Bone Marrow Chimeras Generated from Humanized Sickle Cell Disease Mouse, Exhibiting MHC Class II Downregulation and Immune-Dysregulation
Authors: Malik Nida , Hamidi Asra , Raman Surabhi , Joshi Sunil 
Published: December 2024

Previously, our lab has demonstrated that the humanized Sickle cell disease (SCD) mouse (Berkeley model (STOCK Hbatm1Paz Hbbtm1TowTg(HBA-HBBs)41Paz/J) have significantly decreased MHC Class II and upregulation of inflammatory cytokines along with the diseases pathology progression. The SCD, is an inherited blood disorder characterized by hardened, sticky C-shaped RBCs is a major cause of morbidity and mortality and affects over 100,000 Americans and millions of people of worldwide. Clinical consequences include severe acute and chronic pain, impaired immunity, progressive multi-organ damage and early mortality. APCs such as DCs, B cells and macrophages express MHC II bind antigen that is recognized by TCR expressed on CD4+ Th cells. Thus, the interaction between receptor-ligand clusters on Th and APCs led to the activation of naïve CD4+ Th cells, stimulating the release of cytokines that influenced the activity of many cell types, including the NKT, CD8+ CTLs and the APCs that activated them. We created bone marrow chimeras using sickling mice donors (human β-globin locus-transgenic (β-YAC) mice and Berk-SCD mice as donors and Pepboy mice as recipients). Bone marrow chimerism was confirmed after 3 weeks of bone marrow transfer. All chimeric Pepboy mice were screened for sickle cell phenotype and were confirmed by Hemoglobin Gel Electrophoresis assay. All chimeric Pepboy mice were confirmed having SCD. We used one homozygous mouse's bone marrow to generate 15 Pepboy chimeric mice. As controls, we also made chimera from heterozygous and wild type mouse. We kept half of the chimeric mice in a conventional facility and remaining half were kept in SPF. At various time points (6, 8, 10, 12, 14, 16, 20 weeks), we checked the expression of various immune markers by taking 0.2 ml blood from each mouse and stained for MHC I, MHC II, CD3, CD4, CD8, CD19, B220, IL-6, TNFa, IL-4 after Fc-block. Using multicolor flow cytometer, expression of these biomarkers was measured. Most strikingly, there was decrease in the MHC II expression significantly as disease pathology progresses. Surprisingly, the MHC II downregulation was restricted to only mice boarding in the conventional (“DIRTY”) facility. These mice were very sick as measured on the basis of their appearance, body weight and blood profile. We performed the autopsy of very sick mice and perform liver pathology by making histopathology slides. Pathological changes were characterized by a hyper-inflammatory state with the activation of innate-immune response, upregulation of TLR4, and loss of antigen-presentation machinery, loss of MHC II expression, with disease progression. However, we could not find any loss of MHC II and presence of hyperinflammation in mice, which were kept in SPF. The presence of pathogens in conventional facility along with lack of MHC II causes the Immune-dysregulation. We propose that people with SCD must be carefully and sincerely treated for infectious diseases. There is a great disparity in the treatment of these SCD patients as they are mostly African / African-American and South-east / middle-east Asian population. Further investigation is underway regarding pathology of human SCD based on age, sex and demographic regions. Our study also provides an easier and homogenous system by making bone marrow chimeras from one mouse to 15 mice. In this way, there was less or no variation in the pathology.