Cytokine production by newborns: influence of sex and season of birth.

BACKGROUND: Immune signatures at birth could be associated with clinical outcomes and will improve our understanding of immunity prenatal programming. METHODS: Data come from 235 newborns from the cohort study NELA. Production of cytokines was determined using Luminex technology. Associations between cytokine concentrations with sex and season of birth were examined by multivariate regression models. RESULTS: Umbilical cord blood cells produced high levels of inflammatory cytokines, moderate levels of Th1/Th2/Tr-related cytokines, and low levels of Th17 cytokines. Compared to females, male newborn cells secreted higher levels of Th2 (peptidoglycan-stimulated IL-13, odds ratio [OR] = 2.26; 95% CI 1.18, 4.31, p value = 0.013) and Th17 (polyinosinic:polycytidylic acid-stimulated IL-23, OR = 1.82, 95% CI 1.01, 3.27, p value = 0.046) and lower levels of Th1 (olive-stimulated IL-2, OR = 0.56, 95% CI 0.31, 0.99, p value = 0.047) cytokines. Also, children born during warm seasons showed decreased innate cytokine response to peptidoglycan (IL-6, OR = 0.28, 95% CI 0.15, 0.52, p value < 0.001) compared to those born in cold seasons; meanwhile, adaptive immunity cytokines were more frequently secreted by children born during warm seasons in response to allergen extracts (IL-10, OR = 2.11, 95% CI 1.12, 3.96, p value = 0.020; IL-17F, OR = 3.31, 95% CI 1.83, 5.99, p value < 0.001). CONCLUSION: Newborns showed specific cytokines signatures influenced by sex and season of birth. IMPACT: There is a limited number of population-based studies on the immune status at birth and the influence of prenatal and perinatal factors on it. Characterization of cytokine signatures at birth related to the prenatal environment could improve our understanding of immunity prenatal programming. Newborns exhibit specific unstimulated and stimulated cytokine signatures influenced by sex and season of birth. Unstimulated and stimulated cytokine signatures in newborns may be associated with the development of related clinical outcomes later in life.

Cytokine profiles in cord blood in relation to prenatal traffic-related air pollution: The NELA cohort.

BACKGROUND: Outdoor air pollution may disturb immune system development. We investigated whether gestational exposure to traffic-related air pollutants (TRAP) is associated with unstimulated cytokine profiles in newborns. METHODS: Data come from 235 newborns of the NELA cohort. Innate response-related cytokines (IL-6, IFN-α, IL1-ß, and TNF-α), Th1-related (IFN-γ and IL-2), Th2-related (IL-4, IL-5, and IL-13), Th17-related (IL-17 and IL-23), and immunomodulatory cytokine IL-10 were quantified in the supernatant of unstimulated whole umbilical cord blood cells after 7 days of culture using the Luminex technology. Dispersion/chemical transport modeling was used to estimate long-term (whole pregnancy and trimesters) and short-term (15 days before delivery) residential exposures to traffic-related nitrogen dioxide (NO2 ), particulate matter (PM2.5 and PM10 ), and ozone (O3 ). We fitted multivariable logistic regression, Bayesian kernel machine regression (BKMR), and weighted quantile sum (WQS) regression models. RESULTS: NO2 during the whole pregnancy increased the odds of detection of IL-1ß (OR per 10 µg/m3 increase = 1.37; 95% CI, 1.02, 1.85) and IL-6 (OR per 10 µg/m3 increase = 1.32; 95% CI 1.00, 1.75). Increased odds of detected concentrations of IL-10 was found in newborns exposed during whole pregnancy to higher levels of NO2 (OR per 10 µg/m3 increase = 1.30; 95% CI 0.99, 1.69), PM10 (OR per 10 µg/m3 increase = 1.49; 95% CI 0.95, 2.33), and PM2.5 (OR per 5 µg/m3 increase = 1.56; 95% CI 0.97, 2.51). Exposure to O3 during the whole pregnancy increased the odds of detected IL-13 (OR per 10 µg/m3 increase = 1.22; 95% CI 1.01, 1.49). WQS model revealed first and third trimesters of gestation as windows of higher susceptibility. CONCLUSIONS: Gestational exposure to TRAP may increase detection of pro-inflammatory, Th2-related, and T regulatory cytokines in newborns. These changes might influence immune system responses later in life.

The Nutrition in Early Life and Asthma (NELA) birth cohort study: Rationale, design, and methods.

BACKGROUND: Primary prevention strategies for asthma are lacking. Its inception probably starts in utero and/or during the early postnatal period as the developmental origins of health and disease (DOHaD) paradigm suggests. OBJECTIVES: The main objective of Nutrition in Early Life and Asthma (NELA) cohort study is to unravel whether the following factors contribute causally to the developmental origins of asthma: (1) maternal obesity/adiposity and foetal growth; (2) maternal and child nutrition; (3) outdoor air pollution; (4) endocrine disruptors; and (5) maternal psychological stress. Maternal and offspring biological samples are used to assess changes in offspring microbiome, immune system, epigenome and volatilome as potential mechanisms influencing disease susceptibility. POPULATION: Randomly selected pregnant women from three health areas of Murcia, a south-eastern Mediterranean region of Spain, who fulfilled the inclusion criteria were invited to participate at the time of the follow-up visit for routine foetal anatomy scan at 19-22 weeks of gestation, at the Maternal-Fetal Medicine Unit of the "Virgen de la Arrixaca" University Clinical Hospital over a 36-month period, from March 2015 to April 2018. DESIGN: Prospective, population-based, maternal-child, birth cohort study. METHODS: Questionnaires on exposures and outcome variables were administered to mothers at 20-24 gestation week; 32-36 gestation week; and delivery. Children were surveyed at birth, 3 and 18 months of age and currently at 5 years. Furthermore, physical examinations were performed; and different measurements and biological samples were obtained at these time points. PRELIMINARY RESULTS: Among the 1350 women invited to participate, 738 (54%) were finally enrolled in the study and 720 of their children were eligible at birth. The adherence was high with 612 children (83%) attending the 3 months' visit and 532 children (72%) attending the 18 months' visit. CONCLUSION: The NELA cohort will add original and unique knowledge to the developmental origins of asthma.

Prenatal and Perinatal Environmental Influences Shaping the Neonatal Immune System: A Focus on Asthma and Allergy Origins.

It is suggested that programming of the immune system starts before birth and is shaped by environmental influences acting during critical windows of susceptibility for human development. Prenatal and perinatal exposure to physiological, biological, physical, or chemical factors can trigger permanent, irreversible changes to the developing immune system, which may be reflected in cord blood of neonates. The aim of this narrative review is to summarize the evidence on the role of the prenatal and perinatal environment, including season of birth, mode of delivery, exposure to common allergens, a farming environment, pet ownership, and exposure to tobacco smoking and pollutants, in shaping the immune cell populations and cytokines at birth in humans. We also discuss how reported disruptions in the immune system at birth might contribute to the development of asthma and related allergic manifestations later in life.

Air pollution from traffic during pregnancy impairs newborn's cord blood immune cells: The NELA cohort.

BACKGROUND: Hazards of traffic-related air pollution (TRAP) on the developing immune system are poorly understood. We sought to investigate the effects of prenatal exposure to TRAP on cord blood immune cell distributions; and to identify gestational windows of susceptibility. METHODS: In-depth immunophenotyping of cord blood leukocyte and lymphocyte subsets was performed by flow cytometry in 190 newborns embedded in the Nutrition in Early Life and Asthma (NELA) birth cohort (2015-2018). Long-term (whole pregnancy and trimesters) and short-term (15-days before delivery) residential exposures to traffic-related nitrogen dioxide (NO2), particulate matter (PM2.5 and PM10), and ozone (O3) were estimated using dispersion/chemical transport modelling. Associations between TRAP concentrations and cord blood immune cell counts were assessed using multivariate Poisson regression models. RESULTS: Mean number of natural killer (NK) cells decreased 15% in relation to higher NO2 concentrations (≥36.4 µg/m3) during whole pregnancy (incidence relative risk (IRR), 0.85; 95% CI, 0.72, 0.99), with stronger associations in the first trimester. Higher PM2.5 concentrations (≥13.3 µg/m3) during whole pregnancy associated with a reduced mean number of cytotoxic T cells (IRR, 0.88; 95% CI, 0.78, 0.99). Newborns exposed to higher PM10 (≥23.6 µg/m3) and PM2.5 concentrations during the first and third trimester showed greater mean number of helper T type 1 (Th1) cells (P < 0.05). Decreased number of regulatory T (Treg) cells was associated with greater short-term NO2 (IRR, 0.90; 95% CI, 0.80, 1.01) and PM10 (IRR, 0.88; 95% CI, 0.77, 0.99) concentrations. CONCLUSIONS: Prenatal exposure to TRAP, particularly in early and late gestation, impairs fetal immune system development through disturbances in cord blood leukocyte and lymphocyte distributions.

WNT Signaling in Tumors: The Way to Evade Drugs and Immunity.

WNT/ß-catenin signaling is involved in many physiological processes. Its implication in embryonic development, cell migration, and polarization has been shown. Nevertheless, alterations in this signaling have also been related with pathological events such as sustaining and proliferating the cancer stem cell (CSC) subset present in the tumor bulk. Related with this, WNT signaling has been associated with the maintenance, expansion, and epithelial-mesenchymal transition of stem cells, and furthermore with two distinctive features of this tumor population: therapeutic resistance (MDR, multidrug resistance) and immune escape. These mechanisms are developed and maintained by WNT activation through the transcriptional control of the genes involved in such processes. This review focuses on the description of the best known WNT pathways and the molecules involved in them. Special attention is given to the WNT cascade proteins deregulated in tumors, which have a decisive role in tumor survival. Some of these proteins function as extrusion pumps that, in the course of chemotherapy, expel the drugs from the cells; others help the tumoral cells hide from the immune effector mechanisms. Among the WNT targets involved in drug resistance, the drug extrusion pump MDR-1 (P-GP, ABCB1) and the cell adhesion molecules from the CD44 family are highlighted. The chemokine CCL4 and the immune checkpoint proteins CD47 and PD-L1 are included in the list of WNT target molecules with a role in immunity escape. This pathway should be a main target in cancer therapy as WNT signaling activation is essential for tumor progression and survival, even in the presence of the anti-tumoral immune response and/or antineoplastic drugs. The appropriate design and combination of anti-tumoral strategies, based on the modulation of WNT mediators and/or protein targets, could negatively affect the growth of tumoral cells, improving the efficacy of these types of therapies.

Regulatory T Cells in Allergy and Asthma.

The immune system's correct functioning requires a sophisticated balance between responses to continuous microbial challenges and tolerance to harmless antigens, such as self-antigens, food antigens, commensal microbes, allergens, etc. When this equilibrium is altered, it can lead to inflammatory pathologies, tumor growth, autoimmune disorders, and allergy/asthma. The objective of this review is to show the existing data on the importance of regulatory T cells (Tregs) on this balance and to underline how intrauterine and postnatal environmental exposures influence the maturation of the immune system in humans. Genetic and environmental factors during embryo development and/or early life will result in a proper or, conversely, inadequate immune maturation with either beneficial or deleterious effects on health. We have focused herein on Tregs as a reflection of the maturity of the immune system. We explain the types, origins, and the mechanisms of action of these cells, discussing their role in allergy and asthma predisposition. Understanding the importance of Tregs in counteracting dysregulated immunity would provide approaches to diminish asthma and other related diseases in infants.

Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival.

Having found previously that leukemic cells with multidrug resistant (MDR) phenotype, but not their sensitive counterparts, exhibit collateral sensitivity to cold stress in a P-gp-dependent manner, our aim was to study the signaling pathways involved in this phenomenon in sensitive (L1210) and resistant cells (L1210R and CBMC-6). It was observed that the acquisition of MDR phenotype by leukemic cells or their transfection with the extrussion pump, P-gp, modifies the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. More specifically, cold stress provoked the activation of c-Jun N-terminal kinase (JNK) in sensitive cells, while attenuated JNK signaling was observed in MDR cells. This effect was also observed, although with less intensity, in P-gp-transfected cells. Using pharmacological inhibitors to determine the role of MAPK in leukemic cell survival in physiological conditions or under cold stress, a dual temperature-dependent role was observed for JNK in MDR cell survival. At 37°C JNK is necessary for the survival of parental, resistant and P-gp-transfected cells; however, the use of inhibitors of either extracellular signal-regulated protein kinase (ERK) or JNK significantly counteracts cold-induced death of resistant and P-gp-transfected cells, supporting a role for ERK and JNK in cold-stress induced cell death. Finally, a connectivity model concerning MAPK is proposed, summarizing how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. These findings on MDR cells may assist in the design of specific therapeutic strategies to complement current chemotherapy.

Dock10 regulates CD23 expression and sustains B-cell lymphopoiesis in secondary lymphoid tissue.

Dock10, a guanine nucleotide exchange factor for the Rho GTPases Rac1 and Cdc42, affects cell morphology, membrane protrusive activity, and cell movement. Dock10 is prominently expressed in lymphoid tissue and upregulated by IL-4 in B cells. To investigate the physiological role of Dock10, WT mice and Dock10 KO mice were used. KO mice showed decreased numbers of B cells in spleen, both follicular B cells and marginal zone B cells, and in peripheral blood, but not in bone marrow. The antiapoptotic effect of IL-4 in vitro, the migratory response to CXCL13 or CCL21 in vitro, and the whole genome expression profile were intact in spleen B cells from KO mice. CD23, the low-affinity receptor for immunoglobulin E, was overexpressed on follicular B cells from KO mice, suggesting that Dock10 negatively regulates membrane CD23 expression. Negative regulation of CD23 expression by Dock10 could play a role in B cell maturation and function.

Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells is one of the main causes of chemotherapy failure in cancer, and, usually, is due to the increased expression of P-glycoprotein (MDR-1, P-gp, ABCB1), a pump that expels chemotherapeutics from the cell and/or regulates apoptosis. Thus, it is fundamental to find drugs or stress stimuli with a capacity to induce apoptosis in such cells and to identify the mechanisms involved. We address this matter in human cells and establish new daunomycin (DNM)-resistant cell lines (IM-9R) by exposing the parental lymphoblastic cells (IM-9) to increasing doses of the anti-neoplastic drug, daunomycin. The resistance level of IM-9R cell lines, MDR-1 expression and functionality, collateral sensitivity and Bcl-2 and caspases protein expression are analyzed. As a result, we show for the first time that, unlike the parental cells, human lymphoblastic resistant cells exhibit collateral sensitivity to cold stress, confirming that this phenomenon is not exclusive to murine leukemic cells, but a broader one associated with the acquisition of drug resistance. Furthermore, the new resistant cell lines undergo a significant increase in active caspase-3 and -9 levels and drastic changes in Bcl-2 family protein expression during the process of MDR phenotype acquisition.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL-6 secretion.

BACKGROUND: Bacterial infections are common complications arising in patients with cirrhosis and ascites. Translocation of bacterial DNA is a dynamic process that is associated with an increased inflammatory response and a poor prognosis in this setting. The aim of this study was to study whether peritoneal macrophages remain in a chronic primed status to allow a rapid response to subsequent events of bacterial translocation. PATIENTS AND METHODS: Peritoneal monocyte-derived macrophages were isolated from 25 patients with cirrhosis and non-infected ascites and compared with donor's blood monocytes. Activation cell-surface markers were screened using flow-cytometry, and the phosphorylation state of ERK 1/2, p38 MAP Kinase, PKB/Akt and transcription factors c-Jun and p65 NFκB were evaluated using Western blot. Synthesis of tumour necrosis factor alpha, interleukin 6 (IL-6) and interleukin-10 (IL-10) at baseline and in response to bacterial stimuli was evaluated using ELISA. RESULTS: A high expression of CD54, CD86 and HLA-DR at baseline was displayed by peritoneal macrophages. Increased phosphorylated levels of ERK1/2, protein kinase B (PKB) and c-Jun, together with IL-6 production, were observed in peritoneal macrophages at baseline compared with donors' blood monocytes. A positive correlation was established between basal IL-6 levels and extracellular signal-regulated kinase (ERK) phosphorylation in peritoneal macrophages from patients with cirrhosis (r=0·9; P=0·005). Addition of lipopolysaccharide induced higher phosphorylation levels of all studied signalling intermediates than synthetic-oligodeoxydinucleotides, but similar end-stage p65 NFκB. CONCLUSIONS: A sustained immune response is present in ascitic fluid of cirrhotic patients, even in the temporal absence of bacterial antigens. This would facilitate a fast response, probably controlled by IL-6, against repeated bacterial-DNA translocation or in liver chronic inflammation.

Synthetic oligodeoxynucleotides induce MAP kinases activation in murine TIB-73 hepatocytes.

AIMS: In this work we aimed to investigate the expression of TLR9 protein in the murine hepatocyte cell line TIB-73, compared to macrophage-like J774 cells, by Western blot analysis, and the role played by ERK 1/2 MAP kinase in the intracellular signals triggered by stimulation with CpG and non-CpG phosphodiester-ODN, and their more stable phosphorothioate-modified analogues. RESULTS: TIB-73 hepatocytes express TLR9 protein. CpG and non-CpG ODN stimulation activated ERK 1/2 MAPK signal pathway in both hepatocytes and J774 murine macrophages. As expected, their phosphorothioate-CpG and non-CpG ODN analogues induced higher levels of ERK1/2 phosphorylation in TIB-73 cells, even higher than that induced in J774 cells under the same conditions. Phosphorylation of ERK 1/2 induced by synthetic ODN is dose-response dependent, being maximal at 100 microg/ml. Pretreatment of hepatocytes with an inhibitor of MEK-1 abrogated phosphorylation of ERK1/2 kinase. CONCLUSIONS: TIB-73 hepatocytes constitutively express TLR9 and respond to synthetic ODN stimulation through a high ERK1/2 phosphorylation independent of CpG motifs. Slight differences were found on ERK1/2 activation when using phosphorothioate versus phosphodiester oligonucleotides.

Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their susceptibility to the host immune response.

The use of antineoplastic drugs for cancer treatment is frequently associated with the acquisition of a multidrug-resistant (MDR) phenotype that renders tumoural cells insensitive to antineoplastics. It remains elusive whether the acquisition of the MDR phenotype alters immunological parameters that could influence the cell sensitivity to an eventual host immune response. We report that immunisation of syngeneic mice with gamma-irradiated L1210S (parental line) and L1210R (MDR phenotype) cells results in a significant rejection of subsequently implanted L1210R-based tumours, but not of the L1210S ones. Notably, L1210R tumours display a twofold reduction in vivo proliferative capacity and are less aggressive in terms of mouse survival than their sensitive counterparts. Also, analysis of surface expression of molecules involved in antigen presentation and cytokine activity revealed a slight increase in IFN-gamma receptor expression, a decrease of Fas molecule, and a fourfold up-regulation of MHC class I molecules in L1210R cells. Nonetheless, both cell lines were able to induce a cytotoxic response in syngeneic mice and were equally susceptible to cytotoxicity by splenic cells. Together, these findings indicate that acquisition of drug resistance by L1210 cells is accompanied by pleiotropic changes that result in reduced tumour proliferative capacity and tumorigenicity in syngeneic mice. Hence, immunological studies of MDR tumours may assist in the design of specific therapeutic strategies that complement current chemotherapy treatments.

Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes.

Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.

Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors.

The main goal of our study has been to analyze the efficiency of new anticancer drugs, specifically histone deacetylase inhibitors, in tumor cells bearing a multidrug resistance phenotype. We report that the histone deacetylase inhibitors, Trichostatin A and Suberoylanilide Hydroxamic Acid (SAHA), dramatically reduce cell viability and promote apoptosis in different drug-resistant cells, affecting in a much lesser extent to their parental drug-sensitive counterparts. The differential effects induced by Trichostatin A and SAHA between drug-sensitive and drug-resistant cells are reflected on the main characteristics of the resistant phenotype. Thus, reverse transcription-PCR and Western immunoblots confirm that both histone deacetylase inhibitors promote endogenous down-regulation of P-glycoprotein, which is overexpressed in the drug-resistant cells. Transfection of drug-sensitive cells with the P-glycoprotein cDNA ruled out the a priori possible association between apoptosis and down-regulation of P-glycoprotein induced by the histone deacetylase inhibitors. The results suggest a therapeutic potential of histone deacetylase inhibitors in the treatment of cancers with acquired resistance.