Fetal-Derived B-1 Cells Can Reduce Atherosclerotic Progression in the Clonal Hematopoiesis Condition

B-1 cells, an innate-immune-like B-cell subset, mainly develop from fetal progenitors. B-1 cells secrete natural IgM antibodies (NAbs), which bind Oxidized-LDL (ox-LDL) that is expressed in the atherosclerotic lesion and apoptotic cells. Therefore, NAbs secreted by B-1 cells clear apoptotic cells and cell debris and play key roles in preventing atherosclerosis (ASC) and chronic inflammation caused by aging (Inflammaging). While several investigations showed the atheroprotective effect of B-cells in the standard atherosclerotic model, it is unclear whether B-1 cells can reduce atherosclerosis in the clonal hematopoiesis-related atherosclerosis model. Ldlr-null mice transplanted with clonal hematopoiesis (CH) bone marrow cells (Tet2-KO mouse BM) on high-fat diets are an advanced model that reflects the status of human patients with severe atherosclerosis and CH (CH-ASC model). It has been reported that CH is observed in at least 10% of people older than 70 years and increases the risk of atherosclerotic cardiovascular diseases. We hypothesize that fetal-derived young B-1 cells prevent the progression of atherosclerosis in CH mice by secreting NAbs that reduce chronic inflammation and that aging of B-1 cells may alter their function.

We repeatedly transferred sorted peritoneal cavity (PerC) B-1 cells into CH-ASC model mice with high-fat diets and evaluated atherosclerotic regions as well as immune profiles. The area of aortic plaque formation measured by oil-red staining was significantly reduced in the CH-ASC + B-1 therapeutic transfer group (CH-ASC+B1) compared to CH-ASC alone (30.8 ± 4.7% vs 18.3 ± 6.7%, p<0.03). Plaque formation in the aortic valve also exhibited a reduction in the CH-ASC+B1 group (234.8 ± 37.1 vs 360.3 ± 62.2 sqm, p<0.05). PerC Macrophage (Mac) in the CH-ASC+B1 showed marked increase of M2 polarization (3.2 ± 0.35 x10e5 vs 0.47 ± 0.10 x 10e5, p< 0.01). Plasma anti-PC IgM Abs were measured by ELISA, showing that CH-ASC+B1 plasma before the evaluation had significantly higher than that of CH-ASC (2821 ± 1988 vs 654 ± 611). Interestingly, PerC from CH-ASC showed a dramatic increase of Tet2-KO-derived B-1a cells that are not normally seen in the post-transplant recipient. However, B-1a cells from CH-ASC+B1 PerC had normal frequency of B-1a cells (41.2 ± 6.2 % vs 19.8 ± 9.7 %, p<0.05), suggesting that Tet2-null B-1a cells may not have atheroprotective property.

In addition to the secretion of anti-PC IgM, we explored the impact of B-1a cells on M1/M2 polarization. We sorted B-1a cells and Mac from the PerC and stimulated Mac by Interferon-γ + LPS (Mac-I/L) with or w/o the presence of B-1a cells. Twenty-four hours after stimulation, B-1a and Mac were sorted, and cytokine profiles were measured by qPCR. TNF-α and IL-1β were markedly increased in Mac-I/L, whereas Mac-I/L with B-1a demonstrated restoration down to M0 level, indicating that B-1a cells can suppress Mac activation by cell-to-cell mediated function.

Taken together, our data demonstrated that young B-1 cell injection successfully reduced atherosclerotic lesions in CH mice and that the ability of anti-PC IgM Ab secretion was reduced in CH mice. Also, our data indicates the critical role of fetal-derived B-1 cells well-functioning in adult mice and the possibility that the alteration of B-1 cell function causes age-associated diseases.