Mouton AJ, DeLeon-Pennell KY, Rivera Gonzalez OJ, Flynn ER, Freeman TC, Saucerman JJ, Garrett MR, Ma Y, Harmancey R, Lindsey ML.
PMID: 29868933 | DOI: 10.1007/s00395-018-0686-x
In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.
Biochimica et biophysica acta. Molecular basis of disease
Ha, S;Yang, Y;Kim, BM;Kim, J;Son, M;Kim, D;Yu, HS;Im, D;Chung, HY;Chung, KW;
PMID: 35772632 | DOI: 10.1016/j.bbadis.2022.166474
A high-fat diet (HFD) is a major risk factor for chronic kidney disease. Although HFD promotes renal injury, characterized by increased inflammation and oxidative stress leading to fibrosis, the underlying mechanism remains elusive. Here, we investigated the role and mechanism of protease-activating receptor 2 (PAR2) activation during HFD-induced renal injury in C57/BL6 mice. HFD for 16 weeks resulted in kidney injury, manifested by increased blood levels of blood urea nitrogen, increased levels of oxidative stress with inflammation, and structural changes in the kidney tubules. HFD-fed kidneys showed elevated PAR2 expression level in the tubular epithelial region. To elucidate the role of PAR2, PAR2 knockout mice and their littermates were administered HFD. PAR2 deficient kidneys showed reduced extent of renal injury. PAR2 deficient kidneys showed significantly decreased levels of inflammatory gene expression and macrophage infiltration, followed by reduced accumulation of extracellular matrix proteins. Using NRK52E kidney epithelial cells, we further elucidated the mechanism and role of PAR2 activation during renal injury. Palmitate treatment increased PAR2 expression level in NRK52E cells and scavenging of oxidative stress blocked PAR2 expression. Under palmitate-treated conditions, PAR2 agonist-induced NF-κB activation level was higher with increased chemokine expression level in the cells. These changes were attenuated by the depletion of oxidative stress. Taken together, our results suggest that HFD-induced PAR2 activation is associated with increased levels of renal oxidative stress, inflammatory response, and fibrosis.
Xin Y, Kim J, Okamoto H, Ni M, Wei Y, Adler C, Murphy AJ, Yancopoulos GD, Lin C, Gromada J.
PMID: 27667665 | DOI: 10.1016/j.cmet.2016.08.018
Pancreatic islet cells are critical for maintaining normal blood glucose levels, and their malfunction underlies diabetes development and progression. We used single-cell RNA sequencing to determine the transcriptomes of 1,492 human pancreatic α, β, δ, and PP cells from non-diabetic and type 2 diabetes organ donors. We identified cell-type-specific genes and pathways as well as 245 genes with disturbed expression in type 2 diabetes. Importantly, 92% of the genes have not previously been associated with islet cell function or growth. Comparison of gene profiles in mouse and human α and β cells revealed species-specific expression. All data are available for online browsing and download and will hopefully serve as a resource for the islet research community.
Solá, P;Mereu, E;Bonjoch, J;Casado-Peláez, M;Prats, N;Aguilera, M;Reina, O;Blanco, E;Esteller, M;Di Croce, L;Heyn, H;Solanas, G;Benitah, SA;
PMID: 37291218 | DOI: 10.1038/s43587-023-00431-z
Skin aging is characterized by structural and functional changes that contribute to age-associated frailty. This probably depends on synergy between alterations in the local niche and stem cell-intrinsic changes, underscored by proinflammatory microenvironments that drive pleotropic changes. The nature of these age-associated inflammatory cues, or how they affect tissue aging, is unknown. Based on single-cell RNA sequencing of the dermal compartment of mouse skin, we show a skew towards an IL-17-expressing phenotype of T helper cells, γδ T cells and innate lymphoid cells in aged skin. Importantly, in vivo blockade of IL-17 signaling during aging reduces the proinflammatory state of the skin, delaying the appearance of age-related traits. Mechanistically, aberrant IL-17 signals through NF-κB in epidermal cells to impair homeostatic functions while promoting an inflammatory state. Our results indicate that aged skin shows signs of chronic inflammation and that increased IL-17 signaling could be targeted to prevent age-associated skin ailments.
Muhl, L;Mocci, G;Pietilä, R;Liu, J;He, L;Genové, G;Leptidis, S;Gustafsson, S;Buyandelger, B;Raschperger, E;Hansson, EM;Björkegren, JLM;Vanlandewijck, M;Lendahl, U;Betsholtz, C;
PMID: 36283392 | DOI: 10.1016/j.devcel.2022.09.015
Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.
Gao, F;Li, C;Danopoulos, S;Al Alam, D;Peinado, N;Webster, S;Borok, Z;Kohbodi, GA;Bellusci, S;Minoo, P;
PMID: 35385750 | DOI: 10.1016/j.celrep.2022.110608
The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.
Figeac, F;Tencerova, M;Ali, D;Andersen, T;Appadoo, D;Kerckhofs, G;Ditzel, N;Kowal, J;Rauch, A;Kassem, M;
| DOI: 10.1093/stmcls/sxab011
The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high fat diet (HFD) and insulinopenia induced by treatment with streptozotocin (STZ), we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by - 28.4±7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06±38.71%, and increased density of SCA1+ (+74.99± 29.19%) but not Runx2 +osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82± 33.05%), senescence gene signature (≈106.66± 34.03%) and LAMIN B1 - senescent cell density (+225.18± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9± 6.2% which was inversely correlated with glucose levels (R 2=0.48, p<0.004) and callus adipose tissue area (R 2=0.3711, p<0.01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycaemia per se and not just insulin levels is detrimental for bone healing.
Mertz, E;Makareeva, E;Mirigian, L;Leikin, S;
| DOI: 10.1002/jbm4.10701
Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.
Acta neuropathologica communications
Seeker, LA;Bestard-Cuche, N;Jäkel, S;Kazakou, NL;Bøstrand, SMK;Wagstaff, LJ;Cholewa-Waclaw, J;Kilpatrick, AM;Van Bruggen, D;Kabbe, M;Baldivia Pohl, F;Moslehi, Z;Henderson, NC;Vallejos, CA;La Manno, G;Castelo-Branco, G;Williams, A;
PMID: 37217978 | DOI: 10.1186/s40478-023-01568-z
The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies.
Carr MJ, Toma JS, Johnston APW, Steadman PE, Yuzwa SA, Mahmud N, Frankland PW, Kaplan DR, Miller FD.
PMID: - | DOI: 10.1016/j.stem.2018.10.024
Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymalprecursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.
Sol�-Boldo L, Raddatz G, Sch�tz S, Mallm JP, Rippe K, Lonsdorf AS, Rodr�guez-Paredes M, Lyko F
PMID: 32327715 | DOI: 10.1038/s42003-020-0922-4
Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.
Bulstrode, H;Girdler, GC;Gracia, T;Aivazidis, A;Moutsopoulos, I;Young, AMH;Hancock, J;He, X;Ridley, K;Xu, Z;Stockley, JH;Finlay, J;Hallou, C;Fajardo, T;Fountain, DM;van Dongen, S;Joannides, A;Morris, R;Mair, R;Watts, C;Santarius, T;Price, SJ;Hutchinson, PJA;Hodson, EJ;Pollard, SM;Mohorianu, I;Barker, RA;Sweeney, TR;Bayraktar, O;Gergely, F;Rowitch, DH;
PMID: 36174572 | DOI: 10.1016/j.neuron.2022.09.002
Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNβ) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNβ treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.
