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1.
Proc Natl Acad Sci U S A ; 120(49): e2315096120, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38011564

RESUMO

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.


Assuntos
Hidradenite Supurativa , Humanos , Hidradenite Supurativa/genética , Pele/metabolismo , Epigenômica , Epigênese Genética , Células-Tronco/metabolismo , Cromatina/metabolismo
2.
J Cell Sci ; 135(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35099001

RESUMO

Mitochondrial dysfunction causes severe congenital cardiac abnormalities and prenatal/neonatal lethality. The lack of sufficient knowledge regarding how mitochondrial abnormalities affect cardiogenesis poses a major barrier for the development of clinical applications that target mitochondrial deficiency-induced inborn cardiomyopathies. Mitochondrial morphology, which is regulated by fission and fusion, plays a key role in determining mitochondrial activity. Dnm1l encodes a dynamin-related GTPase, Drp1, which is required for mitochondrial fission. To investigate the role of Drp1 in cardiogenesis during the embryonic metabolic shift period, we specifically inactivated Dnm1l in second heart field-derived structures. Mutant cardiomyocytes in the right ventricle (RV) displayed severe defects in mitochondrial morphology, ultrastructure and activity. These defects caused increased cell death, decreased cell survival, disorganized cardiomyocytes and embryonic lethality. By characterizing this model, we reveal an AMPK-SIRT7-GABPB axis that relays the reduced cellular energy level to decrease transcription of ribosomal protein genes in cardiomyocytes. We therefore provide the first genetic evidence in mouse that Drp1 is essential for RV development. Our research provides further mechanistic insight into how mitochondrial dysfunction causes pathological molecular and cellular alterations during cardiogenesis.


Assuntos
Dinaminas , Proteínas Ribossômicas , Animais , Dinaminas/genética , Dinaminas/metabolismo , Coração/embriologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
3.
J Cell Sci ; 135(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35946425

RESUMO

Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. Here, we specifically inactivated Dicer1 in the endocardium during cardiogenesis and found that Dicer1 deletion caused congenital mitral valve stenosis and regurgitation, whereas it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. We thus reveal miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.


Assuntos
MicroRNAs , Valva Tricúspide , Matriz Extracelular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Valva Mitral , Valva Tricúspide/anormalidades
4.
J Immunol ; 209(8): 1513-1522, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36165181

RESUMO

Activated naive (aNAV) B cells have been shown to be the precursor of the CD11c+T-bet+ IgD-CD27- double-negative (DN)2 or atypical memory (aMEM) B cells in systemic lupus erythematosus (SLE). To determine factors that maintain resting naive (rNAV) B cells, the transcriptomic program in naive (IGHD+IGHM +) B cells in human healthy control subjects (HC) and subjects with SLE was analyzed by single-cell RNA-sequencing analysis. In HC, naive B cells expressed IL-4 pathway genes, whereas in SLE, naive B cells expressed type I IFN-stimulated genes (ISGs). In HC, aNAV B cells exhibited upregulation of the gene signature of germinal center and classical memory (cMEM) B cells. In contrast, in SLE, aNAV B cells expressed signature genes of aMEM. In vitro exposure of SLE B cells to IL-4 promoted B cell development into CD27+CD38+ plasmablasts/plasma and IgD-CD27+ cMEM B cells. The same treatment blocked the development of CD11c+Tbet+ aNAV and DN2 B cells and preserved DN B cells as CD11c-Tbet- DN1 B cells. Lower expression of IL-4R and increased intracellular IFN-ß in naive B cells was correlated with the accumulation of CD21-IgD- B cells and the development of anti-Smith and anti-DNA autoantibodies in patients with SLE (n = 47). Our results show that IL-4R and type I IFN signaling in naive B cells induce the development of distinct lineages of cMEM versus aMEM B cells, respectively. Furthermore, diminished IL-4R signaling shifted activated B cell development from the DN1 to the DN2 trajectory in patients with SLE. Therapies that enhance IL-4R signaling may be beneficial for ISGhi SLE patients.


Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Autoanticorpos/metabolismo , Humanos , Imunoglobulina D/metabolismo , Interleucina-4/metabolismo , RNA/metabolismo
5.
Clin Immunol ; 257: 109842, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37981105

RESUMO

Cardinal features of lupus include elevated B cell activation and autoantibody production with a female sex preponderance. We quantified interactions of sex and genetic variation on the development of autoimmune B-cell phenotypes and autoantibodies in the BXD2 murine model of lupus using a cohort of backcrossed progeny (BXD2 x C57BL/6J) x BXD2. Sex was the key factor leading to increased total IgG, IgG2b, and autoantibodies. The percentage of T-bet+CD11c+ IgD+ activated naive B cells (aNAV) was higher in females and was associated with increased T-bet+CD11c+ IgD- age-related B cells, Fas+GL7+ germinal center B cells, Cxcr5-Icos+ peripheral T-helper cells, and Cxcr5+Icos+ follicular T-helper cells. IFN-ß was elevated in females. Variation in aNAV cells was mapped to Chr 7 in a locus that shows significant interactions between the female sex and heterozygous B/D variant. Our results suggest that activation of naive B cells forms the basis for the female-predominant development of autoantibodies in lupus-susceptible BXD2 mice.


Assuntos
Linfócitos B , Lúpus Eritematoso Sistêmico , Animais , Feminino , Humanos , Masculino , Camundongos , Autoanticorpos , Cruzamentos Genéticos , Centro Germinativo , Lúpus Eritematoso Sistêmico/genética , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores , Caracteres Sexuais
6.
J Am Soc Nephrol ; 33(4): 747-768, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35110364

RESUMO

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.


Assuntos
Cistos , Doenças Renais Policísticas , Animais , Cílios/metabolismo , Cistos/genética , Rim/metabolismo , Camundongos , Mutação , Doenças Renais Policísticas/metabolismo
7.
Clin Immunol ; 244: 109130, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36189576

RESUMO

Here, we report a case of atopic dermatitis (AD) in a patient who received biweekly doses of dupilumab, an antibody against the IL-4 receptor α chain (IL-4Rα). Single cell RNA-sequencing showed that naïve B cells expressed the highest levels of IL4R compared to other B cell subpopulations. Compared to controls, the dupilumab-treated patient exhibited diminished percentages of IL4R+IGHD+ naïve B cells and down-regulation of IL4R, FCER2 (CD23), and IGHD. Dupilumab treatment resulted in upregulation of genes associated with apoptosis and inhibition of B cell receptor signaling and down-regulation of class-switch and memory B cell development genes. The dupilumab-treated patient exhibited a rapid decline in COVID-19 anti-spike and anti-receptor binding domain antibodies between 4 and 8 and 11 months post COVID-19 vaccination. Our data suggest that intact and persistent IL-4 signaling is necessary for maintaining robust survival and development of naïve B cells, and maintaining a long term vaccine response.


Assuntos
Tratamento Farmacológico da COVID-19 , Receptores de Interleucina-4 , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Vacinas contra COVID-19 , Humanos , Interleucina-4 , RNA , Receptores de Antígenos de Linfócitos B
8.
J Immunol ; 204(7): 1982-1987, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32122998

RESUMO

GFP is frequently used as a marker for tracking donor cells adoptively transplanted into recipient animals. The human ubiquitin C promoter (UBC)-driven-GFP transgenic mouse is a commonly used source of donor cells for this purpose. This mouse was initially generated in the C57BL/6 inbred strain and has been backcrossed into the BALB/cBy strain for over 11 generations. Both the C57BL/6 inbred and BALB/cBy congenic UBC-GFP lines are commercially available and have been widely distributed. These UBC-GFP lines can be a convenient resource for tracking donor cells in both syngenic MHC-matched and in allogenic MHC-mismatched studies as C57BL/6 (H-2b) and BALB/cBy (H-2d) have disparate MHC haplotypes. In this report, we surprisingly discover that the UBC-GFP BALB/cBy congenic mice still retain the H-2b MHC haplotype of their original C57BL/6 founder, suggesting that the UBC-GFP transgene integration site is closely linked to the MHC locus on chromosome 17. Using linear amplification-mediated PCR, we successfully map the UBC-GFP transgene to the MHC locus. This study highlights the importance and urgency of mapping the transgene integration site of transgenic mouse strains used in biomedical research. Furthermore, this study raises the possibility of alternative interpretations of previous studies using congenic UBC-GFP mice and focuses attention on the necessity for rigor and reproducibility in scientific research.


Assuntos
Cromossomos/genética , Proteínas de Fluorescência Verde/genética , Complexo Principal de Histocompatibilidade/genética , Mutagênese Insercional/genética , Transgenes/genética , Ubiquitina C/genética , Animais , Haplótipos/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes
9.
J Immunol ; 205(2): 346-358, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32554431

RESUMO

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19-/- ) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)-IL-17 to Tfh-IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19-/- mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19-/- GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra + GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh-IL-17 cells. IL-23 acts indirectly on Il17ra + GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.


Assuntos
Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina G/metabolismo , Interleucina-23/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p19/genética , Switching de Imunoglobulina , Imunoglobulina G/genética , Interferon gama/metabolismo , Interleucina-23/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
J Immunol ; 201(8): 2203-2208, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30201809

RESUMO

In systemic lupus erythematosus (SLE), type I IFNs promote induction of type I IFN-stimulated genes (ISG) and can drive B cells to produce autoantibodies. Little is known about the expression of distinct type I IFNs in lupus, particularly high-affinity IFN-ß. Single-cell analyses of transitional B cells isolated from SLE patients revealed distinct B cell subpopulations, including type I IFN producers, IFN responders, and mixed IFN producer/responder clusters. Anti-Ig plus TLR3 stimulation of SLE B cells induced release of bioactive type I IFNs that could stimulate HEK-Blue cells. Increased levels of IFN-ß were detected in circulating B cells from SLE patients compared with controls and were significantly higher in African American patients with renal disease and in patients with autoantibodies. Together, the results identify type I IFN-producing and -responding subpopulations within the SLE B cell compartment and suggest that some patients may benefit from specific targeting of IFN-ß.


Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/fisiologia , Negro ou Afro-Americano , Interferon Tipo I/metabolismo , Interferon beta/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Insuficiência Renal Crônica/imunologia , Autoanticorpos/sangue , Circulação Sanguínea , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Interferon Tipo I/genética , Espaço Intracelular , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/epidemiologia , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , Análise de Célula Única , Transcriptoma , Estados Unidos/epidemiologia
11.
J Am Soc Nephrol ; 30(5): 767-781, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948627

RESUMO

BACKGROUND: Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. METHODS: As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell-negative CD45+ innate immune cells in mouse, rat, pig, and human kidney tissue. RESULTS: We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. CONCLUSIONS: Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata/genética , Molécula 1 de Adesão Intercelular/imunologia , Macrófagos/metabolismo , Análise de Variância , Animais , Biomarcadores/metabolismo , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Parabiose , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA , Especificidade da Espécie
12.
J Immunol ; 199(8): 2618-2623, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28904124

RESUMO

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-ß is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb-/- T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-ß. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1 Together, these data suggest that T1 B cell expression of IFN-ß plays a key role in regulating responsiveness to external factors.


Assuntos
Linfócitos B/imunologia , Interferon beta/metabolismo , Nefrite Lúpica/imunologia , Células Precursoras de Linfócitos B/imunologia , Baço/imunologia , Animais , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular , Sobrevivência Celular , Suscetibilidade a Doenças , Interferon beta-1a/genética , Interferon beta-1a/metabolismo , Interferon-alfa , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Análise de Célula Única , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo
13.
Proc Natl Acad Sci U S A ; 113(1): E51-60, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26699484

RESUMO

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Ubiquitina Tiolesterase/fisiologia , Proteases Específicas de Ubiquitina/fisiologia , Animais , Contagem de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Endopeptidases/genética , Endopeptidases/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Letais , Hematopoese/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/fisiologia , Transativadores , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/genética
14.
Clin Breast Cancer ; 24(4): e310-e318, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38492995

RESUMO

INTRODUCTION: PD-L1 immunohistochemistry (IHC) is being used as a predictive marker of the benefit derived from immunotherapy in several cancer types, including breast cancer. However, the insight gleaned of the prognostic and predictive value of PD-L1 status and its correlation with molecular characteristics during breast cancer progression remains limited. METHODS: We performed an PD-L1 (22C3) assay in pre-treatment primary and metastatic tumor sections from 33 patients with breast carcinoma, matched for post neoadjuvant chemotherapy (p-NACT). PD-L1 expression was evaluated using 3 scoring methods: immune cell (IC) and tumor cell (TC) with a 1% as the cutoff value, and combined positive scores (CPS) with a 1 as the cutoff value. Twenty-two samples from 11 patients had successful fluorescence in situ hybridization (FISH)-based molecular data available for analysis. RESULTS: In the 33 pre-treatment primary tumors, PD-L1 IC, TC, and CPS showed positive correlation with stromal tumor infiltrate lymphocytes (sTIL), histological grade 3, and triple negative breast carcinoma (TNBC). In the matched metastatic tumors, only PD-L1 IC showed a positive correlation with sTIL. The primary tumors showed a higher PD-L1 expression than the matched metastatic tumors by IC and CPS. Negative to positive conversion by CPS was identified in the metastatic tumors from lung, pleura and liver. p-NACT tumors also showed a trend of lower PD-L1 expression compared to the pre-treatment tumors. Six patients had matched samples for molecular and PD-L1 comparison, and none of them showed consistent gene alterations or PD-L1 expression among the primary, p-NACT and metastatic tumors. CONCLUSION: Our study showed a decrease in PD-L1 expression and disconnected molecular features during breast cancer progression. Repeating PD-L1 IHC testing could be considered in some specific metastatic sites if primary tumors were negative. Further studies are needed to identify other predictive factors for immune checkpoint inhibitor (ICI) therapy in patients with breast carcinoma.


Assuntos
Antígeno B7-H1 , Biomarcadores Tumorais , Neoplasias da Mama , Humanos , Feminino , Antígeno B7-H1/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Adulto , Prognóstico , Idoso , Imuno-Histoquímica , Terapia Neoadjuvante/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia
15.
Neurosurgery ; 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38747605

RESUMO

BACKGROUND AND OBJECTIVES: Vein of Galen malformation (VOGM), the result of arteriovenous shunting between choroidal and/or subependymal arteries and the embryologic prosencephalic vein, is among the most severe cerebrovascular disorders of childhood. We hypothesized that in situ analysis of the VOGM lesion using endoluminal tissue sampling (ETS) is feasible and may advance our understanding of VOGM genetics, pathogenesis, and maintenance. METHODS: We collected germline DNA (cheek swab) from patients and their families for genetic analysis. In situ VOGM "endothelial" cells (ECs), defined as CD31+ and CD45-, were obtained from coils through ETS during routine endovascular treatment. Autologous peripheral femoral ECs were also collected from the access sheath. Single-cell RNA sequencing of both VOGM and peripheral ECs was performed to demonstrate feasibility to define the transcriptional architecture. Comparison was also made with a published normative cerebrovascular transcriptome atlas. A subset of VOGM ECs was reserved for future DNA sequencing to assess for somatic and second-hit mutations. RESULTS: Our cohort contains 6 patients who underwent 10 ETS procedures from arterial and/or venous access during routine VOGM treatment (aged 12 days to ∼6 years). No periprocedural complications attributable to ETS occurred. Six unique coil types were used. ETS captured 98 ± 88 (mean ± SD; range 17-256) experimental ECs (CD31+ and CD45-). There was no discernible correlation between cell yield and coil type or route of access. Single-cell RNA sequencing demonstrated hierarchical clustering and unique cell populations within the VOGM EC compartment compared with peripheral EC controls when annotated using a publicly available cerebrovascular cell atlas. CONCLUSION: ETS may supplement investigations aimed at development of a molecular-genetic taxonomic classification scheme for VOGM. Moreover, results may eventually inform the selection of personalized pharmacologic or genetic therapies for VOGM and cerebrovascular disorders more broadly.

16.
Nat Commun ; 15(1): 3140, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38605083

RESUMO

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.


Assuntos
Edição de Genes , Rim , Animais , Suínos , Humanos , Animais Geneticamente Modificados , Xenoenxertos , Transplante Heterólogo , Rejeição de Enxerto/genética
17.
JCI Insight ; 8(9)2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36928191

RESUMO

Emerging data indicate an association between environmental heavy metal exposure and lung disease, including lower respiratory tract infections (LRTIs). Here, we show by single-cell RNA sequencing an increase in Pparg gene expression in lung macrophages from mice exposed to cadmium and/or infected with Streptococcus pneumoniae. However, the heavy metal cadmium or infection mediated an inhibitory posttranslational modification of peroxisome proliferator-activated receptor γ (PPARγ) to exacerbate LRTIs. Cadmium and infection increased ERK activation to regulate PPARγ degradation in monocyte-derived macrophages. Mice harboring a conditional deletion of Pparg in monocyte-derived macrophages had more severe S. pneumoniae infection after cadmium exposure, showed greater lung injury, and had increased mortality. Inhibition of ERK activation with BVD-523 protected mice from lung injury after cadmium exposure or infection. Moreover, individuals residing in areas of high air cadmium levels had increased cadmium concentration in their bronchoalveolar lavage (BAL) fluid, increased barrier dysfunction, and showed PPARγ inhibition that was mediated, at least in part, by ERK activation in isolated BAL cells. These observations suggest that impaired activation of PPARγ in monocyte-derived macrophages exacerbates lung injury and the severity of LRTIs.


Assuntos
Lesão Pulmonar , PPAR gama , Camundongos , Animais , PPAR gama/metabolismo , Pulmão/metabolismo , Cádmio/toxicidade , Cádmio/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Macrófagos Alveolares/metabolismo
18.
Front Immunol ; 14: 1082078, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37256130

RESUMO

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.


Assuntos
Macrófagos , Monócitos , Camundongos , Animais , Macrófagos/metabolismo , Monócitos/metabolismo , Rim/metabolismo , Receptores de Quimiocinas/metabolismo , Modelos Animais de Doenças , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo
19.
Res Sq ; 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36711785

RESUMO

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

20.
JCI Insight ; 7(20)2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36066976

RESUMO

The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney-resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury. While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA-Seq (scRNA-Seq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNA-Seq and spatial transcriptomics revealed 7 distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature, suggesting distinct functions. Specific protein markers were identified for 2 clusters, allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation was lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs was not fully restored for at least 28 days after injury. The change in KRM localization confirmed a long-hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.


Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Humanos , Macrófagos/metabolismo , Rim/metabolismo , Injúria Renal Aguda/metabolismo , Citometria de Fluxo , Insuficiência Renal Crônica/metabolismo
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