Lineage Tracing Mouse Models Research Articles

Origins of pathological myofibroblasts in lung fibrosis: insights from lineage tracing mouse models in the single-cell RNA sequencing era.

Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease with a poor prognosis. Chronic microinjuries, mainly caused by environmental factors to an aging alveolar epithelium, would lead to the aberrant differentiation and accumulation of aberrant mesenchymal cells with a contractile phenotype, known as fibrosis-associated myofibroblasts, which trigger abnormal extracellular matrix accumulation and fibrosis. The origin of those pathological myofibroblasts in pulmonary fibrosis is not fully understood to date. Lineage tracing methods using mouse models have opened new avenues for studying cell fate in a pathological context. This review aims to present a nonexhaustive list of different potential sources of those harmful myofibroblasts during lung fibrosis, based on these in vivo approaches, and considering the normal and fibrotic lung cellular atlas recently established by single-cell RNA sequencing.

Multiple waves of fetal-derived immune cells constitute adult immune system.

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

PDC-like cells are pre-DC2 and require KLF4 to control homeostatic CD4 T cells.

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes.

Multilineage commitment of Sca-1+ cells in reshaping vein grafts.

Vein graft failure remains a significant clinical problem. Similar to other vascular diseases, stenosis of vein grafts is caused by several cell lines; however, the sources of these cells remain unclear. The objective of this study was to investigate the cellular sources that reshape vein grafts. By analyzing transcriptomics data and constructing inducible lineage-tracing mouse models, we investigated the cellular components of vein grafts and their fates. The sc-RNAseq data suggested that Sca-1+ cells were vital players in vein grafts and might serve as progenitors for multilineage commitment. By generating a vein graft model in which the venae cavae from C57BL/6J wild-type mice were transplanted adjacent to the carotid arteries of Sca-1(Ly6a)-CreERT2; Rosa26-tdTomato mice, we demonstrated that the recipient Sca-1+ cells dominated reendothelialization and the formation of adventitial microvessels, especially at the perianastomotic regions. In turn, using chimeric mouse models, we confirmed that the Sca-1+ cells that participated in reendothelialization and the formation of adventitial microvessels all had a non-bone-marrow origin, whereas bone-marrow-derived Sca-1+ cells differentiated into inflammatory cells in vein grafts. Furthermore, using a parabiosis mouse model, we confirmed that non-bone-marrow-derived circulatory Sca-1+ cells were vital for the formation of adventitial microvessels, whereas Sca-1+ cells derived from local carotid arteries were the source of endothelium restoration. Using another mouse model in which venae cavae from Sca-1 (Ly6a)-CreERT2; Rosa26-tdTomato mice were transplanted adjacent to the carotid arteries of C57BL/6J wild-type mice, we confirmed that the donor Sca-1+ cells were mainly responsible for smooth muscle cells commitment in the neointima, particularly at the middle bodies of vein grafts. In addition, we provided evidence that knockdown/knockout of Pdgfrα in Sca-1+ cells decreased the cell potential to generate SMCs in vitro and decreased number of intimal SMCs in vein grafts. Our findings provided cell atlases of vein grafts, which demonstrated that recipient carotid arteries, donor veins, non-bone-marrow circulation, and the bone marrow provided diverse Sca-1+ cells/progenitors that participated in the reshaping of vein grafts.

Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.

Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h-/- mice to lethally irradiated Ldlr-/- mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr-/- or Ch25h-/-;Ldlr-/- mice. The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage-tracing mouse model, Myh11ERT2CREmT/mG;Ldlr-/-, adoptively transferred with wild-type or Ch25h-/- mice bone marrow followed by 12 weeks of Western diet feeding. We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB-mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC-mediated modulation of liver X receptor or SREBP (sterol regulatory element-binding protein) transcriptional activity. Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

CD34+ cell atlas of main organs implicates its impact on fibrosis.

CD34+ cells are believed being progenitors that may be used to treat cardiovascular disease. However, the exact identity and the role of CD34+ cells in physiological and pathological conditions remain unclear. We performed single-cell RNA sequencing analysis to provide a cell atlas of normal tissue/organ and pathological conditions. Furthermore, a genetic lineage tracing mouse model was used to investigate the role of CD34+ cells in angiogenesis and organ fibrosis. Single-cell RNA sequencing analysis revealed a heterogeneous population of CD34+ cells in both physiological and pathological conditions. Using a genetic lineage tracing mouse model, we showed that CD34+ cells not only acquired endothelial cell fate involved in angiogenesis, but also, CD34+ cells expressing Pi16 may transform into myofibroblast and thus participate in organ fibrosis. A heterogeneous CD34+ cells serve as a contributor not only to endothelial regeneration but also a wound healing response that may provide therapeutic insights into fibrosis.

Role of smooth muscle-derived S100A4 in an atherosclerotic mouse model

Abstract Background S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages). Methods Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE−/− background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq). Results We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt showed a reduction in macrophages and dendritic cells and an increase in total SMCs, likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical SMC markers and maintenance of the contractile phenotype. Conclusion We report an intrinsic function of S100A4 in the establishment of SMC fate within the plaque and, surprisingly, a global impact on the atherosclerotic plaque microenvironnement and inflammation status. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation

Prx1+ MPCs Accumulate in the Dura Mater of Wild-Type and p21-/- Mice Followed by a Specific Reduction in p21-/- Dural MPCs.

Epidural fat contains a population of mesenchymal progenitor cells (MPCs), and this study explores the behavior of these cells on the adjacent dura mater during growth and in response to injury in a p21 knockout mouse model. p21-/- mice are known to have increased cell proliferation and enhanced tissue regeneration post-injury. Therefore, it is hypothesized that the process by which epidural fat MPCs maintain the dura mater can be accelerated in p21-/- mice. Using a Prx1 lineage tracing mouse model, the epidural fat MPCs are found to increase in the dura mater over time in both C57BL/6 (p21+/+ ) and p21-/- mice; however, by 3weeks post-tamoxifen induction, few MPCs are observed in p21-/- mice. These endogenous MPCs also localize to dural injuries in both mouse strains, with MPCs in p21-/- mice demonstrating increased proliferation. When epidural fat MPCs derived from p21-/- mice are transplanted into dural injuries in C57BL/6 mice, these MPCs are found in the injury site. It is demonstrated that epidural fat MPCs play a role in dural tissue maintenance and are able to directly contribute to dural injury repair. This suggests that these MPCs have the potential to treat injuries and/or pathologies in tissues surrounding the spinal cord.

Abstract P057: Adipocyte Progenitor Cells In Perivascular Adipose Tissues: The Effect Of Sex And Anatomical Location

Perivascular adipose tissue (PVAT) modifies vascular function due to its capacity to synthesize vasoactive products and its mechanical properties. During cardiovascular diseases (CVD), changes in adipocyte populations affect PVAT function. In obesity, expansion of adipose tissue (AT) differs between sex in rodents and humans in a site-dependent manner. Consequently, men are more susceptible to obesity-associated hypertension. However, how adipocyte progenitors (APC) contribute to those differences in PVAT depots is currently unknown. Our objective was to evaluate the distribution of APCs among different AT sites in both sexes under normotensive conditions using a genetic lineage tracing mouse model. We hypothesize that APC population distribution is affected by sex and anatomical location. PVAT from abdominal (abPVAT) and thoracic (atPVAT) aorta, mesenteric arteries (mesPVAT), and non-PVAT subscapular (BAT), perigonadal (GON), and subcutaneous (SCAT) were collected from 30-week-old female and male double transgenic mice ( PDGFRa -CreER T2 /R26-LSL-tdTomato; n=13). In this model, tamoxifen administration (5d, 150 mg/kg) induces PDGFRa + cells well-defined APC populations, to express the fluorescent tdTomato reporter. To harvest APC, AT was minced, digested [Liberase™ (50μg/ml)] for 1hr at 37°C, and then filtered with a 40μm cell strainer. The cell suspension was incubated with conjugated antibodies anti CD45, and CD31. Zombie NIR was used to exclude dead cells. Next, flow cytometry was performed to determine APC population distribution defined as CD45 - , CD31 - , and tdTomato + ; results are shown as % APC±SEM. PDGFRα + are present in all AT tissues. Among PVAT sites, abPVAT had fewer APC (1.17±0.25) compared with GON (5.6±0.25; P<0.05). APC populations in BAT (5.61±0.23), atPVAT (2.74±0.23), MesPVAT (3.72±0.25), and SCAT (2.65±0.25) did not differ. There were no sex differences in APC populations in PVAT; however, in GON, males (1.28±0.75; n=4) had fewer APC compared to females (9.92±0.31; n=9) (P<0.01). These data show that APC is in a lower proportion in abPVAT than in other depots, which can clarify the underlying process of differences in PVAT expansion; our future experiments will study how APC can be affected by hypertensive conditions.

Club cells employ regeneration mechanisms during lung tumorigenesis

The high plasticity of lung epithelial cells, has for many years, confounded the correct identification of the cell-of-origin of lung adenocarcinoma (LUAD), one of the deadliest malignancies worldwide. Here, we employ lineage-tracing mouse models to investigate the cell of origin of Eml4-Alk LUAD, and show that Club and Alveolar type 2 (AT2) cells give rise to tumours. We focus on Club cell originated tumours and find that Club cells experience an epigenetic switch by which they lose their lineage fidelity and gain an AT2-like phenotype after oncogenic transformation. Single-cell transcriptomic analyses identified two trajectories of Club cell evolution which are similar to the ones used during lung regeneration, suggesting that lung epithelial cells leverage on their plasticity and intrinsic regeneration mechanisms to give rise to a tumour. Together, this study highlights the role of Club cells in LUAD initiation, identifies the mechanism of Club cell lineage infidelity, confirms the presence of these features in human tumours, and unveils key mechanisms conferring LUAD heterogeneity.

7-OR: Distinct Developmental Origins of Brown Adipose Tissue at Different Anatomical Locations

Brown adipose tissue (BAT) is a thermogenic organ that promotes metabolic fitness. In humans, there are two spatiotemporally distinct phases of BAT ontogeny: the prenatally developed dorsal-anterior-located interscapular and retroperitoneal BAT; and the postnatally emerged anterior cervical and supraclavicular BAT. It has been established in rodents that the dorsal-anterior-located BAT depots predominately arise from a Pax3+/Myf5+ lineage in the epaxial dermomyotome that also forms the trunk muscle. However, the developmental origins of the anterior cervical and supraclavicular BAT that adult humans have remain unknown. In this study, using lineage tracing mouse models, we found that these neck BAT depots are not derived from the Pax3+ or Myf5+ precursor cells. Knock out of PPARγ, the master regulator of adipogenesis, in Myf5+ cells caused severe atrophy of interscapular BAT, but did not affect the anterior cervical and supraclavicular depots. Instead, we identified that roughly half of the anterior cervical and supraclavicular brown adipocytes are progeny cells of a developmental lineage that also gives rise to neck muscle cells. PPARγ deficiency in this lineage did not perturb iBAT development, but caused paucity in the anterior cervical and supraclavicular regions, thus sensitizing mice to cold-induced decrease in body temperature. In summary, our results suggest that there are region-specific myogenic precursors that contribute to BAT development at different anatomical locations. Future studies to understand the pathophysiological regulation of these developmentally distinct BAT depots could inspire new approaches to measure BAT mass and to activate BAT activity in humans for the treatment of obesity, diabetes and related metabolic disorders. Disclosure H.Ruan: None. Z.Zhang: None. Z.Huang: None. Funding American Diabetes Association (1-18-IBS-167)

A novel lineage-tracing mouse model for studying early MmuPV1 infections

Human papillomaviruses are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe, and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time. Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

Characterization of Pulmonary Fibroblast Response to Aspergillosis fumigatus Exposure and Clinical Implications on the Development of Invasive Aspergillosis

Aspergillosis fumigatus(AF) is a ubiquitous environmental mold that spreads by producing airborne conidia (spores). Playing a crucial role in the recycling of environmental carbon and nitrogen, A. fumigatus is inhaled abundantly by animals and humans alike. Unfortunately, it is also responsible for one of the worst life‐threatening pneumonias known as invasive aspergillosis with a mortality rate above 30%, even with treatment. Healthy individuals that are immunocompetent can quickly clear inhaled AF spores before their germination via immune cells. However, in immunocompromised individuals the inability to clear the fungus allows the spores to germinate releasing a plethora of proteolytic enzymes as they infiltrate the tissue resulting in severe damage to the lung. Recent studies have shown that the proper response of the immune system hinges upon complex cellular cross‐talk between resident interstitial cells and circulating immune cells. Tissue resident fibroblasts, which are responsible for the production and maintenance of the extracellular matrix (ECM), have been recently suggested to play a pivotal role in regulating immune response. We hypothesize that A. fumigatus‐induced damage to the lungs would result in activation of pulmonary fibroblasts (PFs) and aid in protective inflammatory response. Utilizing our recently developed in vivo cre‐loxP lineage tracing mouse models we have interrogated and manipulated all activated lung fibroblasts during AF infection, clearing, and reinfection. Data from our lab shows that PFs can be activated following acute inhalation of AF spores even when they are cleared from the lungs of immunocompetent mice before pathological tissue damage occurs. Furthermore, repeated AF infections which are pathologically asymptomatic result in the accumulation of activated PFs. Here we show for the first time that fibroblast activation occurs in both transient and chronic pulmonary infections revealing a role in the immunomodulatory response and subsequent development of pulmonary disease.

Abstract 240: Role Of Smooth Muscle-derived Vascular Progenitor Cells In Atherosclerosis

Purpose: Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis progression by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to VV expansion to drive disease progression. Methods: We generated a highly specific lineage tracing mouse model in order to track AdvSca1-SM cells in vivo even if they differentiate into other cell types. Lineage tracing mice were placed on either normal chow or Western diet for 8 or 16 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry. Results: scRNA-Seq and flow cytometry revealed that AdvSca1-SM cells in atherosclerosis primarily differentiate into mature SMCs, modulated SMCs, and myofibroblasts. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells. Additionally, despite previous evidence of SMCs gaining a macrophage-like phenotype in atherosclerosis, we identified only rare instances of AdvSca1-SM cells contributing to macrophage populations. Conclusions: As in our findings in acute vascular injury, AdvSca1-SM cells in atherosclerosis predominantly differentiate into SMCs and myofibroblasts. Future studies on advanced lesions will define the functional role of AdvSca1-SM cells in atherosclerotic plaque progression.

HSC-independent definitive lymphopoiesis persists into adult life

Abstract The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive type of erythromyeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and considered to be transient during fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adoptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adoptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology. Supported by grant from NIH R01AI121197.

Abstract 317: Resident Vascular Adventitial Progenitor Cells Of Smooth Muscle Cell-lineage Adopt Profibrotic Phenotype And Contribute To Cardiac Fibrosis

Activated cardiac myofibroblasts are the major contributors to ECM deposition in pathological cardiovascular fibrosis, which is the characteristic feature of cardiovascular diseases. However, due to potential heterogeneity of myofibroblasts, the origin of these cells remains controversial. Using highly specific smooth muscle cell lineage-tracing mouse models, we discovered the smooth muscle cell origin of a subpopulation of resident vascular adventitial progenitor cells, defined by the expression of the stem cell marker Sca1 (AdvSca1-SM cells), which rapidly proliferate and adopt a myofibroblast phenotype in response to acute vascular injury. Further, we identified a specific gene signature of active hedgehog/Wnt/β-catenin/ Klf4 signaling in AdvSca1-SM cells and validated a Gli1 -CreERT2-ROSA26-YFP (Gli1) reporter mouse model to be a faithful lineage tracing system for AdvSca1-SM cells. However, the function of AdvSca1-SM cells in cardiac fibrosis is unknown. Using immunofluorescent staining and label-free second harmonic generation (SHG) imaging, we observed the expansion and migration of AdvSca1-SM cells in close association with perivascular and interstitial cardiac fibrosis in pressure overload-induced cardiac fibrosis in Gli1 reporter mice. We performed single cell RNA sequencing (scRNA-seq) to examine the phenotype of AdvSca1-SM cells in Angiotensin II (AngII) model. Our data showed that, upon AngII challenge, AdvSca1-SM cells differentiate along a profibrotic trajectory, which is characterized by loss of expression of Klf4 , the lncRNA, Meg3 , and stemness genes and up-regulation of myofibroblast genes. Importantly, AngII-induced profibrotic transcriptomic changes of AdvSca1-SM cells were recapitulated in human ventricular tissues exhibiting a gene signature of cardiac hypertrophy, emphasizing the translational significance of this phenotypic transition. Connectivity map analysis of the scRNA-seq data identified statins as potential candidates for inhibition of the profibrotic transition of AdvSca1-SM cells. In agreement, simvastatin induced the expression of stemness genes and inhibited TGFβ-induced up-regulation of αSMA and down-regulation of Klf4 in cultured AdvSca1-SM cells.

Pathogen-associated T follicular helper cell plasticity is critical in anti-viral immunity.

T follicular helper (Tfh) cells are critical in providing help for B cells in the germinal center reaction. Tfh cell plasticity, especially with regard to their expression of effector Th cytokines, has been described, but lacks in-depth analysis with genetic approaches. In this study, we systemically compared transcriptomic profiles of Tfh cells derived from various types of immune responses and found gene clusters corresponding to effector Th cells were differentially induced in response to pathogens or immune responses. Of special interest, a subset of Tfh cells producing IFN-γ was generated in an influenza virus infection, partially dependent on the innate cytokine IL-12. Lineage-tracing mouse model revealed unique developmental regulation of IFN-γ+ Tfh cells, while selective ablation of these cells impaired the induction of IgG2c+ germinal center B cells and the control of influenza infection. These results indicate that pathogen-associated Tfh cell plasticity is necessary for host immunity, which has implications in vaccine design.

Role of Macrophages and Mast Cells as Key Players in the Maintenance of Gastrointestinal Smooth Muscle Homeostasis and Disease.

The gut contains the largest macrophage pool in the body, with populations of macrophages residing in the mucosa and muscularis propria of the gastrointestinal (GI) tract. Muscularis macrophages (MMs), which are located within the muscularis propria, interact with cells essential for GI function, such as interstitial cells of Cajal, enteric neurons, smooth muscle cells, enteric glia, and fibroblast-like cells, suggesting that these immune cells contribute to several aspects of GI function. This review focuses on the latest insights on the factors contributing to MM heterogeneity and the functional interaction of MMs with other cell types essential for GI function. This review integrates the latest findings on macrophages in other organs with increasing knowledge of MMs to better understand their role in a healthy and diseased gut. We describe the factors that contribute to (muscularis macrophage) MM heterogeneity, and the nature of MM interactions with cells regulating GI function. Finally, we also describe the increasing evidence suggesting a critical role of another immune cell type, the mast cell, in normal and diseased GI physiology.

3236 – HSC-INDEPENDENT ADAPTIVE LYMPHOPOIESIS PERSISTS INTO ADULT LIFE

The stem cell theory that all blood cells are derived from hematopoietic stem cells (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive types of erythro-myeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and is considered to be transient during the fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at the pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adaptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adaptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpins the immune system and challenges the paradigm of HSC theory in hematology. The stem cell theory that all blood cells are derived from hematopoietic stem cells (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive types of erythro-myeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and is considered to be transient during the fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at the pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adaptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adaptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpins the immune system and challenges the paradigm of HSC theory in hematology.