BACKGROUND
aspergillus fumigatus is a ubiquitous filamentous fungus found in soil and compost heaps, as well as in most indoor environments  <cit> .
it achieves widespread dispersal by asexual reproduction through the release of haploid conidia , but can also reproduce sexually  <cit> .
although a. fumigatus is not the most prevalent fungal species worldwide, it is one of the most ubiquitous fungi due to the large number of airborne conidia it releases.
estimates of  <dig> to  <dig> colony forming units of a. fumigatus per cubic metre have been reported for indoor and outside air, and this widespread distribution ensures that all humans are likely to inhale at least hundreds of conidia each day  <cit> .
the small size of the a. fumigatus conidia  allows them to reach the innermost areas of the lung, including the alveoli  <cit> .
a. fumigatus conidia have been shown to be efficiently internalized by cultured murine alveolar macrophages  <cit>  and the human alveolar type ii pneumocyte cell line, a <dig>  <cit> .
in immune compromised individuals, a. fumigatus can cause a spectrum of diseases which range from local hypersensitivity reactions to often fatal systemic mycoses  <cit> .
although infections by a. fumigatus have been described in other sites of the body, the respiratory tract is the main route of entry and site of infection.
the three predominant forms of disease caused by a. fumigatus are: allergic bronchopulmonary aspergillosis , which is prevalent in up to 5% of asthmatic and 10% of cystic fibrosis patients; aspergilloma, a condition in which fungal mycelia grow as a mass in pre-existing lung cavities; and invasive pulmonary aspergillosis , a life-threatening systemic mycosis in immunocompromised individuals  <cit> .
despite the importance of the host's response in these conditions, the mechanisms involved in each of these diseases are still not completely understood.
the bronchial epithelium is the first point of contact and barrier to inhaled environmental particulates.
a recent electron microscopy study by amitani and kawanami  <cit> , using an organ culture model, showed three possible pathways by which a. fumigatus conidia invade the epithelial barrier:  penetration of hyphae through the intercellular spaces in the epithelium;  direct penetration of hyphae through epithelial cells; and  internalization of conidia within epithelial cells.
once internalized, conidia are ingested by acidified phagolysosomes and are degraded, although a small number of internalized conidia may survive and germinate within the phagolysosomes  <cit> .
hence, uptake of a. fumigatus conidia into lung epithelial cells may represent a mechanism of immune evasion.
clearly, the cellular response of the bronchial epithelium following internalization of a. fumigatus conidia is important to understand, particularly since the interaction may be important in mediating the allergenic properties of the mould as well as the host immune response.
this would be particularly pertinent to diseases such as asthma, where aberrant epithelial responses to injury or stress may be important drivers of disease pathogenesis  <cit> .
the aim of the present study was to assess the cell-specific transcriptional response of cultured human bronchial epithelial cells following direct interaction with a. fumigatus conidia.
to assess the response of those cells in direct interaction with conidia, we used fluorescence-activated cell sorting  to specifically isolate cells associated with a recombinant strain of a. fumigatus that constitutively expresses green fluorescent protein  from an epithelial culture.
we then applied whole-genome expression microarray technology to assess transcriptional differences between these cell populations.
RESULTS
a. fumigatus conidia are internalized by human bronchial epithelial cell monolayers
to determine if human bronchial epithelial cells could internalize a. fumigatus conidia, 16hbe14o- cell monolayers were co-cultured with gfp-expressing a. fumigatus conidia and internalization was assessed by scanning confocal microscopy.
as shown in the representative 3-dimensional reconstruction, the yz and xz planes of the images demonstrate that a population of 16hbe14o- cells contained intracellular a. fumigatus conidia following  <dig> hrs of co-incubation .
the internalization process did not affect the cell surface expression of the epithelial specific adherens junction protein, e-cadherin, suggesting that the epithelium was not stimulated.
to further confirm the internalization of a. fumigatus conidia by 16hbe14o- cells, we measured immunolabelling of extracellular and internalized a. fumigatus conidia  <cit> .
as demonstrated by the representative images in figure  <dig>  following  <dig> hrs of co-culture ~50% of a. fumigatus conidia were found to be internalized and therefore were not labelled by the anti-a.
fumigatus antibody.
the images also revealed that there was wide variation in the number of conidia internalized, with some cells interacting with several conidia while others did not appear to interact with any.
to further characterize the interaction, we used the nystatin protection assay as an independent method to measure conidia internalization by cultured cells over time  <cit> .
the number of internalized a. fumigatus conidia over a time course of  <dig> mins,  <dig> hrs and  <dig> hrs was 38%, 30% and 41%, respectively .
these differences were not statistically significant.
overall, our results indicate that 16hbe14o- cells internalize more than a third of the a. fumigatus conidia that bind to the cells.
isolation of human bronchial epithelial cells following interaction with a. fumigatus conidia using facs
we took advantage of the observation that not all epithelial cells contained conidia and used facs to separate the 16hbe14o- cells into two populations: epithelial cells directly interacting with gfp-a.
fumigatus conidia, and those with no direct contact.
as shown in figure 4a-c, we were able to easily distinguish cells from conidia by gating on forward  and side  scatter.
identified conidia and cells were further analyzed by determining gfp fluorescence intensity as demonstrated for a. fumigatus conidia alone , 16hbe14o- cells alone  and co-incubated a. fumigatus conidia and 16hbe14o- cells .
other gates were set based on the fluorescence intensities of gfp and pe texas red, and this permitted selection of cells associated with conidia .
as demonstrated in figure  <dig>  positive and negative sorted cell populations were re-analyzed to determine the average sorting accuracy.
the average sorting accuracy was calculated to be  <dig> %, although there was a statistically significant difference in sorting accuracy depending on whether positive or negative cell populations were examined .
confocal microscopy of the two cell populations  confirmed the facs data.
functional genomics analysis reveals differential gene expression between bronchial epithelial cells directly interacting with conidia compared to cells alone
expression data were obtained from ~ <dig>  gene probes on each of eight arrays  and submitted to the gene expression omnibus .
hierarchical clustering demonstrated that 16hbe14o- cells positive for gfp-expressing a. fumigatus conidia  and negative cells showed a high level of concordance, as indicated by their similar gene expression clustering profiles .
furthermore, these arrays formed pairs based on their co-incubation sample, indicating that positive and negative populations sorted from each co-incubated sample were generally more similar to each other than to other sorted populations, reflecting their shared heritage.
using genespring gx  <dig> .
<dig> software,  <dig>  probes exhibited significant expression in either positive or negative samples.
of these,  <dig> showed differential expression between positive and negative paired populations , based on a paired t-test p-value of  <dig>  and a fold change of  <dig>  or greater.
none of these genes retained significant p-values following multiple-testing correction implemented within the genespring software .
within this list of genes of interest,  <dig> were up-regulated in positive sorted samples, while  <dig> showed down-regulated expression in conidia-positive cells compared to conidia-negative cells.
the complete list of genes is provided in additional file  <dig>  to determine the major biological themes found in this dataset, we looked for gene ontology  classifications that were over-represented in these two gene lists using goeast  <cit> .
in both cases, specific go categories were significantly over-represented, with p-values below 10- <dig> .
the up-regulated go list was enriched for terms associated with nucleosome organization, chromatin assembly, as well as chemokines and glutathione, important for inflammation and repair processes.
the down-regulated go list was enriched for terms associated with mitosis, cell cycle progression, and intracellular organelle biology.
the annotated genes showing the greatest up- or down-regulation are shown in table  <dig>  and include matrix metalloproteinases, chemokines, and glutathione s-transferase.
selected terms, belonging to the biological process , molecular function , and cellular component  ontologies, are indicated, and the p-value for enrichment in the appropriate list is shown.
the fold change is the ratio of the expression level in the two conditions, with positive numbers representing up-regulation in positive samples, while negative numbers indicate down-regulation in the positive samples, compared to negative samples.
the paired t-test p-value for differential expression is also shown.
probes lacking annotations were excluded from this list.
we used gene set enrichment analysis  to establish further evidence in support of our findings and thus compensate for the lack of statistical significance after multiple hypothesis testing  <cit> .
leading edge analysis of  <dig>  gene sets resulted in  <dig> sets significantly associated with our up-regulated gene data and  <dig> sets significantly associated with our down-regulated gene data .
these gene sets are provided in additional file  <dig> and are consistent with the go classifications  obtained from our original list of  <dig> differentially expressed genes.
discussion
this study demonstrates that cultured 16hbe14o- human bronchial epithelial cells are capable of rapidly internalizing aspergillus fumigatus conidia.
the number of conidia taken up by individual cells was variable and we exploited this variability to successfully separate cells that were directly interacting with and/or had internalized spores from those that had not.
a microarray-based gene expression analysis on these two populations identified  <dig> genes that were up-regulated and  <dig> genes that were down-regulated in epithelial cells that were directly interacting with and/or had internalized conidia, relative to cells from the same co-incubated sample.
our data suggest that the direct interaction and/or internalization of common fungal spores such as aspergillus fumigatus conidia has measurable, if modest, effects on the transcriptome of human bronchial epithelial cells.
the importance of professional phagocytes in the clearance of inhaled a. fumigatus conidia has motivated multiple studies on the internalization of conidia by these cells  <cit> .
fewer studies have investigated a. fumigatus internalization by non-professional phagocytes such as epithelial cells  <cit> .
we and others have demonstrated the uptake of a. fumigatus conidia in the type-ii pneumocyte cell line, a <dig>  <cit> , primary tracheal epithelial cells, and umbilical vein endothelial cells   <cit> .
these studies showed that a <dig> cells and huvecs internalized 30% and 50%, respectively, of bound a. fumigatus conidia following  <dig> hrs of co-culture  <cit> .
in the present study, we show that 16hbe14o- cells internalized between 30-50% of a. fumigatus conidia within  <dig> hrs of exposure, consistent with previous studies focused on other non-professional phagocyte cell lines  <cit> .
this also compares favourably with botterel et al.
, who showed that co-incubation of a. fumigatus with primary human nasal epithelial cells for  <dig> hrs yielded an internalization rate of about 20%  <cit> .
these studies also demonstrated delayed germination but long term survival of internalized conidia, suggesting that normally non-phagocytic epithelial cells may act as reservoirs for invading a. fumigatus conidia  <cit> .
for our quantitative studies on a. fumigatus conidia internalization by 16hbe14o- cells we used a relatively high multiplicity of infection  of  <dig> conidia per human cell.
the high moi was chosen to maximize the number of infected cells and while this represents a much greater dose than would be found in the lung, it is in line with previous in vitro studies  <cit> .
using facs and gfp-expressing a. fumigatus conidia we were able to sort epithelial cells derived from a single culture that contained bound and/or internalized conidia from those without conidia, allowing us to examine their gene expression profiles using a paired analysis.
to our knowledge, this sorting of otherwise identical cells based on their association with or without a pathogen is a novel application, one that could be applied to a range of pathogens and different cell types.
although facs analysis could not discriminate the number of a. fumigatus conidia associated with each 16hbe14o- cell, fluorescence microscopy indicated that many of the positively sorted 16hbe14o- cells were associated with high numbers of spores.
numerous studies have applied whole genome transcriptional profiling to interactions between mammalian hosts and microbes  <cit> ; however, to our knowledge, there is no study on changes to the transcriptome of human bronchial epithelial cells as a consequence of interacting with fungal conidia.
by analyzing the transcriptomes of 16hbe14o- cells co-incubated with a. fumigatus conidia,  <dig> genes were found to be differentially expressed between the positive and negative cell populations although the magnitude of the fold changes in gene expression were generally small.
the reasons for this may be related in part to the methodology employed: sorting based on fluorescence intensity is not 100% accurate, and the conidia-positive population contained a proportion of negative cells and vice-versa.
this would reduce the magnitude of the observed differences in gene expression between two populations.
nevertheless, the modest transcriptional response following direct interaction with a. fumigatus conidia may also indicate that the host cell does not respond dramatically to conidial uptake thereby providing a protected niche for conidial development.
this is supported by other data.
for example, wasylnka et al.
showed that interaction and even internalization of a. fumigatus conidia by a <dig> cells had little effect on cellular physiology  <cit> .
moreover, binding of a. fumigatus conidia to a <dig> cells has been shown to induce only minor up-regulation of immune response effectors  <cit> .
a recent report indicates that germ tube growth, rather than interaction with conidia, leads to an activation of immune responses in a <dig> cells  <cit> .
in addition, aimanianda et al.
showed that the lack of immune response in macrophages and dendritic cells to fungal conidia was due to the presence of the surface hydrophobin protein layer  on conidia  <cit> .
loss of the rodlet layer during germination unmasked fungal antigens and resulted in an innate immune response in professional phagocytes.
our data indicate that, like professional phagocytes, epithelial cells may not mount a strong immunological response to direct interaction with conidia .
nevertheless, gene ontology  analysis of the list of  <dig> genes demonstrated a coherent and significant response in cells exposed to conidia, and this response was largely re-capitulated using gene set enrichment analysis  on our entire microarray data set.
the go terms enriched for both the up- and down-regulated genes point to fundamental changes associated with the response to fungal spores.
for example, chemokine activity and receptor binding ontologies were prevalent in up-regulated genes, consistent with their established role in responding to fungal invasion  <cit> .
the list of down-regulated genes was enriched for many go terms relating to mitosis and cell cycle progression, indicating that cells reduce their rate of proliferation in response to direct interaction with conidia which may be part of a general stress response.
cell cycle arrest in response to fungal alkaloids has been previously reported  <cit> .
many of the individual genes displaying the highest fold changes belonged to the ontologies identified by go term enrichment analysis, lending further support to the relevance of the identified themes.
amongst those showing the highest degree of up-regulation in response to direct conidial interaction are two chemokines  and two matrix metalloproteinases , as well as a glutathione transferase .
ccl <dig> is an inflammatory chemokine responsible for recruitment of leukocytes to sites of infection, and promotes a th <dig> phenotype in lymphocytes  <cit> .
ccl <dig> is strongly chemotactic for eosinophils  <cit> .
the expression of ccl <dig> has previously been shown to be increased in mouse lungs and rat alveolar macrophages following exposure to a. fumigatus conidia  <cit> .
positive associations between mmps and a. fumigatus have been reported previously in a study involving conidial infection of corneas  <cit> , and mmp <dig> expression has been shown to be higher in sputum cells of asthmatics relative to controls  <cit> .
microsomal glutathione s-transferase  <dig> , found to be up-regulated in cells interacting with a. fumigatus conidia, is the prototypical member of the glutathione s-transferase gene family  <cit>  and has a role in protecting cell from oxidative damage  <cit> .
the experiments in this study used submerged monolayer cultures of a human bronchial epithelial cell line, 16hbe14o-.
some characteristics and responses of these cells are likely to be different from both primary cell cultures and airway cells in situ in the lung.
nevertheless, our approach has the advantage in that it enables us to identify airway cell responses independently of other cell types .
CONCLUSIONS
we demonstrate the use of facs to isolate human airway epithelial cells that bind and/or internalize a. fumigatus conidia in co-culture systems for subsequent assessment of the specific transcriptional responses of cells directly interacting with conidia.
the ability to identify differentially expressed genes within a single culture using facs has potential uses to further investigate the response of other cell types to specific interactions with pathogens.
our ability to better understand the interaction of inhaled a. fumigatus conidia with airway epithelial cells will help provide insights into the mechanisms of several diseases associated with this ubiquitous mould.

 aspergillus fumigatus is a ubiquitous filamentous fungus found in soil and compost heaps, as well as in most indoor environments. it achieves widespread dispersal by asexual reproduction through the release of haploid conidia, but can also reproduce sexually. although a. fumigatus is not the most prevalent fungal species worldwide, it is one of the most ubiquitous fungi due to the large number of airborne conidia it releases. estimates of to colony forming units of a. fumigatus per cubic metre have been reported for indoor and outside air, and this widespread distribution ensures that all humans are likely to inhale at least hundreds of conidia each day. the small size of the a. fumigatus conidia allows them to reach the innermost areas of the lung, including the alveoli. a. fumigatus conidia have been shown to be efficiently internalized by cultured murine alveolar macrophages and the human alveolar type ii pneumocyte cell line, a. in immune compromised individuals, a. fumigatus can cause a spectrum of diseases which range from local hypersensitivity reactions to often fatal systemic mycoses. although infections by a. fumigatus have been described in other sites of the body, the respiratory tract is the main route of entry and site of infection. the three predominant forms of disease caused by a. fumigatus are: allergic bronchopulmonary aspergillosis, which is prevalent in up to 5% of asthmatic and 10% of cystic fibrosis patients; aspergilloma, a condition in which fungal mycelia grow as a mass in pre-existing lung cavities; and invasive pulmonary aspergillosis, a life-threatening systemic mycosis in immunocompromised individuals. despite the importance of the host's response in these conditions, the mechanisms involved in each of these diseases are still not completely understood. the bronchial epithelium is the first point of contact and barrier to inhaled environmental particulates. a recent electron microscopy study by amitani and kawanami, using an organ culture model, showed three possible pathways by which a. fumigatus conidia invade the epithelial barrier: penetration of hyphae through the intercellular spaces in the epithelium; direct penetration of hyphae through epithelial cells; and internalization of conidia within epithelial cells. once internalized, conidia are ingested by acidified phagolysosomes and are degraded, although a small number of internalized conidia may survive and germinate within the phagolysosomes. hence, uptake of a. fumigatus conidia into lung epithelial cells may represent a mechanism of immune evasion. clearly, the cellular response of the bronchial epithelium following internalization of a. fumigatus conidia is important to understand, particularly since the interaction may be important in mediating the allergenic properties of the mould as well as the host immune response. this would be particularly pertinent to diseases such as asthma, where aberrant epithelial responses to injury or stress may be important drivers of disease pathogenesis. the aim of the present study was to assess the cell-specific transcriptional response of cultured human bronchial epithelial cells following direct interaction with a. fumigatus conidia. to assess the response of those cells in direct interaction with conidia, we used fluorescence-activated cell sorting to specifically isolate cells associated with a recombinant strain of a. fumigatus that constitutively expresses green fluorescent protein from an epithelial culture. we then applied whole-genome expression microarray technology to assess transcriptional differences between these cell populations.  a. fumigatus conidia are internalized by human bronchial epithelial cell monolayers to determine if human bronchial epithelial cells could internalize a. fumigatus conidia, 16hbe14o- cell monolayers were co-cultured with gfp-expressing a. fumigatus conidia and internalization was assessed by scanning confocal microscopy. as shown in the representative 3-dimensional reconstruction, the yz and xz planes of the images demonstrate that a population of 16hbe14o- cells contained intracellular a. fumigatus conidia following hrs of co-incubation. the internalization process did not affect the cell surface expression of the epithelial specific adherens junction protein, e-cadherin, suggesting that the epithelium was not stimulated. to further confirm the internalization of a. fumigatus conidia by 16hbe14o- cells, we measured immunolabelling of extracellular and internalized a. fumigatus conidia. as demonstrated by the representative images in figure following hrs of co-culture ~50% of a. fumigatus conidia were found to be internalized and therefore were not labelled by the anti-a. fumigatus antibody. the images also revealed that there was wide variation in the number of conidia internalized, with some cells interacting with several conidia while others did not appear to interact with any. to further characterize the interaction, we used the nystatin protection assay as an independent method to measure conidia internalization by cultured cells over time. the number of internalized a. fumigatus conidia over a time course of mins, hrs and hrs was 38%, 30% and 41%, respectively. these differences were not statistically significant. overall, our results indicate that 16hbe14o- cells internalize more than a third of the a. fumigatus conidia that bind to the cells. isolation of human bronchial epithelial cells following interaction with a. fumigatus conidia using facs we took advantage of the observation that not all epithelial cells contained conidia and used facs to separate the 16hbe14o- cells into two populations: epithelial cells directly interacting with gfp-a. fumigatus conidia, and those with no direct contact. as shown in figure 4a-c, we were able to easily distinguish cells from conidia by gating on forward and side scatter. identified conidia and cells were further analyzed by determining gfp fluorescence intensity as demonstrated for a. fumigatus conidia alone, 16hbe14o- cells alone and co-incubated a. fumigatus conidia and 16hbe14o- cells. other gates were set based on the fluorescence intensities of gfp and pe texas red, and this permitted selection of cells associated with conidia. as demonstrated in figure positive and negative sorted cell populations were re-analyzed to determine the average sorting accuracy. the average sorting accuracy was calculated to be %, although there was a statistically significant difference in sorting accuracy depending on whether positive or negative cell populations were examined. confocal microscopy of the two cell populations confirmed the facs data. functional genomics analysis reveals differential gene expression between bronchial epithelial cells directly interacting with conidia compared to cells alone expression data were obtained from ~ gene probes on each of eight arrays and submitted to the gene expression omnibus. hierarchical clustering demonstrated that 16hbe14o- cells positive for gfp-expressing a. fumigatus conidia and negative cells showed a high level of concordance, as indicated by their similar gene expression clustering profiles. furthermore, these arrays formed pairs based on their co-incubation sample, indicating that positive and negative populations sorted from each co-incubated sample were generally more similar to each other than to other sorted populations, reflecting their shared heritage. using genespring gx. software, probes exhibited significant expression in either positive or negative samples. of these, showed differential expression between positive and negative paired populations, based on a paired t-test p-value of and a fold change of or greater. none of these genes retained significant p-values following multiple-testing correction implemented within the genespring software. within this list of genes of interest, were up-regulated in positive sorted samples, while showed down-regulated expression in conidia-positive cells compared to conidia-negative cells. the complete list of genes is provided in additional file to determine the major biological themes found in this dataset, we looked for gene ontology classifications that were over-represented in these two gene lists using goeast. in both cases, specific go categories were significantly over-represented, with p-values below 10-. the up-regulated go list was enriched for terms associated with nucleosome organization, chromatin assembly, as well as chemokines and glutathione, important for inflammation and repair processes. the down-regulated go list was enriched for terms associated with mitosis, cell cycle progression, and intracellular organelle biology. the annotated genes showing the greatest up- or down-regulation are shown in table and include matrix metalloproteinases, chemokines, and glutathione s-transferase. selected terms, belonging to the biological process, molecular function, and cellular component ontologies, are indicated, and the p-value for enrichment in the appropriate list is shown. the fold change is the ratio of the expression level in the two conditions, with positive numbers representing up-regulation in positive samples, while negative numbers indicate down-regulation in the positive samples, compared to negative samples. the paired t-test p-value for differential expression is also shown. probes lacking annotations were excluded from this list. we used gene set enrichment analysis to establish further evidence in support of our findings and thus compensate for the lack of statistical significance after multiple hypothesis testing. leading edge analysis of gene sets resulted in sets significantly associated with our up-regulated gene data and sets significantly associated with our down-regulated gene data. these gene sets are provided in additional file and are consistent with the go classifications obtained from our original list of differentially expressed genes. discussion this study demonstrates that cultured 16hbe14o- human bronchial epithelial cells are capable of rapidly internalizing aspergillus fumigatus conidia. the number of conidia taken up by individual cells was variable and we exploited this variability to successfully separate cells that were directly interacting with and/or had internalized spores from those that had not. a microarray-based gene expression analysis on these two populations identified genes that were up-regulated and genes that were down-regulated in epithelial cells that were directly interacting with and/or had internalized conidia, relative to cells from the same co-incubated sample. our data suggest that the direct interaction and/or internalization of common fungal spores such as aspergillus fumigatus conidia has measurable, if modest, effects on the transcriptome of human bronchial epithelial cells. the importance of professional phagocytes in the clearance of inhaled a. fumigatus conidia has motivated multiple studies on the internalization of conidia by these cells. fewer studies have investigated a. fumigatus internalization by non-professional phagocytes such as epithelial cells. we and others have demonstrated the uptake of a. fumigatus conidia in the type-ii pneumocyte cell line, a, primary tracheal epithelial cells, and umbilical vein endothelial cells. these studies showed that a cells and huvecs internalized 30% and 50%, respectively, of bound a. fumigatus conidia following hrs of co-culture. in the present study, we show that 16hbe14o- cells internalized between 30-50% of a. fumigatus conidia within hrs of exposure, consistent with previous studies focused on other non-professional phagocyte cell lines. this also compares favourably with botterel et al., who showed that co-incubation of a. fumigatus with primary human nasal epithelial cells for hrs yielded an internalization rate of about 20%. these studies also demonstrated delayed germination but long term survival of internalized conidia, suggesting that normally non-phagocytic epithelial cells may act as reservoirs for invading a. fumigatus conidia. for our quantitative studies on a. fumigatus conidia internalization by 16hbe14o- cells we used a relatively high multiplicity of infection of conidia per human cell. the high moi was chosen to maximize the number of infected cells and while this represents a much greater dose than would be found in the lung, it is in line with previous in vitro studies. using facs and gfp-expressing a. fumigatus conidia we were able to sort epithelial cells derived from a single culture that contained bound and/or internalized conidia from those without conidia, allowing us to examine their gene expression profiles using a paired analysis. to our knowledge, this sorting of otherwise identical cells based on their association with or without a pathogen is a novel application, one that could be applied to a range of pathogens and different cell types. although facs analysis could not discriminate the number of a. fumigatus conidia associated with each 16hbe14o- cell, fluorescence microscopy indicated that many of the positively sorted 16hbe14o- cells were associated with high numbers of spores. numerous studies have applied whole genome transcriptional profiling to interactions between mammalian hosts and microbes; however, to our knowledge, there is no study on changes to the transcriptome of human bronchial epithelial cells as a consequence of interacting with fungal conidia. by analyzing the transcriptomes of 16hbe14o- cells co-incubated with a. fumigatus conidia, genes were found to be differentially expressed between the positive and negative cell populations although the magnitude of the fold changes in gene expression were generally small. the reasons for this may be related in part to the methodology employed: sorting based on fluorescence intensity is not 100% accurate, and the conidia-positive population contained a proportion of negative cells and vice-versa. this would reduce the magnitude of the observed differences in gene expression between two populations. nevertheless, the modest transcriptional response following direct interaction with a. fumigatus conidia may also indicate that the host cell does not respond dramatically to conidial uptake thereby providing a protected niche for conidial development. this is supported by other data. for example, wasylnka et al. showed that interaction and even internalization of a. fumigatus conidia by a cells had little effect on cellular physiology. moreover, binding of a. fumigatus conidia to a cells has been shown to induce only minor up-regulation of immune response effectors. a recent report indicates that germ tube growth, rather than interaction with conidia, leads to an activation of immune responses in a cells. in addition, aimanianda et al. showed that the lack of immune response in macrophages and dendritic cells to fungal conidia was due to the presence of the surface hydrophobin protein layer on conidia. loss of the rodlet layer during germination unmasked fungal antigens and resulted in an innate immune response in professional phagocytes. our data indicate that, like professional phagocytes, epithelial cells may not mount a strong immunological response to direct interaction with conidia. nevertheless, gene ontology analysis of the list of genes demonstrated a coherent and significant response in cells exposed to conidia, and this response was largely re-capitulated using gene set enrichment analysis on our entire microarray data set. the go terms enriched for both the up- and down-regulated genes point to fundamental changes associated with the response to fungal spores. for example, chemokine activity and receptor binding ontologies were prevalent in up-regulated genes, consistent with their established role in responding to fungal invasion. the list of down-regulated genes was enriched for many go terms relating to mitosis and cell cycle progression, indicating that cells reduce their rate of proliferation in response to direct interaction with conidia which may be part of a general stress response. cell cycle arrest in response to fungal alkaloids has been previously reported. many of the individual genes displaying the highest fold changes belonged to the ontologies identified by go term enrichment analysis, lending further support to the relevance of the identified themes. amongst those showing the highest degree of up-regulation in response to direct conidial interaction are two chemokines and two matrix metalloproteinases, as well as a glutathione transferase. ccl is an inflammatory chemokine responsible for recruitment of leukocytes to sites of infection, and promotes a th phenotype in lymphocytes. ccl is strongly chemotactic for eosinophils. the expression of ccl has previously been shown to be increased in mouse lungs and rat alveolar macrophages following exposure to a. fumigatus conidia. positive associations between mmps and a. fumigatus have been reported previously in a study involving conidial infection of corneas, and mmp expression has been shown to be higher in sputum cells of asthmatics relative to controls. microsomal glutathione s-transferase, found to be up-regulated in cells interacting with a. fumigatus conidia, is the prototypical member of the glutathione s-transferase gene family and has a role in protecting cell from oxidative damage. the experiments in this study used submerged monolayer cultures of a human bronchial epithelial cell line, 16hbe14o-. some characteristics and responses of these cells are likely to be different from both primary cell cultures and airway cells in situ in the lung. nevertheless, our approach has the advantage in that it enables us to identify airway cell responses independently of other cell types.  we demonstrate the use of facs to isolate human airway epithelial cells that bind and/or internalize a. fumigatus conidia in co-culture systems for subsequent assessment of the specific transcriptional responses of cells directly interacting with conidia. the ability to identify differentially expressed genes within a single culture using facs has potential uses to further investigate the response of other cell types to specific interactions with pathogens. our ability to better understand the interaction of inhaled a. fumigatus conidia with airway epithelial cells will help provide insights into the mechanisms of several diseases associated with this ubiquitous mould. 
thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line following interaction with a. fumigatus conidia. in allergic and immunocompromised individuals a. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. previous studies have demonstrated that a. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. we used fluorescence-activated cell sorting to separate 16hbe14o- cells having bound and/or internalized a. fumigatus conidia expressing green fluorescent protein from cells without spores. we demonstrate a methodology using facs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as a. fumigatus with human cells derived from individuals with or without underlying disease susceptibility. after fac-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of genes showing differential expression in cells with conidia. in addition, the differentially expressed genes were significantly enriched for gene ontology terms including: chromatin assembly, g-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process; cell cycle phase, mitosis, and intracellular organelle. aspergillus fumigatus is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia, which are easily respirable. total rna was then isolated and the transcriptome of 16hbe14o- cells was evaluated using agilent whole human genome microarrays. immunofluorescent staining and nystatin protection assays demonstrated that 16hbe14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. specifically, these 16hbe14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes.
 aspergillus fumigatus is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia, which are easily respirable. in allergic and immunocompromised individuals a. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. previous studies have demonstrated that a. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line following interaction with a. fumigatus conidia. we used fluorescence-activated cell sorting to separate 16hbe14o- cells having bound and/or internalized a. fumigatus conidia expressing green fluorescent protein from cells without spores. total rna was then isolated and the transcriptome of 16hbe14o- cells was evaluated using agilent whole human genome microarrays.  immunofluorescent staining and nystatin protection assays demonstrated that 16hbe14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. after fac-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of genes showing differential expression in cells with conidia. specifically, these 16hbe14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes. in addition, the differentially expressed genes were significantly enriched for gene ontology terms including: chromatin assembly, g-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process; cell cycle phase, mitosis, and intracellular organelle.  we demonstrate a methodology using facs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as a. fumigatus with human cells derived from individuals with or without underlying disease susceptibility. 