ABSTRACT
Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.
Subject(s)
Apyrase , Receptors, Chimeric Antigen , T-Lymphocytes, Regulatory , Humans , Apyrase/immunology , Apyrase/metabolism , T-Lymphocytes, Regulatory/immunology , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Cytotoxicity, Immunologic , Islets of Langerhans/immunology , Islets of Langerhans/metabolism , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/therapy , HLA-A2 Antigen/immunology , HLA-A2 Antigen/genetics , HLA-A2 Antigen/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Insulin-Secreting Cells/immunology , Insulin-Secreting Cells/metabolism , Antigens, CDABSTRACT
Alloxan (AL)-generated Reactive Oxygen Species (ROS) selectively destroy insulin-producing pancreatic ß-cells. A previous genome-wide scan (GWS) using a cohort of 296 F2 hybrids between NOD (AL-sensitive) and ALR (AL-resistant) mice identified linkages contributing to ß-cell susceptibility or resistance to AL-induced diabetes on Chromosomes (Chr) 2, 3, 8, and a single nucleotide polymorphism in mt-Nd2 of the mitochondrial genome (mtDNA). AL treatment of congenic and consomic NOD mouse stocks confirmed resistance linked to both the mtDNA and the Chr 8 locus from ALR [NOD.mtALR.ALR-(D8Mit293-D8Mit137)]. To identify possible epistatic interactions, the GWS analysis was expanded to 678 F2 mice. ALR-derived diabetes-resistance linkages on Chr 8 as well as the mt-Nd2 a allele were confirmed and novel additional linkages on Chr 4, 5, 6, 7, and 13 were identified. Epistasis was observed between the linkages on Chr 8 and 2 and Chr 8 and 6. Furthermore, the mt-Nd2 genotype affected the epistatic interactions between Chr 8 and 2. These results demonstrate that a combination of nuclear-cytoplasmic genome interactions regulates ß-cell sensitivity to ROS-mediated ALD.
ABSTRACT
The number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross ß-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms. IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.
Subject(s)
Dental Caries , Streptococcus mutans , Adhesins, Bacterial/metabolism , Aging , Amyloid/chemistry , Amyloidogenic Proteins/metabolism , Biofilms , Humans , Streptococcus mutans/metabolismABSTRACT
Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic ß cells can be infected by SARS-CoV-2 and cause ß cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in ß cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic ß cells in patients who succumbed to COVID-19 and selectively infects human islet ß cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces ß cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic ß cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce ß cell killing.
Subject(s)
COVID-19/virology , Diabetes Mellitus/virology , Insulin-Secreting Cells/virology , Neuropilin-1/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Virus Internalization , A549 Cells , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Antigens, CD/metabolism , Apoptosis , Apoptosis Regulatory Proteins/metabolism , COVID-19/complications , COVID-19/diagnosis , Case-Control Studies , Diabetes Mellitus/diagnosis , Diabetes Mellitus/metabolism , Female , Host-Pathogen Interactions , Humans , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Male , Middle Aged , Receptors, Transferrin/metabolism , SARS-CoV-2/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic ß cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human ß cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing ß-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal ß cells, adult α cells, and other non-ß cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, ß cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human ß cells should advance ß-cell replacement and other therapeutic strategies for diabetes.
Subject(s)
Cell Differentiation/genetics , Eye Proteins/metabolism , Gene Expression Regulation/genetics , Homeodomain Proteins/metabolism , Insulin-Secreting Cells/cytology , Nerve Tissue Proteins/metabolism , Diabetes Mellitus, Type 2/physiopathology , Humans , Insulin Secretion/genetics , RNA, Small Interfering/metabolism , Transcriptome , Homeobox Protein SIX3ABSTRACT
Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet ß-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet ß-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to ß-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
Subject(s)
Cell Differentiation/genetics , Insulin-Secreting Cells/physiology , Islets of Langerhans/embryology , Islets of Langerhans/growth & development , Swine , Animals , Animals, Newborn , Cells, Cultured , Embryo, Mammalian , Female , Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Glucagon-Secreting Cells/cytology , Glucagon-Secreting Cells/physiology , Humans , Islets of Langerhans/cytology , Mice , Organogenesis/genetics , Pregnancy , Swine/embryology , Swine/genetics , Swine/growth & development , Transcription Factors/genetics , Transcription Factors/metabolism , TranscriptomeABSTRACT
Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC). Results:Ncf1-mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules. Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells. Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Diabetes Mellitus, Type 1/immunology , Lymphocyte Activation/immunology , NADPH Oxidases/immunology , Animals , Autoimmunity/immunology , Cross-Priming/immunology , Mice , Mice, Inbred NOD , Mice, Mutant StrainsABSTRACT
Type 1 diabetes (T1D) has a strong genetic component. The insulin dependent diabetes (Idd)22 locus was identified in crosses of T1D-susceptible NOD mice with the strongly T1D-resistant ALR strain. The NODcALR-(D8Mit293-D8Mit137)/Mx (NOD-Idd22) recombinant congenic mouse strain was generated in which NOD mice carry the full Idd22 confidence interval. NOD-Idd22 mice exhibit almost complete protection from spontaneous T1D and a significant reduction in insulitis. Our goal was to unravel the mode of Idd22-based protection using in vivo and in vitro models. We determined that Idd22 did not impact immune cell diabetogenicity or ß cell resistance to cytotoxicity in vitro. However, NOD-Idd22 mice were highly protected against adoptive transfer of T1D. Transferred CTLs trafficked to the pancreatic lymph node and proliferated to the same extent in NOD and NOD-Idd22 mice, yet the accumulation of pathogenic CTLs in the islets was significantly reduced in NOD-Idd22 mice, correlating with disease resistance. Pancreatic endothelial cells from NOD-Idd22 animals expressed lower levels of adhesion molecules, even in response to inflammatory stimuli. Lower adhesion molecule expression resulted in weaker adherence of T cells to NOD-Idd22 endothelium compared with NOD-derived endothelium. Taken together, these results provide evidence that Idd22 regulates the ability of ß cell-autoreactive T cells to traffic into the pancreatic islets and may represent a new target for pharmaceutical intervention to potentially prevent T1D.
Subject(s)
Chemotaxis, Leukocyte/genetics , Diabetes Mellitus, Type 1/genetics , Islets of Langerhans/pathology , T-Lymphocytes, Cytotoxic/pathology , Adoptive Transfer , Animals , Autoimmunity/genetics , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Line , Chemotaxis, Leukocyte/physiology , Crosses, Genetic , Cytotoxicity Tests, Immunologic , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/prevention & control , Disease Resistance , Endothelial Cells/immunology , Endothelial Cells/metabolism , Female , Islets of Langerhans/immunology , Mice , Mice, Congenic , Mice, Inbred NOD , Mice, Inbred Strains , Mice, SCID , Specific Pathogen-Free Organisms , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
CD1d-restricted natural killer T (NKT) cells are a unique lymphocyte population that makes important contributions to host defense against numerous microbial pathogens. The powerful immunomodulatory effects of these cells can be exploited in mice by cognate antigens for multiple therapeutic purposes, including for protection from infectious diseases and as adjuvants to improve vaccines against microbial organisms. These applications have potential to treat and prevent infectious diseases in livestock species that express NKT cells, including pigs. In this study, immune tissues from commercial swine of mixed genetic background were compared for NKT cell frequency, cytokine secretion and subset ratios. Pigs were also injected with the model antigen hen-egg lysozyme (HEL) in conjunction with one of three glycosphingolipids, alpha-galactosylceramide (αGC), OCH and C-glycoside that selectively activate NKT cells, to assess the adjuvant potential of each. There was significant variation between individual pigs for all NKT cell parameters measured. The NKT cell agonists elicited HEL-specific immune responses of different quality, but only αGC increased the systemic concentration of NKT cells. Peripheral blood NKT cell frequency measured prior to treatment was a poor predictor of how individual animals responded to NKT cell therapy. However, our results show that although NKT cells vary considerably between pigs, there exists considerable potential to harness these cells to protect swine from infectious diseases.