Fabienne Willems

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.

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.

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.

Human cytomegalovirus elicits fetal γδ T cell responses in utero

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. γδ T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex–unrestricted activity. We show that upon CMV infection in utero, fetal γδ T cells expand and become differentiated. The expansion was restricted to Vγ9-negative γδ T cells, irrespective of their Vδ chain expression. Differentiated γδ T cells expressed high levels of IFN-γ, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vγ8Vδ1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) δ1–CALGELGDDKLIF/CDR3γ8–CATWDTTGWFKIF. Public Vγ8Vδ1-TCR–expressing cell clones produced IFN-γ upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated γδ T cells and public Vγ8Vδ1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal γδ T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

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.

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3‐kinases (PI3K) are known to regulate Toll‐like receptor (TLR)‐mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll‐IL‐1 receptor domain‐containing adapter‐inducing IFN‐β (TRIF)‐dependent signaling, which induces IFN‐β gene expression downstream of TLR3 and TLR4. First, treatment of monocyte‐derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN‐β expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA‐binding activity of NF‐κB, but not interferon response factor (IRF)‐3, the key transcription factors required for TLR‐mediated IFN‐β synthesis. In parallel, wortmannin‐treated DC exhibited enhanced levels of IκB kinase (IKK)‐α/β phosphorylation and IκB‐α degradation with a concomitant increase in NF‐κB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF‐κB‐dependent promoters as well as IFN‐β promoter activities without interfering with transcription at the positive regulatory domain III‐I. Furthermore, wortmannin enhanced NF‐κB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF‐mediated NF‐κB transcriptional activity. Finally, in co‐immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF‐dependent NF‐κB activity, and thereby increases IFN‐β synthesis elicited by TLR3 or TLR4 ligands.

Phenotype and function of neonatal DC.

Newborns face complex physical and immunological changes before and after birth. Although the uterus is a sterile environment for the fetus, it also contains non‐self material from the mother. Birth involves the transition from the sterile intra‐uterine environment to an environment rich in microbes and requires rapid induction of appropriate responses to control these microbes. In this review we focus on the similarities and differences of human and murine neonatal DC and their reaction to various stimuli. A better understanding of the newborn immune system – in particular, the DC–T‐cell interaction – will be beneficial for the development of improved strategies to prevent or treat infections in this vulnerable population and prepare the immune system to cope with allergens and tumors later in life.

Oxidative stress up-regulates IL-8 and TNF-α synthesis by human dendritic cells

In addition to their damaging effects, reactive oxygen intermediates exert a regulatory role on gene expression and cell apoptosis. In this study, we evaluated the effects of oxidative stress on human dendritic cells (DC), a cell type which is critical for the initiation of the immune response. For this purpose, we tested the effects of H2O2 on DC derived from adherent peripheral blood mononuclear cells cultured in the presence of granulocyte‐macrophage colony‐stimulating factor and IL‐4. Despite a moderate increase of DC apoptosis in the presence of H2O2, we observed that H2O2 stimulated the production of IL‐8 and TFN‐α by DC in a dose‐dependent manner. The induction of cytokine synthesis was found to depend on the oxidative properties of H2O2 as it was inhibited by the addition of catalase, and to require de novo protein synthesis as it was not observed in the presence of cycloheximide. These data suggest that DC could contribute to innate immunity through an enhanced production of inflammatory cytokines in response to oxidative stress.

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.