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1.
Blood ; 143(26): 2763-2777, 2024 Jun 27.
Article in English | MEDLINE | ID: mdl-38603633

ABSTRACT

ABSTRACT: Inflammatory responses must be tightly coordinated with the activation of emergency myelopoiesis to produce potent myeloid cells that fight infection without causing excessive host damage. Here, we show that granulocyte-macrophage colony-stimulating factor (GM-CSF) programs myeloid-committed progenitors to produce trained macrophages (increased cytokine response), but programs the upstream noncommitted LKS+ progenitors (defined as Lin- c-Kit+ Sca-1+ cells) to produce tolerized macrophages (decreased cytokine response). In myeloid progenitors, GM-CSF strongly activates signal transducer and activator of transcription 5 (STAT5), Ras-Raf-extracellular signal regulated kinase (ERK), and Akt-mTOR signaling pathways, which are essential to establish a training program, whereas in LKS+ progenitors, GM-CSF induces NF-κB translocation to the nucleus to establish a tolerization program. These differences arise from higher GM-CSF receptor expression in myeloid progenitors compared with LKS+ cells. We demonstrate that ß-catenin regulation of NF-κB nuclear translocation is central in this process. In myeloid progenitors, glycogen synthase kinase 3 (GSK3) inactivation by strong ERK and phosphatidylinositol 3 kinase (PI3K)-Akt signaling increases cytoplasmic ß-catenin levels to block NF-κB nuclear translocation. In contrast, when ERK and PI3K-Akt signaling are weak, active GSK3 causes a decrease in ß-catenin, allowing NF-κB nuclear translocation in LKS+ progenitors. Finally, GM-CSF-induced LKS+ tolerization takes place in several murine models of trained immunity and in human CD34+ CD38- progenitors. Our study reveals that in addition to activating myelopoiesis, GM-CSF also programs early and immediate myeloid progenitors to produce opposing immune memory phenotypes. We propose that the inflammatory response from immediate myeloid progenitors may be balanced by the tolerized phenotype of early progenitors, thus providing a mechanism for appropriate resolution of inflammation and protection against a prolonged cytokine storm.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Myelopoiesis , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor , Animals , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Mice , Phenotype , Signal Transduction , NF-kappa B/metabolism , Immunologic Memory , Mice, Inbred C57BL , Macrophages/metabolism , Macrophages/immunology , Immunity, Innate , Myeloid Progenitor Cells/metabolism , Myeloid Progenitor Cells/immunology , beta Catenin/metabolism , beta Catenin/genetics
2.
Stem Cell Rev Rep ; 20(3): 845-851, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38183535

ABSTRACT

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage- Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes.


Subject(s)
Stem Cells , Toll-Like Receptor 2 , Mice , Animals , Toll-Like Receptor 2/genetics , Cell Differentiation/physiology , Retina , Mammals
3.
Stem Cells ; 40(10): 949-962, 2022 10 21.
Article in English | MEDLINE | ID: mdl-35861517

ABSTRACT

During an infection, hematopoiesis is altered to increase the output of mature myeloid cells to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, more mechanistic studies are needed to reveal whether pattern recognition receptor (PRR) signaling initiates myeloid development directly, or indirectly through the production of cytokines by HSPCs that can act in an autocrine/paracrine manner, or by a combination of both direct and indirect mechanisms. In this study, we have used an in vitro model of murine HSPCs to study myeloid differentiation in response to the TLR2 ligand Pam3CSK4 and showed that, besides indirect mechanisms, TLR2 stimulation of HSPCs promotes myelopoiesis directly by initiating a MyD88-dependent signaling. This direct differentiation program involves a combined activation of the transcription factors PU.1, C/EBPß, and IRF7 driven by TBK1 and PI3K/mTOR. Notably, downstream of MyD88, the activated TBK1 kinase can activate mTOR directly and IRF7 induction is mediated by both TBK1 and mTOR. TLR2 signaling also induces NF-κB dependent IL-6 production that may further induce indirect myeloid differentiation. Our results have identified the direct signaling pathways and the transcription factors involved in macrophage development from HSPCs in response to TLR2 engagement, a critical process to trigger a rapid immune response during infection.


Subject(s)
Myeloid Differentiation Factor 88 , Toll-Like Receptor 2 , Mice , Animals , Toll-Like Receptor 2/metabolism , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Interleukin-6/metabolism , Ligands , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Macrophages/metabolism , Cytokines/metabolism , TOR Serine-Threonine Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases
4.
Curr Opin Hematol ; 29(4): 201-208, 2022 07 01.
Article in English | MEDLINE | ID: mdl-35285448

ABSTRACT

PURPOSE OF REVIEW: Myeloid cells - granulocytes, monocytes, macrophages and dendritic cells (DCs) - are innate immune cells that play key roles in pathogen defense and inflammation, as well as in tissue homeostasis and repair. Over the past 5 years, in part due to more widespread use of single cell omics technologies, it has become evident that these cell types are significantly more heterogeneous than was previously appreciated. In this review, we consider recent studies that have demonstrated heterogeneity among neutrophils, monocytes, macrophages and DCs in mice and humans. We also discuss studies that have revealed the sources of their heterogeneity. RECENT FINDINGS: Recent studies have confirmed that ontogeny is a key determinant of diversity, with specific subsets of myeloid cells arising from distinct progenitors. However, diverse microenvironmental cues also strongly influence myeloid fate and function. Accumulating evidence therefore suggests that a combination of these mechanisms underlies myeloid cell diversity. SUMMARY: Consideration of the heterogeneity of myeloid cells is critical for understanding their diverse activities, such as the role of macrophages in tissue damage versus repair, or tumor growth versus elimination. Insights into these mechanisms are informing the design of novel therapeutic approaches.


Subject(s)
Monocytes , Myeloid Cells , Animals , Dendritic Cells , Granulocytes , Humans , Inflammation , Macrophages , Mice
5.
F1000Res ; 102021.
Article in English | MEDLINE | ID: mdl-33537121

ABSTRACT

This editorial summarises the organisation, activities, and scientific content of the 6th European Student Council Symposium (ESCS) organised by the International Society for Computational Biology Student Council (ISCB-SC). The event was held on September 6, 2020, as a satellite event preceding the ISCB's  19th European Conference in Computational Biology. Both events were first planned to be held in-person in Sitges, Spain, but moved virtually as a strategy to face the SARS-CoV2 sanitary crisis. This completely unforeseen situation has posed several challenges that have been successfully addressed thanks to the robust ISCB Student Council community structure and the strong commitment of the organisers. Despite all the obstacles and challenges, we have found that virtuality has several advantages that can continue to be kept to improve in-person meetings in the future and make conferences more inclusive allowing a larger audience to participate.


Subject(s)
COVID-19 , RNA, Viral , Computational Biology , Humans , SARS-CoV-2 , Students
6.
Front Immunol ; 12: 790309, 2021.
Article in English | MEDLINE | ID: mdl-34975887

ABSTRACT

More mechanistic studies are needed to reveal the hidden details of in vivo-induced trained immunity. Here, using a Candida albicans live vaccine mouse model we show that vaccination protects mice against a secondary infection and increases the number of bone marrow, and especially, splenic trained monocytes. Moreover, vaccination expands and reprograms hematopoietic stem and progenitor cells (HSPCs) early during infection and mobilize them transiently to the spleen to produce trained macrophages. Trained HSPCs are not only primed for myeloid cell production but also reprogramed to produce a greater amount of proinflammatory cytokines in response to a second challenge. Additionally, their adoptive transfer is sufficient to protect mice against reinfection. Mechanistically, autocrine GM-CSF activation of HSPCs is responsible for the trained phenotype and essential for the vaccine-induced protection. Our findings reveal a fundamental role for HSPCs in the trained immune protective response, opening new avenues for disease prevention and treatment.


Subject(s)
Candida albicans/immunology , Candidiasis/prevention & control , Fungal Vaccines/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/drug effects , Reinfection/prevention & control , Vaccination , Animals , Cytokines/biosynthesis , Female , Hematopoietic Stem Cells/physiology , Macrophages/physiology , Male , Mice , Mice, Inbred C57BL , Myelopoiesis
7.
Nature ; 585(7823): 96-101, 2020 09.
Article in English | MEDLINE | ID: mdl-32814898

ABSTRACT

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation1-3. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells4-6. Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72-/- mice show marked early activation of the type I interferon response, and C9orf72-/- myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein-a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72-/- myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72-/- immune cells as well as splenomegaly and inflammation in C9orf72-/- mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.


Subject(s)
C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Inflammation/metabolism , Inflammation/prevention & control , Membrane Proteins/metabolism , Myeloid Cells/metabolism , Aging/immunology , Amyotrophic Lateral Sclerosis/genetics , Animals , C9orf72 Protein/deficiency , Dendritic Cells/cytology , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Humans , Inflammation/genetics , Inflammation/immunology , Interferon Type I/biosynthesis , Interferon Type I/immunology , Membrane Proteins/antagonists & inhibitors , Mice , Myeloid Cells/immunology , Neoplasms/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology
8.
mBio ; 11(3)2020 06 23.
Article in English | MEDLINE | ID: mdl-32576672

ABSTRACT

Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage. In this study, we used an HSPC transplantation model to investigate the possible direct interaction of ß-glucan and its receptor (dectin-1) on HSPCs in vivo Purified HSPCs from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into dectin-1-/- mice (CD45.2 alloantigen), which were then injected with ß-glucan (depleted zymosan). As recipient mouse cells do not recognize the dectin-1 agonist injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted HSPCs differentiated into macrophages in response to depleted zymosan in the spleens and bone marrow of recipient mice. Functionally, macrophages derived from HSPCs exposed to depleted zymosan in vivo produced higher levels of inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin 6 [IL-6]). These results demonstrate that trained immune responses, already described for monocytes and macrophages, also take place in HSPCs. Using a similar in vivo model of HSPC transplantation, we demonstrated that inactivated yeasts of Candida albicans induce differentiation of HSPCs through a dectin-1- and MyD88-dependent pathway. Soluble factors produced following exposure of HSPCs to dectin-1 agonists acted in a paracrine manner to induce myeloid differentiation and to influence the function of macrophages derived from dectin-1-unresponsive or ß-glucan-unexposed HSPCs. Finally, we demonstrated that an in vitro transient exposure of HSPCs to live C. albicans cells, prior to differentiation, is sufficient to induce a trained phenotype of the macrophages they produce in a dectin-1- and Toll-like receptor 2 (TLR2)-dependent manner.IMPORTANCE Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections. Understanding host defense is essential to design novel therapeutic strategies to boost immune protection against Candida albicans In this article, we delve into two new concepts that have arisen over the last years: (i) the delivery of myelopoiesis-inducing signals by microbial components directly sensed by hematopoietic stem and progenitor cells (HSPCs) and (ii) the concept of "trained innate immunity" that may also apply to HSPCs. We demonstrate that dectin-1 ligation in vivo activates HSPCs and induces their differentiation to trained macrophages by a cell-autonomous indirect mechanism. This points to new mechanisms by which pathogen detection by HSPCs may modulate hematopoiesis in real time to generate myeloid cells better prepared to deal with the infection. Manipulation of this process may help to boost the innate immune response during candidiasis.


Subject(s)
Cell Differentiation , Hematopoietic Stem Cells/cytology , Lectins, C-Type/metabolism , Macrophages/cytology , Stem Cells/cytology , Animals , Candida albicans/immunology , Candidiasis/immunology , Female , Hematopoietic Stem Cells/drug effects , Immunity, Innate , Lectins, C-Type/genetics , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/metabolism , Signal Transduction , Stem Cells/drug effects , Toll-Like Receptor 2/immunology , beta-Glucans/administration & dosage
9.
Cells ; 9(5)2020 05 25.
Article in English | MEDLINE | ID: mdl-32466296

ABSTRACT

Microbial recognition by pattern recognition receptors (PRRs) expressed on hematopoietic stem and progenitor cells (HSPCs) not only activates myelopoiesis but also programs the function of the monocytes and macrophages they produce. For instance, changes in HSPC programming modify the ability of macrophages derived from them to produce inflammatory cytokines. While HSPCs exposed to a TLR2 agonist give rise to tolerized macrophages (lower proinflammatory cytokine production), HSPCs treated with Dectin-1 ligands produce trained macrophages (higher proinflammatory cytokine production). However, nothing is known about the impact of HSPC exposure to microbes on the function of antigen presenting cells (APCs). In this study we evaluated whether treatment of murine bone marrow HSPCs with a TLR2 or Dectin-1 ligand impacts the antigen presenting capacity of APCs derived from them in vitro. Following activation with microbial ligands or Candida albicans yeasts, APCs derived from TLR2/Dectin-1-programed HSPCs exhibit altered expression of MHCII (signal 1), co-stimulatory molecules (CD40, CD80 and CD86; signal 2) and cytokines (TNF-α, IL-6, IL-12 p40 and IL-2; signal 3). Moreover, APCs derived from TLR2/Dectin-1-programed HSPCs prime enhanced Th1 and Th17 responses, which are important for antifungal defense, in CD4 T cell cocultures. Overall, these results demonstrate for the first time that microbial detection by bone marrow HSPCs can modulate the adaptive immune response by inducing the production of APCs with an altered phenotype.


Subject(s)
Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/immunology , Hematopoietic Stem Cells/metabolism , Lectins, C-Type/metabolism , Lymphocyte Activation/immunology , Signal Transduction , Toll-Like Receptor 2/metabolism , Animals , Antigen-Presenting Cells/drug effects , CD4-Positive T-Lymphocytes/drug effects , Candida albicans/immunology , Cytokines/metabolism , Hematopoietic Stem Cells/drug effects , Histocompatibility Antigens Class II/metabolism , Lipopeptides/pharmacology , Lymphocyte Activation/drug effects , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin/immunology , Signal Transduction/drug effects , Th1 Cells/drug effects , Th1 Cells/immunology , Th17 Cells/drug effects , Th17 Cells/immunology , Zymosan/pharmacology
10.
Rev. esp. drogodepend ; 44(4): 69-78, oct.-dic. 2019. graf
Article in Spanish | IBECS | ID: ibc-187264

ABSTRACT

El grupo de autoayuda para alcohólicos más extendido en todo el mundo es Alcohólicos Anónimos (AA). Por esta razón, se decidió estudiar los motivos de los alcohólicos para seguir acudiendo a las reuniones de esta comunidad. Así pues, se realizó un estudio cualitativo exploratorio con cuatro personas con más de diez años de abstinencia que forman parte de un grupo de AA de Cataluña, a partir de grabar, transcribir y analizar una entrevista semiestructurada. Los resultados indican que todos los motivos de los participantes para seguir acudiendo a las reuniones de la comunidad son: miedo a recaer, para ayudar, para seguir creciendo y por la relación con el grupo


The most widespread group of self-help for alcoholics in the world is Alcoholics Anonymous (AA). For this reason, it was decided to study the motives of alcoholics to continue attending the meetings of this community. Thus, a qualitative exploratory study was conducted with four people with more than ten years of abstinence who are part of an AA group in Catalonia, by recording, transcribing and analyzing a semi-structured interview. The results indicate that all participants agreed that the reasons to continue attending community meetings are: fear of relapse, to help, to continue growing and because of the relationship with the group


Subject(s)
Humans , Female , Adult , Middle Aged , Alcoholism/prevention & control , Alcoholism/psychology , Alcoholics Anonymous/organization & administration , Self-Help Groups , Qualitative Research , Data Analysis
11.
Front Immunol ; 10: 1642, 2019.
Article in English | MEDLINE | ID: mdl-31379841

ABSTRACT

Classical and non-classical monocytes, and the macrophages and monocyte-derived dendritic cells they produce, play key roles in host defense against pathogens, immune regulation, tissue repair and many other processes throughout the body. Recent studies have revealed previously unappreciated heterogeneity among monocytes that may explain this functional diversity, but our understanding of mechanisms controlling the functional programming of distinct monocyte subsets remains incomplete. Resolving monocyte heterogeneity and understanding how their functional identity is determined holds great promise for therapeutic immune modulation. In this review, we examine how monocyte origins and developmental influences shape the phenotypic and functional characteristics of monocyte subsets during homeostasis and in the context of infection, inflammation, and cancer. We consider how extrinsic signals and transcriptional regulators impact monocyte production and functional programming, as well as the influence of epigenetic and metabolic mechanisms. We also examine the evidence that functionally distinct monocyte subsets are produced via different developmental pathways during homeostasis and that inflammatory stimuli differentially target progenitors during an emergency response. We highlight the need for a more comprehensive understanding of the relationship between monocyte ontogeny and heterogeneity, including multiparametric single-cell profiling and functional analyses. Studies defining mechanisms of monocyte subset production and maintenance of unique monocyte identities have the potential to facilitate the design of therapeutic interventions to target specific monocyte subsets in a variety of disease contexts, including infectious and inflammatory diseases, cancer, and aging.


Subject(s)
Monocytes/immunology , Animals , Dendritic Cells/immunology , Gene Ontology , Humans , Inflammation/immunology , Macrophages/immunology
12.
Commun Biol ; 2: 73, 2019.
Article in English | MEDLINE | ID: mdl-30820468

ABSTRACT

Restoration of cognitive function in old mice by transfer of blood or plasma from young mice has been attributed to reduced C-C motif chemokine ligand 11 (CCL11) and ß2-microglobulin, which are thought to suppress neurogenesis in the aging brain. However, the specific role of the hematopoietic system in this rejuvenation has not been defined and the importance of neurogenesis in old mice is unclear. Here we report that transplantation of young bone marrow to rejuvenate the hematopoietic system preserved cognitive function in old recipient mice, despite irradiation-induced suppression of neurogenesis, and without reducing ß2-microglobulin. Instead, young bone marrow transplantation preserved synaptic connections and reduced microglial activation in the hippocampus. Circulating CCL11 levels were lower in young bone marrow recipients, and CCL11 administration in young mice had the opposite effect, reducing synapses and increasing microglial activation. In conclusion, young blood or bone marrow may represent a future therapeutic strategy for neurodegenerative disease.


Subject(s)
Aging/physiology , Bone Marrow Transplantation/methods , Cognition/physiology , Learning/physiology , Memory/physiology , Rejuvenation/physiology , Age Factors , Animals , Chemokine CCL11/blood , Hippocampus/cytology , Hippocampus/physiology , Male , Mice, Congenic , Mice, Inbred C57BL , Mice, Transgenic , Neurogenesis/physiology , beta 2-Microglobulin/metabolism
13.
Nat Med ; 24(12): 1887-1898, 2018 12.
Article in English | MEDLINE | ID: mdl-30478421

ABSTRACT

Treatment of prostate cancer (PC) by androgen suppression promotes the emergence of aggressive variants that are androgen receptor (AR) independent. Here we identify the transcription factor ONECUT2 (OC2) as a master regulator of AR networks in metastatic castration-resistant prostate cancer (mCRPC). OC2 acts as a survival factor in mCRPC models, suppresses the AR transcriptional program by direct regulation of AR target genes and the AR licensing factor FOXA1, and activates genes associated with neural differentiation and progression to lethal disease. OC2 appears active in a substantial subset of human prostate adenocarcinoma and neuroendocrine tumors. Inhibition of OC2 by a newly identified small molecule suppresses metastasis in mice. These findings suggest that OC2 displaces AR-dependent growth and survival mechanisms in many cases where AR remains expressed, but where its activity is bypassed. OC2 is also a potential drug target in the metastatic phase of aggressive PC.


Subject(s)
Adenocarcinoma/drug therapy , Hepatocyte Nuclear Factor 3-alpha/genetics , Homeodomain Proteins/genetics , Prostatic Neoplasms, Castration-Resistant/drug therapy , Receptors, Androgen/genetics , Transcription Factors/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Androgens/genetics , Androgens/metabolism , Animals , Cell Line, Tumor , Disease Progression , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Homeodomain Proteins/antagonists & inhibitors , Humans , Male , Mice , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathology , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Signal Transduction , Transcription Factors/antagonists & inhibitors , Xenograft Model Antitumor Assays
14.
Article in English | MEDLINE | ID: mdl-30234030

ABSTRACT

We have previously demonstrated that Candida albicans induces differentiation of hematopoietic stem and progenitor cells (HSPCs) toward the myeloid lineage both in vitro and in vivo in a TLR2- and Dectin-1-dependent manner, giving rise to functional macrophages. In this work, we used an ex vivo model to investigate the functional consequences for macrophages derived from HSPCs in vivo-exposed to Pam3CSK4 (a TLR2 agonist) or C. albicans infection. Short in vivo treatment of mice with Pam3CSK4 results in a tolerized phenotype of ex vivo HSPC-derived macrophages, whereas an extended Pam3CSK4 treatment confers a trained phenotype. Early during candidiasis, HSPCs give rise to macrophages trained in their response to Pam3CSK4 and with an increased fungicidal activity; however, as the infection progresses to higher fungal burden, HSPC-derived macrophages become tolerized, while their fungicidal capacity is maintained. These results demonstrate that memory-like innate immune responses, already described for monocytes and macrophages, also take place in HSPCs. Interestingly, extended Pam3CSK4 treatment leads to an expansion of spleen HSPCs and myeloid cells, and drastically reduces the fungal burden in the kidney and spleen during systemic C. albicans infection. This protection against tissue invasion is abrogated by immunodepletion of HSPCs, suggesting their protective role against infection in this model. In addition, HSPCs produce in vitro cytokines and chemokines in response to C. albicans and Pam3CSK4, and these secretomes are capable of inducing myeloid differentiation of HSPCs and modulating peritoneal macrophage cytokine responses. Taken together, these data assign an active role for HSPCs in sensing pathogens during infection and in contributing to host protection by diverse mechanisms.


Subject(s)
Candida albicans/immunology , Candidiasis/immunology , Cell Differentiation , Hematopoietic Stem Cells/physiology , Macrophages/immunology , Macrophages/microbiology , Toll-Like Receptor 2/agonists , Animals , Colony Count, Microbial , Kidney/microbiology , Lipopeptides/metabolism , Mice , Spleen/microbiology , Toll-Like Receptor 2/metabolism
15.
J Vis Exp ; (137)2018 07 29.
Article in English | MEDLINE | ID: mdl-30102291

ABSTRACT

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of "contaminating" progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.


Subject(s)
Bone Marrow/metabolism , Myeloid Progenitor Cells/metabolism , Animals , Cell Differentiation , Flow Cytometry , Mice
16.
Eur Cytokine Netw ; 28(3): 102-110, 2017 Sep 01.
Article in English | MEDLINE | ID: mdl-29187337

ABSTRACT

Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage in vitro and also in vivo following infection. In this study, we used an in vitro model of HSPC differentiation to investigate the functional consequences (cytokine production) that exposing HSPCs to various pathogen-associated molecular patterns (PAMPs) and Candida albicans cells have on the subsequently derived macrophages. Mouse HSPCs (Lin- cells) were cultured with GM-CSF to induce macrophage differentiation in the presence or absence of the following pattern recognition receptor (PRR) agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or inactivated C. albicans yeasts (which activate several PRRs, mainly TLR2 and Dectin-1). Our data show that only pure TLR2 ligand exposure (transient and continuous) impacts the inflammatory function of GM-CSF-derived macrophages, because Pam3CSK4-exposed HSPCs generate macrophages with a diminished ability to produce inflammatory cytokines. Interestingly, the Pam3CSK4-induced tolerance of macrophages (by transient exposure of HSPCs) is reinforced by subsequent exposure to C. albicans cells in GM-CSF-derived macrophages; however, the induced tolerance is partially reversed in M-CSF-derived macrophages. Therefore, the ability of macrophages to produce inflammatory cytokines is extremely dependent on how the HSPCs from which they are derived receive and integrate multiple microenvironmental signals (PRR ligands and/or CSFs).


Subject(s)
Macrophages/cytology , Macrophages/metabolism , Receptors, Pattern Recognition/metabolism , Animals , Antigens, Ly/metabolism , Cell Differentiation/physiology , Cells, Cultured , Cytokines/metabolism , Escherichia coli , Female , Flow Cytometry , Major Histocompatibility Complex/physiology , Mice , Mice, Inbred C57BL , Receptors, Pattern Recognition/agonists , Signal Transduction/physiology
17.
Immunity ; 47(5): 890-902.e4, 2017 11 21.
Article in English | MEDLINE | ID: mdl-29166589

ABSTRACT

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.


Subject(s)
Dendritic Cells/physiology , Granulocyte Precursor Cells/physiology , Monocytes/physiology , Myeloid Progenitor Cells/physiology , Animals , Antigens, Ly/analysis , Cell Differentiation , Leukosialin/analysis , Mice , Sequence Analysis, RNA , Transcriptome
18.
Nat Immunol ; 18(8): 872-873, 2017 07 19.
Article in English | MEDLINE | ID: mdl-28722717
19.
J Immunol ; 198(1): 375-382, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27872213

ABSTRACT

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection, but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal ß-glucans stimulate IFN-ß production by dendritic cells (DCs) following detection by the Dectin-1 receptor, but the effects of ß-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal ß-glucan particle-stimulated DCs. We demonstrate that ß-glucan-stimulated DCs induce CD8 T cell proliferation, activation marker (CD44 and CD69) expression, and production of IFN-γ, IL-2, and granzyme B. Moreover, we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by ß-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically, type I IFNs promote Ag presentation on MHC I molecules, CD86 and CD40 expression, and the production of IL-12 p70, IL-2, IL-6, and TNF-α by ß-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation, although, in the context of LPS stimulation, this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection, which may be useful in the rational design of vaccines directed against pathogens and tumors.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Interferon Type I/immunology , Lymphocyte Activation/immunology , Animals , Autocrine Communication , Blotting, Western , Coculture Techniques , Flow Cytometry , Fungal Proteins/immunology , Lipopolysaccharides/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/immunology , beta-Glucans/immunology
20.
Microbes Infect ; 18(5): 354-63, 2016 05.
Article in English | MEDLINE | ID: mdl-26828664

ABSTRACT

TLRs represent an attractive target for the stimulation of myeloid cell production by HSPCs. We have previously demonstrated that HSPCs use TLR2 to sense Candida albicans in vivo and induce the production of macrophages. In this work, we used an in vitro model of HSPCs differentiation to investigate the functional consequences for macrophages of exposure of HSPCs to various PAMPs and C. albicans cells. Mouse HSPCs (Lin(-) cells) were cultured with M-CSF to induce macrophage differentiation, in the presence or absence of the following PRR agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or C. albicans yeasts (which activate several PRRs, but principally TLR2 and Dectin-1). Our data show that these PAMPs differentially impact the anti-microbial function of the macrophages produced by the exposed HSPCs. Pure TLR2 and TLR4 ligands generate macrophages with a diminished ability to produce inflammatory cytokines. In contrast, HSPCs activation in response to C. albicans leads to the generation of macrophages that are better prepared to deal with the infection, as they produce higher amounts of inflammatory cytokines and have higher fungicidal capacity than control macrophages. Therefore, the tailored manipulation of the differentiation process may help to boost the innate immune response to infection.


Subject(s)
Candida albicans/immunology , Hematopoietic Stem Cells/physiology , Lectins, C-Type/metabolism , Macrophages/immunology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Animals , Cell Differentiation , Female , Lipopeptides/immunology , Lipopolysaccharides/immunology , Macrophage Colony-Stimulating Factor/metabolism , Mice , Mice, Inbred C57BL , Zymosan/immunology
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