Chronic pulmonary LPS exposure in cystic fibrosis drives maladaptive pro-inflammatory hematopoietic stem and progenitor cells Free

Persistent lung inflammation characterizes cystic fibrosis (CF), mainly driven by myeloid cells (neutrophils and monocytes) that can contribute to tissue damage and disease progression. Hematopoietic stem and progenitor cells (HSPCs) are activated during inflammation to enhance myelopoiesis. While this myeloid response is critical for healthy responses during acute infections, chronic inflammatory signals can cause maladaptive epigenetic reprogramming of HSPCs, skewing hematopoiesis towards a prolonged myeloid bias that can lead to hyperinflammatory states. Although this mechanism is implicated in other chronic diseases, its role in CF is not well understood. We hypothesize that CF-associated inflammation epigenetically and functionally reprograms HSPCs, promoting a pathological immune bias that sustains chronic inflammation.

Wild-type (WT) and CF-KO mice (n=6–8/group) were nebulized with LPS for 5 weeks and euthanized at baseline (T0), 24 hours (T1), or 6 weeks (T2) after the last LPS exposure. HSPC frequencies in the bone marrow (BM) were evaluated. Single cell RNA-seq was performed on BM Lin^-Sca⁺Kit⁺ cells and ATAC-sequencing (ATAC-seq) was performed on BM Lin^-Sca⁺Kit⁺ Flt3^-CD48^- cells sorted from n = 4 mice per group. Serum cytokine concentrations were measured at T0. To assess long-term trained immunity, BM from CD45.2 WT or CF-KO mice (T0 or T2) was transplanted into lethally irradiated CD45.1 WT recipients. Donor-derived HSCs were isolated and profiled by ATAC-seq 3 months post-transplant.

At baseline, long-term hematopoietic stem cells (LT-HSC) in CF mice were increased in frequency with enhanced myeloid differentiation. A statistically significant increase was observed in CF LT-HSCs, specifically in the CD41⁺ subpopulation, which is skewed toward myeloid differentiation. Additionally, CF BM had an increased frequency of multi-potent progenitors (MPPs) with erythroid/megakaryocytic potential (MPP2) and monocyte/neutrophil potential (MPP3), but not lymphoid potential (MPP4). Granulocyte-monocyte progenitors (GMPs) were also elevated in CF. Consistent with increased myelopoiesis, CF mouse peripheral blood had higher numbers of monocytes and neutrophils than WT. By scRNA-seq of LSK, we confirmed that LT-HSC and GMP frequencies are higher in CF. CF LT-HSCs exhibited a myeloid-biased signature, characterized by increased myeloid-specific differentially expressed genes (DEGs) (e.g., Jun, Fos, Klf2, Runx1) with DEGs enriched for TNF-α signaling via NF-κB (e.g., Cebpb, Egr1, Atf3, Plaur, Tnfaip8), which was confirmed by bulk RNA-seq. TNF-α (13.66 pg/ml ± 8.43 vs 5.34 pg/ml ±1.52) and other pro-inflammatory cytokines were elevated in the serum of CF vs WT mice. There were no ATACseq differences observed at T0. Chronic LPS in CF lungs led to non-resolving inflammation, increased number of myeloid cells with a pro-inflammatory profile, lung remodeling, and decreased lung function. In contrast, WT mice lungs return to homeostasis by T2. As seen at T0, T1 and T2 CF mice retained a higher frequency of CD41⁺LT-HSCs and higher frequencies of GMPs. At T2, colony forming assays revealed that GMPs from CF mice had an increased tendency to differentiate into granulocytes compared to WT GMPs (n=3/genotype). Bulk RNA-seq revealed that CF HSCs kept a distinct pro-inflammatory, myeloid-skewed epigenetic signature, predominantly enriched for TNF-α/NF-κB signaling whereas WT HSC were very similar to baseline. This was associated with enhanced lung inflammation in CF mice compared with WT. Post-transplantation, WT HSCs from T2 donors resembled those from T0, showing resolution of inflammatory programming. Conversely, CF HSCs from T2 donors retained their inflammatory epigenetic profile, indicating persistent changes in long-term HSCs.

CF-associated inflammation causes lasting epigenetic reprogramming of HSCs, promoting maladaptive myeloid skewed hematopoiesis that likely contributes to persistent inflammation in CF. The TNF-α/NF-κB signaling pathway appears central in acquiring this maladaptive response in CF. Baseline differences in HSPC composition and cytokine profiles suggest pre-existing priming of HSCs in CF mice, which may enhance their response to chronic inflammatory stimuli like LPS.