CD14-independent responses induced by a synthetic lipid A mimetic.

CRX-527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates, which are considered as potential vaccine adjuvants or stand-alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane-bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527. First, we found that CRX-527 induces NF-kappaB and interferon regulatory factor-3 (IRF-3) activation in human embryonic kidney cells transfected with TLR4 and MD-2 genes alone, whereas the responses to LPS require either co-transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte-derived DC, which are devoid of mCD14 respond to CRX-527 but not to LPS in serum-free medium. Furthermore, we found that, in contrast to LPS, CRX-527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14-dependent responses are defective. Finally, we demonstrated that splenocytes from CD14-deficient mice produce cytokines in response to CRX-527 but not to LPS. We conclude that the lipid A mimetic CRX-527 does not require the CD14 co-receptor to elicit TLR4-mediated responses.

A defect in nucleosome remodeling prevents IL-12(p35) gene transcription in neonatal dendritic cells.

To gain insight into the inability of newborns to mount efficient Th1 responses, we analyzed the molecular basis of defective IL-12(p35) expression in human neonatal monocyte-derived dendritic cells (DCs). Determination of IL-12(p35) pre-mRNA levels by real-time RT-PCR revealed that transcriptional activation of the gene in lipopolysaccharide-stimulated neonatal DCs was strongly impaired compared with adult DCs. We next showed that p50/p65 and p65/p65 dimers interact with kB#1 site, a critical cis-acting element of the IL-12(p35) promoter. We found that LPS-induced p65 activation was similar in adult and newborn DCs. Likewise, in vitro binding activity to the Sp1#1 site, previously shown to be critical for IL-12(p35) gene activation, did not differ in adults and newborns. Since the accessibility to this Sp1#1 site was found to depend on nucleosome remodeling, we used a chromatin accessibility assay to compare remodeling of the relevant nucleosome (nuc-2) in adult and neonatal DCs. We observed that nuc-2 remodeling in neonatal DCs was profoundly impaired in response to lipopolysaccharide. Both nuc-2 remodeling and IL-12(p35) gene transcription were restored upon addition of recombinant interferon-gamma. We conclude that IL-12(p35) transcriptional repression in neonatal DCs takes place at the chromatin level.

Lysosome-Dependent Activation of Human Dendritic Cells by the Vaccine Adjuvant QS-21.

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Dendritic cells exposed to nacystelyn are refractory to maturation and promote the emergence of alloreactive regulatory t cells.

BACKGROUND: Drugs blocking dendritic cell (DC) maturation might be useful in transplantation by inhibiting the induction of primary alloimmune responses and promoting the emergence of regulatory T lymphocytes (Treg). We investigated the effects of Nacystelyn (NAL), an N-acetyl-L-cysteine derivative, on human DCs, paying attention to the T-cell responses elicited by NAL-treated DCs in vitro. METHODS: Lipopolysaccharide (LPS) was used to induce the maturation of DCs naturally present in blood or generated from human monocytes cultured in interleukin-4 and granulocyte-macrophage colony-stimulating activity. We first analyzed the consequences of NAL on cytokine production and expression of major histocompatibility complex class II and costimulatory molecules. Monocyte-derived DCs were then used as stimulators in mixed leukocyte cultures with naive CD4 T cells. Cytokine levels were measured in culture supernatants; the phenotype of T cells and their capacity to inhibit the proliferation of third-party T-cell responders was determined at the end of the culture. RESULTS: NAL proved to be a potent inhibitor of DC maturation in whole blood experiments and on monocyte-derived DCs. Alloreactive T cells stimulated with DCs pretreated with LPS in the presence of NAL produced much less interferon-gamma but similar levels of interleukin-13 compared with DCs treated with LPS alone. Immature DCs induced Treg, which was not observed with mature DCs. DCs cultured with LPS in the presence of NAL were as efficient as immature DCs to generate alloreactive T cells with regulatory activity. CONCLUSIONS: NAL is a potent inhibitor of DC maturation, which might be useful to promote allograft acceptance by inducing the differentiation of allospecific Treg.

Adjuvant activity on human cells in vitro.

Efficient vaccines against intracellular microbes or tumors will be based on innovative adjuvants able to induce efficient activation of dendritic cells. Indeed, natural or synthetic products activating Toll-like receptors (TLR) on dendritic cells (DCs) are currently in development for this purpose. Herein, we describe in vitro assays on human cells which might be useful for the preclinical screening and assessment of potential DC activators.

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-alpha, 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-1beta 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.

Interferon regulatory factor 7-mediated responses are defective in cord blood plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDC) are specialized in massive production of type I interferons (IFN) upon viral infections. Activation of IFN regulatory factor (IRF)-7 is critically required for the synthesis of type I IFN in pDC. IRF-7 is highly expressed by resting pDC and translocates into the nucleus to initiate type I IFN transcription. In a previous work, we observed an impaired IFN-alpha production in enriched cord blood pDC following a TLR9 stimulation using CpG oligonucleotides. Herein, we show that highly purified pDC from cord blood exhibit a profound defect in their capacity to produce IFN-alpha/beta in response to TLR9 as well as to TLR7 ligation or human CMV or HSV-1 exposure. Microarray experiments indicate that expression of the majority of type I IFN subtypes induced by a TLR7 agonist is reduced in cord blood pDC. We next demonstrated a reduced nuclear translocation of IRF-7 in cord blood pDC following CpG and HSV stimulation as compared to adult pDC. We conclude that impaired IRF-7 translocation in cord blood pDC is associated with defective expression of type I IFN genes. Our data provide a molecular understanding for the decreased ability of cord blood pDC to produce type I IFN upon viral stimulation.

IL-27 synthesis induced by TLR ligation critically depends on IFN regulatory factor 3.

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3(-/-) upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.

Interferon regulatory factor 3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells.

The synthesis of interferon-beta (IFNbeta) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3-dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNbeta and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNbeta synthesis because IFNbeta signaling was functional in neonatal moDCs. However, addition of exogenous IFNbeta failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNbeta and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.

Preferential production of the IL-12(p40)/IL-23(p19) heterodimer by dendritic cells from human newborns.

Human newborns present impaired T helper type 1 cell responses, associated with a defect in the synthesis of IL-12 by dendritic cells (DC). IL-23 is a heterodimeric cytokine structurally related to IL-12, implicated in protective and autoimmune responses. We recently showed that upon activation neonatal T cells up-regulate a functional IL-23 receptor and that this cytokine polarizes the differentiation of naive T cells. We therefore investigated the capacity of neonatal DC to secrete IL-23. Lipopolysaccharide (LPS) stimulation induced the transcription of IL-23(p19) mRNA in both adult and neonatal DC, in sharp contrast to the repressed IL-12(p35) gene expression observed in neonatal cells. In comparison to adult DC, neonatal DC produced similar levels of IL-23 protein, in reponse to Toll-like receptor (TLR)-2- and TLR-3 ligands, and higher levels in response to TLR-4- or TLR-8 ligands. The same profile was observed in neonatal mononuclear cells. The supernatant of LPS-stimulated DC induced the secretion of IL-17 by polyclonally activated neonatal CD8(+) T cells, confirming the IL-23 bioactivity. Altogether, these observations strongly suggest that IL-23 could play a role in the immune system of human newborns. In particular, a functional IL-23/IL-17 axis might compensate a suboptimal IL-12/IFN-gamma pathway in early life.

Interferon regulatory factor 3 is involved in Toll-like receptor 4 (TLR4)- and TLR3-induced IL-12p35 gene activation.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.

Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes.

Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA(+)CD4(+) T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-alpha and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4(+) T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-gamma by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-kappaB and NFAT transcription factors. Overall, responses of neonatal CD4(+) T cells to PTX were similar to those of adult CD45RA(+)CD4(+) naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen-presenting cells.

IL-23 up-regulates IL-10 and induces IL-17 synthesis by polyclonally activated naive T cells in human.

Interleukin (IL)-23 is a heterodimeric cytokine of the IL-12 family. Human IL-23 is known to induce interferon (IFN)-gamma production and proliferation in T cells, preferentially in the CD45RO+ memory subset. Yet, its role in the differentiation of human naive T cells remains largely unknown. We investigated the effect of recombinant human (rh)IL-23 on cord blood CD4+ and CD8+ T cells during polyclonal activation. The IL-23 receptor complex was not detectable in resting naive T cells. Nevertheless, both IL-23 receptor subunits, IL-12Rbeta1 and IL-23R, were rapidly induced after activation in both naive CD4+ and CD8+ T cells. In both cell types, rhIL-23 enhanced IFN-gamma production. This effect was demonstrable as early as 2 days after activation, illustrating that a functional IL-23 receptor is rapidly induced in naive T cells upon activation. In naive CD8+ T cells, rhIL-23 specifically induced the secretion of IL-17, a pro-inflammatory cytokine. Moreover, rhIL-23 significantly increased the production of IL-10 in both naive CD4+ and CD8+ T cells. IL-17 and IL-10 levels were not affected by the addition of rhIL-12. We conclude that IL-23 induces a specific cytokine profile, remarkably distinct from IL-12, in activated human naive T cells.

Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites.

To get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region. First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line. On stimulation with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) -310 to -160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2). Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the -396/-241 region contained critical cis-acting elements. Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-gamma-inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line. Moreover, we found that in DCs, LPS and IFN-gamma synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis. Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.

Blood plasmacytoid dendritic cell responses to CpG oligodeoxynucleotides are impaired in human newborns.

Plasmacytoid dendritic cells (pDCs) respond to unmethylated cytosine-phosphate-guanosine (CpG) motifs present in bacterial DNA or unmethylated synthetic oligodeoxynucleotides (CpG). In order to assess the function of pDCs in human newborns, interferon-alpha (IFN-alpha) production induced by CpG 2216 and phenotypic maturation of pDCs in response to CpG 2006 were compared in cord blood and adult blood. We first observed that neonatal pDCs displayed decreased up-regulation of CD80, CD83, CD86, and CD40, whereas HLA-DR and CD54 up-regulation did not differ significantly between adults and neonates. We then found that the production of IFN-alpha in response to CpG was dramatically impaired in cord blood. This neonatal defect was detected both at protein and mRNA levels and was still present in blood of 4-day-old babies. Further experiments on enriched pDCs confirmed that these cells are intrinsically deficient in CpG-induced IFN-alpha production at birth. These findings might be relevant to the increased susceptibility of human newborns to infections as well as to the use of CpG oligodeoxynucleotides as vaccine adjuvants in the neonatal period.

Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood.

Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity. To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic-polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively. Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood. Moreover, Poly (I:C)-induced IFN-alpha production was found to be significantly impaired in cord blood. Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood. We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates. Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses.

Bordetella pertussis toxin induces the release of inflammatory cytokines and dendritic cell activation in whole blood: impaired responses in human newborns.

Bordetella pertussis toxin (PTX), a key component of acellular pertussis vaccines, is known to be endowed with adjuvant properties. In experiments designed to get insights into the interactions between PTX and circulating immune cells, we first observed that addition of PTX to adult whole blood induced the release of IL-12 and TNF-alpha as well as maturation of myeloid dendritic cells (DC). These effects were still present with a toxin mutant devoid of ADP-ribosyltransferase activity but not with a formaldehyde-inactivated toxin. These findings indicate that cytokine production and DC maturation require the intact structure of PTX but not its enzymatic activity. Secondly, studies on DC generated in vitro by culturing monocytes with IL-4 and GM-CSF showed that PTX directly stimulates MHC class II and costimulatory molecules up-regulation, cytokine synthesis and NF-kappa B activation. Finally, comparison of data obtained in adult vs. cord blood revealed deficient responses of neonatal DC to PTX. These data suggest new applications of PTX and PTX mutants as vaccine adjuvants.