IFN-γ mRNA Research Articles

Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

S3I-201, a selective stat3 inhibitor, ameliorates clinical symptoms in a mouse model of experimental autoimmune encephalomyelitis through the regulation of multiple intracellular signalling in Th1, Th17, and treg cells.

CD4+T cells, specifically Th cells (Th1 and Th17) and regulatory T cells (Tregs), play a pivotal role in the pathogenesis of multiple sclerosis (MS), a demyelinating autoimmune disease of the CNS. STAT3 inhibitors are potential therapeutic targets for several immune disorders. In this study, we investigated the role of a well-known STAT3 inhibitor, S3I-201, in experimental autoimmune encephalomyelitis (EAE), a model of MS. Following induction of EAE, mice were intraperitoneally administered S3I-201 (10 mg/kg) each day, beginning on day 14 and continuing till day 35 and were evaluated for clinical signs. Flow cytometry was used to investigate further the effect of S3I-201 on Th1 (IFN-γ, STAT1, pSTAT1, and T-bet), Th17 (IL-17A, STAT3, pSTAT3, and RORγt), and regulatory T cells (Treg, IL-10, TGF-β1, and FoxP3) expressed in splenic CD4+ T cells. Moreover, we analyzed the effects of S3I-201 on mRNA and protein expression of IFN-γ, T-bet, IL-17A, STAT1, STAT3, pSTAT1, pSTAT3, RORγ, IL-10, TGF-β1, and FoxP3 in the brains of EAE mice. The severity of clinical scores decreased in S3I-201-treated EAE mice compared to vehicle-treated EAE mice. S3I-201 treatment significantly decreased CD4+IFN-γ+, CD4+STAT1+, CD4+pSTAT1+, CD4+T-bet+, CD4+IL-17A+, CD4+STAT3+, CD4+pSTAT3+, and CD4+RORγt+ and increased CD4+IL-10+, CD4+TGF-β1+, and CD4+FoxP3+ in the spleens of EAE mice. Additionally, S3I-201 administration in EAE mice significantly decreased the mRNA and protein expression of Th1 and Th17 and increased those of Treg. These results suggest that S3I-201 may have novel therapeutic potential against MS.

CXCR3 antagonist NBI-74330 mitigates joint inflammation in Collagen-Induced arthritis model in DBA/1J mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. We used the CXCR3-specific antagonist NBI-74330 to block T-cell-mediated signaling in a DBA/1J mouse model of collagen-induced arthritis (CIA). After CIA induction, DBA/1J mice were treated with NBI-74330 (100mg/kg) daily from day 21 until day 34 and evaluated for arthritic score and histopathological changes. Furthermore, using flow cytometry, we investigated the effects of NBI-74330 on Th1 (IFN-γ, TNF-α, T-bet, STAT4, Notch-3, and RANKL), Th17 (IL-21, IL-17A, STAT3, and RORγt), and Th22 (IL-22) cells in splenic CD4+ and CXCR3+T-cells. We also used RT-PCR to assess the effect of mRNA levels of IFN-γ, TNF-α, T-bet, RANKL, IL-17A, RORγt, and IL-22 in knee tissues. The IFN-γ, TNF-α, and IL-17A serum protein levels were measured using ELISA. Compared to vehicle-treated CIA mice, the severity of arthritic scores and histological severity of inflammation decreased significantly in NBI-74330-treated CIA mice. Moreover, compared to vehicle-treated CIA mice, the percentages of CD4+IFN-γ+, CD4+TNF-α+, CD4+T-bet+, CD4+STAT4+, CD4+Notch-3+, CXCR3+IFN-γ+, CXCR3+TNF-α+, CXCR3+T-bet+, CXCR3+STAT4+, CXCR3+Notch-3+, CD4+RANKL+, CD4+IL-21+, CD4+IL-17A+, CD4+STAT3+, CD4+RORγt+, and CD4+IL-22+ cells decreased in NBI-74330-treated CIA mice. Furthermore, NBI-74330-treatment downregulated IFN-γ, TNF-α, T-bet, RANKL, STAT3, IL-17A, RORγt, and IL-22 mRNA levels. Serum IFN-γ, TNF-α, and IL-17A levels were significantly lower in NBI-74330-treated CIA mice than in vehicle-treated CIA mice. This study demonstrates the antiarthritic effects of NBI-74330 in CIA mice. Therefore, these data suggest that NBI-74330 could be considered a potential RA treatment.

Evaluation of the in vivo and in vitro interleukin-12 p40 and p35 subunit response in yellowtail (Seriola quinqueradiata) to heat-killed Lactobacillus plantarum strain L-137 (HK L-137) supplementation, and immersion challenge with Lactococcus garvieae

Dietary supplementation of immunostimulants might be effective to reduce the economic losses due to infectious diseases and the use of antibiotics in aquaculture. To investigate the immune response of interleukin-12 (IL-12) in yellowtail Seriola quinqueradiata to heat-killed Lactobacillus plantarum strain L-137 (HK L-137), we performed a leukocyte culture, feeding trial with diets containing L-137 and an immersion challenge with Lactococcus garvieae. IL-12 (IL-12p70) is a heterodimeric cytokine composed of IL-12p35 and IL-12p40 subunits. In the yellowtail-leukocyte culture, HK L-137 treatment stimulated the mRNA expression of one IL12p35 subunit (p35a) and all IL12p40 subunits (p40a, p40b, and p40c) in a dose-dependent manner. Furthermore, mRNA expression of type-I helper (Th-1) cytokine (tumor necrosis factor α, TNF-α, and interferon γ, IFN-γ) was also stimulated by HK L-137. After 6 weeks of feeding yellowtails with diets containing 0, 20, and 100 ppm of HK L-137, the mRNA expression of p35a and p40b in the spleen leukocytes increased with the dietary concentration of HK L-137, and that of p40b, p40c, and ifng in the head kidney leukocytes were the highest in the 20 ppm HK L-137 group. Survival rates in the 20 ppm HK L-137 group after immersion challenge with L. garvieae were significantly higher than the control (0 ppm of HK L-137). The 100 ppm HK L-137 group did not significantly suppress mortality. HK L-137 showed immunostimulant activity by increasing the expression of il-12, tnfa, and ifng mRNA in both in vitro and in vivo tests in yellowtail. Our results suggest that dietary supplementation with 20 ppm HK L-137 is the most efficient dose for improving immunity in yellowtail. Furthermore, a high dose of HK L-137 and/or long-term feeding of a diet containing HK L-137 might suppress the immune response, which probably decreases the survival rate of fish. To maintain a high immune response in yellowtail, the optimal dietary concentration of HK L-137 and/or feeding regime should be investigated further.

COVID-19 adenovirus vector vaccine induces higher interferon and pro-inflammatory responses than mRNA vaccines in human PBMCs, macrophages and moDCs

BackgroundDuring the COVID-19 pandemic multiple vaccines were rapidly developed and widely used throughout the world. At present there is very little information on COVID-19 vaccine interactions with primary human immune cells such as peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages and dendritic cells (moDCs). MethodsHuman PBMCs, macrophages and moDCs were stimulated with different COVID-19 vaccines, and the expression of interferon (IFN-λ1, IFN-α1), pro-inflammatory (IL-1β, IL-6, IL-8, IL-18, CXCL-4, CXCL-10, TNF-α) and Th1-type cytokine mRNAs (IL-2, IFN-γ) were analyzed by qPCR. In addition, the expression of vaccine induced spike (S) protein and antiviral molecules were studied in primary immune cells and in A549 lung epithelial cells. ResultsAdenovirus vector (Ad-vector) vaccine AZD1222 induced high levels of IFN-λ1, IFN-α1, CXCL-10, IL-6, and TNF-α mRNAs in PBMCs at early time points of stimulation while the expression of IFN-γ and IL-2 mRNA took place at later times. AZD1222 also induced IFN-λ1, CXCL-10 and IL-6 mRNA expression in monocyte-derived macrophages and DCs in a dose-dependent fashion. AZD1222 also activated the phosphorylation of IRF3 and induced MxA expression. BNT162b2 and mRNA-1273 mRNA vaccines failed to induce or induced very weak cytokine gene expression in all cell models. None of the vaccines enhanced the expression of CXCL-4. AZD1222 and mRNA-1273 vaccines induced high expression of S protein in all studied cells. ConclusionsAd-vector vaccine induces higher IFN and pro-inflammatory responses than the mRNA vaccines in human immune cells. This data shows that AZD1222 readily activates IFN and pro-inflammatory cytokine gene expression in PBMCs, macrophages and DCs, but fails to further enhance CXCL-4 mRNA expression.

Glabridin ameliorates DNFB induced atopic dermatitis by suppressing MAPK/NF κB signaling pathways in mice

Purpose: To investigate the anti-inflammatory mechanism of action of glabridin (GBD) isolated from glycyrrhizae radix et rhizoma (Glycyrrhiza glabra L.) using atopic dermatitis (AD) mouse model. Methods: Nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were investigated. The dorsal skin and serum of 2,4-dinitrofluorobenzene (DNFB)-induced AD mice were used to determine the levels of various inflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-5, IL-6, IL-13, and immunoglobulin E (IgE) by enzyme-linked immunosorbent assay (ELISA). The relative levels of inflammation-associated mRNA and signaling pathways were determined by real time-polymerase chain reaction (RT-PCR) and western blotting, respectively. Hematoxylin and eosin (H&E) staining was used to assess the effect of GBD on tissue thickening and inflammatory cell infiltration in mice with AD. Results: Glabridin significantly relieved dorsal skin thickening, scabbing, and bleeding, and reduced the infiltration of inflammatory cells in mice with AD (p < 0.05). The levels of IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-13, and IgE were significantly reduced (p < 0.05 or 0.01). Moreover, the relative levels of IL-6 and IFN-γ mRNA showed a significant decrease (p < 0.01). Furthermore, GBD inhibited phosphorylation of MAPK signaling pathway and activation of NF-κB (p < 0.05 or 0.01). Conclusion: Glabridin exerts an inhibitory effect on AD by blocking MAPK and NF-κB signaling pathways. This finding highlights the relationship between traditional Chinese medicine and modern AD therapeutic methods, which should be further investigated for its potential to be developed as an external anti-AD medicine.

Abstract 6352: Combination treatment with recombinant murine IL-12 and anti-PD-1 antibody enhanced anti-tumor efficacy through the activation of cytotoxic T cell response

Abstract Introduction: Immune checkpoint inhibitors (ICIs) are widely used in cancer therapy. Among them, anti-PD-1 antibodies are the most used and have been approved by FDA for the treatment of many types of cancer (1). In our previous studies, combination of anti-PD-1 antibody with IL-12 demonstrated significant synergistic effect on tumor growth inhibition. The elevation of IFN-γ mRNA expression suggested the potential roles of CD8 T cells and NK cells in this process. In this study, we used two syngeneic mice tumor models to investigate the mechanisms of synergistic effect by the combination of IL-12 and anti-PD-1 antibody. Methods: We used syngeneic mice tumor models with mice cancer cell lines CT-26 and MC-38. The mice inoculated with tumor cells and were treated until tumors grew to 100 mm3. The mice were divided into three groups to receive the treatment, including rmIL-12 alone, anti-PD1-antibody alone, and the combination of rmIL-12 and anti-PD1-antibody (BIW x 3 weeks). At the end of the study, the mice were sacrificed and tumor resection and splenectomy were performed. The tumor infiltrating lymphocyte subpopulations within the tumor microenvironment were analyzed by flow cytometry and gene expression profile by RNA Seq. Results: The combination of rmIL-12 and anti-PD-1 antibody significantly inhibited tumor growth compared to the treatment with rmIL-12 and anti-PD-1 antibody alone. The combination group demonstrated more CD8+ T cell in the tumor microenvironment via flow cytometry. In addition, RNA Seq showed the combination group increased CD8+ T cells, NK cells, and Th1 populations. The levels of cytotoxicity related genes (IFN-γ, Granzyme B, Perforin) were also higher in combination group. Conclusion: Combination of rmIL-12 and anti-PD-1 antibody increased immune cells infiltration in the tumor microenvironment and enhanced anti-tumor response suggesting the potential of this combination for cancer treatment. Citation:(1). Watson, G.A. et al. (2020) “Novel strategies in immune checkpoint inhibitor drug development: How far are we from the paradigm shift?,” British Journal of Clinical Pharmacology, 86(9), pp. 1753-1768. https://doi.org/10.1111/bcp.14355. Citation Format: Yeng-Jey Yang, Pei-Yi Tsou, Shiuh-Dih Chou, Meng-Na Lee, San-Chi Chen. Combination treatment with recombinant murine IL-12 and anti-PD-1 antibody enhanced anti-tumor efficacy through the activation of cytotoxic T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6352.

Study of the proviral load levels and mRNA expression of cytokines in peripheral blood mononuclear cells and somatic milk cells in cattle with different BLV infection profiles.

The retrovirus bovine leukemia virus (BLV) might produce abnormal immune function, associated with susceptibility to developing other infectious diseases, including mastitis. This study aimed to determine the proviral load and cytokines gene expression in peripheral blood mononuclear cells (PMBC) and milk somatic cells (SC) in BLV-infected and non-infected cattle. Of 27 BLV-infected cows in PBMC, 17 (62.96%) had a high proviral load (HPL), and 10 (37.04%) had a low proviral load (LPL). All SC samples had low proviral load (LPL-SC). Higher IFN-γ and IL-10 expression, and lower IL-12 and IL-6 expression, were found in PBMC from BLV-infected compared to BLV non-infected cattle. Moreover, higher IFN-γ, IL-12, and IL-6 expression, and lower IL-10 expression were observed in cattle with LPL-PBMC compared to HPL-PBMC. In milk samples, lower IFN-γ and higher IL-12 mRNA expression were observed in LPL-SC compared to BLV non-infected cattle in SC. IL-10 and IL-6 expression mRNA was significantly lower in LPL-SC than in SC from BLV non-infected cattle. This study shows that milk SC maintains lower proviral load levels than PBMC. This first report on Th1 and Th2 cytokines expression levels in SC may be relevant to future control strategies for BLV infection, mastitis, and udder health management.

Immunometabolic interference between cancer and COVID-19.

Even though cancer patients are generally considered more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the mechanisms driving their predisposition to severe forms of coronavirus disease 2019 (COVID-19) have not yet been deciphered. Since metabolic disorders are associated with homeostatic frailty, which increases the risk of infection and cancer, we asked whether we could identify immunometabolic pathways intersecting with cancer and SARS-CoV-2 infection. Thanks to a combined flow cytometry and multiomics approach, here we show that the immunometabolic traits of COVID-19 cancer patients encompass alterations in the frequency and activation status of circulating myeloid and lymphoid subsets, and that these changes are associated with i) depletion of tryptophan and its related neuromediator tryptamine, ii) accumulation of immunosuppressive tryptophan metabolites (i.e., kynurenines), and iii) low nicotinamide adenine dinucleotide (NAD+) availability. This metabolic imbalance is accompanied by altered expression of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), with a distinctive downregulation of IL-6 and upregulation of IFNγ mRNA expression levels. Altogether, our findings indicate that cancer not only attenuates the inflammatory state in COVID-19 patients but also contributes to weakening their precarious metabolic state by interfering with NAD+-dependent immune homeostasis.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdriapolymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in anEscherichia colihost expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominantrproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived fromrprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution:This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1β (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

Response to Bovine Viral Diarrhea Virus in Heifers Vaccinated with a Combination of Multivalent Modified Live and Inactivated Viral Vaccines

Bovine viral vaccines contain both live or inactivated/killed formulations, but few studies have evaluated the impact of vaccinating with either live or killed antigens and re-vaccinating with the reciprocal. Commercial dairy heifers were utilized for the study and randomly assigned to three treatment groups. Treatment groups received a commercially available modified-live viral (MLV) vaccine containing BVDV and were revaccinated with a commercially available killed viral (KV) vaccine containing BVDV, another group received the same KV vaccine and was revaccinated with the same MLV vaccine, and yet another group served as negative controls and did not receive any viral vaccines. Heifers in KV/MLV had higher virus neutralizing titers (VNT) at the end of the vaccination period than heifers in MLV/KV and control groups. The frequency of IFN-γ mRNA positive CD4+, CD8+, and CD335+ populations, as well as increased mean fluorescent intensity of CD25+ cells was increased for the MLV/KV heifers as compared to KV/MLV and controls. The data from this study would suggest that differences in initial antigen presentation such as live versus killed could augment CMI and humoral responses and could be useful in determining vaccination programs for optimizing protective responses, which is critical for promoting lifetime immunity.

Alterations in the Expression of IFN Lambda, IFN Gamma and Toll-like Receptors in Severe COVID-19 Patients.

Contradictory results have been reported regarding interferon (IFN) lambda (λ1-3) and IFN gamma (γ) production in COVID-19 patients. To gain insight into the roles played by these IFNs in SARS-CoV-2 infection, IFNλ1-3 and IFNγ mRNA expression was evaluated in peripheral blood mononuclear cells (PBMCs) (n = 32) and in cells of paired bronchoalveolar lavages (BALs) (n = 12). Lower IFNλ1-3 values (p < 0.001 for IFNλ1 and 3 and p = 0.013 for IFNλ2) in the PBMCs of severely ill patients were found compared to healthy donors (n = 15). Reduced levels of IFNγ were also detected in patients' PBMCs (p < 0.01) and BALs (p = 0.041) compared to healthy donors. The presence of secondary bacterial infections was associated with decreased IFNλ amounts in PBMCs (p = 0.001, p = 0.015 and p = 0.003, respectively) but increased concentrations of IFNλ3 (p = 0.022) in BALs. Patients with alterations in C-reactive protein, lactate dehydrogenase and D-dimer levels had decreased IFNλ1 and 3 (p = 0.003 and p < 0.001) and increased IFNγ (p = 0.08) in PBMCs. Analyzing Toll-like receptors (TLRs) involved in IFN production, we found that TLR3 was highly expressed (p = 0.033) in patients with bacterial superinfections, while TLR7 and 8 (p = 0.029 and p = 0.049) were reduced in BALs of deceased patients. Overall, severe COVID-19 might be characterized by dysregulation in IFNγ, IFNλ and TLR3, 7 and 8 production.

Mild heat stress–induced adaptive immune response in blood mononuclear cells and leukocytes from mesenteric lymph nodes of primiparous lactating Holstein cows

Heat stress negatively affects the metabolism and physiology of the bovine gut. However, it is not known whether heat stress induces an inflammatory response in mesenteric lymph nodes (MLN), the primary origin of gut immune cells, and thus contributes to inflammatory processes in the circulation. Therefore, our objective was to elucidate the effects of chronic heat stress on the systemic activation of acute-phase response in blood, proinflammatory cytokine production in peripheral blood mononuclear cells (PBMC), and the activation of the toll-like receptor signaling (TLR) 2/4 pathway in MLN leucocytes and their chemokines and chemokine receptor profiles in Holstein cows. Primiparous Holstein cows (n = 30; 169 ± 9 d in milk) were exposed to a temperature-humidity index (THI) of 60 [16°C, 63% relative humidity (RH)] for 6 d. Thereafter, cows were evenly assigned to 3 groups: heat-stressed (HS; 28°C, 50% RH, THI = 76), control (CON; 16°C, 69% RH, THI = 60), or pair-feeding (PF; 16°C, 69% RH, THI = 60) for 7 d. On d 6, PBMC were isolated and on d 7 MLN. Plasma haptoglobin, TNFα, and IFNγ concentrations increased more in HS than CON cows. Concomitantly, TNFA mRNA abundance was higher in PBMC and MLN leucocytes of HS than PF cows, whereas IFNG mRNA abundance tended to be higher in MLN leucocytes of HS than PF cows, but not for chemokines (CCL20, CCL25) or chemokine receptors (ITGB7, CCR6, CCR7, CCR9). Furthermore, the TLR2 protein expression tended to be more abundant in MLN leucocytes of HS than PF cows. These results suggest that heat stress induced an adaptive immune response in blood, PBMC, and MLN leukocytes involving the acute-phase protein haptoglobin, proinflammatory cytokine production, and TLR2 signaling in MLN leucocytes. However, chemokines regulating the leucocyte trafficking between MLN and gut seem not to be involved in the adaptive immune response to heat stress.

IFNAR signaling of neuroectodermal cells is essential for the survival of C57BL/6 mice infected with Theiler’s murine encephalomyelitis virus

BackgroundTheiler’s murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus that causes encephalitis followed by chronic demyelination in SJL mice and spontaneous seizures in C57BL/6 mice. Since earlier studies indicated a critical role of type I interferon (IFN-I) signaling in the control of viral replication in the central nervous system (CNS), mouse strain-specific differences in pathways induced by the IFN-I receptor (IFNAR) might determine the outcome of TMEV infection.MethodsData of RNA-seq analysis and immunohistochemistry were used to compare the gene and protein expression of IFN-I signaling pathway members between mock- and TMEV-infected SJL and C57BL/6 mice at 4, 7 and 14 days post-infection (dpi). To address the impact of IFNAR signaling in selected brain-resident cell types, conditional knockout mice with an IFNAR deficiency in cells of the neuroectodermal lineage (NesCre±IFNARfl/fl), neurons (Syn1Cre±IFNARfl/fl), astrocytes (GFAPCre±IFNARfl/fl), and microglia (Sall1CreER±IFNARfl/fl) on a C57BL/6 background were tested. PCR and an immunoassay were used to quantify TMEV RNA and cytokine and chemokine expression in their brain at 4 dpi.ResultsRNA-seq analysis revealed upregulation of most ISGs in SJL and C57BL/6 mice, but Ifi202b mRNA transcripts were only increased in SJL and Trim12a only in C57BL/6 mice. Immunohistochemistry showed minor differences in ISG expression (ISG15, OAS, PKR) between both mouse strains. While all immunocompetent Cre-negative control mice and the majority of mice with IFNAR deficiency in neurons or microglia survived until 14 dpi, lack of IFNAR expression in all cells (IFNAR−/−), neuroectodermal cells, or astrocytes induced lethal disease in most of the analyzed mice, which was associated with unrestricted viral replication. NesCre±IFNARfl/fl mice showed more Ifnb1, Tnfa, Il6, Il10, Il12b and Ifng mRNA transcripts than Cre−/−IFNARfl/fl mice. IFNAR−/− mice also demonstrated increased IFN-α, IFN-β, IL1-β, IL-6, and CXCL-1 protein levels, which highly correlated with viral load.ConclusionsIfi202b and Trim12a expression levels likely contribute to mouse strain-specific susceptibility to TMEV-induced CNS lesions. Restriction of viral replication is strongly dependent on IFNAR signaling of neuroectodermal cells, which also controls the expression of key pro- and anti-inflammatory cytokines during viral brain infection.

Dietary coated essential oil and organic acid mixture supplementation improves health of broilers infected with avian pathogenic Escherichia coli.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is a very prevalent disease in poultry farms in China. The exploration of effective non-antibiotic substances is of great significance for the control of APEC infections. This experiment evaluated the efficacy of coated essential oil and organic acid (EOA) supplementation to prevent E. coli O78 infection in broiler chickens. A total of 288 one-day-old male broiler chicks were randomly distributed into 4 groups with 6 replicates per group. Chickens were fed a diet either supplemented with EOA (500mg/kg feed) or not, and either uninfected or infected with E. coli O78 intratracheally. Results showed that E.coli O78 infection reduced body weight gain, increased mortality and the ratio of feed to gain along with cecal and liver E.coli load, damaged gut mucosa, induced local and systemic inflammation, and altered cecal microbial composition, diversity and function (P<0.05). Supplemental EOA improved feed conversion efficiency, lowered gross lesion scores and cecal E.coli population, enhanced intestinal goblet cells and serum IgG concentration, and tended to decrease serum IL-12 production (P<0.05). Essential oil and organic acid addition downregulated IFN-γ mRNA, tended to decrease mucin-2 mRNA levels while upregulating IL-10 mRNA, and tended to increase ZO-1 gene expression in the jejuna of infected birds at 7d after E.coli O78 challenge (P<0.05). The 16S rRNA gene sequencing indicated that both EOA addition and E.coli O78 challenge altered the diversity and composition of the cecal microbiota community. Furthermore, infected birds fed EOA showed decreased Bacteroidetes and genus Lactobacillus abundance compared with the infected control. LEfSe analysis showed that Firmicutes, Ruminococcaceae, Clostridiales, Clostridia, Lactobacillus, Lactobacilaceae, and cc-115 were enriched in the non-infected but EOA-treated group (P< 0.05). Collectively, dietary EOA supplementation could mildly alleviate E.coli-induced gut injury and inflammation.

Characterization of TIM-3 Expression and Its Correlation with TNF-α and IFN-γ in Patients with Surgically Resected Lung Adenocarcinoma.

T cell immunoglobulin and mucin-containing protein-3 (TIM-3) is an important immune checkpoint, but its role in lung cancer is still not clear. In this study, we investigated TIM-3 protein expression and its correlation with TNF-α and IFN-γ by examining the tissues of patients with lung adenocarcinoma. We detected the mRNA quantity of TIM-3, TNF-α, and IFN-γ in 40 surgically resected specimens from patients with lung adenocarcinoma by real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression of TIM-3, TNF-α, and IFN-γ was assessed in normal tissues, paracarcinoma tissues, and tumor tissues by western blotting, respectively. The relevance between the expression and clinicopathological information of the patients was analyzed. The results showed that the expression level of TIM-3 was higher in tumor tissues than normal tissues and paracancerous tissues (P < 0.05). On the contrary, the expression of TNF-α and IFN-γ in tumor tissues was lower than normal tissues and paracarcinoma tissues (P < 0.05). However, the expression levels of IFN-γ mRNA were not observed to be significantly different between cancerous tissues and adjacent tissues. While TIM-3 protein expression in cancer tissues of patients with lymph node metastasis was higher than in patients without metastasis, the expression of TNF-α and IFN-γ was lower (P < 0.05). Importantly, the expression of TIM-3 was negatively correlated with the expression of TNF-α and IFN-γ, and the expression of TNF-α was found to be positively correlated with IFN-γ in the patient. The high expression of TIM-3, the low expression of TNF-α and IFN-γ, and the synergistic effect of TNF-α and IFN-γ in patients with lung adenocarcinoma were closely related to poor clinicopathological characteristics. Overexpression of TIM-3 may play an important role in the relationship between TNF-α and IFN-γ secretion and poor clinicopathological characteristics.

Performance of T-Track® TB, a Novel Dual Marker RT-qPCR-Based Whole-Blood Test for Improved Detection of Active Tuberculosis.

Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p < 0.001) than that of QFT-Plus. The overall agreement of T-Track® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.

IL17A mRNA Staining Distinguishes Palmoplantar Psoriasis from Hyperkeratotic Palmoplantar Eczema in Diagnostic Skin Biopsies

Acral dermatoses, including hyperkeratotic palmoplantar eczema (HPE), palmoplantar psoriasis (PP), and mycosis fungoides palmaris et plantaris (MFPP), can be challenging to diagnose clinically and histopathologically. In this setting, cytokine biomarkers may be able to help provide diagnostic clarity. Therefore, we evaluated IL-17A, IFN-γ, and IL-13 expression in PP, HPE, and MFPP and compared their expression profiles with nonacral sites. We used biopsy specimens from the Yale Dermatopathology database, selecting cases of HPE (n= 12), PP (n= 8), MFPP (n= 8), normal acral skin (n= 9), nonacral eczema (n= 10), and nonacral psoriasis (n= 10) with classic clinical and histopathologic features. IL17A mRNA expression by RNA in situ hybridization differentiated PP (median score 63.1 [interquartile range 9.4-104.1]) from HPE (0.8 [0-6.0]; P= 0.003), MFPP (0.6 [0-2.6]; P= 0.003), and normal acral skin (0 [0-0]; P < 0.001). Unexpectedly, both PP and HPE showed co-expression of IFNG and IL13 mRNA. In contrast, nonacral psoriasis and eczema showed divergent patterns of IFNG and IL13 mRNA expression. Taken together, we show that IL17A mRNA expression may be a useful biomarker of PP, and we further show that acral dermatoses exhibit distinct immunology compared to nonacral sites, with implications for clinical management.

Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.