BACKGROUND
recurrent airway obstruction  is a respiratory disease characterized by periods of airway obstruction caused by hyperresponsiveness to inhaled organic molds and endotoxins  <cit> .
clinically, affected horses exhibit a chronic, spontaneous cough, nasal discharge, and increased respiratory efforts associated with an elevation in maximal transpulmonary pressure change compared to healthy horses or horses with inflammatory airway disease   <cit> .
diagnosis is based on history, clinical signs, and diagnostic tests.
endoscopic evaluation of rao-affected horses reveals excessive mucopurulent exudate in the tracheobronchial tree  <cit> .
cytological analysis of bronchoalveolar lavage fluid  is characterized by non-septic inflammation with increase in mucus and neutrophils   <cit> .
various pulmonary function tests allow quantification of the degree of airway obstruction  <cit> .
the immunological basis for rao is controversial.
a number of studies found that cytokine profiles are consistent with th <dig> type response - <dig>  il-13)  <cit> .
other studies, however, suggest that a th <dig> response and cytokines  are responsible for neutrophil recruitment in rao  <cit> .
a study performed with horses affected by summer pasture-associated obstructive pulmonary disease  revealed that the expression of th <dig> and th <dig> cytokines varies throughout the year  <cit> .
the type and amount of key cytokines and other intracellular regulatory and transcription factors that are expressed upon contact with an antigen modulate the inflammatory response.
characterization of key interactions and pathways would be helpful in understanding the inflammatory response in rao horses and whether it fits the rodent derived th1/th <dig> paradigm.
several studies suggest a strong genetic basis with a complex mode of inheritance for rao.
segregation and genomic analyses performed on two swiss warmblood families have led to the conclusion that the mode of inheritance of rao is characterized by major gene effects, and that these genes differ between families.
in the first of these families, rao was transmitted in an autosomal recessive mode and the major association was found on equine chromosome  <dig> , whereas in the second, it was transmitted in an autosomal dominant mode and the major association was found on eca <dig>  <cit> .
interestingly, horses from both families showed no phenotypical differences in the expression of rao, including clinical scores, endoscopic mucus scores, balf and tracheo-bronchial secretion cytology, response to methacholine challenge and values of arterial oxygenation  <cit> .
these results suggest genetic heterogeneity for the clinical phenotype rao.
proteomic and peptidomic analyses shed light on the metabolic status of biological systems and represent new approaches in the study of complex diseases like asthma and lung cancer in humans  <cit>  and animal models of human diseases  <cit> .
recent research in proteomics improved disease phenotype characterization based on peripheral blood biomarkers or balf cytokines in human suffering from asthma and chronic obstructive pulmonary disease  <cit> .
one of the major challenges in proteomic analysis is the large amount of data generated, which makes bioinformatics software capable of processing the information indispensable  <cit> .
for the present study, we used genomic and proteomic data previously collected from healthy and rao-affected horses, and performed a comparison using bioinformatics software .
the tool "path explorer" was used to search for documented molecular interactions based on the ingenuity® knowledge base.
this database contains millions of documented and published molecular interactions .
proteins present in balf from rao-affected horses and controls were identified by mass spectrometry  <cit>  and these data were imported into ipa®.
information about eight candidate genes for rao identified in a family-based whole-genome scan study  <cit>  was also imported into ipa® to identify documented pathways linking these candidate genes to the balf proteins identified with proteomics.
thus, this study compares genomic and proteomic data within the framework of ipa® in order to 1) identify the number of interactions between candidate genes for rao and proteins detected by proteomic analyses and 2) characterize the interacting proteins and pathways involved.
RESULTS
the following candidate genes  <cit>  were investigated for interactions with the set of proteins detected in balf: interleukin  <dig> receptor , il-21r, chemokine  ligand  <dig> , il- <dig>  prostaglandin e receptor  <dig> , phosphodiesterase 4d , suppressor of cytokine signaling  <dig>  and il-7r.
ipa® identified only a few direct interactions between the eight candidate genes and balf proteins.
products of the following four gene candidates, socs <dig>  il-7r, ptger <dig>  and pde4d, were predicted to directly interact with proteins identified in balf.
direct interactions between protein products of candidate genes and detected proteins were the following: socs <dig> interacts with il-4r, il-7r with forkhead box p <dig> , and ptger <dig> as well as pde4d with arrestin beta  <dig> .
arrb <dig> and il-4r are downregulated and foxp <dig> is upregulated in rao horses according to proteomic analysis.
no other interactions were identified.
from a total of  <dig> proteins identified in the proteomics study,  <dig>  were reported to have indirect interactions with il-4r,  <dig>  with il-21r,  <dig>  with ccl <dig>  and  <dig>  with il- <dig>
to identify the maximum number of possible interactions affiliated with il-4r, ipa® proposed  <dig> "connecting proteins" based on known pathways: tumor necrosis factor , interferon gamma , interleukin  <dig> , guanine nucleotide binding protein , beta polypeptide 2-like  <dig> , signal-regulatory protein alpha , and phosphatase and tensin homolog  .
according to proteomics results,  <dig>  of the  <dig> interacting proteins are downregulated in rao horses, while  <dig>  are upregulated.
six proteins  show controversial expression from the proteomics results .
twenty  interacting proteins are located in the extracellular space,  <dig>  in the cell membrane,  <dig>  in the cytoplasm, and  <dig>  in the nucleus .
extracellular proteins are involved in lungs' innate immunity  and in acquired immunity , amongst other functions .
one extracellular protein in particular, lymphotoxin beta , also operates as a member of the tumor necrosis factor family.
the majority of cell-membrane proteins participate in immune processes such as adhesive interactions of granulocytes , while others are immunoglobulins  or proteins of the major histocompatibility complex class ii .
such heterogeneity of functions can also be seen in cytoplasmic proteins.
these proteins are involved in the induction of nf-κb , protection of cells from oxidative stress , or participate in cell lysis and in cell-mediated immune responses .
proteins present in the nucleus, on the other hand, are assumed to play a role in the regulatory mechanisms of apoptosis, cancerogenesis , immune-regulation  and regulation of nf-κb activity .
figure  <dig> illustrates the different types of interactions between il-4r and other proteins.
interleukin  <dig> receptor
upregulated in rao: igkc; cfb; lamb3; c3; serpina1; c5; muc20; itgam; ighm; hladqb1; rps6ka4; snx9; actb; nod2; gls; thop1; ftl; ald7h7a1; gzmb; igj; hnrnpa3; rb1; foxp3; prkdc;
interleukin  <dig> receptor
upregulated in rao: c5;serpina1; cfb; itgam; hnrnpa3; rb1; actb; nod2; foxp3; aldh7a1; ftl; gls; gzmb; thop1; hla-dqb1; itgam
chemokine  ligand 24
upregulated in rao: c5; igj; c3; ighm; gzmb; thop1; rb1; prkdc; foxp3;
interleukin 27
mucin  <dig>  cell surface associated ;
apart from cd <dig> ligand , which is only present in the il-21r pathway, "connecting proteins" proposed by ipa and indirectly interacting proteins present in these two pathways always appear in the il-4r pathway.
protein product of il- <dig> was found in three indirect interactions, namely hla-dqb <dig>  c <dig>  and foxp <dig> .
discussion
the main conclusions drawn from this comparative analysis of genomic and proteomic data are as follows: 1) four of the eight candidate genes, namely socs <dig>  il-7r, ptger <dig>  and pde4d, are implicated in direct interactions with proteins identified in balf; 2) il-4r, il-21r and ccl <dig> are related to the identified balf proteins by a large number of indirect interactions; 3) these indirect interactions highlight intracellular regulatory mechanisms that might play central roles in rao pathophysiology ; 4) the interaction of socs <dig> with il-4r might explain how genomic heterogeneity in rao-affected horses results in the same phenotype.
socs <dig> encodes for a protein which directly interacts with il-4r.
this gene is one of the main rao-candidates in swiss warmblood family  <dig>  <cit>  and is located in a region of eca <dig> associated with the disease.
socs <dig> downregulates the cytokine signal transduction by terminating intracellular signaling in a variety of ways.
this can happen with the ubiquitination and subsequent degradation of the janus kinase  and signal transducers and activators of transcription  receptors.
socs <dig> is predominantly produced in th <dig> cells and inhibits differentiation toward the th <dig> cell type.
in humans, its role has been implicated in the pathogenesis of allergic asthma  <cit> .
interestingly, the fact that socs <dig> and il-4r directly interact might give a molecular explanation as to why the disease is clinically indistinguishable in both families  <cit> .
genetic variations in il4r and socs <dig> may have various effects during rna replication or splicing, or may influence protein function and stability.
a molecular explanation for disease expression might be that a socs <dig> variant could lead to a protein with a different biological activity that could modulate il-4r function and lead to the same effect as an il-4r mutation.
as il-4r and socs <dig> are located upstream in the signaling cascades, genetic mutations of the coding genes might lead to the same phenotype, thus explaining genetic heterogeneity in rao.
ptger <dig> and pde4d are located in a region of eca <dig> associated with rao in both swiss warmblood family  <dig> and  <dig>  they directly interact with an important intracellular molecule that was detected during proteomic analysis, namely arrb <dig>  arrb <dig> not only inhibits g protein-coupled receptor signaling, but also upregulates gene transcription of b-cell cll/lymphoma  <dig>  this anti-apoptotic factor enhances the survival of cd4+ t cells and might be one explanation for autoimmunity in humans suffering from multiple sclerosis  <cit> .
finally, il-7r, a candidate gene from eca <dig>  directly interacts with foxp <dig> which is a regulatory t-cell transcription factor implicated in the pathophysiology of human asthma.
il-4r was examined in this study both as an rao-gene candidate and as one of the proteins detected by proteomic evaluation of balf.
il-4r was previously identified as an rao candidate gene on eca <dig> in swiss warmblood family  <dig>  <cit> .
also, il-4r was found to have the greatest number of indirect interactions with the proteins detected in the balf according to the pathways suggested by ipa®.
another rao-gene candidate from the same chromosomal region, il-21r, shares homologies with the il-4r alpha chain, and functions along a similar pathway.
since these two genes might be the most promising of all rao-candidate genes, focusing on the il-4r-pathway is likely to produce valuable insights into the signaling cascades ultimately leading to the disease.
the three most crucial "connecting proteins" to this pathway are the tnf-family, ifn-γ, and il- <dig>  and thus deserve special attention.
the tnf-family consists of three members: tnf-α, lymphotoxin-α , and lymphotoxin-β .
a large number of proteins identified by proteomic analysis of balf  interact with the tnf-family in the il-4r pathway and it is noteworthy that ltb was also identified in balf.
these cytokines are mostly membrane-bound, produced by macrophages and t cells, and fulfill multiple functions within immune response mechanisms.
mast cells can also produce large amounts of tnf-α  <cit>  and interactions between these cells may be involved in neutrophil recruitment to the airway mucosa.
important functions of tnf-α that may play a role in the pathophysiology of rao, are neutrophil recruitment and induction of proinflammatory cytokines via activation of the nfκb pathway.
the proteins itgam, nod <dig>  rps6ka <dig>  and foxp <dig> that were detected in the proteomic analysis substantiate the importance of the tnf-family and nfκb pathways.
tnf-proteins interact with receptors of the tumor necrosis factor receptor family , which in turn communicate with an intracellular signaling pathway known as tumor necrosis factors receptor associated factors .
upregulation of tnf-α leads to a rapid externalization of granules containing p-selectin , to an increased expression of e-selectin, and to a strong expression of the intercellular adhesion molecule  <dig>  in endothelial cells.
itgam is located on the surface of neutrophils and interacts with icam- <dig>  these mechanisms are critical events in neutrophil extravasation.
on an intracellular level, tnf-receptor activation leads to apoptosis or to signals that promote new gene expression through activation of nfκb.
nod <dig> is a cytoplasmic protein that recognizes bacterial proteoglycans and then activates nfκb.
both nuclear factors rps6ka <dig> and foxp <dig> repress transcription of nfκb.
foxp <dig> is a transcription factor expressed by regulatory t cells.
these cells are able to modulate the activation of other t cells  and play a key role in immune homeostasis  <cit> .
in humans affected by asthma, foxp <dig> protein expression within cd4+cd25high t-cells is significantly decreased compared to controls  <cit> .
cytokines like il- <dig> and tnf-α are induced by nfκb activation  <cit>  and may play a role in neutrophil recruitment during an rao crisis.
this is illustrated by a study that detected high levels of nfκb activity in bronchial cells of rao-affected horses in comparison to healthy horses  <cit> .
therefore, our data highlight several factors that all deserve further investigations in order to evaluate their impact in rao-pathophysiology, especially regarding the neutrophils recruitment, one of the key mechanisms leading to this disease.
the ipa® platform generates pathways based on experimentally identified genes or proteins reported in the literature.
in this study, for each pathway,  <dig> balf proteins identified using proteomics and one candidate gene were specified so that ipa® could indentify interactions.
furthermore, "connecting proteins" were added by the software in order to expand pathways.
this approach presupposes that the likelihood of a candidate gene playing a pivotal role in the pathophysiology of rao increases if that gene stands in multiple interactions with proteins detected in the proteomic analysis.
the "connecting proteins" tnf, ifn-γ, and il- <dig> act at the origin of signaling cascades and display pleiotropic effects.
hence their effective concentrations may be low or undetectable, which would explain why they were not detected in the proteomic analysis.
protein expression might also differ depending on whether tissue samples  or balf were examined.
furthermore, the heterogeneity and dynamic nature of rao suggest that additional proteomic analysis of balf from a wide range of naturally-occurring cases of rao is warranted before results from this study may be generalized and the implicated proteins and their effects on the lower airways of horses can be confirmed.
another issue is that ipa® builds connections based on literature from humans, mice, rats and cell cultures.
although fundamental immune and inflammatory pathways are conserved, there may be some important interspecies differences.
furthermore, transcriptomic and metabolomic analyses might provide much-needed information about smaller molecules  and their regulatory effects on the pathophysiology of rao, and thus constitute another important research target.
for instance, microarray assays and differential display polymerase chain reaction  have already been used to explore differential gene expression in rao  <cit> .
ultimately, integration of information gained from all of these approaches may be needed to better understand the complex molecular pathogenesis of rao.
CONCLUSIONS
the integration of proteomic and genomic data on rao, collected by two independent approaches, has produced a new set of data that identifies novel interactions which emphasize a central role for il-4r and socs <dig> and implicate downstream intracellular signaling cascades.
indeed, these insights formulate the first molecular hypotheses on a proteomic level to explain the observation of genetic heterogeneity in rao.
this study thus illustrates the value of bioinformatics software for the analysis of large and complex sets of data and offers new approaches for the study of complex immunological diseases such as rao.

 recurrent airway obstruction is a respiratory disease characterized by periods of airway obstruction caused by hyperresponsiveness to inhaled organic molds and endotoxins. clinically, affected horses exhibit a chronic, spontaneous cough, nasal discharge, and increased respiratory efforts associated with an elevation in maximal transpulmonary pressure change compared to healthy horses or horses with inflammatory airway disease. diagnosis is based on history, clinical signs, and diagnostic tests. endoscopic evaluation of rao-affected horses reveals excessive mucopurulent exudate in the tracheobronchial tree. cytological analysis of bronchoalveolar lavage fluid is characterized by non-septic inflammation with increase in mucus and neutrophils. various pulmonary function tests allow quantification of the degree of airway obstruction. the immunological basis for rao is controversial. a number of studies found that cytokine profiles are consistent with th type response - il-13). other studies, however, suggest that a th response and cytokines are responsible for neutrophil recruitment in rao. a study performed with horses affected by summer pasture-associated obstructive pulmonary disease revealed that the expression of th and th cytokines varies throughout the year. the type and amount of key cytokines and other intracellular regulatory and transcription factors that are expressed upon contact with an antigen modulate the inflammatory response. characterization of key interactions and pathways would be helpful in understanding the inflammatory response in rao horses and whether it fits the rodent derived th1/th paradigm. several studies suggest a strong genetic basis with a complex mode of inheritance for rao. segregation and genomic analyses performed on two swiss warmblood families have led to the conclusion that the mode of inheritance of rao is characterized by major gene effects, and that these genes differ between families. in the first of these families, rao was transmitted in an autosomal recessive mode and the major association was found on equine chromosome, whereas in the second, it was transmitted in an autosomal dominant mode and the major association was found on eca. interestingly, horses from both families showed no phenotypical differences in the expression of rao, including clinical scores, endoscopic mucus scores, balf and tracheo-bronchial secretion cytology, response to methacholine challenge and values of arterial oxygenation. these results suggest genetic heterogeneity for the clinical phenotype rao. proteomic and peptidomic analyses shed light on the metabolic status of biological systems and represent new approaches in the study of complex diseases like asthma and lung cancer in humans and animal models of human diseases. recent research in proteomics improved disease phenotype characterization based on peripheral blood biomarkers or balf cytokines in human suffering from asthma and chronic obstructive pulmonary disease. one of the major challenges in proteomic analysis is the large amount of data generated, which makes bioinformatics software capable of processing the information indispensable. for the present study, we used genomic and proteomic data previously collected from healthy and rao-affected horses, and performed a comparison using bioinformatics software. the tool "path explorer" was used to search for documented molecular interactions based on the ingenuity® knowledge base. this database contains millions of documented and published molecular interactions. proteins present in balf from rao-affected horses and controls were identified by mass spectrometry and these data were imported into ipa®. information about eight candidate genes for rao identified in a family-based whole-genome scan study was also imported into ipa® to identify documented pathways linking these candidate genes to the balf proteins identified with proteomics. thus, this study compares genomic and proteomic data within the framework of ipa® in order to 1) identify the number of interactions between candidate genes for rao and proteins detected by proteomic analyses and 2) characterize the interacting proteins and pathways involved.  the following candidate genes were investigated for interactions with the set of proteins detected in balf: interleukin receptor, il-21r, chemokine ligand, il- prostaglandin e receptor, phosphodiesterase 4d, suppressor of cytokine signaling and il-7r. ipa® identified only a few direct interactions between the eight candidate genes and balf proteins. products of the following four gene candidates, socs il-7r, ptger and pde4d, were predicted to directly interact with proteins identified in balf. direct interactions between protein products of candidate genes and detected proteins were the following: socs interacts with il-4r, il-7r with forkhead box p, and ptger as well as pde4d with arrestin beta. arrb and il-4r are downregulated and foxp is upregulated in rao horses according to proteomic analysis. no other interactions were identified. from a total of proteins identified in the proteomics study, were reported to have indirect interactions with il-4r, with il-21r, with ccl and with il- to identify the maximum number of possible interactions affiliated with il-4r, ipa® proposed "connecting proteins" based on known pathways: tumor necrosis factor, interferon gamma, interleukin, guanine nucleotide binding protein, beta polypeptide 2-like, signal-regulatory protein alpha, and phosphatase and tensin homolog. according to proteomics results, of the interacting proteins are downregulated in rao horses, while are upregulated. six proteins show controversial expression from the proteomics results. twenty interacting proteins are located in the extracellular space, in the cell membrane, in the cytoplasm, and in the nucleus. extracellular proteins are involved in lungs' innate immunity and in acquired immunity, amongst other functions. one extracellular protein in particular, lymphotoxin beta, also operates as a member of the tumor necrosis factor family. the majority of cell-membrane proteins participate in immune processes such as adhesive interactions of granulocytes, while others are immunoglobulins or proteins of the major histocompatibility complex class ii. such heterogeneity of functions can also be seen in cytoplasmic proteins. these proteins are involved in the induction of nf-κb, protection of cells from oxidative stress, or participate in cell lysis and in cell-mediated immune responses. proteins present in the nucleus, on the other hand, are assumed to play a role in the regulatory mechanisms of apoptosis, cancerogenesis, immune-regulation and regulation of nf-κb activity. figure illustrates the different types of interactions between il-4r and other proteins. interleukin receptor upregulated in rao: igkc; cfb; lamb3; c3; serpina1; c5; muc20; itgam; ighm; hladqb1; rps6ka4; snx9; actb; nod2; gls; thop1; ftl; ald7h7a1; gzmb; igj; hnrnpa3; rb1; foxp3; prkdc; interleukin receptor upregulated in rao: c5;serpina1; cfb; itgam; hnrnpa3; rb1; actb; nod2; foxp3; aldh7a1; ftl; gls; gzmb; thop1; hla-dqb1; itgam chemokine ligand 24 upregulated in rao: c5; igj; c3; ighm; gzmb; thop1; rb1; prkdc; foxp3; interleukin 27 mucin cell surface associated; apart from cd ligand, which is only present in the il-21r pathway, "connecting proteins" proposed by ipa and indirectly interacting proteins present in these two pathways always appear in the il-4r pathway. protein product of il- was found in three indirect interactions, namely hla-dqb c and foxp. discussion the main conclusions drawn from this comparative analysis of genomic and proteomic data are as follows: 1) four of the eight candidate genes, namely socs il-7r, ptger and pde4d, are implicated in direct interactions with proteins identified in balf; 2) il-4r, il-21r and ccl are related to the identified balf proteins by a large number of indirect interactions; 3) these indirect interactions highlight intracellular regulatory mechanisms that might play central roles in rao pathophysiology; 4) the interaction of socs with il-4r might explain how genomic heterogeneity in rao-affected horses results in the same phenotype. socs encodes for a protein which directly interacts with il-4r. this gene is one of the main rao-candidates in swiss warmblood family and is located in a region of eca associated with the disease. socs downregulates the cytokine signal transduction by terminating intracellular signaling in a variety of ways. this can happen with the ubiquitination and subsequent degradation of the janus kinase and signal transducers and activators of transcription receptors. socs is predominantly produced in th cells and inhibits differentiation toward the th cell type. in humans, its role has been implicated in the pathogenesis of allergic asthma. interestingly, the fact that socs and il-4r directly interact might give a molecular explanation as to why the disease is clinically indistinguishable in both families. genetic variations in il4r and socs may have various effects during rna replication or splicing, or may influence protein function and stability. a molecular explanation for disease expression might be that a socs variant could lead to a protein with a different biological activity that could modulate il-4r function and lead to the same effect as an il-4r mutation. as il-4r and socs are located upstream in the signaling cascades, genetic mutations of the coding genes might lead to the same phenotype, thus explaining genetic heterogeneity in rao. ptger and pde4d are located in a region of eca associated with rao in both swiss warmblood family and they directly interact with an important intracellular molecule that was detected during proteomic analysis, namely arrb arrb not only inhibits g protein-coupled receptor signaling, but also upregulates gene transcription of b-cell cll/lymphoma this anti-apoptotic factor enhances the survival of cd4+ t cells and might be one explanation for autoimmunity in humans suffering from multiple sclerosis. finally, il-7r, a candidate gene from eca directly interacts with foxp which is a regulatory t-cell transcription factor implicated in the pathophysiology of human asthma. il-4r was examined in this study both as an rao-gene candidate and as one of the proteins detected by proteomic evaluation of balf. il-4r was previously identified as an rao candidate gene on eca in swiss warmblood family. also, il-4r was found to have the greatest number of indirect interactions with the proteins detected in the balf according to the pathways suggested by ipa®. another rao-gene candidate from the same chromosomal region, il-21r, shares homologies with the il-4r alpha chain, and functions along a similar pathway. since these two genes might be the most promising of all rao-candidate genes, focusing on the il-4r-pathway is likely to produce valuable insights into the signaling cascades ultimately leading to the disease. the three most crucial "connecting proteins" to this pathway are the tnf-family, ifn-γ, and il- and thus deserve special attention. the tnf-family consists of three members: tnf-α, lymphotoxin-α, and lymphotoxin-β. a large number of proteins identified by proteomic analysis of balf interact with the tnf-family in the il-4r pathway and it is noteworthy that ltb was also identified in balf. these cytokines are mostly membrane-bound, produced by macrophages and t cells, and fulfill multiple functions within immune response mechanisms. mast cells can also produce large amounts of tnf-α and interactions between these cells may be involved in neutrophil recruitment to the airway mucosa. important functions of tnf-α that may play a role in the pathophysiology of rao, are neutrophil recruitment and induction of proinflammatory cytokines via activation of the nfκb pathway. the proteins itgam, nod rps6ka and foxp that were detected in the proteomic analysis substantiate the importance of the tnf-family and nfκb pathways. tnf-proteins interact with receptors of the tumor necrosis factor receptor family, which in turn communicate with an intracellular signaling pathway known as tumor necrosis factors receptor associated factors. upregulation of tnf-α leads to a rapid externalization of granules containing p-selectin, to an increased expression of e-selectin, and to a strong expression of the intercellular adhesion molecule in endothelial cells. itgam is located on the surface of neutrophils and interacts with icam- these mechanisms are critical events in neutrophil extravasation. on an intracellular level, tnf-receptor activation leads to apoptosis or to signals that promote new gene expression through activation of nfκb. nod is a cytoplasmic protein that recognizes bacterial proteoglycans and then activates nfκb. both nuclear factors rps6ka and foxp repress transcription of nfκb. foxp is a transcription factor expressed by regulatory t cells. these cells are able to modulate the activation of other t cells and play a key role in immune homeostasis. in humans affected by asthma, foxp protein expression within cd4+cd25high t-cells is significantly decreased compared to controls. cytokines like il- and tnf-α are induced by nfκb activation and may play a role in neutrophil recruitment during an rao crisis. this is illustrated by a study that detected high levels of nfκb activity in bronchial cells of rao-affected horses in comparison to healthy horses. therefore, our data highlight several factors that all deserve further investigations in order to evaluate their impact in rao-pathophysiology, especially regarding the neutrophils recruitment, one of the key mechanisms leading to this disease. the ipa® platform generates pathways based on experimentally identified genes or proteins reported in the literature. in this study, for each pathway, balf proteins identified using proteomics and one candidate gene were specified so that ipa® could indentify interactions. furthermore, "connecting proteins" were added by the software in order to expand pathways. this approach presupposes that the likelihood of a candidate gene playing a pivotal role in the pathophysiology of rao increases if that gene stands in multiple interactions with proteins detected in the proteomic analysis. the "connecting proteins" tnf, ifn-γ, and il- act at the origin of signaling cascades and display pleiotropic effects. hence their effective concentrations may be low or undetectable, which would explain why they were not detected in the proteomic analysis. protein expression might also differ depending on whether tissue samples or balf were examined. furthermore, the heterogeneity and dynamic nature of rao suggest that additional proteomic analysis of balf from a wide range of naturally-occurring cases of rao is warranted before results from this study may be generalized and the implicated proteins and their effects on the lower airways of horses can be confirmed. another issue is that ipa® builds connections based on literature from humans, mice, rats and cell cultures. although fundamental immune and inflammatory pathways are conserved, there may be some important interspecies differences. furthermore, transcriptomic and metabolomic analyses might provide much-needed information about smaller molecules and their regulatory effects on the pathophysiology of rao, and thus constitute another important research target. for instance, microarray assays and differential display polymerase chain reaction have already been used to explore differential gene expression in rao. ultimately, integration of information gained from all of these approaches may be needed to better understand the complex molecular pathogenesis of rao.  the integration of proteomic and genomic data on rao, collected by two independent approaches, has produced a new set of data that identifies novel interactions which emphasize a central role for il-4r and socs and implicate downstream intracellular signaling cascades. indeed, these insights formulate the first molecular hypotheses on a proteomic level to explain the observation of genetic heterogeneity in rao. this study thus illustrates the value of bioinformatics software for the analysis of large and complex sets of data and offers new approaches for the study of complex immunological diseases such as rao. 
research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. recurrent airway obstruction is a severe chronic respiratory disease affecting horses worldwide, though mostly in the northern hemisphere. the goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid collected from healthy and rao-affected horses. interleukin receptor and chemokine ligand also showed a large number of interactions with the group of detected proteins. the gene with the greatest number of indirect interactions with the set of proteins identified is interleukin receptor, whose protein has also been detected in balf. protein products of other genes like that of socs revealed direct interactions with the il-4r protein. the interacting proteins nod rps6ka and foxp found in several pathways are reported regulators of the nfκb pathway.
 recurrent airway obstruction is a severe chronic respiratory disease affecting horses worldwide, though mostly in the northern hemisphere. environmental as well as genetic factors strongly influence the course and prognosis of the disease. research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. the goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid collected from healthy and rao-affected horses. the analysis was carried out with ingenuity pathway analysis® bioinformatics software.  the gene with the greatest number of indirect interactions with the set of proteins identified is interleukin receptor, whose protein has also been detected in balf. interleukin receptor and chemokine ligand also showed a large number of interactions with the group of detected proteins. protein products of other genes like that of socs revealed direct interactions with the il-4r protein. the interacting proteins nod rps6ka and foxp found in several pathways are reported regulators of the nfκb pathway.  the pathways generated with il-4r highlight possible important intracellular signaling cascades implicating, for instance, nfκb. furthermore, the proposed interaction between socs and il-4r could explain how different genes can lead to identical clinical rao phenotypes, as observed in two swiss warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit. 