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Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

Issuing time:2023-01-10 11:04

Abstract

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression.

Introduction

IL-1β stands out as initiator of inflammation and blocking its activity in humans is applied in clinical treatments against diseases with an inflammatory component1. When dysregulated, chronic inflammation through autoimmune conditions and infections is linked to increased risk of hematological malignancies1. Chronic IL-1β administration biases differentiation of normal HSC to the myeloid lineage and reduces their self-renewal properties through a cell-autonomous effect2,3,4. However, IL-1 receptor 1 knockout (IL-1r1-KO) mice have unaffected blood production and normal stem and progenitor bone marrow (BM) compartments, suggesting that IL-1β-induced myeloid priming of HSC occurs under conditions of injury or infection only and tonic IL-1 signaling has none or small basal hematopoietic effects2,5,6. In turn, enhanced IL-1β and other members of its signaling pathway, including IL-1RAP, MyD88, IRAK1, IRAK4 and TRAF6, have been involved in AML and/or myelodysplastic syndrome (MDS)6,7,8,9,10,11,12,13,14. Furthermore, altered inflammation affects cells from the BM microenvironment, and thereby contributes to malignant hematopoiesis8. In this regard, we reported a causal association between high IL-1β-induced damage to the HSC microenvironment and the onset of myeloproliferative neoplasms (MPN)15.

Hence, IL-1β blockade shows promising therapeutic value in experimental models of MPN, chronic myeloid leukemia (CML), juvenile myelomonocytic leukemia, and AML10,15,16,17. Currently, IL-1β blockade with the human monoclonal antibody (mAb) canakinumab is being tested in a Phase II clinical trial for the treatment of low or intermediate risk MDS and CML (NTC04239157). Another interesting IL-1 blocking agent is the FDA-approved anakinra, the recombinant form of the naturally occurring IL-1 receptor antagonist (IL-1RN), which competes with IL-1 for IL-1r1. Anakinra is currently used as therapy against autoimmune diseases1 and it has shown therapeutic value in experimental models of MPN and CML15,17. However, little is known about the participation of the endogenous IL-1 repressor IL-1RN in healthy and/or malignant hematopoiesis. IL-1RN production follows after IL-1 by roughly the same cell types of hematopoietic and non-hematopoietic origin18. In the BM, these include cells from the BM microenvironment, like BM Nestin+ mesenchymal stromal cells (MSC)15 that represent a niche component that controls HSC function15,19,20,21,22. Despite these intriguing precedents, it is unclear to what extent repression of the IL-1-signaling pathway by endogenous IL-1RN may influence HSC behavior in healthy and diseased hematopoiesis.

Here, we find that low IL-1RN has prognostic value of poor survival in AML patients, it is a common event in AML patients, and it characterizes the lower maturation/differentiation profiles according to the French-American-British (FAB) classification of AML (M0-M3). IL-1RN boost or IL-1β blockade have therapeutic potential for AML patients based on their low IL-1RN. To study the effect of low IL-1RN in the hematopoietic system, we used the IL-1rn-KO strain, which displays IL-1β-induced bias in HSC differentiation towards the myeloid lineage whilst hampering B cell development, via NFκB activation under steady-state conditions. This phenotype is reminiscent of pre-leukemic disease and becomes apparent in the absence of injury or infection. Thus, we demonstrate a critical role for balanced IL-1rn and IL-1β on steady-state HSC function in vivo. Low IL-1rn is present in an experimental NRASG12D model of biased pre-leukemic myelopoiesis and further loss of IL-1β repression through IL-1rn genetic deletion promotes myeloproliferation, which relies on the BM hematopoietic and stromal compartments. Conversely, treatment with exogenous IL-1rn reverts pre-leukemic myeloproliferation. Our data support that loss of repression of IL-1β through low IL-1RN may originate and worsen hematopoietic disease, and predicts poor survival in AML patients.

Results

Low IL-1RN predicts AML progression in patients

To study the expression of IL-1RN in human AML, we reanalyzed publicly available arrays from purified AML blasts in a cohort of 381 AML patients (GSE14468)23,24,25 and found that low IL1RN was associated with reduced survival rate (Fig. 1a). We separately analyzed more differentiated AML profiles according to FAB and found that low IL1RN was a prognostic marker of reduced survival in M4-M5 AML patients (n = 164) (Fig. 1a). There were few M0-M3 AML patients with high IL1RN expression (n = 7/217). Indeed, we identified the lower maturation/differentiation M0-M3 patients according to FAB as those patients with lowest IL1RN expression, versus M4-M5 (Fig. 1b). M0-M3 patients displayed high IL1B to IL1RN ratio (Supplementary Fig. S1A). In a cohort of 19 matched-pair diagnosis-relapsed AML patients (dbGaP accession phs001027)26, we correlated low IL1RN expression in RNA-Seq data from AML blasts with poorer prognosis (Supplementary Fig. S1B), and reduced expression of IL1RN at relapse versus diagnosis (Supplementary Fig. S1C).

We studied the potential contribution of CD34+ progenitors to unbalanced IL-1RN in AML in a cell-intrinsic fashion. Expression levels of IL1RN were reduced in circulating CD34+ progenitors from patients versus healthy controls (Fig. 1c), which was particularly evident in the more undifferentiated FAB categories of AML (M0–M3) (Fig. 1d). Gene expression ratios of all IL1B, CASP1 and IRAK1, to IL1RN were higher in AML patients (Fig. 1e; Supplementary Fig. S1D), suggesting that reduced IL-1RN associates with activation of IL-1β pathway. This was confirmed at the protein level with higher ratio of IL-1β to IL-1RN in CD34+ progenitors from AML patients versus healthy donors (Fig. 1f). As previously described, we confirmed higher expression of IL1RAP gene and higher frequency of CD34+ progenitors expressing IL-1RAP in AML patients compared to healthy donors13 (Supplementary Fig S1E, F).

As a surrogate of IL-1β signaling pathway activation, we studied NFκB activation by phospho-flow and nuclear translocation in CD34+ progenitors from AML patients and healthy controls. The percentage of peripheral blood (PB) CD34+ progenitors activated through NFκB activation was higher in AML patients (Supplementary Fig. S1G), as well as the nuclear translocation of the p50/p65 NFκB heterodimer (Fig. 1g; Supplementary Fig. S1H). To confirm the functional role of increased IL-1β signaling in human AML pathogenesis, immunodeficient NSG-SGM3 mice were transplanted with CD34+ progenitors isolated from the BM of AML patients, and treated in vivo with either human IL-1β or vehicle. IL-1β treatment promoted expansion of human AML cells in PB and BM of NSG-SGM3 mice (Fig. 1h, i). We compared the therapeutic potential for AML patients in NSG-SGM3 xenografts of boosting IL-1RN and blocking IL-1β. Short-term treatments with IL-1RN or with the human IL-1β mAb canakinumab were efficient in reducing the expansion of human leukemic cells, i.e. myeloid cells, to similar extents in the PB of NSG-SGM3 mice transplanted with CD34+ progenitors isolated from the BM of AML patient samples (Fig. 1j; Supplementary Fig. S1I). Long-term treatment with IL-1RN was followed up and confirmed reduced numbers of human leukemic cells, i.e. CD45+ leukocytes, CD33+ myeloid blasts and CD34+ progenitors in the BM of IL-1RN-treated NSG-SGM3 mice (Fig. 1k; Supplementary Fig. S1J). Thus, IL-1RN boost has therapeutic potential for AML patients.

Deletion of IL-1rn induces myelopoiesis in the absence of immunogenic stimulus

To determine the role of IL-1rn deficiency in hematopoiesis, we characterized the IL-1rn-KO mouse strain, which has not been used before in hematopoiesis. Adult IL-1rn-KO mice had higher cellularity in BM, and increased circulating neutrophils with no abnormalities in spleen (Fig. 2a; Supplementary Fig. S2A, B). The abundance, fractions and proliferation of Lin−Sca-1+c-Kit+ LSK cells were similar between WT and IL-1rn-KO mice (Supplementary Fig. S2C). Fluorescence-activated cell sorting (FACS) analysis of the stem and progenitor cell subsets corresponding to HSC and multipotent progenitors MPP1-MPP527,28,29 revealed reduction of HSC and MPP5, and increase of MPP2 in the BM of IL-1rn-KO mice (Fig. 2b; Supplementary Fig. S2D). Absolute numbers of common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) were unchanged, whereas megakaryocyte erythroid progenitors (MEP) and granulocyte-monocyte progenitors (GMP) were reduced in the BM of IL-1rn-KO versus WT mice (Supplementary Fig. S3A). Numbers of colonies formed ex vivo by hematopoietic stem and progenitor cells (HSPC) were higher in IL-1rn-KO mice (Fig. 2c). Analysis of IL-1rn-KO mice also revealed increased numbers of apoptotic cells in total BM (Supplementary Fig. S3B) but not in HSPC (Supplementary Fig. S3C). We found impact of IL-1rn abrogation in the differentiation bias of hematopoiesis towards the myeloid lineage with significant impairment of B lymphoid development (Fig. 2d; Supplementary Fig. S3D, E). These results indicate that IL-1rn represses HSPC differentiation into the myeloid lineage and enables B cell differentiation under steady-state conditions.

Fig. 2: Deletion of IL-1rn triggers IL-1β-induced myelopoiesis in the absence of immunogenic stimulus.

Production of IL-1β in the BM was described under conditions of injury or infection2. Our data showed presence of IL-1β in healthy BM and higher IL-1β BM levels in IL-1rn-KO versus WT mice resulting from the absence of IL-1rn, under steady-state conditions (Fig. 2e). There were no changes in other BM cytokines (Supplementary Fig. S3F). We then asked whether IL-1rn genetic deletion resulted in globally enhanced IL-1r1. IL-1r1-expressing cells in the BM were rare but increased in frequency in IL-1rn-KO mice (Fig. 2f). The myeloid bias of progenitors from IL-1rn-KO mice could be prevented in vitro (Supplementary Fig. S3G) and in vivo by treatment with IL-1RN (Fig. 2g, h) or mAb against IL-1β (Fig. 2i, j), but not mAb against IL-1α (Supplementary Fig. S3H). Thus, under steady-state conditions, IL-1rn prevents IL-1β-driven HSPC differentiation into the myeloid lineage at the expense of lymphoid development.

Both IL-1rn-KO long term (LT)-HSC and short term (ST)-HSC upregulated the expression of Il1b gene, whilst Il1r1 was expressed by HSPC and higher in LT-HSC from IL-1rn-KO versus WT mice (Fig. 2k). This suggested engagement of IL-1β-positive feedback loop for IL-1β expression30 in HSPC in the absence of any immunogenic trigger, due to IL-1rn loss. Il1rap gene expression was unchanged in LT-HSC and ST-HSC but reduced in MPP from IL-1rn-KO versus WT mice (Fig. 2k). Myeloid cells, selectively granulocytes, were expanded in the BM of IL-1rn-KO mice (Fig. 2d; Supplementary Fig. S3E) but, unlike HSPC and similarly to monocytes, they showed no engagement of IL-1β-positive feedback loop through IL-1r1 (Fig. 2l). Il1rap gene expression was similar in granulocytes and monocytes from IL-1rn-KO and WT mice (Fig. 2l). Compared to WT HSPC, mRNA levels of Il1rn were 170- and 20-fold higher in WT granulocytes and monocytes, respectively (Fig. 2m).

To identify specific changes in HSPC driven by IL-1rn loss, we next performed gene expression profiling by RNA sequencing (RNA-Seq) of WT and IL-1rn-KO LT-HSC, ST-HSC and MPP. We identified impact of IL-1rn abrogation on the transcriptional programs of the LT-HSC and the ST-HSC compartments, with 1433 and 2612 differentially expressed genes, respectively (adjusted p < 0.05). The effect on MPP was smaller, with 181 genes differentially expressed (Supplementary Fig. S4A). Gene set enrichment analysis (GSEA) for LT-HSC revealed coordinated changes in a variety of genes associated with the immune system, the immune response and leukocyte differentiation (Supplementary Fig. S4B, C). GSEA of ST-HSC dysregulated genes identified changes in similar gene programs (Supplementary Fig. S4D, E). These changes were related to increased NFκB transcription factor activity (Fig. 3a, b), and activation of myeloid differentiation genes including the transcription factor PU.1 (Spi1) and some of its target genes like Cebpa and Csf2rb (Fig. 3a, c) in IL-1rn-KO HSPC compared to WT HSPC, particularly in LT-HSC and ST-HSC. Various RNA-Seq hits involving genes related to myeloid differentiation (Spi1, Csf2rb) and genes previously found abnormally expressed in myeloid neoplasias (Axl, Stat3, Tlr1) were confirmed by qRT-PCR (Supplementary Fig. S4F–H)31,32,33. The myeloid bias of progenitors from IL-1rn-KO mice could be reverted in vivo by treatment with bortezomib, a proteasome inhibitor that prevents NFκB activation (Fig. 3d).

Fig. 3: IL-1β-induced myelopoiesis after IL-1rn deletion is mediated by NFκB activation.

Myeloid differentiation bias at the expense of B cell development, reduced self-renewal of primitive HSC and expansion of committed MPP seem consistent hematopoietic effects of chronic (20 day) high dose (0.5 μg) IL-1β treatment across studies despite variability2,3,4, and in vivo deletion of IL-1rn phenocopies these results under steady-state (Fig. 2b, d). IL-1β-induced hematopoietic effects are dependent on transcriptional programs activated by Spi1 and Cebpa2,3,4. We compared our RNA-Seq datasets with these previous publicly available datasets studying the transcriptional programs activated by IL-1β treatment in C57BL/6 WT mice using LSK Flt3−CD48−CD150+ HSC in a native microenvironment (GSE165810)3 or LSK Flt3−CD48+CD150− MPP3, with or without YFP expression, in the context of busulfan conditioning and transplantation (GSE166629)4. We found a partial cell type-specific overlap, with a particularly remarkable transcriptional activation of shared genes induced both by IL-1rn deletion in LT-HSC and ST-HSC, and by IL-1β treatment in LSK Flt3−CD48−CD150+ HSC (Supplementary Fig. S4I, J). Of note, the upregulated genes common to all cell subsets and experimental conditions were enriched in NFκB targets, including Cebpd, Csf2rb, Csf2rb2 or Spi1 (Supplementary Fig. S4I, J).

Taken together, these results suggest that IL-1β and IL-1rn control myeloid output mainly through the LT-HSC and ST-HSC compartments and transcriptional control of myeloid differentiation pathways which are at least partially dependent on NFκB activation, with contribution of these cells to Il1b production in the absence of immunogenic stimuli. IL-1rn-KO mice develop an IL-1β-induced phenotype reminiscent of early hematopoietic disease.

Deletion of IL-1rn causes damage to the BM stroma in the absence of immunogenic stimulus

Adult IL-1rn-KO mice showed incipient reticulin fibrosis despite non-detectable changes in collagen deposits in the BM (Fig. 4a; Supplementary Fig. S5A). BM MSC numbers, identified as stromal (CD45−CD31−Ter119−) cells positive for CD6334, CD105 or CD105 Vcam-135, and Nestin expression21,36, which partially overlap21,34,35,36, were reduced in IL-1rn-KO mice (Fig. 4b; Supplementary Fig. S5B, C). Reanalysis of publicly available single-cell transcriptional data of mouse BM stromal populations (GSE108892)34 showed that Cd63 expression largely overlaps with expression of Lepr, Cxcl12, Kitl and Il1rn (Supplementary Fig. S5D). Therefore, BM CD63+ stromal cells were chosen for most follow-up studies. Reduced numbers of BM CD63+ cells coincided with increased levels of membrane IL-1r1 (Fig. 4c), suggesting potential IL-1β contribution. IL-1β-induced damage to the stromal compartment was confirmed by in vivo treatment with IL-1RN or mAb against IL-1β, which rescued numbers of BM CD63+ stromal cells in IL-1rn-KO mice (Fig. 4d, e).

Fig. 4: Deletion of IL-1rn causes IL-1β-induced damage to the bone marrow stroma in the absence of immunogenic stimulus.

To better understand BM MSC alterations, genome-wide expression was profiled by RNA-Seq in sorted CD45−CD31−Ter119−CD63+ cells. Some of the most downregulated genes in BM CD63+ cells derived from IL-1rn-KO mice included MSC- (Lepr, Adipoq) and HSC regulatory- (Cxcl12, Vcam1, Angpt1) genes (Fig. 4f). These changes were confirmed by qRT-PCR (Fig. 4g). qRT-PCR data also revealed engagement of IL-1β-positive feedback loop in BM CD63+ stromal cells from IL-1rn-KO mice through increased Il1b expression compared to WT BM CD63+ stromal cells and that these cells produce high levels of Il1rn in the BM of adult WT mice (Fig. 4g); 2.7-fold higher than WT granulocytes (Fig. 2m). Taken together, absence of IL-1rn leads to IL-1β-induced damage to the stromal compartment of the BM, with contribution of BM CD63+ stromal cells to Il1b production.

Identification of the sources of IL-1β and IL-1rn in the bone marrow at the single-cell level

To provide further insights into the main sources of IL-1β and IL-1rn in the mouse BM at the single-cell level, we performed single-cell (sc)RNA-Seq and RNA fluorescent in situ hybridization (FISH) in sorted cells. After integrating the WT and IL-1rn-KO scRNA-Seq data from FACS-sorted CD11b+ cells by the Canonical Cross-correlation Analysis (CCA) algorithm of Seurat, unbiased cell clustering analysis was performed (Fig. 5a). WT cells were then used for annotation according to ImmGen37 followed by application of the maximum enrichment score ±0.02 to label the clusters29 using the cell type signatures imported from GSE137539 dataset for neutrophil-like subsets38 and according to GSE131834 dataset for monocyte-like clusters39, and subsequent manual curation. This resulted in 10 clusters that encompass neutrophils at different stages of maturation from G0 to G4, common monocyte progenitors (CMoP I), two subsets of monocytes I and II, and minor fractions of HSC and dendritic cell precursors (PreDC II) (Fig. 5a). CD11b+ myeloid cell proportions were similar in the BM of WT and IL-1rn-KO mice (Fig. 5a), with only minor expansions of G3 neutrophils and CMoP, and reductions in G4 neutrophils and type II monocytes (Fig. 5a). Ingenuity Pathway Analysis (IPA) of “Diseases and Functions” of the different CD11b+ myeloid clusters from IL-1rn-KO versus WT mice revealed significant enrichment in pathways related to inflammation, proliferation, migration, endocytosis and survival (Supplementary Fig. S6A). Inflammation was a major pathway activated in CD11b+ myeloid clusters from IL-1rn-KO versus WT mice, with the exceptions of G1 neutrophils that showed no changes and type I monocytes that showed a reduced inflammatory signature (Supplementary Fig. S6A). Among the differentially expressed genes responsible for these enriched signatures, we found upregulation of several major pro-inflammatory genes (Hif1a, Csf2rb, Myd88, Cxcr2, Lmo4)40,41,42,43,44 as well as downregulation of anti-inflammatory genes (Nfkbia, Cebpb)45,46. A few genes that control inflammation (Lyn)47 were upregulated in CD11b+ myeloid clusters from IL-1rn-KO versus WT mice (Supplementary Fig. S6B).

Neutrophils and particularly the most mature G4 cells were the most abundant CD11b+ cell subset expressing Il1b and Il1rn, and expressed the highest levels of both cytokines, followed by monocytes (Fig. 5a, b). Cytokine-expressing neutrophils and monocytes express mainly Il1b or Il1rn as evidenced by both scRNA-Seq (Fig. 5b, c) and RNA fluorescent in situ hybridization (FISH) (Fig. 5d; Supplementary Fig. S7A), with small fractions of cells particularly within G4 neutrophils, expressing both cytokines (Fig. 5c).

Similarly, WT and IL-1rn-KO scRNA-Seq data from FACS-sorted LSK cells were integrated using the CCA algorithm of Seurat, which was followed by unbiased cell clustering (Fig. 6a). WT cells from each cluster were used for annotation. Gene modules imported from E-MTAB-9208 dataset were scored over LSK clusters29, and cluster assignment to each cell type was obtained using the maximum enrichment score ±0.2 as previously described29 and subsequent manual curation. This resulted in 6 annotated clusters, namely HSC, HSC-MPP1, MPP2, MPP3, MPP4 and MPP5 (Fig. 6a). The proportions of BM LSK cells after clustering of the scRNA-Seq data showed overall similar changes to the immunophenotypic analysis of BM LSK in IL-1rn-KO versus WT mice (Fig. 2b), with qualitative reduction in HSC and MPP5, and expansion of MPP2 (Fig. 6a). Unlike FACS quantification, the scRNA-Seq data showed qualitative expansion of the MPP3 transcriptional state and reduction in lymphoid-biased MPP4 in the BM LSK compartment of IL-1rn-KO mice versus WT (Fig. 6a). IPA of “Diseases and Functions” of the different LSK clusters from IL-1rn-KO versus WT mice revealed significant enrichment in pathways related to tumorigenesis and cell death which were activated in HSC, HSC-MPP1, MPP4 and MPP5 (Supplementary Fig. S8A). MPP2 and MPP3 behaved differently and showed significant activation of pathways related to survival (Supplementary Fig. S8A). Further, tumorigenic pathways were activated in MPP2 but slightly reduced in MPP3 (Supplementary Fig. S8A). Looking into the differentially expressed genes responsible for these enriched signatures, we found upregulation of important genes known to be involved in AML (Nmt1, Ifitm3, Crip1, Cd52)48,49,50,51 and downregulation of genes whose reduced expression is a common feature of acute myeloid leukemogenesis (Junb)52. We also found upregulation of genes with antiproliferative role (Ifitm1)53 and downregulation of genes whose loss promote cell exhaustion (Hlf)54 in LSK clusters from IL-1rn-KO versus WT mice (Supplementary Fig. S8B).

Fig. 6: Identification of the sources of IL-1β and IL-1rn in the bone marrow at the single-cell level within the hematopoietic stem and progenitor cell compartment.

Expression of Il1b and Il1rn was not detectable by scRNA-Seq in LSK cells (Supplementary Fig. S8C), but RNA-FISH showed that LT-HSC expressed the highest levels of Il1b among LSK subsets, followed by ST-HSC and MPP (Fig. 6b; Supplementary Fig. S9A). Some of the Il1b-expressing LT-HSC also had detectable levels of Il1rn expression (Fig. 6b; Supplementary Fig. S9A).

The same procedure was used for the annotation of the scRNA-Seq data from FACS-sorted CD63+ stromal cells with gene signatures obtained from GSE128423 dataset35. We identified 5 clusters, namely arteriolar cells, fibroblasts, MSC, pericytes and sinusoidal cells (Fig. 7a). Arteriolar and sinusoidal cells were reduced whereas pericytes were increased within the CD63+ stromal compartment from IL-1rn-KO mice versus WT (Fig. 7a). IPA of “Diseases and Functions” of the CD63+ stromal cluster fibroblasts from IL-1rn-KO versus WT mice revealed significant enrichment in pathways related to differentiation into adipocytes, chondrocytes and osteocytes that were activated whereas gene sets coordinating functions like proliferation and inflammation were reduced, as opposed to MSC (Supplementary Fig. S10A). Among the differentially expressed genes responsible for the enriched differentiation signatures, we found important genes involved in adipocyte (Nr1d1, Zbtb16, Nfia)55,56,57, chondrocyte (Hspg2, Bmp4, Col3a1)58,59,60 and osteocyte lineage differentiation (Fbn1, Twist1, Spp1)61,62,63 (Supplementary Fig. S10B). All arteriolar, sinusoidal and pericyte clusters showed significant enrichment and activation in pathways associated with angiogenesis (Supplementary Fig. S10C). In addition, the sinusoidal cluster displayed activation of pathways related to inflammation, atherosclerosis and recruitment of leukocytes (Supplementary Fig. S10C). We checked the differentially expressed genes responsible for these enriched signatures and found upregulation of major pro-inflammatory genes in the CD63+ sinusoidal cluster from IL-1rn-KO mice versus WT (Il1b, Il6, Nlrp3, Csf1, Ccl4, Ccl6, Ccl9, Osm) (Supplementary Fig. S10D).

Fig. 7: Identification of the sources of IL-1β and IL-1rn in the bone marrow at the single-cell level within the CD63+ stromal cell compartment.

We found cells expressing Il1b, Il1rn or both within the CD63+ stromal cell compartment both by scRNA-Seq (Fig. 7b-c) and RNA-FISH (Fig. 7d; Supplementary Fig. S11A). scRNA-Seq was able to resolve that Il1b/Il1rn are mainly produced by MSC and arteriolar cells within the CD63+ stromal cell compartment, and most cytokine-expressing MSC express Il1rn only whereas arteriolar cells express mainly Il1b or both, with a small fraction expressing Il1rn only (Fig. 7a–c). A small fraction of sinusoidal cells also expresses Il1b only (Fig. 7a–c; Supplementary Fig. S11A).

Together, these data highlight that IL-1rn buffers mostly paracrine IL-1β feed-forward loops through its exclusive expression from subsets of CD63+ MSC, neutrophils and monocytes, and a small fraction of CD63+ arteriolar cells in the BM of WT healthy mice under steady-state conditions. Autocrine responses are rare and were observed mainly in subsets of LT-HSC and CD63+ arteriolar cells, and a small proportion of neutrophils mainly within G4, with expression of both cytokines simultaneously.

Low IL-1rn is a hallmark of pre-leukemic myelopoiesis in vivo

The reduction of IL-1RN in AML patients and the effect of IL-1rn loss in HSPC suggest its role in hematological diseases. To address this possibility, we used the Mx1-Cre NRASG12D mouse model of aberrant pre-leukemic myelopoiesis64,65, from now on referred to as NRAS-G12D+. As previously described, pre-leukemic NRAS-G12D+ HSPC outcompete WT cells (Supplementary Fig. S12A)65. Primary NRAS-G12D+ mice display a mild hematopoietic phenotype, characterized as chronic myelomonocytic leukemia66,67 with increased circulating white blood cells, particularly myeloid cells. In the BM, numbers of c-Kit+ progenitors and LSK HSPC were higher in NRAS-G12D+ mice, with abnormal relative amounts of all LSK subsets (Supplementary Fig. S12B–E)64. Detailed analysis revealed reduction of HSC, MPP5 and MPP1, and an increase of MPP4 in the BM of NRAS-G12D+ mice (Supplementary Fig. S12F). More committed CLP and GMP progenitors were expanded in the BM of NRAS-G12D+ versus control mice (Supplementary Fig. S12G).

RAS transduces IL-1β signaling through MyD88, and oncogenic RAS results in IL-1β amplification through persistent activation of the autocrine feedback loop in other types of cancer68. IL-1β levels in NRAS-G12D+ BM were increased, while levels of IL-1rn were reduced in the BM of diseased mice (Fig. 8a, b). At this stage, BM hypercellularity was prominent, with expansion of cell subsets previously described to produce IL-1-β including neutrophils, monocytes and LSK cells (Fig. 8c). Expression of Il1b was not induced in granulocytes or monocytes from the BM of NRAS-G12D+ mice compared to healthy controls, and these hematopoietic cells did not show reduced expression of Il1rn either (Fig. 8d). In contrast, all HSPC subsets expressed higher levels of Il1b measured by qRT-PCR, and Il1rn was reduced in NRAS-G12D+ versus control MPP only (Fig. 8e). Il1rap gene expression was unchanged in any of the hematopoietic cell populations studied (Fig. 8d, e), but the fraction of cells expressing membrane IL-1RAP was higher in NRAS-G12D+ versus control LT-HSC, MPP and monocytes (Fig. 8f; Supplementary Fig. S12H). To better understand the specific contribution of HSPC to the IL-1β-induced inflammatory environment of the BM in NRAS-G12D+ mice, gene expression profiling was performed by RNA-Seq in LT-HSC, ST-HSC and MPP. The transcriptional programs of both LT-HSC and MPP were influenced by expression of NRAS-G12D, with 894 and 651 differentially expressed genes, respectively. The effect of NRAS-G12D expression on ST-HSC was smaller, with 340 genes differentially expressed (Supplementary Fig. S12I). PCA showed coherent clustering of replicates and revealed that LT-HSC are most influenced by NRAS-G12D expression (Supplementary Fig. S12J). All NRAS-G12D+ HSPC subsets exhibited increased NFκB transcription factor calculated activity (Fig. 8g). The myeloid bias of progenitors from NRAS-G12D+ mice could be improved in vivo by treatment with bortezomib (Fig. 8h). Taken together, the biased myelopoiesis in NRAS-G12D+ mice was at least partially dependent on NFκB activation in HSPC, and HSPC contributed to loss of repression of IL-1β pathway through increased expression of Il1b and membrane IL-1RAP, and reduced expression of Il1rn in the case of MPP.

Fig. 8: Low IL-1rn is present in a mouse model of pre-leukemic myelopoiesis.

We then studied the stromal compartment and found reduction in BM CD63+ stromal cell numbers (Fig. 9a) together with increased apoptotic rates in NRAS-G12D+ mice (Fig. 9b). BM CD63+ stromal cells were sorted and qRT-PCR analyses revealed no changes for Il1b and Il1rap but reduced expression of Il1rn in BM CD63+ stromal cells from diseased mice (Fig. 9c). Reduced numbers of Nestin+ stromal cells and activation of apoptosis at the transcriptomic level was confirmed using Nes-gfp mice that had previously received NRAS-G12D+ BM, compared to recipients of control BM (Supplementary Fig. S13A, B). Hence, damage to the microenvironment is present in the NRAS-G12D+ mouse model of pre-leukemic myelopoiesis, and MSC contribute to the low IL-1rn in BM.

Fig. 9: Damage to the bone marrow stroma in a mouse model of pre-leukemic myelopoiesis.

To minimize potential confounding effects derived from Cre expression in osteolineage cells69 and from transient activation of IFNα signaling through polyI:polyC injection70, we used a conditional model that constitutively expresses NRASG12D in the hematopoietic system under the control of Vav-Cre67. This mouse model reproduced the main hematopoietic abnormalities of Mx1-Cre NRASG12D mice including increased circulating white blood cells particularly monocytes, hypercellularity in the BM, and expansion of immunophenotypically defined LSK HSPC, particularly MPP4, and CLP (Supplementary Fig. S14A–F). Analysis of publicly available gene expression profiling by RNA-Seq of Lin−c-Kit+Sca-1− (LK) progenitors (PRJNA774277)67 showed that NFκB transcription factor calculated activity was higher in Vav-Cre NRASG12D than in control mice (Supplementary Fig. S14G).

Deletion of IL-1rn from the hematopoietic or stromal compartments promotes pre-leukemic myelopoiesis, whereas exogenous IL-1rn protects against it

To address the role of IL-1rn in hematopoietic cells under pre-leukemic myelopoiesis, we generated mixed-chimera systems using WT and IL-1rn-KO mice; WT and NRAS-G12D+ mice; or IL-1rn-KO and NRAS-G12D+ mice, as donors of BM to WT recipients (Fig. 10a). Deletion of IL-1rn in hematopoietic cells exerted a synergistic effect with presence of NRASG12D in neighboring hematopoietic cells to promote myeloid output (Fig. 10b). B cells showed no changes in presence of NRASG12D irrespective of WT or IL-1rn deficient competitor cells (Fig. 10c).

Fig. 10: Deletion of IL-1rn from the hematopoietic or stromal compartments promotes pre-leukemic myelopoiesis, and it is therapeutically targetable.

To investigate the role of IL-1rn deficiency in the stroma under pre-leukemic myelopoiesis, we then performed transplants using WT and IL-1rn-KO mice as recipients of BM from induced disease-free NRASG12D control and Mx1-Cre NRASG12D mice (Fig. 10d). Numbers of circulating myeloid cells, particularly monocytes, were higher in IL-1rn-KO mice transplanted with diseased NRAS-G12D+ cells as compared to WT recipients (Fig. 10e). B cells were expanded only in IL-1rn-KO mice transplanted with diseased NRAS-G12D+ cells (Fig. 10f). These results suggest that loss of IL-1β repression through IL-1rn deletion within either the BM hematopoietic or stromal compartment may play a role in pre-leukemic disease. The data further demonstrate that the reduced numbers of BM MSC and their Il1rn expression in induced Mx1-Cre NRASG12D mice (Fig. 9a, c) contributes to NRAS-G12D+ disease.

Conversely, short-term in vivo treatment of induced Mx1-Cre NRASG12D mice with IL-1RN (Fig. 10g) ameliorated early signs of abnormal myelopoiesis by means of reduced circulating myeloid cells, particularly monocytes (Fig. 10h), with no detected changes in B cells (Fig. 10i). Thus, the low IL-1rn conditions contribute to the biased myelopoiesis in NRAS-G12D+ mice. These data uncover the protective effect of the endogenous IL-1 repressor IL-1rn against pre-leukemic myelopoiesis.

Discussion

Despite the emerging role of IL-1β signaling in the pathogenesis of hematological diseases1,6,7,8,9,10,11,12,15,16, little is known about the potential contribution of its endogenous counteracting cytokine IL-1RN to healthy and/or malignant hematopoiesis. Here, we find that low expression of IL1RN in purified blasts correlated with negative prognosis in a publicly available big cohort of 381 global AML patients, and from those in 164 M4-M5 AML patients, at diagnosis time23,24,25 as well as in matched-pair diagnosis-relapsed AML patients26. We provide translational data in primary CD34+ progenitors from AML patients showing IL-1RN deregulation in newly diagnosed AML patients, particularly those with lower differentiation profiles, making the study of the prognostic value of IL-1RN not possible in this group. IL-1RN deregulation was confirmed in a publicly available cohort of AML patients that used purified AML blasts23,24,25. These data are of particular interest considering AML heterogeneity, as it indicates that low IL-1RN is a hallmark of AML that may contribute to disease aggravation and poor outcome. Future studies aimed at uncovering the underlying molecular mechanisms for IL-1RN reduction in AML will be relevant, and should include methylation in CpG sites as it was previously involved in relapsed versus diagnosis paired AML samples26.

NFκB may be activated by multiple signals but in patients, unbalanced IL-1RN in AML CD34+ progenitors associated with higher numbers of cells activated through NFκB as well as higher nuclear translocation of the p50/p65 NFκB heterodimer. CD34+ cells are a mixture of LT-HSC, ST-HSC and progenitors, so these changes may reflect the differential cell composition and/or selective changes per cell subset, between health and AML. These data are indicative of the enrichment of this pathway in AML CD34+ cells and are consistent with previous observations of NFκB activation in human CD34+CD38– leukemia stem cells71. Chronic IL-1β treatment of NSG-SGM3 mice transplanted with CD34+ progenitors isolated from the BM of AML patients promoted AML cell expansion, in agreement with previous results6. Conversely, boosting IL-1RN improved signs of disease in the NRASG12D-driven myeloid pre-leukemia mouse model and in mouse xenografts from AML patients. IL-1RN boost or IL-1β blockade through treatment with canakinumab, currently under Phase II trial for the treatment of low and intermediate risk MDS and CML (NTC04239157), reduced to similar extent the expansion of human myeloid cells, in NSG-SGM3 mice. Pharmacologically, canakinumab provides advantage over anakinra considering their half-lives of 26 days and 4-6 h, respectively1. However, the CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) raised safety concerns due to higher incidence of deaths due to fatal infection in the treated versus the placebo group72. Anakinra provides the advantages of potentially better regulated physiological responses and opportunity to quickly discontinue IL-1β blockade in case of infection. Efforts towards prolonging its half-life to some extent, to prevent burden of daily injections, by fusion protein technology and/or development of biodegradable polymers to increase its steady-state sustained release are underway73.

Functionally, loss of IL-1rn in vivo biases HSC differentiation into the myeloid lineage and induces excess myeloid lineage expansion reminiscent of early hematological disease, through IL-1r1 and IL-1β overactivation in HSC but not IL-1α, in the absence of injury or infection. These findings are consistent with previous observations upon long-term administration of high IL-1β doses2, but further demonstrate that IL-1rn represses HSC differentiation into the myeloid lineage driven by IL-1β under steady-state conditions. Our mouse studies showed that abnormal myelopoiesis after IL-1rn deletion is mediated mainly through transcriptional activation of IL-1β-induced myeloid differentiation pathways at least partially dependent on NFκB activation in HSPC, which partially overlap with the transcriptional programs activated by long-term administration of high IL-1β doses as shown by reanalysis of publicly available datasets3,4 and are consistent with our findings in AML patients. Our results suggest that these effects are mediated mainly through the LT-HSC and ST-HSC compartments, with selective reduction in more primitive HSC and MPP5, and increase in MPP2 numbers and colonies ex vivo. Further immunophenotypic characterization of the BM of IL-1rn-KO mice showed that the myeloid lineage was expanded at the further expense of reduced numbers of MEPs and GMPs, whereas lymphoid lineage repression in the absence of IL-1rn did not involve active coordinated transcriptomic repression in LT-HSC or ST-HSC, or changes in immunophenotypically defined CLP numbers. Our scRNA-Seq analyses confirmed overall the immunophenotypic changes in the proportions of BM HSPC cells but showed qualitative expansion of MPP3 transcriptional state and reduction in lymphoid-biased MPP4 cells in the BM LSK compartment of IL-1rn-KO versus WT mice. Analysis of gene sets enriched at single-cell resolution revealed significant activation in pathways related to tumorigenesis and cell death in the HSPC subsets that were reduced i.e. HSC, HSC-MPP1, MPP4 and MPP5. Conversely, the expanded MPP2 and MPP3 compartments showed significant activation of pathways related to survival, whereas tumorigenic pathways were activated in MPP2 but slightly reduced in MPP3. These opposing and cell type-specific responses in HSPC subsets may be main factors underlying the chronic nature of the hematopoietic abnormalities in IL-1rn-KO mice, which will require additional investigation.

The expanded myeloid compartment in IL-1rn-KO mice may contribute to higher levels of IL-1β in the BM, as previously suggested under IL-1β administration2. Unlike myeloid cells, LT-HSC and ST-HSC showed sustained engagement of an IL-1β-positive feedback loop through IL-1r1, suggesting that HSPC are active modulators of inflammation rather than passive receivers. Of note, Il1rap gene expression was unchanged in LT-HSC and ST-HSC but reduced in MPP from IL-1rn-KO versus WT mice, which could contribute to the limited effects of IL-1rn deletion on MPP. Together, our data suggest that LT-HSC and ST-HSC play a key role in the chronic inflammatory process driven by IL-1rn deletion through IL-1β production and sustained supply of effector inflammatory cells, in the absence of immunogenic triggers. Selective regulatory mechanisms of Il1b expression and responsiveness in HSC, MPP, progenitors and fully differentiated myeloid cells, should be subject of future work.

The sc-RNA-Seq analysis and annotation of BM CD11b+ myeloid cells showed similar proportions of cell types in the BM of WT and IL-1rn-KO mice, with only a slight increase in more immature G3 neutrophils and CMoP, and reduction in more differentiated G4 neutrophils and type II monocytes. In general, inflammation was a major pathway activated in all IL-1rn-KO CD11b+ myeloid clusters except in G1 neutrophils that showed no changes and type I monocytes that displayed a reduced inflammatory signature. Thus, despite showing no engagement of an IL-1β-positive feedback loop, effector inflammatory cells are activated in the BM of IL-1rn-KO mice in the absence of immunogenic triggers. The potential functional consequences of this phenomenon on hematopoiesis should be studied in future work.

IL-1rn deletion induced alterations to the BM microenvironment, including fibrosis, reduced MSC numbers and their HSC-regulatory activity at the transcriptomic level, which were caused by IL-1β. This is in accordance with our previous observations in the IL-1β inflammatory conditions driven by JAK2-V617F+ hematopoietic cells15. In view of the overlap with other relevant MSC markers and high expression of Il1rn, we used BM CD63+ stromal cells as a surrogate for BM MSC. The sc-RNA-Seq analysis and annotation of BM CD63+ cells underscored that they are indeed comprised by MSC, but also related-cell subsets i.e. fibroblasts, arteriolar and sinusoidal cells, and pericytes. Arteriolar and sinusoidal cells were reduced, and pericytes increased within the CD63+ stromal compartment from IL-1rn-KO mice versus WT. Analysis of gene sets enriched at single-cell resolution highlighted the complex effects of IL-1rn deletion on the CD63+ stromal compartment, activating gene signatures of differentiation and restricting proliferation in fibroblasts, as opposed to MSC, and stimulating endothelial cell development in vascular and perivascular clusters. Thus, our data indicate that both changes in cell composition and selective gene expression shifts may underlie alterations to the BM microenvironment in the absence of IL-1rn. The strong cell type-specific responses within the CD63+ stromal compartment will require functional characterization.

Normal BM MSC switch between their pro- and anti-inflammatory phenotypes to initiate inflammation at early stages of damage sensing and to promote its resolution at advanced stages, respectively74. Our data suggest that BM CD63+ stromal cells contribute to inflammation with Il1b expression in the absence of IL-1rn with no need of injury or infection sensing. Interestingly, our scRNA-Seq in CD63+ BM stromal cells from WT mice was able to resolve that Il1b/Il1rn are mainly produced by MSC and arteriolar cells, and a small fraction of sinusoidal cells. Most cytokine-expressing MSC in fact express Il1rn only whereas arteriolar cells express mainly Il1b or both, with a small fraction expressing Il1rn only. Sinusoidal cells express Il1b only and they may be particularly involved in the induction of inflammation in the absence of IL-1rn, together with MSC.

In the BM of WT mice, our data at single-cell resolution further highlight that IL-1rn buffers mostly paracrine IL-1β feed-forward loops through its exclusive expression from subsets of CD63+ MSC, neutrophils and monocytes, and a small fraction of CD63+ arteriolar cells. Autocrine responses are rare and mainly in subsets of LT-HSC and CD63+ arteriolar cells, and a small fraction of neutrophils mainly within G4, with expression of both cytokines. Selective regulatory mechanisms of Il1rn expression from different cell sources should be investigated in future work.

The hematopoietic phenotype observed in IL-1rn-KO mice is reminiscent of that observed in patients of the rare syndrome named deficiency in IL-1RN (DIRA). DIRA is a rare congenital disease caused by loss of function mutations in the IL1RN locus that results in death from severe auto-inflammation and multiorgan failure if not treated with anakinra75. DIRA patients suffer from hematopoietic abnormalities including leukocytosis and monocytosis, anemia, thrombocytosis, extramedullary hematopoiesis with enlargement of liver and spleen, BM fibrosis and osteosclerosis75,76, reminiscent of early hematopoietic disease.

In further support of our data in human AML, we provide multiple evidence demonstrating unbalanced IL-1rn in aberrant myelopoiesis in mouse. We used the NRAS-G12D+ model of pre-leukemic myelopoiesis Mx1-Cre NRASG12D, based on IL-1β amplification mediated by oncogenic RAS described in other types of cancer68. This paradigm was true for NRAS-G12D+ HSPC but no other major producers of IL-1β within the differentiated cell compartment, i.e. granulocytes and monocytes. Consistent with previous findings in human AML reproduced here13,14, the percentage of LT-HSC, MPP and monocytes expressing membrane IL-1RAP was higher in pre-leukemic mice compared to controls. Whereas MPP were resistant to IL-1β-positive feedback loop in IL-1rn deficient mice, they expressed higher levels of Il1b in the presence of oncogenic NRAS, comparable to LT-HSC and ST-HSC. Thus, the high IL-1β levels in the BM of diseased mice were result of a HSPC-selective gene regulation event together with expansion of IL-1β-producing hematopoietic cell types, i.e. HSPC, granulocytes and monocytes, in NRAS-G12D+ disease. Of note, MPP were the most expanded HSPC subset in NRAS-G12D+ mice and this was coincident with their abnormally high Il1b expression, and selective downregulation of Il1rn across all hematopoietic cell types studied, including LT-HSC and ST-HSC. Thus, both human AML CD34+ cells and mouse pre-leukemic MPP participate to unbalanced IL-1RN. Like IL-1β, selective regulation of IL-1rn production across hematopoietic cells was apparent and its underlying mechanism should be subject of future work. Congruent with our findings in AML patients and IL-1rn-KO mice, low IL-1rn contributed to the biased myelopoiesis in NRAS-G12D+ mice through pathways at least partially dependent on NFκB activation, which was confirmed in two different mouse models, i.e. Mx1-Cre NRASG12D and Vav-Cre NRASG12D. Our data showed that BM CD63+ stromal cells contributed to the lower levels of IL-1rn in the BM of NRAS-G12D+ mice by their cellular fragility, as demonstrated by both their increased apoptotic rates and reduced cell numbers, and selective downregulated expression of Il1rn. Absence of IL-1rn from either the hematopoietic or the stromal compartments induced faster progression of NRASG12D-driven pre-malignant hematopoiesis. Thus, both compartments are relevant for IL-1rn production to repress myelopoiesis under neoplastic hematopoiesis. Our data are consistent with the idea that inflammation damages the BM microenvironment, which then establishes abnormal communication with HSPC and thereby plays a role in abnormal hematopoiesis8,15. We further suggest that this abnormal communication comprises deficient control of IL-1β-induced inflammation by BM MSC and arteriolar cells in the presence of a pre-leukemic lesion. The study of the potential role for IL-1RN anti-inflammatory properties of BM MSC and arteriolar cells in human AML will be of interest.

Together, loss of repression by IL-1rn resulting in enhanced stimulation of IL-1β signaling may underlie both pre-leukemic lesion and AML progression. Low IL-1rn and subsequent high IL-1β promotes myeloproliferation and inhibits the most primitive HSC, with the contribution of HSC to the low IL-1rn and high IL-1β. These conditions may pose a pressuring environment for the long-term selection of clones with lighter sensitivity to the inhibitory effect of low IL-1rn and high IL-1β. In theory, the potential benefit of this process would be to prevent the exhaustion of the HSC pool. However, HSC clones that thrive and expand in the presence of an otherwise inhibitory signal meet the criteria of functionally malignant leukemia stem cells, and selection of these clones would result in aggressive malignancy. Further work is required to validate this intriguing hypothesis, but if it holds true it could explain the association of low IL-1RN both with myeloproliferation at early or mild stages of disease and with a more primitive state and poor survival in AML patients at diagnose or relapse who are experiencing late or aggressive transformation events. It would also provide grounds to the therapeutic potential of IL-1RN at both stages.

Collectively, our data support that endogenous IL-1RN represses myelopoiesis in HSPC under steady-state and protects from neoplasia, whereas low IL-1RN is a hallmark of AML with prognostic value for reduced survival in AML patients, it contributes to myeloproliferation in the presence of a pre-leukemic lesion and provides a mechanistic rationale for IL-1RN boost or IL-1β blockade therapeutic potential in AML patients (Supplementary Fig. S15). We depict a potential origin for high IL-1β in human AML, and a type of patient that may be particularly good responder to anti-IL-1β therapies over patients that present with high IL-1β resulting from primary lesions in the signaling pathway downstream of IL-1R1.

Article classification: Biological abstract

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