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Gamma delta (γδ) T cells play a crucial role in anti-tumor immunity due to their cytotoxic properties. However, the role and extent of γδ T cells in production of pro-tumorigenic interleukin- 17 (IL-17) within the tumor microenvironment (TME) of colorectal cancer (CRC) remains controversial. In this study, we re-analyzed nine published human CRC whole-tissue single-cell RNA sequencing (scRNA-seq) datasets, identifying 18,483 γδ T cells out of 951,785 total cells, in the neoplastic or adjacent normal tissue of 165 human CRC patients. Our results confirm that tumor-infiltrating γδ T cells exhibit high cytotoxicity-related transcription in both tumor and adjacent normal tissues, but critically, none of the γδ T cell clusters showed IL-17 production potential. We also identified various γδ T cell subsets, including Teff, TRM, Tpex, and Tex, and noted an increased expression of cytotoxic molecules in tumor-infiltrating γδ T cells compared to their normal area counterparts. Our work demonstrates that γδ T cells in CRC primarily function as cytotoxic effector cells rather than IL-17 producers, mitigating the concerns about their potential pro-tumorigenic roles in CRC, highlighting the importance of accurately characterizing these cells for cancer immunotherapy research and the unneglectable cross-species discrepancy between the mouse and human immune system in the study of cancer immunology.
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Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4+ naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4+ naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined populations of immune cells in the gut microphysiological system demonstrated the usefulness of GuMI physiomimetic platform to study microbe-epithelial-immune interactions in healthy and disease conditions.
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Faecalibacterium prausnitzii , Sistemas Microfisiológicos , Humanos , Faecalibacterium prausnitzii/fisiología , Receptor Toll-Like 1 , Citocinas , InflamaciónRESUMEN
Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we establish a gut epithelium-microbe-immune microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), with CD4+ naïve T cells circulating underneath the colonic epithelium. Multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines being secreted into both apical and basolateral compartments. In contrast, the absence of APCs does not allow reliable detection of these cytokines. In the presence of APCs, F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, but no significant change on the secreted cytokines. In contrast, integration of CD4+ naïve T cells reverses this effect by decreasing the transcription of TLR1, IFNA1, and indoleamine 2,3-dioxygenase, and increasing the F. prausnitzii-induced secretion of pro-inflammatory cytokines such as IL-8, MCP-1/CCL2, and IL1A. These results highlight the contribution of individual innate immune cells in the regulation of the immune response triggered by the gut commensal F. prausnitzii. The successful integration of defined populations of immune cells in this gut microphysiological system demonstrated the usefulness of the GuMI physiomimetic platform to study microbe-epithelial-immune interactions in health and disease.
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Metabolic and inflammatory disorders such as autoimmune and neurodegenerative diseases are increasing at alarming rates. Many of these are not tissue-specific occurrences but complex, systemic pathologies of unknown origin for which no cure exists. Such complexity obscures causal relationships among factors regulating disease progression. Emerging technologies mimicking human physiology, such as microphysiological systems (MPSs), offer new possibilities to provide clarity in systemic metabolic and inflammatory diseases. Controlled interaction of multiple MPSs and the scalability of biological complexity in MPSs, supported by continuous multiomic monitoring, might hold the key to identifying novel relationships between interorgan crosstalk, metabolism, and immunity. In this perspective, I aim to discuss the current state of modeling multiorgan physiology and evaluate current opportunities and challenges.
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Fenómenos Fisiológicos Celulares , Dispositivos Laboratorio en un Chip , HumanosRESUMEN
The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.
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Bacteroides thetaiotaomicron/fisiología , Colon/citología , Células Epiteliales/fisiología , Mucosa Intestinal/citología , Técnicas de Cocultivo/métodos , Humanos , OrganoidesRESUMEN
Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ- and organ-immune-specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson's disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell-derived cerebral MPS in a systemically circulated common culture medium containing CD4+ regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo-like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD.
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Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Encéfalo/metabolismo , Humanos , Hígado/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor ß (TGF-ß), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-ß signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.
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Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/virología , Tracto Gastrointestinal/virología , Infecciones por VIH/virología , VIH-1/fisiología , Cadenas alfa de Integrinas/metabolismo , Transcripción Genética/genética , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , ADN Viral/metabolismo , Tracto Gastrointestinal/inmunología , Regulación de la Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Humanos , Linfocitos Intraepiteliales/metabolismo , Linfocitos Intraepiteliales/virología , Provirus/fisiología , ARN Viral/metabolismo , Proteínas Ribosómicas/genética , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/virología , Latencia del VirusRESUMEN
The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.
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Alternativas a las Pruebas en Animales , Bienestar del Animal , Desarrollo de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Dispositivos Laboratorio en un Chip , Animales , Industria Farmacéutica , Humanos , Modelos BiológicosRESUMEN
Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 T cells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFAs exacerbated CD4+ T cell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.
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Ácidos Grasos Volátiles/metabolismo , Tracto Gastrointestinal/metabolismo , Hígado/metabolismo , Biomimética/métodos , Microbioma Gastrointestinal/fisiología , Homeostasis , Humanos , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/metabolismo , Modelos Biológicos , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
Innate immune responses to Zika virus (ZIKV) are dampened in the lower female reproductive tract (LFRT) compared to other tissues, but the mechanism that underlies this vulnerability is poorly understood. Using tissues from uninfected and vaginally ZIKV-infected macaques and mice, we show that low basal expression of RNA-sensing pattern recognition receptors (PRRs), or their co-receptors, in the LFRT contributes to high viral replication in this tissue. In the LFRT, ZIKV sensing provides limited protection against viral replication, and the sensors are also minimally induced after vaginal infection. While IFNα/ß receptor signaling offers minimal protection in the LFRT, it is required to prevent dissemination of ZIKV to other tissues, including the upper FRT. Our findings support a role for RNA-sensing PRRs in the dampened innate immunity against ZIKV in the LFRT compared to other tissues and underlie potential implications for systemic dissemination upon heterosexual transmission of ZIKV in women.
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Genitales Femeninos/inmunología , Inmunidad Innata/inmunología , ARN Viral/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Femenino , Regulación Viral de la Expresión Génica , Genitales Femeninos/metabolismo , Genitales Femeninos/virología , Humanos , Inmunidad Innata/genética , Macaca mulatta , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Viral/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 3/metabolismo , Vagina/inmunología , Vagina/metabolismo , Vagina/virología , Replicación Viral/genética , Replicación Viral/inmunología , Virus Zika/genética , Virus Zika/fisiología , Infección por el Virus Zika/genética , Infección por el Virus Zika/virologíaRESUMEN
HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.
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Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/fisiología , Antígeno Ki-1/metabolismo , Tejido Linfoide/virología , Recto/virología , Activación Transcripcional , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa , Biomarcadores/sangre , Biomarcadores/metabolismo , Brentuximab Vedotina , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Estudios de Cohortes , ADN Viral/sangre , ADN Viral/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Humanos , Inmunoconjugados/farmacología , Hibridación in Situ , Antígeno Ki-1/antagonistas & inhibidores , Antígeno Ki-1/sangre , Antígeno Ki-1/química , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , ARN Viral/sangre , ARN Viral/metabolismo , Recto/efectos de los fármacos , Recto/metabolismo , Recto/patología , Solubilidad , Activación Transcripcional/efectos de los fármacos , Carga Viral/efectos de los fármacosRESUMEN
Determining the magnitude of local immune response during mucosal exposure to viral pathogens is critical to understanding the mechanism of viral pathogenesis. We previously showed that vaginal inoculation of lymphocytic choriomeningitis virus (LCMV) fails to induce a robust innate immune response in the lower female reproductive tract (FRT), allowing high titer viral replication and a delay in T-cell-mediated viral control. Despite this immunological delay, LCMV replication remained confined mainly to the FRT and the draining iliac lymph node. Here, we show that rectal infection with LCMV triggers type I/III interferon responses, followed by innate immune activation and lymphocyte recruitment to the colon. In contrast to vaginal exposure, innate immunity controls LCMV replication in the colon, but virus rapidly disseminates systemically. Virus-induced inflammation promotes the recruitment of LCMV target cells to the colon followed by splenic viral dissemination by infected B cells, and to a lesser extent by CD8 T cells. These findings demonstrate major immunological differences between vaginal and rectal exposure to the same viral pathogen, highlighting unique risks associated with each of these common routes of sexual viral transmission.
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Infecciones por Arenaviridae/inmunología , Linfocitos B/inmunología , Colon/inmunología , Linfocitos/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Vagina/inmunología , Animales , Linfocitos B/virología , Movimiento Celular , Colon/virología , Femenino , Inmunidad Innata , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recto/metabolismo , Vagina/virologíaRESUMEN
The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.
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Linfocitos T CD4-Positivos/inmunología , Separación Celular/métodos , Tracto Gastrointestinal/inmunología , Infecciones por VIH/inmunología , Biopsia/métodos , Recuento de Linfocito CD4 , Quimiocinas/análisis , Tracto Gastrointestinal/virología , VIH-1/inmunología , HumanosRESUMEN
The single greatest challenge to an HIV cure is the persistence of latently infected cells containing inducible, replication-competent proviral genomes, which constitute only a small fraction of total or infected cells in the body. Although resting CD4+ T cells in the blood are a well-known source of viral rebound, more than 90% of the body's lymphocytes reside elsewhere. Many are in gut tissue, where HIV DNA levels per million CD4+ T cells are considerably higher than in the blood. Despite the significant contribution of gut tissue to viral replication and persistence, little is known about the cell types that support persistence of HIV in the gut; importantly, T cells in the gut have phenotypic, functional, and survival properties that are distinct from T cells in other tissues. The mechanisms by which latency is established and maintained will likely depend on the location and cytokine milieu surrounding the latently infected cells in each compartment. Therefore, successful HIV cure strategies require identification and characterization of the exact cell types that support viral persistence, particularly in the gut. In this review, we describe the seeding of the latent HIV reservoir in the gut mucosa; highlight the evidence for compartmentalization and depletion of T cells; summarize the immunologic consequences of HIV infection within the gut milieu; propose how the damaged gut environment may promote the latent HIV reservoir; and explore several immune cell targets in the gut and their place on the path toward HIV cure.
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Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , Tejido Linfoide/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Latencia del Virus/inmunología , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Tejido Linfoide/citología , Replicación Viral/inmunologíaRESUMEN
Understanding the host immune response to vaginal exposure to RNA viruses is required to combat sexual transmission of this class of pathogens. In this study, using lymphocytic choriomeningitis virus (LCMV) and Zika virus (ZIKV) in wild-type mice, we show that these viruses replicate in the vaginal mucosa with minimal induction of antiviral interferon and inflammatory response, causing dampened innate-mediated control of viral replication and a failure to mature local antigen-presenting cells (APCs). Enhancement of innate-mediated inflammation in the vaginal mucosa rescues this phenotype and completely inhibits ZIKV replication. To gain a better understanding of how this dampened innate immune activation in the lower female reproductive tract may also affect adaptive immunity, we modeled CD8 T cell responses using vaginal LCMV infection. We show that the lack of APC maturation in the vaginal mucosa leads to a delay in CD8 T cell activation in the draining lymph node and hinders the timely appearance of effector CD8 T cells in vaginal mucosa, thus further delaying viral control in this tissue. Our study demonstrates that vaginal tissue is exceptionally vulnerable to infection by RNA viruses and provides a conceptual framework for the male to female sexual transmission observed during ZIKV infection.
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Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Vagina/inmunología , Replicación Viral/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/fisiología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Linfocitos T CD8-positivos/patología , Femenino , Inmunidad Innata , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/patología , Ratones , Ratones Noqueados , Vagina/patología , Vagina/virología , Replicación Viral/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/patologíaRESUMEN
Corneal wound healing is often affected by TGF-ß-mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-ß2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-ß2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-ß/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.
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The intestinal epithelium is composed of diverse cell types, most abundant being the enterocytes. Among other functions, they maintain the intestinal barrier and play a critical role in the absorption of nutrients, drugs and toxins. This study describes the development and characterization of human intestinal epithelial cells (HUIEC), a spontaneously arising cell line established by selective trypsinization and cloning of the intestinal epithelium, resulting in a uniform population of highly epithelial cells with a strong growth potential.
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Técnicas de Cultivo Celular por Lotes/métodos , Separación Celular/métodos , Células Epiteliales/citología , Células Epiteliales/fisiología , Mucosa Intestinal/citología , Diferenciación Celular/fisiología , Línea Celular , Células Epiteliales/clasificación , HumanosRESUMEN
Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-ß could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-ß inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-ß inhibitor.