RELA governs a network of islet-specific metabolic genes necessary for beta cell function.

AIMS/HYPOTHESIS: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. METHODS: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (ßp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (ßNEMOKO mice), as well as ßA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. RESULTS: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as ßp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in ßp65KO islets. CONCLUSIONS/INTERPRETATION: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.

The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation.

BACKGROUND: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. METHODS: We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. RESULTS: Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation. CONCLUSION: We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.

Spatial and Temporal Control of CRISPR-Cas9-Mediated Gene Editing Delivered via a Light-Triggered Liposome System.

The CRISPR-Cas9 and related systems offer a unique genome-editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with the CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with the CRISPR-Cas9 system. We demonstrated its efficient protein release by respectively assessing the targeted knockout of the eGFP gene in human HEK293/GFP cells and the TNFAIP3 gene in TNFα-induced HEK293 cells. We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). These findings indicate that light-triggered liposomes may have new options for precise control of CRISPR-Cas9 release and editing.

A zebrafish functional genomics model to investigate the role of human A20 variants in vivo.

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.

A20 as an immune tolerance factor can determine islet transplant outcomes.

Islet transplantation can restore lost glycemic control in type 1 diabetes subjects but is restricted in its clinical application by a limiting supply of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signaling thresholds. Here, we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for ~45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon Tregs and was antigen specific, and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signaling, graft hyperinflammation, and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators, with no impact on glucose-stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signaling thresholds to favor immune tolerance and promotes islet allogeneic survival. Clinically, this would allow for reduced immunosuppression and support the use of alternate islet sources.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

ß Cell Replacement Therapy: The Next 10 Years.

ß cell replacement with either pancreas or islet transplantation has progressed immensely over the last decades with current 1- and 5-year insulin independence rates of approximately 85% and 50%, respectively. Recent advances are largely attributed to improvements in immunosuppressive regimen, donor selection, and surgical technique. However, both strategies are compromised by a scarce donor source. Xenotransplantation offers a potential solution by providing a theoretically unlimited supply of islets, but clinical application has been limited by concerns for a potent immune response against xenogeneic tissue. ß cell clusters derived from embryonic or induced pluripotent stem cells represent another promising unlimited source of insulin producing cells, but clinical application is pending further advances in the function of the ß cell like clusters. Exciting developments and rapid progress in all areas of ß cell replacement prompted a lively debate by members of the young investigator committee of the International Pancreas and Islet Transplant Association at the 15th International Pancreas and Islet Transplant Association Congress in Melbourne and at the 26th international congress of The Transplant Society in Hong Kong. This international group of young investigators debated which modality of ß cell replacement would predominate the landscape in 10 years, and their arguments are summarized here.

Equipping the islet graft for self defence: targeting nuclear factor κB and implications for tolerance.

PURPOSE OF REVIEW: Clinical islet transplantation does not enjoy the success seen for solid organ transplants, indicating a need for new therapeutic approaches to improve patient outcomes. This has prompted investigation into islet autonomous factors and pathways that may represent druggable targets. These have the potential to synergize with approaches aimed at generating graft-specific tolerance. RECENT FINDINGS: There are emerging data that nuclear factor κB (NF-κB) activation can prevent and or overcome tolerance, whereas dampening NF-κB activation in immune cells is associated with prolonged allograft survival. In islet cells, NF-κB plays a central role in triggering the inflammatory transcriptional response that is often associated with reduced islet function and contributes to poor transplant outcomes. SUMMARY: Targeting intraislet NF-κB represents a promising target in islet transplantation. Here we will discuss the current state of the knowledge on the role of NF-κB activation in the context of islet transplantation and the implications of targeting NF-κB for tolerance induction.

MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD.

Influenza A virus (IAV) infections lead to severe inflammation in the airways. Patients with chronic obstructive pulmonary disease (COPD) characteristically have exaggerated airway inflammation and are more susceptible to infections with severe symptoms and increased mortality. The mechanisms that control inflammation during IAV infection and the mechanisms of immune dysregulation in COPD are unclear. We found that IAV infections lead to increased inflammatory and antiviral responses in primary bronchial epithelial cells (pBECs) from healthy nonsmoking and smoking subjects. In pBECs from COPD patients, infections resulted in exaggerated inflammatory but deficient antiviral responses. A20 is an important negative regulator of NF-κB-mediated inflammatory but not antiviral responses, and A20 expression was reduced in COPD. IAV infection increased the expression of miR-125a or -b, which directly reduced the expression of A20 and mitochondrial antiviral signaling (MAVS), and caused exaggerated inflammation and impaired antiviral responses. These events were replicated in vivo in a mouse model of experimental COPD. Thus, miR-125a or -b and A20 may be targeted therapeutically to inhibit excessive inflammatory responses and enhance antiviral immunity in IAV infections and in COPD.

Targeted deletion of Traf2 allows immunosuppression-free islet allograft survival in mice.

AIMS/HYPOTHESIS: Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. METHODS: T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. RESULTS: Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. CONCLUSION/INTERPRETATION: Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

Nuclear factor κB-inducing kinase activation as a mechanism of pancreatic ß cell failure in obesity.

The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic ß cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB-inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of ß cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive ß cell-intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to ß cell failure. These studies reveal that NIK contributes a central mechanism for ß cell failure in diet-induced obesity.

TRAF2 regulates peripheral CD8(+) T-cell and NKT-cell homeostasis by modulating sensitivity to IL-15.

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15.

Emerging roles for A20 in islet biology and pathology.

A20 is most characteristically described in terms relating to inflammation and inflammatory pathologies. The emerging understanding of inflammation in the etiology of diabetes mellitus lays the framework for considering a central role for A20 in this disease process. Diabetes mellitus is considered a major health issue, and describes a group of common metabolic disorders pathophysiologically characterized by hyperglycemia. Within islets of Langherhans, the endocrine powerhouse of the pancreas, are the insulin-producing pancreatic beta-cells. Loss of beta-cell mass and function to inflammation and apoptosis is a major contributing factor to diabetes. Consequently, restoring functional beta-cell mass via transplantation represents a therapeutic option for diabetes. Unfortunately, transplanted islets also suffers from loss of beta-cell function and mass fueled by a multifactorial inflammatory cycle triggered by islet isolation prior to transplantation, the ischemic environment at transplantation as well as allogeneic or recurrent auto-immune responses. Activation of the transcription factor NF-kappaB is a central mediator of inflammatory mediated beta-cell dysfunction and loss. Accordingly, a plethora of strategies to block NF-kappaB activation in islets and hence limit beta-cell loss have been explored, with mixed success. We propose that the relatively poor efficacy of NF-kappaB blockade in beta-cells is due to concommittant loss of the important, NF-kappaB regulated anti-apoptotic and anti-inflammatory protein A20. A20 has been identified as a beta-cell expressed gene, raising questions about its role in beta-cell development and function, and in beta-cell related pathologies. Involvement of apoptosis, inflammation and NF-kappaB activation as beta-cell factors contributing to the pathophysiology of diabetes, coupled with the knowledge that beta-cells express the A20 gene, implies an important role for A20 in both normal beta-cell biology as well as beta-cell related pathology. Genome wide association studies (GWAS) linking single nucleotide polymorphisms in the A20 gene with the occurrence of diabetes and its complications support this hypothesis. In this chapter we review data supporting the role of A20 in beta-cell health and disease. Furthermore, by way of their specialized function in metabolism, pancreatic beta-cells also provide opportunities to explore the biology of A20 in scenarios beyond inflammation.

Acute phase proteins are major clients for the chaperone action of α2-macroglobulin in human plasma.

Extracellular protein misfolding is implicated in many age-related diseases including Alzheimer's disease, macular degeneration and arthritis. In this study, putative endogenous clients for the chaperone activity of α2-macroglobulin (α2M) were identified after human plasma was subjected to physiologically relevant sheer stress at 37 °C for 10 days. Western blot analysis showed that four major acute phase proteins: ceruloplasmin, fibrinogen, α1-acid glycoprotein and complement component 3, preferentially co-purified with α2M after plasma was stressed. Furthermore, the formation of complexes between α2M and these putative chaperone clients, detected by sandwich ELISA, was shown to be enhanced in response to stress. These results support the hypothesis that α2M plays an important role in extracellular proteostasis by sequestering misfolded proteins and targeting them for disposal, particularly during acute phase reactions.

Low-dose rapamycin unmasks the protective potential of targeting intragraft NF-κB for islet transplants.

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 ß-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.

Human islets express a marked proinflammatory molecular signature prior to transplantation.

In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-κB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-κB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1ß and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG(-/-) recipients. Thus, human islets can provide a reservoir of NF-κB-dependent inflammatory factors that have the potential to contribute to the anti-islet-graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-κB, and transplanted them into allogenic recipients. These NF-κB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-κB-activated islets were rejected with a faster tempo as compared to non-NF-κB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.