RESUMO
The innate immune system is the first line of host defense. Innate immune activation utilizes pattern recognition receptors to detect pathogens, pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), and homeostatic alterations and drives inflammatory signaling pathways and regulated cell death. Cell death activation is critical to eliminate pathogens and aberrant or damaged cells, while excess activation can be linked to inflammation, tissue damage, and disease. Therefore, there is increasing interest in studying cell death mechanisms to understand the underlying biology and identify therapeutic strategies. However, there are significant technical challenges, as many cell death pathways share key molecules with each other, and genetic models where these cell death molecules are deleted remain the gold standard for evaluation. Furthermore, extensive crosstalk has been identified between the cell death pathways pyroptosis, apoptosis, necroptosis, and the more recently characterized PANoptosis, which is defined as a prominent, unique innate immune, lytic, and inflammatory cell death pathway initiated by innate immune sensors and driven by caspases and RIPKs through PANoptosomes. PANoptosomes are multi-protein complexes assembled by innate immune sensor(s) in response to pathogens, PAMPs, DAMPs, cytokines, and homeostatic changes that drive PANoptosis. In this article, we provide methods for molecularly defining distinct cell death pathways, including PANoptosis, using both genetic and chemical approaches through western blot, LDH assay, and microscopy readouts. This procedure allows for the assessment of cell death on the cell population and single-cell levels even without access to genetic models. Having this comprehensive workflow that is more accessible to all labs will improve our ability as a scientific community to accelerate discovery. Using these protocols will help identify new innate immune sensors that drive PANoptosis and define the molecular mechanisms and regulators involved to establish new targets for clinical translation. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction and quantification of cell death using live cell imaging Alternate Protocol 1: Quantification of cell death using LDH Alternate Protocol 2: Assessment of cell death complexes in single cells using immunofluorescence staining Basic Protocol 2: Analysis of cell death mechanisms by immunoblots (western blots).
Assuntos
Morte Celular , Imunidade Inata , Humanos , Animais , Necroptose/imunologia , CamundongosRESUMO
The complement system is an important component of transplant rejection. Sertoli cells, an immune regulatory testicular cell, survive long-term when transplanted across immunological barriers; thus, understanding the mechanisms behind this unique survival would be of great benefit to the transplantation field. This study focused on Sertoli cell inhibition of complement as relevant in xenotransplantation. Neonatal pig Sertoli cells (NPSCs) survived activated human complement in vitro while neonatal pig islet (NPI) aggregates and pig aortic endothelial cell (PAEC) survival were diminished to about 65% and 12%, respectively. PAECs cultured in NPSC-conditioned media and human complement demonstrated a 200% increase in survival suggesting that NPSCs secrete complement-inhibiting substances that confer protection. Bioinformatic and molecular analyses identified 21 complement inhibitors expressed by NPSCs with several significantly increased in NPSCs compared to NPIs or PAECs. Lastly, RNA sequencing revealed that NPSCs express 25 other complement factors including cascade components and receptors. Overall, this study identified the most comprehensive Sertoli cell complement signature to date and indicates that the expression of a variety of complement inhibitors ensures a proper regulation of complement through redundant inhibition points. Understanding the regulation of the complement system should be further investigated for extending xenograft viability.
Assuntos
Proteínas do Sistema Complemento , Rejeição de Enxerto , Células de Sertoli , Humanos , Masculino , Inativadores do Complemento , Proteínas do Sistema Complemento/metabolismo , Rejeição de Enxerto/metabolismo , Xenoenxertos , Células de Sertoli/metabolismo , Transplante Heterólogo , Suínos , AnimaisRESUMO
An effective treatment and possible cure for type 1 diabetes is transplantation of pancreatic islets. Unfortunately, transplanted islets are rejected by the immune system with humoral-mediated responses being an important part of rejection. Sertoli cells (SC), an immune regulatory cell shown to survive as allografts long-term without immunosuppressants, have the potential to be used as a cell-based gene therapy vehicle to deliver endogenous insulin-a possible alternative to islets. Previously, we transduced a mouse SC line to produce human insulin. After transplantation into diabetic mice, these cells consistently produced low levels of insulin with graft survival of 75% at 50 days post-transplantation. The object of this study was to assess humoral immune regulation by these engineered SC. Both nontransduced and transduced SC survived exposure to human serum with complement in vitro. Analysis of allografts in vivo at 20 and 50 days post-transplantation revealed that despite IgG antibody detection, complement factor deposition was low and grafts survived through 50 days post-transplantation. Furthermore, the transduced SC secreted elevated levels of the complement inhibitor C1q binding protein. Overall, this suggests SC genetically engineered to express insulin maintain their ability to prevent complement-mediated killing. Since inhibiting complement-mediated rejection is important for graft survival, further studies of how SC modifies the immune response could be utilized to advance the use of genetically engineered SC or to prolong islet allograft survival to improve the treatment of diabetes.
Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Masculino , Humanos , Camundongos , Animais , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Experimental/metabolismo , Células de Sertoli/metabolismo , Insulina/metabolismo , Insulina Regular Humana , Proteínas do Sistema Complemento/metabolismo , Imunidade , Aloenxertos , Rejeição de EnxertoRESUMO
Background Emerging research suggests hyperglycemia can increase intestinal permeability. Ginger and its bioactive compounds have been reported to benefit diabetic animals due to their anti-inflammatory and antioxidant properties. In this study, we revealed the beneficial effect of gingerol-enriched ginger (GEG) on intestinal health (i.e., barrier function, mitochondrial function, and anti-inflammation) in diabetic rats. Methods Thirty-three male Sprague Dawley rats were assigned to three groups: low-fat diet (control group), high-fat-diet (HFD) + streptozotocin (single low dose 35 mg/kg body weight (BW) after 2 weeks of HFD feeding) (DM group), and HFD + streptozotocin + 0.75% GEG in diet (GEG group) for 42 days. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted at baseline and prior to sample collection. Total pancreatic insulin content was determined by ELISA. Total RNA of intestinal tissues was extracted for mRNA expression using qRT-PCR. Results Compared to the DM group, the GEG group had improved glucose tolerance and increased pancreatic insulin content. Compared to those without GEG (DM group), GEG supplementation (GEG group) increased the gene expression of tight junction (Claudin-3) and antioxidant capacity (SOD1), while it decreased the gene expression for mitochondrial fusion (MFN1), fission (FIS1), biogenesis (PGC-1α, TFAM), mitophagy (LC3B, P62, PINK1), and inflammation (NF-κB). Conclusions Ginger root extract improved glucose homeostasis in diabetic rats, in part, via improving intestinal integrity and mitochondrial dysfunction of GI health.
Assuntos
Diabetes Mellitus Experimental , Zingiber officinale , Masculino , Ratos , Animais , Estreptozocina , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Antioxidantes/farmacologia , NF-kappa B/metabolismo , Claudina-3 , Superóxido Dismutase-1/metabolismo , Ratos Sprague-Dawley , Dieta Hiperlipídica/efeitos adversos , Insulina/metabolismo , Mitocôndrias/metabolismo , Extratos Vegetais/uso terapêutico , Anti-Inflamatórios/farmacologia , Glucose/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismoRESUMO
The testis must create and maintain an immune privileged environment to protect maturing germ cells from autoimmune destruction. The establishment of this protective environment is due, at least in part, to Sertoli cells. Sertoli cells line the seminiferous tubules and form the blood-testis barrier (BTB), a barrier between advanced germ cells and the immune system. The BTB compartmentalizes the germ cells and facilitates the appropriate microenvironment necessary for spermatogenesis. Further, Sertoli cells modulate innate and adaptive immune processes through production of immunoregulatory compounds. Sertoli cells, when transplanted ectopically (outside the testis), can also protect transplanted tissue from the recipient's immune system and reduce immune complications in autoimmune diseases primarily by immune regulation. These properties make Sertoli cells an attractive candidate for inflammatory disease treatments and cell-based therapies. Conversely, the same properties that protect the germ cells also allow the testis to act as a reservoir site for infections. Interestingly, Sertoli cells also have the ability to mount an antimicrobial response, if necessary, as in the case of infections. This review aims to explore how Sertoli cells act as a double-edged sword to both protect germ cells from an autoimmune response and activate innate and adaptive immune responses to fight off infections.
Assuntos
Barreira Hematotesticular , Células de Sertoli , Células Germinativas , Humanos , Masculino , Espermatogênese , Testículo/metabolismoRESUMO
Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células de Sertoli/metabolismo , Animais , Humanos , Masculino , Camundongos , Células de Sertoli/citologiaRESUMO
The acute cell-mediated immune response presents a significant barrier to xenotransplantation. Immune-privileged Sertoli cells (SC) can prolong the survival of co-transplanted cells including xenogeneic islets, hepatocytes, and neurons by protecting them from immune rejection. Additionally, SC survive as allo- and xenografts without the use of any immunosuppressive drugs suggesting elucidating the survival mechanism(s) of SC could be used to improve survival of xenografts. In this study, the survival and immune response generated toward neonatal pig SC (NPSC) or neonatal pig islets (NPI), nonimmune-privileged controls, was compared after xenotransplantation into naïve Lewis rats without immune suppression. The NPSC survived throughout the study, while NPI were rejected within 9 days. Analysis of the grafts revealed that macrophages and T cells were the main immune cells infiltrating the NPSC and NPI grafts. Further characterization of the T cells within the grafts indicated that the NPSC grafts contained significantly more cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) regulatory T cells (Tregs) at early time points than the NPI grafts. Additionally, the presence of increased amounts of interleukin 10 (IL-10) and transforming growth factor (TGF) ß and decreased levels of tumor necrosis factor (TNF) α and apoptosis in the NPSC grafts compared to NPI grafts suggests the presence of regulatory immune cells in the NPSC grafts. The NPSC expressed several immunoregulatory factors such as TGFß, thrombospondin-1 (THBS1), indoleamine-pyrrole 2,3-dioxygenase, and galectin-1, which could promote the recruitment of these regulatory immune cells to the NPSC grafts. In contrast, NPI grafts had fewer Tregs and increased apoptosis and inflammation (increased TNFα, decreased IL-10 and TGFß) suggestive of cytotoxic immune cells that contribute to their early rejection. Collectively, our data suggest that a regulatory graft environment with regulatory immune cells including CD4 and CD8 Tregs in NPSC grafts could be attributed to the prolonged survival of the NPSC xenografts.