2. Capítulo La chagra en el espectro de la antropología del conocimiento: Mapeando
2.2. Los mecanismos culturales de producción y transformación del CET en la
In the present study we demonstrate that CD14++CD16- monocytes produce more IL- 1β and PGE2 upon stimulation with HK C. albicans yeast, compared with CD14+CD16+ monocytes. IL-1β is a central cytokine involved in the induction of Th17 responses, and CD14++CD16- monocytes induce a greater IL-17A production in CD4 lymphocytes. These differences in cytokine production were associated with an increased expression of MR on the cell membrane of CD14++CD16- compared to CD14+CD16+ monocytes. Regarding the innate host defense, there was no large difference in Candida-induced IL-6 production, and both monocyte subpopulations are able to phagocytose and kill C. albicans, and inhibit at similar rates the germination of yeasts into hyphae. Thus, while both CD14++CD16- and CD14+CD16+ monocytes play a role in host defense against C. albicans, it is only CD14++CD16- monocytes that are able to induce protective Th17 responses.
Host defense against Candida species is a dynamic interplay between innate immunity and adaptive immune mechanisms, with cellular T-helper responses playing a central role. The first step in the activation of host defense is represented by fungal recognition by various pattern recognition receptors such as TLRs and C-type lectin
45
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Monocyte subsetsFigure 4. Stimulation of monocytes with PRR-specific stimuli
(A,B,D,E) Monocytes were stimulated in the presence of CD4+ lymphocytes and 10% serum for
seven days. IL-17A production was determined in cell culture supernatants using ELISA. (A) IL-17A production upon stimulation with RPMI, serotype A mannan (MR ligand) (100 μg/mL), MP65 (antigen) (1 μg/mL) or a combination of MP65 and serotype A mannan. Data represent mean + SEM from six healthy volunteers. (B) IL-17A production upon stimulation with RPMI, heat-killed C. albicans yeast (1×105/mL) in the absence or presence of S. cerevisiae mannan. Experiments
were performed with cells isolated from four healthy volunteers. Data for each volunteer are presented. (C) Monocytes were stimulated for 24 hours with RPMI or heat-killed C. albicans yeast (1×106/mL). The concentration of PGE
2 was determined in cell culture supernatants using
ELISA. Data represent mean + SEM from six healthy volunteers. (D) IL-17A production upon stimulation with RPMI, heat-killed C. albicans yeast (1×105/mL) in the absence or presence of
diclofenac. Experiments were performed with cells isolated from four healthy volunteers. Data for each volunteer are presented. (E) IL-17A production upon stimulation with RPMI, or heat-killed C. albicans yeast (1×106/mL) in the absence or presence of IL-1RA (10 µg/mL) was
determined in cell culture supernatants using ELISA. Data represent mean + SEM from four healthy volunteers. (A,C,E) Data were analyzed using the Wilcoxon signed rank test (* = p ≤ 0.05).
46 Chapter 3
receptors (e.g. dectin-1 and MR), followed by the release of proinflammatory cytokines. This is followed by phagocytosis and killing of the fungus by tissue macrophages, as well as by inflammatory neutrophils and monocytes migrating into the tissues 30. These activated monocytes are also responsible for the induction of a Th17 response, both through the production of IL-β and IL-6 31, as well as through cell contact with recruited CD4 T cells 32,33.
If initial innate immune mechanisms are not able to eliminate the infection, adaptive cellular immune mechanisms, represented by Th1 (IFN-γ production) and Th17 (IL-17A production) responses, will contribute to elimination of infection at later stages 34. In the present study we assessed the differential antifungal properties of CD14++CD16- and CD14+CD16+ monocytes in initiating both the innate and adaptive cellular immune response to the fungal pathogen C. albicans.
The proinflammatory cytokine response showed an important difference between the two monocyte subsets: CD14++CD16- monocytes released more IL-1β than CD14+CD16+ monocytes. In addition to its direct proinflammatory effects, IL-1β also has an important role in the induction of Th17 responses 35. Part of this effect is due to the potent induction by IL-1β of PGE2, a pivotal factor in the induction of Th17 differentiation 36,37. The PGE
2 response was indeed strongly enhanced in CD14++CD16+ monocytes and not in CD14+CD16+ monocytes, which is in line with our recent data showing a crucial role of PGE2 for Candida-induced IL-17 29. Indeed, blocking PGE
2 production reduced IL-17 synthesis induced by CD14++CD16- monocytes. IL-17A is the most important effector cytokine of the Th17 response 38, and plays an important role in Candida host-defense 39. Mice deficient in IL-17R have an increased susceptibility to both systemic 40 and mucosal 41 candidiasis, and patients with defective Th17 responses suffer from chronic mucocutaneous candidiasis 15,42. In line with their induction of IL-1β and PGE2, it was mainly CD14++CD16-, but not CD14+CD16+ monocytes, that induced IL-17A production from CD4+ lymphocytes after stimulation with C. albicans. We have previously demonstrated that C. albicans only induces IL-17 production in CD4+ memory T cells 17, and here we demonstrate that CD14++CD16- monocytes are the main blood monocyte subpopulation to initiate this response 43.
How can the difference in the capacity to induce IL-17A responses between the two monocyte populations be explained? In a recent study, we have identified the PRRs responsible for the induction of a Th17 response after stimulation with C. albicans 17. We identified that, in addition to antigen presentation, the engagement of MR by Candida mannans is a central event in the induction of an antifungal Th17 response. This pathway was amplified by recognition of β–glucans by dectin-1, and of an unidentified fungal component by TLR2 17. We therefore analyzed the distribution of several PRRs on the monocyte subpopulations using flow cytometric analysis. While expression of TLR2, TLR4 and dectin-1 was similar between the two monocyte subpopulations, a striking difference was found in the distribution of the MR, which was present on only 66% of CD14+CD16+ monocytes, versus on 91% of CD14++C16- monocytes. To assess whether this may explain the differences in the induction of IL-17A, cells were stimulated with various specific receptor ligands. While specific stimulation of dectin-1, TLR2 or TLR4 with purified ligands did not induce IL-17A production, stimulation of CD14++CD16- (but not CD14+CD16+) monocytes with mannans and MPs isolated from C. albicans, which are known to be MR ligands 30, induced a high Th17 response in
47
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Monocyte subsetsCD4-lymphocytes co-cultured with CD14++CD16- monocytes. Interestingly, MP65 alone only induced little IL-17 production, but together with mannan, IL-17 production was significantly increased in CD14++CD16- monocytes, indicating that both mannoproteins and mannans are necessary for an effective anti-fungal immune response. Since it is known that Candida stimulation of the MR results in a Th17 response, either directly 17 or via the production of PGE
2 27,29, the difference in MR expression might explain the difference in the IL-17 inducing capacity between both monocyte subsets. Indeed, blocking the MR decreased IL-17 production induced by CD14++CD16- monocytes. While these data support a role of MR in the differences between the two monocyte subsets, a role for other PRRs such as dendritic cell-specific intercellular adhesion molecule-3- grabbing non-integrin 44, dectin-2 45,46, galectin-3 47-49, or macrophage-inducible C-type lectin 50,51 cannot be completely ruled out, although their expression on monocytes is known to be very low or absent.
In addition to Th17 cells, other cellular responses such as Th1-IFN-γ also play an important role for host defense to Candida 30. No significant differences in the induction of Th1 responses were seen between CD14++CD16- and CD14+CD16+ monocytes, although the latter tended to induce higher IFN-γ production from the lymphocytes after stimulation with HK C. albicans yeast. Monocyte derived IL-18 is an important inducer of IFN-γ production 52, unfortunately in our experiments IL-18 production was undetectable (data not shown). Both monocyte subpopulations released very low amounts of IL-10 after challenge with C. albicans. However, CD14+CD16+ monocytes produced less IL-10 in response to S. aureus than CD14++CD16- monocytes. This is in agreement with previous studies which found that the CD14+CD16+ monocytes produced less IL-10 mRNA upon LPS stimulation 3,4.
Another observation in this study is that both CD14++CD16- and CD14+CD16+ monocytes displayed vigorous innate antifungal mechanisms, and no large differences in terms of IL-6 production, phagocytosis and killing, or inhibition of yeast-hyphal germination were observed. Some earlier studies have proposed that CD14+CD16+ monocytes have a more pronounced proinflammatory profile based on higher production of TNF-α upon stimulation with TLR4 stimuli such as LPS 5 or dormant Aspergillus conidia 13. In this study, CD14++CD16- monocytes produced more TNF-α upon Candida compared with CD14+CD16+ monocytes. However, CD14+CD16+ monocytes are not the major source of TNF-α in all infections: in patients with erysipelas infected with group A streptococci, the increase in the CD14+CD16+ cells is associated with diminished TNF-α production 53, and germinated Aspergillus induce similar TNF-α production in CD14++CD16+ and CD14+CD16- monocytes 13.
In conclusion, in the present study we report an assessment of the anti- Candida host defense properties of the two monocyte subpopulations of human blood, the CD14++CD16- and CD14+CD16+ monocytes. While both monocyte subpopulations displayed equally effective phagocytosis and killing of Candida, only CD14++CD16- monocytes were able to initiate effective antifungal Th17 responses in human CD4 cells. This effect was mediated by a pathway involving enhanced expression of the MR, followed by higher production of IL-1β and PGE2 by CD14++CD16- monocytes. These findings may have important consequences in the design of future immunotherapeutic strategies aimed to enhance host defense against Candida infections.
48 Chapter 3
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