RESUMO
Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.
Assuntos
Melanoma Experimental/imunologia , Células Supressoras Mieloides/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.
Assuntos
Autorrenovação Celular , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/patologia , Leucemia/patologia , Mielopoese , Células-Tronco Neoplásicas/patologia , Animais , Reprogramação Celular , Citocinas/metabolismo , Granulócitos/citologia , Granulócitos/patologia , Fatores Reguladores de Interferon/metabolismo , Macrófagos/citologia , Macrófagos/patologia , Camundongos , Imagem Molecular , Nicho de Células-Tronco/fisiologia , beta Catenina/metabolismoRESUMO
Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.
Assuntos
Envelhecimento , Plaquetas/imunologia , Inflamação/imunologia , Mitocôndrias/imunologia , Trombose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Plaquetas/patologia , Inflamação/patologia , Megacariócitos/imunologia , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Ativação Plaquetária , Trombose/patologiaRESUMO
Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.
Assuntos
Artrite Experimental , Artrite Reumatoide , Doenças Autoimunes , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Citocinas , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.
Assuntos
Senescência Celular/fisiologia , Replicação do DNA/fisiologia , Células-Tronco Hematopoéticas/patologia , Estresse Fisiológico , Animais , Proliferação de Células , Senescência Celular/genética , Dano ao DNA/genética , DNA Ribossômico/genética , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Manutenção de Minicromossomo/genéticaRESUMO
Hematopoietic stem cells (HSCs) are responsible for lifelong production of blood cells. At the same time, they must respond rapidly to acute needs such as infection or injury. Significant interest has emerged in how inflammation regulates HSC fate and how it affects the long-term functionality of HSCs and the blood system as a whole. Here we detail recent advances and unanswered questions at the intersection between inflammation and HSC biology in the contexts of development, aging, and hematological malignancy.
Assuntos
Linhagem da Célula , Doença , Saúde , Células-Tronco Hematopoéticas/citologia , Inflamação/patologia , Envelhecimento/patologia , Animais , HumanosRESUMO
STUDY DESIGN: Cross-sectional cohort analysis of patients with Modic Changes (MC). OBJECTIVE: Our goal was to characterize the molecular and cellular features of MC bone marrow and adjacent discs. We hypothesized that MC associate with biologic cross-talk between discs and bone marrow, the presence of which may have both diagnostic and therapeutic implications. BACKGROUND DATA: MC are vertebral bone marrow lesions that can be a diagnostic indicator for discogenic low back pain. Yet, the pathobiology of MC is largely unknown. METHODS: Patients with Modic type 1 or 2 changes (MC1, MC2) undergoing at least 2-level lumbar interbody fusion with one surgical level having MC and one without MC (control level). Two discs (MC, control) and two bone marrow aspirates (MC, control) were collected per patient. Marrow cellularity was analyzed using flow cytometry. Myelopoietic differentiation potential of bone marrow cells was quantified to gauge marrow function, as was the relative gene expression profiles of the marrow and disc cells. Disc/bone marrow cross-talk was assessed by comparing MC disc/bone marrow features relative to unaffected levels. RESULTS: Thirteen MC1 and eleven MC2 patients were included. We observed pro-osteoclastic changes in MC2 discs, an inflammatory dysmyelopoiesis with fibrogenic changes in MC1 and MC2 marrow, and up-regulation of neurotrophic receptors in MC1 and MC2 bone marrow and discs. CONCLUSION: Our data reveal a fibrogenic and pro-inflammatory cross-talk between MC bone marrow and adjacent discs. This provides insight into the pain generator at MC levels and informs novel therapeutic targets for treatment of MC-associated LBP.
Assuntos
Medula Óssea/patologia , Disco Intervertebral/patologia , Medula Óssea/metabolismo , Estudos de Coortes , Estudos Transversais , Regulação para Baixo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Osteogênese , Regulação para CimaRESUMO
Hematopoiesis is the hierarchical process in which all lineages of blood cells are produced by self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). While the regulatory factors that maintain proper HSC function and lineage output under normal conditions are well understood, significantly less is known about how HSC fate is regulated in response to inflammation or disease. As many blood disorders are associated with overproduction of pro-inflammatory cytokines, significant interest has emerged in understanding the impact of these factors on HSC function. In this review we highlight key advances demonstrating the impact of pro-inflammatory cytokines on the biology of HSCs and the BM niche, and address ongoing questions regarding their role in normal and pathogenic hematopoiesis.
Assuntos
Citocinas/farmacologia , Doenças Hematológicas/patologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Mediadores da Inflamação/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Doenças Hematológicas/tratamento farmacológico , Doenças Hematológicas/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , HumanosAssuntos
Imunidade Inata , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Spliceossomos/genética , Fator de Processamento U2AF/genética , Animais , Citocinas/metabolismo , Humanos , Inflamação , Células K562 , Lipopolissacarídeos , Camundongos , Mutação , Subunidade p50 de NF-kappa B/metabolismo , Fenótipo , Células RAW 264.7 , Splicing de RNARESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.
Assuntos
Hematopoiese Clonal , Células-Tronco Hematopoéticas , Nicho de Células-Tronco , Humanos , Células-Tronco Hematopoéticas/metabolismo , Hematopoiese Clonal/genética , Animais , Medula Óssea/metabolismo , MutaçãoRESUMO
Studying fetal hematopoiesis is challenging as hematopoiesis transitions from the liver to bone marrow. Obtaining human samples is not possible, and small animal models may not provide sufficient biological material. Here, we present a protocol for isolating hematopoietic cells from the nonhuman primate fetal liver and bone. We describe steps for using cells from the same fetus for fluorescence lifetime imaging microscopy to measure metabolism, assessing cellular function, and flow cytometry for immunophenotyping at the single-cell level. For complete details on the use and execution of this protocol, please refer to Nash et al. (2023).1.
Assuntos
Leucócitos , Fígado , Animais , Humanos , Imunofenotipagem , Feto , PrimatasRESUMO
Chronic inflammation, although subtle, puts the body in a constant state of alertness and is associated with many diseases, including cancer and cardiovascular diseases. It leads hematopoietic cells to produce and release proinflammatory cytokines, which trigger specific signaling pathways in hematopoietic stem cells (HSCs) that cause changes in proliferation, differentiation, and migration. This response is essential when HSCs are needed to produce specific blood cells to eliminate an intruder, such as a pathogenic virus, but mutant HSCs can use these proinflammatory signals to their advantage and accelerate the development of hematologic disease or malignancy. Understanding this complex process is vital for monitoring and controlling disease progression in patients. In the 2023 International Society for Experimental Hematology winter webinar, Dr. Eric Pietras (University of Colorado Anschutz Medical Campus, United States) and Dr. Katherine Y. King (Baylor College of Medicine, United States) gave a presentation on this topic, which is summarized in this review article.
Assuntos
Doenças Hematológicas , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Transdução de Sinais , Doenças Hematológicas/metabolismo , Inflamação/patologiaRESUMO
BACKGROUND: Biliary atresia (BA) is a progressive pediatric inflammatory disease of the liver that leads to cirrhosis and necessitates liver transplantation. The rapid progression from liver injury to liver failure in children with BA suggests that factors specific to the perinatal hepatic environment are important for disease propagation. Hematopoietic stem and progenitor cells (HSPCs) reside in the fetal liver and are known to serve as central hubs of inflammation. We hypothesized that HSPCs are critical for the propagation of perinatal liver injury (PLI). METHODS: Newborn BALB/c mice were injected with rhesus rotavirus (RRV) to induce PLI or with PBS as control. Livers were compared using histology and flow cytometry. To determine the effects of HSPCs on PLI, RRV-infected neonatal mice were administered anti-CD47 and anti-CD117 to deplete HSPCs. RESULTS: PLI significantly increased the number of common myeloid progenitors and the number of CD34+ hematopoietic progenitors. Elimination of HSPCs through antibody-mediated myeloablation rescued animals from PLI and significantly increased survival (RRV+isotype control 36.4% vs. RRV+myeloablation 77.8%, Chi-test = 0.003). CONCLUSIONS: HSPCs expand as a result of RRV infection and propagate PLI. Targeting of HSPCs may be useful in preventing and treating neonatal inflammatory diseases of the liver such as BA.
RESUMO
Chronic inflammation is a common feature of aging and numerous diseases such as diabetes, obesity, and autoimmune syndromes and has been linked to the development of hematological malignancy. Blood-forming hematopoietic stem cells (HSC) can contribute to these diseases via the production of tissue-damaging myeloid cells and/or the acquisition of mutations in epigenetic and transcriptional regulators that initiate evolution toward leukemogenesis. We previously showed that the myeloid "master regulator" transcription factor PU.1 is robustly induced in HSC by pro-inflammatory cytokines such as interleukin (IL)-1ß and limits their proliferative activity. Here, we used a PU.1-deficient mouse model to investigate the broader role of PU.1 in regulating hematopoietic activity in response to chronic inflammatory challenges. We found that PU.1 is critical in restraining inflammatory myelopoiesis via suppression of cell cycle and self-renewal gene programs in myeloid-biased multipotent progenitor (MPP) cells. Our data show that while PU.1 functions as a key driver of myeloid differentiation, it plays an equally critical role in tailoring hematopoietic responses to inflammatory stimuli while limiting expansion and self-renewal gene expression in MPPs. These data identify PU.1 as a key regulator of "emergency" myelopoiesis relevant to inflammatory disease and leukemogenesis.
RESUMO
Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+ mesenchymal stromal cells associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors, which promotes myeloid differentiation and hinders haematopoietic regeneration. Moreover, we show how production of interleukin-1ß (IL-1ß) by the damaged endosteum acts in trans to drive the proinflammatory nature of the central marrow, with damaging consequences for the old blood system. Notably, niche deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during ageing.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Medula Óssea/metabolismo , Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco , Interleucina-1/metabolismoRESUMO
Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.