Macrophages RAW264.7 were classically activated by A. fumigatus conidia and produced more ROS than hyphae stimulation
To elucidate the different responses of macrophages against conidia or hyphae, cells were incubated with the moulds. Above all, we found that in the conidia-incubated cells, the expression of iNOS gradually increased within 24 hours (Fig.1a), which meant that macrophages were classically activated (M1 polarization). Based on this, we determined the best co-incubation duration for subsequent experiments, 6 hours for live fungi and 24 hours for inactivated ones. Moreover, the conidia-incubated macrophages showed a concentration-dependent enhancement of M1 polarization (Fig.1b).
In host defense against fungi like alveolar macrophages, reactive oxygen species (ROS) plays an essential role. In this study, intracellular superoxide (O2–) were detected by DHE staining. Macrophages stimulated by conidia revealed a higher production of ROS compared with the control and hyphae group (Fig.1d,1e), which was consistent with the cellular iNOS levels.
Fungal stimulation promotes the proliferation of macrophages RAW264.7
CCK-8 assay was used to measure the cell viability after fungal stimulation. Both conidia and hyphal fragments stimulation exhibited a concentration-dependent rise in the cell proliferation (P<0.05), and the live fungi groups showed a more obvious alteration than the inactivated ones (P<0.05), though no significant difference was found between conidia and hyphae groups (P>0.05) (Fig.1c).
Cytokine profile of A. fumigatus stimulated macrophages RAW264.7
Cytokine profile can intuitively reflect inflammatory reactions of macrophages after different mould particles exposure. As is shown in the Fig.2, the overall secretion trends of TNF-α and IL-6 were concentration-dependent rises, though excessive hyphae exposure could lead to secretion suppression sometimes. The cytokine levels of macrophages stimulated by heat-inactivated fungi were higher than live ones because of different incubation times. Hyphal fragments triggered remarkable releases of TNF-α and IL-6 in both live and inactivated fungal groups. In contrast, minor elevation of cytokines was found after exposing to live conidia for 6 hours. Heat-inactivated conidia induced a remarkably high level of TNF-α release only in the highest concentration (MOI 100) after 24-hour exposure, while IL-6 secretion had been already markedly elevated in a relative lower concentration of stimulation.
Cytokine profile of A. fumigatus stimulated PBMCs
PBMCs include monocytes and lymphocytes. Cytokine profile of PBMCs induced by A. fumigatus conidia and hyphal fragments exhibited a further insight into the host immune response. The overall secretion trends of all the cytokines including human TNF-α, IL-6, IL-4 and IL-1b were increases along with stimuli concentration (Fig.2), which were similar to cytokine profile of macrophages. In most cases, the cytokine concentration of hyphae-incubated PBMCs was higher than that of conidia-incubated cells at the same fungal concentration.
Dynamic T cell subset changes induced by conidia and hyphae
PBMCs exposed to conidia or hyphae were significantly different in T cell subsets (Fig.3). In general, hyphae-incubated cells showed an obvious elevation of CD4/CD8 ratio compared with that of conidia-incubated ones (P<0.05), with the percentage of CD4+ significantly increasing (P<0.05) and CD8+ slightly decreasing (P>0.05). The percentage of helper T cell 1 (Th1%) increased greatly in both LC and HIC group (P<0.05). On the contrary, Th1% showed no significant change in either of hyphae group, despite the overall average of Th1% for four donors decreased sharply in HIH group (P>0.05). In terms of helper T cell 2 (Th2 cell), hyphae group exhibited a higher proportion compared with corresponding conidia group, though the result did not reach statistical significance (P>0.05). Neither group showed any change in helper T cell 17 (Th17) cell differentiation (P>0.05).
Survival of immunocompetent C57BL/6 mice following inhalation of A. fumigatus conidia or hyphal fragments
In murine pulmonary aspergillosis model, the mortality of hyphae-inoculated immunocompetent mice reached 40%, while the mice inoculated with conidia and normal saline all survived to the end point of the assay (Fig.4a). The result was consistent with the previous report of Zhang et al. In addition, amongst all sections made, focal infection of aspergillus was only observed on the pulmonary histopathology of hyphae-inoculated mice 4 days post inoculation (Fig.4b). And positive result about fungal culture of murine lungs also confirmed the aspergillosis.
The immune responses against hyphae and conidia has similarities and differences in murine pulmonary aspergillosis model
A total of 18,992 transcripts were detected by RNA-seq. Among three groups, the difference of expression between control and conidia group was relatively smaller, with only 705 genes significantly differentially regulated (|log2FC|≥ 1, Qvalue≤0.05). On the contrary, the number of differentially expressed genes (DEGs) between hyphae and control group was up to 3977, with 1839 up-regulated and 2138 down-regulated (Fig.5a). However, 2893 significant DEGs (Fig.5a, b) between conidia and hyphae group were focused to analyze. KEGG pathway enrichment analysis showed a series of immunologically relevant pathways were involved, including NOD-like receptor (NLR) signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway and others (Fig.5c). In addition, KEGG pathway classification revealed that signal transduction and immune system were top2 DEGs-intensive categories (Fig.5d).
Further analysis demonstrated that both hyphae and conidia could induce a series of inflammatory responses in murine lungs, but host defense against hyphae was much more intensive. Immunologically relevant pathways mentioned above were activated in both groups, but associated genes expressed more strongly in hyphae group. We subsequently identified a series of differentially expressed essential receptor genes including TLRs, Clecs and NLRs to gain insight into immune responses against aspergillus (Fig.6).
Thereafter, to better understand about immune signaling patterns amongst the murine model, T cell differentiation pathways associated genes were analyzed by qRT-PCR. As is shown in Fig.7, Th17 cell differentiation pathway tended to be activated after intratracheal mould injection in both groups. We found that relative expressions of IL-17a, IL-17f and stat3 were up-regulated. Besides, expressions of IL-17 signaling associated chemokines CXCL1, CXCL2, CXCL5, CXCL10, and CCL2 were likewise significantly increased. When it comes to Th1 and Th2 cell differentiation, RNA-seq revealed that Th2 cell differentiation tended to get activated and Th1 differentiation relatively silenced in hyphae group. The genes related to Th2 cell differentiation including IL-13 and IL-4Ra, were significantly up-regulated while the key genes in Th1 cell differentiation pathway such as stat1and T-bet were down-regulated. However, in conidia group, gene expressions of Th1 and Th2 cell differentiation pathways were not significantly altered except the mild elevation of IL-4Ra, which was not consistent with the result of experiment in vitro. We speculated that the phenomenon might be ascribed to the effective anatomical elimination of conidia in immunocompetent host and only a small portion of them escaped to challenge subsequent immune system[16, 24]. Moreover, Th1 and Th17 cells showed antagonistic action against each other and activation of IL-17 signaling would suppress Th1 cell differentiation. The slight increase of IL-4Ra was speculated may be attributed to the conidia germination over time.