IRF8 restrains a neutrophil alarmin program that drives inflammatory remodeling of the bone marrow niche

Technical Summary: IRF8 Restraints a Neutrophil Alarmin Program Driving Inflammatory Bone Marrow Remodeling

Problem Statement
Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein, yet its pathogenesis extends beyond the malignant clone to include profound remodeling of the bone marrow (BM) niche. This remodeling involves vascular alterations, altered bone turnover, and fibrosis, which are associated with inferior clinical outcomes and suboptimal responses to tyrosine kinase inhibitor (TKI) therapy. While stromal involvement is recognized, the specific mechanisms linking aberrant myelopoiesis to niche inflammation and fibrotic restructuring remain incompletely understood. A critical gap exists in defining the early cellular circuits that translate leukemic hematopoiesis into structural niche changes, particularly because longitudinal human bone marrow access is limited. The transcription factor Interferon Regulatory Factor 8 (IRF8) is known to be suppressed in CML and is a central regulator of myeloid differentiation, but its specific role in governing the reciprocal interactions between immune cells and the stromal niche during chronic-phase CML has not been fully dissected.

Methodology
The study employed a multi-modal approach combining murine models, single-cell genomics, and human clinical validation:

• Murine Models: The authors utilized Irf8-deficient (Irf8^d/d) mice to model the hematopoietic defects of CML. To distinguish between hematopoietic and stromal drivers, they performed bone marrow transplantation experiments using Irf8^d/d donors into Gli1^CreERT;tdTomato reporter recipients (to trace stromal remodeling) and Irf8^+/+ or Irf8^d/d recipients transplanted with BCR-ABL1-transduced wild-type cells (to test niche effects on leukemia progression).
• Single-Cell Transcriptomics (scRNA-seq): Bone marrow and spleen cells from full Irf8^d/d mice and hematopoietic chimeras were subjected to 10x Genomics scRNA-seq. This allowed for the resolution of hematopoietic and stromal populations, pseudotime trajectory analysis of differentiation, and cell-cell interaction (CCI) inference.
• Spatial and Molecular Validation: Techniques included CODEX multiplexed imaging to visualize spatial architecture, flow cytometry for phenotyping, reticulin and collagen staining for fibrosis assessment, and Chromatin Immunoprecipitation sequencing (ChIP-seq) to identify direct IRF8 binding targets.
• Human Validation: The study analyzed human CML patient samples (peripheral blood and bone marrow biopsies) to correlate IRF8 expression, S100A8/A9 abundance, and fibrosis grades. Additionally, human iPSC-derived bone marrow organoids were used to model hypoxic and inflammatory niches.

Key Contributions and Results

1. IRF8 Suppression is a CML-Specific Feature:
   The authors established that IRF8 expression is selectively reduced in human and murine CML compared to controls and other myeloproliferative neoplasms (like Polycythemia Vera). Importantly, IRF8 levels were restored following effective interferon-α therapy, suggesting its suppression marks an active, reversible disease state.

2. IRF8 Deficiency Recapitulates Chronic-Phase CML Phenotypes:
   Irf8^d/d mice exhibited a chronic-phase-like state characterized by:

   • Granulocytic Expansion: Marked expansion of mature Ly6G⁺ neutrophils in the bone marrow and spleen.
   • Extramedullary Hematopoiesis: Splenomegaly with disrupted white pulp architecture and expansion of red pulp hematopoiesis.
   • Megakaryocytic Dysplasia: Increased clustering, micromegakaryocytes, and emperipolesis (neutrophils within megakaryocytes).
   • Early Fibrosis: Mild reticulin fibrosis and collagen I deposition, particularly in the spleen and bone marrow.

3. Identification of a Neutrophil-Stromal Communication Axis:
   scRNA-seq revealed that Irf8 loss drives a coordinated program where inflammatory neutrophils communicate with perivascular stromal cells (specifically CXCL12-abundant reticular [CAR] cells, pericytes, and endothelial cells).

   • Signaling Axis: The dominant communication pathway identified was S100A8/A9 (alarmins) from neutrophils binding to TLR4 on stromal cells. This was coupled with TGF-β–integrin signaling.
   • Stromal Response: Receiving these signals, CAR cells and perivascular cells upregulated pro-fibrotic programs (TGF-β, collagen biosynthesis, myofibroblast differentiation) and hypoxia-associated signatures (HIF-1α).
   • Mechanism of Regulation: ChIP-seq confirmed that IRF8 directly binds the S100a8 locus, acting as a transcriptional repressor. Loss of IRF8 unleashes S100A8/A9 production in neutrophils.

4. Causality and Niche Amplification:

   • Hematopoietic Sufficiency: Transplanting Irf8^d/d hematopoietic cells into wild-type recipients was sufficient to induce stromal remodeling, vascular expansion, and fibrosis in the recipient's niche, proving that hematopoietic IRF8 loss drives the process.
   • Niche Amplification of Leukemia: Conversely, transplanting BCR-ABL1-driven leukemia into an Irf8^d/d (deficient) niche resulted in more severe disease progression, higher S100A8/A9 levels, and more extensive fibrosis compared to wild-type recipients. This indicates that an IRF8-deficient, inflammatory niche amplifies leukemic burden.

5. Clinical Correlation:
   In human CML patients, stromal S100A8 abundance positively correlated with reticulin fibrosis grade. Furthermore, serum lactate dehydrogenase (LDH) levels, a marker of tissue turnover, correlated with fibrosis severity, supporting the relevance of the inflammatory niche axis in human disease.

Significance
The paper claims to identify IRF8 as a critical hematopoietic regulator that restrains a neutrophil alarmin program (S100A8/A9) which, when unleashed, drives inflammatory remodeling of the bone marrow niche. The study proposes a two-component model of CML pathogenesis:

1. Leukemic cells drive initial expansion.
2. Suppressed IRF8 in myeloid cells leads to S100A8/A9 overproduction, which activates TLR4 on stromal cells, triggering a self-reinforcing cycle of hypoxia, inflammation, and fibrosis.

The authors conclude that this IRF8–S100A8/A9–TLR4 axis represents a targetable inflammatory circuit. Therapeutic disruption of this pathway could potentially address the microenvironmental component of disease persistence, particularly in patients with residual fibrosis or inflammatory remodeling despite molecular responses to BCR-ABL1 inhibition. The work positions IRF8 as a "molecular brake" preventing inflammatory hematopoiesis from evolving into self-sustaining stromal pathology.