IFN-γ Transcripts Research Articles

Abstract 602: A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function

Abstract Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder chimeric antigen receptor (CAR) T cell efficacy. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. A dominant negative receptor version (DNR) of TGFBR2 that does not contain signaling domains protects T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs. Here, we report the development of a novel TGF-β signal conversion platform that provides a T cell stimulatory signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of the T cell simulating IL-12 receptors IL-12R-β2 and IL-12R-β1, respectively. Using a single lentiviral vector encoding both chimeric TGF-β receptors (CTBR) and a CAR, we demonstrated that CAR-CTBR T cells were completely protected from TGF-β-mediated SMAD phosphorylation. In addition, CAR-CTBR T cells generated significant amounts of pSTAT4 and pSTAT5 in response to TGF-β exposure, a response that mimics the T cell stimulation effects of IL-12. To further demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on the secretion of IFNγ, a major downstream target of IL-12 signaling. CAR-CTBR cells secreted significantly greater amounts of IFNγ than either control CAR T or CAR-DNR T cells following activation in the presence of TGF-β. Lastly, we utilized a serial restimulation assay to expand CAR, CAR-DNR, and CAR-CTBR T cells in the presence or absence of TGF-β. As expected, TGF-β exposure resulted in a significant inhibition of T cell proliferation in control CAR T cells. By contrast, both CAR-DNR and CAR-CTBR cells were protected from TGF-β-mediated inhibition of expansion. Gene expression analysis following 21 days of weekly antigen-driven expansion revealed specific TGF-β-mediated gene expression changes in CAR-CTBR cells consistent with increased T cell potency, including significant upregulation of IFNγ, IL10, IL18RAP, IL18R1, IL21R and CD62L transcripts. These data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into an IL-12R-like T cell stimulatory signal that has the potential to produce superior CAR T cell responses in vivo. Note: This abstract was not presented at the meeting. Citation Format: Benjamin Boyerinas, Sara Miller, Ryan Murray, Stacie Seidel, Geoffrey Parsons, Kathy Seidl, Kevin Friedman, Richard Morgan. A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2017-602

Pathogenic Role of Associated Adherent-Invasive Escherichia coli in Crohn's Disease.

Several lines of evidence suggest that adherent-invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. Organ cultures of colonic biopsies from patients were set up to assess the effects of LF82 and O83:H1 on the expression of CEACAM6, LAMP1, HLA-DR, ICAM1 by immunohistochemistry and of IL-8, IFNʏ, and TNF-α genes by RT-PCR. Moreover, on Caco2 cells, we analyzed the cell cycle, the expression of MGMT and DNMT1 genes, and DNA damage induced by LF82 and O83:H1, by FACS, RT-PCR, and DAPI staining, respectively. Epithelial and lamina propria mononuclear cells (LPMNC) expression of CEACAM6 and LAMP1 were higher in biopsies cultured in the presence of both O83:H1 and LF82 than in biopsies cultured with non-pathogenic E. coli. Both AIEC strains induced increased expression of ICAM-1 on blood vessels and HLA-DR on LPMNC. We observed higher levels of TNF-α, IFN-γ, and IL-8 transcripts in biopsies cultured with both AIEC strains than in those cultured with NP. Both LF82 and O83:H1, block the cell cycle into S phase, inducing DNA damage, and modulate the expression of DNMT1 and MGMT genes. Our data suggest that LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro-inflammatory cytokines and all the mucosal immune markers investigated. J. Cell. Physiol. 232: 2860-2868, 2017. © 2016 Wiley Periodicals, Inc.

Francisella noatunensis ssp. noatunensis iglC deletion mutant protects adult zebrafish challenged with acute mortality dose of wild-type strain.

The intracellular fish pathogen Francisella noatunensis remains an unsolved problem for aquaculture worldwide and an efficient vaccine is needed. In Francisella sp., IglC is an important virulence factor necessary for intracellular growth and escape from phagolysosomes. Deletion of the intracellular growth locus C (iglC) in Francisella sp. causes attenuation, but vaccine potential has only been attributed to ΔiglC from Francisella noatunensis ssp. orientalis, a warm-water fish pathogen. A ΔiglC mutant was constructed in the cold-water fish pathogen F. noatunensis ssp. noatunensis (Fnn), which causes francisellosis in Atlantic cod; the mutant was assessed in primary head kidney leucocytes from Atlantic cod. Fluorescence microscopy revealed reduced growth, while qPCR revealed an initial increase followed by a reduction in mutant genomes. Mutant-infected cod leucocytes presented higher interleukin 1 beta (il1β) and interleukin 8 (il8) transcription than wild-type (WT)-infected cells. Two doses of mutant and WT were tested in an adult zebrafish model whereupon 3 × 109 CFU caused acute disease and 3 × 107 CFU caused low mortality regardless of strain. However, splenomegaly developed only in the WT-infected zebrafish. Immunization with 7 × 106 CFU of Fnn ΔiglC protected zebrafish against challenge with a lethal dose of Fnn WT, and bacterial load was minimized within 28 d. Immunized fish had lower interleukin 6 (il6) and il8 transcription in kidney and prolonged interferon-gamma (ifng) transcription in spleens after challenge compared with non-immunized fish. Our data suggest an immunogenic potential of Fnn ΔiglC and indicate important cytokines associated with francisellosis pathogenesis and protection.

Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development

TGF-β is known to influence tumour progression. Here we report an additional role of Smad3 in the tumour microenvironment regulating cancer progression. Deletion or inhibition of Smad3 in the tumour microenvironment suppresses tumour growth, invasion and metastasis in two syngeneic mouse tumour models. Smad3−/− bone marrow gives rise to an expanded NK cell population with enhanced tumour-suppressive activities in vivo, and promotes differentiation of NK cells ex vivo. We identify E4BP4/NFIL3 as a direct Smad3 target gene critical for NK cell differentiation. Smad3 suppresses transcription of IFN-γ via E4BP4 in a T-bet independent manner. Therefore disruption of Smad3 enhances both the E4BP4-mediated NK cell differentiation and anti-cancer effector functions in vivo and in vitro. Furthermore, systemic treatment with a Smad3 inhibitor SIS3 effectively suppresses cancer progression. In summary, suppression of NK cell-mediated immunosurveillance via the Smad3-E4BP4 axis contributes to cancer progression. We propose targeting Smad3-dependent tumour microenvironment may represent an effective anti-cancer strategy.

Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P < 0.01). Conversely, iNOS mRNA and protein expression decreased according to refeeding time, with a significant negative correlation between iNOS protein level and mucosal height being recorded during the 48-h refeeding period ( r = -0.898, P < 0.01). We also noted a significant negative correlation between jejunal neuronal nitric oxide synthase and iNOS protein concentrations over this same period ( r = -0.734, P < 0.01). Refeeding also restored the decreased jejunal MI caused by fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation and apoptosis in crypts and villi. These novel findings elucidate the relationship between these NOS isoforms and its impact on recovery from intestinal injury. A mechanism is proposed comprising the up-regulation of nNOS activity by mechanical stimulation due to the presence of food in the intestine, restricting iNOS-associated apoptosis and promoting cell proliferation and gut motility. Our investigation sheds light on the molecular basis behind the repercussions of total parenteral nutrition on intestinal mucosal integrity, and more importantly, the beneficial effects of early enteral feeding.

Combined Single-Cell Measurement of Cytokine mRNA and Protein Identifies T Cells with Persistent Effector Function.

Effective T cell responses entail the coproduction of IFN-γ, TNF-α, and IL-2. Cytokine production is determined by transcriptional and posttranscriptional events. However, increased transcript levels do not always translate into protein production, and therefore simultaneous transcripts and protein measurement are essential for the appropriate analysis of T cell responses. In this study, we optimized flow cytometry-based fluorescence in situ hybridization (Flow-FISH) for IFN-γ to multicolor flow cytometry that allows for single-cell measurement of mRNA and protein levels. This high-throughput analysis detected Ag-specific human T cells of low frequency. We also employed Flow-FISH for single-tube analysis of IFN-γ transcript and protein profile to simultaneously study the responsiveness of different T cell subsets, that is, naive, effector, and memory T cells. Importantly, the simultaneous transcript and protein analysis of IFN-γ and of TNF-α and IL-2 revealed that T cell responses consist of two types: one subtype loses mRNA expression during activation, whereas the other maintains high transcript levels throughout stimulation. High cytokine transcript levels correlated with increased protein production. Intriguingly, this mRNAhi-expressing T cell population also produced higher levels of other cytokines, indicating that Flow-FISH helps identify the best cytokine producers during T cell activation. We conclude that Flow-FISH is a rapid, sensitive, and cost-effective method to determine the quality of T cell responses induced by, for instance, T cell vaccines.

Electro-acupuncture at Acupoint ST36 Ameliorates Inflammation and Regulates Th1/Th2 Balance in Delayed-Type Hypersensitivity.

Increasing evidence indicates anti-allergic and anti-inflammatory effects of electro-acupuncture (EA) therapy. However, its underlying mechanism on delayed-type hypersensitivity (DTH), a classic allergic inflammatory disease, still remains unclear. In this study, we aimed to explore the immunomodulatory mechanism of EA intervention in a mouse model of ovalbumin (OVA)-induced DTH. Mice were randomly divided into four groups: Control, OVA-DTH, DTH + EA, DTH + Sham. "Zusanli" acupoint (ST36) was used for DTH + EA, whereas a non-acupoint (localized 5mm below the "Zusanli" acupoint) was selected for DTH + Sham. Footpad thickness was checked, and the infiltration of inflammatory cells was estimated by hematoxylin and eosin staining. Levels of IgG and IgE in serum of different groups and inflammatory cytokines in the supernatants from homogenized footpads, including IFN-γ, TNF-α, IL-4, and IL-5, were determined by ELISA. Cell proliferation of spleen lymphocytes was assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). The frequency of CD4+IFN-γ+ and CD4+IL-4+ T cells was analyzed with flow cytometry. In addition, the mRNA and protein expression of T-bet and GATA-3 were evaluated by real-time PCR and Western blotting, respectively. Our data showed EA treatment at acupoint ST36 relieved the pathological progression of DTH responses via reduction in footpad swelling, infiltration of inflammatory cells, levels of IgG and IgE as well as decreased production of IFN-γ and TNF-α in homogenized footpad tissue. Moreover, detailed studies were performed revealing that EA attenuated the percentage of CD4+IFN-γ+ T cells and prevented Th cells differentiation into Th1 cells, and this results from inhibiting secretion of IFN-γ and suppressing expression of T-bet, an IFN-γ transcription factor. The results indicated that EA treatment improved Th1-mediated allergic skin inflammation via restoring Th1/Th2 balance by curbing Th1 differentiation. These findings suggested that EA at acupoint ST36 might be a useful and promising therapeutic for allergic inflammatory as well as Th1-mediated inflammation response.

Context-Specific and Immune Cell-Dependent Antitumor Activities of α1-Antitrypsin.

α1-antitrypsin (AAT), a circulating glycoprotein that rises during acute phase responses and healthy pregnancies, exhibits immunomodulatory properties in several T-cell-dependent immune pathologies. However, AAT does not directly interfere with T-cell responses; instead, it facilitates polarization of macrophages and dendritic cells towards M2-like and tolerogenic cells, respectively. AAT also allows NK cell responses against tumor cells, while attenuating DC-dependent induction of autoimmune NK cell activities. Since AAT-treated macrophages bear resemblance to cancer-promoting tumor-associated macrophages (TAMs), it became imperative to examine the possible induction of tumor permissive conditions by AAT. Here, AAT treatment is examined for its effect on tumor development, metastatic spread, and tumor immunology. Systemic AAT treatment of mice inoculated with B16-F10 melanoma cells resulted in significant inhibition of tumor growth and metastatic spread. Using NK cell-resistant RMA cells, we show that AAT interferes with tumor development in a CD8+ T-cell-dependent manner. Unexpectedly, upon analysis of tumor cellular composition, we identified functional tumor-infiltrating CD8+ T-cells alongside M1-like TAMs in AAT-treated mice. Based on the ability of AAT to undergo chemical modifications, we emulated conditions of elevated reactive nitrogen and oxygen species. Indeed, macrophages were stimulated by treatment with nitrosylated AAT, and IFNγ transcripts were significantly elevated in tumors extracted soon after ischemia-reperfusion challenge. These context-specific changes may explain the differential effects of AAT on immune responses towards tumor cells versus benign antigenic targets. These data suggest that systemically elevated levels of AAT may accommodate its physiological function in inflammatory resolution, without compromising tumor-targeting immune responses.

Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence?

Background: Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One mechanism used by pre-malignant and/or malignant cells to escape immune surveillance is the upregulation of inhibitory immune checkpoint molecules, such as PD-1 and PD-L1. Another process associated with aging is genetic or epigenetic modifications of tumor suppressor genes (TSGs). We have reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG(1). We thus examined - on a molecular level - the expression of specific transcription factors (BACH2 and PRDM1) and checkpoint molecules (PD-1 and PD-L1) in the major lymphocytes subsets for their potential as predictive markers of immunosenescence.Methods: Lymphocyte subpopulations (CD3+/CD4+; CD3+/CD8+ and CD19+) were isolated for subsequent molecular analyses using the MACS technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. PD-1 (PDCD1), PD-L1 (CD274), IL4, IFNG, BACH2 and PRDM1 (Blimp1) mRNA transcripts were quantified in the purified subpopulations using qRT-PCR. BACH2 and PRDM1 protein expression were examined by Western blotting. Peripheral blood mononuclear cells were treated by Etoposide (VP16) to induce intracellular oxidative stress. Apoptotic function was assessed by flow cytometry (Annexin-V and Propidium Iodide).Results: Peripheral blood samples were obtained from 40 healthy donors (HDs) between the ages of 20 to 90 yrs, subdivided into <50 yrs (n=21; median: 36yo) and ³50 yrs (n=19; median: 61yo). And 40 blood samples from untreated patients with chronic lymphocytic leukemia (CLL; median: 67yo) were comparatively analyzed.BACH2-deficient mice have been shown to have increased numbers of IL4-producing CD4+ T cells after activation(2), with BACH2 known to repress PRDM1 expression in B andT cells(2,3). We, therefore, examined BACH2 gene expression in the HD groups finding it was significantly downregulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.001; 0.004 and 0.03, respectively). BACH2 expression was further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to HD of similar age (p=0.001; <0.001 and 0.004). In contrast, PRDM1 was significantly upregulated in CD4+ and CD8+ T cells (p=0.003; 0.001) from CLL patients but not in their leukemic B cells. As expected, BACH2 expression was inversely correlated with PRDM1 in CD4+, CD8+ T cells and CD19+ B cells (r=0.57; 0.71 and 0.62, respectively). Western blot analysis demonstrated that BACH2 and Blimp1 (PRDM1) protein expressions in the T and B cell subpopulations were significantly correlated with transcript expression. We also observed that BACH2 downregulation is correlated with increased IL-4 gene expression (r=0.61) but not IFNγ in CD4+ T cells. These observations suggest that BACH2 downregulation in CD4+ T cells could enhance expressed Th2-related genes, such as IL-4 and PRDM1.PD-1 gene expression was significantly upregulated in CD4+, CD8+ T cells (p=0.015 and 0.02) in the older HD group. PD-1 was also significantly upregulated in the same T cells subpopulations isolated from untreated CLL patients (p=0.001 and 0.002) when compared to a similar HD age group. High PD-L1 gene expression was correlated with increased age in HD B cells (p=0.04) with a further increase detected in leukemic B cells compared with the older HD group (p=0.001). We also observed an inverse correlation between BACH2 and PD-1 in CD4+, CD8+ T cells (r=0.59 and 0.68); and between BACH2 and PD-L1 in CD19+ B cells (r=0.66).We further examined VP16-induced apoptosis in isolated lymphocytes subpopulations. As recently reported in BACH2-deficient mice(4), our observation showed that BACH2 downregulation was strongly correlated with resistance to apoptosis in CD4+, CD8+ T cells and leukemic B cells (r=0.61; 0.75 and 0.69, respectively).Conclusion: These data suggest that BACH2, PRDM1, PD-1 and PD-L1 gene expression is correlated with aging and age-related immunosuppression. These effects are even more pronounced in leukemic cells from CLL patients.1. C. Sibille, et al.,(personal data) ; 2. S. Tsukumo, et al. Proc. Natl Acad Sci USA. 2013 ; 3. H. Tanaka et al. J Biol Chem. 2016 ; 4. Muto, A., et al. J Biol Chem. 2002; DisclosuresNo relevant conflicts of interest to declare.

β-1,3/1,6-Glucan-supplemented diets antagonize immune inhibitory effects of hypoxia and enhance the immune response to a model vaccine

The diets of farmed salmon are usually supplemented with immunostimulants to improve health status. Because β-glucan is one of the most common immunostimulants used in diets, here we examined the effect of two β-1,3/1,6-glucan-supplemented diets on the expression of immune response genes of Atlantic salmon. The relative abundances of IFN-α1, Mx, IFN-γ, IL-12, TGF-β1, IL-10, and CD4 transcripts were evaluated in head kidney by qRT-PCR. We assessed the effects of the diets under normoxia and acute hypoxia, as salmon are especially sensitive to changes in the concentration of dissolved oxygen, which can also affect immunity. These effects were also tested on vaccinated fish, as we expected that β-1,3/1,6-glucan-supplemented diets would enhance the adaptive immune response to the vaccine. We found that administration of the Bg diet (containing β-1,3/1,6-glucan) under normoxia had no effects on the expression of the analyzed genes in the kidney of the diet-fed fish, but under hypoxia/re-oxygenation (90 min of acute hypoxia), the βg diet affected the expression of the antiviral genes, IFN-α1 and Mx, preventing their decrease caused by hypoxia. The Bax diet, which in addition to β-1,3/1,6-glucan, contained astaxanthin, increased IL-12 and IFN-γ in kidneys. With fish exposed to hypoxia/reoxygenation, the diet prevented the decrease of IFN-α1 and Mx levels observed after hypoxia. When fish were vaccinated, only the levels of IL-12 and CD4 transcripts increased, but interestingly if fish were also fed the Bax diet, the vaccination induced a significant increase in all the analyzed transcripts. Finally, when vaccinated fish were exposed to hypoxia, the effect of the Bax diet was greatly reduced for all genes tested and moreover, inducible effects completely disappeared for IL-12, IFN-α, and Mx. Altogether, these results showed that the tested β-1,3/1,6-glucan diets increased the levels of transcripts of key genes involved in innate and adaptive immune response of salmon, potentiating the response to a model vaccine and also antagonizing the effects of hypoxia.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility.Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-β1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01).To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Erythropoietin treatment in murine multiple myeloma: immune gain and bone loss.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by osteolytic lesions and monoclonal immunoglobulins. The anemia, accompanying the disease is often treated with recombinant human EPO. Diverse non-erythropoietic effects of EPO have led us to question its combined action on the immune system and bone in the 5T33MM mouse model. EPO administration to MM mice attenuated disease progression as demonstrated by a decrease in serum MM IgG2b, splenic CD138 expressing cells, IL-6 and RORγτ transcripts in bone marrow (BM). IFN-γ transcript levels and macrophages (F4/80+CD11b+) in the BM both increased ~1.5 fold in the EPO-treated MM mice. In-vitro, EPO stimulated phagocytosis of 5T33MM cells (+30%) by BM-derived macrophages. In contrast, high-resolution microCT analysis of distal femurs revealed EPO-associated bone loss in both healthy and 5T33MM mice. EPO significantly increased expression of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in healthy mice, but not in MM mice, likely due to antagonizing effects on MM progression. Thus, in MM, EPO may act as a double-edged-sword stimulating immune response, while accelerating bone resorption, possibly via direct action on BM macrophages. This study supports a prudent approach of treating anemia in MM patients, aiming to maintain EPO-associated anti-MM effects, while considering bone damage.

The kinetics and protection of the antiviral state induced by recombinant iIFN1a in rainbow trout against infectious hematopoietic necrosis virus.

The iIFN1a (intracellular IFN-a1), that is one of the IFN-a1 variants, was shown to be functional intracellularly and act as a novel defense against an infectious hematopoietic necrosis virus (IHNV). To determine its antiviral properties, a recombinant iIFN1a was generated in Escherichia coli. Its antiviral activity against IHNV was 1.69×10(7)U/mg in CHSE-214 cells. Additionally, iIFN1a was capable of inducing comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver tissues at an early time point (6h), that was followed by a rapid decline 24h after induction. The recombinant protein also elicited protection against IHNV in vivo. At 6 and 24h after induction there was 100% protection against the virus, however, at 48 and 72h the protection decreased to 57 and 40%, respectively. The in vivo protection kinetics correlated with the kinetics of gene expression. The results of this study provide details of the antiviral state that was induced by iIFN1a in vivo for the first time. Additionally, this information will facilitate the development of this recombinant protein as a potential anti-viral treatment and/or adjuvant.

Transmembrane protein 147 (TMEM147): another partner protein of Haemonchus contortus galectin on the goat peripheral blood mononuclear cells (PBMC).

BackgroundRecombinant galectins of male and female Haemonchus contortus (rHco-gal-m/f) have been recognized as significant regulators of the functions of goat peripheral blood mononuclear cells (PBMC). In previous research, transmembrane protein 63A (TMEM63A) was identified as a partner protein in the regulation associated with H. contortus infection. However, in the identification of binding partners for galectins of male and female H. contortus (Hco-gal-m/f) by yeast two-hybrid (YTH) screening, it was found that the transmembrane protein 147 (TMEM147) could also bind to Hco-gal-m/f. In this study, the functions of TMEM147 in the regulations of H. contortus galectin on the goat PBMC were investigated.MethodsTo identify Hco-gal-m/f-interacting proteins, a yeast two-hybrid system to detect interactions was used. Co-immunoprecipitation and immunoblotting were used to validate the interaction between recombinant galectins of male H. contortus (rHco-gal-m) and candidate binding protein. The localization of TMEM147 in PBMC was explored by immunofluorescence in confocal imaging studies. Flow cytometry was used to determine the distribution of TMEM147 in T cells, B cells and monocytes in PBMC. The modulatory effects of rHco-gal-m and TMEM147 on cell proliferation, phagocytosis, nitric oxide production, migration, apoptosis and cytokine mRNA transcription were observed by co-incubation of rHco-gal-m and knockdown of the tmem147 gene.ResultsIn this research, it was demonstrated that TMEM147 could bind to rHco-gal-m/f. Immunofluorescence assays showed that TMEM147 was localized to the cell membrane and within the cell membrane in goat PBMC. Flow cytometric analysis revealed that TMEM147 was expressed in all B cells and monocytes in goat PBMC. However, 3.8 % of T cells did not express this protein. Knockdown of the tmem147 gene using RNA interference (RNAi) showed that the interaction of galectin with TMEM147 mainly mediated cell proliferation, cell apoptosis, transcription of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) of goat PBMC. This membrane protein, together with TMEM63A, was also related to the regulation of galectin on phagocytosis and nitric oxide production of goat PBMC. However, it might not be involved in the regulation of galectin on the migration and interferon-γ (IFN-γ) transcription of goat PBMC.ConclusionsOur results showed that TMEM147 was a binding partner of Hco-gal-m/f and mediated the immunological regulation of Hco-gal-m/f on goat PBMC in a manner different to that of TMEM63A.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1640-0) contains supplementary material, which is available to authorized users.

Treatment with synthetic lipophilic tyrosyl ester controls Leishmania major infection by reducing parasite load in BALB/c mice

Synthesized lipophilic tyrosyl ester derivatives with increasing lipophilicity were effective against Leishmania (L.) major and Leishmania infantum species in vitro. These findings prompted us to test in vivo leishmanicidal properties of these molecules and their potential effect on the modulation of immune responses. The experimental BALB/c model of cutaneous leishmaniasis was used in this study. Mice were infected with L. major parasites and treated with three in vitro active tyrosyl esters derivatives. Among these tested tyrosylcaprate (TyC) compounds, only TyC10 exhibited an in vivo anti-leishmanial activity, when injected sub-cutaneously (s.c.). TyC10 treatment of L. major-infected BALB/c mice resulted in a decrease of lesion development and parasite load. TyC10 s.c. treatment of non-infected mice induced an imbalance in interferon γ/interleukin 4 (IFN-γ/IL-4) ratio cytokines towards a Th1 response. Our results indicate that TyC10 s.c. treatment improves lesions' healing and parasite clearance and may act on the cytokine balance towards a Th1 protective response by decreasing IL-4 and increasing IFN-γ transcripts. TyC10 is worthy of further investigation to uncover its mechanism of action that could lead to consider this molecule as a potential drug candidate.

Upregulation of duck interleukin-17A during Riemerella anatipestifer infection

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-β, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1β, IL-6, and IL-17A mRNA expressions, but not TGF-β, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

Functional characterization of the turkey macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in Escherichia coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro-inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-γ and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive.

Tissue-specific transcription profile of cytokine and chemokine genes associated with flavivirus control and non-lethal neuropathogenesis in rabbits

We previously showed that New Zealand White (NZWRs) and cottontail rabbits (CTRs) are a suitable model for studying immune mechanisms behind virus control and non-lethal neuropathogenesis associated with West Nile virus (WNV) and Murray Valley encephalitis virus (MVEV) infections. In the current study, we observed that MVEV infection induced high IFNα, TNFα, IL6, and CXCL10 transcript levels in the brains of weanling NZWRs, unlike infection with the less virulent WNVNSW2011. These transcript levels also correlated with encephalitis severity. Widespread STAT1 protein expression in brain with moderate neuropathology suggests that IFN-I signaling is crucial for limiting neural infection and mediating non-lethal neuropathogenesis. Unlike NZWRs, CTRs limit neuroinvasion without upregulation of many cytokine/chemokine transcripts, suggesting a species-dependent virus control mechanism. However, the common IFNγ, TNFα and IL6 transcript upregulation in specific lymphoid organs suggest some conserved elements in the response against flaviviruses, unique to all rabbits.

Immune response of turkey poults exposed at 1 day of age to either attenuated or wild Salmonella strains

Salmonellosis is a foodborne zoonosis that is most often acquired by consuming poultry products such as eggs and poultry meat. Amongst other measures the vaccination of food-producing poultry is thought to contribute to a reduction in human salmonellosis. In the European Union (EU) in 2014 the licence of a commercially available Salmonella vaccine for chickens and ducks was extended to turkeys. In the present study, we examined the course of infection with a virulent Salmonella enterica ssp. enterica serovar Enteritidis (SE) strain, a virulent S. enterica ssp. enterica serovar Typhimurium (ST) strain, and the respective live vaccine containing attenuated strains of both serovars in turkey poults. Besides collecting microbiological data and detecting invading Salmonella in the caecal mucosa via immunohistochemistry, we also assessed immune reactions in terms of antibody production, influx of CD4-, CD8α- and CD28-positive cells into the caecal mucosa and the expression of four different immune-related proteins. We found that the attenuated strains were able to invade the caecum, but to a lower degree and for a shorter duration of time compared to virulent strains. Infections with virulent Salmonellae also caused an increase in CD4-, CD8α- and CD28-positive cells in the caecal mucosa and an increased transcription of iNOS, IL-8-like chemokines, and IFN-γ. In poults treated with attenuated bacteria we could not detect any evidence of immune responses. In conclusion, the vaccine showed a lower degree of caecal invasion and induced weaker immune reactions compared to the virulent Salmonella strains in turkeys. The efficiency of the vaccine has to be verified in future studies.