Role Of IFN-γ Research Articles

Local up-regulation of interferon-γ (IFN-γ) following disc herniation is involved in the inflammatory response underlying acute lumbar radicular pain

Abstract Aims Lumbar radicular pain after disc herniation may be associated release of pro-inflammatory cytokines from nucleus pulposus (NP) tissue. In the present study we examined the role of interferon-γ (IFN-γ) and cluster of differentiation 68 (CD68) in the acute phase of this process. Methods First, in an animal model mimicking the clinical situation after disc herniation, the role of IFN-γ on the dorsal horn single cell activity and gene expression close to the nerve roots was studied. Second, in patients with severe lumbar radicular pain due to disc herniation, we examined how two single nucleotide polymorphisms (SNPs; rs2069705 and rs2069718) important for the IFN-γ expression influenced the pain and disability measured by visual analogue scale (VAS) and Oswestry Disability Index (ODI). Results The animal data demonstrated a significant increase in the nociceptive activity at the spinal level after local application of NP and IFN-γ onto the nerve-roots. A positive correlation between IFN-γ and CD68 in the NP tissue was also observed. Moreover, the data of the patients revealed that carriers of the IFN-γ SNPs; rs2069705 A allele and rs2069718 G allele had an increased disability score i.e. ODI. Conclusions The present data suggest that IFN-γ through activation of tissue-specific macrophages close to the nerve roots may be important for acute inflammatory pain and disability following lumbar disc herniation.

IFN-γ-induced JAK/STAT, but not NF-κB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells.

Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-γ (IFN-γ) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-γ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-γ-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-γ-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN-γ-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-γ-inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/STAT vs. NF-κB signaling pathways with GC-sensitive IIGs being more NF-κB dependent and GC-insensitive IIGs being more JAK/STAT dependent. Importantly, transfection of cells with siRNA-STAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-γ-induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma.

IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression.

ObjectivePatients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). The immune system plays an important regulatory role in controlling JCV reactivation from latent sites by limiting viral gene expression and replication. However, little is known regarding the molecular mechanisms responsible for this regulation.Methods and ResultsHere, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-γ showed a robust inhibition of T-antigen expression. While potential roles for IFN-β, and to a lesser extent IFN-α have been described for JCV, IFN-γ has not been previously implicated. Further analysis of IFN-γ signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-γ suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity.ConclusionsOur results suggest a novel role for IFN-γ in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML.

Interferon-gamma and nitric oxide synthase 2 mediate the aggregation of resident adherent peritoneal exudate cells: implications for the host response to pathogens.

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2 -/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2 -/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or “group behavior” of APECs are discussed in the context of host resistance to infectious organisms.

Silencing IFN-γ binding/signaling in astrocytes versus microglia leads to opposite effects on central nervous system autoimmunity.

IFN-γ, the hallmark cytokine of Th1 cells, plays an important role in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Thus far, the role of IFN-γ in EAE has been largely studied through its effects on immune cells, whereas much less is known about its effects on CNS cells, especially in vivo. In this study, we dissected the in vivo effects and mechanisms of IFN-γ binding/signaling in astrocytes and microglia, and found that IFN-γ signaling in these cell types has opposite effects in EAE pathogenesis. Silencing IFN-γ binding/signaling in astrocytes alleviated EAE, whereas in microglia, and likely in some infiltrating macrophages, it increased disease severity. Silencing IFN-γ signaling in astrocytes resulted in diminished expression of chemokines and fewer inflammatory cells infiltrating into the CNS, whereas blocking IFN-γ binding/signaling in microglia, probably infiltrating macrophages as well, increased disease severity through augmented activation and proliferation of microglia. Further, blocking IFN-γ binding/signaling in astrocytes alleviated both Th1- and Th17-mediated adoptive EAE, indicating an important role for IFN-γ signaling in astrocytes in autoimmune CNS inflammation. Thus, our study defines novel mechanisms of action of IFN-γ in EAE pathogenesis, and also highlights an opportunity for development of multiple sclerosis therapies directed at CNS cells.

Abstract 219: IFN-γ Plays an Essential Role in Accelerating Cardiovascular Disease of Recipients With MHC Class II-mismatched Allografts.

Background: Organ transplant recipients die from cardiovascular disease (CVD) at a higher rate than the general population. Traditional treatment with statins to mitigate risk factors such as hyperlipidemia has had limited success. Using an animal model of graft-accelerated recipient CVD by combining murine models of atherosclerosis and heterotopic heart transplantation, we aim to better understand its pathogenesis and to explore more effective therapeutic strategies. Methods and Results: C57BL/6 (B6) ApoE-deficient recipient mice were heterotopically transplanted with MHC class II mismatched cardiac allografts from donor BM12 mice. Isograft controls were from donor B6 mice. IFN-γ neutralization antibody or isotype IgG was given subcutaneously at a dose of 250 μg per mouse 1 day prior to transplantation and thereafter 125 μg per mouse, 3 times per week for 12 weeks. Serum levels of cholesterol and triglyceride were measured by Amplex® Red Cholesterol Assay Kit and Triglyceride Determination Kit respectively. Inflammatory cytokines were assessed by both real-time PCR and flow cytometry. Native heart function of host mice was evaluated by echocardiography. Aortic atherosclerotic plaques in the host were examined by Oil Red O staining. MHC class II upregulation and neointimal formation in host coronary arteries were determined by immunofluorescence and EVG staining respectively. We found the levels of cholesterol and triglyceride were not changed by introducing allografts into host mice. However, a higher level of IFN-γ was produced by circulating and splenic CD4 T cells in hosts with allografts that was associated with de novo expression of endothelial MHC class II alloantigens in native coronary arteries. These phenomena were diminished by IFN-γ neutralization. Additionally, IFN-γ depletion reduced the local accumulation of IFN-γ and IP-10 mRNA in the native hearts and neointimal formation of coronary arteries in the hosts of allografts. Most importantly, IFN-γ neutralization prevented impaired cardiac function and increased aortic atherosclerotic burden in host mice with allografts. Conclusions: Our study reveals an essential role of IFN-γ in accelerating recipient CVD due to systemic inflammation from allograft rejection.

Abstract P6-05-03: CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor senescence in breast cancer

Abstract Background: HER-2 a molecular oncodriver in breast tumorigenesis is over expressed in 25% of human breast cancers, and its expression correlates with enhanced tumor aggressiveness. While targeted therapies have improved outcomes many patients become resistant or recur. We have recently established a progressive loss in anti-HER-2 CD4 Th1 responses during disease progression and is associated with outcomes. The pleiotropic Th1 cytokines IFN-γ and TNF-α have diverse effects on tumor epithelial cells. In this study we sought to determine whether these Th1 cytokines induce senescence of HER-2 expressing breast cancer cells and assess the impact of IFN-γ and TNF-α with simultaneous HER-2 and HER-3 blockade. Results: All breast cancer cell lines tested express IFN-γ and TNF-α receptors measured by western blot analysis. The high and intermediate HER-2 expressing cells are sensitive to tumor senescence induction when treated with combinations of IFN-γ and TNF-α in a dose dependent manner. Low HER-2 expressing cells were less sensitive to senescence induction as measured by positive β-galactosidase activity and the expression of p15INK4b and p16INK4a by western blot. Addition of IFN-γ and TNF-α treatment to HER-2-depleted cells by RNAi resulted in an increase senescent phenotype and was increased further when the cells were double HER-2- and HER-3-depleted. To determine whether CD4 Th1 mediated effects on high HER-2 human breast cancer cell lines, we co-cultured increasing number of HER-2 antigen-primed CD4+ T cells with high HER-2 human breast cancer cells in a transwell cell culture system. This resulted in a dose-dependent senescence of breast cancer cells compared with CD4+ T cells primed either with immature dendritic cells (DC) or mature DC plus irrelevant (Class II BRAF) peptide. In addition, SK-BR-3 breast cancer cells incubated with the supernatant of the CD4+ T cells-immature DC or mature DC co-culture demonstrated similar results. CD4+ Th1-elaborated cytokines IFN-γ and TNF-α in the supernatants were confirmed using ELISA. Blocking antibodies against IFN-γ and TNF-α demonstrated reduced senescence induction. Conclusions: Our results establish a role for IFN-γ and TNF-α in inducing tumor senescence in breast cancer. An effective CD4 Th1 responses combined with HER-2 and HER-3 blockade can significantly drive tumor senescence in breast cancer that can be explored as treatment to effectively eliminate residual breast cancer cells and prevent recurrence. Citation Format: Cinthia Rosemblit, Brian J Czerniecki. CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor senescence in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-05-03.

The role of IFN-γ in regulating Nfkbiz expression in epidermal keratinocytes.

Nfkbiz is an inhibitor of nuclear factor κB (IκB) protein localized to the nucleus. We previously found that Nfkbiz gene-disrupted mice showed atopic dermatitis-like lesion, implying the important role of Nfkbiz in skin homeostasis. The purpose of this study was to examine the effect of interferon (IFN)-γ on Nfkbiz expression in keratinocytes. IFN-γ induced Nfkbiz expression at a comparable level to IL-1. Promoter analysis revealed that interferon-stimulated response element (ISRE) located in the Nfkbiz promoter region is important for responding to the stimulation. Interestingly, IFN-γ and IL-1 displayed synergism in terms of inducing Nfkbiz expression. By using selective inhibitors, we found that Janus activated kinase (JAK) 1 and nuclear factor (NF)-κB are important for Nfkbiz expression after IFN-γ stimulation and for synergism between IFN-γ and IL-1. These findings indicate a possible important role of Nfkbiz in modulating the progression of inflammatory diseases in which IFN-γ and IL-1 are abundant.

Insights into the possible role of IFNG and IFNGR1 in Kala-azar and Post Kala-azar Dermal Leishmaniasis in Sudanese patients.

BackgroundLittle is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-γ receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-γ (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan.MethodsSanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test.ResultsTen variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low.ConclusionsUniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0662-5) contains supplementary material, which is available to authorized users.

Loss of EP2 receptor subtype in colonic cells compromise epithelial barrier integrity by altering claudin-4.

Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.

S-16: Function and regulation of IL-17 cytokine family in infection and autoimmunity

CD4+T cells that secrete IL-17 (Th17 cells) play a pathogenic role in many autoimmune diseases, but also function with Th1 cells to mediate protective immunity to infection by promoting recruitment and activation of neutrophils and macrophages respectively. Stimulation of dendritic cells by pathogen-derived molecules promotes maturation and the production of T-cell differentiating cytokines. We have shown that TLR and NLR agonists induce innate IL-1 and IL-18 which synergize with IL-23 to activate memory Th17 cells, but also IL-17 production by γδ T cells without TCR engagement. TLR signalling also leads to IL-12 production, which promotes induction of Th1 cells. Consequently TLR agonists are effective adjuvants for vaccines against infectious disease. We have demonstrated that TLR2 or TLR9 agonists are potent adjuvants for an acellular pertussis vaccine (aP) and considerably enhance its efficacy over the currently licensed aP which is administered with alum as the adjuvant. Whooping cough is a re-emerging infectious disease and the current aP induces Th2 and Th17 responses, but weak Th1 responses. Interestingly, Th2 responses were redundant and protection with alum-adjuvanted aP is mediated by Th17 cells, driven by alum-activated IL-1β. In contrast, immunization with an aP formulation containing a novel TLR2 lipopeptide from Bordetella pertussis or a TLR9 agonist induced strong Th1 and IgG2a responses and a high level of protection against respiratory infection with Bordetella pertussis. In the field of autoimmunity, much of the focus in drug discovery has been on IL-17A production by CD4+ Th17 cells and its induction by IL-23. However CD8+ T cells, gd T cells, NKT cells and other innate lymphoid cells are all capable of secreting IL-17A. There is also evidence that other cytokines, including IL-17F, GM-CSF, IL-21 and IL-22, are produced by Th17 and IL-17A-secreting innate immune cells. Furthermore, the role of IFN-γ, secreted by Th1, NK and type 1 innate lymphoid cells (ILC1) cells, is still unclear, with some evidence that IFN-γ is protective in autoimmunity by inhibiting Th17 cells. However, we have demonstrated in experimental autoimmune encephalomyelitis, a mouse model for multiple sclerosis, that early NK cell-derived IFN-γ plays a pathogenic role through activation of M1 macrophages and VLA4 expression on T cells, required for their migration into the CNS. The induction and function of Th1 and Th17 cells is regulated by cytokines secreted by the other major subtypes of T cells, especially IL-10 and TGF-β production by Treg cells but also by regulatory cells of the innate immune system. The induction of adaptive Treg cells is stimulated by retinoic acid, TGF-β and IL-10 in response to certain virulence factors from pathogens, such as helminth parasites that have evolved sophisticated mechanisms to subvert host protective immunity. Pathogens and pathogen-derived molecules can also promote activation of alternatively activated M2 macrophages, ILC2 and tolerogenic dendritic cells that can suppress Th1 or Th17 cells, either directly or through the induction of Treg cells. We have identified approaches for activation of anti-inflammatory cytokines, regulatory innate immune cells and Treg cells, without Th1 or Th17 cells. These approaches have been effective in attenuating inflammatory disease in pre-clinical models of autoimmunity.

The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling.

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

Stimulation of invariant natural killer T cells by α-Galactosylceramide activates the JAK-STAT pathway in endothelial cells and reduces angiogenesis in the 5T33 multiple myeloma model.

Tumour pathogenesis in multiple myeloma (MM) correlates with a high vascular index. Therefore, targeting angiogenesis is an important therapeutic tool to reduce MM progression. This study aimed to investigate the role of invariant natural killer T (iNKT) cells in angiogenesis and the mechanisms behind the stimulation by α-Galactosylceramide (α-GalCer). We have previously found that α-GalCer could increase the survival of 5T33MM mice and here we demonstrate that α-GalCer reduces the microvessel density. We performed both in vivo and in vitro angiogenic assays to confirm this observation. We found that conditioned medium of α-GalCer stimulated iNKT cells reduced neovascularization in the chick chorioallantoic membrane and in matrigel plug assays. Moreover, we observed a reduction in proliferation, migration and network formation and an induction of apoptosis upon exposure of murine endothelial cell lines to this conditioned medium. We furthermore observed that the JAK-STAT signaling pathway was highly activated in endothelial cells in response to stimulated iNKT cells, indicating the possible role of IFN-γ in the anti-angiogenic process. In conclusion, these results highlight the possibility of recruiting iNKT cells to target MM and angiogenesis. This gives a rationale for combining immunotherapy with conventional anti-tumour treatments in view of increasing their therapeutic potential.

Innate IFN-γ promotes development of experimental autoimmune encephalomyelitis: a role for NK cells and M1 macrophages.

The role of IFN-γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN-γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN-γ during development of EAE. Depletion of NK cells or neutralization of IFN-γ delayed the onset of EAE and was associated with reduced infiltration of IL-17(+) and GM-CSF(+) T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)-immunized IFN-γ(-/-) mice failed to induce EAE, despite producing IL-17 and GM-CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen-4 (VLA-4) expression and failed to infiltrate the CNS. Addition of recombinant IFN-γ to immune cells from the IFN-γ(-/-) mice activated M1 macrophages and restored VLA-4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti-VLA-4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN-γ-producing T cells are not required for development of EAE, but NK cell-derived IFN-γ has a key role in promoting M1 macrophage expansion and VLA-4-mediated migration of encephalitogenic T cells into the CNS.

Further evidence for the role of interferon-gamma on anxiety- and depressive-like behaviors: Involvement of hippocampal neurogenesis and NGF production

A series of evidence suggests that interferon-gamma (IFN-γ) plays an important role in central nervous system (CNS) functions. However, previous studies have obtained inconsistent results regarding the role of IFN-γ in modulating emotion-related behaviors. The present study aimed to evaluate the behavioral profile of IFN-γ knockout (K.O.) mice in models of anxiety and depression. Male C57Bl6 wild type (WT) or IFN-γ K.O. mice were submitted to the following tests: contextual fear conditioning (CFC), elevated plus maze (EPM), open field (OF) and forced swimming test (FST). To explore the possible neurobiological mechanisms involved, we also assessed hippocampal neurogenesis by means of hippocampal doublecortin expression, and the levels of brain-derived neurothophic factor (BDNF) and nerve growth factor (NGF) in the hippocampus and prefrontal cortex. Our results suggested that IFN-γ K.O. mice exhibited an anxiogenic profile in CFC, EPM and OF tests. In FST, the K.O. group spent more time immobile than the WT group. The number of doublecortin positive cells was reduced in the dentate gyrus, and the expression of NGF was down regulated in the prefrontal cortex of IFN-γ K.O. mice. Our results suggest that IFN-γ is involved in CNS plasticity, contributing to the modulation of anxiety and depressive states.

Role of IFN-γ in the regulation of murine hemopoiesis

This study was aimed to explore the role and mechanism of IFN-γ in the regulation of hemopoiesis in mice. Murine IFN-γ fragment was amplified from murine splenic cells with RT-PCR and plasmid pCDH1-mIFN-γ-EF1-copGFP (pCDH-mIFN-γ-GFP) was constructed. Plasmids pCDH-mIFN-γ-GFP and pCDH1-EF1-copGFP (pCDH-GFP) together with packaging plasmids pPACK-A, pPACK-B and pPACK-C were respectively transfected into 293T cells by using a method of calcium phosphate precipitation to produce lentivirus. Bone marrow mononuclear cells (BMMNC) from male C57BL/6J mice were transfected with the lentiviral vector pCDH expressing mIFN-γ and green fluorescent protein (GFP). The cells were cultured in M3434 semi-solid medium for colony formation assay and transplanted into lethally-irradiated mice through caudal vein injection, and the peripheral blood cell counts and GFP were monitored regularly after transplantation. The results showed that lentiviral vector pCDH-mIFN-γ-GFP was constructed successfully and 293T cells transfected with mIFN-γ secreted mIFN-γ. Transfection of mIFN-γ into BMMNC decreased colony formation, colony number of the mIFN-γ group was significantly less than that of the control group. The recovering of circulating blood cell parameters in mIFN-γ transplantation group was significantly later than control group. GFP positive cells could be detected in the peripheral blood at 8 weeks after transplantation. It is concluded that mIFN-γ may inhibit the colony-forming capacity of transduced BMMNC and delay the hematopoietic reconstitution.

61 Interferon-Gamma Interplay With Serotonin Metabolism and Mucosal Permeability in Irritable Bowel Syndrome

Background and aim: A variety of peripheral mechanisms may be involved in the pathophysiology of irritable bowel syndrome (IBS). These include alterations in serotonin metabolism, epithelial permeability, composition of microbiota and immune system. In vitro studies demonstrated that interferongamma (IFN-γ) enhanced paracellular permeability and decreased serotonin reuptake transporter (SERT) expression. In this study we assessed the potential role of IFN-γ in IBS pathophysiology. Material and methods: We included a total of 10 healthy controls (HC) and 26 patients with Rome III IBS. In all cases, we obtained 6 mucosal biopsies from the descending colon. Paraffin-embedded colonic biopsies were used to evaluate IFN-γ mRNA expression. Total RNA was retrotranscribed into cDNA and analysed in real-time PCR assays. Enzyme-linked immunosorbent assays (ELISA) were used to evaluate IFN-γ levels both in colonic biopsy homogenates and into supernatants of cultured biopsies. Supernatants of cultured biopsies were used to treat Caco-2 cell line to evaluate their effect on SERT expression level by real-time RT-PCR assays. Supernatants were also used to evaluate their effect on Caco-2 paracellular permeability. Results: IFN-γ mRNA expression was significantly higher in patients with IBS compared to HC (2-11Ct = 2.57±0.6 vs 2-11Ct = 0.66±0.1; P<0.05). In addition, we showed increased protein levels of IFN-γ in biopsy homogenates of patients with IBS vs HC (increase of 48% in IBS-D; P<0.05). Similarly, we demonstrated a higher concentration of IFN-γ protein in IBS supernatants. The treatment of Caco-2 cells with IBS supernatants induced a significant decrease in SERT expression compared to HC supernatants. IBS supernatants produced an increase in Caco-2 cell permeability. The effects of IBS supernatants on Caco-2 cells were mediated, at least in part, by IFN-γ. Conclusions: We demonstrated an increased expression of IFN-γ in the colonic mucosa of patients with IBS. We also showed increased IFN-γ amount both in biopsies and supernatants of patients with IBS. Epithelial permeability was enhanced by IBS supernatants which induced also a reduction of SERT expression, at least in part via IFN-γ. All together, these data suggest a potential role of IFN-γ in IBS pathophysiology.

OxLDL-induced lipid accumulation in glomerular podocytes: role of IFN-γ, CXCL16, and ADAM10.

Previous studies have shown that lipid accumulation plays an important role in the pathogenesis and development of glomerular sclerosis. oxLDL caused damage in renal mesangial cells, endothelial cells, and podocytes, and podocytes might be the major victim of oxLDL insult. However, the regulatory mechanism of how oxLDL induces the damage of podocytes remains to be elucidated. In this study, oil red staining was used to investigate the lipid accumulation in podocytes. Moreover, the effects of CXCL16 antibody, IFN-γ, and ADAM10 inhibitor on oxLDL intake and CXCL16 expression were also explored to elucidate the regulatory factors of lipid accumulation in podocytes.

Cutting Edge: IFN-γR Signaling in Non–T Cell Targets Regulates T Cell–Mediated Intestinal Inflammation through Multiple Mechanisms

Naive CD4 T cells transferred into lymphopenic mice undergo spontaneous proliferation and induce chronic inflammation in the intestine. Cellular mechanisms regulating the proliferative and inflammatory processes are not fully understood. In this study, we report that IFN-γ signaling in host cells plays a major role in limiting both T cell expansion and T cell-induced intestinal inflammation. However, the role of IFN-γ appears to differ depending on the target cells. IFN-γ signaling in dendritic cells controls T cell expansion, whereas IFN-γ signaling in neutrophils seems to regulate both T cell expansion and inflammation. IFN-γ signaling in nonhematopoietic cells may control inflammation. Therefore, our results suggest novel immunoregulatory functions for IFN-γ to orchestrate colitogenic T cell responses through its distinct action on different non-T cell target cells.

Immune mechanisms in postpartum psychosis

A series of evidence suggests that interferon-gamma (IFN-γ) plays an important role in central nervous system (CNS) functions. However, previous studies have obtained inconsistent results regarding the role of IFN-γ in modulating emotion-related behaviors. The present study aimed to evaluate the behavioral profile of IFN-γ knockout (K.O.) mice in models of anxiety and depression. Male C57Bl6 wild type (WT) or IFN-γ K.O. mice were submitted to the following tests: contextual fear conditioning (CFC), elevated plus maze (EPM), open field (OF) and forced swimming test (FST). To explore the possible neurobiological mechanisms involved, we also assessed hippocampal neurogenesis by means of hippocampal doublecortin expression, and the levels of brain-derived neurothophic factor (BDNF) and nerve growth factor (NGF) in the hippocampus and prefrontal cortex. Our results suggested that IFN-γ K.O. mice exhibited an anxiogenic profile in CFC, EPM and OF tests. In FST, the K.O. group spent more time immobile than the WT group. The number of doublecortin positive cells was reduced in the dentate gyrus, and the expression of NGF was down regulated in the prefrontal cortex of IFN-γ K.O. mice. Our results suggest that IFN-γ is involved in CNS plasticity, contributing to the modulation of anxiety and depressive states.