Glucagon-like peptide-1 therapy in people with obesity restores natural killer cell metabolism and effector function.

OBJECTIVE: People with obesity (PWO) have functionally defective natural killer (NK) cells, with a decreased capacity to produce cytokines and kill target cells, underpinned by defective cellular metabolism. It is plausible that the changes in peripheral NK cell activity are contributing to the multimorbidity in PWO, which includes an increased risk of cancer. This study investigated whether therapy with long-acting glucagon-like peptide-1 (GLP-1) analogues, which are an effective treatment for obesity, could restore NK cell functionality in PWO. METHODS: In a cohort of 20 PWO, this study investigated whether 6 months of once weekly GLP-1 therapy (semaglutide) could restore human NK cell function and metabolism using multicolor flow cytometry, enzyme-linked immunosorbent assays, and cytotoxicity assays. RESULTS: These data demonstrate that PWO who received GLP-1 therapy have improved NK cell function, as measured by cytotoxicity and interferon-γ/granzyme B production. In addition, the study demonstrates increases in a CD98-mTOR-glycolysis metabolic axis, which is critical for NK cell cytokine production. Finally, it shows that the reported improvements in NK cell function appear to be independent of weight loss. CONCLUSIONS: The restoration, by GLP-1 therapy, of NK cell functionality in PWO may be contributing to the overall benefits being seen with this class of medication.

Heightened metabolic responses in NK cells from patients with neuroblastoma suggests increased potential for immunotherapy.

High risk neuroblastoma is responsible for 15% of deaths in pediatric cancer patients. The introduction of anti-GD2 immunotherapy has significantly improved outcomes but there is still only approximately a 50% 5 year event-free-survival for these children and improvements in treatments are urgently required. Anti-GD2 immunotherapy uses the patients' own immune system to kill cancer cells. In particular, Natural Killer (NK) cells kill antibody coated tumor cells by a process called antibody dependent cellular cytotoxicity (ADCC). However, our previous work has highlighted metabolic exhaustion of NK cells in circulating blood of adult cancer patients, identifying this as a potential therapeutic target. In this study, we investigated circulating NK cells in patients newly diagnosed with neuroblastoma. We found evidence of activation of NK cells in vivo by the cancer itself. While some evidence of NK cell dysfunction was observed in terms of IFNγ production, most results indicated that the NK cell compartment remained relatively intact. In fact, some aspects of metabolic and functional activities were actually increased in patients compared to controls. Glycolytic responses, which we show are crucial for ADCC, were actually enhanced in patients and CD16, the NK cell receptor that mediates ADCC, was also expressed at high levels in some patients. Overall, the data suggest that patient NK cells could be harvested at diagnosis for subsequent beneficial autologous use during immunotherapy. Enhancing glycolytic capacity of cell therapies could also be a strategic goal of future cell therapies for patients with neuroblastoma and indeed other cancers.

Increased extracellular vesicles mediate inflammatory signalling in cystic fibrosis.

RATIONALE: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways. OBJECTIVES: To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF. METHODS: EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs. RESULTS: A significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001-0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls. CONCLUSION: This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

Nucleic acid cytokine responses in obese children and infants of obese mothers.

Almost a third of Irish children are now overweight and the country ranks 58th out of 200 countries for its proportion of overweight youths. With the rising obesity epidemic, and the impaired immune responses of this population, it is vital to understand the effects that obesity has on the immune system and to design future therapeutics, adjuvants and vaccines with overweight and obese populations in mind. Many current vaccines use adjuvants that have been found to be less effective at stimulating the immune response in children compared with adults and there is now substantial effort to design paediatric-focused adjuvants. Additionally, vaccine responses have been shown to be less effective in obese populations indicating that this is a particularly vulnerable population. We have recently identified cytosolic nucleic acids (CNAs), as novel candidate adjuvants for childhood vaccines. Here we investigated whether immune responses to these candidate adjuvants were adversely affected in infants born to overweight or obese mothers, and in overweight and obese children. Type I Interferon (IFN) and proinflammatory cytokines such as Tumor Necrosis Factor α (TNFα) are vital for driving innate and adaptive immune responses. We found that childhood obesity conferred no significant adverse effect on CNA-induced Type I IFN responses when compared with lean children. Similarly, Type I IFN responses were intact in the cord blood of babies delivered from overweight and obese mothers, when compared with lean mothers. There was also no significant impact of obesity on CNA-induced TNFα responses in children or from cord blood of infants born to overweight/obese mothers. In all cases, there was a tendency towards decreased production of innate cytokine Type I Interferon and TNFα, however there was no significant negative correlation. Interestingly, high maternal BMI showed weak and moderate positive correlation with IL-12p70 and IFNγ, respectively, in response to CNA stimulation. This study demonstrates that future adjuvants can be tailored for these populations through the use of activators of CNA sensors.

Engagement of Fas differentially regulates the production of LPS-induced proinflammatory cytokines and type I interferons.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype.

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production. Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells. ULBP2 expression was however linked to expression of mature CD57(+) NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated "mature" NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.

IL-18 Immunotherapy for Neovascular AMD: Tolerability and Efficacy in Nonhuman Primates.

PURPOSE: Age-related macular degeneration is the most common form of central retinal blindness in the elderly. Of the two end stages of disease, neovascular AMD-although the minority form-is the most severe. Current therapies are highly successful at controlling progression of neovascular lesions; however, a significant number of patients remain refractory to treatment and the development of alternative and additive therapies to anti-VEGFs is essential. METHODS: In order to address the translational potential of interleukin (IL)-18 for use in neovascular AMD, we initiated a nonhuman primate tolerability and efficacy study for the use of intravitreally (IVT) administered clinical grade human IL-18 (SB-485232). Cynomolgus monkeys were injected IVT with increasing doses of human IL-18 (two each at 1000, 3000, and 10,000 ng per eye). In tandem, 21 monkeys were administered nine laser burns in each eye prior to receiving IL-18 as an IVT injection at a range of doses. Fundus fluorescein angiography (FFA) was performed on days 8, 15, and 22 post injection and the development of neovascular lesions was assessed. RESULTS: We show intravitreal, mature, recombinant human IL-18 is safe and can reduce choroidal neovascular lesion development in cynomolgus monkeys. CONCLUSIONS: Based on our data comparing human IL-18 to current anti-VEGF-based therapy, clinical deployment of IL-18 for neovascular AMD has the potential to lead to a new adjuvant immunotherapy-based treatment for this severe form of central blindness.

IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common form of central retinal blindness globally. Distinct processes of the innate immune system, specifically activation of the NLRP3 inflammasome, have been shown to play a central role in the development of both "dry" and neovascular ("wet") forms of the disease. We show that the inflammatory cytokine interleukin-18 (IL-18) can regulate choroidal neovascularization formation in mice. We observed that exogenous administration of mature recombinant IL-18 has no effect on retinal pigment epithelial (RPE) cell viability, but that overexpression of pro-IL-18 or pro-IL-1ß alone can cause RPE cell swelling and subsequent atrophy, a process that can be inhibited by the promotion of autophagy. A direct comparison of local and systemic administration of mature recombinant IL-18 with current anti-VEGF (vascular endothelial growth factor)-based therapeutic strategies shows that IL-18 treatment works effectively alone and more effectively in combination with anti-VEGF therapy and represents a novel therapeutic strategy for the treatment of wet AMD.

Btk regulates macrophage polarization in response to lipopolysaccharide.

Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\-)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/-) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/-) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/-) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/-) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor ß-activated kinase 1 (TAK1) recruitment.

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Absence of SHIP-1 results in constitutive phosphorylation of tank-binding kinase 1 and enhanced TLR3-dependent IFN-beta production.

Autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, result from a loss of tolerance to self-antigens and immune-mediated injury precipitated by the overproduction of type I IFN and inflammatory cytokines. We have identified the inositol 5' phosphatase SHIP-1 as a negative regulator of TLR3-induced type I IFN production. SHIP-1-deficient macrophages display enhanced TLR-induced IFN-beta production, and overexpression of SHIP-1 negatively regulates the ability of TLR3 and its adaptor, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta, to induce IFN-beta promoter activity, indicating that SHIP-1 negatively regulates TLR-induced IFN-beta production. Further dissection of the IFN-beta pathway implicates TANK-binding kinase 1 (TBK1) as the target for SHIP-1. Critically, in the absence of SHIP-1, TBK1 appears to be hyperphosphorylated both in unstimulated cells and following TLR3 stimulation. In addition, TBK1 appears to be constitutively associated with Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta and TNFR-associated factor 3 in SHIP-1 deficient cells, whereas in wild-type cells this association is inducible following TLR3 stimulation. In support of a role for SHIP-1 in regulating complex formation, confocal microscopy demonstrates that TBK1 distribution in the cell is significantly altered in SHIP-1-deficient cells, with more prominent endosomal staining observed, compared with wild-type controls. Taken together, our results point to SHIP-1 as a critical negative regulator of IFN-beta production downstream of TLR3 through the regulation of TBK1 localization and activity.

Proteomic analysis of protein complexes in Toll-like receptor biology.

Purification of protein complexes and identification of the constituent components therein have been made relatively simple by the recent advances in proteomics. Uniting good biochemical and protein chemistry techniques with protein identification by mass spectrometry (MS) has resulted in advances in this field that are unprecedented. Our knowledge of Toll-like receptor (TLR) biology has been considerably advanced through the use of such techniques, with key intermediates such as TRAF3, TANK, RIP1 all being identified using proteomic strategies. Applying these techniques to key questions in TLR -biology will undoubtedly serve to further advance the field.