Active systemic lupus erythematosus is associated with a reduced cytokine production by B cells in response to TLR9 stimulation

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break in self-tolerance reflected by a production of antinuclear autoantibodies. Since autoantibody production can be activated via nucleic acid Toll-like receptor 9 (TLR9), the respective pathway has been implicated in the development of SLE and pathogenic B cell responses. However, the response of B cells from SLE patients to TLR9 stimulation remains incompletely characterized.

Methods

In the current study, the response of B cells from SLE patients and healthy donors upon TLR9 stimulation was analyzed in terms of proliferation and cytokine production and correlated with the lupus disease activity and anti-dsDNA titers.

Results

B cells from SLE patients showed a reduced response to TLR9 agonist compared to B cells from healthy donors in terms of proliferation and activation. B cells from SLE patients with higher disease activity produced less interleukin (IL)-6, IL-10, vascular endothelial growth factor, and IL-1ra than B cells from healthy donors. Further analyses revealed an inverse correlation of cytokines produced by TLR9-stimulated B cells with lupus disease activity and anti-dsDNA titer, respectively.

Conclusion

The capacity of B cells from lupus patients to produce cytokines upon TLR9 engagement becomes less efficient with increasing disease activity, suggesting that they either enter an exhausted state or become tolerant to TLR stimulation for cytokine production when disease worsens.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0477-1) contains supplementary material, which is available to authorized users.     

DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas lpr/lpr mice in vivo

Introduction  

B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.     

Sex Differences in Monocyte Activation in Systemic Lupus Erythematosus (SLE)

Introduction

TLR7/8 and TLR9 signaling pathways have been extensively studied in systemic lupus erythematosus (SLE) as possible mediators of disease. Monocytes are a major source of pro-inflammatory cytokines and are understudied in SLE. In the current project, we investigated sex differences in monocyte activation and its implications in SLE disease pathogenesis.

Methods

Human blood samples from 27 healthy male controls, 32 healthy female controls, and 25 female patients with SLE matched for age and race were studied. Monocyte activation was tested by flow cytometry and ELISA, including subset proportions, CD14, CD80 and CD86 expression, the percentage of IL-6-producing monocytes, plasma levels of sCD14 and IL-6, and urine levels of creatinine.

Results

Monocytes were significantly more activated in women compared to men and in patients with SLE compared to controls in vivo. We observed increased proportions of non-classic monocytes, decreased proportions of classic monocytes, elevated levels of plasma sCD14 as well as reduced surface expression of CD14 on monocytes comparing women to men and lupus patients to controls. Plasma levels of IL-6 were positively related to sCD14 and serum creatinine.

Conclusion

Monocyte activation and TLR4 responsiveness are altered in women compared to men and in patients with SLE compared to controls. These sex differences may allow persistent systemic inflammation and resultant enhanced SLE susceptibility.     

Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis

Background

Deficiency in clearance of self nuclear antigens, including DNA, is the hallmark of systemic lupus erythematosus (SLE), a chronic autoimmnue disease characterized by the production of various autoantibodies, immune complex deposition and severe organ damage. Our previous studies revealed that administration of syngeneic BALB/c mice with activated lymphocyte-derived DNA (ALD-DNA) could induce SLE disease. Mannose-binding lectin (MBL), a secreted pattern recognition receptor with binding activity to DNA, has been proved to be a modulator of inflammation, but whether MBL takes responsibility for DNA clearance, modulates the DNA-mediated immune responses, and is involved in the development of DNA-induced SLE disease remain poorly understood.

Methodology/Principal Findings

The levels of serum MBL significantly decreased in lupus mice induced by ALD-DNA and were negatively correlated with SLE disease. MBL blunted macrophage M2b polarization by inhibiting the MAPK and NF-κB signaling while enhancing the activation of CREB. Furthermore, MBL suppressed the ability of ALD-DNA–stimulated macrophages to polarize T cells toward Th1 cells and Th17 cells. Importantly, MBL supplement in vivo could ameliorate lupus nephritis.

Conclusion/Significance

These results suggest MBL supplement could alleviate SLE disease and might imply a potential therapeutic strategy for DNA-induced SLE, which would further our understanding of the protective role of MBL in SLE disease.     

Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Introduction

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.

Methods

Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.

Results

Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).

Conclusions

This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.     

Inhibitor of IκB kinase activity,BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

Multidimensional single cell based STAT phosphorylation profiling identifies a novel biosignature for evaluation of systemic lupus erythematosus activity

Introduction

Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE.

Methods

Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored.

Results

We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells.

Conclusions

The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.     

Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

Introduction

More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.

Methods

Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.

Results

Circulating CD14⁺ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.

Conclusions

Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.     

Detection of Circulating B Cells Producing Anti-GPIb Autoantibodies in Patients with Immune Thrombocytopenia

Background

We previously reported that an enzyme-linked immunospot (ELISPOT) assay for detecting anti-GPIIb/IIIa antibody-secreting B cells is a sensitive method for identifying patients with immune thrombocytopenia (ITP). Here we assessed the clinical significance of measuring circulating B cells producing antibodies to GPIb, another major platelet autoantigen.

Methods

Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were simultaneously measured using ELISPOT assays in 32 healthy controls and 226 consecutive thrombocytopenic patients, including 114 with primary ITP, 25 with systemic lupus erythematosus (SLE), 30 with liver cirrhosis, 39 with post-hematopoietic stem cell transplantation (post-HSCT), and 18 non-ITP controls (aplastic anemia and myelodysplastic syndrome).

Results

There were significantly more circulating anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells in primary ITP, SLE, liver cirrhosis, and post-HSCT patients than in healthy controls (P<0.05 for all comparisons). For diagnosing primary ITP, the anti-GPIb ELISPOT assay had 43% sensitivity and 89% specificity, whereas the anti-GPIIb/IIIa ELISPOT assay had 86% sensitivity and 83% specificity. When two tests were combined, the sensitivity was slightly improved to 90% without a reduction in specificity. In primary ITP patients, the anti-GPIb antibody response was associated with a low platelet count, lack of Helicobacter pylori infection, positive anti-nuclear antibody, and poor therapeutic response to intravenous immunoglobulin.

Conclusion

The ELISPOT assay for detecting anti-GPIb antibody-secreting B cells is useful for identifying patients with ITP, but its utility for diagnosing ITP is inferior to the anti-GPIIb/IIIa ELISPOT assay. Nevertheless, detection of the anti-GPIb antibody response is useful for subtyping patients with primary ITP.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

Increased expression of Toll-like receptors by monocytes and natural killer cells in ANCA-associated vasculitis

Introduction

Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo.

Methods

Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed.

Results

In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4⁺ monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC.

Conclusions

In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.     

TLR4 Asp299Gly and Thr399Ile Polymorphisms: No Impact on Human Immune Responsiveness to LPS or Respiratory Syncytial Virus

Background

A broad variety of natural environmental stimuli, genotypic influences and timing all contribute to expression of protective versus maladaptive immune responses and the resulting clinical outcomes in humans. The role of commonly co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms Asp299Gly and Thr399Ile in this process remains highly controversial. Moreover, what differential impact these polymorphisms might have in at risk populations with respiratory dysfunction, such as current asthma or a history of infantile bronchiolitis, has never been examined. Here we determine the importance of these polymorphisms in modulating LPS and respiratory syncytial virus (RSV) - driven cytokine responses. We focus on both healthy children and those with clinically relevant respiratory dysfunction.

Methodology

To elucidate the impact of TLR4 Asp299Gly and Thr399Ile on cytokine production, we assessed multiple immune parameters in over 200 pediatric subjects aged 7–9. Genotyping was followed by quantification of pro- and anti-inflammatory cytokine responses by fresh peripheral blood mononuclear cells upon acute exposure to LPS or RSV.

Principal Findings

In contrast to early reports, neither SNP influenced immune responses evoked by LPS exposure or RSV infection, as measured by the intermediate phenotype of pro- and anti-inflammatory cytokine responses to these ubiquitous agents. There is no evidence of altered sensitivity in populations with “at risk” clinical phenotypes.

Conclusions/Significance

Genomic medicine seeks to inform clinical practice. Determination of the TLR4 Asp299Gly/Thr399Ile haplotype is of no clinical benefit in predicting the nature or intensity of cytokine production in children whether currently healthy or among specific at-risk groups characterized by prior infantile broncholitis or current asthma.     

A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype

Background

Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize “danger” signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol.

Principal Findings

Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1.

Significance

Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies.     

Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

Background

Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs).

Methods

Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state.

Results

HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam₃CSK₄, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β₂-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5.

Conclusion

Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.     

The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

Background

Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed.

Methods

TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry).

Results

VTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC₅₀, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs.

Conclusions

VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.     

Dysfunctional interferon-α production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus

Background  

It is well known that interferon (IFN)-α is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-α producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum.     

Acquisition of Adult-Like TLR4 and TLR9 Responses during the First Year of Life

Background

Characteristics of the human neonatal immune system are thought to be responsible for heightened susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells, many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life.

Methodology/Principal Findings

Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life for mDCs and 6–9 months of life for monocytes and pDCs. In response to LPS, production of TNF-α, IP-10 and IL-12p70 reached adult levels between 6–9 months of life. In response to CpG stimulation, production of type I IFN-dependent chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1β and IL-10, a situation that was not observed for 3 month-old infants.

Conclusions

The first year of life represents a critical period during which adult-like levels of TLR responses are reached for most but not all cytokine responses.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

Objectives

To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect.

Methods

Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using ³H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay.

Results

TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67⁺) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages.

Conclusions

IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation.