M-CSF instructs myeloid lineage fate in single haematopoietic stem cells.

Under stress conditions such as infection or inflammation the body rapidly needs to generate new blood cells that are adapted to the challenge. Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival, expansion and differentiation of lineage-committed progenitors, but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. Although genetic changes in transcription factor balance can sensitize HSCs to cytokine instruction, the initiation of HSC commitment is generally thought to be triggered by stochastic fluctuation in cell-intrinsic regulators such as lineage-specific transcription factors, leaving cytokines to ensure survival and proliferation of the progeny cells. Here we show that macrophage colony-stimulating factor (M-CSF, also called CSF1), a myeloid cytokine released during infection and inflammation, can directly induce the myeloid master regulator PU.1 and instruct myeloid cell-fate change in mouse HSCs, independently of selective survival or proliferation. Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1(+) cells with myeloid gene signature and differentiation potential. In vivo, high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced a PU.1-dependent myeloid differentiation preference. Our data demonstrate that lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity. This fundamentally changes the current view of how HSCs respond to environmental challenge and implicates stress-induced cytokines as direct instructors of HSC fate.

M-CSF directs myeloid and NK cell differentiation to protect from CMV after hematopoietic cell transplantation.

Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies.

C/EBPß-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPß targets, and C/EBPß deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPß-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.

M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation.

Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation.

Progressive replacement of embryo-derived cardiac macrophages with age.

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.

BACKGROUND: Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. METHODS: Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. RESULTS: The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. CONCLUSIONS: Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.