Date Published: November 18, 2015
Publisher: Public Library of Science
Author(s): Wafa Markikou-Ouni, Sima Drini, Narges Bahi-Jaber, Mehdi Chenik, Amel Meddeb-Garnaoui, Alain Haziot.
Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS) that can be observed during human leishmaniasis, suggests that under certain circumstances LipESP may play a role in disease progression.
Leishmaniasis is a heterogeneous group of diseases caused by an intracellular protozoan parasite of the Leishmania genus, transmitted by a sandfly vector and associated with considerable morbidity and mortality throughout the world . Depending on the parasite species and the host immunological response, infection with Leishmania results in a spectrum of disease manifestations ranging from self-healing cutaneous lesions to fatal visceral disease. After inoculation of infective metacyclic promastigotes into the dermis of a mammalian host, Leishmania parasites preferentially infect macrophages and DCs, both being major antigen presenting cells (APCs). While macrophages are the main host cell for Leishmania parasites and the main effector cells able to destroy them, DCs play a critical role in the initiation and differentiation of the adaptative immune responses to parasites leading to the control of infection or progression of disease [2–4]. To escape from the innate immune response, parasites have evolved subversion mechanisms that allow them to survive and grow inside phagocytic cells. Among these mechanisms, the inhibition of protective cytokines production, interference with effective antigen presentation, or with host cell signaling events that lead to the generation of effectors molecules and activation/deactivation of DCs and macrophages functions by parasite factors [2, 3, 5–7]. Some Leishmania excreted/secreted molecules are key mediators of the host-parasite interaction and are involved in these processes. Such molecules are therefore very important for parasite virulence and pathogenicity protecting the parasite from the early action of the host immune system . Some of these molecules include, the secreted form of the metalloprotease GP63, the promastigote surface antigen-2 (PSA-2), the secreted acid phosphatase (sAcP), the kinetoplast membrane protein-11 (KMP-11), heat shock protein HSP-70 and cysteines proteases [9–13]. These proteins are involved in parasite survival, attachment of promastigotes to the macrophages, inhibition of antigen presentation resulting in reduced T cell activation and modulation of a number of host cell signaling molecules including blocking protein kinase C signaling, activation of protein tyrosine phosphatases and inactivation of transcription factors resulting in inhibition of cytokine production and microbicidal functions [14–19]. Most of these studies describing the interactions between Leishmania molecules and cells of the innate immune system were reported for macrophages but very little is known about the involvement of such molecules in modulating DCs functions. It has been shown that products secreted by Leishmania (L.) major promastigotes inhibit murine splenic DCs motility . More recently, modulation of DCs phenotype and cytokine secretion by excreted/secreted antigens from L. major and L. donovani has been reported . Molecules excreted and secreted by Leishmania parasites have been targets of interest for decades. Several studies have shown that these molecules play important roles in the infection process and modulation of local and systemic host immune factors, and that some of them such as PSA-2, KMP-11 and cysteine proteinases could also be a source of vaccine antigens against leishmaniasis [9, 22–29]. In order to identify Leishmania excreted/secreted proteins, we have previously used antibodies generated against promastigote culture supernatants to screen a L. major cDNA library allowing the isolation of different clones . Among all the proteins revealed by sequence analysis, we have selected four and identified their orthologues in L. infantum using BLAST searches. LinJ.17.0090 encoded for EF-1α which plays an essential role in protein biosynthesis . It was described as an Src homology domain containing tyrosine phosphatase (SHP-1) binding protein and SHP-1 activator and was proposed as a virulence factor since it was associated with macrophage deactivation [30–33]. In addition, a phosphoproteomic analysis of differentiating L. donovani parasites has shown that EF-1α has been identified in both promastigotes and amastigotes stages . Furthermore, based on peptide quantification, a Leishmania exosome analysis has revealed the presence of EF-1α . LinJ.13.0990 encoded for a putative protein: proteasome regulatory ATPase subunit. It showed a protein family signature: the AAA domain (ATPases Associated with a wide variety of cellular Activities). Members of the ATPase superfamily are known to be involved in essential processes of protein degradation and DNA replication by using the energy from ATP hydrolysis to remodel their respective substrates . LinJ.15.0460 and LinJ.23.0070 encoded both for unknown proteins with no conserved domains. We have termed the corresponding proteins LiP15 and LiP23, respectively, in regards to their clone number identified in our previous study . Here, we report a first characterization of LiEF-1α, LiAAA-ATPase, LiP15 and LiP23 based on the analysis of their immunomodulatory effects on in vitro differentiation and maturation of human DCs and on cytokine production by human DCs and monocytes.
Leishmania excreted/secreted proteins likely play crucial roles in parasite virulence as well as host-parasite interactions, more particularly through the modulation of the host immune response [8, 12, 35, 37, 38]. In this paper, we examined the immunomodulatory effects of four potential L. infantum excreted/secreted proteins: LiEF-1α, LiAAA-ATPase, LiP15 and LiP23 on differentiation and maturation of two major phagocyte populations, DCs and monocytes playing critical roles during infection. The first aim of our study was to evaluate the ability of these proteins to interfere with DCs differentiation. We showed that LipESP were able to significantly reduce CD1a expression, with the most important effect observed for LiEF-1α, LiAAA-ATPase and LiP15. CD1a is member of the CD1 group I molecules, a family of cell surface glycoproteins that directly bind a variety of lipids and present them for T cell recognition at the surface of APCs [39, 40]. In humans three groups of CD1 isoforms have been identified, group 1 (CD1a, CD1b, CD1c), group 2 (CD1d), and group 3 (CD1e) while mice have only CD1d, making the in vivo analysis of group 1 CD1-restricted T cells difficult. However, the development of humanized mouse in which the human CD1 system is present and group 1 CD1 transgenic mouse models as well as the expansion of CD1-tetramer technology have facilitated the study of T cell reactivity to CD1/lipid complexes and provided evidence that group 1 CD1-restricted T cells participate in adaptive immune responses during human infection [41–43]. Most studies examining microbial antigen presentation by group 1 CD1 molecules have focused on Mycobacterium tuberculosis (Mtb). It was shown that group 1 CD1-restricted T cells produce IFN-γ and TNF-α upon encountering mycobacterial antigens supporting the role of these cells in protective immunity against Mtb infection [41, 44, 45]. Both mycobacterial infection and immunization with Mtb lipids elicit group 1 CD1-restricted Mtb lipid-specific T cell responses in human group 1 CD1 transgenic mice . Interestingly, these cells exhibit rapid secondary responses, similar to conventional T cells suggesting that they could serve as targets for the development of novel vaccines . However little is known about the involvement of group 1 CD1-restricted T cells in other microbial infection including leishmaniasis. The engagement of CD1 molecules by human T cells and functional consequences on T cell activation during Leishmania infection is still largely unknown. To our knowledge, no Leishmania-derived glycolipid antigen presented by this pathway has been identified to date in humans. However some data support the involvement of the CD1 pathway during Leishmania infection. Studies including ours have shown that live L. major, L. donovani [46–48] and L. amazonensis  promastigotes were able to significantly downregulate CD1a expression on human DCs. It was also shown that Leishmania-induced down-regulation of CD1 expression was not mediated by LPG or other phosphoglycans . There are evidences that Leishmania-induced CD1 down-regulation is associated with a reduced ability of DCs to present antigen and to stimulate a CD1-restricted T cell response. Indeed, CD1 down regulation induced by L. donovani in DCs was associated with a reduced ability of DCs to stimulate an Mtb restricted T lymphocyte response . A lower production of IFN-γ was observed in the supernatants of autologous cultures in which DCs differentiated in the presence of L. amazonensis parasites were used as APC . These results suggest that Leishmania-CD1 down-regulation may be associated with a down-regulation of the Th1-adaptative immune response and therefore with the establishment of a disease-promoting immune response. However, L. infantum did not alter CD1a expression in infected DCs, but, in contrast, up-regulated CD1d cell surface expression . These cells were efficiently recognized and killed by NKT cells that produce IFN-γ and a cytotoxic response which may facilitate the development of Th1 responses against Leishmania . These contradictory results could be explained by the differences between the biology of the Leishmania species. Based on all these data and our results, we can suggest that through their capacity to down-regulate CD1a expression, LipESP could be associated with an impairment of CD1-restricted T cells activation by DCs. We also showed that HLA-DR expression was down regulated in LiP23-stimulated cells while CD86 expression was up regulated in LiEF-1α-stimulated cells, during DCs differentiation. Reduced levels of MHC class II but high CD86 levels were observed during DCs differentiation in the presence of live L. amazonensis, L. donovani, L. major and M. tuberculosis [47, 49, 51]. These changes were associated with a reduced capability to induce proliferation and IFN-γ secretion by T lymphocytes [49, 51]. Our results suggest that LiP23 and LiEF-1α, when present during DCs differentiation, could interfere with antigen presentation and optimal costimulatory activity and consequently have an impact on T cell activation, which could promote parasite survival. The second part of this study was to assess the ability of LipESP to induce DCs maturation. Stimulation of immature DCs with LiP23, LiP15 and LiAAA-ATPase resulted in a significant increase in HLA-DR, CD80, and CD86 surface expression, an indication of DCs maturation. LiP15 was the only protein also able to induce a significant up-regulation of CD40 expression. LiEF-1α only induced a CD86 up-regulation. Previous studies mainly using live parasites showed that both L. major and L. donovani were either able to induce up-regulation of CD80, CD86, CD40 and HLA-DR molecules [52–54] or had no effect on the expression of these molecules in immature DCs [21, 47, 52, 55, 56]. Effects of some parasite components on costimulatory molecules expression by human DCs were also reported. Leishmania eukaryotic initiation factor (LeIF), an exosomal protein, was able to up-regulate CD80 expression in human DCs . However, phosphoglycans family of virulence-associated antigens was involved in inhibition of DCs maturation  whereas Leishmania exosomes did not alter the expression of HLA-DR, CD80, or CD86 in immature DCs . Interestingly, it has been suggested that changes in the protein cargo of Leishmania exosomes may influence the impact of these vesicles on myeloid cell function . In addition to induction of costimulatory molecule expression, we also demonstrated that LipESP activated DCs but not monocytes for a significant IL-12p70 production. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. LipESP also induced significant levels of TNF-α and IL-10 in both cell populations with significantly higher cytokine-inducing capacities for LiP15 and LiAAA-ATPase. In vitro infection studies have mainly showed that in the absence of other stimuli, Leishmania parasites can trigger relatively weak or no cytokine production by DCs [21, 48, 52–54]. Similarly to our results, IL-12 production by infected DCs can be markedly enhanced by the addition of exogenous stimuli such as IFN-γ, IFN-γ/LPS, and CD40L [52–54]. LeIF, LPG and KMP-11 were described as inducing IL-12 production in DCs but not or to a lesser extent in monocytes [37, 38, 57, 58]. NF-kB was involved in the differential production of IL-12 between DCs and macrophages [38, 58, 59]. Recently, it was suggested that the regulation of type I IFN-associated signaling pathways was involved in L. major-induced expression of IL-12 in DCs . Leishmania-induced IL-12 and TNF-α in matured DCs, was associated with the capacity of these cells to induce Th1 responses and IFN-γ production which are critical for resistance and cure of leishmaniasis [35, 38, 52, 53]. Leishmania promastigotes or amastigotes infected DCs, were able to induce a Th1 response with IFN-γ production by autologous T lymphocytes from leishmaniasis patients [52, 53]. Leishmania HSP100-/- exosomes promoted the differentiation of naïve CD4 lymphocytes into Th1 cells . More recently, presentation of KMP-11 antigen by DCs to autologous T cells from visceral leishmaniasis patients resulted in a significant IFN-γ production by CD4+ T cells . Our results showed that among LipESP, LiP15 and LiAAA-ATPase were the most efficient to induce DCs maturation and suggest that these proteins could be involved in T lymphocyte activation and IFN-γ production upon antigen presentation by DCs. However, LiEF-1α only induced a weak up-regulation of CD86 and cytokine production suggesting partial DCs maturation. Interestingly, partially matured DCs conditioned by inflammatory mediators or low concentrations of TLR ligands have been shown to instruct Th2-cell responses. It has been shown that partially matured DCs injected into mice before L. major infection were associated to the development of a Th2 response whereas fully matured DCs induced a Th1 response, suggesting that the differentiation stage of DCs determines Th1/Th2 differentiation . More recently, it was demonstrated that T. brucei antigens induced partial DCs maturation that was associated with the differentiation of Th2-cell responses in vitro and in vivo [62–64]. Whether LiEF-1α is associated with the generation of a Th2 response, needs to be further investigated. LipESP also induced a significant IL-10 production in DCs and monocytes. IL-10 promotes the differentiation of tolerogenic DCs which play an important role in activating regulatory T (Treg) cells . Treg cells play key roles in regulating the balance of Th1/Th2 immunity and in preventing excessive damages during the inflammatory responses and have been described during Leishmania infection [66, 67]. Furthermore, a recent study suggested that Treg cells are induced by L. major excreted/secreted antigens . However, it seems unlikely that the LipESP-induced DCs profiles may correspond to tolerogenic DCs, except for the one induced by LiEF-1α. Indeed, tolerogenic DCs were characterized by low levels of MHC Class II and costimulatory molecule expression along with production of IL-10 and impairment of IL-12 production. Finally, we observed that LPS-induced cytokine production during DCs maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment, suggesting that the presence of LipESP leads to a reduced ability to respond to inflammatory stimuli. Altered DCs responsiveness to exogenous stimuli has been reported by our group and by others in the presence of Leishmania antigens and live parasites [21, 35, 48, 49]. Stimulation of LPS/TNF-α matured DCs with Leishmania excreted/secreted antigens induced a decrease in IL-10 and IL-12p70 productions . Leishmania exosomes containing virulence factors such as LiEF-1α inhibited cytokine production by CD40L-matured DCs and by Leishmania-infected or IFN-γ-treated monocytes, suggesting that exosomes are able to modulate the immune response to make it permissive for infection . Considering the presence of LPS during bacterial superinfections that can be observed in leishmaniasis [69, 70], it is tempting to speculate that LipESP could benefit from the presence of LPS to immunomodulate DCs functions to the parasite advantage.