Differential Cytokine Gene Expression Research Articles

Differential gene expression of cytokines, receptors, and miRNAs in individuals living with HIV-1 and vaccinated against yellow fever

Between 2016 and 2018, Brazil faced a yellow fever (YF) outbreak, which led to an expansion of vaccination coverage. The coexistence of the YF outbreak and the HIV-1 epidemic in Brazil raised concerns regarding the immune response and vaccine effectiveness in individuals living with HIV (PLWH). The aim of this study was to investigate the immune response to YF vaccination in PLWH and HIV-uninfected individuals as controls. Transcript levels of immunomodulatory molecules, including IL-6, IL-10, IL-21, TGF-β, CD19, CD163, miR-21, miR-146, and miR-155, were measured using RTqPCR. TCD4+ cells were evaluated by cytometry, and neutralizing antibody (Nab) titers were detected by a micro plaque-reduction neutralization test. The findings of our study revealed several noteworthy observations. First, there was a notable reduction in the circulation of TCD4+ cells postvaccination. Among people living with HIV (PLWH), we observed an increase in the expression of IL-10 following vaccination, while IL-6 expression was diminished in PLWH with lower TCD4+ counts. Furthermore, we identified the downregulation of CD19 and TGF-β, along with the upregulation of IL-21 and CD163. Notably, we observed positive correlations between the levels of IL-10/IL-21, IL-10/CD163, and IL-6/CD19. Additionally, there was a positive correlation between miRNAs 146 and 155. It is important to emphasize that all participants exhibited robust neutralizing antibody responses after receiving 17DD YF vaccination. In this context, the gene expression data presented can be useful for biomarker studies of protective antibodies induced by YF vaccination. This study sheds light on immune mechanisms in individuals living with HIV and YF vaccination.

Analysis of differential gene expression of pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases.

A subset of individuals with COVID-19 can suffer from a severe form of the disease requiring breathing support for respiratory failure and even death due to disease complications. COVID-19 disease severity can be attributed to numerous factors, where several studies have associated changes in the expression of serum pro-inflammatory cytokines with disease severity. However, very few studies have associated the changes in expression of pro-inflammatory changes in the nasopharyngeal milieu with disease severity. Therefore, in the current study, we performed differential gene expression analysis of various pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases. For this retrospective, cross-sectional study, a total of 118 nasopharyngeal swab samples, previously collected from mild and severe (based on the WHO criteria) COVID-19 patients were used. A real-time qPCR was performed to determine the viral loads and also evaluate the mRNA expression of eight cytokines (IL-1, IL-2, IL-4, IL-6, IL-10, IFN-γ, TGF-β1, and TNF-α). Subsequently, an unpaired T-test was applied to compare the statistical difference in mean expression of viral loads and each cytokine between the mild and severe groups, while the Pearson correlation test was applied to establish a correlation between disease severity, viral load, and cytokines expression. Similarly, a multivariable logistic regression analysis was performed to assess the relationship between different variables from the data and disease severity. Out of 118 samples, 71 were mild, while 47 were severe. The mean viral load between the mild and severe groups was comparable (mild group: 27.07± 5.22; severe group: 26.37 ±7.89). The mRNA expression of cytokines IL-2, IL-6, IFN- γ, and TNF-α was significantly different in the two groups (p<0.05), where the Log2 normalized expression of IL-2, IL-6, IFN- γ, and TNF-α was found to be 2.2-, 16-, 2.3-, and 1.73-fold less in the severe group as compared to the mild group. Furthermore, we also observed a significant positive correlation between all the cytokines in the severe group. The multivariate analysis showed a significant relationship between age, IL-6, and disease severity. This decreased expression of certain cytokines (IL-2, IL-6, TNF-α, and IFN-γ) in the nasopharyngeal milieu may be considered early biomarkers for disease severity in COVID-19 patients.

Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust.

Air pollution is a known environmental health hazard. A major source of air pollution includes diesel exhaust (DE). Initially, research on DE focused on respiratory morbidities; however, more recently, exposures to DE have been associated with neurological developmental disorders and neurodegeneration. In this study, we investigated the effects of sub-chronic inhalation exposure to DE on neuroinflammatory markers in two inbred mouse strains and both sexes, including whole transcriptome examination of the medial prefrontal cortex. We exposed aged male and female C57BL/6J (B6) and DBA/2J (D2) mice to DE, which was cooled and diluted with HEPA-filtered compressed air for 2 h per day, 5 days a week, for 4 weeks. Control animals were exposed to HEPA-filtered air on the same schedule as DE-exposed animals. The prefrontal cortex was harvested and analyzed for proinflammatory cytokine gene expression (Il1β, Il6, Tnfα) and transcriptome-wide response by RNA-seq. We observed differential cytokine gene expression between strains and sexes in the DE-exposed vs. control-exposed groups for Il1β, Tnfα, and Il6. For RNA-seq, we identified 150 differentially expressed genes between air and DE treatment related to natural killer cell-mediated cytotoxicity per Kyoto Encyclopedia of Genes and Genomes pathways. Overall, our data show differential strain-related effects of DE on neuroinflammation and neurotoxicity and demonstrate that B6 are more susceptible than D2 to gene expression changes due to DE exposures than D2. These results are important because B6 mice are often used as the default mouse model for DE studies and strain-related effects of DE neurotoxicity warrant expanded studies.

Immune Transcriptome and Secretome Differ between Human CD71+ Erythroid Cells from Adult Bone Marrow and Fetal Liver Parenchyma.

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchyma CECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchyma CECs only. These observations suggest that there might be undiscovered roles in immune response for CECs.

Heterogeneous Nature of Trained Innate Immune Cells in Health and Disease.

Heterogeneous Nature of Trained Innate Immune Cells in Health and Disease.

Cytokine Expression in Peripheral Blood Mononuclear Cell Cultures Obtained from Cattle with Different Stages of Natural Mycobacterium bovis Infection

In bovine tuberculosis (bTB), cellular, humoral, or both types of immune responses have been observed. The purpose of this study was to examine the immune status of tuberculous cows based on the differential cytokine gene expression associated with Th1 (IFN-γ, IL-2), or Th2 (IL-4, IL-10) responses. Twenty-three (23) cows belonging to a dairy herd located in a rural region of the State of Hidalgo, México, were selected for the study. Single Intradermal Comparative Cervical Tuberculin (SICCT) Test, Interferon-Gamma (IFN-γ) Release Assay (BOVIGAM), and Enzyme-Linked Immunosorbent Assay (ELISA) were used for detection of cattle infected by M. bovis. Thirteen cows were positive to all the tests (Group 1); ten cows were positive only to ELISA (Group 2), and the remaining Group (Group 3, control) included cows negative to all the tests. Peripheral blood mononuclear cells (PBMC) from animals were in vitro stimulated by bovin purified protein derivative (PPD), avian PPD, and Concanavalin A (Con A) mitogen for 72h. Changes in the levels of expression of mRNA of the respective cytokines was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using β-actin gene as internal control. In group 1, PPD bovis and Con A-stimulated cells exhibited high production of IFN-γ, IL-2 and IL-4, but not IL-10. In contrast, PPD avium-stimulated cells displayed a low production of cytokine transcripts. In group 2, cells showed a significant production of IL-10 in response to bovine PPD (P&lt; 0.001). In the control group, a high production of IFN-γ and IL-2 was observed only in Con A-stimulated cells. Post-mortem examinations in animals of group 1 showed slight and medium lesions in lymph nodes, whereas in group 2, the lesions were more extensive. Results indicate differences on gene expression levels of cytokines considered to determine balance in Th1/Th2 response among the evaluated groups. In addition, high levels of antibodies against M. bovis and high IL-10 expression in PBMC together are indicators of progressive bTB when both tuberculin test and IFN-γ assay are negative in tuberculous anergic cattle. Inclusion of serology and IL-10 cytokine expression in in the diagnosis checklist improves detection of infected cattle to help control bovine tuberculosis.

Modulation of the Expression of TLR2, TLR4, TLR9, IFNy and IL-10 in THP1 Cell Line Following Infection with Leishmanai donovani Isolates from Different Clinical forms of Leishmaniasis

Introduction: Protozoa of the genus Leishmania cause a wide range of pathologies from self-healing skin lesions to visceral pathology. The outcome of infection depends on the species of the infecting Leishmania parasite, the quality and quantity of the host immune response and the host genetic background. Objective: This study aimed to determine the differential expression of TL2, TL4, TL9, IFN-γ and IL-10 cytokine genes of Human THP1 cell line following in vitro infection with Leishmania donovani isolates from different clinical forms. Methods: Human THP1 cells were infected by live promastigotes of leishmania donovani isolates from visceral (VL), cutaneous (CL), Post Kala-Azar dermal Leishmaniasis (PKDL) and mucosal Leishmaniasis (ML) Patients. The expression of Tolls like receptor TL2, TL4 and TL9 and the expression of IFN-γ and IL-10 cytokine were measured using Real Time PCR. Results: A significant increase in the expression of TLR 2, TLR4 and TLR9 by THP-1 was detected following infection of THP-1 cells with L. donovani isolates from ML and PKDL patients. A high expression IL-10 mRNA by THP-1 cells was detected in cells infected by Leishmania donovani isolates from mucosal lesions. Conclusion: Leishmania donovani isolates from different clinical forms of Leishmaniasis induce different cytokine responses.

Differences in Cytokine Gene Expression after a Stimulation with Escherichia Coli and Porphyromonas Gingivalis or Lipopolysaccharides Derived from these Bacteria

Monocytes are important cells in innate immunity. The early stage of the innate immunity is regulated by various cytokines produced by monocytes. We conducted a preliminary study to investigate TNFα expression by stimulating THP-1 cells with several bacterial species. The TNFα mRNA levels significantly varied, with the most potent stimulatory effects observed with P. gingivalis. In the present study, we focused on P. gingivalis and compared differences in cytokine expression profiles after the stimulation of THP-1 with E. coli. Bacterial antigen stimulation increased various cytokine gene expressions in THP-1. P. gingivalis had significantly more potent effects on the mRNA expressions of TNFα, IL-1β, and IL-10, but not of IL-12p40, than E. coli. This result suggests the potent ability of P. gingivalis to induce inflammation. THP-1 stimulated with LPS derived from both bacterial species showed that E. coli had significantly more potent effects on the expressions of TNFα, IL-1β, and IL-12p40 than P. gingivalis. The differences in the bacterial antigens and the LPS stimulation effects suggest involvements of different receptors, such as TLR-2 and -4, which recognize bacterial components. The present results suggest that the P. gingivalis somatic cell antigen stimulates a number of pattern recognition receptors at the same time as the synthesis of bacterial components, except LPS. The potent virulence of P. gingivalis and persistence of infection might be affected by differences in cytokine production. Pro-inflammatory responses are dependent not only on the bacterial type, but also bacterial components.

Effects of equine metabolic syndrome on inflammation and acute-phase markers in horses

Obesity and metabolic disorders are associated with systemic low-grade chronic inflammation, both in humans and animals. The aim of the study is to assess the effects of obesity and hyperinsulinemia on individual components of the acute-phase reaction in equine metabolic syndrome (EMS) horses. Eight mixed-breed EMS and six control, age-matched horses of both sexes were included in the study. Animals were classified as EMS or control based on the assessment of BCS, cresty neck score, and basal insulin >50 μU/mL and/or insulin responses to the oral sugar test (OST) >60 μU/mL. Peripheral venous blood was collected. The expression of proinflammatory cytokines, the concentration of circulating cytokines, and acute-phase proteins (serum amyloid A, C-reactive protein, haptoglobin, activin A, and procalcitonin) were measured. The data were analyzed using the Mann-Whitney test, whereas correlations were examined using Spearman's correlation coefficient. The tests were statistically significant if P ≤ 0.05. There were no differences in cytokine gene expression, circulating cytokine concentrations, or concentrations of acute-phase proteins between the EMS and the control groups. There was a strong correlation between the basal concentration of insulin and the serum concentrations of IL-6 (r = 0.71, P < 0.05). Activin A was positively correlated with post-OST insulin concentrations (r = 0.707, P = 0.05), indicating that this marker of inflammation could warrant further investigation in horses with EMS.

Proinflammatory cytokine changes in bronchoalveolar lavage fluid cells isolated from pigs infected solely with porcine reproductive and respiratory syndrome virus or co-infected with swine influenza virus

IntroductionThe study evaluated the patterns of local innate immune response in bronchoalveolar lavage fluid (BALF) cells of pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) alone or co-infected with swine influenza virus (SIV).Material and MethodsThe study was performed on 26 seven-week-old pigs in three groups: PRRSV-infected (n = 11), PRRSV and SIV-infected (n = 11), and control (n = 4). BALF was collected post euthanasia at 2 and 4 dpi (three piglets per inoculated group) and at 21 dpi (all remaining pigs). Expression of IFN-α, IFN-γ, IL-1β, IL-6, IL-8, and IL-10 mRNA was quantified in BALF cells. PRRSV RNA was quantified in BALF samples using a commercial real-time RT-PCR kit.ResultsThe three cytokines IFN-α, IFN-γ, and IL-1β presented significant expression changes in all experimental pigs. In PRRSV-infected animals IL-8 also did, but in co-infected subjects IL-6 and IL-10 were the additional upregulated cytokines. The highest number of differentially expressed genes was observed at 4 dpi, and significant differences in cytokine gene expression did not occur between the experimental groups at any other time point. The mean PRRSV load in the BALF of PRRSV-infected pigs was higher than that of co-infected pigs at each time point, having statistical significance only at 4 dpi.ConclusionThe results of the study indicate that infection with PRRSV alone as well as with SIV interferes with innate and adaptive immune response in the infected host. They also showed that co-infection demonstrates additive effects on IL-6 and IL-10 mRNA expression levels.

Differential Expression of Cytokine in Kadaknath and Commercial Chicken Against Coccidial Challenge

In the present investigation the relative expression of interleukins (IL)-1â, IL-2, IL-6, IL-17 and interferon (IFN)-Y genes were explored in response to coccidial challenge in Kadaknath (n=12), Cari-Vishal (n=12) and Cobb (n=12) broiler chicken using quantitative PCR. The intraepithelial lymphocytes cell of control, T1 and T2 on 4, 7 and 14d post infection (pi) were used as templates for expression of five inflammatory cytokines. The relative mean fold expression for ILâ1 ranged from 9.91± 0.14 (Kadaknath) to 119.01±0.29 (Cobb). The highest and lowest relative mean fold expression for IL-2 was observed to be 242.19±0.40 and 6.25±0.03 in Cobb at d4 and d14 pi, respectively. For IL-6 gene, the lowest relative mean fold expression was observed in Kadaknath (8.11±0.05) while the highest value was recorded in Cobb (99.04±0.92). The relative mean fold expression for IL-17 was 11.04±0.29 in Kadaknath and 197.40±0.37 in Cobb. For IFN-Y gene, the lowest relative mean fold expression was observed in Kadaknath (8.11±0.19), while the highest value was recorded in Cobb (84.74±0.25). In Cobb and Cari-Vishal broilers most cytokines (IL-1â, IL-6, IL-17 and IFN-Y) were observed to be transcribed to higher levels than Kadaknath chicken. It is concluded that the differential expression of cytokine genes in three genetic groups showed different degree of mucosal immune response to Eimeria infection and it depend upon the genetic background/genotype of birds, coccidial dosage and age of infection.

Molecular variants of Leishmania (Viannia) braziliensis trigger distinct patterns of cytokines and chemokines expression in golden hamster

Cutaneous leishmaniasis (CL) mainly caused by Leishmania braziliensis is a chronic inflammatory disease widely spread in Brazil. Genetic variant strains of this parasite have been associated with atypical clinical manifestations of CL in an endemic area in Brazil. Furthermore, these strains have presented distinct biological behaviors in golden hamster, suggesting differential activation of the immune response. In the present study we proposed to evaluate the localized immune response in golden hamsters infected with known molecular variant strains of L. braziliensis, in distinct time points post-infection (PI). Detailed analyses of the mRNA expression of cytokines and chemokines in hamster-skin lesions were performed. Heat map matrix and hierarchical cluster analysis were carried out to segregate the strains due to mRNA expression. Distinct patterns of immune response were found in both time points, more evident in the recent-phase disease (30 days-PI). At this time point, the genetic variant strains expressed high levels of tnfα, il12 and tgfβ whilst the non-variant strain expressed ifnγ, il6, il4, il10, il13 and ccl17. The hierarchical clustering highlights this distinct pattern in which all genetic variant strain was grouped in the cluster I and the non-variant strain grouped into the cluster II. At late-phase disease (60 days-PI) all isolates expressed high levels of il4 and il10. The non-variant strain shown a significant reduced expression of ifnγ, il6, ccl17, and ccl22 whilst distinct patterns were observed for the genetic variant strains. For the first time, a large panel of cytokines and chemokines mRNA-expression was analyzed in experimental trials using golden hamsters as animal model and genetic variant strains of L. braziliensis. Our findings suggest that genetic variant strains of L. braziliensis are able to trigger differential gene expression of cytokines and chemokines in the skin lesion from infected hamsters. The parasite intrinsic ability to activate distinct pathways in the host-parasite interaction may be associated to the large spectrum of clinical manifestation observed in CL-patients.

Controllable and uncontrollable stress differentially impact pathogenicity and survival in a mouse model of viral encephalitis

Intranasal instillation of vesicular stomatitis virus (VSV) into mice given controllable stress (modeled by escapable foot shock, ES) resulted in enhanced pathogenicity and decreased survival relative to infected mice given uncontrollable stress (modeled by inescapable foot shock, IS) and non-shocked control mice. Survival likely reflected differential cytokine gene expression that may have been regulated by miR146a, a predicted stress-responsive upstream regulator. Controllability also enhanced the accumulation of brain T resident memory cells that persisted long after viral clearance. The unexpected facilitatory effect of ES on antiviral neuroimmune responses and pathogenicity may arise from differential immunoactivating and immunosuppressive effects of uncontrollable and controllable stress.

Dynamic expression of cytokine and transcription factor genes during experimental Fasciola gigantica infection in buffaloes

BackgroundDetermining the mechanisms involved in the immune-pathogenesis of the tropical liver fluke, Fasciola gigantica, is crucial to the development of any effective therapeutic intervention. Here, we examined the differential gene expression of cytokines and transcription factors in the liver of F. gigantica-infected buffaloes, over the course of infection.MethodsWater buffaloes (swamp type) were infected orally with 500 F. gigantica encysted metacercariae. Liver tissue samples were collected 3, 10, 28, 42, 70 and 98 days post-infection (dpi). Levels of gene expression of nine cytokines (IFN-γ, TGF-β, IL-1β, IL-4, IL-6, IL-10, IL-12B, IL-13 and IL-17A) and four transcription factors (T-bet, GATA-3, Foxp3 and ROR-γτ) were determined using quantitative real-time PCR (qRT-PCR). We evaluated any correlation between gene expression of these immune-regulatory factors and the severity of liver pathology.ResultsHistopathological examination revealed that cellular infiltration, hemorrhage and fibrosis without calcification in the liver parenchyma of infected buffaloes, increased over the course of infection. This progressive pathology was attributed to dysregulated and excessive inflammatory responses induced by infection. The early infection phase (3–10 dpi) was marked by a generalized immunosuppression and elevated TGF-β expression in order to facilitate parasite colonization. A mixed Th1/Th2 immune response was dominant from 28 to 70 dpi, to promote parasite survival while minimizing host tissue damage. During late infection (98 dpi), the response was biased towards Th1/Treg in order to inhibit the host’s Th2 protective response and promote chronic infection. Both IL-10 and IL-17A and the Th17/Treg balance, played key roles in mediating the inflammatory and immunoregulatory mechanisms in the liver during chronic fasciolosis.ConclusionsOur data showed distinct CD4+ T helper (Th) polarization and cytokine dysregulation in response to F. gigantica infection in water buffaloes over the course of infection. Characterizing the temporal expression profiles for host immune genes during infection should provide important information for defining how F. gigantica adapts and survives in the liver of buffaloes and how host immune responses influence F. gigantica pathogenicity.

Airborne nitro-PAHs induce Nrf2/ARE defense system against oxidative stress and promote inflammatory process by activating PI3K/Akt pathway in A549 cells

Ambient particulate matter (PM) is a worldwide health issue of concern. However, limited information is available regarding the toxic contributions of the nitro-derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs). This study intend to examine whether 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) could activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) antioxidant defense system, and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway participates in regulating pro-inflammatory responses in A549 cells. Firstly, 1-NP and 3-NF concentration-dependently induced cellular apoptosis, reactive oxygen species (ROS) generation, DNA damage, S phase cell cycle arrest and differential expression of related cytokine genes. Secondly, 1-NP and 3-NF activated the Nrf2/ARE defense system, as evidenced by increased protein expression levels and nuclear translocation of transcription factor Nrf2, elevated Nrf2/ARE binding activity, up-regulated expression of the target gene heme oxygenase-1 (HO-1). Significantly increased protein expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylation level of Akt indicated that the PI3K/Akt pathway was activated during pro-inflammatory process. Further, both PI3K inhibitor (LY294002) and Akt inhibitor (MK-2206) reversed the elevated TNF-α expression to control level. Our results suggested that Nrf2/ARE pathway activation might cause an initiation step in cellular protection against oxidative stress caused by nitro-PAHs, and the PI3K/Akt pathway participated in regulating inflammatory responses.

Gene Expression Analysis of Inflammatory Cytokines in Korean Psoriatic Patients.

BackgroundAlthough phenotypic heterogeneity of psoriasis is suggested by the alternate activation of either T-helper (Th)1-related or Th17-related cytokines, little is known about the mRNA levels of inflammatory cytokines.ObjectiveTo investigate whether there is differential expression of Th1-related and Th17-related inflammatory cytokine genes 1) between psoriatic patients and healthy controls, and 2) between patients with different psoriasis phenotypes.MethodsTwenty-five patients with psoriasis (10 with guttate psoriasis and 15 with plaque psoriasis) and 5 healthy volunteers were enrolled in this study. The mRNA levels of circulating cytokines (interleukin [IL]-2, IL-12p40, interferon-γ, IL-17A, IL-22, and IL-23R) were measured by real-time reverse transcription polymerase chain reaction.ResultsThe comparison between psoriatic and healthy control samples revealed that IL-12p40, IL-17A, and IL-22 mRNA levels were significantly higher (approximately 4∼6 folds) in the patients with psoriasis. The mRNA levels of these six cytokines in the blood did not differ between the guttate and plaque psoriasis groups.ConclusionWe found that the mRNA levels of blood inflammatory cytokines (IL-12p40, IL-17A, and IL-22) were significantly elevated in patients with psoriasis compared to the levels in healthy controls, but they did not significantly differ between patients with guttate and plaque type psoriasis.

CpG Oligodeoxynucleotides Induce Differential Cytokine and Chemokine Gene Expression Profiles in Dapulian and Landrace Pigs.

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) mimic the immunostimulatory activity of microbial DNA by interacting with Toll-like receptor 9 (TLR9) to activate both the innate and adaptive immune responses in different species. However, few studies have been published to compare the effects of CpG ODN on different pig breeds. Therefore, in this study, whole blood gene expression profiles of DPL and Landrace pigs treated with CpG ODN were studied using RNA-seq technology. Five Hundred differentially expressed genes (DEGs) were identified between the two breeds. DPL pigs had significantly higher number of immune-relevant DEGs than the Landrace pigs after CpG ODN treatment. Pathway analysis showed that cytokine-cytokine receptor interaction and chemokine signaling pathway were the major enriched pathways of the immune-relevant DEGs. Further in vitro experiments showed that PBMCs of the DPL pigs had significantly higher levels of TLR9 mRNA than those of the Landrace pigs, both before and after CpG ODN stimulation. Cytokine and chemokine induction in the PBMCs of both breeds were also measured after CpG ODN stimulation. Our data showed that mRNA levels of cytokines (IFNα, IL8, IL12 p40) and chemokines (CXCL9, CXCL13) were significantly higher in the PBMCs of the DPL pigs than those of the Landrace pigs. Taken together, our data provide new information regarding the pig breed difference in response to CpG ODN stimulation and that higher levels of TLR9 mRNA in DPL pigs may be a major contributor for disease resistance.

Differential gene and protein expression of chemokines and cytokines in synovial fluid of patients with arthritis.

BackgroundPsoriatic arthritis (PsA), an inflammatory musculoskeletal disease, develops in approximately 30% of patients with psoriasis. Previously, chemokine (C-X-C motif) ligand 10 (CXCL10) was identified as a predictive biomarker of PsA in patients with psoriasis and was reduced after development of PsA. The purpose of the present study was to explore messenger RNA (mRNA) and protein expression of CXCL10 and its receptor, chemokine (C-X-C motif) receptor 3 (CXCR3), in the joints of patients with PsA to gain insight into their role in the pathogenesis of the disease.MethodsSera from 47 patients with PsA and 33 healthy control subjects were compared for expression of CXCL10 by Luminex assay. Synovial fluid (SF) was obtained from patients with PsA (n = 40), osteoarthritis (OA; n = 14), gout (n = 8), and rheumatoid arthritis (RA; n = 11) during clinical care. SF mRNA and protein expression of CXCL10, interleukin-17A (IL-17A), CXCR3, TBX21, RORC and/or interferon γ (IFNγ) were compared among the above-mentioned disease groups, as well as in paired SF and serum samples from patients with PsA using real-time polymerase chain reaction and Luminex assays, respectively.ResultsSerum CXCL10 was significantly higher in patients with PsA than in control subjects (p = 0.0007). CXCL10, IL-17A, and TBX21 expression were elevated in SF cells of patients with PsA compared with those of patients with OA and gout, but not those of patients with RA. CXCR3 and RORC were elevated in PsA SF cells compared with all other patient groups. Concordant results were obtained for CXCL10 and IL-17A protein expression. IFNγ was elevated in PsA SF compared with OA SF (p = 0.015). CXCL10 protein expression was substantially increased in SF (median 7283.9 pg/ml, interquartile range [IQR] 1330–10,362 pg/ml) compared with paired serum samples (median 282.06, IQR 180.7–395.8 pg/ml; p = 0.001), whereas IFNγ was significantly reduced (SF median 6.03 pg/ml, IQR 4.47–8.94 pg/ml; versus serum median 23.70 pg/ml, IQR 3.2–104.6 pg/ml; p = 0.001).ConclusionsCXCL10 may have an important etiological role in PsA that is analogous to that in RA, and it is a candidate biomarker to distinguish PsA from healthy individuals and from patients with OA and gout.

Differential Cytokine Gene Expression in Granulomas from Lungs and Lymph Nodes of Cattle Experimentally Infected with Aerosolized Mycobacterium bovis.

The hallmark lesion of tuberculosis in humans and animals is the granuloma. The granuloma represents a distinct host cellular immune response composed of epithelioid macrophages, lymphocytes, and multinucleated giant cells, often surrounding a caseous necrotic core. Within the granuloma, host-pathogen interactions determine disease outcome. Factors within the granulomas such as cytokines and chemokines drive cell recruitment, activity, function and ultimately the success or failure of the host’s ability to control infection. Hence, an understanding of the granuloma-level cytokine response is necessary to understand tuberculosis pathogenesis. In-situ cytokine expression patterns were measured using a novel in situ hybridization assay, known as RNAScope® in granulomas of the lungs, tracheobronchial lymph nodes and caudal mediastinal lymph nodes of cattle experimentally infected with Mycobacterium bovis via aerosol exposure. In spite of microscopic morphological similarities, significant differences were seen between late stage granulomas of the lung compared to those of the tracheobronchial lymph nodes for IL-17A, IFN-γ, TGF-β, IL10 and IL-22 but not for TNF-α. Additionally, significant differences were noted between granulomas from two different thoracic lymph nodes that both receive afferent lymphatics from the lungs (i.e., tracheobronchial and caudal mediastinal lymph nodes) for TNF-α, IL-17A, IFN-γ, TGF-β and IL-10 but not for IL-22. These findings show that granuloma morphology alone is not a reliable indicator of granuloma function as granulomas of similar morphologies can have disparate cytokine expression patterns. Moreover, anatomically distinct lymph nodes (tracheobronchial vs caudal mediastinal) differ in cytokine expression patterns even when both receive afferent lymphatics from a lung containing tuberculoid granulomas. These findings show that selection of tissue and anatomic location are critical factors in assessing host immune response to M. bovis and should be considered carefully.

Abstract 2340: PD-1 blockade suppresses gastric cancer development by promoting antitumor immunity in mice

Abstract Since advanced gastric cancer is difficult to cure with chemo and/or radiation therapy, new therapeutic approaches are clearly needed. Targeting immune checkpoints such as CTLA-4 and PD-1/PD-L1 has shown promising antitumor immunity in many malignancies by modulating T cell activity. PD-L1 and to a lesser extent of PD-L2 are expressed in human gastric cancer and expression of PD-L1 is associated with poor prognosis. Therefore, developing immunotherapies is an attractive strategy for the treatment of gastric cancer. We evaluated the efficacy of anti-PD-1 therapy in a mouse model of gastric cancer. Gastrin-deficient (GAS-KO) mice have been shown to develop large antral tumors with the combination of N-methyl-N-nitrosourea (MNU) and Helicobacter felis (H. felis) infection. Tumors developed in GAS-KO mice exhibited increased expression of PD-L1 and higher mutation rates than those in wild-type mice. To study the effect of anti-PD-1 antibody in tumor initiation or an early stage of progression, GAS-KO mice were induced with H.felis/MNU to develop tumors, one week later (10 weeks post-MNU) treated with anti-PD-1 antibody or isotype control (10 mg/kg IP qw) and sacrificed at 30 weeks post-MNU. We confirmed that GAS-KO mice treated with isotype control had moderate tumor burdens. Of interest, however, less and much smaller tumors were observed in mice treated with anti-PD-1. To compare immune cell populations in spleen tissue and peripheral blood from each group of mice, flow cytometric analysis was performed. Anti-PD-1-treated mice showed an increase in T cells including cytotoxic CD8+ T cells and a decrease in regulatory T cells and myeloid-derived suppressor cell (MDSC) populations compared with controls. In accordance with this, CD3-positive T lymphocytes were more frequently found in gastric tumors of anti-PD-1-treated mice than controls. Furthermore, qRT-PCR analysis revealed differential cytokine gene expression profiles including Il6, Il1b, and Il23 following anti-PD-1 treatment. Together, these data implicate that PD-1 blockade promotes immune infiltrates and therefore potentiates antitumor immune responses, suggesting targeting PD-1 as a potential new anticancer strategy in gastric cancer. Understanding anti-PD-1 therapy at the cellular and molecular level could provide new strategies for developing highly efficacious combination therapies. Citation Format: Woosook Kim, Yoku Hayakawa, James G. Fox, Timothy C. Wang. PD-1 blockade suppresses gastric cancer development by promoting antitumor immunity in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2340.