BACKGROUND
atherosclerosis is an inflammatory disease with complex multifactorial pathophysiology.
in addition to the chronic inflammation in the vessel wall causing vascular endothelial dysfunction, the progression of the disease may be enhanced by persistent systemic infection, low-grade inflammation, and metabolic disturbances of major organs such as liver and adipose tissue .
at is a multi-functional metabolic organ composed of a heterogeneous cell population.
by storing and releasing fatty acids and secreting several important adipokines, at plays a significant role in both energy metabolism and homeostatic regulation of the endocrine system.
the at dysfunction, followed by the spill-over of free fatty acids  into the circulation via enhanced lipolysis and dysregulated expression of adipokines, modifies immune responses and contributes to metabolic disorders such as metabolic syndrome and type  <dig> diabetes  <cit> .
at depots are dispersed throughout the body and are closely associated with several organs  <cit> .
recent studies have reported many site-specific physiological characteristics in at depots, i.e.
differences in cell populations, microvasculature, innervation, and regional variation in metabolic functions including adipokine profiles, lipase activities, uptake of fatty acids, and the insulin response  <cit> .
these differences have a major impact on the proinflammatory potential of the various at depots.
in humans, visceral at has been shown to generate angiotensin, interleukin  - <dig>  and plasminogen activator inhibitor - <dig> at higher levels and leptin and adiponectin at lower levels than subcutaneous at  <cit> .
differences in hormone-sensitive lipase activity and maximum lipolytic capacity between omental and subcutaneous at depots have been reported; however, these phenomena seem to be strongly correlated to the adipocyte size and their capacity to expand  <cit> .
in obese humans, perivisceral at has been reported to contain more saturated fatty acids  and monounsaturated fatty acids  than subcutaneous at  <cit> .
in mice, high-fat feeding results in a continuous increase in adipocyte size both in subcutaneous and visceral fat.
interestingly, large adipocytes contain relatively more stearic acid and oleic acid, and these cells produce more tumour necrosis factor -α, leptin, angiotensinogen, adipsin, and pai- <dig> compared with small adipocytes  <cit> .
although at has considerable pro-inflammatory and pro-atherogenic potential, and contributes to systemic innate immune responses, the impact of chronic systemic infections on at is rarely investigated.
many pathogens related to common chronic infections, such as gastritis, periodontitis, and pulmonary infections, are lipopolysaccharide  containing gram-negative species.
although lps promotes inflammation mainly through its effects on the monocyte/macrophage function, an in vitro study has demonstrated that subcutaneous human adipocytes secrete large quantities of tnf-α when activated by lps  <cit> .
szalowska et al.
<dig> have shown that lps activation of human omental fat results in up-regulated expression of inflammation- and angiogenesis-related genes  <cit> .
the periodontal pathogen aggregatibacter actinomycetemcomitans and the persistent pulmonary infection agent chlamydia pneumoniae are gram-negative pathogens and associated with an increased risk for cardiovascular diseases  <cit> .
both pathogens have been found in human atherosclerotic lesions  <cit> , accelerate atherosclerosis in mouse model  <cit> , and are highly common among general population  <cit> .
we have recently shown in apolipoprotein e -deficient mouse model that systemic a. actinomycetemcomitans and c. pneumoniae infections may induce broad range pro-atherogenic effects including alterations in macrophage cholesterol homeostasis, endothelial function  <cit> , and, moreover, in the liver morphology, fatty acid composition, and inflammation marker profile  <cit> .
in the present study, we investigated the effects of systemic recurrent a. actinomycetemcomitans and chronic c. pneumoniae infections on epididymal and inguinal at depots in apoe-deficient mice.
in addition, combined infection model was used to study the impact of pathogen burden on these depots.
apoe-deficient mice display a proatherogenic plasma lipoprotein profile and they develop atherosclerotic lesions on a chow diet.
in addition, due to decreased levels of lps-neutralizing high-density lipoproteins, apoe-deficient mice show increased susceptibility to lps-mediated inflammation compared to wild-type mice  <cit> .
in order to include both visceral and subcutaneous at and to identify their putative depot-specific pro-inflammatory and pro-atherogenic potential, we utilized both epididymal and inguinal at depots.
we focused on molecules associated with inflammation and lipid homeostasis, which were investigated on the level of gene expression and cellular fat composition.
methods
mice
male apolipoprotein  e-deficient mice  were purchased from charles river laboratories.
the mice were fed regular mouse chow ab libitum and maintained in a germ-free environment at the national public health institute animal facilities .
animal care and experimentation were conducted under animal care and use committee of national public health institute ethical authorization and were in accordance with the guidelines of the council of europe.
bacterial cultures, infection procedure, and serum analyses
the bacteria cultures and experimental design have been previously reported  <cit> .
briefly, the clinical a. actinomycetemcomitans strain at445b  was cultivated in a tryptic soy-serum-bacitracin-vancomycin medium at 37°c with 5% co <dig> for 48 h. the c. pneumoniae isolate, kajaani  <dig>  a finnish epidemic strain, was isolated and cultured as previously described  <cit> .
the infection procedure is presented in table  <dig>  the experimental design included chronic c. pneumoniae infection model, which was achieved by three consecutive inoculations, recurrent a. actinomycetemcomitans infection model mimicking repeated systemic exposure to the pathogen, and a combination of these two infection models to imitate the effects of infection burden.
chronic c. pneumoniae infection was induced in younger mice e in order to see the potential effect of persistence.
viable c. pneumoniae  was administered intranasally as 2 ×  <dig> inclusion forming units in 40 μl of sucrose-phosphate-glutamic acid buffer , and viable a. actinomycetemcomitans  was administered intravenously  as 1 ×  <dig> colony forming units in 50 μl of  <dig> % nacl.
the 10-week experiment included four groups: 1) recurrent aa infection group  with ten aa inoculations; 2) the chronic cpn infection group  with three cpn inoculations; 3) the combined chronic cpn and recurrent aa infection group  with three cpn inoculations and ten aa inoculations; and 4) the control group  with three spg inoculations and ten  <dig> % nacl inoculations.
the black squares represent cpn inoculations and the black circles aa inoculations.
the controls received the respective vehicle .
†, sacrifice; aa, a. actinomycetemcomitans; cpn, c. pneumoniae.
the serum levels of nonesterified fatty acids , the lps activity , and the serum levels of tnfα  were measured as described in our previous publications  <cit> .
preparation of adipose tissues
inguinal and epididymal at depots were carefully dissected from sacrificed animals.
the tissue specimens were stabilized with rnalater™  or snap-frozen in liquid nitrogen.
all the samples were stored at -70°c.
isolation and quality evaluation of rna
epididymal and inguinal at homogenization was performed with a mixer mill mm  <dig>  and total rna was extracted using the rneasy lipid tissue mini kit .
dnase treatment was performed on a column using the rnase-free dnase set  and again after rna extraction using the dna-free™ kit .
the rna concentration was determined using a nanodrop  <dig> spectrophotometer .
the quality of total rna was assessed by an agilent  <dig> bioanalyzer using the eukaryote total rna nano kit .
adipose tissue rna microarray processing
inguinal and epididymal at gene expression was analyzed using the illumina mouse wg- <dig> v <dig>  platform.
three biological replicates were used and the high quality total rna  was amplified using the illumina totalprep rna amplification kit according to the manufacturer’s instructions.
briefly, the total rna was reverse-transcribed using t <dig> oligo primers following the second strand cdna synthesis.
after purification, the cdna was in vitro transcripted into crna using biotin-labeled nucleoside triphosphates.
the quality of biotinylated crna was assessed by an agilent  <dig> bioanalyzer and  <dig>  μg of crna from each sample was hybridized at 58°c overnight using the illumina whole-genome gene expression direct hybridization assay system with the six-sample beadchip platform.
the signal was developed using streptavidin-cy <dig> and the beadchips were scanned with an illumina beadarray reader.
microarray data analyses
the microarray data has been deposited in miame-compliant format in gene expression omnibus , accession number gse <dig>  gene ontology  enrichment analysis for microarray data was performed using the fisher's exact test, where the observed frequency of each present go term was compared with the frequency in a reference gene set .
p-values were corrected for multiple hypotheses using the benjamini-hochberg false discovery rate  <cit> .
the analyses were performed using the r base package version  <dig> .
<dig>  the maximum of  <dig> most enriched go terms were reported for both epididymal and inguinal at and ranked according to the p-value.
to limit the data and focus on the biologically meaningful pathways and molecular functions, go terms referring to cellular components were excluded.
the fold-change  limitation for the presented up- and down-regulated genes was  <dig>  except when observing de novo lipogenesis-associated genes where all results are present.
quantitative real-time pcr
epididymal and inguinal at cdna was synthesized from total rna using the improm-ii™ reverse transcription system .
the amount of total rna in each reverse transcription  reaction was 240 ng for the inguinal at and 500 ng for the epididymal at.
the number of mice in each group was  <dig>  except for the control group where  <dig> mice were used.
primers  were designed using beacon designer  and national center for biotechnology information primer-blast  for cd <dig>  glyceraldehyde-3-phosphate dehydrogenase , monocyte chemoattractant protein  <dig> , macrophage migration inhibitory factor , mannose receptor c type  <dig> , myeloperoxidase , and secretory leukocyte peptidase inhibitor .
primer sequences are presented in additional file 1: table s <dig>  quantitative real-time pcr  was performed using the mx <dig> real-time qpcr system .
the analyses were conducted in  <dig>  μl reaction mixtures, each containing 2 μl of 1: <dig> dilution of cdna synthesized in rt reaction,  <dig>  μl of brilliant sybr® green qpcr master mix ,  <dig> nm of rox reference dye , and optimized concentrations of primers .
the amplifications were performed using the following thermocycling program: initial denaturation at 95°c for 15 min, 40 cycles of 15 s at 95°c and 1 min at 60°c followed by melting curve analysis for 1 min at 95°c, gradual decrease to 55°c, 30 s at 55°c, gradual increase to 95°c, and 30 s at 95°c.
all samples were analyzed as duplicates and the accepted intra-assay coefficient of variation for threshold cycle was ≤  <dig> %.
the results were analyzed using mx <dig> real-time qpcr system  software and expressed as a log <dig> fold change  to the respective control mice .
gapdh was used as a normalization gene and the software utilizes modified comparative quantitation method introduced by pfaffl   <cit> .
adipose tissue fatty acid analysis
total lipids were extracted from 30–80 mg of epididymal and inguinal adipose tissue with hexane-isopropanol   <cit>  and trans-methylated by heating with acidic methanol   <cit> .
the resulting fatty acid methyl ester compositions were analyzed by gas–liquid partition chromatography using a hewlett packard  <dig> gas chromatograph  with a db- <dig> gc column .
the samples were split injected with hydrogen as a carrier gas and the temperature program was from 160°c to 230°c.
the relative amounts of the methylated fatty acids were expressed as percentages of weight normalized to 100%.
statistical analyses
the at fatty acid data were expressed as mean percentages of weight with standard deviations .
the gene expression data were expressed as log <dig> fold change  medians with interquartile ranges .
the significance of the differences between the experimental and control mouse groups was assessed using the non-parametric mann–whitney u test.
bivariate correlations were determined using the spearman test.
analyses were performed using pasw statistics  <dig> .
RESULTS
infection-enriched biological pathways in the inguinal at
infection-enriched biological pathways  and the associated up- and down-regulated genes in the inguinal at transcriptome are presented in tables  <dig>   <dig> and  <dig>  compared with the control mice, the expression of “protein binding” pathway  was increased  most significantly in aa group .
in addition, the inflammation-related “antigen binding” , “antibacterial humoral response” , and “antimicrobial humoral response”  pathways were significantly enriched .
a. actinomycetemcomitans
infection-enriched go terms in the inguinal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
c. pneumoniae
infection-enriched go terms in the inguinal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
c. pneumoniae
and recurrent
a. actinomycetemcomitans
infections-enriched go terms in the inguinal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
compared with the control mice, most of the enriched go terms in the inguinal at transcriptome of cpn group were pathways associated with development- and morphogenesis .
the most affected pathway was “negative regulation of cellular process” pathway .
the most significantly enriched pathway in the inguinal at transcriptome of cpn + aa group was “protein binding”  followed by inflammation-related “antigen binding” , “antibacterial humoral response” , and “antimicrobial humoral response”  pathways .
lipid homeostasis-related “regulation of lipoprotein lipid oxidation”  and “lipoprotein lipid oxidation”  pathways were also enriched.
infection-enriched biological pathways in the epididymal at
table  <dig> presents the aa infection-enriched biological pathways in the epididymal at transcriptome.
compared with the control mice, in the epididymal at, infection enriched the following humoral antibody response and inflammation-associated pathways: “antigen binding” , “antibacterial humoral response” , “antimicrobial humo-ral response” , “immunoglobulin mediated immune response” , “humoral immune response mediated by circulating immunoglobulin” , and “inflammatory response” .
the lipid homeostasis-related “monocarboxylic acid metabolic process” , “fatty acid biosynthetic process” , “regulation of lipid metabolic process” , “negative regulation of lipid metabolic process” , “triglyceride metabolic process” , and “regulation of lipid biosynthetic process”  were also enriched.
a. actinomycetemcomitans
infection-enriched go terms in the epididymal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
table  <dig> presents the cpn infection-enriched pathways in the epididymal at transcriptome.
the enriched pathways included “very long-chain fatty acid metabolic process” , “long-chain fatty acid metabolic process” , “long-chain fatty acid-coa ligase activity” , and “very long-chain fatty acid-coa ligase activity”  pathways.
the most affected pathway was “defense response” .
c. pneumoniae
infection-enriched go terms in the epididymal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
table  <dig> presents the cpn + aa infections-enriched pathways in the epididymal at transcriptome.
the “protein binding” pathway  included the highest number of affected genes.
inflammation-related “antigen binding” , “antibacterial humoral response” , and “antimicrobial humoral response”  pathways were enriched.
there were no enrichments in lipid metabolism-related pathways.
c. pneumoniae
and recurrent
a. actinomycetemcomitans
infections-enriched go terms in the epididymal at transcriptome
a
up-regulated
b
b
ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate.
bfold change limit  <dig> .
number of biological replicates is  <dig>
go, gene ontology.
the maximum of  <dig> go categories is shown.
inflammation- and lipid homeostasis-related up- and down-regulated genes in the inguinal at
in aa group, most of the “protein binding” pathway-associated up-regulated genes belonged to immunoglobulin variables .
the most up-regulated genes were s <dig> calcium binding protein a <dig> , s <dig> calcium binding protein a <dig> , and lactotransferrin .
lipid homeostasis-associated leptin  was down-regulated .
in cpn group, infection induced down-regulation of acetyl-coenzyme a acyltransferase  <dig> , elovl family member  <dig>  elongation of long chain fatty acids , and nuclear receptor subfamily  <dig>  group d, member  <dig>  .
in cpn + aa group, the majority of up-regulated genes were immunoglobulin variables .
in addition, s100a <dig> , s100a <dig> , and ltf  were up-regulated.
lep  and synuclein δ  were down-regulated .
inflammation- and lipid homeostasis-related up- and down-regulated genes in the epididymal at
in aa group, the enriched “monocarboxylic acid metabolic process” pathway  included four up-regulated genes: pyruvate carboxylase , elovl <dig> , stearoyl-coenzyme a desaturase  <dig> , and insulin induced gene  <dig>  .
insig <dig>  cell death-inducing dna fragmentation factor, alpha subunit-like effector a  and thyroid hormone responsive  were associated with the “regulation of lipid metabolic process” pathway .
in addition, fatty acid binding protein  <dig>  and immunoglobulin variables were up-regulated .
in cpn group, the “defense response” pathway  included four defensin subtypes, 5′ nucleotidase, ecto , and chemokine  ligand  <dig>  .
the up-regulated solute carrier family  <dig> , member  <dig>  was associated with the lipid homeostasis-related pathways.
in cpn + aa group, a large number of up-regulated genes were immunoglobulin variables and the most up-regulated was ig gamma-2a chain c region secreted form-like  .
lipid homeostasis -associated arachidonate 12-lipoxygenase  was also up-regulated.
the expression of de novo lipogenesis-associated genes
figure  <dig> presents the expression of de novo lipogenesis -associated genes in the at depot transcriptomes among the different infection models .
in the epididymal transcriptome of aa-infected mice, the up-regulated genes were pcx , fatty acid synthase , scd <dig> , and elovl <dig>  .
fasn and elovl <dig> were down-regulated in the cpn group in both at depots  .
the combined infection model had little impact on de novo lipogenesis associated gene expression .
alterations in qpcr-analyzed mrna expression levels
the relative mrna expression levels of neutrophil activation-  and macrophage phenotype-  and activation-  related genes in inguinal and epididymal at are presented in table  <dig>  in the inguinal at, the relative expression levels of genes encoding slpi and mpo were significantly higher in the aa group  than in control mice .
cpn infection did not have a significant effect on the selected gene expression levels.
<dig>
fold change
a
p-value
b
mcp-1
mrc-1
cd68
mif
slpi
mpo
mcp-1
mrc-1
cd68
mif
slpi
amedian log <dig> fold change  to the control mice.
the control mice have been set into the value of  <dig>
bthe mann–whitney u test with the control mice as the reference group.
aa group, recurrent a. actinomycetemcomitans infection; cpn group, chronic c. pneumoniae infection; cpn + aa group, combined recurrent a. actinomycetemcomitans and chronic c. pneumoniae infections; iqr, interquartile range; mcp- <dig>  monocyte chemoattractant protein 1; mif, macrophage migration inhibitory factor, mrc- <dig>  mannose receptor c type 1; mpo, myeloperoxidase; slpi, secretory leukocyte peptidase inhibitor.
in the epididymal at, the relative mrna expression levels of mcp- <dig>  mif, and slpi in the aa group, mrc- <dig> and cd <dig> in cpn group, and mif in aa + cpn group were significantly altered but between - <dig> to  <dig> log2fc .
the cpn + aa combination induced a reduction in the mcp- <dig> mrna levels .
changes in at fatty acid composition
the proportions of fatty acids in the epididymal and inguinal at are presented in figure  <dig>  in the inguinal at, aa infection induced a significantly higher proportion of sfas  and a lower proportion of pufas  compared to the control mice.
the proportion of mufas was greater in the cpn group .
in the epididymal at, the proportions of sfas were higher among all infected mice but the difference reached statistical significance only in the aa group .
the proportion of pufas was significantly lower in the aa  and cpn + aa groups  .
when the two at depots were compared, the proportion of sfas was lower in the epididymal at than in the inguinal at in the control group  and the aa + cpn group .
associations between serum and at parameters
the associations between different serum and at variables were determined by correlation analysis  and presented as graphs in figure  <dig>  serum lps activity correlated positively with the serum ffa concentration  and with the proportion of sfa in the epididymal at , and negatively with both inguinal  and epididymal  proportions of pufa.
discussion
in the present study, we examined the inflammation- and lipid homeostasis-related effects of systemic a. actinomycetemcomitans and c. pneumoniae infections on at derived from chow-fed apoe-deficient mice.
these effects were examined on gene expression and fat composition level in both inguinal and epididymal at depots.
recurrent aa infection affected both at depots significantly: inflammation-related biological pathways were enriched in inguinal at and both inflammation- and lipid homeostasis-related pathways were enriched in epididymal at.
at fat distribution was altered and sfas were increased at the expense of pufas in both epididymal and inguinal at among all a. actinomycetemcomitans-infected mice.
the impact of chronic c. pneumoniae infection on at depots was less prominent.
the mouse models used in the present study were designed to display chronic infections.
our aa model is characterized by endotoxemia and systemic inflammation.
the observed increased humoral immune responses can be somewhat expected, because we have previously shown that the serum levels of lps activity, tnfα, and igg antibodies against a. actinomycetemcomitans after  <dig> intravenous injections of live bacteria are significantly higher in the aa group compared with the control group  <cit> .
the persistence of c. pneumonia after three inoculations was also shown as the pathogen was recovered from the lungs of the mice  <cit> .
since atherosclerosis is a multiorgan disease, our aim was to broaden the study from the lipid depots in heart and liver to adipose tissue, which plays an important role in sustaining systemic inflammation and proatherogenic lipid profile.
experimental acute endotoxemia has been shown to induce general insulin resistance and local and adaptive inflammation in human subcutaneous at  <cit> .
in our study, aa infection enriched inflammation-related pathways in the inguinal at.
qpcr analysis verified up-regulated expression of mpo and slpi, and the most up-regulated genes in the microarray analysis were s100a <dig> and ltf.
these innate immunity-associated gene products are considered anti-inflammatory mediators and they exhibit various antimicrobial functions such as promotion of oxidative activity responses  <cit> .
human slpi and ltf have been shown to co-localize in secondary granules and they are co-released after neutrophil activation  <cit> .
interestingly, increased plasma slpi levels were associated with progressive metabolic dysfunction in men  <cit> .
despite the up-regulation in neutrophil activation-related genes, we did not observe changes in the mrna expression of genes associated with classically or alternatively activated macrophages.
the response of aa infection in the epididymal at was quite distinct.
as in inguinal at, the humoral antibacterial response and immunoglobulin-related pathways were enriched.
however, the myeloid cell-specific signs of enhanced oxidative stress were absent, expression of slpi was somewhat down-regulated and, instead, several lipid homeostasis-related biological pathways were enriched.
on at fatty acid level, we observed a potentially adverse increase in the proportion of sfas among all aa-infected mice.
mengting et al.
<dig> reported that compared with lean mice, obese mice have a higher level of sfas in their epididymal at  <cit> .
an interesting study by suganami et al.
<dig> demonstrated that sfas released by hypertrophied adipocytes activate the nf-κb pathway in the macrophages with ensuing tnf-α release  <cit> .
the subsequent lipolytic release of sfas creates a positive feed-back loop between adipocytes and macrophages.
importantly, the macrophage-derived tnf-α also resulted in nf-κb-dependent release of proinflammatory cytokines from the adipocytes.
both lps and sfa have been shown to generate endoplasmic reticulum stress in primary human adipocytes  <cit> .
elevated levels of sfas in both epididymal and inguinal at depots in the aa and cpn + aa groups suggested an increased proinflammatory potential of these at depots.
in addition, because pufas have been reported to inhibit the tlr dimerization induced by sfas  <cit> , the significantly decreased proportion of pufas among all a. actinomycetemcomitans-infected mice may intensify the effects of increased sfas.
the proportion of pufas also showed a significant inverse association with lps serum activity.
this result may be due to lps-triggered enhanced oxidative stress in the at, followed by the peroxidation of pufas.
in addition to intracellular effects, lps has been shown to elevate the levels of circulating ffas in rodents in vivo <cit> .
this association was also found in our study as there was a strong positive correlation between serum lps activity and the ffa level.
lipid homeostasis-related biological pathways were enriched and four important de novo lipogenesis-related genes were moderately up-regulated in the epididymal at in aa group.
the observed up-regulation of pcx and fasn could partially explain the increase in the sfa proportion.
however, up-regulation of de novo lipogenesis genes was not observed in cpn and cpn + aa groups, indicating the involvement of other regulatory pathways affecting sfa levels in the at.
chronic infection with c. pneumoniae did not enhance the pathways associated with humoral antibody response, and moreover, the effects on the at depot fatty acid distribution were less evident than with aa infection.
however, the “defense response” pathway which included four up-regulated defensins was enriched in the epididymal at depot.
previously, c. pneumoniae infection in vitro was reported to induce human β-defensin  <dig> production mediated by the tlr <dig> pathway in monocytes  <cit> .
although both c. pneumoniae and a. actinomycetemcomitans are gram-negative pathogens, the effects of cpn infection differed considerably from those of aa infection.
this result may be due to the distinct infection routes and c. pneumoniae being an obligatory intracellular pathogen.
in addition, the difference in the infection time points may also influence the outcome and, altogether, the character of chronic infection differs from the recurrent one.
the combined infection model displayed a humoral antibody response and changes in at depot fa distribution similar to those with single aa infection.
nevertheless, indicators of the potentially amplified de novo lipogenesis in epididymal at remained absent.
the major limitation of the study was the small number of mice available for the microarray analysis, which resulted in non-significant gene fc-results after fdr correction.
although the apoe-deficient mouse model has been utilized widely in atherosclerosis research, it possesses certain drawbacks, such as a somewhat non-physiological lipoprotein profile  <cit> .
the adipocytes of apoe-deficient mice are smaller, contain fewer lipids, and accumulate less triglycerides from very low-density lipoproteins and chylomicrons compared with adipocytes derived from wild-type mice  <cit> .
in addition, because apoe has been shown to protect against lps-derived sepsis, apoe deficiency actually exposes mice to the deleterious and possibly proatherogenic effects of lps  <cit> .
nevertheless, the augmented susceptibility to lps may produce a more human-like response, because rodents are generally more resistant to lps-mediated effects than humans.
our study design included only males and therefore we could only draw gender-specific conclusions.
it is noteworthy that, in mice, approximately 20% of at genes exhibit sexual dimorphism , and the enriched functional categories with fc > <dig> include “lipid metabolism”  <cit> .
CONCLUSIONS
the present study introduces in vivo evidence that systemic infection of a. actinomycetemcomitans activates inflammation-related biological pathways and alters the lipid homeostasis adversely in both inguinal and epididymal at depots in apoe-deficient mice.
the results suggest that repeated endotoxemia promotes at dysfunction as shown by accumulation of saturated fat in the tissue and spill-over of free fatty acids into the circulation possibly via induced lipolysis.
these observations may be useful when studying the potential mechanism linking adipose tissue to the proposed atherogenicity of the chronic infections.
abbreviations
aa: aggregatibacter actinomycetemcomitans; apoe: apolipoprotein e; at: adipose tissue; cpn: chlamydia pneumoniae; fc: fold change; fdr: false discovery rate; ffa: free fatty acid; go: gene ontology; iqr: interquartile range; lps: lipopolysaccharide; mufa: monounsaturated fatty acid; pai 1: plasminogen activator inhibitor-1; pufa: polyunsaturated fatty acid; qpcr: quantitative polymerase chain reaction; sd: standard deviation; sfa: saturated fatty acid; spg: sucrose-phosphate-glutamic acid buffer; tlr: toll-like receptor; tnf-α: tumour necrosis factor α.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
kh, amt, sl, mj, ptk, ml, ps, ga, and pjp designed the study.
kh performed experiments, analyzed and interpreted data, initially drafted the manuscript, and prepared tables and figures.
amt, sl, and is performed experiments and edited the manuscript.
mj, ptk, ml, ps, ga, and pjp critically revised the manuscript and contributed to discussion.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
qpcr primer sequences.
click here for file
additional file 2: table s2
differentially expressed genes in the inguinal at transcriptome of recurrent a. actinomycetemcomitans-infected mice.
click here for file
additional file 3: table s3
differentially expressed genes in the inguinal at transcriptome of chronic c. pneumoniae-infected mice.
click here for file
additional file 4: table s4
differentially expressed genes in the inguinal at transcriptome of combined chronic c. pneumoniae and recurrent a. actinomycetemcomitans-infected mice.
click here for file
additional file 5: table s5
differentially expressed genes in the epididymal at transcriptome of recurrent a. actinomycetemcomitans-infected mice.
click here for file
additional file 6: table s6
differentially expressed genes in the epididymal at transcriptome of combined chronic c. pneumoniae-infected mice.
click here for file
additional file 7: table s7
differentially expressed genes in the epididymal at transcriptome of combined chronic c. pneumoniae and recurrent a. actinomycetemcomitans-infected mice.
click here for file
acknowledgements
the authors are grateful for the valuable work of saija perovuo, tiina karvonen, and pirjo laaksonen.
we also thank the personnel at biomedicum genomics  and lilli saarinen at the computational systems biology laboratory .
wihuri research institute is maintained by the jenny and antti wihuri foundation.
this work was supported by the sigrid juselius foundation ; the finnish dental society apollonia ; the paulo foundation ; the jenny and antti wihuri foundation ; the aarno koskelo foundation ; the ida montin foundation ; the finnish atherosclerosis society ; and the research council for the health, academy of finland, grant  <dig> .

 atherosclerosis is an inflammatory disease with complex multifactorial pathophysiology. in addition to the chronic inflammation in the vessel wall causing vascular endothelial dysfunction, the progression of the disease may be enhanced by persistent systemic infection, low-grade inflammation, and metabolic disturbances of major organs such as liver and adipose tissue. at is a multi-functional metabolic organ composed of a heterogeneous cell population. by storing and releasing fatty acids and secreting several important adipokines, at plays a significant role in both energy metabolism and homeostatic regulation of the endocrine system. the at dysfunction, followed by the spill-over of free fatty acids into the circulation via enhanced lipolysis and dysregulated expression of adipokines, modifies immune responses and contributes to metabolic disorders such as metabolic syndrome and type diabetes. at depots are dispersed throughout the body and are closely associated with several organs. recent studies have reported many site-specific physiological characteristics in at depots, i.e. differences in cell populations, microvasculature, innervation, and regional variation in metabolic functions including adipokine profiles, lipase activities, uptake of fatty acids, and the insulin response. these differences have a major impact on the proinflammatory potential of the various at depots. in humans, visceral at has been shown to generate angiotensin, interleukin - and plasminogen activator inhibitor - at higher levels and leptin and adiponectin at lower levels than subcutaneous at. differences in hormone-sensitive lipase activity and maximum lipolytic capacity between omental and subcutaneous at depots have been reported; however, these phenomena seem to be strongly correlated to the adipocyte size and their capacity to expand. in obese humans, perivisceral at has been reported to contain more saturated fatty acids and monounsaturated fatty acids than subcutaneous at. in mice, high-fat feeding results in a continuous increase in adipocyte size both in subcutaneous and visceral fat. interestingly, large adipocytes contain relatively more stearic acid and oleic acid, and these cells produce more tumour necrosis factor -α, leptin, angiotensinogen, adipsin, and pai- compared with small adipocytes. although at has considerable pro-inflammatory and pro-atherogenic potential, and contributes to systemic innate immune responses, the impact of chronic systemic infections on at is rarely investigated. many pathogens related to common chronic infections, such as gastritis, periodontitis, and pulmonary infections, are lipopolysaccharide containing gram-negative species. although lps promotes inflammation mainly through its effects on the monocyte/macrophage function, an in vitro study has demonstrated that subcutaneous human adipocytes secrete large quantities of tnf-α when activated by lps. szalowska et al. have shown that lps activation of human omental fat results in up-regulated expression of inflammation- and angiogenesis-related genes. the periodontal pathogen aggregatibacter actinomycetemcomitans and the persistent pulmonary infection agent chlamydia pneumoniae are gram-negative pathogens and associated with an increased risk for cardiovascular diseases. both pathogens have been found in human atherosclerotic lesions, accelerate atherosclerosis in mouse model, and are highly common among general population. we have recently shown in apolipoprotein e -deficient mouse model that systemic a. actinomycetemcomitans and c. pneumoniae infections may induce broad range pro-atherogenic effects including alterations in macrophage cholesterol homeostasis, endothelial function, and, moreover, in the liver morphology, fatty acid composition, and inflammation marker profile. in the present study, we investigated the effects of systemic recurrent a. actinomycetemcomitans and chronic c. pneumoniae infections on epididymal and inguinal at depots in apoe-deficient mice. in addition, combined infection model was used to study the impact of pathogen burden on these depots. apoe-deficient mice display a proatherogenic plasma lipoprotein profile and they develop atherosclerotic lesions on a chow diet. in addition, due to decreased levels of lps-neutralizing high-density lipoproteins, apoe-deficient mice show increased susceptibility to lps-mediated inflammation compared to wild-type mice. in order to include both visceral and subcutaneous at and to identify their putative depot-specific pro-inflammatory and pro-atherogenic potential, we utilized both epididymal and inguinal at depots. we focused on molecules associated with inflammation and lipid homeostasis, which were investigated on the level of gene expression and cellular fat composition. methods mice male apolipoprotein e-deficient mice were purchased from charles river laboratories. the mice were fed regular mouse chow ab libitum and maintained in a germ-free environment at the national public health institute animal facilities. animal care and experimentation were conducted under animal care and use committee of national public health institute ethical authorization and were in accordance with the guidelines of the council of europe. bacterial cultures, infection procedure, and serum analyses the bacteria cultures and experimental design have been previously reported. briefly, the clinical a. actinomycetemcomitans strain at445b was cultivated in a tryptic soy-serum-bacitracin-vancomycin medium at 37°c with 5% co for 48 h. the c. pneumoniae isolate, kajaani a finnish epidemic strain, was isolated and cultured as previously described. the infection procedure is presented in table  the experimental design included chronic c. pneumoniae infection model, which was achieved by three consecutive inoculations, recurrent a. actinomycetemcomitans infection model mimicking repeated systemic exposure to the pathogen, and a combination of these two infection models to imitate the effects of infection burden. chronic c. pneumoniae infection was induced in younger mice e in order to see the potential effect of persistence. viable c. pneumoniae was administered intranasally as 2 × inclusion forming units in 40 μl of sucrose-phosphate-glutamic acid buffer, and viable a. actinomycetemcomitans was administered intravenously as 1 ×  colony forming units in 50 μl of % nacl. the 10-week experiment included four groups: 1) recurrent aa infection group with ten aa inoculations; 2) the chronic cpn infection group with three cpn inoculations; 3) the combined chronic cpn and recurrent aa infection group with three cpn inoculations and ten aa inoculations; and 4) the control group with three spg inoculations and ten % nacl inoculations. the black squares represent cpn inoculations and the black circles aa inoculations. the controls received the respective vehicle. †, sacrifice; aa, a. actinomycetemcomitans; cpn, c. pneumoniae. the serum levels of nonesterified fatty acids, the lps activity, and the serum levels of tnfα were measured as described in our previous publications. preparation of adipose tissues inguinal and epididymal at depots were carefully dissected from sacrificed animals. the tissue specimens were stabilized with rnalater™ or snap-frozen in liquid nitrogen. all the samples were stored at -70°c. isolation and quality evaluation of rna epididymal and inguinal at homogenization was performed with a mixer mill mm and total rna was extracted using the rneasy lipid tissue mini kit. dnase treatment was performed on a column using the rnase-free dnase set and again after rna extraction using the dna-free™ kit. the rna concentration was determined using a nanodrop spectrophotometer. the quality of total rna was assessed by an agilent bioanalyzer using the eukaryote total rna nano kit. adipose tissue rna microarray processing inguinal and epididymal at gene expression was analyzed using the illumina mouse wg- v platform. three biological replicates were used and the high quality total rna was amplified using the illumina totalprep rna amplification kit according to the manufacturer’s instructions. briefly, the total rna was reverse-transcribed using t oligo primers following the second strand cdna synthesis. after purification, the cdna was in vitro transcripted into crna using biotin-labeled nucleoside triphosphates. the quality of biotinylated crna was assessed by an agilent bioanalyzer and  μg of crna from each sample was hybridized at 58°c overnight using the illumina whole-genome gene expression direct hybridization assay system with the six-sample beadchip platform. the signal was developed using streptavidin-cy and the beadchips were scanned with an illumina beadarray reader. microarray data analyses the microarray data has been deposited in miame-compliant format in gene expression omnibus, accession number gse gene ontology enrichment analysis for microarray data was performed using the fisher's exact test, where the observed frequency of each present go term was compared with the frequency in a reference gene set. p-values were corrected for multiple hypotheses using the benjamini-hochberg false discovery rate. the analyses were performed using the r base package version. the maximum of most enriched go terms were reported for both epididymal and inguinal at and ranked according to the p-value. to limit the data and focus on the biologically meaningful pathways and molecular functions, go terms referring to cellular components were excluded. the fold-change limitation for the presented up- and down-regulated genes was except when observing de novo lipogenesis-associated genes where all results are present. quantitative real-time pcr epididymal and inguinal at cdna was synthesized from total rna using the improm-ii™ reverse transcription system. the amount of total rna in each reverse transcription reaction was 240 ng for the inguinal at and 500 ng for the epididymal at. the number of mice in each group was except for the control group where mice were used. primers were designed using beacon designer and national center for biotechnology information primer-blast for cd glyceraldehyde-3-phosphate dehydrogenase, monocyte chemoattractant protein, macrophage migration inhibitory factor, mannose receptor c type, myeloperoxidase, and secretory leukocyte peptidase inhibitor. primer sequences are presented in additional file 1: table s quantitative real-time pcr was performed using the mx real-time qpcr system. the analyses were conducted in  μl reaction mixtures, each containing 2 μl of 1: dilution of cdna synthesized in rt reaction,  μl of brilliant sybr® green qpcr master mix, nm of rox reference dye, and optimized concentrations of primers. the amplifications were performed using the following thermocycling program: initial denaturation at 95°c for 15 min, 40 cycles of 15 s at 95°c and 1 min at 60°c followed by melting curve analysis for 1 min at 95°c, gradual decrease to 55°c, 30 s at 55°c, gradual increase to 95°c, and 30 s at 95°c. all samples were analyzed as duplicates and the accepted intra-assay coefficient of variation for threshold cycle was ≤  %. the results were analyzed using mx real-time qpcr system software and expressed as a log fold change to the respective control mice. gapdh was used as a normalization gene and the software utilizes modified comparative quantitation method introduced by pfaffl. adipose tissue fatty acid analysis total lipids were extracted from 30–80 mg of epididymal and inguinal adipose tissue with hexane-isopropanol and trans-methylated by heating with acidic methanol. the resulting fatty acid methyl ester compositions were analyzed by gas–liquid partition chromatography using a hewlett packard gas chromatograph with a db- gc column. the samples were split injected with hydrogen as a carrier gas and the temperature program was from 160°c to 230°c. the relative amounts of the methylated fatty acids were expressed as percentages of weight normalized to 100%. statistical analyses the at fatty acid data were expressed as mean percentages of weight with standard deviations. the gene expression data were expressed as log fold change medians with interquartile ranges. the significance of the differences between the experimental and control mouse groups was assessed using the non-parametric mann–whitney u test. bivariate correlations were determined using the spearman test. analyses were performed using pasw statistics.  infection-enriched biological pathways in the inguinal at infection-enriched biological pathways and the associated up- and down-regulated genes in the inguinal at transcriptome are presented in tables  and compared with the control mice, the expression of “protein binding” pathway was increased most significantly in aa group. in addition, the inflammation-related “antigen binding”, “antibacterial humoral response”, and “antimicrobial humoral response” pathways were significantly enriched. a. actinomycetemcomitans infection-enriched go terms in the inguinal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. c. pneumoniae infection-enriched go terms in the inguinal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. c. pneumoniae and recurrent a. actinomycetemcomitans infections-enriched go terms in the inguinal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. compared with the control mice, most of the enriched go terms in the inguinal at transcriptome of cpn group were pathways associated with development- and morphogenesis. the most affected pathway was “negative regulation of cellular process” pathway. the most significantly enriched pathway in the inguinal at transcriptome of cpn + aa group was “protein binding” followed by inflammation-related “antigen binding”, “antibacterial humoral response”, and “antimicrobial humoral response” pathways. lipid homeostasis-related “regulation of lipoprotein lipid oxidation” and “lipoprotein lipid oxidation” pathways were also enriched. infection-enriched biological pathways in the epididymal at table  presents the aa infection-enriched biological pathways in the epididymal at transcriptome. compared with the control mice, in the epididymal at, infection enriched the following humoral antibody response and inflammation-associated pathways: “antigen binding”, “antibacterial humoral response”, “antimicrobial humo-ral response”, “immunoglobulin mediated immune response”, “humoral immune response mediated by circulating immunoglobulin”, and “inflammatory response”. the lipid homeostasis-related “monocarboxylic acid metabolic process”, “fatty acid biosynthetic process”, “regulation of lipid metabolic process”, “negative regulation of lipid metabolic process”, “triglyceride metabolic process”, and “regulation of lipid biosynthetic process” were also enriched. a. actinomycetemcomitans infection-enriched go terms in the epididymal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. table  presents the cpn infection-enriched pathways in the epididymal at transcriptome. the enriched pathways included “very long-chain fatty acid metabolic process”, “long-chain fatty acid metabolic process”, “long-chain fatty acid-coa ligase activity”, and “very long-chain fatty acid-coa ligase activity” pathways. the most affected pathway was “defense response”. c. pneumoniae infection-enriched go terms in the epididymal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. table  presents the cpn + aa infections-enriched pathways in the epididymal at transcriptome. the “protein binding” pathway included the highest number of affected genes. inflammation-related “antigen binding”, “antibacterial humoral response”, and “antimicrobial humoral response” pathways were enriched. there were no enrichments in lipid metabolism-related pathways. c. pneumoniae and recurrent a. actinomycetemcomitans infections-enriched go terms in the epididymal at transcriptome a up-regulated b b ap-values are corrected for multiple hypotheses using benjamini-hochberg false discovery rate. bfold change limit. number of biological replicates is go, gene ontology. the maximum of go categories is shown. inflammation- and lipid homeostasis-related up- and down-regulated genes in the inguinal at in aa group, most of the “protein binding” pathway-associated up-regulated genes belonged to immunoglobulin variables. the most up-regulated genes were s calcium binding protein a, s calcium binding protein a, and lactotransferrin. lipid homeostasis-associated leptin was down-regulated. in cpn group, infection induced down-regulation of acetyl-coenzyme a acyltransferase, elovl family member elongation of long chain fatty acids, and nuclear receptor subfamily group d, member. in cpn + aa group, the majority of up-regulated genes were immunoglobulin variables. in addition, s100a, s100a, and ltf were up-regulated. lep and synuclein δ were down-regulated. inflammation- and lipid homeostasis-related up- and down-regulated genes in the epididymal at in aa group, the enriched “monocarboxylic acid metabolic process” pathway included four up-regulated genes: pyruvate carboxylase, elovl, stearoyl-coenzyme a desaturase, and insulin induced gene. insig cell death-inducing dna fragmentation factor, alpha subunit-like effector a and thyroid hormone responsive were associated with the “regulation of lipid metabolic process” pathway. in addition, fatty acid binding protein and immunoglobulin variables were up-regulated. in cpn group, the “defense response” pathway included four defensin subtypes, 5′ nucleotidase, ecto, and chemokine ligand. the up-regulated solute carrier family, member was associated with the lipid homeostasis-related pathways. in cpn + aa group, a large number of up-regulated genes were immunoglobulin variables and the most up-regulated was ig gamma-2a chain c region secreted form-like. lipid homeostasis -associated arachidonate 12-lipoxygenase was also up-regulated. the expression of de novo lipogenesis-associated genes figure  presents the expression of de novo lipogenesis -associated genes in the at depot transcriptomes among the different infection models. in the epididymal transcriptome of aa-infected mice, the up-regulated genes were pcx, fatty acid synthase, scd, and elovl. fasn and elovl were down-regulated in the cpn group in both at depots. the combined infection model had little impact on de novo lipogenesis associated gene expression. alterations in qpcr-analyzed mrna expression levels the relative mrna expression levels of neutrophil activation- and macrophage phenotype- and activation- related genes in inguinal and epididymal at are presented in table  in the inguinal at, the relative expression levels of genes encoding slpi and mpo were significantly higher in the aa group than in control mice. cpn infection did not have a significant effect on the selected gene expression levels. fold change a p-value b mcp-1 mrc-1 cd68 mif slpi mpo mcp-1 mrc-1 cd68 mif slpi amedian log fold change to the control mice. the control mice have been set into the value of bthe mann–whitney u test with the control mice as the reference group. aa group, recurrent a. actinomycetemcomitans infection; cpn group, chronic c. pneumoniae infection; cpn + aa group, combined recurrent a. actinomycetemcomitans and chronic c. pneumoniae infections; iqr, interquartile range; mcp- monocyte chemoattractant protein 1; mif, macrophage migration inhibitory factor, mrc- mannose receptor c type 1; mpo, myeloperoxidase; slpi, secretory leukocyte peptidase inhibitor. in the epididymal at, the relative mrna expression levels of mcp- mif, and slpi in the aa group, mrc- and cd in cpn group, and mif in aa + cpn group were significantly altered but between - to log2fc. the cpn + aa combination induced a reduction in the mcp- mrna levels. changes in at fatty acid composition the proportions of fatty acids in the epididymal and inguinal at are presented in figure  in the inguinal at, aa infection induced a significantly higher proportion of sfas and a lower proportion of pufas compared to the control mice. the proportion of mufas was greater in the cpn group. in the epididymal at, the proportions of sfas were higher among all infected mice but the difference reached statistical significance only in the aa group. the proportion of pufas was significantly lower in the aa and cpn + aa groups. when the two at depots were compared, the proportion of sfas was lower in the epididymal at than in the inguinal at in the control group and the aa + cpn group. associations between serum and at parameters the associations between different serum and at variables were determined by correlation analysis and presented as graphs in figure  serum lps activity correlated positively with the serum ffa concentration and with the proportion of sfa in the epididymal at, and negatively with both inguinal and epididymal proportions of pufa. discussion in the present study, we examined the inflammation- and lipid homeostasis-related effects of systemic a. actinomycetemcomitans and c. pneumoniae infections on at derived from chow-fed apoe-deficient mice. these effects were examined on gene expression and fat composition level in both inguinal and epididymal at depots. recurrent aa infection affected both at depots significantly: inflammation-related biological pathways were enriched in inguinal at and both inflammation- and lipid homeostasis-related pathways were enriched in epididymal at. at fat distribution was altered and sfas were increased at the expense of pufas in both epididymal and inguinal at among all a. actinomycetemcomitans-infected mice. the impact of chronic c. pneumoniae infection on at depots was less prominent. the mouse models used in the present study were designed to display chronic infections. our aa model is characterized by endotoxemia and systemic inflammation. the observed increased humoral immune responses can be somewhat expected, because we have previously shown that the serum levels of lps activity, tnfα, and igg antibodies against a. actinomycetemcomitans after intravenous injections of live bacteria are significantly higher in the aa group compared with the control group. the persistence of c. pneumonia after three inoculations was also shown as the pathogen was recovered from the lungs of the mice. since atherosclerosis is a multiorgan disease, our aim was to broaden the study from the lipid depots in heart and liver to adipose tissue, which plays an important role in sustaining systemic inflammation and proatherogenic lipid profile. experimental acute endotoxemia has been shown to induce general insulin resistance and local and adaptive inflammation in human subcutaneous at. in our study, aa infection enriched inflammation-related pathways in the inguinal at. qpcr analysis verified up-regulated expression of mpo and slpi, and the most up-regulated genes in the microarray analysis were s100a and ltf. these innate immunity-associated gene products are considered anti-inflammatory mediators and they exhibit various antimicrobial functions such as promotion of oxidative activity responses. human slpi and ltf have been shown to co-localize in secondary granules and they are co-released after neutrophil activation. interestingly, increased plasma slpi levels were associated with progressive metabolic dysfunction in men. despite the up-regulation in neutrophil activation-related genes, we did not observe changes in the mrna expression of genes associated with classically or alternatively activated macrophages. the response of aa infection in the epididymal at was quite distinct. as in inguinal at, the humoral antibacterial response and immunoglobulin-related pathways were enriched. however, the myeloid cell-specific signs of enhanced oxidative stress were absent, expression of slpi was somewhat down-regulated and, instead, several lipid homeostasis-related biological pathways were enriched. on at fatty acid level, we observed a potentially adverse increase in the proportion of sfas among all aa-infected mice. mengting et al. reported that compared with lean mice, obese mice have a higher level of sfas in their epididymal at. an interesting study by suganami et al. demonstrated that sfas released by hypertrophied adipocytes activate the nf-κb pathway in the macrophages with ensuing tnf-α release. the subsequent lipolytic release of sfas creates a positive feed-back loop between adipocytes and macrophages. importantly, the macrophage-derived tnf-α also resulted in nf-κb-dependent release of proinflammatory cytokines from the adipocytes. both lps and sfa have been shown to generate endoplasmic reticulum stress in primary human adipocytes. elevated levels of sfas in both epididymal and inguinal at depots in the aa and cpn + aa groups suggested an increased proinflammatory potential of these at depots. in addition, because pufas have been reported to inhibit the tlr dimerization induced by sfas, the significantly decreased proportion of pufas among all a. actinomycetemcomitans-infected mice may intensify the effects of increased sfas. the proportion of pufas also showed a significant inverse association with lps serum activity. this result may be due to lps-triggered enhanced oxidative stress in the at, followed by the peroxidation of pufas. in addition to intracellular effects, lps has been shown to elevate the levels of circulating ffas in rodents in vivo. this association was also found in our study as there was a strong positive correlation between serum lps activity and the ffa level. lipid homeostasis-related biological pathways were enriched and four important de novo lipogenesis-related genes were moderately up-regulated in the epididymal at in aa group. the observed up-regulation of pcx and fasn could partially explain the increase in the sfa proportion. however, up-regulation of de novo lipogenesis genes was not observed in cpn and cpn + aa groups, indicating the involvement of other regulatory pathways affecting sfa levels in the at. chronic infection with c. pneumoniae did not enhance the pathways associated with humoral antibody response, and moreover, the effects on the at depot fatty acid distribution were less evident than with aa infection. however, the “defense response” pathway which included four up-regulated defensins was enriched in the epididymal at depot. previously, c. pneumoniae infection in vitro was reported to induce human β-defensin production mediated by the tlr pathway in monocytes. although both c. pneumoniae and a. actinomycetemcomitans are gram-negative pathogens, the effects of cpn infection differed considerably from those of aa infection. this result may be due to the distinct infection routes and c. pneumoniae being an obligatory intracellular pathogen. in addition, the difference in the infection time points may also influence the outcome and, altogether, the character of chronic infection differs from the recurrent one. the combined infection model displayed a humoral antibody response and changes in at depot fa distribution similar to those with single aa infection. nevertheless, indicators of the potentially amplified de novo lipogenesis in epididymal at remained absent. the major limitation of the study was the small number of mice available for the microarray analysis, which resulted in non-significant gene fc-results after fdr correction. although the apoe-deficient mouse model has been utilized widely in atherosclerosis research, it possesses certain drawbacks, such as a somewhat non-physiological lipoprotein profile. the adipocytes of apoe-deficient mice are smaller, contain fewer lipids, and accumulate less triglycerides from very low-density lipoproteins and chylomicrons compared with adipocytes derived from wild-type mice. in addition, because apoe has been shown to protect against lps-derived sepsis, apoe deficiency actually exposes mice to the deleterious and possibly proatherogenic effects of lps. nevertheless, the augmented susceptibility to lps may produce a more human-like response, because rodents are generally more resistant to lps-mediated effects than humans. our study design included only males and therefore we could only draw gender-specific conclusions. it is noteworthy that, in mice, approximately 20% of at genes exhibit sexual dimorphism, and the enriched functional categories with fc > include “lipid metabolism”.  the present study introduces in vivo evidence that systemic infection of a. actinomycetemcomitans activates inflammation-related biological pathways and alters the lipid homeostasis adversely in both inguinal and epididymal at depots in apoe-deficient mice. the results suggest that repeated endotoxemia promotes at dysfunction as shown by accumulation of saturated fat in the tissue and spill-over of free fatty acids into the circulation possibly via induced lipolysis. these observations may be useful when studying the potential mechanism linking adipose tissue to the proposed atherogenicity of the chronic infections. abbreviations aa: aggregatibacter actinomycetemcomitans; apoe: apolipoprotein e; at: adipose tissue; cpn: chlamydia pneumoniae; fc: fold change; fdr: false discovery rate; ffa: free fatty acid; go: gene ontology; iqr: interquartile range; lps: lipopolysaccharide; mufa: monounsaturated fatty acid; pai 1: plasminogen activator inhibitor-1; pufa: polyunsaturated fatty acid; qpcr: quantitative polymerase chain reaction; sd: standard deviation; sfa: saturated fatty acid; spg: sucrose-phosphate-glutamic acid buffer; tlr: toll-like receptor; tnf-α: tumour necrosis factor α. competing interests the authors declare that they have no competing interests. authors’ contributions kh, amt, sl, mj, ptk, ml, ps, ga, and pjp designed the study. kh performed experiments, analyzed and interpreted data, initially drafted the manuscript, and prepared tables and figures. amt, sl, and is performed experiments and edited the manuscript. mj, ptk, ml, ps, ga, and pjp critically revised the manuscript and contributed to discussion. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 qpcr primer sequences. click here for file additional file 2: table s2 differentially expressed genes in the inguinal at transcriptome of recurrent a. actinomycetemcomitans-infected mice. click here for file additional file 3: table s3 differentially expressed genes in the inguinal at transcriptome of chronic c. pneumoniae-infected mice. click here for file additional file 4: table s4 differentially expressed genes in the inguinal at transcriptome of combined chronic c. pneumoniae and recurrent a. actinomycetemcomitans-infected mice. click here for file additional file 5: table s5 differentially expressed genes in the epididymal at transcriptome of recurrent a. actinomycetemcomitans-infected mice. click here for file additional file 6: table s6 differentially expressed genes in the epididymal at transcriptome of combined chronic c. pneumoniae-infected mice. click here for file additional file 7: table s7 differentially expressed genes in the epididymal at transcriptome of combined chronic c. pneumoniae and recurrent a. actinomycetemcomitans-infected mice. click here for file acknowledgements the authors are grateful for the valuable work of saija perovuo, tiina karvonen, and pirjo laaksonen. we also thank the personnel at biomedicum genomics and lilli saarinen at the computational systems biology laboratory. wihuri research institute is maintained by the jenny and antti wihuri foundation. this work was supported by the sigrid juselius foundation; the finnish dental society apollonia; the paulo foundation; the jenny and antti wihuri foundation; the aarno koskelo foundation; the ida montin foundation; the finnish atherosclerosis society; and the research council for the health, academy of finland, grant.
the transcriptomics data revealed significant enrichment in inflammation-associated biological pathways in both at depots derived from the aa and cpn + aa treated mice compared with the control group. the proportion of saturated fatty acids was higher in the inguinal at in aa and cpn + aa groups and in the epididymal at in aa group. the proportion of polyunsaturated fatty acids was significantly lower among all aa-infected groups in both depots. chronic cpn infection displayed only minor effects on transcriptomics and fatty acids of the at depots. microarray data were analyzed using gene ontology enrichment analysis. chronic infections have been demonstrated to maintain low-grade systemic inflammation and associate with atherosclerosis. the adverse changes in adipose tissues during chronic infection may promote atherosclerosis. we studied the inflammation- and lipid homeostasis-related effects of aggregatibacter actinomycetemcomitans and chlamydia pneumoniae infections on the epididymal and inguinal adipose tissue transcriptomes and fatty acid distribution in apolipoprotein e-deficient mice. systemic infection with a. actinomycetemcomitans activates inflammation-related biological pathways and modulates cellular lipid homeostasis. chow-fed apoe-deficient mice were exposed to 1) chronic intranasal infection with c. pneumoniae, 2) recurrent intravenous infection with a. actinomycetemcomitans, 3) a combination of both types of infection, or 4) infection with the vehicle. epididymal and inguinal at gene expression was analyzed using an illumina mouse wg- v platform and quantitative pcr.
 chronic infections have been demonstrated to maintain low-grade systemic inflammation and associate with atherosclerosis. we studied the inflammation- and lipid homeostasis-related effects of aggregatibacter actinomycetemcomitans and chlamydia pneumoniae infections on the epididymal and inguinal adipose tissue transcriptomes and fatty acid distribution in apolipoprotein e-deficient mice. chow-fed apoe-deficient mice were exposed to 1) chronic intranasal infection with c. pneumoniae, 2) recurrent intravenous infection with a. actinomycetemcomitans, 3) a combination of both types of infection, or 4) infection with the vehicle. epididymal and inguinal at gene expression was analyzed using an illumina mouse wg- v platform and quantitative pcr. microarray data were analyzed using gene ontology enrichment analysis. at fatty acid analysis was performed using gas–liquid chromatography.  the transcriptomics data revealed significant enrichment in inflammation-associated biological pathways in both at depots derived from the aa and cpn + aa treated mice compared with the control group. the proportion of saturated fatty acids was higher in the inguinal at in aa and cpn + aa groups and in the epididymal at in aa group. the proportion of polyunsaturated fatty acids was significantly lower among all aa-infected groups in both depots. chronic cpn infection displayed only minor effects on transcriptomics and fatty acids of the at depots.  systemic infection with a. actinomycetemcomitans activates inflammation-related biological pathways and modulates cellular lipid homeostasis. the adverse changes in adipose tissues during chronic infection may promote atherosclerosis. a. actinomycetemcomitansc. pneumoniaeadipose tissueapoe-deficient micetranscriptomefatty acid distribution 