Recombinant IFN-γ Research Articles

Abstract LB-232: BiTE antibody constructs can mediate bystander tumor cell killing

Abstract Recent clinical data demonstrate the significance of T cells in anti-tumor activity. For instance, the CD19/CD3 bispecific T cell engager (BiTE) blinatumomab is a proven means of harnessing T cells for cancer treatment. BiTE antibody constructs comprise an anti-CD3 scFv (single chain variable fragment) linked to an scFv binding a tumor-associated antigen (TAA). One potential challenge for TAA-targeted therapeutics is that treatment may only eliminate TAA-expressing tumor cells and heterogeneity of TAA expression becomes a potential means of resistance. To prevent escape of TAA-negative tumor cells, a treatment modality with a bystander effect on TAA-negative cells may be desirable. To evaluate the potential of BiTE antibodies to mediate bystander cell killing, mixtures of TAA-positive and -negative (bystander) cells were co-cultured with human T cells and the effect of BiTE antibodies tested. Lysis of TAA-expressing and bystander cells was evaluated using both imaging and viability assays. For this study, we used BiTE antibodies recognizing either epidermal growth factor receptor (EGFR) or CD33. In the presence of TAA-positive cells, T cells were activated and bystander cells lysed. In the absence of TAA-positive cells, bystander cells were not killed. Bystander cell lysis was also observed in a xenograft mouse model with subcutaneous tumors comprising EGFR-positive and -negative cancer cells, and human T-cells. The mechanism of BiTE-mediated bystander killing was further investigated. In the presence of TAA-positive cells, T cells released many cytokines, including IFN-γ and TNFα. However, exposure of bystander cells to just the soluble factors released by T cells did not induce their lysis, suggesting that a direct interaction between BiTE-activated T cells and bystander cells was required. BiTE treatment induced the expression on bystander cells of intercellular adhesion molecule 1 (ICAM-1), a protein involved in formation of cytolytic T cell synapses with target cells. ICAM-1 upregulation on bystander cells was also observed following exposure to recombinant IFN-γ and TNFα. These findings suggest that exposure of bystander cells to cytokines secreted by BiTE-activated T cells caused ICAM-1 expression on bystander cells leading to their improved attachment and cytolytic synapse formation. Blockade of ICAM-1 by an antibody partially protected bystander cells from lysis. Our data suggest a model where BiTE-activated T cells secrete cytokines that cause upregulation of ICAM-1 on TAA-negative cells. This can then lead to T cell binding and T cell-induced bystander cell lysis. This mechanism is not expected to cause systemic cell death because only those cells proximal to the activated T cell in the tumor environment would be exposed to sufficiently high concentrations of ICAM-1-inducing cytokines. However, this locally confined bystander cell lysis may be sufficient to enable effective treatment of tumors that are heterogeneous for TAA expression. Citation Format: Sandra L. Ross, Marika Mulen, Patricia L. McElroy, Julie Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara L. Arvedson. BiTE antibody constructs can mediate bystander tumor cell killing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-232. doi:10.1158/1538-7445.AM2015-LB-232

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95.

α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored. α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD4 ; and dexamethasone. α-GalCer (10(-6) -10(-8) M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 μg·kg(-1) , i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD4 and NSAIDs. α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD4 , NSAIDs and aminoguanidine, and modified by dexamethasone.

Mo1719 Epithelial NF-κB Activation via TNF Signaling May Be Involved in the Development of Colitis-Associated Carcinogenesis

Background & Aim: Prolonged inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease may promote carcinogenesis in the epithelia. It has been reported that activation of NF-κB pathway in both intestinal epithelial cells and myeloid cells are significant for the development of colitis-associated cancer (CAC). We previously reported that specific up-regulation of the type 2 receptor for tumor necrosis factor (TNFR2) rather than TNFR1 expression was observed in the inflamed colonic epithelia and further in the CAC. It is known that TNFR2 signaling may induce NF-κB activation, but the role of its expression in the setting of CAC has not been elucidated. Here we analyzed TNFR2 signaling in the colonic epithelial cells. Methods & Results: As previously observed in animal models of colitis and CAC, the expression of TNFR2 was up-regulated in an epithelial cell line, MOC1, which was derived from murine colonic ‘non-cancer' tissue, when stimulated with recombinant (r) IFN-γ. MOC1 cells incubated with both rIFN-γ and rTNF showed that NFκB pathway was activated rather than apoptosis pathway. Furthermore, the activated NFκB in MOC1 cells was associated with the expression of myosin light chain kinase (MLCK) as well as disrupted tight junction (TJ) in a rTNF dose-dependent manner. Such MLCK upregulation and TJ disruption in MOC1 cells were abrogated by either anti-TNF mAb (MP6XT22), TNFR2-specific siRNA or even MLCK inhibitor (ML-7). Using an animal model of CAC involving azoxymethane (AOM) and dextran sodium sulfate (DSS), the colonic lamina propria was found to have pro-tumorigenic cytokine production such as IL-1β, IL-6 and MIP-2 in association with epithelial NF-κB activation, TNFR2 and MLCK up-regulations and epithelial TJ disruption. AOM and DSS-administered mice with either MP6-XT22 or ML7 treatment failed to show significant abrogation of colitis severity, however such treatments revealed the restored epithelial TJ and decreased pro-tumorigenic cytokine production in the colonic tissues in association with the reduced CAC development. Conclusions: Our studies showed that epithelial NF-κB activation via TNFR2 signaling in the context of IBD may be involved in the epithelial permeabilization and pro-tumorigenic cytokine production that result in the induction of CAC development.

Vascular smooth muscle cells contribute to APOL1-induced podocyte injury in HIV milieu

Clinical reports have demonstrated that higher rates of non-diabetic glomerulosclerosis in African Americans can be attributed to two coding sequence variants (G1 and G2) in the APOL1 gene; however, the underlying mechanism is still unknown. Kidney biopsy data suggest enhanced expression of APOL1/APOL1 variants (Vs) in smooth muscle cells (SMCs) of renal vasculature. Since APOL1 is a secretory protein of relatively low molecular weight (41kDa), SMCs may be a contributory endocrine/paracrine source of APOL1 wild type (WT)/APOL1Vs in the glomerular capillary perfusate percolating podocytes. In the present study, we tested the hypothesis that an HIV milieu stimulated secretion of APOL1 and its risk variants by arterial SMCs contributes to podocyte injury. Human umbilical artery smooth muscle cells (HSMCs)-treated with conditioned media (CM) of HIV-infected peripheral mononuclear cells (PBMC/HIV-CM), CM of HIV-infected U939 cells, or recombinant IFN-γ displayed enhanced expression of APOL1. Podocytes co-cultured in trans-wells with HSMCs-over expressing APOL1WT showed induction of injury; however, podocytes co-cultured with HSMC-over expressing either APOL1G1 or APOL1G2 showed several folds greater injury when compared to HSMC-over expressing APOL1WT. Conditioned media collected from HSMC-over-expressing APOL1G1/APOL1G2 (HSMC/APOL1G1-CM or HSMC/APOL1G2-CM) also displayed higher percentages of injured podocytes in the form of swollen cells, leaky lysosomes, loss of viability, and enhanced sensitivity to adverse host factors when compared to HSMC/APOL1WT-CM. Notably, HSMC/APOL1WT-CM promoted podocyte injury only at a significantly higher concentrations compared to HSMC/APOL1G1/G2-CM. We conclude that HSMCs could serve as an endocrine/paracrine source of APOL1Vs, which mediate accelerated podocyte injury in HIV milieu.

Mechanisms of interferon-beta-induced inhibition ofToxoplasma gondiigrowth in murine macrophages and embryonic fibroblasts: role of immunity-related GTPase M1

The apical complex of Toxoplasma gondii enables it to invade virtually all nucleated cells in warm-blooded animals, including humans, making it a parasite of global importance. Anti-T. gondii cellular defence mechanisms depend largely on interferon (IFN)-γ production by immune cells. However, the molecular mechanism of IFN-β-mediated defence remains largely unclear. Here, mouse peritoneal macrophages and murine embryonic fibroblasts (MEFs) primed with recombinant IFN-β and IFN-γ showed different pathways of activation. Treatment of these cells with IFN-β or IFN-γ inhibited T. gondii (type II PLK strain) growth. Priming macrophages with IFN-β had no effect on inflammatory cytokine expression, inducible nitric oxide synthase or indoleamine 2,3-dioxygenase, nor did it have an effect on their metabolites, nitric oxide and kynurenine respectively. In contrast, IFN-γ stimulation was characterized by classical macrophage activation and T. gondii elimination. IFN-β activation recruited the immunity-related GTPase M1 (IRGM1) to the parasitophorous vacuole in the macrophages and MEFs. Anti-toxoplasma activities induced by IFN-β were significantly reduced after IRGM1 knockdown in murine macrophages and in IRGM1-deficient MEFs. Thus, this study unravels an alternative pathway of macrophage activation by IFN-β and provides a mechanistic explanation for the contribution of IRGM1 induced by IFN-β to the elimination of T. gondii.

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice.

BackgroundRespiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.MethodsNormal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.ResultsRSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.ConclusionsMMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0176-8) contains supplementary material, which is available to authorized users.

TIR-Domain-Containing Adaptor-Inducing Interferon-β (TRIF) Mediates Antibacterial Defense during Gram-Negative Pneumonia by Inducing Interferon-γ

Klebsiella pneumoniae is an important cause of Gram-negative pneumonia and sepsis. Mice deficient for TIR-domain-containing adaptor-inducing interferon-β (TRIF) demonstrate enhanced bacterial growth and dissemination during Klebsiella pneumonia. We show here that the impaired antibacterial defense of TRIF mutant mice is associated with absent interferon (IFN)-γ production in the lungs. IFN-γ production by splenocytes in response to K. pneumoniae in vitro was critically dependent on Toll-like receptor 4 (TLR4), the common TLR adaptor myeloid differentiation primary response gene (MyD88) and TRIF. Reconstitution of TRIF mutant mice with recombinant IFN-γ via the airways reduced bacterial loads in lungs and distant body sites to levels measured in wild-type mice, and partially restored pulmonary cytokine levels. The IFN-γ-induced, improved, enhanced antibacterial response in TRIF mutant mice occurred at the expense of increased hepatocellular injury. These data indicate that TRIF mediates antibacterial defense during Gram-negative pneumonia, at least in part, by inducing IFN-γ at the primary site of infection.

Effect of rCaIFN-γ pretreatment on propofol–isoflurane suppression of NK cytotoxic activity in the peripheral blood of dogs

In dogs, natural killer (NK) cell cytotoxic activity, which is an index of antitumor immunity, decreases after general anesthesia. In this study, we examined whether the decrease in NK cytotoxic activity can be controlled with interferon-gamma (IFN-γ) treatment. Beagles were divided into 2 groups: a treated group (n = 6) that received recombinant canine IFN-γ (rCaIFN-γ) and an untreated control group (n = 6). Blood samples were taken before and at 24, 120, and 192 hours after anesthesia. NK cytotoxic activity toward canine thyroid cancer cells was measured in isolated lymphocytes with the Rose Bengal assay. The decrease in NK cytotoxic activity after anesthesia was significantly inhibited by administration of rCaIFN-γ before propofol–isoflurane anesthesia. Further studies are necessary to evaluate the clinical implications of rCaIFN-γ treatment in tumor recurrence and morbidity.

Editor’s Letter

<h3>Background</h3> Interferon-gamma (IFN-γ) plays an important role in the development of lupus nephritis (LN). Regulation of IFN-γ signaling that occurs in disease remission and in active LN is herein addressed. <h3>Objectives</h3> To study the impact of IFN-γ on PBMC obtained from patients with LN in remission as compared to active LN. <h3>Methods</h3> Sixteen patients fulfilling the ACR classification criteria for systemic lupus erythematosus were recruited. All patients had a history of LN of whom 10 were in remission (as defined by EULAR criteria) and 6 had active LN (as defined by SLEDAI-2K or BILAG). Healthy subjects (n=10) were included as a control group. Sera and PBMC were obtained from each individual. Flow cytometry, western blots and real time RT-PCR were used in processing and detection of cell subtypes, protein and mRNA levels. Recombinant human IFN-γ (rhIFN-γ) and anti-IFN-γ neutralizing antibody were used in vitro. Mann-Whitney and student t-tests were used for statistical analysis. <h3>Results</h3> In active LN there was a significant 2-fold increase in CD4⁺CD69⁺ activated T cells as compared to healthy subjects and patients in remission. Reactivity to interferon-gamma receptor was determined by the phosphorylation of its predominant transcription factor, signal transducer and activator of transcription 1 (STAT1) in the cells that were incubated with rhIFN-γ or with media alone. In active LN, 3- and 6-fold increase in pSTAT1 occurred with rhIFN-γ incubation during 24h and 48h, respectively, and healthy subjects responded likewise. In patients in remission, pSTAT1 increase was even higher (by 8- and 10-fold at 24h and 48h, respectively). After 24h incubation with rhIFN-g all groups had elevated (mRNA) expression of SOCS1, but at 48h, it significantly decreased in healthy subjects and in patients in remission by 34% and 50%, respectively. Further, at 24h the frequency of CD4⁺CD127^lowFoxP3⁺ regulatory T cells (e.g. Tregs) increased by 27–30% in active LN and in healthy subjects, and in remission it was minimally changed. At 48h, the frequency of Tregs significantly decreased in healthy subjects (to baseline levels before rhIFN-γ challenge) and in patients in remission (by 24%, p=0.003). A challenge of PBMC from LN patients in remission with sera (at 1% concentration) derived from patients with active LN resulted in 7.5-fold increase in pSTAT1 expression and 20–30% decrease in Tregs, however, combination of sera and anti-IFN-g neutralizing antibody resulted in 5-fold decrease in pSTAT1 expression and significantly diminished the decrease in Tregs. <h3>Conclusions</h3> In LN in remission a challenge with IFN-γ could lead to immune activation and a risk of flare-up, as it results in a decrease in both SOCS1 and Tregs and a robust STAT1 phosphorylation. In active LN, STAT1 phosphorylation is less diminishing, as both SOCS1 and Tregs are saturated, which could affect their suppressive effectiveness. <h3>Disclosure of Interest</h3> None declared

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Clinical implications of interferon-γ genetic and epigenetic variants.

Interferon-γ (IFN-γ) is an integral and critical molecule of the immune system, with multiple functions, mostly related to the T helper type 1 (Th1) response to infection. It is critical for defence against mycobacterial infection and is of increasing interest in defence against fungi. In this article, we review the genetic and epigenetic variants affecting IFN-γ expression and investigate its role in disease, with an emphasis on fungal diseases such as invasive and chronic pulmonary aspergillosis. Over 347 IFN-γ gene variants have been described, in multiple ethnic populations. Many appear to confer a susceptibility to disease, especially tuberculosis (TB) and hepatitis, but also some non-infectious conditions such as aplastic anaemia, cervical cancer and psoriasis. Several epigenetic modifications are also described, increasing IFN-γ expression in Th1 lymphocytes and reducing IFN-γ expression in Th2 lymphocytes. Recombinant IFN-γ administration is licensed for the prophylaxis of infection (bacterial and fungal) in patients with the phagocyte functional deficiency syndrome chronic granulomatous disease, although the benefits appear limited. Interferon-γ therapy is given to patients with profound defects in IFN-γ and interleukin-12 production and appears to be beneficial for patients with invasive aspergillosis and cryptococcal meningitis, but the studies are not definitive. A high proportion of patients with chronic pulmonary aspergillosis are poor producers of IFN-γ in response to multiple stimuli and could also benefit from IFN-γ administration. The investigation and management of patients with possible or demonstrated IFN-γ deficiency in adulthood is poorly studied and could be greatly enhanced with the integration of genetic data.

Type II interferon promotes differentiation of myeloid-biased hematopoietic stem cells.

Interferon gamma (IFNγ) promotes cell division of hematopoietic stem cells (HSCs) without affecting the total HSC number. We postulated that IFNγ stimulates differentiation of HSCs as part of the innate immune response. Here, we report that type II interferon signaling is required, both at baseline and during an animal model of LCMV infection, to maintain normal myeloid development. By separately evaluating myeloid-biased and lymphoid-biased HSC subtypes, we found that myeloid-biased HSCs express higher levels of IFNγ receptor and are specifically activated to divide after recombinant IFNγ exposure in vivo. While both HSC subtypes show increased expression of the transcription factor C/EBPβ after infection, only the myeloid-biased HSCs are transiently depleted from the marrow during the type II interferon-mediated immune response to Mycobacterium avium infection, as measured both functionally and phenotypically. These findings indicate that IFNγ selectively permits differentiation of myeloid-biased HSCs during an innate immune response to infection. This represents the first report of a context and a mechanism for discriminate utilization of the alternate HSC subtypes. Terminal differentiation, at the expense of self-renewal, may compromise HSC populations during states of chronic inflammation.

Abstract 2895: Small cell lung cancer cells express the late stage gBK tumor antigen: a possible immunotarget for the terminal disease

Abstract Big Potassium (BK) ion channels have several splice variants. One splice variant initially described within human glioma cells is called the glioma BK channel (gBK). This isoform consists of 33 amino acids inserted into the intracellular region of the BK ion channel. Using a gBK-specific antibody, we detected gBK within 3 human small cell lung cancer (SCLC) lines. Patch clamping revealed that functional membrane channels were found on the SCLC cells. Prolonged exposure to BK channel activators caused the SCLC cells to swell within 20 minutes and resulted in their cytotoxicity within 5 hours. Transfection of BK-negative HEK cells with gBK produced functional gBK channels. Quantitative RT-PCR analysis using primers specific for gBK, but not with a lung-specific marker, Sox11, confirmed that advanced, late-stage human SCLC tissues strongly expressed gBK mRNA. Normal human lung tissue and early, lower stage SCLC resected tissues very weakly expressed this transcript. Immunofluorescence using the gBK antibodies confirmed that SCLC cells taken at the time of autopsy intensely displayed this protein. gBK may represent a late-stage marker for SCLC. HLA-A*0201 restricted human CTL were generated in vitro by one-way mixed lymphocyte reactions or mixed lymphocyte dendritic cell reactions, the latter using gBK peptide pulsed dendritic cells. The exposure of SCLC cells to interferon-γ (IFN-γ) increased the expression of HLA; these treated cells were killed by the CTL better than non-IFN-γ treated cells even though the IFN-γ treated SCLC cells displayed diminished gBK protein expression. Prolonged incubation with recombinant IFN-γ slowed the in vitro growth and migration of the SCLC cells, suggesting it might inhibit tumor growth in vivo. Immunotherapy targeting gBK might possibly impede advancement to terminal state of SCLC. Citation Format: Neil T. Hoa, Lisheng Ge, Rajeev B. Tajhya, Christine Beeton, Andrew N. Cornforth, Amir Abolhoda, Nils Lambrecht, Maria Dacosta-Iyer, Yi Ouyang, Michelle J. Hickey, Kate L. Erickson, Carol A. Kruse, Martin R. Jadus. Small cell lung cancer cells express the late stage gBK tumor antigen: a possible immunotarget for the terminal disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2014-2895

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.

Pro-inflammatory interferon gamma signaling is directly associated with stroke induced neurodegeneration.

The delayed immune response to stroke is responsible for the increased neural injury that continues to occur after the initial ischemic event. This delayed immune response has been linked to the spleen, as splenectomy prior to middle cerebral artery occlusion (MCAO) is neuroprotective. Interferon gamma (IFNγ) is linked to the splenic response, which enhances neural injury following MCAO. IFNγ activates the expression of the inflammatory chemokine interferon-inducible protein 10 (IP-10). This study was designed to determine the role of IFNγ signaling in the inflammatory response following MCAO. Expression of IP-10 increased in the brain and the spleen following MCAO. Splenectomy inhibited the increase of IP-10 in the brain post-MCAO, while recombinant IFNγ administration to splenectomized rats returned IP-10 levels in the brain to levels found in rats after MCAO only. Systemic administration of an IFNγ neutralizing antibody to MCAO-treated rats reduced infarct volume and IP-10 levels in the brain. T cell infiltration was reduced in the MCAO-damaged brains of IFNγ antibody-treated animals relative to those that received isotype control antibodies. Additionally, inhibiting IFNγ signaling with splenectomy or an IFNγ neutralizing antibody blocked the induction of IP-10 expression and decreased neurodegeneration following MCAO. Targeting this pro-inflammatory pathway following stroke could be a promising stroke therapeutic.

Type II interferon promotes differentiation of myeloid-biased hematopoietic stem cells

Interferon gamma (IFNγ) promotes cell division of hematopoietic stem cells (HSCs) without affecting the total HSC number. We postulated that IFNγ stimulates differentiation of HSCs as part of the innate immune response. Here, we report that type II interferon signaling is required, both at baseline and during an animal model of LCMV infection, to maintain normal myeloid development. By separately evaluating myeloid-biased and lymphoid-biased HSC subtypes, we found that myeloid-biased HSCs express higher levels of IFNγ receptor and are specifically activated to divide after recombinant IFNγ exposure in vivo. While both HSC subtypes show increased expression of the transcription factor C/EBPβ after infection, only the myeloid-biased HSCs are transiently depleted from the marrow during the type II interferon-mediated immune response to Mycobacterium avium infection, as measured both functionally and phenotypically. These findings indicate that IFNγ selectively permits differentiation of myeloid-biased HSCs during an innate immune response to infection. This represents the first report of a context and a mechanism for discriminate utilization of the alternate HSC subtypes. Terminal differentiation, at the expense of self-renewal, may compromise HSC populations during states of chronic inflammation.

Optimization of conditions for expression of recombinant interferon-γ in E.coli.

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.

Endocytic and NBT-reduction activities and TNF expression by macrophages and monocytes of the armadillo (Dasypus novemcinctus)

Summary The armadillo could be considered as an experimental model for leprosy studies. Mycobacterium leprae main host cells are macrophages and Schwann cells. However, endocytosis and germicidal activities of armadillo macrophages and monocytes have not yet been evaluated. The aim of this research was to evaluate endocytosis and NBT-reduction activities of monocyte-derived macrophages (MDM) and monocytes from non-infected armadillos, as well as to determine tumor necrosis factor (TNF) levels during stimulation with phorbol myristate acetate (PMA). Mononuclear cells of peripheral blood were purified using the ficollhypaque technique. The monocytes obtained were transformed to MDM after being incubated for 8 days in an RPMI-1640 medium supplement with fetal calf serum at 37°C. Endocytosis and NBT-reduction were evaluated by yeast ingestion. TNF production by stimulation with PMA was evaluated through a bioassay with L-929 cells. The results revealed an endocytosis of 71.77 ± 4.11% and 60.66 ± 6.9% (P<0.001) for armadillo MDM and monocytes respectively, whereas NBT-reduction was 16.57 ± 4.79% and 17.66 ± 4.08%, respectively. With the addition of recombinant human IFNγ for 24 h, endocytosis and NBT reduction by MDM were 89.92 ± 6.17% (P<0.001) and 36.42 ± 4.31% (P<0.001), respectively. Low levels of TNF are produced (10 U/ml) when MDM and armadillo monocytes are stimulated with 25 and 50 ng/ml of PMA, in comparison with human U-937 cells. In conclusion, MDM and armadillo monocytes show diminished activities in oxidative bactericidal mechanisms and in TNF production when stimulated with PMA.

Interferon-gamma enhances phagocytosis, the production of reactive oxygen species and pro-inflammatory cytokines – implications for innate and acquired immunity.

Neutrophils and interferon-γ (IFN-γ) are known critical components of innate and acquired immune responses. However, recent studies have demonstrated that these two immune functions are not entirely isolated. Treatment of neutrophils with IFN-γ elicits a variety of responses depending on the stimulus and on environmental conditions. In view of the physiological and pathophysiological importance of the regulatory activity of IFN-γ in neutrophil functions and the diverse, sometimes conflicting results reported, we decided to investigate the effects of this cytokine on phagocytosis, the production of reactive oxygen species (ROS) and the release of lysosome enzymes, as mediated by different types of immune receptors in mouse neutrophils. We found increased phagocytic capacity of mouse neutrophils, which was accompanied by the up-regulation of FcγR, CR, and the dectin-1 receptor. The increase in ROS production mediated by these receptors with the activation of NADPH oxidase is correlated with the increased expression of p47 phox and gp91 phox mRNA. In addition to enhanced phagocytosis, we also observed the release of some lysosomal enzymes. The increased production of reactive oxygen species may be important for the enhanced capacity for killing phagocytosed microorganisms but may also favor the induction of oxidative stress in adjacent cells. In agreement with this potential inflammatory role, we also demonstrated the increased release of the inflammatory cytokines TNF-α and IL-6 from neutrophils treated with recombinant IFN-γ. These results show that IFN-γ can act as a signaling molecule for neutrophils, modulating their functions in innate as well as acquired immunity. This Research Highlight discusses the findings of our recent study and active research endeavors.

Transient transfection of CHO cells using linear polyethylenimine is a simple and effective means of producing rainbow trout recombinant IFN-γ protein.

A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32°C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10µg per 5ml of RPMI1640 culture medium, with cells subsequently cultured at 32°C for 7days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations.