell disease (Wallace KL, Marshall MA, et al. Blood. 2009; 114(3):667-76). NF-κB is known to control many genes involved in inflammation.

Given that NF-κB plays a role in the formation of many inflammatory mediators we hypothesized that NF-κB is highly activated in the iNKT cells of sickle cell patients during vaso-occlusive pain crises and that inhibition of NF-κB would decrease inflammation mediated by iNKT cells. Our aims were: 1) to measure NF-κB activation in iNKT cells of sickle cell patients at steady state and during pVOC; and 2) to determine if inhibition of NF-κB in cultured human iNKT cells blocks activation of specific iNKT cell markers as measured by changes in gene expression and cellular protein expression.

Blood was collected from 8 SCD patients at steady state and during pVOC. The active phosphorylated form of p65 NF-κB was identified by fluorescence-activated cell sorting (FACS) with anti-phospho-NF-κB p65 (Cell Signaling, 93H1). For the cultured human iNKT cell studies we purified iNKT cells from healthy donor whole blood. After 13 days of expansion in culture we pre-incubated the cells with either vehicle or NF-κB inhibitors, Bay 11-7082 or IKK Inhibitor VII, then activated the cells with plate-bound anti-CD3. We used RT qPCR to evaluate the mRNA for iNKT cell markers of activation GM-CSF (WBC growth factor), T-bet (Th1 transcription factor) and IFN-γ (Th1 cytokine) in triplicate wells of iNKT cells. We harvested cells at 2, 6 and 12 hours for mRNA evaluation by RT qPCR with normalization to the housekeeping gene RNA polymerase IIA. We also harvested iNKT cells at 24 hours for analysis of protein expression of iNKT cell activation markers phospho-NF-κB, T-bet, and CD69 (early surface glycoprotein) by FACS.

In 8 of 8 adult sickle cell disease patients expression of phospho-NF-κB p65 was greater in iNKT cells (but not conventional T cells) during pVOC than at steady state (P < 0.001). In cultured iNKT cells NF-κB inhibition with either Bay 11-7082 or IKK Inhibitor VII produced a dose-dependent decrease in mRNA induction of activation marker such as GM-CSF, T-bet and IFN-γ upon iNKT cell activation. We also found reduced protein expression of activation markers in iNKT cells such as phospho-NF-κB, T-bet, and CD69 as determined by FACS. These data support our hypothesis that NF-κB plays a key role in iNKT cell activation and that inhibition of NF-κB decreases iNKT cell inflammatory markers.

Our findings indicate that NF-κB is a major regulator of iNKT cell activation during sickle cell disease vaso-occlusive crisis and that inhibition of NF-κB activation could reduce inflammation in a sickle cell crisis.

Field:NKT Therapeutics: Consultancy.