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Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naive B cells.

ABSTRACT: Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B-cell proliferation and Ab production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naïve B-cell class-switch recombination (CSR) and cytokine production. Gene expression levels of most TLRs was similar between newborn and adult B cells, except that newborn naïve B cells expressed more TLR9 than adult naïve B cells. Neonatal naïve B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn naïve B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn naïve B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early-life immunization.

SUBMITTER: Pettengill MA

PROVIDER: S-EPMC4963298 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells.

Pettengill Matthew A MA van Haren Simon D SD Li Ning N Dowling David J DJ Bergelson Ilana I Jans Jop J Ferwerda Gerben G Levy Ofer O

Innate immunity 20160601 6

Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B-cell proliferation and Ab production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naïve B-cell class-switch recombination (CSR) and cytoki ...[more]

PMID: 27252169

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Project description:IntroductionNeonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production.MethodsWhole newborn and adult blood was treated with PTX (50-200 ?M), DEX (10-10-10-7 M), or AZI (2.5-20 ?M), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity.ResultsPTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation. PTX preferentially inhibited pro-inflammatory cytokines especially TNF. DEX inhibited IL-10 in newborn, and TNF, IL-1?, IL-6 and interferon-? in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1?, IL-6 and IL-10, and LPS-induced IL-1? and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1?, and IL-6, and R848-induced IL-1? and interferon-?, while (PTX+AZI) synergistically decreased induction of TNF, IL-1?, and IL-6. Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA.ConclusionsAge, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.

Project description:IntroductionPrenatal smoking exposure may lead to permanent changes in neonatal inflammation and immune response that have lifelong implications, including increased risks for atopy and respiratory disorders.MethodsThe effect of maternal smoking on neonatal biomarkers of inflammation and immune response was assessed among 3459 singletons and twins in the Upstate KIDS Study. The following inflammatory biomarkers were measured using newborn dried blood spots (DBSs): interleukin (IL)-1?, IL-1 receptor antagonist, IL-6, IL-8, C-reactive protein, and tumor necrosis factor alpha. Immunoglobulins (IgE, IgA, IgM, and IgG subclasses) were also assessed. We used generalized estimating equations to calculate mean differences (?) in biomarker levels by timing of pregnancy smoking, cigarette load, and secondhand smoke exposure after adjusting for sociodemographic and lifestyle factors including maternal body mass index.ResultsOf the 344 (12%) women reporting smoking during pregnancy, about 40% continued throughout pregnancy and 13% reported smoking more than 1 pack per day. After covariate adjustment and Bonferroni correction for multiple comparisons, maternal smoking throughout pregnancy remained significantly associated with increased levels of IL-8 (? = 0.20, 95% confidence interval: 0.07, 0.32; p < .003). No significant associations were found with cigarette load or secondhand smoke exposure. Higher IgG3 levels were also associated with maternal smoking throughout pregnancy, although the association became nominally significant after adjustment for covariates (? = 0.09; 95% confidence interval: 0.0007, 0.17; p < .05).ConclusionsMaternal smoking throughout pregnancy was independently associated with increased IL-8 levels in newborns. Importantly, neonates of women who stopped smoking anytime in pregnancy did not have increased IL-8 levels.ImplicationsThis study evaluated a range of inflammatory biomarkers and immunoglobulins in association with maternal smoking and timing/duration of smoking along with secondhand smoke exposure. By using DBSs, we present data from a large cohort of children born in Upstate New York. Our findings suggest that early differences in immunoregulation of neonates exposed to maternal smoking for full duration in utero may already be detected at birth.

Project description:BackgroundNewborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells ("innate memory"). However, little is known regarding the impact of age on BCG-induced innate responses.ObjectiveEstablish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts.Design/methodsHuman neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay.ResultsCytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults.ConclusionsBCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

Project description:The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1?. Treatment of PBMCs with IL-6 induced production of TNF-?, CXCL8, and CCL2, but not IL-1?. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1? and CXCL8, but not TNF-? production compared with TLR ligands alone. This enhanced production of IL-1? and CXCL8 paralleled increased p65 NF-?B activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1?, TNF-? and CXCL8 with reduced p65 NF-?B activation. IL-6/IL-1? co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1? production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-?B phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.

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Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naive B cells.

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Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells.

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