document: intracellular pathogens have evolved over many millions of years to coexist in the face of a wide array of host defense mechanisms . in  vitro studies of the host - pathogen interaction 
   exclude the inflammatory milieu , but a major challenge in studying the host response in  vivo is dissecting pathogen - specific responses from the general inflammatory or common host response ( jenner and young , 2005 ) that occurs as a result of tissue insult during the infectious process . 
   this latter response is in part driven by activation of components of the complement cascade ( ricklin et  al . , 2010 ) , the coagulation pathway ( oikonomopoulou et  al . , 2012 ) , and the acute phase response ( suffredini et  al . , 1999 ) , which 
   all come into play within minutes of infection , and is compounded by rapid changes to the inflammatory cytokine and chemokine environment ( oghumu et  al . 
   , 2010 ; sadik and luster , 2012 ; soehnlein and lindbom , 2010 ) . 
   each of these stimuli may have profound effects on phagocyte function ( galvan et  al . 
   , 2012 ; hoffmann et  al . , 2010 ) and reflect , in the broader sense , the normal and/or essential response of phagocytes to inflammatory insult . 
   hence , responses that directly reflect intracellular infection are difficult to distinguish from dominant inflammatory signatures by conventional experimental approaches . 
   we hypothesized that it might be possible to use the transcriptomic response of uninfected cells exposed to inflammation as a measure of the baseline or normal in  vivo response to insult . following this argument 
   , transcriptomic networks specifically observed in infected cells , but not in neighboring uninfected cells , would represent cell - intrinsic pathogen - induced changes . 
   in contrast , transcriptomic networks restricted to uninfected cells would serve to identify responses potentially beneficial to the host , the lack of which ( in infected cells ) might favor pathogen survival . 
   we tested the above hypothesis in a tractable experimental model of leishmania infection , a pathogen associated with a significant global health burden ( alvar et  al . , 2012 ) . 
   leishmania infect a variety of myeloid cells ( including tissue - resident macrophages , inflammatory monocytes , dendritic cells [ dcs ] , and neutrophils ) , but in models of experimental visceral leishmaniasis , leishmania donovani amastigotes have a predilection for resident tissue macrophages , including kupffer cells ( kcs ) , the resident tissue macrophage lining the hepatic sinusoids ( beattie et  al . , 2010 ; murray , 2001 ) . in this study 
   , we discovered that expression of an rxra - centered transcriptomic pathway , which is downregulated in kc in response to inflammation , is aberrantly maintained in infected cells . 
   pharmacological manipulation of rxr activity perturbed the transcriptomic network of infected kcs , leading to enhanced leishmanicidal activity . 
   this study demonstrates that aberrant expression of the rxra - centered transcriptional pathway in leishmania - infected kcs favors parasite survival and validates the use of cells exposed to inflammation as a means to discover pathogen - regulated transcriptomic pathways in  vivo . 
   in the livers of uninfected ( lysm - cre  mt / mg)f1 mice , kcs were obvious from their large size , expression of membrane gfp , and their continued and intimate contact with liver sinusoidal endothelial cells , as has been previously described ( lee et  al . 
   four - dimensional ( 4d ) intravital two - photon imaging further allowed the fine membrane structure and lamellipodia dynamics of steady - state kcs to be observed in exceptional detail ( figure  1a ; movie s1 ) . the continued extension and retraction of kc lamellipodia manifested itself as small fluctuations of cellular volume ( figure  1b ) and surface area over time ( figure  1c ) . 
   in contrast , monocytes and/or neutrophils were identified as moving rapidly through the sinusoidal spaces , whereas liver sinusoidal endothelial cells ( lsecs ) lined the sinusoidal lumen ( figures 1f and 1 g ) . 
   shape factor , a quantitative measure of how close a cell is to a perfect sphere ( shape factor  =  1 ) , showed small fluctuations over time for kcs ( figure  1d ) but was always lower than that for either lsecs or monocytes and/or neutrophils ( figure  1h ) . 
   the effects of kc membrane fluctuation can also be represented by a velocity , which was calculated by measuring the movement around the centroid point of the cell in three dimensions . 
   thus , although kcs do not actively migrate along the sinusoids , velocity as measured here provides an indication of the extent of membrane activity . 
   in contrast , nonmotile lsecs have an even lower velocity ( limited membrane activity ) . 
   many of the monocytes and/or neutrophils that were examined actively migrated along the sinusoids within the time frame of observation , and their velocity was not surprisingly high ( figure  1i ) 
   . as the early response of kcs to infection with l.  donovani amastigotes in  vivo is largely uncharacterized , we examined kc behavior before and after infection in live mice . 
   we injected ( lysm - cre  mt / mg)f1 mice with transgenic td - tomato - expressing  l.  donovani amastigotes ( beattie et  al . , 2010 ) at a dose that resulted in approximately 20% of kcs becoming infected over a 2  hr time period ( a time by which most parasites have been cleared from the blood by kcs and other tissue phagocytes ; figures 2a and 2b ; gorak et  al . , 1998 ) . 
   the binding of amastigotes to the kc membrane ( figure  2a ) was followed by complete phagocytosis within 20  s ( movie s2 ) . 
   neither injection of live , nor of heat - killed , amastigotes resulted in detectable changes in kc volume ( figure  2c ) or surface area ( figure  2d ) , irrespective of whether or not the kcs contained visible amastigotes . 
   in contrast , injection of live , but not dead , amastigotes induced a rapid increase in the average shape factor that was significant for kcs that contained live parasites and showed a trend in this direction for kcs that did not contain amastigotes ( figure  2e ) . when velocity was measured as an indicator of membrane activity ( figure  2f ) 
   , a significant decrease was observed in both infected and uninfected kcs following injection of live amastigotes , suggesting that both populations of cells were responding to the inoculated parasites through rapid activation . 
   such activation was not observed following injection of dead amastigotes ( figure  2f ) . 
   collectively , these data were indicative of a cellular response to active infection , rather than a response merely to nonspecific debris that may be present within the amastigote preparations . 
   the observation that uninfected kcs in the immediate vicinity of infected cells also responded to the infection by loss of membrane activity ( figure  2f ) suggested that the above changes in kc behavior were the result of either signals provided from infected kcs and operating rapidly in trans or from the activation of serum - derived mediators of inflammation , rather than being a direct consequence of intracellular parasitism per se . in either case , the capacity to activate this response was absent from heat - killed amastigote preparations . as we wished to examine the transcriptional events that accompanied the observed change in kc behavior , we developed a method to identify and isolate kcs based on whether they contained parasites ( herein termed infected ) or not ( herein termed inflamed ) . 
   flow cytometry analysis of hepatic mononuclear cell preparations with gating on forward and side scatter ( figure  3a ) , complement receptor of the immunologlobulin superfamily ( crig ) ( helmy et  al . , 2006 ) , and gr-1 ( figure  3b ) demonstrated that both infected and inflamed populations of kcs could be observed and discriminated by the presence of fluorescent amastigotes ( figure  3c ) . both infected and inflamed kcs showed heterogeneous expression of cd11b but relatively uniform expression of f4/80 ( figure  3d ) . 
   backgating on the infected and inflamed kcs showed that both populations contained both crig gr-1 cells and crig gr-1 cells ( figure  3e ) . to confirm that a proportion of kcs express gr-1 in  situ , we labeled liver sections with f4/80 , crig , and gr-1 ( figures 3f3i ) and observed variable expression of gr-1 on kcs , in addition to gr-1 neutrophils that did not express crig or f4/80 and were readily identifiable from their irregular - shaped nuclei ( figures 3h and 3i ) . 
   we then used fluorescence - activated high - speed cell sorting to isolate kcs and separate them into either infected or uninfected cell populations . 
   we identified kcs from infected mice at 2  hr and 12  hr postinfection ( p.i . ) , as described above , based on forward and side scatter ( size and granularity ) ( figure  3j ) and expression of crig , gr-1 ( figure  3k ) , and f4/80 ( figure  3l ) and then further sorted these based on whether or not they contained parasites ( figures 3m3o ) 
   . cytospins of the sorted populations demonstrated that the cells had macrophage - like morphology , that neutrophils were not included in the analysis , and the presence of amastigotes within the infected population ( figure  3q ) , but not in the inflamed population ( figure  3p ) . 
   kcs were also isolated from sham - infected mice to serve as a control population that exhibited steady - state activity . to determine whether there were underlying transcriptional events that differed between kcs infected with amastigotes in an inflammatory setting and those induced in kcs exposed to inflammatory stimuli alone , transcriptomic profiles for each population , in addition to kcs from sham - infected controls , were generated by microarray analysis . instead of performing a direct comparison of the differences in gene expression between the infected and inflamed kcs 
   , we generated a list of genes differentially expressed between infected kcs and sham - infected kcs ( using a cutoff of > 3-fold difference ) . 
   this approach eliminated the possibility that any of the regulated genes were induced or suppressed due to the isolation procedure , as the sham - infected kcs were subjected to the same digestion and sort process . 
   the same analysis was then performed for the inflamed kcs . as a result of this analysis 
   , we therefore generated two gene lists ( infected and inflamed kcs ) for 2  hr p.i . and two gene lists for 12  hr p.i . 
   at 2  hr p.i . , the expression of 1,188 genes ( 909 up and 279 down ) was differentially regulated between sham - treated and infected kcs , whereas only 353 genes ( 327 up and 26 down ) were differentially regulated between sham - treated and inflamed kcs . by 12  hr p.i . 
   ,  the number of genes showing changes in expression was substantially increased ( 2,135 between sham - treated and infected kcs , of which 1,337 had increased , and 798 had decreased , expression ) . string analysis of these genes showed that although there were some interactions between these genes , there were a large number of genes that were outside of the central network ( figure  s1 ) . 
   although substantially fewer genes ( 1,288 ) were differentially expressed at a messenger rna ( mrna ) level between the sham - treated and inflamed kcs overall ( 713 upregulated and 575 downregulated ) , string analysis for these genes showed a higher degree of connectivity between the genes within this list and a substantial network connection that did not appear to be present in the infected cells ( figure  s1 ) . 
   the top 20 genes ( based on fold change compared to sham - infected kcs for each comparison , or according to relative expression ) are provided in table s1 . 
   we interpret these data as indicating that there are genes that are normally regulated as part of the inflammatory response that are not regulated in infected cells . 
   to determine the identity of genes and gene pathways that were common between the infected and inflamed cells , and those that were unique to either population , we performed a subtractive analysis of the gene lists ( figure  4a ) . 
   from this analysis , we could readily identify genes that were either commonly regulated in both infected and inflamed cells or uniquely regulated in either infected or inflamed kcs at both 2  hr and 12  hr p.i . 
   for all genes identified as being selectively or commonly expressed in infected kcs and inflamed kcs , we have provided the top 20 genes ranked by expression level ( table s2 ) . 
   the gene lists were then subjected to pathway and gene ontology ( go ) analysis ( see experimental procedures for detail ) . 
   a total of 1,088 genes were uniquely regulated in their expression at 12  hr p.i . 
   genes that showed differentially regulated expression in both infected and inflamed cells were enriched for go terms suggestive of responsiveness to inflammation , including host defense and inflammatory response and cytokine - cytokine receptor interaction and chemokine activity . 
   significantly , we identified 241 genes that were differentially regulated in their expression in inflamed cells , but not in cells that contained intracellular amastigotes , in spite of exposure of these cells to the same inflammatory insult 
   . ingenuity pathway analysis ( ipa ) was then used to identify genes that fell within ipa - generated pathways at a higher frequency than would normally be expected to occur for a randomly selected set of genes . 
   this analysis identified 8 interacting networks within the inflamed gene list , in which genes involved in cell - to - cell signaling and the inflammatory response ( ipa network score of 38 ) , cell death ( ipa network score of 37 ) , antigen presentation ( ipa network score of 35 ) , cancer ( ipa network score of 26 ) , immune cell trafficking and cell cycle ( ipa network score of 23 ) , and cell morphology and cellular compromise ( ipa network score of 10 ) were all overrepresented . 
   the network that showed the highest level of gene enrichment was that of the lipid metabolism and small - molecule biochemistry pathway ( figure  4c ) , which had an ipa network score of 40 . the fold change and the expression values for this pathway 
   are shown in table 1 . as the above networks were not regulated in infected cells 
   , we hypothesized that they might represent genes and gene pathways for which homeostasis was favorable to parasite survival and , inter alia , that they represented genes potentially associated with antimicrobial defense regulated by inflammation . to test this hypothesis , we selected the most substantially represented network for further proof - of - concept studies ( figure  4c ) . 
   ipa identified that , 11% ( 27/241 genes ) of genes selectively regulated in their expression in inflamed kcs were found within the lipid metabolism and small - molecule biochemistry pathway ( figure  4c ) , which had retinoid x receptor  ( rxra ) as a key hub ( with rxra itself being downregulated 3.5-fold in inflamed cells ) . 
   we then sought to confirm the level of mrna accumulation in infected and inflamed kcs in independent samples by quantitative real - time pcr ( figure  5 ) . 
   although rxra mrna expression was not significantly different in infected and inflamed cells ( figure  5b ) , other genes within this network were validated as being differentially expressed between infected and inflamed cells , including cebpa ( figure  5c ) , vamp4 ( figure  5d ) , lxr/nr1h3 ( figure  5f ) , and lxr/nr1h2 ( figure  5 g ) . as infection with leishmania resulted in maintenance or upregulation of expression of genes within the rxra - associated gene network 
   , we reasoned that if this was associated with parasite survival , then forced down modulation of rxr activity might sufficiently perturb the entire rxra - related gene network within infected cells to generate an enhanced antimicrobial effect . 
   we therefore tested this approach by targeted pharmacological intervention , using a small - molecule antagonist hx531 , specific for rxr ( ebisawa et  al . 
   , 1999 ; yamauchi et  al . , 2001 ) . to first establish that hx531 treatment had a direct effect on the gene network in infected kcs , we treated mice with hx531 or saline , infected the mice with l.donovani , and isolated infected or inflamed kcs to examine gene expression by quantitative real - time pcr ( figure  5a ) . 
   treatment had no significant effect on the expression of rxra at an mrna level ( figure  5b ) , but treatment resulted in a significant modulation of the expression of a number of network - associated genes , including cebpa ( figure  5c ) ,  vamp4 ( figure  5d ) , vdr ( figure  5e ) , lxr ( but not lxr ) ( figures 5f and 5 g ) , and rara ( figure  5h ) . 
   peroxisome proliferator - activated receptor  ( ppar ) was not regulated in infected cells ( figure  5i ) and showed no change in expression following hx531 treatment . 
   we also examined the effect of hx531 treatment on a number of rxr - related genes that were not represented within the identified network ( figure  4c ) , including abca1 ( figure  5j ) , abcg1 ( figure  5k ) , apoe ( figure  5l ) , cyp27a1 ( figure  5 m ) , and cyp27b1 ( figure  5n ) . 
   treatment had no measurable effect on the expression of these genes in kcs , with the exception of abca1 , where treatment appeared to block upregulation in uninfected cells ( figure  5j ) . 
   hx531 treatment blocked the upregulation of il-1 in infected kcs ( figure  5o ) but had no effect on inducible nitric oxide synthase ( inos ) mrna levels in infected or inflamed kcs ( figure  5p ) . to confirm that genes within this network were not subject to regulation merely by phagocytosis , we performed rt - pcr for a subset of genes within this network on kc isolated from mice injected with latex beads . 
   as shown in figure  s2 , uptake of latex did not affect mrna levels for rxra ( figure  s2a ) , lxr ( figure  s2b ) , cebpa ( figure  s2c ) , or vamp4 ( figure  s2d ) , relative to kc isolated from control - injected mice . to determine the effect that the perturbation of the rxr pathway would have on infection , and hence to examine the potential of the expression profiling approach that we have used for the discovery of important host response pathways , we treated mice with hx351 prior to and for 4  days following infection with  td - tomato l.  donovani amastigotes ( figure  6a ) . using a flow cytometric approach to quantify the number of parasites in the tissues , we first assessed splenic and hepatic parasite burden at 2  hr p.i . 
   , when there was no difference in the presence or absence of treatment , and at 96  hr p.i . 
   treatment significantly reduced the number of parasites present in both the liver and the spleen at 4  days postinfection relative to the saline - treated control animals . to examine whether perturbation in this pathway could also be achieved by stimulating , rather than inhibiting , rxr , we treated mice with lg268 ( an rxr agonist ) . 
   lg268 also reduced the number  of parasites present in the spleen but not the liver . to determine whether rxr perturbation could be operating through effects on lxr , we treated mice with gw3965 ( an lxr agonist ) . 
   gw9395 treatment had no effect on parasite burden in either tissue ( figures 6b and 6c ) , suggesting that lxr is not involved in control of parasite load mediated through the rxr network . in order to examine 
   if the therapeutic effect observed reflected leishmanistatic or leishmanicidal activity , we also calculated the fold change in parasite numbers in the 2 organs at 4  days p.i . 
   , parasite numbers increased over a 96  hr period , and this growth was inhibited after drug treatment . 
   in contrast , in liver , parasite numbers did not increase significantly over 96  hr in nontreated mice and were reduced to below the level seen at 2  hr p.i . in drug - treated mice , 
   suggestive of leishmanicidal activity ( figure  6e ) . to confirm this observation , we used a histological approach in the liver and further showed that the percentage of infected kcs was significantly reduced in the lg268-treated animals and showed a trend in this direction in hx531-treated mice but not in gw3965-treated mice ( figures 6f and 6 g ) . 
   although not significant in the single experiment shown in figure  6 , replicate experiments with mice treated with hx531 confirmed that this drug enhanced the leishmanicidal activity of kcs ( p  = 0.0028 ; n  = 11 mice for saline controls and 8 mice for hx531 treatment ) . in conclusion 
   , these data indicate that pharmacological manipulation of rxr function was sufficient to perturb the gene network that we identified as being aberrantly expressed in infected kcs . 
   furthermore , it was unlikely that the effects of hx531 or lg268 were due to direct effects on lxr , as stimulation of lxr had no measurable biological effect . 
   we have examined the early in  vivo response of liver macrophages to infection with leishmania , independently studying the activation of infected and uninfected cells exposed to inflammatory signals within the infected tissue microenvironment . 
   our approach provides a number of unique insights into the transcriptomic response induced by l.  donovani infection in  vivo and provides proof of concept that the use of cells exposed to inflammation is a powerful method for the discovery of pathogen - regulated gene pathways . 
   first , the isolation of kc from infected mice revealed a transcriptional response in infected kcs that is somewhat distinct from that observed by others using different types of mononuclear host cells ( including bone marrow - derived macrophages , monocytes , and dcs ) . 
   it remains to be determined whether this reflects cell specificity in host response or differences due to the analysis being performed on cells infected in  vitro versus in  vivo . in most previous in  vitro experiments , the possible influence of uninfected cells on gene expression profile , 
   e.g. , by dilution of the signal associated with infected cells , was not taken into account ( buates and matlashewski , 2001 ; chaussabel et  al . , 2003 ; 
   , 2009 ; rodriguez et  al . , 2004 ) . in one series of studies , 
   dcs infected in  vitro with l.  amazonensis were sorted to allow microarrays to be conducted only on infected cells ( giraud et  al . , 2012 ; lecoeur et  al . , 
   while these studies revealed unique signatures not evident from bulk population analysis , data were not provided to show whether the uninfected dc within the culture system had also made bystander responses ( e.g. , mediated by cytokine release from infected dcs in culture ) . 
   furthermore , the transcriptomic signatures generated by microarray in this and indeed most other studies ( including those cited above ) have not been validated fully at the protein level . 
   second , our data demonstrate significant quantitative and qualitative differences in transcriptomic responses compared to previous in  vitro gene expression studies . in  vitro experiments have usually been interpreted as indicating that visceralizing species of leishmania result in a relatively silent infection of macrophages ( zhang et  al . , 2010 ) , significant suppression of gene expression ( buates and matlashewski , 2001 ) , or a signature distinct from that associated with interferon ( ifn)- activation ( rodriguez et  al . , 2004 ) . 
   the tendency of l.  donovani to induce a weak inflammatory response in  vitro may be species specific , with l.  donovani metacyclics inducing lower fold changes in inflammatory gene expression compared to l.  major metacyclics in one study ( chaussabel et  al . , 2003 ) . 
   alternatively , there may be life cycle stage - specific determinants of gene expression , as shown for l.  mexicana ( aebischer et  al . , 2005 ) . 
   in an additional study , l.  amazonensis amastigotes were also shown to induce significant gene expression changes in  vitro , including changes to the sterol biosynthesis pathway ( osorio y forta et  al . , 2009 ) . 
   importantly , the impact of l.  chagasi on host macrophage response was significantly modulated by the addition of t  cells to the culture in  vitro ( ettinger and wilson , 2008 ) , suggesting that factors external to the infected macrophage impact the response of the infected cell . 
   our data support and extend these observations by adding the full in  vivo complexity of the microenvironment created in the infected liver . 
   third , we show that uninfected kcs also undergo significant biological and gene transcriptional changes associated with the inoculation into mice of live l.  donovani amastigotes . 
   the activation of uninfected kcs at early time points , with further downstream transcriptional consequences seen at 12  hr , suggests that soluble mediators or signals operating in trans facilitate global activation of macrophages within the infected tissue . 
   although this activation shows both qualitative and quantitative differences between infected and inflamed kcs , many of the genes that have been associated with cell - autonomous defense against intracellular protozoa stimulated by interferons ( macmicking , 2012 ) are induced to similar levels in infected and uninfected kcs at 12  hr postinfection . 
   this would place such genes in the category of commonly - regulated genes within the framework of our analysis . 
   such genes include the guanylate - binding proteins gbp2 and gbp5 , indoleamine 2,3-dioxygenase 1 ( ido1 ) , immunity - related gtpase family m members 1 and 2 ( irgm1 and irgm2 ) , and immunoresponsive gene 1 ( irg1 ) . 
   these data are somewhat intuitive , given that ifn is a soluble mediator , capable of trans - activating neighboring cells . 
   collectively , our comparative analysis of the gene expression profiles of infected and inflamed kcs taken from the livers of infected mice suggests that a degree of caution should be exercised when interpreting whether genes observed to alter their expression in response to infection truly represent autonomously regulated genes associated with the presence of intracellular pathogens . 
   fourth , we have identified and validated the importance of a gene network centered around rxra that is associated with the survival of amastigotes within the liver of l.  donovani - infected  mice . within this rxra network , 
   although lipid metabolism centered around rxr has not previously been implicated in l.  donovani pathogenesis in mice , other studies have observed changes in lipid metabolism genes associated with leishmania infection . 
   for example , changes in components of the sterol biosynthesis pathway were induced by macrophage infection with l.  amazonensis ( osorio y forta et  al . 
   previous studies have characterized a role for two heterodimeric partners of rxr in the control of leishmania major infection , the vitamin d receptor ( vdr ) ( ehrchen et  al . 
   , 2007 ; whitcomb et  al . , 2012 ) and the liver x receptor ( lxr ) ( bruhn et  al . , 
   in addition , upregulation of vdr was previously reported following in  vitro infection of macrophages with l.  chagasi ( rodriguez et  al . , 
   2004 ) as well as l.  donovani and l.  major ( chaussabel et  al . , 2003 ) , suggesting that vdr is an important component of the host response to leishmania infection . 
   we believe that by targeting rxr as the hub of the identified network , we have successfully perturbed an entire gene network that controls l.  donovani survival in kcs . 
   perturbation of rxr in either direction via activation with the agonist lg268 or suppression with the antagonist hx531 had similar effects on the growth of l.  donovani in  vivo . 
   the mechanism by which this network controls parasite survival is currently subject to further investigation . 
   however , we speculate that the gene network may regulate the usage of lipids and cholesterol by the parasite while it is present in the phagolysosome of the host cell ( mcconville and naderer , 2011 ) . alternatively , 
   macrophage sensing of leishmania - derived lipids may be having profound effects on the nature of the innate immune response that is initiated ( nagy et  al . , 2012 ) . finally , and of broader significance 
   , we have described an approach to study host - pathogen interactions , which is capable of revealing important host response pathways that would be invisible in studies of the interaction between macrophages and pathogens in  vitro . 
   importantly , we show that manipulation of such pathways in  vivo can impact parasite survival , reducing parasite load at early stages after liver infection . 
   targeting of nuclear receptors for therapy is a burgeoning field of medicine , with rxr therapeutics showing efficacy in clinical settings of cutaneous t  cell lymphoma ( duvic et  al . , 2001 ; khuri et  al . , 2001 ) and a preclinical model of alzheimer s disease ( cramer et  al . , 2012 ) . 
   rxr antagonists , including hx531 , have also shown efficacy in preclinical models of diet - induced obesity and type 2 diabetes ( yamauchi et  al . , 2001 ) . 
   in this respect , while it is tempting to suggest that our findings may also set the foundation for the use of rxr - mediated therapies for leishmaniasis , detailed investigations into the mechanism of parasite growth arrest and/or killing are now required . 
   mt / mg ( muzumdar et  al . , 2007 ) and lysmcre ( clausen et  al . , 1999 ) 
   mice were bred and housed under specific pathogen - free conditions and used at 612  weeks of age . 
   the ethiopian strain of leishmania donovani ( lv9 ) and tandem tomato fluorescent protein expressing lv9 ( tdtom.lv9 ) ( beattie et  al . , 2010 ) were maintained by serial passage in rag-1 mice . 
   amastigotes were isolated from infected spleens ( smelt et  al . , 1997 ) , and 
   mice were infected with 3   10 
   l.  donovani amastigotes intravenously ( i.v . ) via the tail vein in 200  l of rpmi 1640 ( gibco ) . 
   parasites were killed via heat treatment at 56c for 15  min , followed by labeling with pkh26 red fluorescent cell linker kit ( sigma ) . for latex bead experiments , 1  m fluoresbrite polychromatic red microbeads ( polysciences ) 
   were washed twice in saline , and 3   10 beads per mouse were injected i.v . 
   all experiments were approved by the university of york animal procedures and ethics committee and performed under uk home office license ( immunity and immunopathology of leishmaniasis ref # ppl 60/3708 ) . 
   mice were anesthetized , and surgery was performed similar to that previously described ( beattie et  al . , 2010 ) , except that anesthesia was maintained by inhalation of 4% isofluorane ( abbott laboratories ) . 
   images were acquired on an inverted lsm 780 multiphoton microscope ( zeiss ) maintained at 36c by a blacked - out environmental chamber ( solent scientific ) . 
   images were acquired with a 40 1.1 water immersion objective and fluorescence excitation provided by a chameleon xr ti : sapphire laser ( coherent ) tuned to 870  nm . for 4d analysis , 2035  m 
   z stacks were acquired with a z distance of 2  m approximately every 1530 s. real - time injection was performed via tail vein catheter ( strategic applications incorporated , infusion technologies ) . 
   in addition to collecting baseline data for animals before infection , individual kcs were imaged prior to and for 2  hr directly following infection of mice with l.  donovani . 
   data were rendered and analyzed using volocity software ( perkinelmer ) , and cell analysis was performed automatically with manual checking . 
   confocal microscopy was performed on 20  m frozen sections as previously described ( beattie et  al . , 2011 ) . 
   slides were blinded before imaging on a zeiss lsm 510 axioplan microscope ( zeiss ) . 
   hepatic mononuclear cells were prepared from the livers of tdtom.lv9- or rpmi - injected c57bl/6 mice . 
   we conducted four independent infection experiments to generate samples for analysis . in each experiment , n  = 16 mice , and at each time point studied ( 2  hr and 12  hr postinfection ) , we isolated kcs from the livers of four mice treated with rpmi and four mice infected with l.  donovani . 
   liver mononuclear cells were pooled prior to sorting . for real - time pcr analysis , replicates represent pooled samples from independently sorted samples . 
   livers were perfused with 0.4  mg / ml of warmed liberase tl ( roche ) then digested at 37c for 45  min . 
   digested livers were passed over 100  m cell strainers , washed in pbs containing 2% fetal calf serum ( fcs ) , and resuspended in 33% percoll for centrifugation at 693   g for 12  min . 
   isolated cells were blocked with anti cd16/32 then labeled with f4/80-alexa 488 , gr-1 pecy7 ( ebioscience ) , and crig ( helmy et  al . , 2006 ) that was conjugated to atto 647h via lightning - link conjugation ( innova biosciences ) . 
   a small fraction of cells ( approximately 3,000 sorted cells ) were spun onto glass slides , fixed in methanol , and stained with giemsa for morphological analysis . 
   rna was isolated from purified kc and amplified via agilent low input quick amp labeling kit ( agilent technologies ) . 
   amplified rna was then assayed with agilent sureprint g3 mouse gene expression 860k microarray chips that were scanned with an agilent c scanner with surescan high - resolution technology ( agilent ) . 
   comparison of the gene expression data between the rpmi controls and the infected or inflamed samples was performed using the benjamini and hochberg false discovery rate ( fdr ) correction ( pawitan et  al . , 2005a ) , albeit with the caveat that this approach may have low sensitivity with the sample size used ( pawitan et  al . , 2005b ) . 
   this analysis was performed with genespring software ( version 9 ; agilent ) as a standard 5% fdr , with the variances assessed by the software for each t test performed . 
   all data for long intergenic noncoding rna ( lincrna ) probe sets were removed from the analysis at this point . 
   gene ontology analysis was performed using the david ( huang et  al . , 2009 ) and ingenuity pathway analysis software packages ( ingenuity systems ) , and gene maps generated with string ( www.string-db.org ) . 
   mice were pretreated with saline , 100  mg / kg hx531 ( yamauchi et  al . , 2001 ) , 
   30  mg / kg lg268 ( shen et  al . , 2004 ) , or 20  mg / kg gw3965 ( lei et  al . , 2013 ) 26  hr prior to infection and then every 24  hr until 72  hr for hx531 and lg268 or every 12  hr for gw3965 . 
   at 96  hr postinfection , the livers and spleens were collected , and the parasite burden was determined both by flow cytometry and in fixed liver slices . 
   data were normalized to the baseline 2  hr time point to determine the survival of parasites relative to the initial dose of infection . 
   liver and spleens were collected , homogenized , and resuspended in a specific volume of rpmi 1640 ( gibco ) . 
   a total of 200  l of each sample was aliquoted in duplicate to a 96-well plate , centrifuged at 2,000   g for 10  min , and the pellets were resuspended in 2  mg / ml saponin for 5  min at room temperature ( rt ) . 
   the centrifugation was repeated and followed by resuspension in 1  mg / ml saponin . 
   the remaining pellets were washed a further two times , and the wells were resuspended in 200  l then spiked with 2  l of 6  m polystyrene microspheres ( polysciences , inc . ) at 4.6   10 particles/l . 
   samples were acquired on a cyan adp analyzer ( beckman coulter ) in which both forward scatter detector neutral density filters had been removed to allow for small - particle detection . 
   the number of parasites was quantitated by determining the ratio of beads to parasites and then multiplying to calculate the total number of parasites in the starting volume . 
   rna was reverse transcribed into complementary dna ( cdna ) with a superscript iii first - strand synthesis kit ( invitrogen ) . 
   oligonucleotides used for the specific amplification of target genes are described in the supplemental experimental procedures . 
   real - time quantitative pcr was performed with fast sybr green pcr master mix in a steponeplus real - time pcr system ( applied biosystems ) . 
   accumulation of target genes was normalized to hprt , -actin ( shih et  al . 
   , 2012 ) , or ribosomal protein l32 ( reis et  al . , 2013 ) and expressed as relative expression via the change in cycle threshold ( ct ) analysis method ( relative expression in test sample versus rpmi controls ) .
summary: summaryintracellular pathogens modulate host cell function to promote their survival . however , in  vitro infection studies do not account for the impact of host - derived inflammatory signals . 
   examining the response of liver - resident macrophages ( kupffer cells ) in mice infected with the parasite leishmania donovani , we identified a transcriptomic network operating in uninfected kupffer cells exposed to inflammation but absent from kupffer cells from the same animal that contained intracellular leishmania . to test 
   the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival , we pharmacologically perturbed the activity of retinoid x receptor alpha ( rxr ) , a key hub within this network , and showed that this intervention enhanced the innate resistance of kupffer cells to leishmania infection . 
   our results illustrate a broadly applicable strategy for understanding the host response to infection in  vivo and identify rxra as the hub of a gene network controlling antileishmanial resistance .