Stanislas Goriely

Deficient IL-12(p35) Gene Expression by Dendritic Cells Derived from Neonatal Monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-γ to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-γ production by allogenic adult CD4+ T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-γ production by T cells.

Cytokine mRNA quantification by real-time PCR

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

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-γ. We conclude that IL-12(p35) transcriptional repression in neonatal DCs takes place at the chromatin level.

How microorganisms tip the balance between interleukin-12 family members

Interleukin-12p70 (IL-12p70) induces T-helper-1-cell responses and IL-23, a related cytokine, is the master switch in several T-cell-mediated inflammatory disorders. IL-27, another member of the IL-12 family, regulates innate and adaptive immune responses. Recently, distinct combinations of transcription factors have been shown to regulate the expression of the genes that encode these three cytokines. Toll-like receptor ligands, in association with other microbial products and endogenous mediators, tip the balance between the expression of IL-12 family members and thereby may control the outcome of T-cell-mediated inflammation. On this basis, we present a novel perspective on the pathogenesis and regulation of inflammatory disorders.

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)‐γ 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‐12Rβ1 and IL‐23R, were rapidly induced after activation in both naive CD4+ and CD8+ T cells. In both cell types, rhIL‐23 enhanced IFN‐γ 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.

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.

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-β 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-β-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.

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‐γ pathway in early life.

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‐α 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‐α/β 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.

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 petussis 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‐α 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 thatPTX directly stimulates MHC class II and costimulatory molecules up‐regulation, cytokine synthesis and NF‐κB 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.