Initial adherence of P. marneffei conidia to human monocytes
Resident tissue macrophages in alveolar sacs serve as the first line of immune defense by detecting the immediate presence of a pathogen and sending an alarm signal, such as cytokine secretion, to other cells. Alveolar macrophages are abundant, making them the most likely to be the first to encounter air-borne pathogens including P. marneffei conidia. Nevertheless, no study has reported the exact route of infection of P. marneffei, except for showing that the fungus can infect tissue macrophages and monocytes. Alveolar macrophages are a type of tissue macrophage derived from peripheral blood monocytes, and the differences between alveolar macrophages and peripheral blood monocytes in the degree of surface marker expression, such as phagocytic markers (CD36, CD44) and co-stimulatory molecules (CD40, CD80, and CD86), have been stated elsewhere (33). The distinction between the functional characteristics of alveolar macrophages and monocytes is still unclear. Taken together, these considerations made us decide to use adherent HM as the study model.
Adherence capability of P. marneffei conidia to HM was found in the present study, which is the first to demonstrate that several PRR, including TLR1, TLR2, TLR4, and TLR6, are involved in the interaction, and contribute to subsequent innate immune cell activation. CD14 was also shown to be essential for the initial interaction of intact P. marneffei conidia with HM. Our results are consistent with earlier findings that TLR2, TLR4, and CD14 play important roles in host response against fungal conidia (23). In that study, macrophages obtained from TLR2-, CD14-knock out mice or TLR4 defective mice showed impairment of TNF-α production on activation with fungal conidia. Involvement of CD14 was not restricted to mouse innate immune response, but was also required for TNF-α production in human antigen presenting cells (17, 23).
The MR has previously been reported to serve as a recognition site for pathogenic fungi, including C. albicans, Pneumocystis carinii (now named P. jiroveci), and P. marneffei yeast (34, 35). In the present study, a very low concentration of MAb against the MR was able to inhibit adhesion and subsequent phagocytosis of P. marneffei conidia by HM, supporting the hypothesis that MR is a common phagocytic receptor for a wide variety of fungal pathogens. However, the binding mediated by MR could depend on the morphotype of fungal pathogen, as reported by Rongrungruang and Levitz, in that unopsonized yeast of P. marneffei do not bind to human HM via MR (11). Recently, dectin-1, a β-glucan receptor expressed on the surface of monocyte/macrophage lineage cells, has been demonstrated to induce biological activation upon β-glucan engagement (13, 36). Hohl and colleagues have shown that β-glucan restrictively expresses on swelling or metabolically active conidia (14). They have further demonstrated that the production of TNF-α and MIP-2 by murine macrophages is initiated by metabolically active conidia, indicating that the β-glucan receptor plays an important role in the innate immune response against restrictive morphotypes of fungal conidia. Our results support the earlier findings of Hohl and colleagues (14), as no direct inhibitory effect from laminarin on the interaction between resting P. marneffei conidia and HM was seen. Furthermore, mannan significantly inhibited conidial adhesion to the target cells, suggesting that mannose-containing molecule(s) may be responsible for macrophage stimulation. Taken together, P. marneffei conidial adherence to HM is mediated by the MR and the β-glucan receptor plays a lesser role in such interactions.
In addition to TLR and MR, adhesion of pathogenic fungi to human leukocytes via integrins has been reported. LFA-1 (CD11a/CD18), CR3 (CD11b/CD18) and p150,95 (CD11c/CD18) have been shown to mediate binding of H. capsulatum yeasts and microconidia to human macrophages (37, 38). In addition, CR3 has also been shown to mediate macrophage binding to other fungi such as Blastomyces dermatitidis and C. albicans (39). In this study, blockade of P. marneffei conidia to HM with MAbs against integrins suggests that attachment of intact conidia of P. marneffei to HM is mediated through CR3 or Mac-1. Further investigation also indicates that CR3 is required for phagocytosis of intact P. marneffei conidia by macrophages. This result strongly supports the possibility that CD18-associated integrins, especially CD11b/CD18, serve as common receptors for pathogenic fungi on human leukocytes (40, 41).
Host response on initial adherence of P. marneffei conidia to human monocytes
In the present study, the ability of P. marneffei conidia to activate HM was revealed by FACS analysis, which showed increasing CD86 and CD40 expression on the cell surface and a significant increase in TNF-α and IL-1β secretion. Increase in the concentrations of TNF-α, IL-1β, and IL-6 production by monocytes infected with conidia or hyphae of A. fumigatus has been reported elsewhere (17, 42). Furthermore, Sisto and colleagues have demonstrated that the spleen and liver of BALB/c mice initially infected with P. marneffei had local production of high concentrations of TNF-α, IL-12 and IFN-γ, which may be responsible for fungal clearance (43). These findings, as well as ours, strongly suggest that this group of proinflammatory cytokines are commonly secreted in the initial phase of fungal infection. With regards to monocyte production of IL-10, exposure to P. marneffei conidia seems to significantly suppress release of IL-10 from human HM. This result is corroborated by a reduction in IL-10 production, associated with enhanced host resistance against a number of infectious fungi, including C. albicans, A. fumigatus, and C. neoformans (44). Previous work on P. marneffei infection in mice has also demonstrated no difference in the concentrations of IL-10 secreted from the suspension of whole spleen as compared to uninfected controls (43).
In the present study MAbs specific to CD14 or TLR4 diminished TNF-α production by conidial-stimulated HM. Our results are consistent with involvement of CD14 in proinflammatory cytokine production by human monocytes in A. fumigatus infection (17, 23). Previously, Meier et al. also demonstrated the importance of TLR4 on macrophages activated by A. fumigatus conidia in TNF-α production (45). These results suggest that at least TLR4 and CD14 are involved in induction of proinflammatory cytokine production. In addition, TNF-α seems to be a major cytokine responsible for control of P. marneffei conidia infection, and its involvement against other pathologic fungal infections has been reported. Fungal pneumonia occurring after TNF-ablation therapy is evidence which supports the possible pivotal role of TNF-α in control of fungal infection (46).
In the present study, the results of MAb blockade on TNF-α production by HM revealed that anti-TLR4 and CD14 MAbs exerted partial inhibitory effects of 20–50%. We also performed a parallel experiment on blockade of IL-1β production. Here inhibitory effects of MAbs were not as conclusive as in the case of TNF-α production, due to a low and varied degree of IL-1β production from HM of certain donors, leading to lower discriminatory power for that data set (data not shown). Notably, we were surprised to see that treatment of P. marneffei-activated HM with MAbs against CD14, TLR4 or CD18 did not cause a decrease in surface expression of co-stimulatory molecules although there was a reduction in TNF-α production upon treatment with anti-TLR4 and -CD14 MAbs. This might be attributable to the fact that these antibodies do not specifically block at the activation motifs which lead to co-stimulatory molecule expression. In addition, blockade of CD18 (integrin β-subunit) alone might not be sufficient to reduce the Ca2+ transducing signal that is critically regulated by the association of CD18 with the unique α-subunit, CD11b.
With regards to TLR1, TLR2 and TLR6, although the MAbs against these TLR were able to inhibit P. marneffei conidia adherence to HM, they failed to block both TNF-α production and the surface expression of co-stimulatory molecules. It is possible that while these MAbs can inhibit the binding sites, they are not capable of activating blockade. One possible explanation is that the blockade of conidial attachment to TLR1 and TLR2 by the MAbs used in this study might occur at motifs other than the ones directly involved in signal transduction leading to TNF-α production. Secondly, Mab blockade of adhesion to TLR 1 and 2 might be through steric hindrance and not directly related to the activation motif. Additionally, we may have to take into account the distribution and degree of expression of each individual TLR on the surface of HM. It should also be noted, as previously reported by Netea and colleagues, that not every TLR that participates in binding of fungal cells involves induction of cytokine production (47). TLR6, but not TLR1, plays a role in recognition and acts as a modulator for the adaptive immune response in C. albicans infection (47). The inductive role of TLR1, TLR2 and TLR6 in proinflammatory cytokine production should be further elucidated by employing a TLR-transfected cell line as a representative model.
The present study also addressed the importance of serum factors in HM stimulation during P. marneffei infection. Previously, Rongrungruang and Levitz demonstrated that, despite an opsonic requirement for optimal adhesion of P. marneffei yeasts to human macrophages, yeast binding to human macrophages is still present when serum is omitted from the adhesion assay (11). They further reported that stimulation of human macrophages, resulting in phagocytosis and respiratory burst, can occur in the absence of opsonins. Similar results were also observed in the case of P. marneffei conidia in our laboratory (data not shown). Taken together, these data confirm that certain cell wall components of both yeast and conidia, independent of serum factors, interact directly with receptors on the surface of macrophages, leading to cell activation and killing of pathogenic fungi.
With respect to proinflammatory cytokine secretion by HM, it was demonstrated in the present study that serum factors are required for the expression of such cytokines. Heat treatment strongly attenuated cellular activation of serum to induce TNF-α and IL-1β production. Thus serum factors, including heat-labile factors such as complements, are required for proinflammatory cytokine production by HM on infection with P. marneffei conidia. However, inactivation of complements by heat treatment does not result in complete loss of leukocyte activation, implying that other, heat-stable, factors available in serum may be involved in cellular activation by P. marneffei conidia. It is also important to note that HM exhibited similar requirements in response to P. marneffei yeasts (11).
During colonization P. marneffei conidia generally interact with macrophages residing in the alveoli through adhesion to the MR, which may induce internalization of P. marneffei conidia by the macrophages. This interaction is mediated by mannan, one of the major components of the fungal cell wall, and its receptor. It is still not known whether β-glucan exists on the surface of conidia, but the β-glucan receptor on the surface of macrophages seems to play a very minor role in the early phase of the interaction. Similar to MR, CD11b and CD18 participate in conidial attachment, although to a lesser extent. Interestingly, the present study reveals for the first time that TNF-α can be induced by a signal derived from the interaction of P. marneffei conidia with CD14 or TLR4. Assessment of the relative contribution of TLR in proinflammatory cytokine production by host cells in response to P. marneffei is currently ongoing, with the hope of providing insight into the molecular basis of signal transduction.