IFN-γ signals a changing of the guards

After acute inflammation or infection, tissue-resident macrophages are replaced by inflammatory DCs and effector macrophages that arise from circulating monocytes. In this issue of Immunity, Goldszmid et?al. (2012) demonstrate a central role for NK cell-derived IFN-γ in signaling and facilitating this transition.     

Effect of amino acid substitutions in the human IFN-γR2 on IFN-γ responsiveness

Patients with interferon-γ receptor (IFN-γR) null mutations have severe infections with poorly pathogenic Mycobacteria. The IFN-γR complex involves two IFN-γR1 and two IFN-γR2 chains, in which several amino acid substitutions, some linked to disease and some apparently naturally occurring, have been described. We developed a model system to study functional effects of genetic variations in IFN-γR2. We retrovirally transduced wild-type IFN-γR2 and IFN-γR2 carrying presently known amino acid substitutions in various human cell lines, and next determined the IFN-γR2 expression pattern as well as IFN-γ responsiveness. We determined that the T58R, Q64R, E147K and K182E variants of IFN-γR2 are fully functional, although the Q64R variant may be expressed higher on the cell membrane. The R114C, T168N and G227R variants were identified in patients that had disseminated infections with non-tuberculous Mycobacteria. Of these genetic variants, T168N was confirmed to be completely non-functional, whereas the novel variant G227R, and the previously reported R114C, were partial functional. The impaired IFN-γ responsiveness of R114C and G227R is mainly due to reduced receptor function, although expression on the cell membrane is reduced as well. We conclude that the T58R, Q64R, E147K and K182E variants are polymorphisms, whereas the R114C, T168N and G227R constitute mutations associated with disease.     

Regulation of tumor growth by IFN-γ in cancer immunotherapy

Tumor immunity involves a concerted interplay between cytokines and effector cells. Extensive efforts have focused on understanding the roles of cytokines and their interactions with effector cells for the production of effective tumor immunity. One cytokine that is well recognized to play a central role in coordinating tumor immune responses is IFN-γ. IFN-γ exerts its biological effects through interaction with an IFN-γ receptor that isubiquitously expressed on nearly all cells. In this review, we discuss the positive and negative effects of IFN-γ signaling in the tumor cell on tumor growth.     

Effect of IFN-γ receptor gene deletion on vaccinia virus virulence

Summary.  Two vaccinia virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-γ receptor (IFN-γR) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-γR in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits. Received March 13, 2000 Accepted August 16, 2000     

IFN-λs

For decades, type I IFNs have been considered indispensable and unique antiviral mediators for the activation of rapid innate antiviral protection. However, the recent discovery of type III IFNs is challenging this paradigm. Since their identification in 2002/2003 by two independent groups, type III IFNs or IFN-λs, also known as IL-28/29, have been the subject of increased study with consequent recognition of their importance in virology and immunology. Initial reports suggested that IFN-λs functionally resemble type I IFNs. Although IFN-λs and classical type I IFNs (IFN-α/β) utilize distinct receptor complexes for signaling, both types of IFNs activate similar intracellular signaling pathways and biological activities, including the ability to induce antiviral state in cells, and both type I and type III IFNs are induced by viral infection. However, different antiviral potency, pattern of their induction and differential tissue expression of their corresponding receptor subunits suggest that the type I and type III IFN antiviral systems do not merely duplicate each other. Recent studies have started to reveal unique biological activities of IFN-λs in and beyond innate antiviral immunity.     

Suppression of Th17 response by Streptococcus thermophilus ST28 through induction of IFN-γ

The proinflammatory cytokine interleukin (IL)-17 plays important roles in various inflammatory diseases, and IL-17-producing T helper 17 cells (Th17) have received much attention. For therapy of Th17-mediated diseases, some reports have indicated the clinical efficacy of lactic acid bacteria, including Streptococcus thermophilus. In this study, we examined the mechanism for the suppressive effects of S. thermophilus ST28 on the Th17 response in murine splenocytes stimulated with transforming growth factor (TGF)-β plus IL-6. Stimulation with TGF-β plus IL-6 increased mRNA expression of IL-17 and its production in the splenocytes, but ST28 markedly suppressed both. Meanwhile, ST28 increased the mRNA expression of interferon (IFN)-γ as well as its production. Anti-IFN-γ completely cancelled the suppressive effect of ST28 on IL-17 production. From these data, it was concluded that IFN-γ induced by ST28 had an important role on the effect. A genomic DNA (10 μg/ml) from ST28 effectively suppressed IL-17 production, probably via the Toll-like receptor 9. Therefore, modulation of Th1/Th17 balance would be one of the mechanisms under which S. thermophilus ST28 exerts an anti-inflammatory effect.     

Anti-tumoral capabilities of effector cells after IFN-α or CpG-motif treatment of cocultured dendritic cells

Introduction Ex vivo expansion of monocyte-derived dendritic cells (mDCs) and subsequent coculture with autologous cytokine-induced killer (CIK) cells is an established system to create specific and non-specific anti-tumoral immunity. mDCs constitute the most frequently applied DC subset in clinical studies. One recently published approach to optimize the immunological functions of the DC/CIK cell system is the replacement of interleukin (IL)-4 by interferon (IFN)-α in the maturation process of the DCs. Materials and Methods The expressions of relevant surface antigens of IL-4-DCs and IFNα-DCs by flow cytometry and the anti-tumoral activation of effector cells cocultured with both types of DCs using cytotoxicity assays were compared. In addition, short-term coculture experiments with both types of DCs and IFNγ-LAK effector cells were performed and compared with standard CIK cell coculture experiments. Results Regarding the expressions of functionally relevant surface markers, no differences could be detected for CD80, CD83, and HLA-DR between IFNα-DCs and IL-4-DCs, whereas the mean fluorescence intensities of CD40, CD86, CD54, and HLA-ABC were decreased and the expression of CD14 was increased for IFNγ-DCs. Moreover, no enhancement of cytotoxicity of cocultured CIK cells against tumor cell lines (A498 and SW480) was detected by the use of IFNα-DCs. Additionally, coculture experiments with IFNγ-LAK cells were performed and unexpectedly higher lysis rates in comparison with the established IL-4-DC/CIK coculture model was observed. Early incubation of the mDCs with several CpG-ODNs failed to increase the anti-tumoral cytotoxicity of the cocultured IFNγ-LAK cells. Conclusions These results demonstrate that in the mDC/CIK cell system, IFNα-DCs are not superior in inducing anti-tumoral cytotoxicity and even moderately inferior regarding the expression of functionally relevant surface markers compared with IL-4-DCs. Michael Erhardt and Ingo G. H. Schmidt-Wolf contributed equally.     

Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

Gamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses upon Mycobacterium bovis infection in cattle by using archived samples from two aerosol inoculation studies. In the first study (10⁴ CFU M. bovis by aerosol, n = 7), M. bovis purified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r = 0.87). In the second study (10⁵ CFU M. bovis by aerosol, n = 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r ≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.     

Study of immunological aspects of aspergillosis in mice and effect of polyene macrolide antibiotic (SJA-95) and IFN-γ: a possible role of IFN-γ as an adjunct in antifungal therapy

New polyene macrolide antibiotic SJA-95 in free as well as liposomal (lip.) forms, with and without interferon-γ (IFN-γ) was studied in mice model of aspergillosis using biological and biochemical parameters viz. colony forming units (CFU) in liver, spleen, kidney, lung and brain, and serum IgG, and interleukin-4 (IL-4). Treatment with free and lip SJA-95 along with IFN-γ prolonged the survival time, reduced CFU in vital organs, decreased serum IgG and IL-4 levels. SJA-95 lip form showed greater antifungal activity as compared to free form. The combined treatment of lip SJA-95 with IFN-γ showed further enhancement in antifungal activity of SJA-95 (lip). The present experimental findings demonstrated IFN-γ might act as a potent modulator in immune reaction during fungal infection and can be a useful adjunctive in antifungal therapy in the management of deep seated systemic mycoses.     

Strain-to-strain difference of V protein of measles virus affects MDA5-mediated IFN-β-inducing potential

Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-β promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-β promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-β promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-β promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of vaccine MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-β-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA.     

Impaired IFN-γ production and proliferation of NK cells in multiple sclerosis

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patients.     

Ameliorated course of glucose-6-phosphate isomerase (G6PI)-induced arthritis in IFN-γ receptor knockout mice exposes an arthritis-promoting role of IFN-γ

The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen.     

IFN-γ induces IFN-α and IFN-β expressions in cultured rat intestinal mucosa microvascular endothelial cells

Although researchers have recently begun to pay more attention to the immunological characteristics of microvascular endothelial cells (MVECs), there are no reports on whether activation of MVECs by interferon-γ (IFN-γ) exerts any influence on the expressions of IFN-α/β. In the present study, we examined the influence of IFN-γ on the expressions of IFN-α/β in rat intestinal mucous MVECs (RIMMVECs). Different concentrations of IFN-γ were used to stimulate cultured RIMMVECs in vitro, and the cells and cell supernatants were collected at different time intervals. The influence of IFN-γ on the expressions of IFN-α/β in the RIMMVECs was examined at the mRNA and protein levels by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that IFN-γ was able to activate RIMMVECs, thereby leading to upregulated expressions of IFN-α/β. The real-time quantitative PCR analyses indicated that the IFN-α/β mRNA expression levels in RIMMVECs achieved their peak values after stimulation with IFN-γ at 20?ng/mL for 6?h and were increased by 14.88- and 3.82-fold, respectively, when compared with the levels in negative control cells. The ELISA analyses revealed that the IFN-α/β protein expression levels achieved their peak values after stimulation with IFN-γ at 40?ng/mL. The expression of IFN-α protein achieved its peak value at 12?h, while the expression of IFN-β protein achieved its peak value after 6?h. The present results suggest that the expression and secretion of IFNs may participate in the immunologic barrier function of MVECs.