BACKGROUND
cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease in immunocompromised patients  <cit> .
macrophages are essential for effective host defense against c. neoformans in humans  <cit> .
however, murine macrophages have been shown to be permissive for intracellular replication of c. neoformans, which can subsequently be extruded from or lyse the macrophages  <cit> .
in this regard, c. neoformans has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of large phagosomes where multiple cryptococcal cells are present  <cit> .
our group and others have recently reported that after c. neoformans ingestion by macrophages, the yeast replicates and is subsequently extruded, in a process whereby both the yeast and macrophages survive  <cit> .
moreover, it was also recently discovered that c. neoformans can spread from an infected to an uninfected murine macrophage cell  <cit> .
here we further extend our extrusion studies to human peripheral blood monocytes  and report that as in murine macrophages, the interaction between human monocytes and c. neoformans leads to ingestion, intracellular replication, and polysaccharide shedding of c. neoformans, followed by cell to cell spread and extrusion of c. neoformans.
the occurrence of phagosomal 'extrusion' in human peripheral blood monocytes suggests a central role for this phenomenon in the propagation and dissemination of this fungal pathogen.
c. neoformans has a novel intracellular strategy that, to date has no precedent in other well-characterized intracellular pathogens.
since c. neoformans is an environmental microbe that does not require a mammalian host for replication or survival, its sophisticated intracellular pathogenic lifestyle suggests that the mechanisms that govern its virulence are unique and distinct from microbes that require such a host, most likely stemming from selection in the environment  <cit> .
in this regard, the discovery of similar interactions between c. neoformans and acanthamoebae castellanii and dictiostelyium discoidum and murine macrophages  <cit>  have led to the hypothesis that the ability of c. neoformans to survive in mammalian cells evolved accidentally, perhaps from interactions with soil predators  <cit> .
a corollary of this hypothesis is that the interactions of c. neoformans with cells from any mammalian species should be similar.
in this study, we explore this corollary by studying c. neoformans interactions with human peripheral blood monocytes and show that these are similar to those described for murine macrophages.
RESULTS
c. neoformans replicates and sheds polysaccharide in human peripheral blood monocytes
c. neoformans replicated in hpbm cells at similar rates to extracellular c. neoformans, that is, every  <dig> to  <dig> h .
to investigate whether polysaccharide-filled vesicles formed following hpbm incubation with c. neoformans, hpbms with and without ingested c. neoformans cells were permeabilized and incubated with conjugated alexa 546-18b <dig>  which binds gxm.
the cells were then examined in a confocal microscope for the presence of cytoplasmic vesicles containing polysaccharide.
as in previous studies, vesicles positive for polysaccharide were identified starting at  <dig> h post infection .
a group of control-uninfected cells gave no positive signal even when overexposed .
cell-to-cell spread and extrusion of c. neoformans by hpbms
to study the occurrence of cell-to-cell spread and extrusion of c. neoformans, we incubated hpbms with the yeast cells.
following ingestion and subsequent imaging, we witnessed that c. neoformans also spread from host human monocyte to another uninfected one  , confirming similar observations made in other studies  <cit> .
we also observed that c. neoformans was extruded from hpbms in a similar fashion, as previously described for murine cells, leading to the survival of the yeast cells and the monocyte, as evidenced by continual budding and pseudopodial movements, respectively  .
overall, out of  <dig> infected cells,  <dig> cell to cell spread events and  <dig> extrusion events were observed.
cell cycle distribution of monocytes is altered after fc- and complement-mediated phagocytosis
previous studies with mouse cells reported an increase in s phase cells after complement and fc-mediated phagocytosis of polystyrene beads, live or heat-killed c. neoformans  <cit> .
thus, we investigated whether the same phenomenon could be observed in primary human monocytes.
we found that the majority of monocytes were in g <dig> phase in our culture conditions  .
just as in cultured j <dig>  cells, monocytes phagocytosed c. neoformans strain  <dig> opsonized with mab 18b <dig> and h <dig> opsonized with human serum.
both fc- and complement-mediated phagocytosis resulted in cell populations that had a significant shift in cell cycle such that the monocytes with ingested c. neoformans had a much greater percentage of cells shifted into s phase relative to the population that did not phagocytose c. neoformans or relative to control cells that were unexposed to c. neoformans .
interestingly, in both phagocytosis assay groups, there was approximately a 20% decrease in the percentage of g <dig>  which was greater compared to our previous report on j <dig>  cells in which a 10% decrease in the percentage of g <dig> was observed   <cit> .
discussion
blood-derived monocytes have been extensively used to study the interaction of c. neoformans with human phagocytic cells  <cit> ).
however, the mechanisms of cryptococcal intracellular pathogenesis have been studied largely with murine cells  <cit> .
in this study, we investigated whether the events that characterized c. neoformans-murine macrophage interactions also occurred in human cells, with particular emphasis on fungal cell exocytosis, host cell cycle response, and intracellular polysaccharide shedding.
this question is important because, in addition to validating observations made with murine cells in human cells, it can support or refute proposals for the emergence of cryptococcal virulence in mammalian hosts.
if c. neoformans virulence for mammals did emerge accidentally from interactions with phagocytic predators in the environment one could posit that its interaction with macrophages from different mammalian species would be similar.
to date c. neoformans interactions with mammalian macrophages have been limited to three species: mice, rats, and humans.
the comparison of c. neoformans interactions with murine and rat macrophages was not revealing in this regard because the latter were a non-permissive host for cryptococcal replication  <cit> .
furthermore, there are mouse strain differences in murine macrophage permissiveness to cryptococcal replication that correlate with strain susceptibility to cryptococcosis  <cit> .
human monocytes are known to be permissive to c. neoformans intracellular replication  <cit> , but the outcome of this interaction has not been explored.
the major finding of this study is that the interaction of c. neoformans with human monocytes parallels that described with murine macrophages with regards to replication time, fungal cell exocytosis, phagocytosis-triggered cell cycle progression and intracellular polysaccharide shedding.
these observations support the hypothesis that the mechanism of intracellular aggression for c. neoformans is conserved between amoebae to mice to humans
cell replication is affected by external stimuli, such as growth factors, cell-cell contact, and cell adhesion to the extracellular matrix  <cit> .
the fact that there was a 2-fold greater increase in human monocytes going to s phase  than in murine tissue macrophages  suggests that monocytes have a higher replication potential, which is consistent with the fact that they are less differentiated blood macrophage precursors.
the consequence of phagocytic cell replication for the outcome of infection is not known.
a greater ability to replicate could increase the number of effector cells as an outcome that could be advantageous to the host.
on the other hand, the observation that c. neoformans growth in monocytes can exceed growth in media in the presence of opsonizing antibody  <cit> , raising the possibility that host cell replication could be a disadvantage, since this would also increase the number of host cells available for intracellular infection and possibly, potentiate dissemination.
indeed, extrapulmonary dissemination in mice has been associated with macrophage ingestion in mice  <cit> .
induced stimulation of monocyte cell cycle progression following phagocytosis of c. neoformans could influence the outcome of infection by generating additional uninfected effector cells at the site of infection, as previously proposed by luo, et.
al.,  <cit> .
in this study we observed phagosomal extrusion in both forms, i.e.
where single c. neoformans cells were extruded and complete extrusion, where all c. neoformans cells were extruded concomitantly.
single cell exocytosis from human monocytes was observed by ma et al  <cit>  but phagosome extrusion has not been previously reported in human cells.
the significance of the observation that cell-to-cell spread and extrusion of c. neoformans occurred in human monocytes is that these events might contribute to disease pathogenesis, especially in immuno-compromised individuals where the proper cell-mediated immune response is lacking.
even though spreading of macrophages-ingested c. neoformans to other cell types has not been demonstrated it is nevertheless possible that it could take place and thereby contribute to the overall pathogenicity of c. neoformans.
further, human monocytes might function as 'trojan horses' and deliver c. neoformans to the central nervous system, as described for hiv  <cit> .
our study, like all in vitro studies, has several limitations.
first, human monocytes are macrophage precursors and consequently not fully differentiated.
this could account for the significantly higher proportion of cells that underwent cell cycle progression upon c. neoformans phagocytosis relative to what was observed previously for murine macrophages.
second, isolated cells in tissue culture conditions could behave differently than in the body and consequently, one must be cautious in extrapolating these findings to in vivo situations.
in this regard, the interaction of human monocytes with cryptococcus is known to be highly dependent on the conditions of the experiment  <cit> .
third, we opsonized c. neoformans with a murine igg <dig>  an isotype that is known to engage human fc receptors and promote phagocytosis.
however, murine and human igg could engage different types of receptors and it is conceivable that different results would be obtained with human igg mabs that are unfortunately not available.
despite the limitations inherent in this system, we believe the similarities between c. neoformans-macrophage interactions for human and mouse cells is a significant result from the viewpoint of understanding the origin and range of cryptococcal virulence.
this finding supports the continued use of mice and mouse cells for studies of certain c. neoformans-host interactions.
furthermore, the demonstration that the same phenomena that define cryptococcal pathogenesis in mouse cells also occur in human cells, provides strong support for central corollary of the 'ready-made virulence' hypothesis  <cit> , namely, that the virulence of certain environmentally-acquired fungi is conserved across mammalian species.
given that humans and rodents diverged over  <dig> million years ago  <cit> , the similarities in the intracellular pathogenesis of c. neoformans in mouse and human cells suggest two possibilities, which are not mutually exclusive.
first, c. neoformans could be endowed with an ancient intracellular pathogenic mechanism that predated the mammalian radiation.
second, c. neoformans has a non-specific intracellular mechanism that allows it to survive and replicate in phylogenetically different phagocytes.
these possibilities cannot be distinguished based on the available information.
the fact that rat macrophages are not as permissive to c. neoformans replication as murine and human cells appears to be a function of more powerful antifungal mechanisms, which inhibit fungal growth  <cit> .
given that protozoa branched earlier than animals and fungi from the eukaryotic tree of life  <cit>  and that fungi predate the emergence of animals in the evolutionary record, the similarities between the intracellular pathogenic strategy of c. neoformans for animals and protista are consistent with the view that cryptococcal virulence evolved to facilitate resistance to environmental predators to survive against said predators.
in summary, we establish that the interaction of c. neoformans with human monocytes is very similar to that described earlier for murine cells.
the continuity in the phenomena observed for c. neoformans interactions with primate and murine cells highlights the importance of comprehensively studying the pathogenic strategy of c. neoformans in light of the innate immune defense.
CONCLUSIONS
in summary, we establish that the interaction of c. neoformans with human monocytes is very similar to that described earlier for murine cells.
the continuity in the phenomena observed for c. neoformans interactions with primate and murine cells highlights the importance of comprehensively studying the pathogenic strategy of c. neoformans in light of the innate immune defense.

 cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease in immunocompromised patients. macrophages are essential for effective host defense against c. neoformans in humans. however, murine macrophages have been shown to be permissive for intracellular replication of c. neoformans, which can subsequently be extruded from or lyse the macrophages. in this regard, c. neoformans has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of large phagosomes where multiple cryptococcal cells are present. our group and others have recently reported that after c. neoformans ingestion by macrophages, the yeast replicates and is subsequently extruded, in a process whereby both the yeast and macrophages survive. moreover, it was also recently discovered that c. neoformans can spread from an infected to an uninfected murine macrophage cell. here we further extend our extrusion studies to human peripheral blood monocytes and report that as in murine macrophages, the interaction between human monocytes and c. neoformans leads to ingestion, intracellular replication, and polysaccharide shedding of c. neoformans, followed by cell to cell spread and extrusion of c. neoformans. the occurrence of phagosomal'extrusion' in human peripheral blood monocytes suggests a central role for this phenomenon in the propagation and dissemination of this fungal pathogen. c. neoformans has a novel intracellular strategy that, to date has no precedent in other well-characterized intracellular pathogens. since c. neoformans is an environmental microbe that does not require a mammalian host for replication or survival, its sophisticated intracellular pathogenic lifestyle suggests that the mechanisms that govern its virulence are unique and distinct from microbes that require such a host, most likely stemming from selection in the environment. in this regard, the discovery of similar interactions between c. neoformans and acanthamoebae castellanii and dictiostelyium discoidum and murine macrophages have led to the hypothesis that the ability of c. neoformans to survive in mammalian cells evolved accidentally, perhaps from interactions with soil predators. a corollary of this hypothesis is that the interactions of c. neoformans with cells from any mammalian species should be similar. in this study, we explore this corollary by studying c. neoformans interactions with human peripheral blood monocytes and show that these are similar to those described for murine macrophages.  c. neoformans replicates and sheds polysaccharide in human peripheral blood monocytes c. neoformans replicated in hpbm cells at similar rates to extracellular c. neoformans, that is, every to h. to investigate whether polysaccharide-filled vesicles formed following hpbm incubation with c. neoformans, hpbms with and without ingested c. neoformans cells were permeabilized and incubated with conjugated alexa 546-18b which binds gxm. the cells were then examined in a confocal microscope for the presence of cytoplasmic vesicles containing polysaccharide. as in previous studies, vesicles positive for polysaccharide were identified starting at h post infection. a group of control-uninfected cells gave no positive signal even when overexposed. cell-to-cell spread and extrusion of c. neoformans by hpbms to study the occurrence of cell-to-cell spread and extrusion of c. neoformans, we incubated hpbms with the yeast cells. following ingestion and subsequent imaging, we witnessed that c. neoformans also spread from host human monocyte to another uninfected one, confirming similar observations made in other studies. we also observed that c. neoformans was extruded from hpbms in a similar fashion, as previously described for murine cells, leading to the survival of the yeast cells and the monocyte, as evidenced by continual budding and pseudopodial movements, respectively. overall, out of infected cells, cell to cell spread events and extrusion events were observed. cell cycle distribution of monocytes is altered after fc- and complement-mediated phagocytosis previous studies with mouse cells reported an increase in s phase cells after complement and fc-mediated phagocytosis of polystyrene beads, live or heat-killed c. neoformans. thus, we investigated whether the same phenomenon could be observed in primary human monocytes. we found that the majority of monocytes were in g phase in our culture conditions. just as in cultured j cells, monocytes phagocytosed c. neoformans strain opsonized with mab 18b and h opsonized with human serum. both fc- and complement-mediated phagocytosis resulted in cell populations that had a significant shift in cell cycle such that the monocytes with ingested c. neoformans had a much greater percentage of cells shifted into s phase relative to the population that did not phagocytose c. neoformans or relative to control cells that were unexposed to c. neoformans. interestingly, in both phagocytosis assay groups, there was approximately a 20% decrease in the percentage of g which was greater compared to our previous report on j cells in which a 10% decrease in the percentage of g was observed. discussion blood-derived monocytes have been extensively used to study the interaction of c. neoformans with human phagocytic cells ). however, the mechanisms of cryptococcal intracellular pathogenesis have been studied largely with murine cells. in this study, we investigated whether the events that characterized c. neoformans-murine macrophage interactions also occurred in human cells, with particular emphasis on fungal cell exocytosis, host cell cycle response, and intracellular polysaccharide shedding. this question is important because, in addition to validating observations made with murine cells in human cells, it can support or refute proposals for the emergence of cryptococcal virulence in mammalian hosts. if c. neoformans virulence for mammals did emerge accidentally from interactions with phagocytic predators in the environment one could posit that its interaction with macrophages from different mammalian species would be similar. to date c. neoformans interactions with mammalian macrophages have been limited to three species: mice, rats, and humans. the comparison of c. neoformans interactions with murine and rat macrophages was not revealing in this regard because the latter were a non-permissive host for cryptococcal replication. furthermore, there are mouse strain differences in murine macrophage permissiveness to cryptococcal replication that correlate with strain susceptibility to cryptococcosis. human monocytes are known to be permissive to c. neoformans intracellular replication, but the outcome of this interaction has not been explored. the major finding of this study is that the interaction of c. neoformans with human monocytes parallels that described with murine macrophages with regards to replication time, fungal cell exocytosis, phagocytosis-triggered cell cycle progression and intracellular polysaccharide shedding. these observations support the hypothesis that the mechanism of intracellular aggression for c. neoformans is conserved between amoebae to mice to humans cell replication is affected by external stimuli, such as growth factors, cell-cell contact, and cell adhesion to the extracellular matrix. the fact that there was a 2-fold greater increase in human monocytes going to s phase than in murine tissue macrophages suggests that monocytes have a higher replication potential, which is consistent with the fact that they are less differentiated blood macrophage precursors. the consequence of phagocytic cell replication for the outcome of infection is not known. a greater ability to replicate could increase the number of effector cells as an outcome that could be advantageous to the host. on the other hand, the observation that c. neoformans growth in monocytes can exceed growth in media in the presence of opsonizing antibody, raising the possibility that host cell replication could be a disadvantage, since this would also increase the number of host cells available for intracellular infection and possibly, potentiate dissemination. indeed, extrapulmonary dissemination in mice has been associated with macrophage ingestion in mice. induced stimulation of monocyte cell cycle progression following phagocytosis of c. neoformans could influence the outcome of infection by generating additional uninfected effector cells at the site of infection, as previously proposed by luo, et. al.,. in this study we observed phagosomal extrusion in both forms, i.e. where single c. neoformans cells were extruded and complete extrusion, where all c. neoformans cells were extruded concomitantly. single cell exocytosis from human monocytes was observed by ma et al but phagosome extrusion has not been previously reported in human cells. the significance of the observation that cell-to-cell spread and extrusion of c. neoformans occurred in human monocytes is that these events might contribute to disease pathogenesis, especially in immuno-compromised individuals where the proper cell-mediated immune response is lacking. even though spreading of macrophages-ingested c. neoformans to other cell types has not been demonstrated it is nevertheless possible that it could take place and thereby contribute to the overall pathogenicity of c. neoformans. further, human monocytes might function as'trojan horses' and deliver c. neoformans to the central nervous system, as described for hiv. our study, like all in vitro studies, has several limitations. first, human monocytes are macrophage precursors and consequently not fully differentiated. this could account for the significantly higher proportion of cells that underwent cell cycle progression upon c. neoformans phagocytosis relative to what was observed previously for murine macrophages. second, isolated cells in tissue culture conditions could behave differently than in the body and consequently, one must be cautious in extrapolating these findings to in vivo situations. in this regard, the interaction of human monocytes with cryptococcus is known to be highly dependent on the conditions of the experiment. third, we opsonized c. neoformans with a murine igg an isotype that is known to engage human fc receptors and promote phagocytosis. however, murine and human igg could engage different types of receptors and it is conceivable that different results would be obtained with human igg mabs that are unfortunately not available. despite the limitations inherent in this system, we believe the similarities between c. neoformans-macrophage interactions for human and mouse cells is a significant result from the viewpoint of understanding the origin and range of cryptococcal virulence. this finding supports the continued use of mice and mouse cells for studies of certain c. neoformans-host interactions. furthermore, the demonstration that the same phenomena that define cryptococcal pathogenesis in mouse cells also occur in human cells, provides strong support for central corollary of the'ready-made virulence' hypothesis, namely, that the virulence of certain environmentally-acquired fungi is conserved across mammalian species. given that humans and rodents diverged over million years ago, the similarities in the intracellular pathogenesis of c. neoformans in mouse and human cells suggest two possibilities, which are not mutually exclusive. first, c. neoformans could be endowed with an ancient intracellular pathogenic mechanism that predated the mammalian radiation. second, c. neoformans has a non-specific intracellular mechanism that allows it to survive and replicate in phylogenetically different phagocytes. these possibilities cannot be distinguished based on the available information. the fact that rat macrophages are not as permissive to c. neoformans replication as murine and human cells appears to be a function of more powerful antifungal mechanisms, which inhibit fungal growth. given that protozoa branched earlier than animals and fungi from the eukaryotic tree of life and that fungi predate the emergence of animals in the evolutionary record, the similarities between the intracellular pathogenic strategy of c. neoformans for animals and protista are consistent with the view that cryptococcal virulence evolved to facilitate resistance to environmental predators to survive against said predators. in summary, we establish that the interaction of c. neoformans with human monocytes is very similar to that described earlier for murine cells. the continuity in the phenomena observed for c. neoformans interactions with primate and murine cells highlights the importance of comprehensively studying the pathogenic strategy of c. neoformans in light of the innate immune defense.  in summary, we establish that the interaction of c. neoformans with human monocytes is very similar to that described earlier for murine cells. the continuity in the phenomena observed for c. neoformans interactions with primate and murine cells highlights the importance of comprehensively studying the pathogenic strategy of c. neoformans in light of the innate immune defense. 
the interaction between macrophages and c. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. c. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. in this study we extended studies of c. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes. this study demonstrated that c. neoformans can shed polysaccharide within human monocytes, spread from cell to cell, and be extruded from them. furthermore, human monocytes responded to ingestion of c. neoformans with cell cycle progression from g to s. given that these hosts diverged over million years ago, the similar pathogenic strategies for c. neoformans in murine and human cells supports the hypothesis that the mechanism that underlies the mammalian intracellular pathogenesis of c. neoformans originated from interactions with a third host, possibly soil amoeboid predators, before the mammalian radiation. cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. however, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. similarities between mouse and human cells support the suitability of mouse cells for the study of intracellular pathogenesis mechanisms.
 cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. the interaction between macrophages and c. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. c. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. however, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. in this study we extended studies of c. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes.  this study demonstrated that c. neoformans can shed polysaccharide within human monocytes, spread from cell to cell, and be extruded from them. furthermore, human monocytes responded to ingestion of c. neoformans with cell cycle progression from g to s.  similarities between mouse and human cells support the suitability of mouse cells for the study of intracellular pathogenesis mechanisms. given that these hosts diverged over million years ago, the similar pathogenic strategies for c. neoformans in murine and human cells supports the hypothesis that the mechanism that underlies the mammalian intracellular pathogenesis of c. neoformans originated from interactions with a third host, possibly soil amoeboid predators, before the mammalian radiation. 