BACKGROUND
macrophages - cells that phagocytose microbes, unhealthy and cancerous cells - are at the heart of human aging and pathology from infectious and noninfectious aetiologies.
as immune sentinels, macrophages exhibit a variety of pro- and anti-inflammatory phenotypes.
at the cellular level these phenotypes are determined not only by genetic lineage but are also highly plastic to changing tissue environments  <cit> .
the interplay between these phenotypic drivers underlies many macrophage-mediated pathologies.
for example, the complex infectious dynamics between hiv- <dig> and macrophages within particular tissue niches not only prevents virus eradication in patients on antiretrovirals  <cit> , but is a likely source of low grade neuroinflammation leading to neurocognitive decline  <cit> .
gene-edited macrophages can be used to study genes with known host-pathogen interactions, but in the absence of genetically tractable blood derived macrophages, macrophages derived from genetically modified pluripotent stem cells  provide a suitable alternative model system.
these stem cell models have the advantage of reproducibly producing large numbers of edited cells under controlled conditions  <cit> .
however, one challenge with stem cell models is the intersection of biological and technical  heterogeneity that needs to be accounted for, making the case for single-cell sequencing.
in addition, with such highly plastic cells it is important to be able to study the context of genetic modifiers, by controlling signaling microenvironments and cell interactions.
it’s this context that is crucially lost with many single-cell sequencing approaches, and so a technical goal would be to be able to include the effects of multiple environmental, signaling and intervention variables on cell population phenotypes and dynamics.
to allow for a genotype-by-environment investigation we cultured over  <dig> crispr-edited macrophages using a novel microfluidic platform that allows time and dose control over each individual cell’s microenvironment  <cit>  .
a mixture of wild-type and samhd <dig> knockout monocytes, were generated from hues- <dig> human embryonic stem cells  <cit> .
these were differentiated into macrophages that resemble blood monocyte derived macrophages, both phenotypically  and transcriptomically  <cit> , while sharing ontogeny with specific tissue-resident macrophages such as microglia of the central nervous system  <cit> .
the need for and importance of such tissue-resident models has recently been reviewed by sattentau and stevenson  <cit> .
as the protein of interest in these cells, samhd <dig> is a poorly understood dntpase that has emerged as a potent hiv- <dig> restriction factor in non-cycling cells  <cit> .
its primary physiological role is believed to be the maintenance of genome integrity by limiting the dntp pool when dna replication is not required, which is in keeping with observed samhd <dig> downregulation in several cancers  <cit> .
of direct relevance to innate immunity is its congenital loss of function associated with aicardi-goutieres syndrome, a neurodegenerative disease linked to dysregulated inflammation  <cit> .
in order to gain better insights into samhd <dig> biology within macrophages, we set out to study the knockout of this gene in our model.
rna sequencing at a single-cell resolution was necessary to, at a minimum, rule out technical contributions from unwanted cell populations that can occur with imperfect stem cell model differentiation.
furthermore, as samhd <dig> has a direct association with inflammation signaling, we sought to do this in a way that would not only study aspects of inflammatory activation but also cell autonomous effects separate from the influences of macrophage paracrine signaling.fig.
<dig> macrophage culture and subtypes.
a a single micro-volume culture chamber from the polaris™ microfluidics chip, containing a macrophage.
the media conditions per chamber can be modified to study microenvironmental perturbations.
b visualization of the major cell differences — such as with this multi-dimensional scaling of the transcriptomic rank correlations — demonstrated the separation of cells cultured for one hour , eight hours , and a reproducible subcluster present across both time points .
c&d formal clustering confirmed this subcluster  in addition to a third subcluster emerging after eight hours.
the inner 50% of cells in each cluster are shown in colour for dimensions one and three to better convey relative cluster positions and densities.
e&f cluster three significantly reduced its proportion in the context of lps and standard media.
95% confidence intervals for change in proportions are shown
per microfluidic chip, up to  <dig> individual macrophages were isolated at random from a mixture of differentially stained wild-type and knockout cells, and cultured in isolation under different inflammatory signaling environments.
these culture environments comprised four possible combinations of exposure or non-exposure to lipopolysaccharide , and standard or conditioned media.
conditioned medium was derived from bulk macrophage cultures, to simulate the intercellular signaling component that is important in the control of macrophage inflammatory activation.
in total this provided eight cell populations per chip , with each chip cultured for either one or eight hours in order to account for early temporal changes in response.
details of the experimental work flow are provided in additional file 1: figure s <dig>  cellular phenotypes such as motility and morphology were tracked by live-cell imaging, before the cdna from cells was harvested for single-cell rna sequencing.
all of the aggregate 16-fold combinations of genotype, environment and time were repeated in at least nine replicate runs to ensure robustness of conclusions.
details of the experimental and imaging methods, including the sequencing quality control are provided in the methods and additional file  <dig>
RESULTS
identifying different macrophage states
exploratory rna-seq data analysis  revealed a clear differentiation between one- and eight-hour cultured cells.
it also highlighted a subgroup of cells present at both time points that enriched for a gene set previously demonstrated to be differentially expressed in macrophages treated with the inflammatory tgfb cytokine .
this provided initial evidence for the potential biological relevance of the observed heterogeneity among the macrophages.
to more formally study this latent/emergent  heterogeneity and the gene expression differences underlying it, we developed a novel hybrid model-driven and non-parametric clustering method constrained to report only cell clusters that were well represented across replicates.
we provide further details in the methods, with all analysis code and results provided in the additional files  <dig> and  <dig> respectively.
our more formal clustering analysis confirmed the existence of the lower-abundance cell state at both time points  but also a reproducible third state  emerging after eight hours.
a search for associations between proportions of the cell states and culture conditions revealed a lower abundance of cluster three when in standard media with lps , suggesting another inflammatory sub-phenotype in the cells.
with these results demonstrating heterogeneity that would otherwise be missed with traditional sequencing, we then asked if any of these cell types resembled the tissue-resident phenotype of interest.
identifying the macrophage phenotype of interest
to better understand these latent cell states, we used our modification on the heskes rank product method  <cit>  to estimate upper and lower p-value bounds on differential and heterogeneous gene expression.
as detailed in the methods, we defined differential expression as a global/overall shift in gene expression, while heterogeneous/context-specific expression was defined as highly variable expression across cell states or culture microenvironments.
we selected this non-parametric gene ranking approach not only for its focus on result reproducibility, but for its speed and ease of data fusion that make it well suited to single-cell analysis.
compared against one-hour cultures, most cells in eight-hour cultures  progressed towards an anti-inflammatory transcriptional signature consistent with the tissue-resident macrophage phenotype of interest.
this is evidenced, for example, by the expression of il1rn, encoding il- <dig> receptor antagonist, which increased 18-fold over time in cluster two .
recombinant il1rn is used to treat severe inflammatory conditions mediated by its ligand, the archetypal pro-inflammatory cytokine il- <dig>  <cit> .
in contrast to other cells, cluster one cells did not move towards a transcriptional profile consistent with a tissue-resident phenotype, and tended to maintain up- or down-regulation of cluster-specific genes.
this pattern included lower expression of gapdh and tpi <dig>  genes involved in glycolysis, a pathway known to vary across macrophage activation phenotypes  <cit> .
compared with the other clusters, cluster one expressed higher levels of foxp <dig> across all culture conditions .
foxp <dig> is a transcription factor involved in maintaining embryonic stem cell pluripotency that must be turned off for complete monocyte differentiation into macrophages  <cit> .
concerned that selective expression of foxp <dig> might represent technical differentiation heterogeneity, we used the imaging data to search for other contributing factors.
while we did not find evidence for cell motility or morphology associations, we noted that cluster one cells were more likely to have come into contact with the culture chamber retention beads used to prevent cells from escaping .
we also noted that cells cultured towards the edges of the chips were more likely to touch retention beads , but that the chip edge positions were enriched for in cluster one, seemingly independent of the bead association .
as cell imaging was performed hourly, this may in part reflect false negatives for detecting bead contact of cells in a less mature and adherent state.
notwithstanding the evidence for both differentiation and environmental factors underlying cluster one, the low correlation between cluster one and off-chip bulk tissue samples  led us to conclude that it may represent a potentially interesting but low-abundance phenotype that is unrelated to our main question of samhd <dig> biology in tissue-resident macrophages.fig.
<dig> cell cluster gene expression.
in each plot, yellow indicates increased and magenta indicates reduced gene expression.
a-b heatmaps of the top  <dig> gene expression results, ranked by statistical significance, are shown for clusters one and two over time  and cluster three versus other cells, broken down by culture condition .
the numbers provided in parentheses in this and other heatmaps are -log <dig> p-values for differential and heterogeneous  expression respectively.
results that are globally significant after 5% false discovery rate  correction are marked with an asterisk.
c the differential expression results for cluster one versus other cells at one and eight hours.
d a cumulative proportion plot for foxp <dig> expression broken down by cell clusters.
as in other plots, clusters one, two and three are plotted in red, blue and green respectively.
each line plots the cumulative proportion of cells at or below a certain expression level.
cluster one demonstrates greater expression, with approximately half of cluster two and three cells having no detectable expression
a comparison of gene expression in cluster three versus other cells  was consistent with a shift towards macrophages with a tissue remodelling phenotype.
this was evidenced, for example, by greater expression of timp <dig>  an inhibitor of extracellular matrix degradation, and chi3l <dig>  a secreted glycoprotein also thought to drive tissue remodelling and a known genetic risk factor for asthma severity  <cit> .
since cluster three’s transcriptional phenotype most closely resembles that of cluster two , it seems plausible that this represents an emerging secondary phenotype that requires macrophage signaling when cells are exposed to lps .
future studies over longer culture periods will help clarify the microenvironmental dependence and persistence of this cluster.
we focus the remainder of our observations on the main body of cells , resembling the desired tissue-resident phenotype.
while no cluster two enrichment for particular motility or morphology imaging features were found over these short culture periods, we briefly note an imaging subtype of cells observed to be phagocytosing the retention beads.
phagocytosing macrophages did not assume a distinct transcriptomic cluster 一 likely due to short culture times 一 nevertheless they did enrich for oxidative stress and mitochondrial genes, examples being prdx <dig> and mt-co <dig> .
this unexpected result suggests an avenue for single-cell studies to explore the temporal dynamics of phagocytosis  <cit> .
changes in macrophage behavior  with samhd <dig> knockout
after filtering our data to focus on the cell subtype of interest , we tested for varying knockout and wild-type differential and heterogeneous expression over time.
the most striking feature of the globally significant knockout effects was that they were predominantly microenvironment specific .
not only does this stress the importance of studying gene-environment interactions in cellular genetics models with known phenotypic plasticity, but in this work allows us to comment on macrophage signaling contributions.
specifically, we note a highly significant association with sod <dig> expression, a gene that encodes the cytosolic isozyme of superoxide dismutase.
sod <dig> is a superoxide radical scavenger that may confer some protection against hiv- <dig> neuropathy as part of the oxidative stress response  <cit> , but which has no described associations with hiv- <dig> restriction factors.
as with samhd <dig> loss of function, gain-of-function mutations in sod <dig> are associated with neuroinflammation and degeneration, clinically manifesting as amyotrophic lateral sclerosis, which is likely a result of toxic protein aggregates  <cit> .
overall, sod <dig> transcript levels in this study were similar in wild-type and samhd <dig> knockout cells.
however, whereas sod <dig> expression in wild-type cells stimulated with lps increased over time as expected, expression in knockout cells increased over time in the absence of lps activation.
not only does this point to a qualitative difference in expression but 一 as can be seen from the barely detectable expression changes in standard media  一 the magnitude of the difference is much greater in conditioned media.
this sod <dig> oxidative stress response thus has both a notable genetic and signaling component, which may be dampened by identifying and blocking the augmenting signaling factor.fig.
<dig> samhd <dig> knockout gene expression  heatmap of the difference between knockout and wild-type expression over time, broken down by culture condition.
block colours and numbers in parentheses share the same meaning as in the fig.
<dig> heatmaps.
b globally significant msigdb signatures correlating with knockout sod <dig> expression.
c the expression of sod <dig> and its top co-expressors after eight hours across all cell clusters.
red, blue and green lines correspond to clusters one, two and three respectively.
widths indicate expression level
further exploring this oxidative stress response, we noticed a change in sod1’s coexpression  in knockouts versus wild-types .
analysing which genes tend to coexpress with sod <dig> allows for biological contextualisation of this effect, as genes are only expressed when required, and so expression correlation implies coregulation.
a key difference here being the increased coexpression resolution offered by single-cell analysis to generate gene networks in both perturbed and unperturbed cells, compared with conventional sequencing that is limited to only studying average gene correlations with perturbation.
we asked whether there are genes with which sod <dig> alters its rank  correlation across cells when samhd <dig> is knocked out.
fig.
3b lists the globally significant signatures from msigdb v <dig>   <cit> , consisting of gene sets more correlated with sod <dig> in knockout versus wild-type cells.
in other words, these highlight genes and pathways coregulating with the altered oxidative stress response in the knockout macrophages.
in addition to the expected enrichment for reactive oxygen species  modulators in this list, we note a striking enrichment for proteasome genes.
the proteosome is known to be activated and dysregulated with sod <dig> mutation, leading to aberrant cellular proteostasis  <cit> .
however, it is plausible in these cells that the altered proteostasis is simply a result of the macrophages’ stress response.
in particular we note an enrichment for proteasome genes involved with the activation of nf-kappab , a pro-inflammatory transcription factor on which multiple macrophage signaling pathways converge  <cit> .
a similar proteostasis theme is observed for the regulation of ornithine decarboxylase, a macrophage anti-inflammatory enzyme  <cit>  that metabolises a well described marker of macrophage pro- versus anti-inflammatory phenotype polarisation.
with these pathway associations validating this approach by supporting known biology of increased inflammation with samhd <dig> knockout - albeit within the context of oxidative stress and altered proteostasis - we asked if other pathways in fig.
3b point to unexpected biological themes.
a striking pathway result was the repeated enrichment for proteasome genes involved in the degradation of cell cycle proteins.
while the direct causal relationship between cell cycle and samhd <dig> is considered to be via its cyclin-dependent kinase phosphorylation and inhibition  <cit> , these results point to an additional reverse relationship.
a tumor suppressor role for samhd <dig> has been proposed based on its maintenance of genome integrity and cancer associations with downregulation  <cit> .
however, the pathways in fig.
3b counter-intuitively point to p <dig> dependent and independent g <dig> arrest as part of the dna damage response.
these results highlight a necessary fine balance in samhd <dig> activity in terms of cell cycle control, with too little samhd <dig> arresting cell cycle progression via other tumour suppressors in response to dna damage.
this relationship between samhd <dig> and g <dig> arrest has been demonstrated in dividing fibroblasts that turn senescent  <cit> , though an important difference here is that it suggests the wild-type g <dig>  macrophages shift to a g <dig> block with samhd <dig> knockout.
finally, and perhaps most interesting in terms of the hiv- <dig> host-pathogen relationship, is the highly significant pathway enrichment for the degradation of apobec3g as part of the altered stress response in samhd <dig> knockouts.
this hiv- <dig> restriction factor causes proviral dna hypermutation via cytidine deamination, and so shares no known overlap in antiviral activity with samhd <dig>  <cit> .
discussion and
CONCLUSIONS
as single-cell methods continue to mature, so will the ability to move beyond simple ‘snapshots’ of cell populations towards studying the determinants of population dynamics.
we expect that one area of demand for this type of single-cell functional genomics will be cellular genetics models, with large-scale efforts already underway to generate stem cell banks to support tissue-specific insights into genetic variants  <cit> .
even with the simplest such models, single-cell sequencing will prove useful to rule out heterogeneity unrelated to the cell phenotypes of interest.
in our macrophage model, for example, we have seen that while the majority of cells adopted a tissue-resident phenotype, cells that behave quite differently can be present.
as the co-culture and genetic complexity of cellular genetic models increases, so will the demand for methods to confidently map heterogeneity with high replicability across multiple culture or laboratory conditions.
automated and standardized microfluidics present a decided advantage in this regard.
in this proof-of-principle work we have been able to generate complex experimental designs within chips over multiple iterations.
as all the conditions studied were replicated at least nine times, we have been able to develop and implement statistical models that focus on highly replicated cell behaviors that could easily be integrated with results from other laboratories.
perhaps the most distinct advantage of such lab-on-chip microfluidics is the ability to perform imaging, temporal and microenvironmental analyses of cell population dynamics.
in this study these variables allowed us, for example, to comment on the altered oxidative stress response  with samhd <dig> knockout and macrophage signaling.
by blocking the signaling component we observed a significant reduction in this knockout effect.
interestingly, sod <dig> and samhd <dig> have known respective gain and loss of function associations with neuroinflammation that are in keeping with effects noticed in this model.
we speculate that targeting the observed signaling component may provide an avenue for the treatment of these and other neurodegenerative diseases influenced by innate immunity.
microglia , for example, are not only implicated in hiv associated neurocognitive disease , but also in alzheimer’s disease via aberrant inflammatory signalling  <cit> .
future work with the polaris™, assaying single-cell macrophage supernatants, may prove useful in narrowing down this as yet unidentified signaling factor.
in this study, single-cell sod <dig> coexpression pathway analysis enriched for altered proteostasis.
the most widely observed biological association with this altered proteostasis was a cell cycle  block as part of the dna damage response.
while an increased dntp pool with samhd <dig> loss of function is known to reduce genome integrity, the sod <dig> association with these pathways point to an oxidative stress contribution to dna breaks triggering g <dig> checkpoint genes.
sod <dig> may also directly contribute to this arrest via its anti-apoptotic signaling  <cit> .
perhaps most intriguing is how these associations could provide insights into activating latent viruses within therapeutically intractable reservoirs such as macrophages and resting cd4+ t cells, where samhd <dig> is highly expressed  <cit> .
triggering apoptosis signaling has been proposed as one therapeutic strategy for hiv- <dig> activation  <cit> , which would require sod <dig> apoptosis inhibition to be minimised.
our knockout cells strongly suppressed sod <dig> expression when activated in conditioned media, suggesting that promoting macrophage activation in combination with samhd <dig> inhibition would, at least in vitro, be the most effective strategy to purge latent viruses.
infection of the macrophages with hiv- <dig> in these microfluidic chips to directly study these effects is one promising approach to study this.
such hiv- <dig> infection studies with other macrophage knockouts such as the hiv- <dig> restriction factor apobec3g, may prove particularly enlightening, as results from this study suggest a previously unappreciated connection between samhd <dig> loss of function and apobec3g degradation.
understanding the conditions under which samhd <dig> inhibition also results in reduced apobec3g levels would be of direct relevance to therapies aimed at viral activation.
under these situations, samhd <dig> inhibition might have the desired activation response, but reduced apobec3g would enhance the ability of the activated viruses to reinfect other cells.

 macrophages - cells that phagocytose microbes, unhealthy and cancerous cells - are at the heart of human aging and pathology from infectious and noninfectious aetiologies. as immune sentinels, macrophages exhibit a variety of pro- and anti-inflammatory phenotypes. at the cellular level these phenotypes are determined not only by genetic lineage but are also highly plastic to changing tissue environments. the interplay between these phenotypic drivers underlies many macrophage-mediated pathologies. for example, the complex infectious dynamics between hiv- and macrophages within particular tissue niches not only prevents virus eradication in patients on antiretrovirals, but is a likely source of low grade neuroinflammation leading to neurocognitive decline. gene-edited macrophages can be used to study genes with known host-pathogen interactions, but in the absence of genetically tractable blood derived macrophages, macrophages derived from genetically modified pluripotent stem cells provide a suitable alternative model system. these stem cell models have the advantage of reproducibly producing large numbers of edited cells under controlled conditions. however, one challenge with stem cell models is the intersection of biological and technical heterogeneity that needs to be accounted for, making the case for single-cell sequencing. in addition, with such highly plastic cells it is important to be able to study the context of genetic modifiers, by controlling signaling microenvironments and cell interactions. it’s this context that is crucially lost with many single-cell sequencing approaches, and so a technical goal would be to be able to include the effects of multiple environmental, signaling and intervention variables on cell population phenotypes and dynamics. to allow for a genotype-by-environment investigation we cultured over crispr-edited macrophages using a novel microfluidic platform that allows time and dose control over each individual cell’s microenvironment. a mixture of wild-type and samhd knockout monocytes, were generated from hues- human embryonic stem cells. these were differentiated into macrophages that resemble blood monocyte derived macrophages, both phenotypically and transcriptomically, while sharing ontogeny with specific tissue-resident macrophages such as microglia of the central nervous system. the need for and importance of such tissue-resident models has recently been reviewed by sattentau and stevenson. as the protein of interest in these cells, samhd is a poorly understood dntpase that has emerged as a potent hiv- restriction factor in non-cycling cells. its primary physiological role is believed to be the maintenance of genome integrity by limiting the dntp pool when dna replication is not required, which is in keeping with observed samhd downregulation in several cancers. of direct relevance to innate immunity is its congenital loss of function associated with aicardi-goutieres syndrome, a neurodegenerative disease linked to dysregulated inflammation. in order to gain better insights into samhd biology within macrophages, we set out to study the knockout of this gene in our model. rna sequencing at a single-cell resolution was necessary to, at a minimum, rule out technical contributions from unwanted cell populations that can occur with imperfect stem cell model differentiation. furthermore, as samhd has a direct association with inflammation signaling, we sought to do this in a way that would not only study aspects of inflammatory activation but also cell autonomous effects separate from the influences of macrophage paracrine signaling.fig. macrophage culture and subtypes. a a single micro-volume culture chamber from the polaris™ microfluidics chip, containing a macrophage. the media conditions per chamber can be modified to study microenvironmental perturbations. b visualization of the major cell differences — such as with this multi-dimensional scaling of the transcriptomic rank correlations — demonstrated the separation of cells cultured for one hour, eight hours, and a reproducible subcluster present across both time points. c&d formal clustering confirmed this subcluster in addition to a third subcluster emerging after eight hours. the inner 50% of cells in each cluster are shown in colour for dimensions one and three to better convey relative cluster positions and densities. e&f cluster three significantly reduced its proportion in the context of lps and standard media. 95% confidence intervals for change in proportions are shown per microfluidic chip, up to individual macrophages were isolated at random from a mixture of differentially stained wild-type and knockout cells, and cultured in isolation under different inflammatory signaling environments. these culture environments comprised four possible combinations of exposure or non-exposure to lipopolysaccharide, and standard or conditioned media. conditioned medium was derived from bulk macrophage cultures, to simulate the intercellular signaling component that is important in the control of macrophage inflammatory activation. in total this provided eight cell populations per chip, with each chip cultured for either one or eight hours in order to account for early temporal changes in response. details of the experimental work flow are provided in additional file 1: figure s cellular phenotypes such as motility and morphology were tracked by live-cell imaging, before the cdna from cells was harvested for single-cell rna sequencing. all of the aggregate 16-fold combinations of genotype, environment and time were repeated in at least nine replicate runs to ensure robustness of conclusions. details of the experimental and imaging methods, including the sequencing quality control are provided in the methods and additional file  identifying different macrophage states exploratory rna-seq data analysis revealed a clear differentiation between one- and eight-hour cultured cells. it also highlighted a subgroup of cells present at both time points that enriched for a gene set previously demonstrated to be differentially expressed in macrophages treated with the inflammatory tgfb cytokine. this provided initial evidence for the potential biological relevance of the observed heterogeneity among the macrophages. to more formally study this latent/emergent heterogeneity and the gene expression differences underlying it, we developed a novel hybrid model-driven and non-parametric clustering method constrained to report only cell clusters that were well represented across replicates. we provide further details in the methods, with all analysis code and results provided in the additional files and respectively. our more formal clustering analysis confirmed the existence of the lower-abundance cell state at both time points but also a reproducible third state emerging after eight hours. a search for associations between proportions of the cell states and culture conditions revealed a lower abundance of cluster three when in standard media with lps, suggesting another inflammatory sub-phenotype in the cells. with these results demonstrating heterogeneity that would otherwise be missed with traditional sequencing, we then asked if any of these cell types resembled the tissue-resident phenotype of interest. identifying the macrophage phenotype of interest to better understand these latent cell states, we used our modification on the heskes rank product method to estimate upper and lower p-value bounds on differential and heterogeneous gene expression. as detailed in the methods, we defined differential expression as a global/overall shift in gene expression, while heterogeneous/context-specific expression was defined as highly variable expression across cell states or culture microenvironments. we selected this non-parametric gene ranking approach not only for its focus on result reproducibility, but for its speed and ease of data fusion that make it well suited to single-cell analysis. compared against one-hour cultures, most cells in eight-hour cultures progressed towards an anti-inflammatory transcriptional signature consistent with the tissue-resident macrophage phenotype of interest. this is evidenced, for example, by the expression of il1rn, encoding il- receptor antagonist, which increased 18-fold over time in cluster two. recombinant il1rn is used to treat severe inflammatory conditions mediated by its ligand, the archetypal pro-inflammatory cytokine il-. in contrast to other cells, cluster one cells did not move towards a transcriptional profile consistent with a tissue-resident phenotype, and tended to maintain up- or down-regulation of cluster-specific genes. this pattern included lower expression of gapdh and tpi genes involved in glycolysis, a pathway known to vary across macrophage activation phenotypes. compared with the other clusters, cluster one expressed higher levels of foxp across all culture conditions. foxp is a transcription factor involved in maintaining embryonic stem cell pluripotency that must be turned off for complete monocyte differentiation into macrophages. concerned that selective expression of foxp might represent technical differentiation heterogeneity, we used the imaging data to search for other contributing factors. while we did not find evidence for cell motility or morphology associations, we noted that cluster one cells were more likely to have come into contact with the culture chamber retention beads used to prevent cells from escaping. we also noted that cells cultured towards the edges of the chips were more likely to touch retention beads, but that the chip edge positions were enriched for in cluster one, seemingly independent of the bead association. as cell imaging was performed hourly, this may in part reflect false negatives for detecting bead contact of cells in a less mature and adherent state. notwithstanding the evidence for both differentiation and environmental factors underlying cluster one, the low correlation between cluster one and off-chip bulk tissue samples led us to conclude that it may represent a potentially interesting but low-abundance phenotype that is unrelated to our main question of samhd biology in tissue-resident macrophages.fig. cell cluster gene expression. in each plot, yellow indicates increased and magenta indicates reduced gene expression. a-b heatmaps of the top gene expression results, ranked by statistical significance, are shown for clusters one and two over time and cluster three versus other cells, broken down by culture condition. the numbers provided in parentheses in this and other heatmaps are -log p-values for differential and heterogeneous expression respectively. results that are globally significant after 5% false discovery rate correction are marked with an asterisk. c the differential expression results for cluster one versus other cells at one and eight hours. d a cumulative proportion plot for foxp expression broken down by cell clusters. as in other plots, clusters one, two and three are plotted in red, blue and green respectively. each line plots the cumulative proportion of cells at or below a certain expression level. cluster one demonstrates greater expression, with approximately half of cluster two and three cells having no detectable expression a comparison of gene expression in cluster three versus other cells was consistent with a shift towards macrophages with a tissue remodelling phenotype. this was evidenced, for example, by greater expression of timp an inhibitor of extracellular matrix degradation, and chi3l a secreted glycoprotein also thought to drive tissue remodelling and a known genetic risk factor for asthma severity. since cluster three’s transcriptional phenotype most closely resembles that of cluster two, it seems plausible that this represents an emerging secondary phenotype that requires macrophage signaling when cells are exposed to lps. future studies over longer culture periods will help clarify the microenvironmental dependence and persistence of this cluster. we focus the remainder of our observations on the main body of cells, resembling the desired tissue-resident phenotype. while no cluster two enrichment for particular motility or morphology imaging features were found over these short culture periods, we briefly note an imaging subtype of cells observed to be phagocytosing the retention beads. phagocytosing macrophages did not assume a distinct transcriptomic cluster 一 likely due to short culture times 一 nevertheless they did enrich for oxidative stress and mitochondrial genes, examples being prdx and mt-co. this unexpected result suggests an avenue for single-cell studies to explore the temporal dynamics of phagocytosis. changes in macrophage behavior with samhd knockout after filtering our data to focus on the cell subtype of interest, we tested for varying knockout and wild-type differential and heterogeneous expression over time. the most striking feature of the globally significant knockout effects was that they were predominantly microenvironment specific. not only does this stress the importance of studying gene-environment interactions in cellular genetics models with known phenotypic plasticity, but in this work allows us to comment on macrophage signaling contributions. specifically, we note a highly significant association with sod expression, a gene that encodes the cytosolic isozyme of superoxide dismutase. sod is a superoxide radical scavenger that may confer some protection against hiv- neuropathy as part of the oxidative stress response, but which has no described associations with hiv- restriction factors. as with samhd loss of function, gain-of-function mutations in sod are associated with neuroinflammation and degeneration, clinically manifesting as amyotrophic lateral sclerosis, which is likely a result of toxic protein aggregates. overall, sod transcript levels in this study were similar in wild-type and samhd knockout cells. however, whereas sod expression in wild-type cells stimulated with lps increased over time as expected, expression in knockout cells increased over time in the absence of lps activation. not only does this point to a qualitative difference in expression but 一 as can be seen from the barely detectable expression changes in standard media 一 the magnitude of the difference is much greater in conditioned media. this sod oxidative stress response thus has both a notable genetic and signaling component, which may be dampened by identifying and blocking the augmenting signaling factor.fig. samhd knockout gene expression heatmap of the difference between knockout and wild-type expression over time, broken down by culture condition. block colours and numbers in parentheses share the same meaning as in the fig.  heatmaps. b globally significant msigdb signatures correlating with knockout sod expression. c the expression of sod and its top co-expressors after eight hours across all cell clusters. red, blue and green lines correspond to clusters one, two and three respectively. widths indicate expression level further exploring this oxidative stress response, we noticed a change in sod1’s coexpression in knockouts versus wild-types. analysing which genes tend to coexpress with sod allows for biological contextualisation of this effect, as genes are only expressed when required, and so expression correlation implies coregulation. a key difference here being the increased coexpression resolution offered by single-cell analysis to generate gene networks in both perturbed and unperturbed cells, compared with conventional sequencing that is limited to only studying average gene correlations with perturbation. we asked whether there are genes with which sod alters its rank correlation across cells when samhd is knocked out. fig. 3b lists the globally significant signatures from msigdb v, consisting of gene sets more correlated with sod in knockout versus wild-type cells. in other words, these highlight genes and pathways coregulating with the altered oxidative stress response in the knockout macrophages. in addition to the expected enrichment for reactive oxygen species modulators in this list, we note a striking enrichment for proteasome genes. the proteosome is known to be activated and dysregulated with sod mutation, leading to aberrant cellular proteostasis. however, it is plausible in these cells that the altered proteostasis is simply a result of the macrophages’ stress response. in particular we note an enrichment for proteasome genes involved with the activation of nf-kappab, a pro-inflammatory transcription factor on which multiple macrophage signaling pathways converge. a similar proteostasis theme is observed for the regulation of ornithine decarboxylase, a macrophage anti-inflammatory enzyme that metabolises a well described marker of macrophage pro- versus anti-inflammatory phenotype polarisation. with these pathway associations validating this approach by supporting known biology of increased inflammation with samhd knockout - albeit within the context of oxidative stress and altered proteostasis - we asked if other pathways in fig. 3b point to unexpected biological themes. a striking pathway result was the repeated enrichment for proteasome genes involved in the degradation of cell cycle proteins. while the direct causal relationship between cell cycle and samhd is considered to be via its cyclin-dependent kinase phosphorylation and inhibition, these results point to an additional reverse relationship. a tumor suppressor role for samhd has been proposed based on its maintenance of genome integrity and cancer associations with downregulation. however, the pathways in fig. 3b counter-intuitively point to p dependent and independent g arrest as part of the dna damage response. these results highlight a necessary fine balance in samhd activity in terms of cell cycle control, with too little samhd arresting cell cycle progression via other tumour suppressors in response to dna damage. this relationship between samhd and g arrest has been demonstrated in dividing fibroblasts that turn senescent, though an important difference here is that it suggests the wild-type g macrophages shift to a g block with samhd knockout. finally, and perhaps most interesting in terms of the hiv- host-pathogen relationship, is the highly significant pathway enrichment for the degradation of apobec3g as part of the altered stress response in samhd knockouts. this hiv- restriction factor causes proviral dna hypermutation via cytidine deamination, and so shares no known overlap in antiviral activity with samhd. discussion and  as single-cell methods continue to mature, so will the ability to move beyond simple ‘snapshots’ of cell populations towards studying the determinants of population dynamics. we expect that one area of demand for this type of single-cell functional genomics will be cellular genetics models, with large-scale efforts already underway to generate stem cell banks to support tissue-specific insights into genetic variants. even with the simplest such models, single-cell sequencing will prove useful to rule out heterogeneity unrelated to the cell phenotypes of interest. in our macrophage model, for example, we have seen that while the majority of cells adopted a tissue-resident phenotype, cells that behave quite differently can be present. as the co-culture and genetic complexity of cellular genetic models increases, so will the demand for methods to confidently map heterogeneity with high replicability across multiple culture or laboratory conditions. automated and standardized microfluidics present a decided advantage in this regard. in this proof-of-principle work we have been able to generate complex experimental designs within chips over multiple iterations. as all the conditions studied were replicated at least nine times, we have been able to develop and implement statistical models that focus on highly replicated cell behaviors that could easily be integrated with results from other laboratories. perhaps the most distinct advantage of such lab-on-chip microfluidics is the ability to perform imaging, temporal and microenvironmental analyses of cell population dynamics. in this study these variables allowed us, for example, to comment on the altered oxidative stress response with samhd knockout and macrophage signaling. by blocking the signaling component we observed a significant reduction in this knockout effect. interestingly, sod and samhd have known respective gain and loss of function associations with neuroinflammation that are in keeping with effects noticed in this model. we speculate that targeting the observed signaling component may provide an avenue for the treatment of these and other neurodegenerative diseases influenced by innate immunity. microglia, for example, are not only implicated in hiv associated neurocognitive disease, but also in alzheimer’s disease via aberrant inflammatory signalling. future work with the polaris™, assaying single-cell macrophage supernatants, may prove useful in narrowing down this as yet unidentified signaling factor. in this study, single-cell sod coexpression pathway analysis enriched for altered proteostasis. the most widely observed biological association with this altered proteostasis was a cell cycle block as part of the dna damage response. while an increased dntp pool with samhd loss of function is known to reduce genome integrity, the sod association with these pathways point to an oxidative stress contribution to dna breaks triggering g checkpoint genes. sod may also directly contribute to this arrest via its anti-apoptotic signaling. perhaps most intriguing is how these associations could provide insights into activating latent viruses within therapeutically intractable reservoirs such as macrophages and resting cd4+ t cells, where samhd is highly expressed. triggering apoptosis signaling has been proposed as one therapeutic strategy for hiv- activation, which would require sod apoptosis inhibition to be minimised. our knockout cells strongly suppressed sod expression when activated in conditioned media, suggesting that promoting macrophage activation in combination with samhd inhibition would, at least in vitro, be the most effective strategy to purge latent viruses. infection of the macrophages with hiv- in these microfluidic chips to directly study these effects is one promising approach to study this. such hiv- infection studies with other macrophage knockouts such as the hiv- restriction factor apobec3g, may prove particularly enlightening, as results from this study suggest a previously unappreciated connection between samhd loss of function and apobec3g degradation. understanding the conditions under which samhd inhibition also results in reduced apobec3g levels would be of direct relevance to therapies aimed at viral activation. under these situations, samhd inhibition might have the desired activation response, but reduced apobec3g would enhance the ability of the activated viruses to reinfect other cells. 
using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of samhd such as an altered oxidative stress response, have a large paracrine signaling component. furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and apobec3g degradation, both associated with the oxidative stress response and altered proteostasis. by combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. studying the genetic and environmental modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. as single-cell methods continue to mature, so will the ability to move beyond simple ‘snapshots’ of cell populations towards studying the determinants of population dynamics. perhaps the most important shortfall with transcriptomic ‘snapshots’ of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context.
 single-cell rna-seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome’s limitations in describing higher functional phenotypes and protein events. perhaps the most important shortfall with transcriptomic ‘snapshots’ of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. studying the genetic and environmental modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages.  we introduce the programmable polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of samhd such as an altered oxidative stress response, have a large paracrine signaling component. furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and apobec3g degradation, both associated with the oxidative stress response and altered proteostasis. interestingly, samhd and apobec3g are both hiv- inhibitors, with no known co-regulation.  as single-cell methods continue to mature, so will the ability to move beyond simple ‘snapshots’ of cell populations towards studying the determinants of population dynamics. by combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. it’s these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant hiv reservoirs. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords single-cell sequencingsingle-cell culturesingle-cell imagingmacrophage heterogeneitysignaling microenvironmentissue-copyright-statement© the author 2017 