IFN-γ KO Mice Research Articles

Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Interleukin-27 and IFNγ regulate the expression of CXCL9, CXCL10, and CXCL11 in hepatitis.

Interleukin-27 (IL-27) belongs to the IL-6/IL-12 family of cytokines, associated with different inflammatory diseases and orchestrates its biological activity via common heterodimeric receptor composed of WSX-1 (IL-27Rα) and gp130. The present study was aimed to investigate the regulation of CXCL9, CXCL10, and CXCL11 chemokines in hepatic cells (human LX-2 cell line derived from normal human stellate cells (HSC), primary human hepatocytes, HSC, and HepG2 cells) and concanavalin A (ConA)-induced liver inflammation. We demonstrated that IL-27, but not IL-6, induced/up-regulated CXCR3 ligand genes (CXCL9, CXCL10, and CXCL11; out of 26 selected genes) in a STAT1-dependent manner in hepatic cells in vitro both at transcript and protein levels. In ConA-induced T cell-mediated hepatic model, we showed that soluble IL-27/IFNγ was elevated following ConA hepatitis in association with increased CXCL9, CXCL10, and CXCL11 expression in the liver. The exogenous IL-27 administration induced CXCR3 ligands in mouse liver at 4 h with any significant effect on recruitment of CXCR3(+) immune cells in the liver. The neutralization of IL-27 during ConA hepatitis differentially modulated (transcript vs protein expression) CXCR3 ligands and IFNγ during ConA-induced hepatitis with down-regulated expression of CXCL9 and CXCL10 at transcript level. The IFNγ, complementary regulated the expression of CXCR3 ligands as their up-regulation during ConA hepatitis, was abolished in IFNγ KO mice. In summary, IL-27 up-regulated the CXCL9, CXCL10, and CXCL11 chemokine expression in hepatic cells. IL-27 regulated CXCR3 ligand expression in IFNγ-dependent manner during acute hepatitis suggesting a complementary role of IL-27 and IFNγ to moderate liver inflammation via regulation of CXCR3 ligands. IL-27 up-regulated CXCR3 ligand expression in human hepatic cells in vitro. IL-27 up-regulated CXCR3 ligand expression and secretion in ConA hepatitis in vivo. CXCR3 ligand expression was down-regulated by blocking IL-27 or IFNγ deficiency. IL-27 modulated liver injury by regulation of CXCR3 ligands in IFNγ-dependent manner.

Oral rapamycin (eRapa) requires IFN-γ and promotes γδ T cell cytotoxicity to prevent carcinogen and inflammation-induced dermal cancer (TUM9P.1003)

Abstract Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, prevents distinct cancers in mice and prolongs life, making it a candidate cancer prevention (and life extension) agent. We hypothesized that eRapa cancer prevention includes immune effects, as mTOR is pivotal in immunity. We tested immune effects in carcinogen (DMBA) and inflammation (TPA)-induced dermal carcinogenesis with eRapa daily at 14 ppm, which protected 100% from skin cancer in wild type (WT) mice. Tumor mTORC1 was not suppressed, supporting eRapa immune effects. γδ T cells and IFN-γ are protective in this model. In δ TCR KO (no γδ T cells) and IFN-γ KO mice, eRapa cancer protection was lost, confirming immune mechanisms. IFN-γ KO mice had significantly reduced CD69+Lamp1+GzB+CXCR3+ epidermal γδ T cells and CXCL10, suggesting IFN-γ is required for epidermal γδ T cell migration (via CXCR3/CXCL10), activation and cytolysis. Bone marrow (BM) chimeras showed eRapa skin cancer protection and maximum γδ T cell epidermal infiltration requires IFN-γ signals in BM (e.g., γδ T cells) and non-BM (e.g., epidermal) cells. Intratumor injections of WT but not Prf1 (perforin) KO γδ T cells regressed DMBA/TPA tumors in δ TCR KO mice on eRapa but not control, showing eRapa boosts anti-tumor γδ T cell cytotoxicity. Our data confirm eRapa cancer prevention through immune mechanisms, challenging the paradigm of mTOR effects solely on cancer cells for prevention, and suggesting novel immune-mediated life extension mechanisms.

Mammalian target of rapamycin inhibition with enterically given rapamycin alters immunity and gut flora in young and aged wild type mice and extends life of immunodeficient mice (IRC8P.452)

Abstract mTOR regulates many important immune processes. Interest in immune effects of mTOR inhibitors is heightened by recent findings of ability to boost specific immunity and vaccine response in elderly humans, treat cancers and autoimmunity, and extend life and healthspan in mice, suggesting important new clinical applications. However, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. We comprehensively analyzed immune effects of long-term rapamycin in mice. Gene expression profiling of purified T and B cells (CD4+PD1+, CD4+PD1-, CD8+PD1+, CD8+PD1-, B220+), CD11b+CD11c- myeloid cells and CD11c+ dendritic cells showed numerous, novel changes in genes affecting differentiation, function, homeostasis, migration, exhaustion, cell death and inflammation. Novel effects on Th9, Th22 and Tfh differentiation were seen as was improved function of exhausted PD1+ T cells (supported by human clinical data) and increased numbers and function of innate lymphoid cells. Immune functions relevant to cancer, infections, aging and inflammation, and innate lymphoid cell effects were validated in vitro and in vivo. eRapa prolonged life in RAG KO mice (without T or B cells) >12x and in IFN-γ KO mice >3x over wild type mice, supporting mTOR suppression immune effects mitigating disease as a mechanism for longevity extension. Metagenomic effects were linked to immune outcomes. Our data demonstrate novel mTOR immune effects meriting further investigations.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

Desiccating stress-induced chemokine expression in the epithelium is dependent on upregulation of NKG2D/RAE-1 and release of IFN-γ in experimental dry eye.

The Th1-associated chemokines CXCL9, CXCL10, and CXCL11 coordinate migration of CXCR3(+) Th1 cells. The objective of this study was to evaluate the role of the innate immune system in stimulating chemokine expression in an experimental model of dry eye and bridge the gap between innate and adaptive immunity. Desiccating stress (DS) induced very early (6 h) expression and production of Th1-associated chemokines in cornea and conjunctiva of C57BL/6 and RAG1 knockout (KO) mice, demonstrating that chemokine expression does not require innate T cells. We then demonstrated that activating the innate immune system prior to adoptive transfer of T cells to RAG1KO mice increased disease severity. Interestingly, lack of induction of chemokines CXCL9, CXCL10, and CXCL11 in IFN-γKO mice provided evidence that their expression requires IFN-γ for induction. Treatment of RAG1KO mice with anti-NK1.1 prevented the increase of CXCL9, CXCL10, and CXCL11 in response to DS, compared with isotype controls. Additionally, DS increased the expression of NKG2D in the conjunctiva. The expression of the NKG2D ligand, retinoic acid early inducible gene 1, also increased at the ocular surface at both the protein and gene levels. Neutralization of NKG2D at the ocular surface decreased the expression of CXCL9, CXCL10, CXCL11, and IFN-γ. In summary, upregulation of CXCL9, CXCL10, and CXCL11 expression in experimental dry eye is T cell-independent, requiring IFN-γ-producing NKG2D(+) NK cells that are activated in response to DS-induced stress signals. This study provides insight into the events that trigger the initial immune response in dry eye pathology.

Oral rapamycin (eRapa) safely prevents carcinogen-induced dermal carcinogenesis through an interferon-γ-dependent mechanism (TUM7P.931)

Abstract eRapa extends life in mice and cancer prevention could be a mechanism. Rapamycin inhibits mTOR, which has significant immune effects that are surprisingly unstudied in cancer therapy or prevention. We hypothesize that cancer prevention by eRapa is mediated by improved cancer immune surveillance, which we tested in the well-established DMBA/TPA carcinogen-induced skin cancer model. Mice got eRapa or control chow, and skin tumors were induced with DMBA/TPA over 24 weeks. eRapa reduced benign (p=.001) and malignant (p=.05) tumors in WT mice. T cells and IFN-γ mediate cancer immune surveillance. eRapa reduced skin tumors in βδ KO mice lacking all T cells (p=.04), but not in IFN-γ KO mice (p=.13), consistent with loss of beneficial eRapa-induced, non-T cell IFN-γ. In support, WT or IFN-γ KO T cell transfer into IFN-γ KO mice did not alter eRapa cancer prevention in DMBA/TPA. In WT mice on DMBA/TPA, eRapa increased IFN-γ-producing natural killer cells (p=.01) that could mediate skin cancer immune surveillance, and decreased CD34+CD49fint skin cancer stem cells (p=.01) and CXCR3+ T cells (p<.001) that contribute to cancer in this model. eRapa reduced skin pAKT with divergent mTORC1/2 effects needing more study. eRapa appeared safe (no increased Tregs or reduced protection in infection and autoimmunity models). A widely applicable, safe and tolerable cancer prevention agent would be highly useful. Understanding its immune mechanisms could improve efficacy and widen applications.

IFNγ protects neonatal neuronal precursor cells during measles virus infection of the brain (VIR1P.1007)

Abstract In the developing brain, self-renewing neural stem/progenitor cells (NSPC) differentiate into neurons or glia. NSPC survival and differentiation can be altered by neurotropic viruses and by the anti-viral immune response. We hypothesize that interferon-gamma (IFNγ), a pro-inflammatory cytokine required for non-cytolytic clearance of many neurotropic viruses, alters NSPC proliferation and cell fate in measles virus (MV)-infected neonates. Two transgenic mouse lines were used: CD46+ express human CD46, a receptor for MV, under the neuron-specific enolase promoter, thereby restricting MV infection to mature neurons; CD46+/IFNγ-KO lack IFNγ. In our model, NSPCs are not infected, allowing us to evaluate the impact of solely the immune response on NSPCs. Flow cytometry and western blot were used to quantify effects on cell number and IFNγ-mediated signaling, respectively. Mice were mock- or MV-infected on postnatal day 2 and sacrificed 3, 7, and 10 days post-infection. Whole brain isolates were double-labeled with BrdU and markers for NSPCs (nestin), early neuronal lineage (CD24), and glial lineage (GFAP, O4). CD24+ cells showed the greatest cell loss at 7 dpi in both genotypes. NSPCs were reduced in CD46+/IFNγ-KO mice at 3 and 7 dpi, but unaffected in CD46+ mice. Thus, IFNγ may protect against NSPC, but not neuronal, loss. Current experiments aim to address the cell fate of NSPCs exposed to the antiviral immune response and define mechanisms of NSPC loss in CD46+/IFNγ-KO neonates.

Systemic Juvenile Idiopathic Arthritis–like Syndrome in Mice Following Stimulation of the Immune System With Freund's Complete Adjuvant: Regulation by Interferon‐γ

Systemic juvenile idiopathic arthritis (JIA) is unique among the rheumatic diseases of childhood, given its distinctive systemic inflammatory character. Inappropriate control of innate immune responses following an initially harmless trigger is thought to account for the excessive inflammatory reaction. The aim of this study was to generate a similar systemic inflammatory syndrome in mice by injecting a relatively innocuous, yet persistent, immune system trigger: Freund's complete adjuvant (CFA), containing heat-killed mycobacteria. Given the central role of interferon-γ (IFNγ) in immune regulation, we challenged wild-type (WT) and IFNγ-knockout (KO) BALB/c mice with CFA, and analyzed their clinical symptoms and biologic characteristics. The production of cytokines and the effects of anticytokine antibodies were investigated. In WT mice, CFA injection resulted in splenomegaly, lymphadenopathy, neutrophilia, thrombocytosis, and increased cytokine expression. In the absence of IFNγ, these symptoms were more pronounced and were accompanied by weight loss, arthritis, anemia, hemophagocytosis, abundance of immature blood cells, and increased levels of interleukin-6 (IL-6), all of which are reminiscent of the symptoms of systemic JIA. CFA-challenged IFNγ-KO mice showed increased expression of IL-17 by CD4+ T cells and by innate γ/δ T cells. Inflammatory and hematologic changes were prevented by treatment with anti-IL-12/IL-23p40 and anti-IL-17 antibodies. Immune stimulation of IFNγ-KO mice with CFA produces a systemic inflammatory syndrome reflecting the clinical, biologic, and histopathologic picture of systemic JIA. The protective function of IFNγ in preventing anemia and overall systemic inflammation is a striking observation. The finding that both adaptive and innate T cells are important sources of IL-17 may be of relevance in the pathogenesis of systemic JIA.

Exacerbation of Invasive Candida albicans Infection by Commensal Bacteria or a Glycolipid Through IFN-γ Produced in Part by iNKT Cells

The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.

Properties of Immature Myeloid Progenitors with Nitric-Oxide-Dependent Immunosuppressive Activity Isolated from Bone Marrow of Tumor-Free Mice

Myeloid derived suppressor cells (MDSCs) from tumor-bearing mice are important negative regulators of anti-cancer immune responses, but the role for immature myeloid cells (IMCs) in non-tumor-bearing mice in the regulation of immune responses are poorly described. We studied the immune-suppressive activity of IMCs from the bone marrow (BM) of C57Bl/6 mice and the mechanism(s) by which they inhibit T–cell activation and proliferation. IMCs, isolated from BM by high-speed FACS, inhibited mitogen-induced proliferation of CD4+ and CD8+ T-cells in vitro. Cell-to-cell contact of T-cells with viable IMCs was required for suppression. Neither neutralizing antibodies to TGFβ1, nor genetic disruption of indolamine 2,3-dioxygenase, abrogated IMC-mediated suppressive activity. In contrast, suppression of T-cell proliferation was absent in cultures containing IMCs from interferon-γ (IFN-γ) receptor KO mice or T-cells from IFN-γ KO mice (on the C57Bl/6 background). The addition of NO inhibitors to co-cultures of T-cells and IMC significantly reduced the suppressive activity of IMCs. IFN-γ signaling between T-cells and IMCs induced paracrine Nitric Oxide (NO) release in culture, and the degree of inhibition of T-cell proliferation was proportional to NO levels. The suppressive activity of IMCs from the bone marrow of tumor-free mice was comparable with MDSCs from BALB/c bearing mice 4T1 mammary tumors. These results indicate that IMCs have a role in regulating T-cell activation and proliferation in the BM microenvironment.

OP0054 Complete freund’s adjuvant induces, in interferon-gamma-deficient mice, a chronic inflammatory disease reminiscent of systemic juvenile idiopathic arthritis

BackgroundSystemic juvenile idiopathic arthritis (sJIA) is a disease characterized by arthritis and systemic features such as fever, rash, lymphadenopathy and leukocytosis. The underlying pathogenic mechanism of sJIA is not understood...

IFN-γ Mediates the Antitumor Effects of Radiation Therapy in a Murine Colon Tumor

Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, a new concept has recently emerged, suggesting that the immune system is required for effective IR therapy. Our work here has identified interferon gamma (IFN-γ) as an essential cytokine for the efficacy of IR therapy. Local IR (15 Gy) to mice bearing Colon38, a colon adenocarcinoma, decreases tumor burden in wild-type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral levels of IFN-γ increased 2 days following IR, which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFN-γ on tumor cells. T cells from IR-treated tumors exhibited a far greater capacity to lyse tumor cells in a (51)Cr release assay, a process that was dependent on IFN-γ. CD8(+) T cells were the predominant producers of IFN-γ, as demonstrated by IFN-γ intracellular staining and studies in IFN-γ reporter mice. Elimination of CD8(+) T cells by antibody treatment reduced the intratumoral levels of IFN-γ by over 90%. More importantly, elimination of CD8(+) T cells completely abrogated the effects of radiation therapy. Our data suggest that IFN-γ plays a pivotal role in mediating the antitumor effects of IR therapy.

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

Abstract A95: Distinct roles of type I and type II interferons in radiation-induced antitumor immunity against B16 melanoma.

Abstract Endogenous type I interferons (IFN-α/β) and interferon gamma (IFN-γ) have both been implicated in immune responses against cancer. Interestingly, studies done using viral infection models have demonstrated that IFN-α/β and IFN-γ play non-redundant roles in the immune system and they act synergistically, enhancing responses that are induced by the two different signaling pathways. However, this phenomenon, known as priming, has not been well-characterized in the context of anti-tumor immunity. Our lab previously demonstrated using a B16 murine melanoma model that single local high dose (15Gy) radiation therapy induced elevated levels of intratumoral IFN-γ, which had several downstream effects like increased MHC class I expression on tumor cells that in turn enhanced recognition by CD8+ T cells. Overall, these IFN-γ;-dependent effects led to increased tumor control, evident by significant decreased rate of tumor growth following treatment. In the current study, we hypothesize that local radiation therapy (RT) increases the levels of IFN-α/β within the tumor, and this is required to enhance IFN-γ responses so that maximal therapeutic potential can be achieved. Indeed, local RT resulted in an early, transient spike in intratumoral IFN-α levels about two days after treatment. Interestingly, an increase in IFN-γ levels was observed four days post-treatment and remained elevated for at least five days. When B16 tumors were grown in mice that lack functional IFN-α/β receptors (IFNABR KO), the subsequent increase in IFN-γ following RT was completely abrogated. The ability of RT in reducing tumor growth was also less effective in IFNABR KO mice than in wild type (WT) mice. To investigate the mechanism involved, we examined the transcription of interferon-inducible genes, including the CXCR3 chemokines CXCL9 (MIG) and CXCL10 (IP-10). Following RT, intratumoral transcript levels of MIG and IP-10 were increased to similar extents in both WT and IFNγKO mice, but remained at basal levels in IFNABR KO mice. This suggests that IFN-α/β, but not IFN-γ, may be contributing to the initial chemoattraction of lymphocytes into radiation-treated tumors. Using flow cytometry analysis of immune cell populations within the tumors, we confirmed that the levels of intratumoral CXCR3 chemokines correlate strongly with tumor infiltration by T lymphocytes. Our data suggest that IFN-α/β play a critical role in the increase of CXCR3 chemokine levels within the tumor after RT. These chemokines are important for the recruitment of activated T cells into the tumor, which then produce IFN-γ, increasing IFNγ-induced immune responses locally. To investigate whether exogenous IFN-α will improve the efficacy of radiation therapy, we genetically-engineered a B16 cell line that can be induced to express IFN-α by exposing these cells, named B16-iIFN-α, to a rapamycin-analog. When inoculated in mice, B16-iIFN-α cells form established tumors similar to that of parental B16 cells. Production of IFN-α by B16-iIFN-α cells can be switched on at specific time points in tumor-bearing mice by treating the mice with the rapamycin-analog. This is a novel and invaluable tool for examining the anti-tumor effects of a local increase in IFN-α, and can be modified to study the effects of other cytokines as well. Our preliminary data demonstrated that radiation and IFN-α combination therapy resulted in promising improvement in tumor growth control compared to either of the treatments alone. We are currently studying the immunological mechanisms involved, as well as fine-tuning the treatment regimen to harness the maximal therapeutic potential of this treatment strategy. Citation Format: Joanne Y.H. Lim, Scott A. Gerber, Edith M. Lord. Distinct roles of type I and type II interferons in radiation-induced antitumor immunity against B16 melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A95.

Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes

BackgroundNeutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.MethodsEAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.ResultsWe discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.ConclusionsThese results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Interferon-γ ablation exacerbates myocardial hypertrophy in diastolic heart failure

Diastolic heart failure (HF) accounts for up to 50% of all HF admissions, with hypertension being the major cause of diastolic HF. Hypertension is characterized by left ventricular (LV) hypertrophy (LVH). Proinflammatory cytokines are increased in LVH and hypertension, but it is unknown if they mediate the progression of hypertension-induced diastolic HF. We sought to determine if interferon-γ (IFNγ) plays a role in mediating the transition from hypertension-induced LVH to diastolic HF. Twelve-week old BALB/c (WT) and IFNγ-deficient (IFNγKO) mice underwent either saline (n = 12) or aldosterone (n = 16) infusion, uninephrectomy, and fed 1% salt water for 4 wk. Tail-cuff blood pressure, echocardiography, and gene/protein analyses were performed. Isolated adult rat ventricular myocytes were treated with IFNγ (250 U/ml) and/or aldosterone (1 μM). Hypertension was less marked in IFNγKO-aldosterone mice than in WT-aldosterone mice (127 ± 5 vs. 136 ± 4 mmHg; P < 0.01), despite more LVH (LV/body wt ratio: 4.9 ± 0.1 vs. 4.3 ± 0.1 mg/g) and worse diastolic dysfunction (peak early-to-late mitral inflow velocity ratio: 3.1 ± 0.1 vs. 2.8 ± 0.1). LV ejection fraction was no different between IFNγKO-aldosterone vs. WT-aldosterone mice. LV end systolic dimensions were decreased significantly in IFNγKO-aldosterone vs. WT-aldosterone hearts (1.12 ± 0.1 vs. 2.1 ± 0.3 mm). Myocardial fibrosis and collagen expression were increased in both IFNγKO-aldosterone and WT-aldosterone hearts. Myocardial autophagy was greater in IFNγKO-aldosterone than WT-aldosterone mice. Conversely, tumor necrosis factor-α and interleukin-10 expressions were increased only in WT-aldosterone hearts. Recombinant IFNγ attenuated cardiac hypertrophy in vivo and modulated aldosterone-induced hypertrophy and autophagy in cultured cardiomyocytes. Thus IFNγ is a regulator of cardiac hypertrophy in diastolic HF and modulates cardiomyocyte size possibly by regulating autophagy. These findings suggest that IFNγ may mediate adaptive downstream responses and challenge the concept that inflammatory cytokines mediate only adverse effects.

Reciprocal Interaction between Macrophages and T cells Stimulates IFN-γ and MCP-1 Production in Ang II-induced Cardiac Inflammation and Fibrosis

BackgroundThe inflammatory response plays a critical role in hypertension-induced cardiac remodeling. We aimed to study how interaction among inflammatory cells causes inflammatory responses in the process of hypertensive cardiac fibrosis.Methodology/Principal FindingsInfusion of angiotensin II (Ang II, 1500 ng/kg/min) in mice rapidly induced the expression of interferon γ (IFN-γ) and leukocytes infiltration into the heart. To determine the role of IFN-γ on cardiac inflammation and remodeling, both wild-type (WT) and IFN-γ-knockout (KO) mice were infused Ang II for 7 days, and were found an equal blood pressure increase. However, knockout of IFN-γ prevented Ang II-induced: 1) infiltration of macrophages and T cells into cardiac tissue; 2) expression of tumor necrosis factor α and monocyte chemoattractant protein 1 (MCP-1), and 3) cardiac fibrosis, including the expression of α-smooth muscle actin and collagen I (all p<0.05). Cultured T cells or macrophages alone expressed very low level of IFN-γ, however, co-culture of T cells and macrophages increased IFN-γ expression by 19.8±0.95 folds (vs. WT macrophage, p<0.001) and 20.9 ± 2.09 folds (vs. WT T cells, p<0.001). In vitro co-culture studies using T cells and macrophages from WT or IFN-γ KO mice demonstrated that T cells were primary source for IFN-γ production. Co-culture of WT macrophages with WT T cells, but not with IFN-γ-knockout T cells, increased IFN-γ production (p<0.01). Moreover, IFN-γ produced by T cells amplified MCP-1 expression in macrophages and stimulated macrophage migration.Conclusions/SignificanceReciprocal interaction between macrophages and T cells in heart stimulates IFN-γ expression, leading to increased MCP-1 expression in macrophages, which results a forward-feed recruitment of macrophages, thus contributing to Ang II-induced cardiac inflammation and fibrosis.

Type II interferon gamma is detrimental to the host in Streptococcus suis infections caused by an epidemic strain (164.6)

Abstract S. suis is a major swine pathogen that can be transmitted to humans causing severe symptoms. A large human outbreak was described in China, where approximately 25% out of 215 infected humans developed an unusual streptococcal toxic shock-like syndrome (STSLS). Albeit increased expression of inflammatory mediators following infection by the Chinese S. suis strain was suggested as responsible for STSLS case severity, the mechanisms involved are still poorly understood. Therefore, we investigated the host innate immune response to infection by either one of 3 strains of S. suis: 89-1591 (Canadian, intermediate virulence), P1/7 (European, high virulence), and SC84 (Chinese, epidemic strain). Using Illumina microarray, qPCR, and Luminex assay, infected mice showed elevated expression of mainly pro-inflammatory chemokine and cytokine genes. Generally, pro-inflammatory genes were expressed at a higher level in mice infected with strain SC84 &amp;gt; P1/7 &amp;gt; 89-1591. Interestingly, IFNγ was expressed at much higher levels only in mice infected with strain SC84, which could potentially explain some of the STSLS symptoms. IFNγ-KO mice infected with epidemic SC84 strain showed significantly better survival than WT mice while no difference was observed in mice infected with highly virulent P1/7 strain. Overall, our results show an important role of IFNγ in S. suis infections and might explain in part the increased virulence of the epidemic SC84 strain responsible for the outbreak in China.

Effects of Gametophytes of Ecklonia Kurome on the Levels of Glucose and Triacylglycerol in db/db, Prediabetic C57BL/6J and IFN-γ KO Mice

We have studied edible algae that have the potential to down-regulate blood glucose. In Japan, Ecklonia species have been believed to improve the circulation of blood. In this study, we used leptin receptor deficient type 2 diabetes model mice (db/db) and prediabetic C57BL/6J mice. We also focused on the role of IFN-γ in the control of blood levels of triacylglycerol and glucose, because it is reportedly engaged in the regulation of energy consumption together with leptin. We report that gametophytes of Ecklonia kurome down-regulate the blood level of glucose and serum level of triacylglycerol in db/db. We also report that gametophytes of Ecklonia kurome down-regulate the level of glucose but not the level of triacylglycerol in prediabetic C57BL/6J mice induced by a high fat diet. They increased the level of triacylglycerol compared to that of control group in C57BL/6J, but not in IFN-γ KO mice. Gametophytes of Ecklonia kurome were administered orally to prediabetic C57BL/6J and IFN-γ KO mice and oral glucose tolerance tests were performed to evaluate the effects of algae. During the administration of the normal diet, we found a higher level of blood glucose in a glucose tolerance test of IFN-γ KO mice compared with that of C57BL/6J. Although a high fat diet induced a higher level of blood glucose compared with a normal diet group in a glucose tolerance test of C57BL/6J mice, this effect of high fat diet was not observed clearly at first but appeared three hours after glucose administration in IFN-γ KO mice. Gametophytes of Ecklonia kurome down-regulated the level of blood glucose in both C57BL/6J and IFN-γ KO mice, when administered a normal diet after making them prediabetic. These results suggest that Ecklonia kurome are effective to down-regulate the blood glucose and IFN-γ is involved in the regulation of blood glucose and triacylglycerol.