Abstract
Introduction. Secondary hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening inflammatory syndrome triggered by autoimmune disease, infection, or malignancy. The pathophysiologic mechanisms of HLH implicate an initial dysfunctional cytotoxic lymphocyte-based immune response that fails to eradicate the immunogenic trigger. Continued antigen stimulation results in the activation of cytotoxic lymphocytes, increased IFNγ secretion and subsequent activation of myeloid immune cells such as macrophages, which infiltrate and damage tissues. Bromodomain and extra-terminal domain (BET) proteins bind to acetylated lysine residues on histones and facilitate the recruitment of transcriptional regulatory complexes to chromatin, playing a pivoltal role in regulating the transcription of genes that drive inflammation. The BET inhibitor OPN-51107 binds to the bromodomain of the BET protein BRD4 and inhibits interaction with acetylated lysines residues. We investigated the effects of BET inhibition on the inflammatory features in the murine model of secondary HLH.
Materials and methods. We utilized a model of secondary HLH in which the disease is induced in C57BL/6 mice by repeated intraperitoneal injection of CpG 1826 and anti-IL-10 receptor monoclonal antibody. Mice with HLH develop weight loss, cytopenia, hepatosplenomegaly, and cytokine storm. Mice were randomized to three groups: healthy control, HLH treated with vehicle, or HLH treated with OPN-51107 (10 mg/kg/day orally starting on day 4). On day 9, mice were sacrificed. Organ weights and blood counts were recorded. Spleens were processed into a single-cell suspension and cultured for six hours with a Golgi block and analyzed by flow cytometry. Plasma IFN-γ, IL-6, and IL-18 were measured by ELISA. Significance was evaluated using one-way ANOVA, applying multiple comparisons where appropriate.
Results. HLH led to the development of hepatomegaly (p<0.0001) and splenomegaly (p<0.0001) in mice treated with vehicle compared to healthy controls, but mice with HLH treated with OPN-51107 demonstrated a significant decrease in hepatomegaly and splenomegaly compared to vehicle-treated mice (liver p= 0.0009, spleen p <0.0001). Anemia is a core feature of HLH, and the red blood cell count (RBC) was significantly reduced in mice with HLH treated with vehicle compared to healthy control (p <0.0001); we observed a significant increase in the average RBC count in mice with HLH treated with BET inhibitor compared to vehicle (p= 0.0035). Plasma levels of IFN-γ significantly increased in mice with HLH treated with vehicle compared to healthy controls (p= 0.0003) but reduced to baseline in mice with HLH treated with BET inhibitor (p= 0.0156). Furthermore, plasma IL-6 levels rose significantly in mice with HLH treated with vehicle compared to healthy control (p <0.0001) but reduced significantly in mice with HLH treated with OPN-51107 compared to vehicle (p= 0.0002). Similarly, plasma IL-18 levels significantly increased in HLH mice treated with vehicle (p <0.0001) but reduced in mice with HLH treated with the BET inhibitor (p=0.0375). Finally, HLH led to the expansion of splenic myeloid cells (CD11b+, p = 0.0308), especially neutrophils (CD11b+/Ly6C+/Ly6G+, p <0.0001) in mice with HLH treated with vehicle, compared to healthy control mice. HLH is also associated with a notable depletion of CD4⁺ T cells (p <0.0001) in HLH mice treated with vehicle, compared to healthy control. Treatment with OPN-51107 significantly restored the prevalence of these immune cell populations (CD11b+, p = 0.0377; CD4+, p = 0.0140) in treated mice with HLH, compared to the vehicle-treated cohort. Furthermore, intracellular TNF-α production in myeloid cells showed significantly higher intracellular median fluorescence in vehicle-treated HLH mice compared to both healthy controls (p <0.0001) and BET inhibitor treated HLH mice (p = 0.0002).
Discussion. We have demonstrated that BET inhibition alleviates features of inflammation, including organomegaly, anemia, increased cytokine production and the splenic infiltration by myeloid effector cells in the murine model of secondary HLH. Our findings support the potential of targeting BET proteins to alleviate the hallmark symptoms of the disease. Additional research into the mechanisms through which BET inhibition exerts its anti-inflammatory effects in a murine HLH model is warranted.
