Pomalidomide Modifies Sickle Cell Related Organ Damage in Transgenic Mice with Sickle Cell Anemia.

We previously reported that Pomalidomide (PL), a novel thalidomide-derived immunomodulatory drug (IMiD), is capable of enhancing erythropoiesis and fetal hemoglobin (HbF) production in a knockout-transgenic (KT) mouse model of sickle cell anemia (SCA). In addition to these hematological properties, PL is known to modulate specific effector functions of the innate and adaptive immune system. PL and other IMiDs potently inhibit the output of the inflammatory mediators TNF-α, IL-1β, and IL-6 in activated monocytes/macrophages, whereas their ability to promote T helper 1 (Th1) lymphocyte differentiation and co-stimulation of both CD4⁺ and CD8⁺ T lymphocytes can lead to increased TNF-α, IL-2, and IFN-γ secretion in both cell culture and human subjects. SCA is an inflammatory disease. It is therefore conceivable that PL could diminish disease severity in SCA by targeting vascular endothelial and innate immune cell activation, whereas preferential induction of a Th1-biased lymphocyte program could have unwanted effects. To test these possibilities in SCA, we evaluated PL's immunomodulatory activities in a relevant KT mouse model.

Animals. Six week old KT homozygous sickle mice were treated daily (Mon-Fri; i.p. injections) for eight weeks with Vehicle (n=8) or PL (10 mg/kg; n=9). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes (Becton-Dickinson). Soluble plasma adhesion molecules (sVCAM-1, sICAM-1, and sE-Selectin) and cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α, G-CSF, and GM-CSF) were measured by ELISA (R&D Systems and SABiosciences). Organ analysis. We focused our analysis on the liver of KT mice because this organ manifests severe sickle cell-related pathology. Livers were removed and divided for H&E paraffin sections, immunohistochemistry, and RNA analysis (storage at -80°C). The area of liver ischemic infarcts (ALI) was measured on H&E tissue sections using an image processing program (Image J, NIH) and averaged from four randomly selected low power optical fields (5x) / animal. Whole liver RNA was pooled from PL animals with low ALI scores (n=2) and vehicle animals (n=3) using Trizol followed by RNeasy column purification. RNA was submitted to SABioscience for analysis using the 440 gene Inflammatory Response and Autoimmunity GEArray. Statistical analysis. One-Way ANOVA/Student-Newman-Kuels and Kruskal-Wallis One-Way ANOVA/Dunn's Method (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant.

The peritoneal cavity of all animals was free of adhesions, exudates and drug, suggesting that daily i.p. injections did not inflame the peritoneal membranes and resulted in complete absorption of the active compound. PL significantly reduced the level of the endothelial cell marker of inflammation, sVCAM-1 (sVCAM-1[ng/ml]: Veh: 1202±36; PL: 962±48; P<0.01), but did not affect sICAM-1 or sE-Selectin. Plasma cytokines in vehicle animals were measured at the assay's lower limit of detection and were not significantly modified by PL, suggesting that PL did not induce overt activation of the TH1 lymphocyte program. Liver histology in vehicle controls revealed scattered tissue infarcts surrounded by a mixed inflammatory cell infiltrate (macrophages [F4/80+], T-lymphocytes [CD3+]). PL reduced the ALI by 60 % (ALI [mm²/LPF: Veh: 0.30±0.05; PL: 0.12±0.06; P=0.01). The ALI score of eight out of nine PL mice was lower than the lowest score in the vehicle group (89% responder rate). Gene array data were consistent with decreased organ inflammation (serum amyloid A2 [-3.57 fold]), modifications in antigen presentation (CD74 [-2.12 fold]), inhibition of T-cell signaling (SLAP-2 [+5.68 fold]; SLP-76 [-1.92 fold]), and reduced T-cell migration (MIP-3β [-2.56 fold]). Tissue protection in the PL group did not correlate with HbF expression, total WBC count, or any other hematological variable.

Pomalidomide modulates vascular and tissue markers of inflammation and protects from sickle cell-induced organ damage in a HbF-independent manner. These data suggest that PL, in addition to its HbF-inducing properties, may exert beneficial anti-inflammatory effects in SCA.

Meiler:Celgene: Research Funding. Wade:Celgene: Research Funding. Moutouh de Parseval:Celgene: Employment. Corral:Celgene: Employment. Swerdlow:Celgene: Research Funding. Kutlar:Celgene: Research Funding.