The Effect of TET2 Loss on Macrophage Phagocytosis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) arises from somatic mutations occurring in hematopoietic stem cells (HSCs) and is linked to myeloid and lymphoid malignancies and increased inflammatory disorders such as atherosclerosis. TET2 mutations, associated with increased inflammation, are among the most frequent in CHIP and play a key role in the development of cardiovascular disease. Macrophages, a central regulator of the innate immune response and inflammation, play a key role in the immune dysfunction seen in CHIP. Phagocytosis contributes to increased pro-inflammatory cytokine production and is a critical homeostatic mechanism in tissues. Dysfunctional phagocytosis is often associated with cardiovascular disease and other inflammatory disorders (Rauch et al., Nature Cardiovascular Research 2023; Jan, Ebert and Jaiswal., Seminars in Hematology 2017). However, it is unclear whether macrophage phagocytosis is dysfunctional in TET2 CHIP-mediated inflammation.

Using Tet2 fl/fl x Vav-Cre mice to model CHIP (referred to as Tet2 KO), we revealed that Tet2 KO bone marrow derived macrophages (BMDMs) exhibit altered phagocytic capabilities, which may play a role in the inflammatory conditions associated with CHIP. Using murine BMDMs, we demonstrated that when compared to WT macrophages, Tet2 KO macrophages increased expression of CD206. CD206, a known pattern recognition receptor, is a marker of M2 polarized macrophages and increased phagocytic competency (Schulz et al., Nature Scientific Reports 2019). This increase of CD206⁺ macrophages in Tet2 KO murine BMDMs has also been linked to atherosclerosis (Rauch et al., Nature Cardiovascular Research 2023).

To understand the phagocytic potential of WT and Tet2 KO, we cultured BMDMs and RAW264.7 macrophages in the presence of IL4 (10ng/ml) or lipopolysaccharides (LPS; 10ng/ml) and used FITC-conjugated IgG beads to model innate immune system stimulation. When macrophages bind IgG opsonized targets using Fc Gamma receptors (FcYRs) they initiate the phagocytosis process. Compared to WT, Tet2-KO BMDMs or RAW264.7 macrophages had significantly increased phagocytosis 8 to 24 hours after the introduction of IgG beads. To uncover potential mechanisms driving increased phagocytosis, we performed RNA-sequencing for CD206⁺ WT and Tet2-KO BMDMs, which showed significantly increased gene set enrichment for leukocyte endothelial migration and Fc-gamma receptor dependent phagocytosis in Tet2-KO when compared to WT.

To further investigate the mechanism of increased phagocytosis, we performed a CRISPR/Cas9 knockout screen using a genome wide library in Cas9+ WT and Tet2-KO BMDMs with stimulation by FITC-Conjugated IgG beads as before. As a control, the screen identified genes such as Gpr84, Ralgapb and Ilf2, which have appeared in prior literature as phagocytic regulators (Haney et al., Nature Genetics 2018; Shi et al., Nature Communications). The top identified genes required for Tet2-KO phagocytosis but not WT phagocytosis included Ly9, Ppm1a, Slc44a1 which are genes involved in immune regulation and inflammation, metabolic homeostasis and lysosomal function.

It is clear that TET2 inactivation within macrophages plays an important role in mediating disease progression. In atherosclerosis, a process known as efferocytosis, the phagocytosis of autophagic cells, is dysregulated in tissue resident macrophages within the aortic wall (Adkar et al., Nature Reviews Cardiology). This inability to clear dying cells can lead to increased plaque formation and atheroma (Rauch et al., Nature Cardiovascular Research 2023). While the role of phagocytosis and its effects on plaque stability is currently still under further investigation, increased phagocytosis in Tet2 inactivated macrophages is a finding with potential implications for the pathophysiology of CHIP-mediated atherosclerosis.