Combinatorial Effect of Chronic Alcohol Drinking and Age Perpetuates Myeloid-Biased Hematopoiesis Via Hematopoietic Stem Cell Senescence

Introduction:

Myeloid cells, such as neutrophils and monocytes, are crucial for innate immune responses, yet their overproduction promotes aged-related frailties. Studies in non-human primates established the role of alcohols in causing myeloid cell overproduction, albeit its impact on aging hematopoietic stem cell (HSC) commitment remains unexplored. Alcohol is catabolized into acetaldehyde and reactive oxygen species (ROS) that inflict DNA damage. Importantly, DNA repair capacity in aged HSC diminishes with age, which correlates with their myeloid bias and decreased self-renewal. However, there is no study establishes a combinatorial effect of alcohol and age in the HSC differentiation. Therefore, we sought to: 1) analyze the impact of chronic alcohol and age in HSC lineage commitment, and 2) elucidate mechanisms underlying changes in HSC differentiation due to chronic alcohol exposures and aging.

Methods:

Young (2 months) and old (22 months) C57BL/6N mice were fed with Liebe de Carli liquid diet containing 5% v/v alcohol or isocaloric control for 4 weeks (4 groups: young control (YC), young alcohol (YA), old control (OC) and old alcohol (OA)). First, hematological parameters and myeloid bias were measured from peripheral blood (PB) collected at baseline and after alcohol feeding using complete blood count with differentials (CBC w/ diff) and flow cytometry (Cytek Aurora). To further elucidate the underlying mechanisms, single-cell RNA-sequencing was performed from 30.000 sorted Lineage^- Sca-1⁺ cKit⁺ (LSK) cells using 3’ Chromium v3 (10X Genomics). We also conducted competitive BM transplantation (BMT) to assess the engraftment potential and lineage commitment of the donor cells from the four groups.

Results:

CBC w/ diff showed significant anemia, thrombocytopenia, and relative reticulocytosis as well as monocytosis and lymphopenia in the OA vs. OC group. However, those differences were not observed between the YA vs. YC groups. While YA mice showed a similar myeloid versus lymphoid (M:L) ratio compared to YC mice, OA mice had a 3-6-fold higher M:L ratio, 2-3 fold higher CD172α⁺ (myeloid) fraction, and 2-3 fold higher neutrophils as well as classical monocytes than OC mice analyzed by flow cytometry. By contrast, the proportion of B cells decreased by 4 times in OA vs. OC groups. Unexpectedly, the percentage of CD4⁺ T cells was slightly elevated in both YA and OA groups. LSK scRNA-seq showed 15 cell clusters including long-term HSC (LT-HSC), myeloid-biased and lymphoid-biased multipotent progenitors (MPP3 and MPP4, respectively), and Eif2ak2 (PKR)-expressing progenitors. Strikingly, the proportion of LT-HSC was lower in OA compared to OC group, whereas the proportion of myeloid-biased MPP3 and PKR expressing progenitors increased in the same group comparison. By looking at the molecular changes inside LT-HSC cluster comparing YA vs. YC groups, it is evident that LT-HSC exhibited signatures of TNFα-NFκB and Inflammatory response pathways. Among those genes are Fos, Junb, Ifitm1 and Ifitm3. Moreover, the LT-HSC in OA exhibited the same signatures in addition to IL2-STAT5 signaling, granulopoiesis, early T lymphocyte development, and megakaryocytes-platelet development as compared to OC group, suggesting increased myeloid lineage priming. Interestingly, LT-HSC cluster in OA mice showed senescence gene expression programs, comprising cell cycle checkpoint, DNA damage-induced senescence, and epigenetic remodeling via histone and DNA methylation mediated by polycomb repressive complex 2 (PRC2). Indeed, aged HSC marker genes, Selp and Itgb3, increased dramatically in the LT-HSC cluster of OA vs. OC but not when comparing YA vs YC. Importantly, competitive BMT revealed no significant defects in the self-renewal capacity of OA and YA at the primary BMT. However, the combinatorial effect of age and alcohol promoted myeloid-biased differentiation maintained up to 16 weeks post-BMT.

Conclusions:

Chronic alcohol exposures and aging synergistically increased myeloid cell production. Mechanistically, it involves acquisition of inflammation and senescence gene signatures in the LT-HSC, as well as expansion of myeloid-primed progenitors. Nevertheless, previous alcohol exposure did not alter the self-renewal capacity, but the myeloid bias remained for 16 weeks. Analysis of the secondary transplant to study potential defects in long-term self-renewal is underway.