Rheumatoid arthritis, a chronic inflammatory joint disease, is strongly associated with HLA-DRB1*01 and *04 alleles that have in common similar 5-amino acid motifs in the third hypervariable region ...of DRB1 (QKRAA, QRRAA, RRRAA), the so called shared epitope (SE). Most patients with RA carry 1 or 2 doses of the SE, with particular genetic combinations at higher risk. In recent work we provided evidence that patients who lack HLA-DRB1*01 and/or *04 alleles can acquire RA susceptibility through fetal, maternal or iatrogenic microchimerism. We also discuss how Mc carrying HLA-DRB1*04 alleles is more likely to be present in the peripheral blood of RA patients compared to Mc carrying HLA-DRB1*01 alleles. We further analyze our results in light of the hierarchy for RA risk with different combinations of the SE. How Mc could contribute to RA susceptibility and whether it also contributes to the hierarchy of risk observed with particular combinations of SE-containing alleles is certainly the beginning of an intriguing story and may offer hope for future therapeutic and/or preventative interventions.
Acute myeloid leukaemia can develop from antecedent conditions such as myeloproliferative neoplasms as well as clonal haematopoiesis through stepwise acquisition of multiple mutations. However, the ...perturbing effects of individual mutations on the entire haematopoietic system remain elusive. To better characterise the mutation-specific haematopoietic disruption, we conducted single-cell RNA sequencing of eight different mutant mouse models with the following mutations: Flt3 internal tandem duplication (n=3), Npm1c (n=2), Idh1 R132H (n=3), Dnmt3a R882H (n=2), Ezh2 knock-out (n=2), Utx knock-out (n=2), Jak2 V617F (n=2) and Calr 52-bp deletion (n=2).
Compared with the wild-type samples, each mutant model showed significant accumulation of distinct subsets of progenitors: granulomonocytic progenitors in the Flt3 mutant, stem/immature cells in the Idh1 mutant, lymphoid and erythroid progenitors in the Ezh2 knock-out, granulocyte progenitors and megakaryocytes in the Utx mutant, late erythroid progenitors in the Jak2 mutant, and stem cells and megakaryocytes in the Calr mutant. Npm1 and Dnmt3a mutants showed smaller effects on the global cell type distribution. Transcriptome-based inference of cellular metabolism further revealed that energy generating glycolysis and TCA-cycle reactions were differentially associated with the cell type abundance, where Jak2 and Calr mutations activated these metabolic processes in the accumulated stages of progenitors, while Idh1, Ezh2 and Utx mutations downregulated these energy generation processes in the abundant cell types.
I will present an integrated analysis of eight preleukemic mouse models, and the different methods which I have utilised to investigate differential abundance of cell populations, metabolic analysis, differential gene expression and differentiation biases of the progenitor cell populations.
Background: BM HSC accumulate during aging but are functionally impaired; however, it remains debated whether this aging results from HSC-intrinsic and/or -extrinsic mechanisms. Myeloid malignancies ...are more frequent in the elderly, but whether the aged microenvironment predisposes to these malignancies is unclear. Myeloid-biased HSC and especially platelet-primed HSC expand in aged mice. However, both platelet-primed and unprimed old HSCs exhibit myeloid bias, possibly suggesting a microenvironmental participation. Moreover, some hallmarks of murine hematopoietic aging (like increased platelets) are present in premature aging (Hutchinson-Gilford progeria syndrome, HGPS) and are exacerbated in age-related myeloproliferative neoplasms (MPN), where we found that niche heterogeneity can influence disease progression (ASH abstract ID 113495). Given that aging increases myelopoiesis in physiology, HGPS and MPN (despite their heterogenous genetic background), we hypothesized that shared microenvironmental alterations influence HSC lineage bias during aging
Results: HSCs, perisinusoidal megakaryocytes (Mk) and myelopoietic cytokines increase in the BM of HGPS mice, resembling normally aged mice. However, WT recipients of HGPS BM cells do not reproduce myeloid bias, indicating that premature aging affects hematopoiesis in HGPS in a non-HSC-autonomous manner. We have investigated niche contributions to lineage bias during physiological aging because HGPSmice do not tolerate myeloablation. Consistently with recent findings (Nat Med 2018;24:782-91) HSC niches decrease near bone (endosteal BM) and expand further from bone in aged mice, but we observed different vascular changes mainly consisting of halved endosteal transition zone vessels with associated Nes-GFP+ cells and 4-fold-increased non-endosteal capillaries with Nes-GFP+ cells. However, contrasting the afore mentioned study but in agreement with other reports, we found aging-related increase of BM noradrenergic innervation, which promotes megakaryopoiesis in WT mice, but not in mice lacking β2 and β3 adrenergic receptors (ARs). Yet, these receptors exhibit opposite effects on microenvironmental regulation of myelopoiesis. β2-AR-agonist promotes Mk differentiation from human CD34+ HSCs co-cultured with MS-5 cells or in WT (but not IL-6 KO) primary murine BM culture. Consequently, adult β2-AR KO mice exhibit decreased Mk-lineage cells, which correlated with reduced BM IL-6. In contrast, adult β3-AR KO mice exhibit increased mature Mk near sinusoids, which correlated with tripled Cxcl12 mRNA expression in non-endosteal BM. Mechanistically β3-AR regulates Cxcl12-dependent HSPC and megakaryocyte localization, nitric oxide (NO) production and expression of myelopoietic cytokines. In vitro, β3-AR-agonist-treated stromal cells decrease human and murine HSPC differentiation into Mk. This effect requires Nos1-dependent NO production, since Nos1-/- mice have high circulating platelets and β3-AR agonist specifically increases lymphoid-biased HSCs in primary BM cultures in a Nos1-dependent manner. Finally, Mk increased in β3-AR KO mice transplanted with MPN BM cells, whereas Mk decreased in MPN mice treated with β3-AR agonist. To test the clinical relevance of our findings, we measured plasma nitrates and myelopoietic cytokines in MPN patients (median age=62) chronically treated only with β3-AR agonist, who show improved myelofibrosis correlated by rescued nestin+ niches (Blood 2016;128:3108), which is consistent with previous results in mice (Nature 2014;512:78-81). We found inverse correlations between the concentration of nitrates and myelopoietic cytokines
Conclusions: microenvironmental aging promotes myeloid bias through similar cytokines (e.g. IL1β, IL6) during premature or physiological aging. In the latter, HSC supporting niches decrease near bone but expand further from bone. Increased BM sympathetic noradrenergic innervation promotes β2-AR-IL-6-dependent myeloid bias. Reduction of endosteal niches decreases β3-AR-NO-dependent inhibition of myelopoietic cytokines. However, chronic treatment with β3-AR-agonist does not rejuvenate overall hematopoiesis but decreases exacerbated megakaryopoiesis in mice and humans with MPNs. Therefore, niche aging promotes myeloid bias and might represent a therapeutic target in age-related myeloproliferative disorders
No relevant conflicts of interest to declare.
Different MPNs have distinct rates of malignant transformation (PMF>PV>ET). Although PV and ET can arise from hematopoietic stem/progenitor cells (HSPCs) with similarly activated JAK-STAT oncogenic ...pathway, the transformation rate into secondary myelofibrosis and leukemia is higher for PV than for ET. However, the underlying reasons are not fully clear. Whereas in some cases secondary mutations might cause transformation, it remains unclear whether distinct bone marrow (BM) microenvironments can influence the progression of MPNs or any preleukemic disorder. Previous studies have suggested that normal BM niches close to bone (endosteal) promote HSPC quiescence, whereas non-endosteal vessels permit transmigration of activated HSPCs. We hypothesized that PV and ET HSPCs might expand in different BM niches, which could thereby influence disease progression.
To address this question, we performed combined two-photon and confocal real-time intravital microscopy in the skull BM of WT mice transplanted with HSPCs from WT mice or MPN mice carrying the same driver mutation (JAK2V617F) but showing phenotypically distinct diseases (ET or PV). ET HSPCs resembled WT HSPCs in their homing and engraftment near bone (22±15 and 26±13 μm, respectively) 3 days after i.v. injection into lethally-irradiated WT mice. In contrast, PV HSPCs located significantly further (31±21 μm) from bone, which was independently confirmed in a distinct PV model. The different homing of ET and PV HSPCs was similarly observed in non-irradiated WT recipients and was therefore independent of myeloablative conditioning. Following engraftment, ET and PV HSPCs preferentially expanded in endosteal and non-endosteal locations, respectively, over 7 months' follow up. Importantly, the asymmetric expansion of HSPCs in ET and PV was confirmed in human BM trephines. Human CD34+ HSPCs were significantly closer to bone in ET than in PV patients (86±2 vs. 109±6 μm). Together, these results suggest that mutated HSPCs preferentially occupy distinct BM niches in ET and PV.
Murine ET and PV HSPCs also differed in their dynamic interactions with the microenvironment. ET HSPCs migrated significantly faster (1 μm/min) than PV HSPCs (0.8 μm/min) and covered longer tracks after 1 h (57±2 and 35±1 μm, respectively). Moreover, ET HSPCs (but not PV HSPCs) migrated faster when closer to bone, suggesting an exploratory strategy of ET HSPCs to find endosteal niches. Separation of endosteal and non-endosteal BM fractions revealed increased abundance of integrin β3+ HSPCs in the endosteal BM of ET mice carrying JAK2V617F or CALRdel52/del52 mutations, but not in different PV models (despite generally sharing oncogenic JAK-STAT activation). Furthermore, competitive gravity adhesion assay and interference reflection microscopy showed that ET HSPCs are more adhesive to ECM substrates of integrin β3, suggesting that integrin β3 might trigger endosteal lodging of ET HSPCs.
Asymmetric HSPC expansion caused differential microenvironmental remodeling possibly explaining differences in the pathophysiology and secondary outcomes of ET and PV. Non-endosteal sinusoids were dilated in different PV (but not ET) models, whereas CD31hiSca1hi arterioles and aberrant bone-forming integrin β1+ blood vessels increased only in ET mice. Similarly, sinusoidal vessels showed increased coverage by the integrin β1 ligand laminin α4 only in ET mice. Consequently, increased bone (μCT), osteoblasts and osteoclasts were found in ET but not PV mice. Increased expression of vascular-derived bone-forming factors (such as Bmp1 and Dll4) downstream of endothelial laminin α4/integrin β1 signaling might trigger osteosclerosis in ET mice.
Finally, we tested whether HSPC location might directly impact ET progression. In a separate study we found that β3-adrenergic receptor (AR) signaling regulates Cxcl12-dependent BM HSPC localization (ASH abstract ID 116015). Therefore, we transplanted WT or ET donor BM cells into WT and β3-AR KO mice. Measurement of peripheral blood counts over 28 weeks showed that the microenvironment lacking β3-AR significantly worsened thrombocytosis and leukocytosis in ET, which correlated with redistribution of HSPCs and their progeny away from bone towards central BM.
Altogether, these results suggest that differential interactions with the microenvironment might impact disease progression in MPNs and possibly in other preleukemic disorders.
Mead:Cell Therapeutics: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding; Elstar: Research Funding; Evotek: Research Funding.
•Ciclopirox Ethanolamine (CPX) is a regulator of HSCs and preserves the stem cell state in culture.•CPX-induced HSC maintenance is mediated by intracellular iron chelation, which restricts ...respiratory capacity and proliferation.
Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood (CB) cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC-specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.
The cause of aging society is the significant prolongation of life expectancy. With the aging process, there is progressive and permanent decrease in functional reserves and deterioration of ...homeostasis control. 1,2,3,4 By degenerative changes, there is imbalance and hence the frequency of falls, and therefore regular physical activity is important to restore, maintain, and maintain independence. 3,4,5,6 The study was performed by a patient aged 86, 56 kg and a height of 149 cm. Equilibrium assessment was performed using static posturography and the Four Squar Step Test (FSST) before and after a 10-day rehabilitation session under the supervision of a physiotherapist. The results of the exercise were improved in the test and posturography test with open eyes, while deterioration was indicated in the eyes with closed eyes.
The cause of aging society is the significant prolongation of life expectancy. With the aging process, there is progressive and permanent decrease in functional reserves and deterioration of ...homeostasis control. 1,2,3,4 By degenerative changes, there is imbalance and hence the frequency of falls, and therefore regular physical activity is important to restore, maintain, and maintain independence. 3,4,5,6 The study was performed by a patient aged 86, 56 kg and a height of 149 cm. Equilibrium assessment was performed using static posturography and the Four Squar Step Test (FSST) before and after a 10-day rehabilitation session under the supervision of a physiotherapist. The results of the exercise were improved in the test and posturography test with open eyes, while deterioration was indicated in the eyes with closed eyes.
Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of ...premalignancy to cancer. To investigate this, we initiated preleukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene
. By combining single-cell RNA sequencing of preleukemia with functional analysis of transformation, we show that
loss results in global variegation of cell identity and accumulation of preleukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. That is, transcriptional variability confers forward momentum to cell fate systems, with differential multistage impact throughout cancer evolution.
Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative ...neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK-STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver.
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