Valérie Gafa

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

IFN‐β modulates the response to TLR stimulation in human DC: Involvement of IFN regulatory factor‐1 (IRF‐1) in IL‐27 gene expression

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN‐β by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN‐β release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA‐DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN‐β treatment suggest that IFN‐β‐primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN‐β‐primed DC to TLR stimulation. While IFN‐β pretreatment increases slightly the expression of maturation markers in TLR2‐ or TLR4‐stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno‐regulating cytokines. Interestingly, IL‐27p28 subunit was induced by IFN‐β alone or during LPS‐induced maturation of DC in a type I IFN‐dependent manner through IFN regulatory factor‐1 (IRF‐1) activation. Taken together, our results shed light on the capacity of IFN‐β to finely tune DC response to invading pathogens.

Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response

ABSTRACT Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-γ) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-γ production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.

EBV stimulates TLR- and autophagy-dependent pathways and impairs maturation in plasmacytoid dendritic cells: implications for viral immune escape.

Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ‐herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA‐DR and that pDCs express EBV‐induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF‐α release. Accordingly, EBV‐infected pDCs are unable to mount a full T‐cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV‐mediated upregulation of the T‐cell inhibitory molecules B7‐H1 and ICOS‐L, could represent an immune‐evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV‐associated diseases.

Sensitization to TLR7 Agonist in IFN-β-Preactivated Dendritic Cells

TLRs interact with a growing list of pathogen-derived products and these interactions drive the activation of innate and adaptive immune responses. Dendritic cells (DC) play a key role in these events expressing a heterogeneous repertoire of TLRs. We have previously demonstrated the production of type I IFNs in DC following bacterial infections and TLR triggering. In this study, we sought to characterize the transcriptome specifically induced in human DC by IFN-β production stimulated upon LPS treatment. To this aim, by using cDNA microarrays, we compared the transcriptome of DC following LPS treatment in the absence or presence of neutralizing anti-type I IFN Abs. Interestingly, we found that the expression of TLR7 was induced during LPS-induced maturation of DC in a type I IFN-dependent manner. The induction of TLR7 in maturing DC was mainly a consequence of the transcriptional activity of IRF-1, whose binding site was located within TLR7 promoter. Moreover, we also demonstrated that “priming” of immature DC, that usually express TLR8 but not TLR7, with exogenous IFN-β induced a functionally active TLR7. In fact, treatment with the TLR7-specific ligand 3M-001 up-regulated the expression of CD83, CD86, and CD38 in IFN-β-primed DC but not in immature DC. Therefore, a robust enhancement in proinflammatory as well as regulatory cytokines was observed. These data suggest that TLR4-mediated type I IFN release activates specific transcription programs in DC amplifying the expression of pathogen sensors to correctly and combinatorially respond to a bacterial as well as viral infection.

IFN‐β improves BCG immunogenicity by acting on DC maturation

Given the variable protective efficacy provided by Mycobacterium bovis bacillus Calmette‐Guérin (BCG), there is an urgent need to develop new vaccines against tuberculosis. As dendritic cells (DC) play a critical role in initiating and regulating a protective T cell response against the pathogens, the comprehension of mycobacterium‐induced modulation of DC functions is critical to pinpoint new, immunological strategies. To this end, a comparative analysis of the effect induced by BCG and Mycobacterium tuberculosis (Mtb) infection on the DC immunophenotype indicated that BCG is less efficient in inducing DC maturation than Mtb. In addition, BCG‐infected DC poorly expressed IFN‐β and displayed a reduced production of IL‐12 as compared with Mtb‐stimulated cells. The impaired expression of IL‐12p35 and IFN‐β is likely a result of the inability of BCG to induce the activation of the IFN regulatory factor‐3. Taking into account these data, we sought to investigate whether the exogenous addition of IFN‐β, a cytokine that exerts important effects on the immune system, could enhance the Th1‐polarizing capacity of BCG‐infected DC. Interestingly, when DC infected by BCG were pretreated in vitro with IFN‐β, they displayed a fully mature phenotype and released a significant amount of bioactive IL‐12p70, which resulted in an enhanced Th1 response. This study demonstrates that IFN‐β potentiates DC immunological functions following BCG infection, thus suggesting IFN‐β as a possible candidate as vaccine adjuvant.

NF-κB is required for STAT-4 expression during dendritic cell maturation

The transcription factor STAT‐4 plays a pivotal role in the IL‐12‐mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT‐4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT‐4 in human monocyte‐derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR‐4, TLR‐2, and TLR‐3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF‐α and IL‐1β. It is interesting that we found that STAT‐4 is tyrosine‐phosphorylated in response to type I IFN but not IL‐12 in human mature DC. Cloning and functional analysis of the STAT‐4 promoter showed that a NF‐κB binding site, localized at –969/–959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF‐κB binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF‐κB/Rel proteins involved in STAT‐4 gene expression in maturing DC. The mutation of this κB site or the overexpression of the repressor IκBα exerted an inhibitory effect on a STAT‐4 promoter‐driven reporter as well as on STAT‐4 expression. Altogether, these results indicate that STAT‐4 can be finely tuned along with DC maturation through NF‐κB activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.

Bystander inhibition of dendritic cell differentiation by Mycobacterium tuberculosis-induced IL-10.

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen‐presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon‐α and granulocyte‐macrophage colony‐stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat‐inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb‐stimulated cells (SN Mtb) remained CD14+ and poorly differentiated into CD1a+ cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin‐1β, interleukin‐6, interleukin‐10 and tumor necrosis factor‐α upon Mtb stimulation. However, only interleukin‐10 neutralization through the addition of soluble interleukin‐10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin‐10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen‐activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin‐10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non‐infected monocytes into DCs through the release of interleukin‐10. These results shed light on new aspects of the host–pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.

Infection of human dendritic cells with a Mycobacterium tuberculosis sige mutant stimulates production of high levels of interleukin-10 but low levels of CXCL10 : Impact on the T-cell response

ABSTRACT The Mycobacterium tuberculosis genome encodes 13 sigma factors. We have previously shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and can attenuate the virulence phenotype. In this work, we focused on extracytoplasmic factor σE and studied the effects induced by the deletion of its structural gene (sigE) in the infection of human monocyte-derived dendritic cells (MDDC). We found that the wild-type M. tuberculosis strain (H37Rv), the sigE mutant (ST28), and the complemented strain (ST29) were able to infect dendritic cells (DC) to similar extents, although at 4 days postinfection a reduced ability to grow inside MDDC was observed for the sigE mutant ST28. After mycobacterium capture, the majority of MDDC underwent full maturation and expressed both inflammatory cytokines, such as tumor necrosis factor alpha, and the regulatory cytokines interleukin-12 (IL-12), IL-18, and beta interferon (IFN-β). Conversely, a higher level of production of IL-10 was observed in ST28-infected MDDC compared to H37Rv- or ST29-infected cell results. However, in spite of the presence of IL-10, supernatants from ST28-infected DC induced IFN-γ production by T cells similarly to those from H37Rv-infected DC culture. On the other hand, IL-10 impaired CXCL10 production in sigE mutant-infected DC and, indeed, its neutralization restored CXCL10 secretion. In line with these results, supernatants from ST28-infected cells showed a decreased capability to recruit CXCR3+ CD4+ T cells compared to those obtained from H37Rv-infected DC culture. Thus, our findings suggest that the sigE mutant-induced secretion of IL-10 inhibits CXCL10 expression and, in turn, the recruitment of activated-effector cells involved in the formation of granulomas.

Expression of Proinflammatory and Regulatory Cytokines via NF-κB and MAPK-Dependent and IFN Regulatory Factor-3-Independent Mechanisms in Human Primary Monocytes Infected by Mycobacterium tuberculosis

Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1β. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-β was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-β gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.