IL-1 in aging and pathologies of hematopoietic stem cells.

ABSTRACT: Defense-oriented inflammatory reactivity supports survival at younger age but might contribute to health impairments in modern, aging societies. The interleukin-1 (IL-1) cytokines are highly conserved and regulated, pleiotropic mediators of inflammation, essential to respond adequately to infection and tissue damage but also with potential host damaging effects when left unresolved. In this review, we discuss how continuous low-level IL-1 signaling contributes to aging-associated hematopoietic stem and progenitor cell (HSPC) functional impairments and how this inflammatory selective pressure acts as a driver of more profound hematological alterations, such as clonal hematopoiesis of indeterminate potential, and to overt HSPC diseases, like myeloproliferative and myelodysplastic neoplasia as well as acute myeloid leukemia. Based on this, we outline how IL-1 pathway inhibition might be used to prevent or treat inflammaging-associated HSPC pathologies.

Aging drives Tet2+/- clonal hematopoiesis via IL-1 signaling.

Clonal hematopoiesis of indeterminate potential (CHIP), also referred to as aging-related clonal hematopoiesis, is defined as an asymptomatic clonal expansion of mutant mature hematopoietic cells in ≥4% of blood leukocytes. CHIP associates with advanced age and increased risk for hematological malignancy, cardiovascular disease, and all-cause mortality. Loss-of-function somatic mutations in TET2 are frequent drivers of CHIP. However, the contribution of aging-associated cooperating cell-extrinsic drivers, like inflammation, remains underexplored. Using bone marrow (BM) transplantation and newly developed genetic mosaicism (HSC-SCL-Cre-ERT; Tet2+/flox; R26+/tm6[CAG-ZsGreen1]Hze) mouse models of Tet2+/-driven CHIP, we observed an association between increased Tet2+/- clonal expansion and higher BM levels of the inflammatory cytokine interleukin-1 (IL-1) upon aging. Administration of IL-1 to mice carrying CHIP led to an IL-1 receptor 1 (IL-1R1)-dependent expansion of Tet2+/- hematopoietic stem and progenitor cells (HSPCs) and mature blood cells. This expansion was caused by increased Tet2+/- HSPC cell cycle progression, increased multilineage differentiation, and higher repopulation capacity compared with their wild-type counterparts. In agreement, IL-1α-treated Tet2+/- hematopoietic stem cells showed increased DNA replication and repair transcriptomic signatures and reduced susceptibility to IL-1α-mediated downregulation of self-renewal genes. More important, genetic deletion of IL-1R1 in Tet2+/- HPSCs or pharmacologic inhibition of IL-1 signaling impaired Tet2+/- clonal expansion, establishing the IL-1 pathway as a relevant and therapeutically targetable driver of Tet2+/- CHIP progression during aging.

A microbiome-macrophage-iron axis guides stressed hematopoietic stem cell fate.

The mammalian microbiota is a recently recognized regulator of hematopoiesis. In this issue of Cell Stem Cell, Zhang et al. (2022) show in mice that microbiota-derived butyrate enhances bone marrow macrophage erythrophagocytosis-dependent iron availability, which supports stress-induced hematopoietic stem cell differentiation and blood regeneration.

IL-1 mediates microbiome-induced inflammaging of hematopoietic stem cells in mice.

Aging is associated with impaired hematopoietic and immune function caused in part by decreased fitness in the hematopoietic stem cell (HSC) population and an increased myeloid differentiation bias. The reasons for this aging-associated HSC impairment are incompletely understood. Here we demonstrate that older specific pathogen free (SPF) wild-type (WT) mice in contrast to young SPF mice produce more interleukin-1a and interleukin-1b (IL-1a/b) in steady-state bone marrow (BM), with most of the IL-1a/b being derived from myeloid BM cells. Furthermore, blood from steady-state older SPF WT mice contains higher levels of microbe-associated molecular patterns, specifically TLR4 and TLR8 ligands. In addition, BM myeloid cells from older mice produce more IL-1b in vitro, and older mice show higher and more durable IL-1a/b responses upon stimulation with lipopolysaccharide in vivo. To test whether HSC aging is driven by IL-1a/b, we evaluated HSCs from IL-1 receptor 1 (IL-1R1) knockout (KO) mice. Indeed, older HSCs from IL-1R1KO mice show significantly mitigated aging-associated inflammatory signatures. Moreover, HSCs from older IL-1R1KO and from germ-free mice maintain unbiased lymphomyeloid hematopoietic differentiation upon transplantation, thus resembling this functionality of young HSCs. Importantly, in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signaling in older WT mice was similarly sufficient to reverse myeloid-biased output of their HSC populations. Collectively, our data define the microbiome/IL-1/IL-1R1 axis as a key, self-sustaining and also therapeutically partially reversible driver of HSC inflammaging.

Inflammation as a regulator of hematopoietic stem cell function in disease, aging, and clonal selection.

Inflammation is an evolutionarily selected defense response to infection or tissue damage that involves activation and consumption of immune cells in order to reestablish and maintain organismal integrity. In this process, hematopoietic stem cells (HSCs) are themselves exposed to inflammatory cues and via proliferation and differentiation, replace mature immune cells in a demand-adapted fashion. Here, we review how major sources of systemic inflammation act on and subsequently shape HSC fate and function. We highlight how lifelong inflammatory exposure contributes to HSC inflamm-aging and selection of premalignant HSC clones. Finally, we explore emerging areas of interest and open questions remaining in the field.

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection.

Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses.

Author Correction: Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells.

Acute myeloid leukemia (AML) remains a clinical challenge due to frequent chemotherapy resistance and deadly relapses. We are exploring the immunotherapeutic potential of peripheral blood Vδ1+ T cells, which associate with improved long-term survival of stem-cell transplant recipients but have not yet been applied as adoptive cell therapy. Using our clinical-grade protocol for expansion and differentiation of "Delta One T" (DOT) cells, we found DOT cells to be highly cytotoxic against AML primary samples and cell lines, including cells selected for resistance to standard chemotherapy. Unlike chemotherapy, DOT-cell targeting did not select for outgrowth of specific AML lineages, suggesting a broad recognition domain, an outcome that was consistent with the polyclonality of the DOT-cell T-cell receptor (TCR) repertoire. However, AML reactivity was only slightly impaired upon Vδ1+ TCR antibody blockade, whereas it was strongly dependent on expression of the NKp30 ligand, B7-H6. In contrast, DOT cells did not show reactivity against normal leukocytes, including CD33+ or CD123+ myeloid cells. Adoptive transfer of DOT cells in vivo reduced AML load in the blood and target organs of multiple human AML xenograft models and significantly prolonged host survival without detectable toxicity, thus providing proof-of-concept for DOT-cell application in AML treatment.

VEGFR2-Mediated Reprogramming of Mitochondrial Metabolism Regulates the Sensitivity of Acute Myeloid Leukemia to Chemotherapy.

Metabolic reprogramming is central to tumorigenesis, but whether chemotherapy induces metabolic features promoting recurrence remains unknown. We established a mouse xenograft model of human acute myeloid leukemia (AML) that enabled chemotherapy-induced regressions of established disease followed by lethal regrowth of more aggressive tumor cells. Human AML cells from terminally ill mice treated with chemotherapy (chemoAML) had higher lipid content, increased lactate production and ATP levels, reduced expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and fewer mitochondria than controls from untreated AML animals. These changes were linked to increased VEGFR2 signaling that counteracted chemotherapy-driven cell death; blocking of VEGFR2 sensitized chemoAML to chemotherapy (re-)treatment and induced a mitochondrial biogenesis program with increased mitochondrial mass and oxidative stress. Accordingly, depletion of PGC-1α in chemoAML cells abolished such induction of mitochondrial metabolism and chemosensitization in response to VEGFR2 inhibition. Collectively, this reveals a mitochondrial metabolic vulnerability with potential therapeutic applications against chemotherapy-resistant AML.Significance: These findings reveal a mitochondrial metabolic vulnerability that might be exploited to kill chemotherapy-resistant acute myeloid leukemia cells. Cancer Res; 78(3); 731-41. ©2017 AACR.

Intra-tumour heterogeneity - going beyond genetics.

Cancer patients die primarily due to disease recurrence after transient treatment responses. The emergence of therapy-resistant escape variants is fuelled by intra-tumour heterogeneity, underpinned by interference and Darwinian evolution among continuously developing sub-clones in the mutating tumour. Novel cancer cell variants build upon the pre-existing genetic landscape and tumour heterogeneity is often ascribed largely to genetic variability. While mutations are required for cancer development and studies of genetic evolution of tumours have improved our understanding of cancer biology, genetics only represents one dimension of the fitness of each cancer cell. Beyond the mutations, several non-genetic factors also add significant variability, resulting in a complex and highly dynamic tumour cell population that can drive disease under almost any condition. This viewpoint article summarizes the genetic basis of intra-tumour heterogeneity, before dissecting four major interdependent non-genetic factors we think critically contribute to the overall variability of tumour cells in all types of cancer: epigenetic regulation, cellular differentiation hierarchies, gene expression stochasticity and tumour microenvironment. We finally present the relevant technological approaches to address the combined contribution of both genetic and non-genetic factors to intra-tumour heterogeneity, focusing on genomic profiling, cellular lineage tracing and single-cell RNA sequencing technologies. This strategy will ultimately allow dissection of the full range and depth of intra-tumour heterogeneity. We thus believe that understanding how cancer genetics synergize with the emerging non-genetic factors will be key for development of therapies able to tackle tumour escape and thereby improve cancer patient survival.

miR-363-5p regulates endothelial cell properties and their communication with hematopoietic precursor cells.

Recent findings have shown that the blood vessels of different organs exert an active role in regulating organ function. In detail, the endothelium that aligns the vasculature of most organs is fundamental in maintaining organ homeostasis and in promoting organ recovery following injury. Mechanistically, endothelial cells (EC) of tissues such as the liver, lungs or the bone marrow (BM) have been shown to produce "angiocrine" factors that promote organ recovery and restore normal organ function. Controlled production of angiocrine factors following organ injury is therefore essential to promote organ regeneration and to restore organ function. However, the molecular mechanisms underlying the coordinated production and function of such "angiocrine" factors are largely undisclosed and were the subject of the present study. In detail, we identified for the first time a microRNA (miRNA) expressed by BM EC that regulates the expression of angiocrine genes involved in BM recovery following irradiation. Using a microarray-based approach, we identified several miRNA expressed by irradiated BMEC. After validating the variations in miRNA expression by semi-quantitative PCR, we chose to study further the ones showing consistent variations between experiments, and those predicted to regulate (directly or indirectly) angiogenic and angiocrine factors. Of the mi-RNA that were chosen, miR-363-5p (previously termed miR-363*) was subsequently shown to modulate the expression of numerous EC-specific genes including some angiocrine factors. By luciferase reporter assays, miR-363-5p is shown to regulate the expression of angiocrine factors tissue inhibitor of metalloproteinases-1 (Timp-1) and thrombospondin 3 (THBS3) at post-transcriptional level. Moreover, miR-363-5p reduction using anti-miR is shown to affect EC angiogenic properties (such as the response to angiogenic factors stimulation) and the interaction between EC and hematopoietic precursors (particularly relevant in a BM setting). miR-363-5p reduction resulted in a significant decrease in EC tube formation on matrigel, but increased hematopoietic precursor cells adhesion onto EC, a mechanism that is shown to involve kit ligand-mediated cell adhesion. Taken together, we have identified a miRNA induced by irradiation that regulates angiocrine factors expression on EC and as such modulates EC properties. Further studies on the importance of miR-363-5p on normal BM function and in disease are warranted.

Bone marrow-derived CD11b+Jagged2+ cells promote epithelial-to-mesenchymal transition and metastasization in colorectal cancer.

Timely detection of colorectal cancer metastases may permit improvements in their clinical management. Here, we investigated a putative role for bone marrow-derived cells in the induction of epithelial-to-mesenchymal transition (EMT) as a marker for onset of metastasis. In ectopic and orthotopic mouse models of colorectal cancer, bone marrow-derived CD11b(Itgam)(+)Jagged2 (Jag2)(+) cells infiltrated primary tumors and surrounded tumor cells that exhibited diminished expression of E-cadherin and increased expression of vimentin, 2 hallmarks of EMT. In vitro coculture experiments showed that the bone marrow-derived CD11b(+)Jag2(+) cells induced EMT through a Notch-dependent pathway. Using neutralizing antibodies, we imposed a blockade on CD11b(+) cells' recruitment to tumors, which decreased the tumor-infiltrating CD11b(+)Jag2(+) cell population of interest, decreasing tumor growth, restoring E-cadherin expression, and delaying EMT. In support of these results, we found that peripheral blood levels of CD11b(+)Jag2(+) cells in mouse models of colorectal cancer and in a cohort of untreated patients with colorectal cancer were indicative of metastatic disease. In patients with colorectal cancer, the presence of circulating CD11b(+)Jag2(+) cells was accompanied by loss of E-cadherin in the corresponding patient tumors. Taken together, our results show that bone marrow-derived CD11b(+)Jag2(+) cells, which infiltrate primary colorectal tumors, are sufficient to induce EMT in tumor cells, thereby triggering onset of metastasis. Furthermore, they argue that quantifying circulating CD11b(+)Jag2(+) cells in patients may offer an indicator of colorectal cancer progression to metastatic levels of the disease.

Endothelial progenitor cells and integrins: adhesive needs.

In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs) to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs) and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4ß1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5ß1, α6ß1, αvß3 and αvß5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. ß2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of this cell population as a relevant clinical agent.

Context- and cell-dependent effects of Delta-like 4 targeting in the bone marrow microenvironment.

Delta-like 4 (Dll4) is a ligand of the Notch pathway family which has been widely studied in the context of tumor angiogenesis, its blockade shown to result in non-productive angiogenesis and halted tumor growth. As Dll4 inhibitors enter the clinic, there is an emerging need to understand their side effects, namely the systemic consequences of Dll4:Notch blockade in tissues other than tumors. The present study focused on the effects of systemic anti-Dll4 targeting in the bone marrow (BM) microenvironment. Here we show that Dll4 blockade with monoclonal antibodies perturbs the BM vascular niche of sub-lethally irradiated mice, resulting in increased CD31(+), VE-Cadherin(+) and c-kit(+) vessel density, and also increased megakaryocytes, whereas CD105(+), VEGFR3(+), SMA(+) and lectin(+) vessel density remained unaltered. We investigated also the expression of angiocrine genes upon Dll4 treatment in vivo, and demonstrate that IGFbp2, IGFbp3, Angpt2, Dll4, DHH and VEGF-A are upregulated, while FGF1 and CSF2 are reduced. In vitro treatment of endothelial cells with anti-Dll4 reduced Akt phosphorylation while maintaining similar levels of Erk 1/2 phosphorylation. Besides its effects in the BM vascular niche, anti-Dll4 treatment perturbed hematopoiesis, as evidenced by increased myeloid (CD11b(+)), decreased B (B220(+)) and T (CD3(+)) lymphoid BM content of treated mice, with a corresponding increase in myeloid circulating cells. Moreover, anti-Dll4 treatment also increased the number of CFU-M and -G colonies in methylcellulose assays, independently of Notch1. Finally, anti-Dll4 treatment of donor BM improved the hematopoietic recovery of lethally irradiated recipients in a transplant setting. Together, our data reveals the hematopoietic (BM) effects of systemic anti-Dll4 treatment result from qualitative vascular changes and also direct hematopoietic cell modulation, which may be favorable in a transplant setting.

The role of fibrin E on the modulation of endothelial progenitors adhesion, differentiation and angiogenic growth factor production and the promotion of wound healing.

Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin α5ß1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-ß1, SDF-1, IL-8 and MIP-1α. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies.

Bone marrow-derived endothelial progenitors expressing Delta-like 4 (Dll4) regulate tumor angiogenesis.

Neo-blood vessel growth (angiogenesis), which may involve the activation of pre-existing endothelial cells (EC) and/or the recruitment of bone marrow-derived vascular precursor cells (BM-VPC), is essential for tumor growth. Molecularly, besides the well established roles for Vascular endothelial growth factor (VEGF), recent findings show the Notch signalling pathway, in particular the ligand Delta-like 4 (Dll4), is also essential for adequate tumor angiogenesis; Dll4 inhibition results in impaired, non-functional, angiogenesis and reduced tumor growth. However, the role of BM-VPC in the setting of Notch pathway modulation was not addressed and is the subject of the present report. Here we show that SDF-1 and VEGF, which are produced by tumors, increase Dll4 expression on recruited BM-VPC. Mechanistically, BM-VPC activated, in a Dll4-dependent manner, a transcriptional program on mature EC suggestive of EC activation and stabilization. BM-VPC induced ICAM-2 and Fibronectin expression on EC, an effect that was blocked by a Dll4-specific neutralizing antibody. In vivo, transplantation of BM-VPC with decreased Dll4 into tumor-bearing mice resulted in the formation of microvessels with decreased pericyte coverage and reduced fibronectin expression. Consequently, transplantation of BM-VPC with decreased Dll4 resulted in impaired tumor angiogenesis, increased tumor hypoxia and apoptosis, and decreased tumor growth. Taken together, our data suggests that Dll4 expression by BM-VPC affects their communication with tumor vessel endothelial cells, thereby modulating tumor angiogenesis by affecting vascular stability.

Butyrate-rich colonic microenvironment is a relevant selection factor for metabolically adapted tumor cells.

The short chain fatty acid (SCFA) butyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.