Deficiency of the innate immune adaptor STING promotes autoreactive T cell expansion in NOD mice.

AIMS/HYPOTHESIS: Stimulator of IFN genes (STING) is a central hub for cytosolic nucleic acid sensing and its activation results in upregulation of type I IFN production in innate immune cells. A type I IFN gene signature seen before the onset of type 1 diabetes has been suggested as a driver of disease initiation both in humans and in the NOD mouse model. A possible source of type I IFN is through activation of the STING pathway. Recent studies suggest that STING also has antiproliferative and proapoptotic functions in T cells that are independent of IFN. To investigate whether STING is involved in autoimmune diabetes, we examined the impact of genetic deletion of STING in NOD mice. METHODS: CRISPR/Cas9 gene editing was used to generate STING-deficient NOD mice. Quantitative real-time PCR was used to assess the level of type I IFN-regulated genes in islets from wild-type and STING-deficient NOD mice. The number of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214-specific CD8+ T cells was determined by magnetic bead-based MHC tetramer enrichment and flow cytometry. The incidence of spontaneous diabetes and diabetes after adoptive transfer of T cells was determined. RESULTS: STING deficiency partially attenuated the type I IFN gene signature in islets but did not suppress insulitis. STING-deficient NOD mice accumulated an increased number of IGRP206-214-specific CD8+ T cells (2878 ± 642 cells in NOD.STING-/- mice and 728.8 ± 196 cells in wild-type NOD mice) in peripheral lymphoid tissue, associated with a higher incidence of spontaneous diabetes (95.5% in NOD.STING-/- mice and 86.2% in wild-type NOD mice). Splenocytes from STING-deficient mice rapidly induced diabetes after adoptive transfer into irradiated NOD recipients (median survival 75 days for NOD recipients of NOD.STING-/- mouse splenocytes and 121 days for NOD recipients of NOD mouse splenocytes). CONCLUSIONS/INTERPRETATION: Data suggest that sensing of endogenous nucleic acids through the STING pathway may be partially responsible for the type I IFN gene signature but not autoimmunity in NOD mice. Our results show that the STING pathway may play an unexpected intrinsic role in suppressing the number of diabetogenic T cells.

IL-17F induces inflammation, dysfunction and cell death in mouse islets.

Type 17 immune responses, typified by the production of the cytokines IL-17A and IL-17F, have been implicated in the development of type 1 diabetes in animal models and human patients, however the underlying pathogenic mechanisms have not been clearly elucidated. While previous studies show that IL-17A enhances inflammatory gene expression and cell death in mouse ß-cells and human islets, the function of IL-17F in pancreatic ß-cells is completely untested to date. Here we show that IL-17F exhibits potent pathogenic effects in mouse ß-cell lines and islets. IL-17F signals via the IL-17RA and -RC subunits in ß-cells and in combination with other inflammatory cytokines induces expression of chemokine transcripts, suppresses the expression of ß-cell identity genes and impairs glucose stimulated insulin secretion. Further IL-17F induces cell death in primary mouse islets. This occurs via Jnk, p38 and NF-κB dependent induction of Nos2 and is completely ablated in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Together these data indicate that IL-17F possesses similar pathogenic activities to IL-17A in mouse ß-cell lines and islets and is likely to be a type 17 associated pathogenic factor in type 1 diabetes.

Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

IL-21 regulates SOCS1 expression in autoreactive CD8+ T cells but is not required for acquisition of CTL activity in the islets of non-obese diabetic mice.

In type 1 diabetes, maturation of activated autoreactive CD8+ T cells to fully armed effector cytotoxic T lymphocytes (CTL) occurs within the islet. At present the signals required for the maturation process are poorly defined. Cytokines could potentially provide the necessary "third signal" required to generate fully mature CTL capable of killing insulin-producing ß-cells. To determine whether autoreactive CTL within islets respond to cytokines we generated non-obese diabetic (NOD) mice with a reporter for cytokine signalling. These mice express a reporter gene, hCD4, under the control of the endogenous regulatory elements for suppressor of cytokine signalling (SOCS)1, which is itself regulated by pro-inflammatory cytokines. In NOD mice, the hCD4 reporter was expressed in infiltrated islets and the expression level was positively correlated with the frequency of infiltrating CD45+ cells. SOCS1 reporter expression was induced in transferred ß-cell-specific CD8+ 8.3T cells upon migration from pancreatic draining lymph nodes into islets. To determine which cytokines induced SOCS1 promoter activity in islets, we examined hCD4 reporter expression and CTL maturation in the absence of the cytokine receptors IFNAR1 or IL-21R. We show that IFNAR1 deficiency does not confer protection from diabetes in 8.3 TCR transgenic mice, nor is IFNAR1 signalling required for SOCS1 reporter upregulation or CTL maturation in islets. In contrast, IL-21R-deficient 8.3 mice have reduced diabetes incidence and reduced SOCS1 reporter activity in islet CTLs. However IL-21R deficiency did not affect islet CD8+ T cell proliferation or expression of granzyme B or IFNγ. Together these data indicate that autoreactive CD8+ T cells respond to IL-21 and not type I IFNs in the islets of NOD mice, but neither IFNAR1 nor IL-21R are required for islet intrinsic CTL maturation.

IL-21 promotes CD8+ CTL activity via the transcription factor T-bet.

CD8(+) T cells are fundamental for immune-mediated clearance of viral infections and contribute to immune pathology in autoimmune diseases such as type 1 diabetes. To execute these functions, CD8(+) T cells must differentiate into CTLs, a process that is precisely regulated by a variety of cytokines, costimulatory molecules, and transcription factors. IL-21 is an IL-2 family cytokine and a growth factor for multiple lymphocyte effector lineages, including cytotoxic CD8(+) T cells. Recent studies demonstrate that loss of IL-21 signaling results in reduced viral clearance in models of lymphocytic choriomeningitis virus infection, and also protection from type 1 diabetes in the NOD model. This is most likely the result of impaired CD8(+) CTL function in the absence of IL-21 signaling. Currently, the mechanisms by which IL-21 promotes CTL differentiation in CD8(+) T cells remain unclear, particularly the identity of the relevant transcription factor(s). We show that IL-21 promotes CTL function in vitro and killing of pancreatic islets in vivo via the use of transgenic mice expressing IL-21 in pancreatic ß cells. We demonstrate that IL-21 induces the expression of the transcription factor T-bet in CD8(+) T cells, predominantly via STAT1, and that T-bet is required for the induction of cytolytic molecules, including perforin and granzyme B in response to IL-21. Finally, we show that IL-21-induced CTL function is T-bet dependent, as T-bet deficiency results in defective IL-21-dependent cytotoxicity in CD8(+) T cells in vitro and in vivo. Thus, IL-21 drives CD8(+) CTL differentiation via the actions of the transcription factor T-bet.

Use of gentamicin-collagen sponges in closure of sternal wounds in cardiothoracic surgery to reduce wound infections.

A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was: Are antibiotic implants like gentamicin-collagen implants useful in preventing sternal wound infections (SWIs)? Altogether, more than 484 papers were found using the reported search; of these, 7 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. We conclude that most randomized controlled trials (RCTs) have been performed with gentamicin-collagen sponges for sternal closure. Two out of four RCTs showed a significant benefit of the implant in a reduction in superficial and deep SWIs in routine and emergency cardiac surgery. One RCT showed a significant reduction in superficial infections in 2005, a follow-up trial by the same group in 2009 showed a reduction in deep infections as well. Another group has shown a reduction in deep and superficial SWI with gentamicin implant, in an RCT on 800 patients, however have not published details of the complete trial. The third trial on 542 patients showed no benefit of the implant, but was not adequately powered. However, the most recent multicentre RCT conducted on 1052 patients showed no benefit of gentamicin-collagen sponges in elective surgery (coronary artery bypass grafting and/or valve surgery) in high-risk patients with diabetes, obesity or both. Concerns were raised that gentamicin sponges dipped in saline 1-2 s prior to application may have lost the gentamicin into the saline, thereby reducing their efficacy and that some surgeons may have been unfamiliar with wound closure with sponges. However, these were robustly refuted by the authors. One RCT showed that gentamicin sponges may cause increased sternal rebleeding if used in double layers. Coagulase-negative staphylococci were predominantly isolated from infected wounds in all the trials except the one from USA, where infections were polymicrobial. Gentamicin resistance among common pathogens causing SWIs can vary from 15 to 80%. Gentamicin-collagen is unlikely to reduce SWIs in high-risk patients and polymicrobial infections.

Overexpression of the Ctla-4 isoform lacking exons 2 and 3 causes autoimmunity.

CTLA-4 is a potent inhibitor of T cell activation, primarily upon binding to its costimulatory ligands (B7.1 and B7.2) expressed on APCs. However, variants of CTLA-4 can also function independently of B7 molecules. 1/4CTLA-4 is a highly conserved isoform encoded by exons 1 and 4 of the Ctla4 gene that lacks the ligand-binding and the transmembrane domains, and as yet, its function is not known. To investigate the function of 1/4CTLA-4, we generated transgenic (Tg) mice overexpressing this variant. Cytokine production by 1/4CTLA-4 Tg T cells was elevated compared with wild type T cells. The frequency of CD44(high) memory T cells in 1/4CTLA-4 Tg mice was increased, and as the mice aged, the frequency further increased. 1/4CTLA-4 Tg mice >1 y old had increased expression of T cell activation markers and developed spontaneous autoimmunity, including elevated production of autoantibodies. In contrast with young 1/4CTLA-4 Tg mice, aged 1/4CTLA-4 Tg mice had elevated frequencies of Foxp3(+) regulatory T cells, but the regulatory T cells from these mice were not able to inhibit colitis development. Collectively, these data suggest that the function of the 1/4CTLA-4 isoform is distinct from that of CTLA-4 in that it enhances T cell activation and promotes autoimmunity rather than inhibiting immune responses.

Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis.

Zbtb20 is a member of the POK family of proteins, which function primarily as transcriptional repressors via interactions mediated by their conserved C(2)H(2) Krüppel type zinc finger and BTB/POZ domains. To define the function of Zbtb20 in vivo, we generated knockout mice by homologous recombination. Zbtb20 null mice display a stark phenotype characterized by postnatal growth retardation, metabolic dysfunction, and lethality. Zbtb20 knockout mice displayed abnormal glucose homeostasis, hormonal responses, and depletion of energy stores, consistent with an energetic deficit. Additionally, increased serum bilirubin and alanine aminotransferase levels were suggestive of liver dysfunction. To identify potential liver-specific Zbtb20 target genes, we performed transcript profiling studies on liver tissue from Zbtb20 knockout mice and wild-type littermate controls. These studies identified sets of genes involved in growth, metabolism, and detoxification that were differentially regulated in Zbtb20 knockout liver. Transgenic mice expressing Zbtb20 in the liver were generated and crossed onto the Zbtb20 knockout background, which resulted in no significant normalization of growth or glucose metabolism but a significant increase in life span compared to controls. These data indicate that the phenotype of Zbtb20 knockout mice results from liver-dependent and -independent defects, suggesting that Zbtb20 plays nonredundant roles in multiple organ systems.

Interleukin-21 is required for the development of type 1 diabetes in NOD mice.

OBJECTIVE: Interleukin (IL)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic (NOD) mouse strain. The aim of this study was to investigate a causal role for IL-21 in type 1 diabetes. RESEARCH DESIGN AND METHODS: We generated IL-21R-deficient NOD mice and C57Bl/6 mice expressing IL-21 in pancreatic beta-cells, allowing the determination of the role of insufficient and excessive IL-21 signaling in type 1 diabetes. RESULTS: Deficiency in IL-21R expression renders NOD mice resistant to insulitis, production of insulin autoantibodies, and onset of type 1 diabetes. The lymphoid compartment in IL-21R-/- NOD is normal and does not contain an increased regulatory T-cell fraction or diminished effector cytokine responses. However, we observed a clear defect in autoreactive effector T-cells in IL-21R-/- NOD by transfer experiments. Conversely, overexpression of IL-21 in pancreatic beta-cells induced inflammatory cytokine and chemokines, including IL-17A, IL17F, IFN-gamma, monocyte chemoattractant protein (MCP)-1, MCP-2, and interferon-inducible protein-10 in the pancreas. The ensuing leukocytic infiltration in the islets resulted in destruction of beta-cells and spontaneous type 1 diabetes in the normally diabetes-resistant C57Bl/6 and NOD x C57Bl/6 backgrounds. CONCLUSIONS: This work provides demonstration of the essential prodiabetogenic activities of IL-21 on diverse genetic backgrounds (NOD and C57BL/6) and indicates that IL-21 blockade could be a promising strategy for interventions in human type 1 diabetes.

Development of nephritis but not sialadenitis in autoimmune-prone BAFF transgenic mice lacking marginal zone B cells.

B cell-activating factor belonging to the TNF family (BAFF) is a B cell survival factor required for B cell maturation. BAFF transgenic (Tg) mice develop autoimmune disorders characterized by autoantibody production, which leads to nephritis and salivary gland destruction (sialadenitis), features reminiscent of systemic lupus erythematosus and Sjögren's syndrome (SS), respectively. Disease in BAFF Tg mice correlates with the expansion of the marginal zone (MZ) B cell compartment and the abnormal presence of MZ-like B cells in the blood, LN and inflamed salivary glands, suggesting a role for these cells in BAFF-induced autoimmunity. Lymphotoxin-beta (LTbeta)-deficient mice show disrupted splenic architecture, lack MZ B cells and some peripheral LN, and are unable to mount T cell-dependent immune responses. BAFF Tg mice lacking LTbeta (LTbetaDelta-BTg) retained these defects, yet still developed nephritis associated with the presence of B-1 B cells in the kidneys. However, in contrast to old BAFF Tg mice, aging LTbetaDelta-BTg mice no longer developed sialadenitis. Thus, autoimmune disorders in BAFF Tg mice are possibly events coordinated by MZ and B-1 B cells at separate anatomical sites.

Targeting BAFF: immunomodulation for autoimmune diseases and lymphomas.

In an effort to develop more effective treatments for inflammatory diseases, immunologists have targeted numerous molecular pathways, but with limited success. Notable exceptions are anti-TNF agents, which have proved efficacious in a proportion of rheumatoid arthritis (RA) patients. Another TNF family member, termed BAFF ("B cell-activating factor belonging to the TNF family"), plays a central role in autoimmune diseases, as well as in B cell maturation, survival, and T cell activation. Agents that block BAFF have proven to be highly effective in the treatment of certain autoimmune conditions in mice. In addition, phase II data in human clinical trials for RA appear very promising. BAFF is also a survival factor for certain B cell lymphomas. Despite the relatively recent identification of BAFF, this molecule has provided considerable new insight into B cell homeostasis and immune function, and represents an important new molecular target for treatment of autoimmune diseases and lymphomas.

BAFF costimulation of Toll-like receptor-activated B-1 cells.

B cell-activating factor belonging to the TNF family (BAFF) and its receptor BAFF-R play critical roles in the maturation and survival of conventional peripheral B cells. However, they appeared to be dispensable for the generation and maintenance of CD5(+) B-1 cells as BAFF(-/-) and BAFF-R(-/-) mice have normal B-1 cell populations. Hence, it is presently unclear if B-1 cells are responsive to BAFF and if BAFF regulates some aspects of B-1 cell function. We show here that BAFF-R and transmembrane activator and CAML interactor (TACI) are the major receptors expressed by B-1 cells. Specifically, we show that BAFF treatment of B-1 cells leads to increased NF-kappaB p100 processing and CD21/CD35 expression. Interestingly, toll-like receptor (TLR) engagement of B-1 cells augmented the surface expression of BAFF receptors and rendered them responsive to BAFF costimulation, as evidenced by their increased proliferation, expression of cell surface activation markers and secretion of the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10. This costimulatory effect is achieved primarily through BAFF-R as BAFF failed to costimulate B-1 cells obtained from A/WySnJ mice which have defective BAFF-R signaling. Thus, as TLR are innate immune receptors and B-1 cells are "innate-like" lymphocytes, our data provide evidence that BAFF plays a role in innate immunity.

Plasminogen activator inhibitor-2 is highly tolerant to P8 residue substitution--implications for serpin mechanistic model and prediction of nsSNP activities.

The serine protease inhibitor (serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis, inflammation, metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of serpins through saturation mutagenesis of the P8 residue for a typical family member, plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the serpin inhibitory mechanism. The importance of this residue for inhibition of the PAI-2 protease target urinary plasminogen activator (urokinase, uPA) is confirmed, although a high degree of tolerance to P8 substitution is observed. Out of 19 possible PAI-2 P8 mutants, 16 display inhibitory activities within an order of magnitude of the wild-type P8 Thr species. Crystal structures of complexes between PAI-2 and RCL-mimicking peptides with P8 Met or Asp mutations are determined, and structural comparison with the wild-type complex substantiates the ability of the S8 pocket to accommodate disparate side-chains. These data indicate that the identity of the P8 residue is not a determinant of efficient RCL insertion, and provide further evidence for functional plasticity of key residues within enzyme structures. Poor correlation of observed PAI-2 P8 mutant activities with a range of physicochemical, evolutionary and thermodynamic predictive indices highlights the practical limitations of existing approaches to predicting the molecular phenotype of protein variants.

BAFF augments certain Th1-associated inflammatory responses.

B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.

B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells.

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using specific mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti- micro responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells.