BACKGROUND
there is substantial clinical heterogeneity in the clinical, radiologic, and histopathologic features within each subtype of idiopathic interstitial pneumonias .
for instance, all forms of iip have a somewhat unpredictable prognosis and many but not all patients can progress to end stage lung disease .
while subtypes of iips differ in clinical, radiographic, and histopathologic presentation , the type of iip often cannot be determined and many of the subtypes of iip have overlapping clinical and laboratory features indicating that the current definitions remain too broad.
idiopathic pulmonary fibrosis , by far the most common form of iip, is histopathologically defined by the presence of the prototypical form of pulmonary fibrosis, usual interstitial pneumonia , a fibrosing interstitial pneumonia characterized by a pattern of heterogeneous, subpleural regions of fibrotic, and remodeled lung that often results in death within 2– <dig> years of diagnosis  <cit> .
other iips, such as respiratory bronchiolitis-associated interstitial lung disease , are more cellular, occur earlier in life, and have a considerably lower mortality  <cit> .
by contrast, idiopathic nonspecific interstitial pneumonia , a pattern of iip that is more likely a syndrome than a disease, is most commonly characterized by interstitial fibrosis but in a more uniform pattern than uip, and carries a better prognosis than ipf/uip  <cit> .
these differences within the subtype and overlaps among subtypes create a distinct challenge to achieve an accurate diagnosis for an individual patient with this potentially life-threatening diagnosis.
the molecular mechanisms that account for the extreme heterogeneity in clinical presentation, radiologic patterns, histopathologic variation, and disease progression are largely unknown.
we hypothesize that biological heterogeneity in iips will be reflected by gene expression patterns in lung tissue from patients with iip, and gene expression patterns will change as a function of disease activity, progression, and severity.
to test this hypothesis, we measured gene expression in lung tissue of patients with iip, and correlated gene expression patterns with diffusing capacity of the lung for carbon monoxide  and forced vital capacity .
RESULTS
demographic characteristics of the lung tissue research consortium  cohort
table  <dig> summarizes demographic and clinical characteristics of the ltrc iips cohort and by subtypes of iips that include at least  <dig> individuals.
included in the table is the portion of the non-diseased control cohort used together with the ltrc cohort.
overall, the iips cohort is older than the controls.
within the iip cohort, individuals within ipf/uip group are the oldest followed by insip, uncharacterized fibrosis, and rb-ild.
there are no significant differences across iip subtypes in gender or racial distribution.
comparison of smoking histories reveals that approximately half of the individuals with iip are former smokers, as compared to controls that are almost 50 % current smokers.
subjects with ipf/uip and rb-ild diagnosis reported higher pack-years compared to subjects with insip, uncharacterized fibrosis, and controls; however, there is variability within each group and therefore there are no statistically significant differences among groups.
we also compared st. george’s score, an indicator of overall lung health  <cit>  in subtypes of iip  and did not find significant differences among groups.
finally, variables associated with lung function, % predicted pre-bronchodilator fvc and dlco reveal better lung function in the rb-ild group compared to other iips.table  <dig> subject demographics and clinical characteristics of the derivation  cohort by iip subcategory
disease groupa
pack years - mean b
aiip = idiopathic interstitial pneumonia, ipf = idiopathic pulmonary fibrosis, uip = usual interstitial pneumonia, insip = idiopathic nonspecific interstitial pneumonia, uf = uncharacterized fibrosis, rb-ild = respiratory bronchiolitis associated interstitial lung disease.
data not reported individually for cryptogenic organizing pneumonia   and desquamative interstitial pneumonia   categories
baverage for current and former smokers
*two-tailed student’s t-test
**fisher’s exact test
***one-way anova
identification of genes associated with the iips clinical subtype
to establish whether there are significant differences in expression in clinical subtypes of iip, we first identified molecular profiles associated with clinically defined subtypes of iip.
for this initial analysis, we used an ancova model that incorporates clinical subtype with at least  <dig> individuals per group , age, gender and smoking status as factors.
venn diagrams in additional file 1: figure s <dig> illustrate overlap of differentially expressed mrnas amongst iip categories versus controls using the 5 % fdr criterion alone or combined 5 % fdr and 2-fold change criteria.
while the statistical interpretation of intersection-union testing among these groups is compromised by differences in the power of individual contrasts and by the common comparator group, a large proportion of the differentially expressed mrna transcripts exhibit differences between control lung and two or more of the iip subgroups.
however, we also observed genes unique to each iip subtype, especially in the ipf/uip group which has the most differentially expressed transcripts overall as well as the most unique transcripts.
this result is likely a combination of ipf/uip being the largest group of iips examined in our study and the fact that ipf/uip has the most remodeled lung of all iips.
examination of  <dig> genes in common to all iips using the  <dig> % fdr and  <dig> fold change criteria reveals genes involved in inflammation , extracellular matrix , wnt signaling , coagulation  and host defense  .
interestingly, defensin defa <dig> is one of the genes we identified as differentially expressed in peripheral blood of severe vs mild ipf/uip categorized by differences in the dlco  <cit> .
we used the high degree of overlap among iips and biological relevance of genes identified in common to all iips as a rationale for inclusion of all iip subtypes in the analysis of lung function variables as opposed to focusing only on ipf/uip; however, we adjust for iip subtype in all further analyses.
identification of genes associated with dlco
we next sought to identify genes whose expression is associated with the dlco measurement.
for this analysis, we used an anova model that incorporates categories of disease based on predicted dlco , iip subtype and smoking status, or an ancova model that incorporates the % predicted dlco measurement, iip subtype and smoking status as factors.
although no significant difference in smoking status exist among iips due to high variability in each iip subtype, differences in smoking histories in these individuals may influence gene expression and we therefore included them in the model.
we did not include age and gender because they are accounted for in the calculation of % predicted lung function variables.
by categorical analysis,  <dig> unique transcripts differentiate mild disease  from severe disease  at 5 % fdr .
when dlco is treated as a continuous variable ,  <dig> genes correlate with changes in % predicted dlco at 5 % fdr .
six hundred fourteen genes not identified in the categorical analysis were found to be associated with changes in dlco in the continuous analysis.
identification of genes associated with fvc
we also identified gene expression changes associated with the fvc measurement, in an analogous manner to the analysis of dlco.
by categorical analysis,  <dig> genes differentiate mild disease  from severe disease  when categorized by % predicted fvc at 5 % fdr .
two thousand four hundred sixty seven genes correlate with changes in % predicted fvc in  <dig> subjects with available fvc data at  <dig> % fdr  with  <dig> of these transcripts not overlapping with categorical analysis.
transcriptional changes in common to decline in dlco and fvc
to focus on genes that are the most likely to be involved in the pathogenesis of iip, we identified differentially expressed transcripts in common to lower measures of dlco and fvc.
we first intersected  <dig> transcripts identified as significant in the categorical analysis of disease severity based on dlco and  <dig> from the analogous analysis of fvc.
fifty eight transcripts that are in common to the two analyses are listed in table  <dig>  while a number of these transcripts, including adamts family members   <cit> , ager  <cit> , hif-1α  <cit> , serpin family members   <cit> , and selectin e   <cit>  have an established role in lung fibrosis, expression of novel iip candidate genes such as rhotekin  <dig>  and peptidase inhibitor  <dig>  is also significantly altered in severe compared to mild disease.
dot plots shown in fig.
<dig> demonstrate decrease in rtkn <dig> but an increase in sele and pi <dig> with a decrease in lung function.
we confirmed expression levels of these three genes in the same set of ltrc samples by qrt-pcr .table  <dig> differentially expressed genes  in severe vs mild disease as characterized both by a decline in % predicted dlco and fvc
fig.
<dig> categorical analysis of changes in gene expression based on lung disease severity categorized by % predicted dlco  or % predicted fvc  for novel iip candidate genes rhotekin  <dig> , selectin e , and peptidase inhibitor  <dig> .
mild, moderate, and severe are defined as percent predicted dlco , or fvc .
p values for severe vs mild groups are shown in table 2
fig.
<dig> qrt-pcr validation of microarray data.
fold changes relative to control lungs for rhotekin  <dig> , selecting e  and pi <dig>  in all iips and mild and severe disease based on either fvc or dlco.
error bars represent standard deviations
given that analysis of expression changes as a function of continuous decrease in lung function variables identified large number of additional genes, we use the overlap between transcripts associated with a decline in dlco or fvc in the continuous analysis  to identify canonical pathways and transcriptional networks that are likely to be important predictors of severity of iips.
canonical pathways analysis of the  <dig> genes identified hepatic fibrosis and hepatic stellate cell activation , acute phase response signaling , and hif-1α signaling  as the most significantly enriched .
a number of other pathways were identified  including il17a, dc and nk crosstalk, lps/il- <dig> mediated inhibition of rxr function, leukocyte extravasation signaling, atherosclerosis signaling, and aryl hydrocarbon receptor signaling.
protein-protein interactome analysis using the innatedb database of interactions suggests that more severe presentation of iips is characterized by an increase in cell cycle progression and apoptosis , increased hypoxia  and dampened innate immune response  .
network analysis in ingenuity pathway analysis identified seven high scoring networks  that largely recapitulate findings from innatedb database.fig.
<dig>  <dig> differentially expressed genes common to fvc and dlco by continuous analysis were evaluated by  canonical pathways and  protein-protein interactome analysis.
pathways and their associated –log p value, as determined by the fisher exact test in ingenuity pathway analysis, are shown as the blue bars.
the ratio of the number of genes mapping to these pathways relative to the total number of genes in the pathway is plotted as orange squares.
the interactome was created using networkanalyst  <cit>  and the innatedb ppi dataset by first importing all  <dig> genes and then reducing the network to zero-order interactome.
the nodes are colored based on their correlation of expression and lung function variables .
the sizes of nodes are proportional to their betweenness centrality values
validation of gene expression changes in an independent cohort
to test the generalizability of our findings, we examined expression of the candidate genes with the strongest associations with dlco and fvc in an independent cohort of iips.
additional file 1: table s <dig> provides characteristics of the replication  cohort.
we primarily focused this analysis on the most pronounced transcriptional changes,  <dig> genes with > <dig> fold change from the categorical analysis of both fvc and dlco .
we used the same anova models as in the derivation cohort, incorporating lung function variable, iip subtype, and smoking, on these  <dig> genes to assess whether there are significant differences in expression between mild and severe disease, as defined by percent predicted dlco  or fvc .
the results of this analysis  demonstrate that extent of transcriptional changes associated with lung function are dampened in this cohort compared to the ltrc cohort.
however, all genes have p < <dig>  for association with either fvc or dlco.
we also performed an analysis to validate  <dig> genes from table  <dig> in the replication cohort in the same manner as previous analysis.
44/ <dig>  genes have nominal p < <dig>  in at least one of the analyses .
we also considered lack of rb-ild samples in the replication cohort as a counfounder of our validation analysis.
this subtype of iips is characterized by better lung function than ipf and insip; presence of rb-ild cases with better lung function creates a wider range of values for fvc and dlco in the ltrc cohort and may potentially allow for identification of more associations despite controlling for iip subtype in the model.
49/ <dig>  genes identified as significant in the categorical analysis of fvc and dlco  remain significant after removal of rb-ild samples from the analysis.
similarly, of the  <dig> genes identified in common to dlco and fvc when considered as continuous variables,  <dig> genes  remain significant in the analysis without rb-ild.
therefore, rb-ild samples do not significantly influence the results of our analysis beyond loss of power with fewer samples.table  <dig> validation of gene expression changes from the ltrc iip cohort in an independent cohort of iips
localization of gene expression changes
to partially address the issue of cell specificity of gene expression profiles identified in whole lung tissue, we performed immunohistochemistry to localize expression of rhotekin  <dig>  one of the genes with most prominent expression change associated with diseases progression and severity, in normal and iip lung.
in histologically normal lung tissue, rhotekin  <dig> is expressed in airway/bronchial epithelia, alveolar epithelia, alveolar macrophages, and submucosal glands .
we next examined different histopathological features  in lung tissue of ipf subjects with more compared to less severe disease.
while we did not observe consistent differences across all subjects we examined, we observed a trend of lower expression of rhotekin  <dig> in ipf airways , honeycomb cysts , and alveolar cystic areas  of individuals with more severe disease.
based on our data, reduced mrna levels of rhotekin  <dig> in severe ipf are most likely a combination of reduced expression in specific cells and loss of healthy airway epithelia that are replaced by honeycomb cysts  <cit> .
localization of rhotekin  <dig> in insip lung tissue also demonstrated a decrease in expression in more severe disease .
these findings corroborate the findings of reduced rhotekin  <dig> expression at the mrna level in more severe iips.fig.
<dig> localization of rhotekin  <dig> expression in ipf lung of patients with mild  and severe  disease.
immunohistochemical staining of airways , honeycomb cysts , and alveolar cysts  lung tissue reveals some decrease in expression of rhotekin  <dig> in airway epithelial cells in patients with severe compared to mild disease accompanied by an increase in expression in hyperplastic alveolar type ii cells in alveolar cysts.
top panels represent rhotekin  <dig> stained sections and bottom panels are corresponding tissue sections incubated with non-immune serum .
tissue sections were counterstained with hematoxylin.
images were taken at 10× and 40×  magnifications
discussion
results of our investigation of global gene expression changes in lungs of patients with iips demonstrate that, at the molecular level, there are more commonalities than differences among different subtypes of iips.
on the other hand, the extent of disease, as characterized by lower measures of fvc and dlco, is associated with marked changes in expression of novel and established genes and pathways involved in iips.
our findings that lung tissue from ipf, insip, rb-ild, and uncharacterized fibrosis share many common expression patterns supports the concept that, despite differences in clinical presentation, these diseases may be related etiologically and pathogenically.
three publications  indicate that ipf/uip and insip, presumed distinct clinical-pathologic processes, may be related etiologically and pathogenically.
two of these studies  <cit>  have used transcriptional profiles of affected lung tissue to create iip molecular signatures, and their findings suggest that dissimilar histological patterns may be related biologically.
in addition to some differences in expression among different iip subtypes, we identified more pronounced changes in gene expression associated with more advanced disease.
this conclusion is supported by previous reports  <cit> .
selman et al.
identified  <dig> differentially expressed genes in rapid compared to slow progressors, including overexpression of genes involved in morphogenesis, oxidative stress, migration/proliferation, and genes from fibroblasts/smooth muscle cells  <cit> .
our group also demonstrated differential expression  of  <dig> transcripts in individuals with progressive ipf  compared to slow progressors  <cit> .
while only less than 10 % of the genes in table  <dig>  were identified by these earlier studies, additional overlap was observed when we considered gene families , suggesting that the present study was successful at confirming published observations but also identifying novel gene transcripts.
two potential explanations for limited overlap are the fact that technologies and genome annotations have changed  and sample size .
among the gene transcripts most strongly associated with disease severity are rhotekin  <dig> , selecting e , and peptidase inhibitor  <dig> .
rhotekin  <dig> is a member of a family of proteins containing a rho-binding domain that are target peptides for the rho-gtpases and are important in lymphocyte development and function  <cit> ; however rhotekin  <dig> is ubiquitously expressed  <cit> .
genetic variants in rhotekin  <dig> have been associated with rheumatoid arthritis  and activation of the nf-κb pathway in japanese  <cit> .
these known roles for rhotekin  <dig>  the observation of decreased expression of rhotekin  <dig> in more severe disease, both in our earlier publication  <cit>  and in the current study, and localization of expression to the lung epithelium in the current study, point to this gene as a candidate that warrants further functional investigation in iips.
selectin e is a member of the selectin family of cell adhesion molecules and plays a central role in adhesion of leukocytes to the endothelium  <cit> .
its expression inhibits bleomycin-induced lung fibrosis  <cit>  but the soluble form of the protein product is increased in serum of patients with pulmonary fibrosis  <cit> .
finally, pi <dig> is a trypsin inhibitor that is developmentally regulated in the lung mesenchyme  <cit>  but otherwise is not well characterized; its role in development is consistent with recapitulation of developmental pathways in lung injury that is a hallmark of iips  <cit> .
one of the strengths of the present study is the use of two independent cohorts of iip for derivation and validation.
however, we were only able to validate the most pronounced transcriptional changes identified in the derivation cohort.
one possible explanation for this is that overall the njh cohort has milder disease, an observation we have previously made  <cit> .
we also considered the possibility that lack of rb-ild cases in the replication cohort contributed to limited validation but ruled this out by showing that exclusion of rb-ild cases from the derivation cohort did not substantially influence the results.
a notable weaknesses of the present study in that, analogous to earlier publications, we have used whole lung tissue with the mixture of cells.
immunohistochemistry analysis of rhotekin  <dig>  one of the genes with strongest correlation with disease severity in our study, demonstrates that reduced expression may be a combination of reduced expression in specific cells and loss of healthy airway epithelia that are replaced by honeycomb cysts.
further future studies will be needed to determine which of the genes identified by our analysis are differentially regulated in specific cell populations.
another weakness of our study is cross-sectional design in which we identify gene expression changes associated with a decline in lung function across a cohort of different individuals.
a study of longitudinal design with measurements of gene expression and lung function over time in same individuals will be needed to validate these findings.
however, the finding that rhotekin  <dig> expression was associated with more rapid disease progression in our earlier study  <cit>  supports the notion that genes identified in our study have direct relevance to disease progression.
CONCLUSIONS
in summary, we identified commonalities and differences in gene among different subtypes of iip.
disease progression, as characterized by lower measures of fvc and dlco, results in marked changes in expression of novel and established genes and pathways involved in iip.
these genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for iip severity.

 there is substantial clinical heterogeneity in the clinical, radiologic, and histopathologic features within each subtype of idiopathic interstitial pneumonias. for instance, all forms of iip have a somewhat unpredictable prognosis and many but not all patients can progress to end stage lung disease. while subtypes of iips differ in clinical, radiographic, and histopathologic presentation, the type of iip often cannot be determined and many of the subtypes of iip have overlapping clinical and laboratory features indicating that the current definitions remain too broad. idiopathic pulmonary fibrosis, by far the most common form of iip, is histopathologically defined by the presence of the prototypical form of pulmonary fibrosis, usual interstitial pneumonia, a fibrosing interstitial pneumonia characterized by a pattern of heterogeneous, subpleural regions of fibrotic, and remodeled lung that often results in death within 2– years of diagnosis. other iips, such as respiratory bronchiolitis-associated interstitial lung disease, are more cellular, occur earlier in life, and have a considerably lower mortality. by contrast, idiopathic nonspecific interstitial pneumonia, a pattern of iip that is more likely a syndrome than a disease, is most commonly characterized by interstitial fibrosis but in a more uniform pattern than uip, and carries a better prognosis than ipf/uip. these differences within the subtype and overlaps among subtypes create a distinct challenge to achieve an accurate diagnosis for an individual patient with this potentially life-threatening diagnosis. the molecular mechanisms that account for the extreme heterogeneity in clinical presentation, radiologic patterns, histopathologic variation, and disease progression are largely unknown. we hypothesize that biological heterogeneity in iips will be reflected by gene expression patterns in lung tissue from patients with iip, and gene expression patterns will change as a function of disease activity, progression, and severity. to test this hypothesis, we measured gene expression in lung tissue of patients with iip, and correlated gene expression patterns with diffusing capacity of the lung for carbon monoxide and forced vital capacity.  demographic characteristics of the lung tissue research consortium cohort table  summarizes demographic and clinical characteristics of the ltrc iips cohort and by subtypes of iips that include at least individuals. included in the table is the portion of the non-diseased control cohort used together with the ltrc cohort. overall, the iips cohort is older than the controls. within the iip cohort, individuals within ipf/uip group are the oldest followed by insip, uncharacterized fibrosis, and rb-ild. there are no significant differences across iip subtypes in gender or racial distribution. comparison of smoking histories reveals that approximately half of the individuals with iip are former smokers, as compared to controls that are almost 50 % current smokers. subjects with ipf/uip and rb-ild diagnosis reported higher pack-years compared to subjects with insip, uncharacterized fibrosis, and controls; however, there is variability within each group and therefore there are no statistically significant differences among groups. we also compared st. george’s score, an indicator of overall lung health in subtypes of iip and did not find significant differences among groups. finally, variables associated with lung function, % predicted pre-bronchodilator fvc and dlco reveal better lung function in the rb-ild group compared to other iips.table subject demographics and clinical characteristics of the derivation cohort by iip subcategory disease groupa pack years - mean b aiip = idiopathic interstitial pneumonia, ipf = idiopathic pulmonary fibrosis, uip = usual interstitial pneumonia, insip = idiopathic nonspecific interstitial pneumonia, uf = uncharacterized fibrosis, rb-ild = respiratory bronchiolitis associated interstitial lung disease. data not reported individually for cryptogenic organizing pneumonia and desquamative interstitial pneumonia categories baverage for current and former smokers *two-tailed student’s t-test **fisher’s exact test ***one-way anova identification of genes associated with the iips clinical subtype to establish whether there are significant differences in expression in clinical subtypes of iip, we first identified molecular profiles associated with clinically defined subtypes of iip. for this initial analysis, we used an ancova model that incorporates clinical subtype with at least individuals per group, age, gender and smoking status as factors. venn diagrams in additional file 1: figure s illustrate overlap of differentially expressed mrnas amongst iip categories versus controls using the 5 % fdr criterion alone or combined 5 % fdr and 2-fold change criteria. while the statistical interpretation of intersection-union testing among these groups is compromised by differences in the power of individual contrasts and by the common comparator group, a large proportion of the differentially expressed mrna transcripts exhibit differences between control lung and two or more of the iip subgroups. however, we also observed genes unique to each iip subtype, especially in the ipf/uip group which has the most differentially expressed transcripts overall as well as the most unique transcripts. this result is likely a combination of ipf/uip being the largest group of iips examined in our study and the fact that ipf/uip has the most remodeled lung of all iips. examination of genes in common to all iips using the % fdr and fold change criteria reveals genes involved in inflammation, extracellular matrix, wnt signaling, coagulation and host defense. interestingly, defensin defa is one of the genes we identified as differentially expressed in peripheral blood of severe vs mild ipf/uip categorized by differences in the dlco. we used the high degree of overlap among iips and biological relevance of genes identified in common to all iips as a rationale for inclusion of all iip subtypes in the analysis of lung function variables as opposed to focusing only on ipf/uip; however, we adjust for iip subtype in all further analyses. identification of genes associated with dlco we next sought to identify genes whose expression is associated with the dlco measurement. for this analysis, we used an anova model that incorporates categories of disease based on predicted dlco, iip subtype and smoking status, or an ancova model that incorporates the % predicted dlco measurement, iip subtype and smoking status as factors. although no significant difference in smoking status exist among iips due to high variability in each iip subtype, differences in smoking histories in these individuals may influence gene expression and we therefore included them in the model. we did not include age and gender because they are accounted for in the calculation of % predicted lung function variables. by categorical analysis, unique transcripts differentiate mild disease from severe disease at 5 % fdr. when dlco is treated as a continuous variable, genes correlate with changes in % predicted dlco at 5 % fdr. six hundred fourteen genes not identified in the categorical analysis were found to be associated with changes in dlco in the continuous analysis. identification of genes associated with fvc we also identified gene expression changes associated with the fvc measurement, in an analogous manner to the analysis of dlco. by categorical analysis, genes differentiate mild disease from severe disease when categorized by % predicted fvc at 5 % fdr. two thousand four hundred sixty seven genes correlate with changes in % predicted fvc in subjects with available fvc data at % fdr with of these transcripts not overlapping with categorical analysis. transcriptional changes in common to decline in dlco and fvc to focus on genes that are the most likely to be involved in the pathogenesis of iip, we identified differentially expressed transcripts in common to lower measures of dlco and fvc. we first intersected transcripts identified as significant in the categorical analysis of disease severity based on dlco and from the analogous analysis of fvc. fifty eight transcripts that are in common to the two analyses are listed in table  while a number of these transcripts, including adamts family members, ager, hif-1α, serpin family members, and selectin e have an established role in lung fibrosis, expression of novel iip candidate genes such as rhotekin and peptidase inhibitor is also significantly altered in severe compared to mild disease. dot plots shown in fig.  demonstrate decrease in rtkn but an increase in sele and pi with a decrease in lung function. we confirmed expression levels of these three genes in the same set of ltrc samples by qrt-pcr.table differentially expressed genes in severe vs mild disease as characterized both by a decline in % predicted dlco and fvc fig. categorical analysis of changes in gene expression based on lung disease severity categorized by % predicted dlco or % predicted fvc for novel iip candidate genes rhotekin, selectin e, and peptidase inhibitor. mild, moderate, and severe are defined as percent predicted dlco, or fvc. p values for severe vs mild groups are shown in table 2 fig. qrt-pcr validation of microarray data. fold changes relative to control lungs for rhotekin, selecting e and pi in all iips and mild and severe disease based on either fvc or dlco. error bars represent standard deviations given that analysis of expression changes as a function of continuous decrease in lung function variables identified large number of additional genes, we use the overlap between transcripts associated with a decline in dlco or fvc in the continuous analysis to identify canonical pathways and transcriptional networks that are likely to be important predictors of severity of iips. canonical pathways analysis of the genes identified hepatic fibrosis and hepatic stellate cell activation, acute phase response signaling, and hif-1α signaling as the most significantly enriched. a number of other pathways were identified including il17a, dc and nk crosstalk, lps/il- mediated inhibition of rxr function, leukocyte extravasation signaling, atherosclerosis signaling, and aryl hydrocarbon receptor signaling. protein-protein interactome analysis using the innatedb database of interactions suggests that more severe presentation of iips is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia and dampened innate immune response. network analysis in ingenuity pathway analysis identified seven high scoring networks that largely recapitulate findings from innatedb database.fig. differentially expressed genes common to fvc and dlco by continuous analysis were evaluated by canonical pathways and protein-protein interactome analysis. pathways and their associated –log p value, as determined by the fisher exact test in ingenuity pathway analysis, are shown as the blue bars. the ratio of the number of genes mapping to these pathways relative to the total number of genes in the pathway is plotted as orange squares. the interactome was created using networkanalyst and the innatedb ppi dataset by first importing all genes and then reducing the network to zero-order interactome. the nodes are colored based on their correlation of expression and lung function variables. the sizes of nodes are proportional to their betweenness centrality values validation of gene expression changes in an independent cohort to test the generalizability of our findings, we examined expression of the candidate genes with the strongest associations with dlco and fvc in an independent cohort of iips. additional file 1: table s provides characteristics of the replication cohort. we primarily focused this analysis on the most pronounced transcriptional changes, genes with > fold change from the categorical analysis of both fvc and dlco. we used the same anova models as in the derivation cohort, incorporating lung function variable, iip subtype, and smoking, on these genes to assess whether there are significant differences in expression between mild and severe disease, as defined by percent predicted dlco or fvc. the results of this analysis demonstrate that extent of transcriptional changes associated with lung function are dampened in this cohort compared to the ltrc cohort. however, all genes have p < for association with either fvc or dlco. we also performed an analysis to validate genes from table  in the replication cohort in the same manner as previous analysis. 44/ genes have nominal p < in at least one of the analyses. we also considered lack of rb-ild samples in the replication cohort as a counfounder of our validation analysis. this subtype of iips is characterized by better lung function than ipf and insip; presence of rb-ild cases with better lung function creates a wider range of values for fvc and dlco in the ltrc cohort and may potentially allow for identification of more associations despite controlling for iip subtype in the model. 49/ genes identified as significant in the categorical analysis of fvc and dlco remain significant after removal of rb-ild samples from the analysis. similarly, of the genes identified in common to dlco and fvc when considered as continuous variables, genes remain significant in the analysis without rb-ild. therefore, rb-ild samples do not significantly influence the results of our analysis beyond loss of power with fewer samples.table validation of gene expression changes from the ltrc iip cohort in an independent cohort of iips localization of gene expression changes to partially address the issue of cell specificity of gene expression profiles identified in whole lung tissue, we performed immunohistochemistry to localize expression of rhotekin one of the genes with most prominent expression change associated with diseases progression and severity, in normal and iip lung. in histologically normal lung tissue, rhotekin is expressed in airway/bronchial epithelia, alveolar epithelia, alveolar macrophages, and submucosal glands. we next examined different histopathological features in lung tissue of ipf subjects with more compared to less severe disease. while we did not observe consistent differences across all subjects we examined, we observed a trend of lower expression of rhotekin in ipf airways, honeycomb cysts, and alveolar cystic areas of individuals with more severe disease. based on our data, reduced mrna levels of rhotekin in severe ipf are most likely a combination of reduced expression in specific cells and loss of healthy airway epithelia that are replaced by honeycomb cysts. localization of rhotekin in insip lung tissue also demonstrated a decrease in expression in more severe disease. these findings corroborate the findings of reduced rhotekin expression at the mrna level in more severe iips.fig. localization of rhotekin expression in ipf lung of patients with mild and severe disease. immunohistochemical staining of airways, honeycomb cysts, and alveolar cysts lung tissue reveals some decrease in expression of rhotekin in airway epithelial cells in patients with severe compared to mild disease accompanied by an increase in expression in hyperplastic alveolar type ii cells in alveolar cysts. top panels represent rhotekin stained sections and bottom panels are corresponding tissue sections incubated with non-immune serum. tissue sections were counterstained with hematoxylin. images were taken at 10× and 40× magnifications discussion results of our investigation of global gene expression changes in lungs of patients with iips demonstrate that, at the molecular level, there are more commonalities than differences among different subtypes of iips. on the other hand, the extent of disease, as characterized by lower measures of fvc and dlco, is associated with marked changes in expression of novel and established genes and pathways involved in iips. our findings that lung tissue from ipf, insip, rb-ild, and uncharacterized fibrosis share many common expression patterns supports the concept that, despite differences in clinical presentation, these diseases may be related etiologically and pathogenically. three publications indicate that ipf/uip and insip, presumed distinct clinical-pathologic processes, may be related etiologically and pathogenically. two of these studies have used transcriptional profiles of affected lung tissue to create iip molecular signatures, and their findings suggest that dissimilar histological patterns may be related biologically. in addition to some differences in expression among different iip subtypes, we identified more pronounced changes in gene expression associated with more advanced disease. this conclusion is supported by previous reports. selman et al. identified differentially expressed genes in rapid compared to slow progressors, including overexpression of genes involved in morphogenesis, oxidative stress, migration/proliferation, and genes from fibroblasts/smooth muscle cells. our group also demonstrated differential expression of transcripts in individuals with progressive ipf compared to slow progressors. while only less than 10 % of the genes in table  were identified by these earlier studies, additional overlap was observed when we considered gene families, suggesting that the present study was successful at confirming published observations but also identifying novel gene transcripts. two potential explanations for limited overlap are the fact that technologies and genome annotations have changed and sample size. among the gene transcripts most strongly associated with disease severity are rhotekin, selecting e, and peptidase inhibitor. rhotekin is a member of a family of proteins containing a rho-binding domain that are target peptides for the rho-gtpases and are important in lymphocyte development and function; however rhotekin is ubiquitously expressed. genetic variants in rhotekin have been associated with rheumatoid arthritis and activation of the nf-κb pathway in japanese. these known roles for rhotekin the observation of decreased expression of rhotekin in more severe disease, both in our earlier publication and in the current study, and localization of expression to the lung epithelium in the current study, point to this gene as a candidate that warrants further functional investigation in iips. selectin e is a member of the selectin family of cell adhesion molecules and plays a central role in adhesion of leukocytes to the endothelium. its expression inhibits bleomycin-induced lung fibrosis but the soluble form of the protein product is increased in serum of patients with pulmonary fibrosis. finally, pi is a trypsin inhibitor that is developmentally regulated in the lung mesenchyme but otherwise is not well characterized; its role in development is consistent with recapitulation of developmental pathways in lung injury that is a hallmark of iips. one of the strengths of the present study is the use of two independent cohorts of iip for derivation and validation. however, we were only able to validate the most pronounced transcriptional changes identified in the derivation cohort. one possible explanation for this is that overall the njh cohort has milder disease, an observation we have previously made. we also considered the possibility that lack of rb-ild cases in the replication cohort contributed to limited validation but ruled this out by showing that exclusion of rb-ild cases from the derivation cohort did not substantially influence the results. a notable weaknesses of the present study in that, analogous to earlier publications, we have used whole lung tissue with the mixture of cells. immunohistochemistry analysis of rhotekin one of the genes with strongest correlation with disease severity in our study, demonstrates that reduced expression may be a combination of reduced expression in specific cells and loss of healthy airway epithelia that are replaced by honeycomb cysts. further future studies will be needed to determine which of the genes identified by our analysis are differentially regulated in specific cell populations. another weakness of our study is cross-sectional design in which we identify gene expression changes associated with a decline in lung function across a cohort of different individuals. a study of longitudinal design with measurements of gene expression and lung function over time in same individuals will be needed to validate these findings. however, the finding that rhotekin expression was associated with more rapid disease progression in our earlier study supports the notion that genes identified in our study have direct relevance to disease progression.  in summary, we identified commonalities and differences in gene among different subtypes of iip. disease progression, as characterized by lower measures of fvc and dlco, results in marked changes in expression of novel and established genes and pathways involved in iip. these genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for iip severity. 
disease progression, as characterized by lower measures of fvc and dlco, results in marked changes in expression of novel and established genes and pathways involved in iips. these genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for iip severity. using gene expression profiling of lung tissue specimens with iip diagnosis and control lungs, we identified genes whose expression is associated with changes in lung function modeled as categorical or continuous variables while adjusting for smoking status and iip subtype; false discovery rate approach was used to correct for multiple comparisons. since lung function is a key determinant of survival, we reasoned that the transcriptional profile in iip lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to iips. protein-protein interactome analysis of genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of iips is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. this analysis identified transcripts that are associated with mild vs severe disease, including those with established role in fibrosis as well as novel iip candidate genes such as rhotekin and peptidase inhibitor. we identified commonalities and differences in gene expression among different subtypes of iips. our findings were validated in an independent cohort of iips and controls at the mrna level and for one gene at the protein level by immunohistochemistry in a subset of samples. idiopathic interstitial pneumonias are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium.
 idiopathic interstitial pneumonias are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. since lung function is a key determinant of survival, we reasoned that the transcriptional profile in iip lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to iips.  using gene expression profiling of lung tissue specimens with iip diagnosis and control lungs, we identified genes whose expression is associated with changes in lung function modeled as categorical or continuous variables while adjusting for smoking status and iip subtype; false discovery rate approach was used to correct for multiple comparisons. this analysis identified transcripts that are associated with mild vs severe disease, including those with established role in fibrosis as well as novel iip candidate genes such as rhotekin and peptidase inhibitor. protein-protein interactome analysis of genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of iips is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. our findings were validated in an independent cohort of iips and controls at the mrna level and for one gene at the protein level by immunohistochemistry in a subset of samples.  we identified commonalities and differences in gene expression among different subtypes of iips. disease progression, as characterized by lower measures of fvc and dlco, results in marked changes in expression of novel and established genes and pathways involved in iips. these genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for iip severity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 