Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulatingCryptococcus neoformansandCandida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture ofC. neoformans. Internalization ofC. neoformansby KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth ofC. neoformansis inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions.

Although neutrophils are typically the first immune cells attracted to an infection site, little is known about how neutrophils dynamically interact with invading pathogens in vivo. Here, with the use of intravital microscopy, we demonstrate that neutrophils migrate to the arrested Cryptococcus neoformans, a leading agent to cause meningoencephalitis, in the brain microvasculature. Following interactions with C. neoformans, neutrophils were seen to internalize the organism and then circulate back into the bloodstream, resulting in a direct removal of the organism from the endothelial surface before its transmigration into the brain parenchyma. C. neoformans infection led to enhanced expression of adhesion molecules macrophage 1 antigen on neutrophils and ICAM-1 on brain endothelial cells. Depletion of neutrophils enhanced the brain fungal burden. Complement C3 was critically involved in the recognition of C. neoformans by neutrophils and subsequent clearance of the organism from the brain. Together, our finding of the direct removal of C. neoformans by neutrophils from its arrested site may represent a novel mechanism of host defense in the brain, in addition to the known, direct killing of microorganisms at the infection sites. These data are the first to characterize directly the dynamic interactions of leukocytes with a microbe in the brain of a living animal.

Significance: Bloodborne parasites develop very sophisticated mechanisms to survive in the bloodstream, causing serious diseases. The mechanisms involved in intravascular clearance of bloodborne parasites are largely unknown. Understanding the underlying mechanisms is fundamental for developing strategies to treat the diseases. In the current study, we reveal that CRIg, a macrophage receptor previously shown to bind C3b/iC3b in vitro, plays an essential role in intravascular clearance of the bloodborne parasites African trypanosomes. More importantly, we demonstrate that CRIg, through interactions with complement deposited on the parasites in vivo, rather than parasites themselves, captures parasites under flow conditions. Thus, CRIg functions as a complement receptor in vivo to capture bloodborne pathogens and may represent a target for treatment.

Neutrophils have been shown to efficiently killCryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils withC. neoformansand the underlying mechanisms in real time. Neutrophils were directly seen to chaseC. neoformanscells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation withC. neoformansresulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing ofC. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils towardC. neoformansbut almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach toC. neoformansbut was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils withC. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing ofC. neoformansin real time.

African trypanosomes cause fatal infections in both humans and livestock. Interferon gamma (IFN-γ) plays an essential role in resistance to African trypanosomes. However, increasing evidence suggests that IFN-γ, when excessively synthesized, also induces immunopathology, enhancing susceptibility to the infection. Thus, production of IFN-γ must be tightly regulated during infections with African trypanosomes to ensure that a robust immune response is elicited without tissue destruction. Early studies have shown that secretion of IFN-γ is downregulated by interleukin 10 (IL-10). More recently, IL-27 has been identified as a negative regulator of IFN-γ production during African trypanosome infections. In this review, we discuss the current state of our understanding of the role of IFN-γ in African trypanosome infections. We have focused on the cellular source of IFN-γ, its beneficial and detrimental effects, and mechanisms involved in regulation of its production, highlighting some recent advances and offering some perspectives on future directions.