Administration Of IL-2 Research Articles

IL-33 Alleviated Brain Damage via Anti-apoptosis, Endoplasmic Reticulum Stress, and Inflammation After Epilepsy.

Interleukin (IL)-33 belongs to a novel chromatin-associated cytokine newly recognized by the IL-1 family, and its specific receptor is the orphan IL-1 receptor (ST2). Cumulative evidence suggests that IL-33 plays a crucial effect on the pathological changes and pathogenesis of central nervous system (CNS) diseases and injuries, such as recurrent neonatal seizures (RNS). However, the specific roles of IL-33 and its related molecular mechanisms in RNS remain confused. In the present study, we investigated the protein expression changes and co-localized cell types of IL-33 or ST2, as well as the effect of IL-33 on RNS-induced neurobehavioral defects, weight loss, and apoptosis. Moreover, an inhibitor of IL-33, anti-IL-33 was performed to further exploited underlying mechanisms. We found that administration of IL-33 up-regulated the expression levels of IL-33 and ST2, and increased the number of its co-localization with Olig-2-positive oligodendrocytes and NeuN-positive neurons at 72 h post-RNS. Noteworthily, RNS-induced neurobehavioral deficits, bodyweight loss, and spatial learning and memory impairment, as well as cell apoptosis, were reversed by IL-33 pretreatment. Additionally, the increase in IL-1β and TNF-α levels, up-regulation of ER stress, as well as a decrease in anti-apoptotic protein Bcl-2 and an increase in pro-apoptotic protein CC-3 induced by RNS are prevented by administration of IL-33. Moreover, IL-33 in combination with Anti-IL-33 significantly inverted the effects of IL-33 or Anti-IL-33 alone on apoptosis, ER stress, and inflammation. Collectively, these data suggest that IL-33 attenuates RNS-induced neurobehavioral disorders, bodyweight loss, and spatial learning and memory deficits, at least in part through mechanisms involved in inhibition of apoptosis, ER stress, and neuro-inflammation.

Anti-inflammatory IL-10 administration rescues depression-associated learning and memory deficits in mice

BackgroundMajor depressive disorder is a widespread mood disorder. One of the most debilitating symptoms patients often experience is cognitive impairment. Recent findings suggest that inflammation is associated with depression and impaired cognition. Pro-inflammatory cytokines are elevated in the blood of depressed patients and impair learning and memory processes, suggesting that an anti-inflammatory approach might be beneficial for both depression and cognition.MethodsWe subjected mice to the learned helplessness paradigm and evaluated novel object recognition and spatial memory. Mice were treated with IL-10 intranasally or/and microglia cells were depleted using PLX5622. Statistical differences were tested using ANOVA or t tests.ResultsWe first established a mouse model of depression in which learning and memory are impaired. We found that learned helplessness (LH) impairs novel object recognition (NOR) and spatial working memory. LH mice also exhibit reduced hippocampal dendritic spine density and increased microglial activation compared to non-shocked (NS) mice or mice that were subjected to the learned helpless paradigm but did not exhibit learned helplessness (non-learned helpless or NLH). These effects are mediated by microglia, as treatment with PLX5622, which depletes microglia, restores learning and memory and hippocampal dendritic spine density in LH mice. However, PLX5622 also impairs learning and memory and reduces hippocampal dendritic spine density in NLH mice, suggesting that microglia in NLH mice produce molecules that promote learning and memory. We found that microglial interleukin (IL)-10 levels are reduced in LH mice, and IL-10 administration is sufficient to restore NOR, spatial working memory, and hippocampal dendritic spine density in LH mice, and in NLH mice treated with PLX5622 consistent with a pro-cognitive role for IL-10.ConclusionsAltogether these data demonstrate the critical role of IL-10 in promoting learning and memory after learned helplessness.

Abstract 1075: Conditionally active de novo IL-2 cytokine mimetics for targeted immunotherapy: de novo split technology

Abstract IL-2 is a highly potent, pleiomorphic cytokine approved for the treatment of melanoma and renal cell carcinoma. The therapeutic potential of this cytokine has been restricted by dose-limiting toxicities and off-tumor effects associated with systemic IL-2 administration. Scientists have long-pursued the development of targeted IL-2 variants to control biodistribution and improve the therapeutic index, e.g. IL-2-antibody fusions or immunocytokines. However, the potential benefit of targeting IL-2 is limited by the high affinity of the cytokine for its cellular receptor, which dominates biodistribution and results in activation of cells outside the tumor microenvironment. To address this challenge, we took advantage of the remarkable stability of Neoleukin-2/15 (Neo-2/15), a fully de novo cytokine mimetic that potently activates the beta and gamma subunits shared by the IL-2 and IL-15 receptors. We demonstrate that Neo-2/15 can be divided into two pieces that cannot signal individually, but that can recover their binding and cell-signaling potential when co-localized. Fusing the split molecules to independent targeting domains creates a modular and highly specific implementation of conditional activation. We demonstrate that if one split fragment is targeted to one tumor-associated antigen, and the complementary fragment is targeted to a second tumor-associated antigen, the ability to bind the IL-2 receptor and trigger signaling is only restored when these split fragments bind to the surface of cells expressing both targets. The split Neo-2/15 molecules have anti-tumor activity in syngeneic cancer models and have the potential to enhance the therapeutic index of cytokine mimetics in immunotherapy. To our knowledge, these are the first designed examples of strictly conditionally-active, targeted, cytokine-like molecules for immunotherapy. *Current affiliation for D-A.S.: Neoleukin Therapeutics Inc, Seattle, WA, USA. Citation Format: Alfredo Quijano-Rubio, Lestat R. Ali, Md A. Bhuiyan, Marc J. Lajoie, Michael Dougan, David Baker, Daniel-Adriano Silva. Conditionally active de novo IL-2 cytokine mimetics for targeted immunotherapy: de novo split technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1075.

IL-33 deficiency protects mice from DSS-induced experimental colitis by suppressing ILC2 and Th17 cell responses.

Recently, IL-33-driven ILC2 response has been shown to participate in a variety of diseases. However, IL-33-driven ILC2 immunity has not been extensively characterized in the context of colitis yet. The RAG-2- and IL-33-deficient mice were used to investigate the role and underlying mechanisms of IL-33-driven ILC2 response in the DSS-induced experimental colitis. Body weight, length of colon, and histological analysis were monitored to evaluate the severity of colitis. Proportions of immune cells were examined by flow cytometry. Levels of cytokines were analyzed by ELISA and q-PCR. Administration of exogenous IL-33 aggravated the DSS-induced colitis, which revealed that IL-33 promoted the generation of ILC2 cells to mediate the inflammation of colon. Consistently, this effect was confirmed in RAG-2-deficient mice without T, B cells. Furthermore, IL-33-deficient mice were used to examine the role of endogenous IL-33 on the pathogenesis of DSS-induced colitis. Interestingly, lack of endogenous IL-33 protected the mice from the DSS-induced colitis. The protective effect is associated with impairments of development of ILC2 as well as Th17 cells. Analysis of their cytokine production profiles revealed that IL-33 deficiency resulted in the reduction of cytokines IL-6 and IL-1β as well as IL-10. These results suggest that IL-33/ILC2 axis is a potential therapeutic target for human colitis. Our findings demonstrate that IL-33 deficiency protects mice from DSS-induced colitis. The protective effect is associated with impairments of ILC2 and Th17 cell development as well as reduction of inflammatory cytokines IL-6 and IL-1β.

Corrigendum: Defective Transcriptional Programming of Effector CD8 T Cells in Aged Mice Is Cell-Extrinsic and Can Be Corrected by Administration of IL-12 and IL-18

[This corrects the article DOI: 10.3389/fimmu.2019.02206.].

Interleukin-10 administration inhibits TNF-α and IL-1β, but not IL-6, secretion of LPS-stimulated peritoneal macrophages

We hypothesize that exogenous administration of anti-inflammatory cytokine interleukin (IL)-10 would affect the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, or tumor necrosis factor (TNF)-α using lipopolysaccharide (LPS)-stimulated macrophages exuded from the peritoneal cavity of female BALB/c mice. The LPS-stimulated macrophages were incubated for 48 hr to investigate the secretions of pro- and anti-inflammatory cytokine. The results indicated that the secretion levels of IL-1β, IL-6, IL-10, and TNF-α by LPS-stimulated macrophages elevated in a time dependent manner during 48-hr incubation period. The amount of cytokines secretion by LPS-stimulated macrophages varied: IL-6 > TNF-α > IL-10 > IL-1β. However, the secretion of IL-10 started to decrease at 18 hr of incubation. Therefore, the macrophage cultures were extra-administrated with various concentrations (0, 75, 150, and 225 pg/ mL) of exogenous IL-10 after 18-hr incubation in order to evaluate the effect of exogenous IL-10 administration on inflammation. IL-10, 1L-1β, IL-6, and TNF-α in LPS-stimulated macrophage cultures before and after exogenous IL-10 administration were determined by ELISA. The results demonstrated that exogenous IL-10 administration inhibited IL-1β (21.3-38.6%) and TNF-α (44.7-66.8%) secretion. However, the administration did not inhibit IL-6 secretion. Low dose (75 pg/mL) of exogenous IL-10 exerted maximal effects. IL-10 levels in LPS-stimulated macrophage cultures after exogenous IL-10 administration increased from 18.4% to 35.5%. Furthermore, only low dose of exogenous IL-10 administration increased endogenous IL-10 secretion (by 10.4%), while higher doses of exogenous IL-10 inhibited endogenous IL-10 production (from 31.7% to 41.9%). The results suggest that low dose administration of exogenous IL-10 might exhibit antiinflammatory effects via inhibiting TNF-α and IL-1β, but not IL-6, secretion and increasing endogenous IL-10 production by LPS-stimulated peritoneal macrophages.

Regulation of avian uncoupling protein (av-UCP) expression by cytokines and hormonal signals in quail myoblast cells

Uncoupling proteins (UCPs), members of the mitochondrial anion carrier family, play a pivotal role in thermogenesis, redox balance, reactive oxygen species and many other cellular processes. They were extensively studied in mammalian species and have been shown to be tightly regulated at transcriptional and translational levels by various environmental and hormonal factors. Such studies are very limited in avian species which represent a unique model because they lack brown adipose tissue and they contain only one UCP (av-UCP) predominantly expressed in the muscle. The present study aimed, therefore, to determine the effects of pro-inflammatory cytokines (IL-6 and TNFα) and energy homeostasis-related hormones (leptin and T3) on the expression of av-UCP and its related transcription factors in quail myoblast (QM7) cells.Leptin treatment for 24 h significantly down-regulated av-UCP, and up-regulated PGC-1α, PPARα, and PPARγ expression in QM7 cells. IL-6 and TNFα administration significantly up-regulated the expression of av-UCP, however T3 had a biphasic effects (up-regulation with low dose and down-regulation with high dose) on av-UCP mRNA levels (P < .05). TNFα significantly induced PPARα and PPARγ mRNA abundances, however T3 and IL-6 down-regulated PPARα expression (P < .05).Together, these data are the first to report cytokine and hormonal regulation of av-UCP in avian muscle cells, suggesting that these effects are mediated through PPARs and PGC-1α, and opening a new vista for future functional and mechanistic studies.

IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors.

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

The contribution from interleukin-27 towards rheumatoid inflammation: insights from gene expression.

We aimed to assess expression of genes encoding the heterodimeric IL-27 cytokine and constituent subunits of the Il-27 receptor in rheumatoid arthritis (RA), including in extra-articular, subcutaneous rheumatoid nodules. Comparing between nodules and joint synovia, significantly elevated expression of IL27A within nodules, and comparable IL27B expression, identified nodules as a significant source of IL-27 in RA. T-lymphocytes were the main source of IL27RA transcript, and IL27RA expression correlated with a number of plasma cytokines, as well as tissue TNF expression in both nodules and RA synovia. In synovia, correlations between IL27A, IL27RA IL17A and CD21L expression, and significantly elevated expression of the genes encoding IL-27, associated the presence of IL-27 with B cell-dominated synovial inflammation. Impact from nodule derived IL-27 on systemic or synovial inflammation in RA remains unknown and further study of these implications is required. Our study raises questions regarding the appropriate circumstances for the blockade or administration of IL-27 as a potential therapeutic adjunct in RA.

Interplay Between the IL-33/ST2 Axis and Bone Marrow ILC2s in Protease Allergen-Induced IL-5-Dependent Eosinophilia.

Background: Eosinophils develop from CD34+ progenitor cells in the bone marrow under the influence of interleukin (IL)-5. Several cell types produce IL-5, including type 2 innate lymphoid cells (ILC2s). The alarmin cytokine IL-33 is known to activate ILC2s in mucosal tissues, but little is known about IL-33-responsive ILC2s in the bone marrow in allergen-induced airway inflammation.Methods: Wild type (WT) and Rag1 deficient (Rag1−/−) mice, which lack mature T and B cells, received intranasal doses of papain to induce acute allergic inflammation. In some experiments, mice were pre-treated with anti-IL-5 prior to the papain challenge. Furthermore, recombinant IL-33 was administered to WT mice, Rag1−/− mice, lymphocyte deficient mice (Rag2−/−Il2rg−/−) and to ex vivo whole bone marrow cultures. Bone marrow eosinophils and ILC2s were analyzed by flow cytometry. Eosinophil count was assessed by differential cell count and secreted IL-5 from bone marrow cells by ELISA.Results: Intranasal administration of papain or IL-33 increased the number of mature eosinophils in the bone marrow despite the absence of adaptive immune cells in Rag1−/− mice. In parallel, an increased number of eosinophils was observed in the airways together with elevated levels of Eotaxin-2/CCL24. Bone marrow ILC2s were increased after papain or IL-33 administration, whereas ILC2s was found to be increased at baseline in Rag1−/− mice compared to WT mice. An upregulation of the IL-33 receptor (ST2) expression on bone marrow ILC2s was observed after papain challenge in both Rag1−/− and WT mice which correlated to increased number of bone marrow eosinophilia. Furthermore, an increased number of ST2+ mature eosinophils in the bone marrow was observed after papain challenge, which was further dependent on IL-5. In addition, bone marrow-derived ILC2s from both mouse strains produced large amounts of IL-5 ex vivo after IL-33 stimulation of whole bone marrow cultures. In contrast, IL-33-induced bone marrow and airway eosinophilia were abolished in the absence of ILC2s in Rag2−/−Il2rg−/− mice and no production of IL-5 was detected in IL-33-stimulated bone marrow cultures.Conclusion: These findings establish bone marrow ILC2s and the IL-33/ST2 axis as promising targets for modulation of uncontrolled IL-5-dependent eosinophilic diseases including asthma.

Donor Lymphocyte-Derived Natural Killer Cells Control MHC Class I-Negative Melanoma.

Natural killer (NK) cells provide a natural defense against MHC-I-negative tumors, such as melanoma. Donor lymphocyte infusion (DLI) containing NK cells, a form of adoptive immunotherapy used after allogenic bone marrow transplantation (allo-BMT), promotes antitumor immune responses but is often associated with life-threatening complications such as graft-versus-host disease (GvHD). Here, we showed that without prior allo-BMT, DLI provoked melanoma control associated with the infiltration and persistence of the transferred NK cells. This allograft acceptance did not correlate with an increase of GvHD; instead it correlated with the expansion and activation of tumor-infiltrating NK cells that expressed the cytotoxic molecules (e.g., IFNγ and granzyme B) and maturation signatures (e.g., CD11bhiCD27lo and KLRGhi/CD43hi). The development of beneficial tumor-infiltrating NK cells of DLI origin required host CD4+ T-cell help in part by producing IL2, as well as by limiting regulatory CD4+ T cells (Treg). IL2 blockade impaired the NK-dependent melanoma control, which could not be rescued by IL2 administration beyond CD4+ T-cell help. Our findings linked NK allograft acceptance-CD4+ T-cell help crosstalk to melanoma development without the need of allo-BMT. We thereby helped define that tumor-infiltrating NK cells of DLI origin may serve as effective therapeutic targets for controlling melanoma.

1741-P: Immune Regulation of Postprandial Glycemia and Whole-Body Insulin Sensitivity through Myeloid Production of IL-10

As feeding increases signals that potently induce gene expression in immune cells, we aimed to elucidate the role of myeloid cells in normal postprandial metabolism. Feeding increased serum levels of LPS, which is known to induce multiple genes in immune cells. Notably, LPS together with insulin potently induced IL-10 in macrophages, which could also be detected in the postprandial portal vein in vivo. In cultured hepatocytes, physiological levels of insulin alone did not suppress gluconeogenic gene expression, although when added together with medium from macrophages stimulated with LPS and insulin, they were markedly suppressed. This suppression was not seen in medium from macrophages of IL-10 knockout mice, showing this to be an IL-10 dependent process. Plasma glucose concentration ad libitum was elevated in mice injected with adenovirus encoding shRNA targeting the IL-10 receptor, and the normal postprandial suppression of gluconeogenic genes in liver was compromised, showing the importance of the IL-10 signal in postprandial glucose metabolism. Furthermore, myeloid-specific knockout of Akt1 and Akt2 resulted in similar phenotypes that were rescued by incrementing mTOR signaling, pointing to an Akt-mTOR dependent mechanism. As whole-body deficiency of IL-10 has been reported to decrease body weight and increase insulin sensitivity, we assessed the effect of myeloid deficiency of IL-10 by conducting bone marrow transfer from IL-10 knockout mice to wild type mice. While these phenotypes were recapitulated, postprandial glucose production was not improved despite markedly increased whole-body insulin sensitivity. These results pointed to the differential actions of myeloid IL-10 where baseline production maintains normal body weight, while postprandial increases in the hepatic-portal system suppresses hepatic gluconeogenic gene expression, collectively showing the need for context-dependent administration of IL-10 based therapies. Disclosure G. Toda: None. K. Soeda: None. N. Arakawa: None. Y. Masuda: None. N. Kobayashi: None. H. Suwanai: None. Y. Izumida: None. T. Yamauchi: Research Support; Self; AeroSwitch, Asahi Mutual Life Insurance Company, Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim International GmbH, Daiichi Sankyo Company, Limited, Kowa Pharmaceutical company,limited., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Corporation Life Sciences Limited, Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Novo Nordisk Inc., NTT Docomo Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, TOSOH CORPORATION. Other Relationship; Self; Covidien Japan Inc. (Medtronic Japan Co., Ltd.), Eli Lilly Japan K.K., Johnson &amp; Johnson, Kissei Pharmaceutical Co., Ltd. T. Kadowaki: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, MSD Corporation, Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott, Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Cosmic Corporation, Daiichi Sankyo, Eli Lilly Japan K.K., FUJIFILM, Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Medscape Education, Medtronic, Mitsubishi Tanabe Pharma Corporation, MSD Corporation, NIPRO Medical Corporation, Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Asahi Mutual Life Insurance. K. Ueki: None. Funding Japan Society for the Promotion of Science (16K15488, 17K16141, 19K17954)

1958-P: Neonatal Il-4 Induces a Persistent Decrease in Adipocyte Maturation

Adipose tissue is a central node for the dynamic regulation of systemic metabolism and energy expenditure. Little is known about adipogenesis in the newborn which is the critical window of adipose tissue development. Emerging data suggest that immune cells, particularly innate immune cells, play an important role in de novo beige adipogenesis and beige fat thermogenesis in mature rodents. In previous studies we showed that administration of low levels of IL-4 in the first week of life to rat pups results in decreased adiposity and improved glucose tolerance in adulthood, suggesting that beige adipogenesis may be enhanced by neonatal IL-4 administration. Developing animals produce higher levels of IL-4 compared to mature animals and use a distinct pre-adipocyte compartment for adipogenesis. In this study, we analyze the effects of IL-4 on adipogenesis in the developing rat. We demonstrate that a short and transient IL-4 exposure in the neonate during postnatal days 1-6 induces acute beige adipogenesis in the inguinal white adipose tissue (iWAT) that is associated with the upregulation of Ucp1 (n=6). Concurrently, adipocyte maturation is decreased with Adipoq downregulated (n=6). Pre-adipocytes from 10 week old mature rats that had been treated with IL-4 as neonates were differentiated in vitro and also show a decrease in Adipoq compared to controls (n=6). These data demonstrate that neonatal IL-4 induces a long term decrease in the maturation potential of pre-adipocytes. Interestingly, these adult differentiated pre-adipocytes show no change in Ucp1 expression after neonatal IL-4 exposure compared to control (n=6). Thus, neonatal IL-4 induces only acute beige adipogenesis, but impairs adipocyte maturation long term. Phenotypically, neonatal IL-4 resulted in decreased iWAT (n=7) and decreased fasting blood glucose in 10 week old animals (n=6) compared to control. Our studies show that the neonatal period is critical for adipocyte development and may influence the later onset of obesity. Disclosure T. Ying: None. P. Seale: None. R.A. Simmons: None. Funding National Institutes of Health (DK114054)

Interleukin-4 Administration or Zinc Supplementation Is Effective in Preventing Zinc Deficiency-Induced Hemolytic Anemia and Splenomegaly.

Nutritional zinc deficiency aggravates inflammation, subsequently causing anemia and splenomegaly in rats; however, the mechanism underlying such splenomegaly remains poorly understood. Therefore, in this study, we aimed to elucidate the mechanisms underlying the splenomegaly and anemia occurring in zinc-deficient rats and investigate whether these effects of zinc deficiency could be reversed by interleukin (IL)-4 administration or zinc supplementation. Five-week-old male Sprague-Dawley rats were fed a standard diet; fed a zinc-deficient diet (n = 7 each) and injected with saline or IL-4; or fed a zinc-deficient diet for 6weeks followed by a standard diet for 4weeks thereafter. White blood cells, segmented neutrophils, platelets, CD4+ T cells, CD11b/c+ granulocytes, CINC/GRO+ cells, and myeloperoxidase-positive cells in the blood and spleen of the zinc-deficient rats were significantly higher than those in all the other groups. Conversely, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, lymphocytes, and CD8+ T cells in the blood of the zinc-deficient rats were significantly lower than those in the other groups. Furthermore, serum aspartate aminotransferase, lactate dehydrogenase, indirect bilirubin concentrations, and erythrocyte osmotic fragility in the zinc-deficient rats were significantly higher than those in the other groups. Moreover, zinc deficiency significantly decreased the GATA1 protein levels in the spleen. Collectively, these results indicate that zinc deficiency aggravates the inflammatory response and causes hemolytic anemia and splenomegaly. Importantly, IL-4 administration and zinc supplementation can reverse the zinc deficiency-induced hemolytic anemia and splenomegaly.

A single administration of Fc-fused recombinant human IL-7 induces expansions of T lymphocytes in healthy adults and cancer patients

Abstract Interleukin-7 (IL-7) is a cytokine that plays important roles in T cell development and homeostasis. IL-7 is a potent agent for promoting T cell reconstruction in lymphopenia patients. In this study, we investigated the effects of GX-I7 (human IL-7 fused to hyFcTM for half-life extension and improved stability) in healthy volunteers (20~60 μg/kg, n=24) and advanced solid cancer patients who had no available effective treatments (60~1,200 μg/kg, n=33). We observed significant increases in the number of circulating CD8+ and CD4+ T cells in healthy volunteers, the peak of which was seen on days 14-21. Importantly, naïve, TEM, and TCM subsets of CD8+ and CD4+ T cells increased, coupled with upregulation of Ki-67. TCR diversity of the naïve T cells tends to be increased in healthy adults by single administration of GX-I7. In addition, cytokine secretion assay using PBMCs revealed that Ag–specific (CMV-specific) T cell responses were maintained after GX-I7 administration in healthy volunteers. Moreover, in advanced solid cancer patients, GX-I7 administration significantly increased the number of all subsets (naïve, TEM, TCM and TEMRA) of both CD8+ and CD4+ T cells in a dose-dependent manner. More importantly, single GX-I7 administration increased the absolute lymphocyte count above the normal range in all patients with lymphopenic conditions (ALC &amp;lt; 1,000/mm3). In conclusion, following GX-I7 administration, dose-dependent increase of ALC and T cell subsets were observed. Our study suggests that GX-I7 as a T cell amplifier provides a unique opportunity for immuno-oncology combination strategies by reconstituting persistent T cell immunity, where anti-tumor effects are mediated by T cells.

Amplification of neoantigen-specific antitumor responses using a long-lasting IL-2 fusion protein

Abstract Low frequency of tumor-specific T cells represents one major reason for failed immunotherapy. IL-2 can boost tumor-reactive T cells but its benefit is limited due to toxicity and potential off-target effects. We developed a novel IL-2/CD25 fusion protein which exhibits more favorable pharmacology when compared to IL-2. At low doses, IL-2/CD25 targets the high affinity IL-2R and selectively boosts Tregs. Here we show that a single administration of high dose mouse IL-2/CD25 in conjunction with cognate peptide and LPS amplified tumor-specific Pmel-1 CD8+ and TRP-1 CD4+ Teff cells and Pmel-1 memory cells while only transiently increasing Tregs. This approach led to effective anti-tumor responses against B16.F10 melanoma. This scheme was modified to amplify endogenous polyclonal melanoma-reactive T cells, which is translationally more relevant. Development of tumor-specific T cells required priming and boosting with a mixture of B16.F10 neoantigens and poly (I:C) followed by application of IL-2/CD25 after the boost. This resulted in largely a CD4+ anti-neoantigen specific T cell response. This immunization scheme delayed B16.F10 growth and extend survival time not only when immunized mice were challenged with B16.F10 but also for mice with pre-established B16.F10. The immune response within the tumor microenvironment was accompanied by increased frequencies of granzyme B-expressing CD4+ and CD8+ T cells. Our data show that limited application of high-dose IL-2/CD25 in the context of neoantigen vaccines offers potential advantages over IL-2 for melanoma treatment by avoiding toxicities seen with IL-2 therapy and minimizing Treg-associated tolerance.

The Wnt-β-catenin-IL-10 signaling axis in intestinal antigen-presenting cells protects mice from colitis-associated colon cancer in response to gut microbiota

Abstract Loss of immune tolerance to gut microflora is inextricably linked to inflammatory bowel disease (IBD) and colitis-associated colon cancer (CAC). Intestinal antigen presenting cells (APCs) play a pivotal role in regulating intestinal inflammation and CAC, but underlying signaling pathways and molecular mechanisms remain largely unknown. In the present study, using a mouse model of CAC, we show that the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6)-β-catenin-IL-10 signaling axis in CD11c+ APCs suppresses CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of Wnt co-receptors LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of tumor-promoting pro-inflammatory factors and decreased expression of the immunosuppressive cytokine, IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor promoting inflammatory factors in CD11c+ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin in CD11c+ APCs or in vivo administration of IL-10 in LRP5/6ΔCD11c mice markedly reduced intestinal inflammation and CAC by suppressing the expression of inflammatory factors. In summary, here we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in regulating CAC in response to the commensal flora and suggest potential means for therapeutic intervention in inflammatory intestinal diseases.

Natural killer cells expanded in vivoor ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses.

The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.

Ex vivo IL-15 replenishment augments bone marrow precursor cell-mediated adaptive immunity via PI3K-Akt pathway.

This study tested the hypothesis that PI3K-Akt activity contributes to the superior immune function of IL-15-administrated bone marrow precursor cells (BMPC). Our previous studies revealed that PI3K-Akt play vital role in dendritic cells (DCs) cross-presentation and DC-based CTL priming. Despite the fact that IL-15 serves multiple functions in its therapeutic potential for the induction and maintenance of T cell response, the exact role of PI3K-Akt in IL-15 increased adaptive immunity is still poorly understood. In this study, we demonstrated that ex vivo IL-15 administration increased BMPC capability of antigen uptake and the expression of costimulatory molecules (such as CD80 and 4-1BB(CD137) ligand [4-1BBL]) and MHC classI molecule via PI3K-Akt pathway. Importantly, PI3K-Akt activity was not only necessary for IL-15 augmented BMPC cross-presentation and CTL priming, but also facilitated IL-15 increased therapeutic potential of the cytolytic capacity and maintenance of BMPC-activated T cells. Thus, these data suggested that PI3K-Akt activity contribute to the superior immune function of IL-15-administrated BMPC and thereby might be therapeutic potential for adaptive immunity.

IL-6 contributes to Nav1.3 up-regulation in trigeminal nerve following chronic constriction injury

ABSTRACTBackground: To verify the hypothesis that the nature of trigeminal neuralgia (TN) is an ectopic impulse induced by sodium channel modulated by cytokines, we conducted an animal study using the infraorbital nerve chronic constriction injury (CCI) model in rats.Method: The expression of Nav1.3 or IL-6 in the infraorbital nerve (ION) and trigeminal ganglion (TG) were detected by western blot and immunocytochemistry after administration of antisense oligodeoxynucleotide sequence (AS), IL-6 or Anti-IL-6.Results: With intrathecal administration of AS or mismatch oligodeoxynucleotide sequence (MM) in the CCI rats, the Nav1.3-IR in ION and TG accounted for 2.2 ± 0.51% and 8.5 ± 3.1% in AS+CCI group vs. 6.9 ± 1.3% and 38.7 ± 4.8% in MM+CCI group (p < 0.05), respectively. While with local administration of IL-6 in those with sham operation, it accounted for 7.4 ± 2.1% and 45.5 ± 3.4% in IL-6+ sham group vs. 1.9 ± 0.67% and 8.1 ± 1.3% in vehicle+sham group (p < 0.05); with local administration of anti-IL-6 in CCI rats, 4.5 ± 0.78% and 32.1 ± 9.6% in Anti-IL-6+ CCI group vs 8.9 ± 2.1% and 61.4 ± 11.2% in vehicle+CCI group (p < 0.05).Discussion: We believe that the emergence of Nav1.3 from the compressed trigeminal nerve might be an important structural basis for the development of the ectopic excitability on the axon and IL-6 may play a role of necessary precondition.