Harmine and exendin-4 combination therapy safely expands human ß cell mass in vivo in a mouse xenograft system.

Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.

Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms.

Prior studies have shown that pancreatic α-cells can transdifferentiate into ß-cells, and that ß-cells de-differentiate and are prone to acquire an α-cell phenotype in type 2 diabetes (T2D). However, the specific human α-cell and ß-cell subtypes that are involved in α-to-ß-cell and ß-to-α-cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into α-ß cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG -expressing human α-cell subclusters [α1, α2, α-ß-transition 1 (AB-Tr1), α-ß-transition 2 (AB-Tr2), and α-ß (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The α1, α2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for α-cell function while AB cells are enriched in genes related to pancreatic progenitor and ß-cell pathways; 4) Trajectory inference analysis of extracted α- and ß-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both α- and ß-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards ß-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards α-cells. 6) Remarkably, in contrast to the events in vitro , the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from α-to-ß-cells in vivo . 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from ß-to-α-cells compatible with dedifferentiation or conversion into α-cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro , in vivo , in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between α- and ß-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo . Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes ß-cell subpopulations with dynamic transcriptome profiles.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts. METHODS: We obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library. RESULTS: First, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (ß-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three ß-cell sub-clusters: an INS pre-mRNA cluster (ß3), an intermediate INS mRNA cluster (ß2), and an INS mRNA-rich cluster (ß1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (ß1) becomes the predominant sub-cluster in vivo. CONCLUSIONS: In summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO's effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor-knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

A novel therapeutic combination of sitagliptin and melatonin regenerates pancreatic ß-cells in mouse and human islets.

Autoimmune-led challenge resulting in ß-cell loss is responsible for the development of type 1 diabetes (T1D). Melatonin, a pineal hormone or sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor, has increased ß-cell mass in various diabetic models and has immunoregulatory property. Both ß-cell regenerative capacity and melatonin secretion decrease with ageing. Thus, we aimed to investigate the therapeutic potential of melatonin combined with sitagliptin on ß-cell regeneration under glucotoxic stress, in the streptozotocin-induced young and old diabetic mouse models, and euglycemic humanized islet transplant mouse model. Our results suggest that combination therapy of sitagliptin and melatonin show an additive effect in inducing mouse ß-cell regeneration under glucotoxic stress, and in the human islet transplant mouse model. Further, in the young diabetic mouse model, the monotherapies induce ß-cell transdifferentiation and reduce ß-cell apoptosis whereas, in the old diabetic mouse model, melatonin and sitagliptin induce ß-cell proliferation and ß-cell transdifferentiation, and it also reduces ß-cell apoptosis. Further, in both the models, combination therapy reduces fasting blood glucose levels, increases plasma insulin levels and glucose tolerance and promotes ß-cell proliferation, ß-cell transdifferentiation, and reduces ß-cell apoptosis. It can be concluded that combination therapy is superior to monotherapies in ameliorating diabetic manifestations, and it can be used as a future therapy for ß-cell regeneration in diabetes patients.

Nrf2 Regulates ß-Cell Mass by Suppressing ß-Cell Death and Promoting ß-Cell Proliferation.

Finding therapies that can protect and expand functional ß-cell mass is a major goal of diabetes research. Here, we generated ß-cell-specific conditional knockout and gain-of-function mouse models and used human islet transplant experiments to examine how manipulating Nrf2 levels affects ß-cell survival, proliferation, and mass. Depletion of Nrf2 in ß-cells results in decreased glucose-stimulated ß-cell proliferation ex vivo and decreased adaptive ß-cell proliferation and ß-cell mass expansion after a high-fat diet in vivo. Nrf2 protects ß-cells from apoptosis after a high-fat diet. Nrf2 loss of function decreases Pdx1 abundance and insulin content. Activating Nrf2 in a ß-cell-specific manner increases ß-cell proliferation and mass and improves glucose tolerance. Human islets transplanted under the kidney capsule of immunocompromised mice and treated systemically with bardoxolone methyl, an Nrf2 activator, display increased ß-cell proliferation. Thus, by managing reactive oxygen species levels, Nrf2 regulates ß-cell mass and is an exciting therapeutic target for expanding and protecting ß-cell mass in diabetes.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

The many lives of Myc in the pancreatic ß-cell.

Diabetes results from insufficient numbers of functional pancreatic ß-cells. Thus, increasing the number of available functional ß-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand ß-cells for diabetes treatment. Nearly all these studies in ß-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced ß-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for ß-cell regeneration. However, recent studies indicate that "gentle" induction of Myc expression enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in ß-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the ß-cell, its potential for ß-cell regeneration, and its physiological importance for neonatal and adaptive ß-cell expansion.

GLP-1 receptor agonists synergize with DYRK1A inhibitors to potentiate functional human ß cell regeneration.

Glucagon-like peptide-1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 inhibitors are widely prescribed diabetes drugs due to their ability to stimulate insulin secretion from remaining ß cells and to reduce caloric intake. Unfortunately, they fail to increase human ß cell proliferation. Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) are able to induce adult human ß cell proliferation, but rates are modest (~2%), and their specificity to ß cells is limited. Here, we provide evidence that combining any member of the GLP1R agonist class with any member of the DYRK1A inhibitor class induces a synergistic increase in human ß cell replication (5 to 6%) accompanied by an actual increase in numbers of human ß cells. GLP1R agonist-DYRK1A inhibitor synergy required combined inhibition of DYRK1A and an increase in cAMP and did not lead to ß cell dedifferentiation. These beneficial effects on proliferation were seen in both normal human ß cells and ß cells derived from individuals with type 2 diabetes. The ability of the GLP1R agonist-DYRK1A inhibitor combination to enhance human ß cell proliferation, human insulin secretion, and blood glucose control extended in vivo to studies of human islets transplanted into euglycemic and streptozotocin-diabetic immunodeficient mice. No adverse events were observed in the mouse studies during a 1-week period. Because of the relative ß cell specificity of GLP1R agonists, the combination provides an improved, although not complete, degree of human ß cell specificity.

Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice.

One of the main unresolved questions in solid organ transplantation is how to establish indefinite graft survival that is free from long-term treatment with immunosuppressive drugs and chronic rejection (i.e., the establishment of tolerance). The failure to achieve this goal may be related to the difficulty in identifying the phenotype and function of the cell subsets that participate in the induction of tolerance. To address this issue, we investigated the suppressive roles of recipient myeloid cells that may be manipulated to induce tolerance to transplanted hearts in mice. Using depleting mAbs, clodronate-loaded liposomes, and transgenic mice specific for depletion of CD11c+, CD11b+, or CD115+ cells, we identified a tolerogenic role for CD11b+CD115+Gr1+ monocytes during the induction of tolerance by costimulatory blockade with CD40L-specific mAb. Early after transplantation, Gr1+ monocytes migrated from the bone marrow into the transplanted organ, where they prevented the initiation of adaptive immune responses that lead to allograft rejection and participated in the development of Tregs. Our results suggest that mobilization of bone marrow CD11b+CD115+Gr1+ monocytes under sterile inflammatory conditions mediates the induction of indefinite allograft survival. We propose that manipulating the common bone marrow monocyte progenitor could be a useful clinical therapeutic approach for inducing transplantation tolerance.

Organ transplantation in rodents: novel applications of long-established methods.

Rodent models of solid organ transplantation have been used for many decades. Standardized operative techniques resulting in highly reproducible survival rates have been developed for several organs. This allowed scientists to investigate many clinically relevant problems, test new drugs and establish novel treatment regimens. Recently, many studies used these models to explore novel issues such as graft modification by pharmaceutical, surgical or genetic engineering methods, post-transplant regeneration, leukocyte trafficking or interactions between the innate and allo-specific arms of the immune response. The results from these studies clearly facilitate a more complex and comprehensive understanding of existing problem. The long-established methods of rodent organ transplantation, combined with the newest achievements in surgical techniques, biotechnology and imaging, will remain indispensable tools of transplantation biology.

Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts.

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+ CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells.

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4(+)CD25(+)CD62L(high) T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4(+)CD25(+) Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3(+)CD4(+)CD25(+) Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.

Increased apoptosis is specific for acute rejection in rat small bowel transplant.

Apoptosis has been associated with several events in solid organ transplantation, including ischemia/reperfusion (IR) injury and acute rejection. To determine whether apoptosis-profiles may distinguish these two conditions, we analyzed apoptosis rates in a rat orthotopic small bowel transplant (SBT) model. SBT was performed in Lewis rats with either freshly harvested or preserved (4 h, in UW at 4 degrees C) syngeneic and allogeneic (Brown-Norway) grafts. Bowel samples were collected 2 h after reperfusion and on small bowel transplant postoperative days (POD) 1, 4, and 7. Apoptosis was detected by measuring levels of histone-associated DNA fragments and caspase 3 expression, and by determining apoptotic body counts. All markers measured 2 h after reperfusion increased profoundly in association with preservation. After a significant decrease on POD 1, apoptosis rates rose again between POD 4 and 7 only in allogeneic grafts. This distinct second increase in apoptosis may be an early and specific sign of acute rejection.

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice.

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in microl h(-1) cm(-2), n = 9 mean+/-s.e.): 4.3+/-0.6, wildtype mice (MCE); 3.5+/-0.6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in microl h(-1) cm(-2)) induced by a 100 mOsm sucrose gradient across MCE cell layers (8.7+/-0.6, n = 8) was significantly greater than that across KMCE (5.7+/-0.7, n = 6, p = 0.007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in microm sec(-1): 74+/-4, n = 19 vs. 44+/-4 microm sec(-1), n = 11, p < 0.001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99+/-1, n = 19 vs. KMCE: 64+/-5, n = 11, p < 0.001). Thus, as in other 'low rate' fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.