Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection

BACKGROUND Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. RESULTS For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. CONCLUSIONS These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

Anti-inflammatory effect of exercise training in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise modalities and independent of weight loss

We investigated the effect of different exercise modalities on high sensitivity-C reactive protein (hs-CRP) and other inflammatory markers in patients with type 2 diabetes and the metabolic syndrome. Eighty-two patients were randomized into 4 groups: sedentary control (A); receiving counseling to perform low-intensity physical activity (B); performing prescribed and supervised high-intensity aerobic (C) or aerobic + resistance (D) exercise (with the same caloric expenditure) for 12 months. Evaluation of leisure-time physical activity and assessment of physical fitness, cardiovascular risk factors and inflammatory biomarkers was performed at baseline and every 3 months. Volume of physical activity increased and HbA1c decreased in Groups B–D. VO2max, HOMA-IR index, HDL-cholesterol, waist circumference and albuminuria improved in Groups C and D, whereas strength and flexibility improved only in Group D. Levels of hs-CRP decreased in all three exercising groups, but the reduction was significant only in Groups C and D, and particularly in Group D. Changes in VO2max and the exercise modalities were strong predictors of hs-CRP reduction, independent of body weight. Leptin, resistin and interleukin-6 decreased, whereas adiponectin increased in Groups C and D. Interleukin-1β, tumor necrosis factor-α and interferon-γ decreased, whereas anti-inflammatory interleukin-4 and 10 increased only in Group D. In conclusion, physical exercise in type 2 diabetic patients with the metabolic syndrome is associated with a significant reduction of hs-CRP and other inflammatory and insulin resistance biomarkers, independent of weight loss. Long-term high-intensity (preferably mixed) training, in addition to daytime physical activity, is required to obtain a significant anti-inflammatory effect.

Caracterización y prevalencia del compromiso ocular en pacientes con enfermedad autoinmune y autoinflamatoria en un centro de referencia reumatológica en Colombia entre los años 2000 a 2015

RESUMEN Objetivo: Estimar la prevalencia de las diferentes enfermedades oftalmológicas que aparecen en el contexto de una enfermedad autoinmune (EAI) en pacientes de un centro de referencia reumatológica en Colombia, según características clínicas y sociodemográficas durante un período de 15 años, comprendido entre los años 2000 a 2015. Métodos: Se realizó un estudio descriptivo, observacional de prevalencia. El tipo de muestreo fue aleatorio estratificado con asignación proporcional en el programa Epidat 3.4. Los datos se analizaron en el programa SPSS v22.0 y se realizó análisis univariado de las variables categóricas, para las variables cuantitativas se realizaron medidas de tendencia central. Resultados: De 1640 historias clínicas revisadas, se encontraron 634 pacientes (38,65%) con compromiso ocular. Si excluimos los pacientes con SS, que por definición presentan ojo seco, 222 pacientes (13,53%) presentaron compromiso oftalmológico. Del total de pacientes, el 83,3% fueron mujeres. La AR fue la enfermedad autoinmune con mayor compromiso oftalmológico con 138 pacientes (62,2%), y en último lugar la sarcoidosis con 1 solo paciente afectado. La QCS fue la manifestación más común en todos los grupos diagnósticos de EAI, con 146 pacientes (63,5%). De 414 pacientes con Síndrome de Sjögren (SS) y QCS 8 presentaron compromiso ocular adicional, siendo la uveítis la segunda patología ocular asociada en pacientes con SS y la primera causa en las espondiloartropatias (71,4 %). Los pacientes con catarata (4,1%) presentaron la mayor prevalencia de uso de corticoide (88.8%). De 222 pacientes, 28 (12,6%) presentaron uveítis. Del total de pacientes, 16 (7,2%) presentaron maculopatía por antimalaráricos y 6 (18,75%) de los pacientes con LES. Los ANAS se presentaron en el 100% los pacientes con trastorno vascular de la retina. Los pacientes con epiescleritis presentaron la mayor proporción de positivización de anticuerpos anti-DNA. La EAI que más presentó epiescleritis fue LES con 4 pacientes (12,5%) El 22% de paciente con anticuerpos anti-RNP presentaron escleritis y 32,1% de los pacientes con uveítis presentaron HLA-B27 positivo. Las manifestaciones oftalmológicas precedieron a las sistémicas entre un 11,1% y un 33,3% de los pacientes. Conclusión: Las enfermedades oculares se presentan con frecuencia en los pacientes colombianos con EAI (38.65%), siendo la AR la enfermedad con mayor compromiso ocular (62,2%) y la QCS la enfermedad ocular con mayor prevalencia en todas las EAI (63,5%). La uveítis se presentó en 28 pacientes (12,6%). Las manifestaciones oftalmológicas pueden preceder a las sistémicas. El examen oftalmológico debe ser incluido en los pacientes con EAI, por ser la enfermedad ocular una comorbilidad frecuente. Adicionalmente, los efectos oftalmológicos de las medicaciones sistémicas utilizadas en EAI deben ser estrechamente monitorizados, durante el curso del tratamiento.

Interleukin-18 and interferon-gamma polymorphisms in Brazilian human immunodeficiency virus-1-infected patients presenting with lipodystrophy syndrome

Cytokines play important roles in the pathogenesis of lipodystrophy syndrome (LS). Single nucleotide polymorphisms (SNPs) at positions -607(C/A) and -137(C/G) in the promoter region of the interleukin-18 (IL-18) gene and at position +874(T/A) of the interferon-gamma (IFN-gamma) gene are related to the expression of these cytokines. To examine whether IL-18 and IFN-gamma polymorphisms are associated with LS, these SNPs were genotyped in 88 human immunodeficiency virus (HIV)-infected patients presenting LS, 79 HIV-infected without LS, and 133 healthy controls. The -607A allele, -607AA genotype, and -137G/-607A and -137C/-607A haplotypes in the IL-18 gene were over-represented in HIV patients presenting LS. The -137G/-607C haplotype was associated with protection against LS. These results indicate that the -607(C/A) SNP is associated with LS development in HIV-infected patients.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Interferon-gamma, interleukin-10, intercellular adhesion molecule-1, and chemokine receptor 5, but not interleukin-4, attenuate the development of periapical lesions

This study examines the role of Th1 (interferon-gamma [IFN-gamma]) and Th2 (interleukin-4 [IL-4] and IL-10) cytokines, an intercellular adhesion molecule (ICAM-1), and a chemokine receptor (CCR5) in the pathogenesis of periapical lesions at different stages of development in knockout mice. For lesion induction, the first molar was opened and inoculated with 4 bacterial strains and left open to the oral environment. After 21 and 42 days, the IFN-gamma, IL-10, ICAM-1, and CCR5 knockout animals presented periapical lesions larger than those of wild-type animals. There was no statistically significant difference between periapical lesions induced in IL-4 knockout and wild-type animals during the periods evaluated. Our findings suggest an important role for IFN-gamma, IL-10, ICAM-1, and CCR5 in the pathogenesis of experimentally induced pulp infection and bone destruction as endogenous suppressor of periapical lesion development, whereas IL-4 appears to present a nonsignificant effect on periapical lesion modulation.

Interleukin-28B polymorphisms, IP-10, viral load and age predict the outcome of therapy in genotype 1 hepatitis C virus under real-life conditions

Background and Aims: IL28B polymorphisms, interferon (IFN)-gamma inducible protein-10 (IP-10) levels and the homeostasis model assessment of insulin resistance (HOMA-IR) score have been reported to predict rapid (RVR) and sustained (SVR) virological response in chronic hepatitis C (CHC), but it is not known whether these factors represent independent, clinically useful predictors. The aim of the study was to assess factors (including IL28B polymorphisms, IP-10 levels and HOMA-IR score) independently predicting response to therapy in CHC under real life conditions.Methods: Multivariate analysis of factors predicting RVR and SVR in 280 consecutive, treatment-naive CHC patients treated with pegylated IFN alpha and ribavirin in a prospective multicenter study.Results: Independent predictors of RVR were HCV RNA < 400,000 IU/ml (OR11.37; 95% CI 3.03-42.6), rs12980275 AA (vs. AG/GG) (OR 7.09; 1.97-25.56) and IP-10 (OR 0.04; 0.003-0.56) in HCV genotype 1 patients and lower baseline γ-glutamyl-transferase levels (OR = 0.02; 0.0009-0.31) in HCV genotype 3 patients. Independent predictors of SVR were rs12980275 AA (OR 9.68; 3.44-27.18), age < 40 yrs (OR = 4.79; 1.50-15.34) and HCV RNA < 400,000 IU/ml (OR 2.74; 1.03-7.27) in HCV genotype 1 patients and rs12980275 AA (OR = 6.26; 1.98-19.74) and age < 40 yrs (OR 5.37; 1.54-18.75) in the 88 HCV genotype 1 patients without a RVR. RVR was by itself predictive of SVR in HCV genotype 1 patients (32 of 33, 97%; OR 33.0; 4.06-268.32) and the only independent predictor of SVR in HCV genotype 2 (OR 9.0, 1.72-46.99; p=0.009) or 3 patients (OR 7.8, 1.43-42.67; p=0.01).Conclusions: In HCV genotype 1 patients, IL28B polymorphisms, HCV RNA load and IP-10 independently predict RVR. The combination of IL28B polymorphisms, HCV RNA level and age may yield more accurate pretreatment prediction of SVR. HOMA-IR score is not associated with viral response.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production.

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor beta (IL-2R beta; CD122) and the polymorphic antigen NK1.1. These "NK-like" T cells are distinguished by their highly skewed V alpha and V beta repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1+ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2R beta+ T cells with a skewed V beta repertoire characteristic of "NK-like" T cells. Furthermore lack of NK1.1 expression on IL-2R beta+ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2R beta+ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that "NK-like" T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.

In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1-.

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-gamma (IFN-gamma) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-gamma dependent. Treatment of resistant C57BL/6 mice with anti-IFN-gamma allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in V beta 8, 7, 2- CD4+ cells in BALB/c mice and NK1.1- CD4+ cells in anti-IFN-gamma treated C57BL/6 mice. These results show that the NK1.1+ CD4+ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.

Induction of interleukin-10 by HIV antigens in peripheral mononuclear cells of health care workers after occupational exposure to HIV-1-positive blood

Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.

TIMP-1 mediates the inhibitory effect of interleukin-6 on the proliferation of a hepatocarcinoma cell line in a STAT3-dependent manner

The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.

Effect of metabolic control on interferon-gamma and interleukin-10 production by peripheral blood mononuclear cells from type 1 and type 2 diabetic patients

The objective of the present study was to evaluate the production of cytokines, interferon-g (INF-g) and interleukin-10 (IL-10), in cultures of peripheral blood mononuclear cells (PBMC) from type 1 and type 2 diabetic patients and to correlate it with inadequate and adequate metabolic control. We studied 11 type 1 and 13 type 2 diabetic patients and 21 healthy individuals divided into two groups (N = 11 and 10) paired by sex and age with type 1 and type 2 diabetic patients. The PBMC cultures were stimulated with concanavalin-A to measure INF-g and IL-10 supernatant concentration by ELISA. For patients with inadequate metabolic control, the cultures were performed on the first day of hospitalization and again after intensive treatment to achieve adequate control. INF-g levels in the supernatants of type 1 diabetic patient cultures were higher compared to type 2 diabetic patients with adequate metabolic control (P < 0.001). Additionally, INF-g and IL-10 tended to increase the liberation of PBMC from type 1 and 2 diabetic patients with adequate metabolic control (P = 0.009 and 0.09, respectively). The increased levels of INF-g and IL-10 released from PBMC of type 1 and 2 diabetic patients with adequate metabolic control suggest that diabetic control improves the capacity of activation and maintenance of the immune response, reducing the susceptibility to infections.

Stress-related hormone norepinephrine induces interleukin-6 expression in GES-1 cells

In the current literature, there is evidence that psychological factors can affect the incidence and progression of some cancers. Interleukin 6 (IL-6) is known to be elevated in individuals experiencing chronic stress and is also involved in oncogenesis and cancer progression. However, the precise mechanism of IL-6 induction by the stress-related hormone norepinephrine (NE) is not clear, and, furthermore, there are no reports about the effect of NE on IL-6 expression in gastric epithelial cells. In this study, we examined the effect of NE on IL-6 expression in immortalized human gastric epithelial cells (GES-1 cells). Using real-time PCR and enzyme-linked immunoassay, we demonstrated that NE can induce IL-6 mRNA and protein expression in GES-1 cells. The induction is through the β-adrenergic receptor-cAMP-protein kinase A pathway and mainly at the transcriptional level. Progressive 5′-deletions and site-directed mutagenesis of the parental construct show that, although activating-protein-1 (AP-1), cAMP-responsive element binding protein (CREB), CCAAT-enhancer binding protein-β (C/EBP-β), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding sites are all required in the basal transcription of IL-6, only AP-1 and CREB binding sites in the IL-6 promoter are required in NE-induced IL-6 expression. The results suggest that chronic stress may increase IL-6 secretion of human gastric epithelial cells, at least in part, by the stress-associated hormone norepinephrine, and provides basic data on stress and gastric cancer progression.