Robust Phagocyte Recruitment Controls the Opportunistic Fungal Pathogen Mucor circinelloides in Innate Granulomas In Vivo

Abstract

Mucormycosis is an emerging fungal infection with extremely high mortality rates in patients with defects in their innate immune response, specifically in functions mediated through phagocytes. However, we currently have a limited understanding of the molecular and cellular interactions between these innate immune effectors and mucormycete spores during the early immune response. Here, the early events of innate immune recruitment in response to infection by Mucor circinelloides spores are modeled by a combined in silico modeling approach and real-time in vivo microscopy. Phagocytes are rapidly recruited to the site of infection in a zebrafish larval model of mucormycosis. This robust early recruitment protects from disease onset in vivo. In silico analysis identified that protection is dependent on the number of phagocytes at the infection site, but not the speed of recruitment. The mathematical model highlights the role of proinflammatory signals for phagocyte recruitment and the importance of inhibition of spore germination for protection from active fungal disease. These in silico data are supported by an in vivo lack of fungal spore killing and lack of reactive oxygen burst, which together result in latent fungal infection. During this latent stage of infection, spores are controlled in innate granulomas in vivo. Disease can be reactivated by immunosuppression. Together, these data represent the first in vivo real-time analysis of innate granuloma formation during the early stages of a fungal infection. The results highlight a potential latent stage during mucormycosis that should urgently be considered for clinical management of patients.

Environmental interactions with amoebae as drivers of bacterial-fungal endosymbiosis and pathogenicity

“…Resting or swollen spores were co-incubated with J774A.1 macrophage-like cells, un-engulfed spores washed away, macrophages lysed, and spores tested for viability. Consistent with our previous observations and others, 40,41 macrophages did not kill phagocytosed dormant or swollen spores ( Figure 2a) . Further, macrophages showed a defect in phagosome acidification as measured by lysotracker staining following uptake of both live resting and pre-swollen spores, suggesting a defect in phagosome maturation ( Figure 2b ).…”

“…We previously showed that the early stages of host-fungus interaction determine disease outcome in the zebrafish model of mucormycosis 41 . Our data suggest that successful control requires both 1) the presence of phagocytes at the site of infection within the first 24 hours and 2) the formation of a granuloma capable of killing spores.…”

“…Given this pressing problem, the focus of Mucorales pathogenesis studies has been on disseminated disease, particularly hyphal resistance to host immunity, a hallmark of pathogenicity. However, we recently showed that the early stages of infection control are critical for predicting disease outcome 41 . Consistent with this, the majority of mucormycosis patients have predisposing factors that impact phagocyte recruitment, including iron overload, diabetes mellitus, neutropenia, organ transplantation, trauma, and corticosteroid therapy 19,22,34,42-50 .…”

“…Recently, we showed that recruitment of phagocytes to the site of infection enables the formation of a granuloma that can contain and control the germination of resting Mucor circinelloides spores 41 . In contrast, a range of Mucorales resting spores fail to elicit pro-inflammatory cytokine responses and do not induce the strong phagocyte chemotaxis required for granuloma formation 33,61-63 .…”

“…However R. microsporus spores are on average 5 μm 68 . Finally, in addition to evading uptake, engulfed Mucorales can survive within the phagolysosome via a mechanism that, at least in part, involves melanin-mediated phagosome maturation arrest through inhibition of LC3 40,41,69-71 .…”