BACKGROUND
vasculitis is characterized by the localized infiltration of inflammatory cells into the intravascular wall and its surroundings, and is accompanied by denaturation and necrosis, leading to potentially life-threatening conditions
<cit> .
the candida albicans water-soluble fraction , a mannoprotein-β-glucan complex obtained from the culture supernatant of c. albicans nbrc <dig>  exhibits vasculitis-inducing activity in mice , which acts as a trigger for the induction of vasculitis in the coronary artery
<cit> .
indeed, caws has strong induction potency for murine vasculitis and induces lethal toxicity in certain mouse strains
<cit> .
unlike polypeptides derived from candida albicans <cit> , however, caws does not display superantigen-like activities because the part of the caws complex that elicits physiological activity is the polysaccharide chain.
dectin- <dig>  a c-type lectin expressed by dendritic cells  and macrophages, functions as the critical dc receptor for α-mannans of caws and plays a pivotal role in host defense against c. albicans by inducing th <dig> cell differentiation
<cit> .
a subtype of caws with a higher ratio of β-mannosyl linkages, named caws <dig>  failed to cause severe vasculitis in dba/ <dig> mice but inhibited the inflammatory response via a competitive association with α-mannan specific lectins, including dectin-2
<cit> : this result indirectly demonstrated dectin- <dig> involvenment in the development caws mediated vasculitis.
intraperitoneal injection of caws induced coronary arteritis in both c57bl/b <dig>  and dba/ <dig> mice
<cit> .
however, only dba/ <dig> mice exhibited severe necrotizing coronary arteritis and aortitis leading to nearly 100% mortality within weeks from stenosis in the left ventricular outflow tract and severe inflammatory changes of the aortic valve with fibrinoid necrosis
<cit> .
although dectin- <dig> may be essential for caws-vasculitis in both b <dig> and dba/ <dig> mice, it remains elusive as to why caws-vasculitis is fatal to dba/ <dig> mice, but not to b <dig> mice.
the complement system, comprised of more than  <dig> serum and cellular proteins, plays a major role in innate immune defenses against infectious agents
<cit> .
complement activation appears to be involved in the pathogenesis of systemic autoimmune diseases
<cit> .
the alternative pathway involving c <dig> convertase , a potent enzymatic protein complex capable of rapidly converting c <dig> into powerful effector fragments , amplifies the initial response, inducing a variety of effector functions
<cit> .
in acute coronary syndrome , the complement cascade is activated and the c <dig> and c <dig> concentration ratio  in serum is a readily available marker of recurrent cardiovascular events
<cit> .
complement activation produces the complement c <dig> , a precursor to the effector molecules, c5a and c5b; c5a acts as a trigger for the cellular immune response and c5b acts as an initiator of the formation of the membrane attack complex
<cit> .
c5a mediates inflammatory responses
<cit>  through the c5a receptor  <dig> ; however, c5a also exerts an anti-inflammatory effect
<cit> , putatively through its second receptor, the c5a-like receptor  <dig> , which may act either as a decoy receptor or by forming the c5ar/c5l2/β-arrestin complex, depending on the cell type, species and disease context
<cit> .
notably, dba/ <dig> mice are defective in c <dig>  though it remains unknown how this c <dig> deficiency may contribute to the fatal effect of caws.
ifng, the only member of the type ii class of interferons, is a soluble homodimer with potent proinflammatory properties that plays critical roles in innate and adaptive immunity against viral and intracellular bacterial infections.
ifng-induced inflammation is involved in aging and aging-associated medical and psychiatric disorders
<cit> .
notably, ifng level was increased in splenocyte culture supernatants following stimulation with lipopolysaccharide
<cit> , by a gram-negative bacterial cell wall component or with caws
<cit> .
thus, it appears that complement factors and ifng are somehow involved in caws-vasculitis; however, no direct data to confirm this have been reported.
in the present study, we report the results of dna microarray analysis of mrnas from pbmcs comparing differences in gene expression patterns in b <dig> and dba/ <dig> mice.
we found that mrna levels of ifng and complement factors such as c <dig>  c <dig>  cfb, cfh, and fcna were conspicuously augmented only in dba/ <dig> mice at  <dig> and  <dig> weeks after caws administration.
moreover, mrna levels of immune-related genes, such as irf <dig>  irf <dig>  irf <dig>  cebpb, ccl <dig>  itgam, icam <dig>  and il-12rb <dig>  whose expression levels are known to be augmented by ifng, were also increased only in dba/ <dig> mice.
based on these results, we propose a molecular model to explain how caws treatment induces fatal vasculitis in dba/ <dig> mice.
RESULTS
induction of arteritis by injection of caws into dba/ <dig> mice
to induce arteritis in b <dig>  and dba/ <dig> mice, we performed intraperitoneal injection  of caws  for  <dig> consecutive days as reported previously
<cit> ; we will call this process as caws administration, hereafter, and set this timing  as  <dig> week .
although the number of surviving mice at  <dig> w,  <dig> w,  <dig> w, and  <dig> w following caws administration was remarkably reduced in dba/ <dig> mice , similar to our previous report
<cit> , few b <dig> mice died during this period , which is also similar to our previous report
<cit> .
at  <dig> w,  <dig> w,  <dig> w, and  <dig> w after caws administration, the surviving mice were sacrificed and mrna isolated from pbmcs.
three mice were sacrificed at each time point .
tissue sections that were obtained from paraffin-embedded hearts fixed with 10% neutral formalin and stained with hematoxylin and eosin  revealed vasculitis in the aorta and coronary artery of dba/ <dig> mice, with fibrotic thickening of the arterial media and adventitia and infiltration of inflammatory cells .
in tissue sections obtained from the hearts of mice at  <dig> w,  <dig> w,  <dig> w, and  <dig> w following caws administration, we observed infiltration of inflammatory cells in the aortic media and adventitia of dba/ <dig> mice, a histological finding typical of aortitis, as early as  <dig> w following caws administration, and this was observed up to the final  <dig> w time point .
b <dig> mice also showed infiltration of inflammatory cells in the aortic media and adventitia, although the onset of aortitis in b <dig> mice occurred later than in dba/ <dig> mice, not appearing until 4– <dig> w following caws administration .
since these results were similar to those of our previous report
<cit> , we concluded that the mice used for dna microarray analysis in this study responded to caws in a similar manner to those described in our previous report.
<cit> .
red vertical arrows indicate the time points  at which three b <dig> mice and three dba/ <dig> mice were sacrificed for mrna purification.
histopathological analysis was performed on aortic roots and coronary arteries collected from dba/ <dig> mice at  <dig> w following caws administration stained with h&e.
enlarged views of the region indicated by rectangles in the left panels are shown in the right panels.
bar =  <dig> μm for left panels and  <dig> μm for right panels.
caws enhanced expression of complement system genes only in dba/ <dig> mice
to identify genes whose expression levels were up- or downregulated in the pbmcs of dba/ <dig> mice, but not in b <dig> mice, following caws treatment, we examined the transcriptome profiles of mrna prepared from dba/ <dig> and b <dig> pbmcs at  <dig> w,  <dig> w,  <dig> w, or  <dig> w following caws administration using agilent’s whole mouse genome microarray .
when the relative intensities of the signals at  <dig> w,  <dig> w,  <dig> w, or  <dig> w were compared with those at  <dig> w following caws administration, the genome-wide expression profiles were found to be distinct between these two mouse strains .
heat map representation of the signal intensities revealed that a number of genes were dramatically up- or downregulated in dba/ <dig> mice compared to b <dig> mice .
the most notable genes that showed enhanced expression following caws treatment in dba/ <dig> mice belong to the “complement system”, in particular the genes encoding c <dig>  c <dig>  complement factor b , complement factor h , and ficolin-a  .
the complement 3a receptor  <dig>  gene showed modestly enhanced expression at  <dig> w,  <dig> w, and  <dig> w following caws administration and conspicuously enhanced expression at  <dig> w .
c5ar <dig> gene also showed enhanced expression at  <dig> w and  <dig> w, but b <dig> mice also showed enhanced expression at this timing, albeit less dramatically.
expression of complement c <dig> , serping <dig> , complement factor i , and hemolytic complement  was extremely low in b <dig> mice at  <dig> w,  <dig> w and  <dig> w, but their expression was detectable in dba/ <dig> mice, displaying a comparatively enhanced ratio in dba/ <dig> mice compared with b <dig> mice.
by contrast, complement c <dig> , decay accelerating factor , and complement receptor  <dig>  showed decreased expression following caws treatment in dba/ <dig> mice, although these genes were weakly expressed in b <dig> .
to determine whether changes in the expression levels of these genes is physiologically significant, a scatter plot was used to analyze the distribution of the expression levels of many genes at a glance.
indeed, c <dig>  c <dig>  cfb, cfh, fcna, and cr <dig> showed expression levels high enough to conduct physiologically significant comparison  in dba/ <dig> mice at  <dig> w or  <dig> w following caws administration .
by contrast, complement genes that showed very low expression levels compared to above described activated complement genes in both b <dig> and dba/ <dig> mice were not deemed significant .
c3ar <dig> mrna levels were low at  <dig> w or  <dig> w and its expression level was enhanced only at  <dig> w following caws administration.
thus, we conclude that the enhanced mrna levels of c <dig>  c <dig>  cfb, cfh, and fcna observed after caws administration in dba/ <dig> mice are significant.
the alternative complement pathway is activated in dba/ <dig> mice following caws administration
we next performed an ontology search using ingenuity pathways analysis .
this powerful query system facilitates the rapid visualization of relationships between sets of relevant genes to predict the putative physiological significance of these genes.
we found that alternative complement pathway genes, including c <dig>  cfb , and cfh , appear to play major roles in activation of the complement system .
by contrast, classical pathway genes, including complement c <dig>  and serping g <dig> , and lectin pathway genes including mannose-binding lectin  appear to play less significant roles because they showed low levels of expression even in dba/ <dig> mice .
the low mrna level of c <dig> measured in dba/ <dig> mice suggests that downstream regulation of the complement system is not important in the induction of vasculitis in the aortas or coronary arteries of dba/ <dig> mice.
to confirm that the microarray signals accurately reflected mrna levels, expression profiling using quantitative reverse transcription-polymerase chain reaction  analysis was performed for c <dig>  c <dig>  cfb, cfh, and fcna genes using the same rna samples used for the microarray analysis.
indeed, all of the genes analyzed by qrt-pcr showed a similar pattern of mrna distribution following caws administration; namely, they showed increased mrna levels of the denoted genes in dba/ <dig> mice compared with b <dig> mice , confirming the accuracy of the microarray analysis data.
caws induced upregulated expression of interferon-related genes only in dba/ <dig> mice
we next examined the expression of interferon-related genes because expression of the ifng gene was enhanced following caws administration in dba/ <dig> but not in b <dig> mice, as revealed by both dna microarray and qrt-pcr analyses .
notably, ifng was the only interferon-related gene that exhibited upregulated dba/2-specific expression following caws administration : moreover, mrna expression of ifng was more conspicuously upregulated than that of other interferon gene subtypes in dba/ <dig> mice .
in the “interferon signaling pathway”, interferon regulatory factor- <dig>  also showed increased expression, although less significantly than interferon itself, in dba/ <dig> mice at  <dig> w and  <dig> w .
notably, the mrna level of irf <dig> was decreased at  <dig> w, and this depressed level persisted throughout the experiment .
this may explain why many of the interferon-stimulated genes were not activated ; the irf9/isgf3g complex binds to the interferon-stimulated response element to activate the transcription of interferon-stimulated genes
<cit> .
in the pathway identified as the “role of prk in interferon induction and antiviral response”, mrna levels of eukaryotic translation factor  <dig> alpha kinase  <dig>  and irf <dig> were enhanced .
eif2aka is a heterotrimeric gtpase that controls the selection of correct start codons on mrna, and thus regulates translation initiation by facilitating the preferential translation of selected transcripts in response to cellular stresses, in this case caws administration
<cit> .
our results suggest that dba/ <dig> mice are more sensitive to this type of stress than b <dig> mice.
in the “acute phase response signaling” pathway , three genes other than c <dig> and cfb were upregulated; ccaat/enhancer-binding protein beta , il- <dig>  and lipopolysaccharide-binding protein .
cebpb, a member of the c/ebp family of basic region-leucine zipper  proteins, is largely expressed in macrophages and is important for the antibacterial activity of macrophages through binding to the regulatory regions of several acute phase and cytokine genes
<cit> .
enhanced expression of cebpb following caws administration suggests that downstream target genes of cebpb, including il- <dig> , may be widely activated.
il- <dig> exerts pleiotropic effects on various target cells, including b cells, eosinophils, and basophils, and overexpression of il- <dig> in vivo significantly increases the number of eosinophils and b cells
<cit> .
lbp is involved in the acute phase immunologic response to gram-negative bacterial infections through binding to bacterial lipopolysaccharide , thereby eliciting immune responses by presenting the lps to the cell surface pattern recognition receptors cd <dig> and tlr4
<cit> .
in this case, caws, rather than lps, may have been recognized as an infectant.
in the “acute phase myeloid leukemia signaling” pathway , two genes were upregulated: spleen focus-forming virus proviral integration  <dig>  and colony stimulating factor  <dig> receptor .
sfpi <dig> is a transcription factor that plays a key role in hematopoietic cell fate decisions through mutual negative regulation that occurs between sfpi1and gata- <dig>  a zinc finger transcription factor that is the central mediator of erythroid gene expression
<cit> .
csf1r is a single pass type i membrane protein that acts as a receptor tyrosine kinase for a cytokine called colony stimulating factor  <dig>  which is a primary growth factor and monocyte/macrophage chemokine that regulates survival, proliferation and differentiation of macrophages and other mononuclear phagocytic lineage cells
<cit> .
enhanced expression of csfr <dig> suggests the activation of macrophages following caws administration.
a scatter plot of irf <dig>  irf <dig>  c <dig>  cebpb, csf1r, trf, ifng, eif2aka, and cfb expression showed expression levels high enough to conduct physiologically significant comparisons  in dba/ <dig> mice at  <dig> w or  <dig> w following caws administration , suggesting that their enhanced mrna levels indicated by the heatmap  are significant.
by contrast, mrna levels of lbp and sfpi <dig> may be too low for comparison between b <dig> and dba/ <dig> mice to be significantly different.
transcription factor genes showing upregulated expression only in dba/ <dig> mouse
some transcription factor genes also showed upregulated expression only in dba/ <dig> mice with mrna levels peaking at  <dig> w ,  <dig> w ,  <dig> w , and  <dig> w/ <dig> w .
irf <dig>  irf <dig>  and cebpb were discussed above.
stat <dig>  a signal transducer and activator of transcription, plays a crucial role in the pathology of atherosclerosis resulting from the effects of interferon-γ produced by t cells.
stat <dig> also induces increased expression of several proinflammatory and pro-atherogenic mediators of signaling mediated by tlr4
<cit> .
expression of nupr <dig>  a highly basic and loosely folded multifunctional protein, is induced in response to several cellular stresses
<cit>  and provides protection against metabolic stress-mediated autophagic-associated cell death resulting from activation of the nupr1-aurora kinase a pathway
<cit> .
t-box  <dig> , a transcriptional regulator required for cardiac development, plays a dual role as both a transcriptional activator and a repressor, promoting or repressing distinct genetic programs within adult heart in response to cellular stress
<cit> .
increased expression with twin peaks observed at  <dig> w and  <dig> w suggests that caws injection altered regulation of tbx <dig>  thereby generating cardiac phenotypes .
zinc finger protein  <dig> , also called nmp <dig> , is a nucleo-cytoplasmic shuttling transcription factor that regulates the expression of collagen and matrix metalloproteinases and represses bone formation induced by parathyroid hormone
<cit> .
ankyrin repeat and socs box-containing protein  <dig>  interacts with mixed lineage leukemia  protein, a key epigenetic regulator of normal hematopoietic development, and overexpression of asb <dig> degrades mll to reduce its transactivation activity
<cit> .
zinc finger protein  <dig> , a member of the tristetraprolin family of tandem ccch finger proteins that bind to au-rich elements in the 3’-untranslated region of mrnas, is a critical modulator of definitive hematopoiesis
<cit> .
zinc finger protein  <dig> , a member of the c2h2-zinc finger  family of proteins, appears to function as a transcriptional and cell cycle/apoptosis regulator and play a role in the establishment and maintenance of pluripotency.
although these proteins play roles in several important cellular events such as hematopoiesis, these functions do not appear to be relevant to the pathogenesis of caws-induced vasculitis.
scatter plot analysis indicated that mrna levels of irf <dig>  cebpb, stat <dig>  nurp <dig>  irf <dig>  and brd <dig> were high enough to conduct physiologically significant comparison between b <dig> and dba/ <dig> mice according to their heatmaps  at both  <dig> w and  <dig> w following caws administration , suggesting that their superficially augmented expression is physiologically significant.
by contrast, mrna levels of asb <dig>  zfp <dig>  zfp <dig>  zfp <dig>  zfp <dig>  and tbx <dig> were too low to conclude that heatmap comparison of these genes between b <dig> and dba/ <dig> mice is physiologically significant.
finally, caws administration resulted in a slight, but comparable increase in dectin- <dig> mrna levels in both b <dig> and dba/ <dig> mice .
moreover, scatter plot analysis indicated that the mrna levels of dectin- <dig> mrna were lower than those of dectin- <dig>  a similar c-type lectin molecule .
discussion
in the present study, we used dna microarray analysis to identify genes whose mrna levels in pbmcs were upregulated in dba/ <dig> mice  but not in b <dig> mice  at  <dig> w,  <dig> w,  <dig> w, and  <dig> w following caws administration .
we found that the mrna levels of c <dig>  c <dig>  cfb, and cfh , which regulate the alternative pathway of the complement system , are conspicuously increased in dba/ <dig> mice at  <dig> w,  <dig> w, and  <dig> w following caws administration, whereas b <dig> mice showed decreased mrna levels of these proteins, and this notable difference was confirmed by qrt-pcr .
we also observed that mrna levels of ifng and interferon-related genes were augmented in the pbmcs of dba/ <dig> mice compared with those of b <dig> mice .
ifng expression is induced upon infection by pathogens such as viruses, bacteria, or parasites, suggesting that some components of caws, possibly the polysaccharide chains of α and β mannoproteins in the cell wall extract, are recognized as pathogens after injection.
ifng enhanced c <dig> mrna and protein expression levels in a human astroglioma cell line
<cit> .
ifng increased steady state mrna levels of both c <dig> and c <dig> in three different human cell types .
ifng stimulation also increased c <dig> and c <dig> protein synthesis
<cit> .
moreover, ifng induced biosynthesis of complement components c <dig>  c <dig>  and cfh at the transcriptional level in human proximal tubular epithelial cells
<cit> .
in mice, cfb mrna expression was synergistically upregulated in murine macrophages
<cit> , and ifng directly stimulated transcription or stabilized complement c <dig> and c <dig> mrnas
<cit> .
transgenic mice over-expressing ifng in the brain exhibited neuroinflammation induced by upregulation of the early components of the complement cascade, including c <dig> and c4a
<cit> .
thus, increased c <dig> and c <dig> mrna expression following caws administration appears to be due to increased ifng expression, which may occur due to recognition of caws as a pathogen.
interferon regulatory factors, including irf <dig> , irf <dig> , and irf <dig> , are ifng-induced genes encoding transcription regulators that induce the expression of other immune-related genes, including genes that mediate the antiviral activity of ifng and play pivotal roles in the regulation of cellular responses, such as inflammation in host defense
<cit> .
indeed, ifng enhanced the expression of itgam
<cit> , icam1
<cit> , and il-12rb1
<cit> .
in particular, irf <dig>  which regulates the expression of ifn-induced genes and type i interferon, is an early signal that promotes inflammation; irf1-deficient mice are protected from autoimmune brain inflammation through regulation of th2-type cytokines
<cit> .
moreover, expression levels of irf <dig> were significantly higher in the synovium of joints with rheumatoid arthritis than in that of those with osteoarthritis
<cit> .
irf <dig> gene activation by reactive oxygen species is an early signal that promotes inflammation after ischemic renal injury
<cit> .
notably, ifng increases the expression of irf <dig> protein in human pbmcs
<cit> , and cebpb , which plays a fundamental role in regulating activated macrophage functions, is necessary for ifng-induced upregulation of irf <dig> mrna in cultured mouse bone marrow-derived macrophages
<cit> .
cebpb is also involved in expression of ccl <dig> mrna  induced by ifng and lipopolysaccharide
<cit> .
the low but similar expression levels of dectin- <dig>  in b <dig> and dba/ <dig> mice suggest that dectin- <dig> plays similar roles in caws-vasculitis in these two mice.
the lethality of caws-vasculitis in dba/ <dig> mice, but not in b <dig> mice, may be due to the increased expression and activity of components in the ifng-complement axis.
dectin- <dig>  a β-glucan specific receptor, may not be involved in caws-vasculitis, because caws is a mannoprotein and lacks the dectin- <dig> ligand, beta- <dig> -glucan
<cit> , which results in no signal being transmitted via the dectin- <dig> route.
a recent report suggests that the mannan-dectin-2/fceric pathway promotes phosphorylation of a protein called linker for activation of b cells , which induces natural killer and t cell-mediated ifng production following c. albicans infection
<cit> .
based on these observations, we propose the following molecular model to explain how caws administration causes lethal vasculitis in dba/ <dig> mice but not in b <dig> mice .
following injection into the mouse body, the α-mannan component of caws binds to the α-mannan receptor dectin- <dig> and is recognized as a pathogen, putatively leading to induced ifng expression.
one downstream pathway, shown by dotted green arrows, may lead to mild inflammation and non-lethal vasculitis in both b <dig> and dba/ <dig> mice.
the other pathway, shown by dotted black arrows, results in enhanced expression of ifng.
ifng then enhances c <dig> and c <dig>  expression, which leads to neutrophil activation and causes a cytokine/chemokine storm, inducing damage and necrosis of vascular endothelial cells
<cit> , severe inflammation and finally, a lethal vasculitis.
ifng also enhances expression of irf <dig>  irf <dig>  and irf <dig>  transcription factors that regulate the expression of immune-related genes that may also lead to severe inflammation of the arteries and subsequent lethal vasculitis.
cebpb expression induced by ifng may play a role in enhanced expression of ifg <dig> and ccl <dig>  which also leads to arterial inflammation, and itgam, icam <dig>  and il-12rb <dig> induced by ifng may also participate in inducing severe inflammation.
it was recently shown that caws-treated dba/ <dig> mice developed left ventricular dilution and dysfunction with macroscopic cardiomegaly and concentric left ventricular hypertrophy
<cit> .
we propose here that this chronic mortality of caws-treated dba/ <dig> mice is due to abnormal activation of the complement system induced by ifng upregulation.
this proposal is supported by recent reports showing that c5a elicits anti-inflammatory action through c5l <dig>  and that inflammation in dba/ <dig> mice deficient in c <dig> cannot be inhibited following caws treatnment
<cit> .
the proposal is also supported by the observation that inflammation cannot be inhibited in a mutant mouse deficient in c <dig> expression with acute pancreatitis and associated lung injury
<cit> .
caws also induced vasculitis in other mouse strains, such as c3h/hen, c3h/hej , dba/ <dig>  a/j, cba/n, c57bl, akr, and balb/c mice
<cit> .
it would be interesting to test whether the ifng-complement axis is also activated following caws administration in these mouse models in the future.
we and others have reported that complement activation is involved in the pathogenesis of systemic autoimmune diseases
<cit> .
in active anti-neutrophil cytoplasmic antibody-associated vasculitis with renal lesions, significantly higher levels of plasma c3a, c5a, soluble c5b- <dig>  and bb, a component of the alternative complement pathway, was observed during active development of the disease
<cit> .
ficolin- <dig> mrna level showed a four-fold increase in the pbmcs of ta patients, and that ficolin- <dig> protein expression was observed in the inflamed regions of surgical aorta specimens, in particular in cd68-positive cells , which are involved in the induction of inflammation
<cit> .
ficolin- <dig> mrna level was also higher in pbmcs isolated from microscopic polyangiitis  patients than in pbmcs isolated from healthy volunteers
<cit> .
moreover, increased expression of ficolin- <dig> protein was also detected by immunostaining in renal specimens taken from mpa patients, and many of the ficolin- <dig> positive cells coincided with cd68-positive cells in the glomeruli of mpa patients
<cit> .
these results suggest that enhanced expression of ficolin- <dig> in monocytes and macrophages is also involved in the pathogenesis of human vasculitis.
CONCLUSIONS
our results suggest that caws administration induces non-lethal caws-vasculitis in both b <dig> and dba/ <dig> mice via a dectin- <dig> pathway, but induces enhanced expression of ifng only in dba/ <dig> mice, which leads to augmented expression of c <dig>  c <dig>  cfb, cfh, and fcna, thereby causing lethal inflammation of the arteries and severe vasculitis.
thus, the ifng-complement axis may play an important role in the pathogenesis of lethal caws-vasculitis, and suggest that this axis may also be important in the pathogenesis of human vasculitis.

 vasculitis is characterized by the localized infiltration of inflammatory cells into the intravascular wall and its surroundings, and is accompanied by denaturation and necrosis, leading to potentially life-threatening conditions. the candida albicans water-soluble fraction, a mannoprotein-β-glucan complex obtained from the culture supernatant of c. albicans nbrc exhibits vasculitis-inducing activity in mice, which acts as a trigger for the induction of vasculitis in the coronary artery. indeed, caws has strong induction potency for murine vasculitis and induces lethal toxicity in certain mouse strains. unlike polypeptides derived from candida albicans, however, caws does not display superantigen-like activities because the part of the caws complex that elicits physiological activity is the polysaccharide chain. dectin- a c-type lectin expressed by dendritic cells and macrophages, functions as the critical dc receptor for α-mannans of caws and plays a pivotal role in host defense against c. albicans by inducing th cell differentiation. a subtype of caws with a higher ratio of β-mannosyl linkages, named caws failed to cause severe vasculitis in dba/ mice but inhibited the inflammatory response via a competitive association with α-mannan specific lectins, including dectin-2: this result indirectly demonstrated dectin- involvenment in the development caws mediated vasculitis. intraperitoneal injection of caws induced coronary arteritis in both c57bl/b and dba/ mice. however, only dba/ mice exhibited severe necrotizing coronary arteritis and aortitis leading to nearly 100% mortality within weeks from stenosis in the left ventricular outflow tract and severe inflammatory changes of the aortic valve with fibrinoid necrosis. although dectin- may be essential for caws-vasculitis in both b and dba/ mice, it remains elusive as to why caws-vasculitis is fatal to dba/ mice, but not to b mice. the complement system, comprised of more than serum and cellular proteins, plays a major role in innate immune defenses against infectious agents. complement activation appears to be involved in the pathogenesis of systemic autoimmune diseases. the alternative pathway involving c convertase, a potent enzymatic protein complex capable of rapidly converting c into powerful effector fragments, amplifies the initial response, inducing a variety of effector functions. in acute coronary syndrome, the complement cascade is activated and the c and c concentration ratio in serum is a readily available marker of recurrent cardiovascular events. complement activation produces the complement c, a precursor to the effector molecules, c5a and c5b; c5a acts as a trigger for the cellular immune response and c5b acts as an initiator of the formation of the membrane attack complex. c5a mediates inflammatory responses through the c5a receptor; however, c5a also exerts an anti-inflammatory effect, putatively through its second receptor, the c5a-like receptor, which may act either as a decoy receptor or by forming the c5ar/c5l2/β-arrestin complex, depending on the cell type, species and disease context. notably, dba/ mice are defective in c though it remains unknown how this c deficiency may contribute to the fatal effect of caws. ifng, the only member of the type ii class of interferons, is a soluble homodimer with potent proinflammatory properties that plays critical roles in innate and adaptive immunity against viral and intracellular bacterial infections. ifng-induced inflammation is involved in aging and aging-associated medical and psychiatric disorders. notably, ifng level was increased in splenocyte culture supernatants following stimulation with lipopolysaccharide, by a gram-negative bacterial cell wall component or with caws. thus, it appears that complement factors and ifng are somehow involved in caws-vasculitis; however, no direct data to confirm this have been reported. in the present study, we report the results of dna microarray analysis of mrnas from pbmcs comparing differences in gene expression patterns in b and dba/ mice. we found that mrna levels of ifng and complement factors such as c c cfb, cfh, and fcna were conspicuously augmented only in dba/ mice at and weeks after caws administration. moreover, mrna levels of immune-related genes, such as irf irf irf cebpb, ccl itgam, icam and il-12rb whose expression levels are known to be augmented by ifng, were also increased only in dba/ mice. based on these results, we propose a molecular model to explain how caws treatment induces fatal vasculitis in dba/ mice.  induction of arteritis by injection of caws into dba/ mice to induce arteritis in b and dba/ mice, we performed intraperitoneal injection of caws for consecutive days as reported previously; we will call this process as caws administration, hereafter, and set this timing as week. although the number of surviving mice at w, w, w, and w following caws administration was remarkably reduced in dba/ mice, similar to our previous report, few b mice died during this period, which is also similar to our previous report. at w, w, w, and w after caws administration, the surviving mice were sacrificed and mrna isolated from pbmcs. three mice were sacrificed at each time point. tissue sections that were obtained from paraffin-embedded hearts fixed with 10% neutral formalin and stained with hematoxylin and eosin revealed vasculitis in the aorta and coronary artery of dba/ mice, with fibrotic thickening of the arterial media and adventitia and infiltration of inflammatory cells. in tissue sections obtained from the hearts of mice at w, w, w, and w following caws administration, we observed infiltration of inflammatory cells in the aortic media and adventitia of dba/ mice, a histological finding typical of aortitis, as early as w following caws administration, and this was observed up to the final w time point. b mice also showed infiltration of inflammatory cells in the aortic media and adventitia, although the onset of aortitis in b mice occurred later than in dba/ mice, not appearing until 4– w following caws administration. since these results were similar to those of our previous report, we concluded that the mice used for dna microarray analysis in this study responded to caws in a similar manner to those described in our previous report.. red vertical arrows indicate the time points at which three b mice and three dba/ mice were sacrificed for mrna purification. histopathological analysis was performed on aortic roots and coronary arteries collected from dba/ mice at w following caws administration stained with h&e. enlarged views of the region indicated by rectangles in the left panels are shown in the right panels. bar = μm for left panels and μm for right panels. caws enhanced expression of complement system genes only in dba/ mice to identify genes whose expression levels were up- or downregulated in the pbmcs of dba/ mice, but not in b mice, following caws treatment, we examined the transcriptome profiles of mrna prepared from dba/ and b pbmcs at w, w, w, or w following caws administration using agilent’s whole mouse genome microarray. when the relative intensities of the signals at w, w, w, or w were compared with those at w following caws administration, the genome-wide expression profiles were found to be distinct between these two mouse strains. heat map representation of the signal intensities revealed that a number of genes were dramatically up- or downregulated in dba/ mice compared to b mice. the most notable genes that showed enhanced expression following caws treatment in dba/ mice belong to the “complement system”, in particular the genes encoding c c complement factor b, complement factor h, and ficolin-a. the complement 3a receptor gene showed modestly enhanced expression at w, w, and w following caws administration and conspicuously enhanced expression at w. c5ar gene also showed enhanced expression at w and w, but b mice also showed enhanced expression at this timing, albeit less dramatically. expression of complement c, serping, complement factor i, and hemolytic complement was extremely low in b mice at w, w and w, but their expression was detectable in dba/ mice, displaying a comparatively enhanced ratio in dba/ mice compared with b mice. by contrast, complement c, decay accelerating factor, and complement receptor showed decreased expression following caws treatment in dba/ mice, although these genes were weakly expressed in b. to determine whether changes in the expression levels of these genes is physiologically significant, a scatter plot was used to analyze the distribution of the expression levels of many genes at a glance. indeed, c c cfb, cfh, fcna, and cr showed expression levels high enough to conduct physiologically significant comparison in dba/ mice at w or w following caws administration. by contrast, complement genes that showed very low expression levels compared to above described activated complement genes in both b and dba/ mice were not deemed significant. c3ar mrna levels were low at w or w and its expression level was enhanced only at w following caws administration. thus, we conclude that the enhanced mrna levels of c c cfb, cfh, and fcna observed after caws administration in dba/ mice are significant. the alternative complement pathway is activated in dba/ mice following caws administration we next performed an ontology search using ingenuity pathways analysis. this powerful query system facilitates the rapid visualization of relationships between sets of relevant genes to predict the putative physiological significance of these genes. we found that alternative complement pathway genes, including c cfb, and cfh, appear to play major roles in activation of the complement system. by contrast, classical pathway genes, including complement c and serping g, and lectin pathway genes including mannose-binding lectin appear to play less significant roles because they showed low levels of expression even in dba/ mice. the low mrna level of c measured in dba/ mice suggests that downstream regulation of the complement system is not important in the induction of vasculitis in the aortas or coronary arteries of dba/ mice. to confirm that the microarray signals accurately reflected mrna levels, expression profiling using quantitative reverse transcription-polymerase chain reaction analysis was performed for c c cfb, cfh, and fcna genes using the same rna samples used for the microarray analysis. indeed, all of the genes analyzed by qrt-pcr showed a similar pattern of mrna distribution following caws administration; namely, they showed increased mrna levels of the denoted genes in dba/ mice compared with b mice, confirming the accuracy of the microarray analysis data. caws induced upregulated expression of interferon-related genes only in dba/ mice we next examined the expression of interferon-related genes because expression of the ifng gene was enhanced following caws administration in dba/ but not in b mice, as revealed by both dna microarray and qrt-pcr analyses. notably, ifng was the only interferon-related gene that exhibited upregulated dba/2-specific expression following caws administration: moreover, mrna expression of ifng was more conspicuously upregulated than that of other interferon gene subtypes in dba/ mice. in the “interferon signaling pathway”, interferon regulatory factor- also showed increased expression, although less significantly than interferon itself, in dba/ mice at w and w. notably, the mrna level of irf was decreased at w, and this depressed level persisted throughout the experiment. this may explain why many of the interferon-stimulated genes were not activated; the irf9/isgf3g complex binds to the interferon-stimulated response element to activate the transcription of interferon-stimulated genes. in the pathway identified as the “role of prk in interferon induction and antiviral response”, mrna levels of eukaryotic translation factor alpha kinase and irf were enhanced. eif2aka is a heterotrimeric gtpase that controls the selection of correct start codons on mrna, and thus regulates translation initiation by facilitating the preferential translation of selected transcripts in response to cellular stresses, in this case caws administration. our results suggest that dba/ mice are more sensitive to this type of stress than b mice. in the “acute phase response signaling” pathway, three genes other than c and cfb were upregulated; ccaat/enhancer-binding protein beta, il- and lipopolysaccharide-binding protein. cebpb, a member of the c/ebp family of basic region-leucine zipper proteins, is largely expressed in macrophages and is important for the antibacterial activity of macrophages through binding to the regulatory regions of several acute phase and cytokine genes. enhanced expression of cebpb following caws administration suggests that downstream target genes of cebpb, including il-, may be widely activated. il- exerts pleiotropic effects on various target cells, including b cells, eosinophils, and basophils, and overexpression of il- in vivo significantly increases the number of eosinophils and b cells. lbp is involved in the acute phase immunologic response to gram-negative bacterial infections through binding to bacterial lipopolysaccharide, thereby eliciting immune responses by presenting the lps to the cell surface pattern recognition receptors cd and tlr4. in this case, caws, rather than lps, may have been recognized as an infectant. in the “acute phase myeloid leukemia signaling” pathway, two genes were upregulated: spleen focus-forming virus proviral integration and colony stimulating factor receptor. sfpi is a transcription factor that plays a key role in hematopoietic cell fate decisions through mutual negative regulation that occurs between sfpi1and gata- a zinc finger transcription factor that is the central mediator of erythroid gene expression. csf1r is a single pass type i membrane protein that acts as a receptor tyrosine kinase for a cytokine called colony stimulating factor which is a primary growth factor and monocyte/macrophage chemokine that regulates survival, proliferation and differentiation of macrophages and other mononuclear phagocytic lineage cells. enhanced expression of csfr suggests the activation of macrophages following caws administration. a scatter plot of irf irf c cebpb, csf1r, trf, ifng, eif2aka, and cfb expression showed expression levels high enough to conduct physiologically significant comparisons in dba/ mice at w or w following caws administration, suggesting that their enhanced mrna levels indicated by the heatmap are significant. by contrast, mrna levels of lbp and sfpi may be too low for comparison between b and dba/ mice to be significantly different. transcription factor genes showing upregulated expression only in dba/ mouse some transcription factor genes also showed upregulated expression only in dba/ mice with mrna levels peaking at w, w, w, and w/ w. irf irf and cebpb were discussed above. stat a signal transducer and activator of transcription, plays a crucial role in the pathology of atherosclerosis resulting from the effects of interferon-γ produced by t cells. stat also induces increased expression of several proinflammatory and pro-atherogenic mediators of signaling mediated by tlr4. expression of nupr a highly basic and loosely folded multifunctional protein, is induced in response to several cellular stresses and provides protection against metabolic stress-mediated autophagic-associated cell death resulting from activation of the nupr1-aurora kinase a pathway. t-box, a transcriptional regulator required for cardiac development, plays a dual role as both a transcriptional activator and a repressor, promoting or repressing distinct genetic programs within adult heart in response to cellular stress. increased expression with twin peaks observed at w and w suggests that caws injection altered regulation of tbx thereby generating cardiac phenotypes. zinc finger protein, also called nmp, is a nucleo-cytoplasmic shuttling transcription factor that regulates the expression of collagen and matrix metalloproteinases and represses bone formation induced by parathyroid hormone. ankyrin repeat and socs box-containing protein interacts with mixed lineage leukemia protein, a key epigenetic regulator of normal hematopoietic development, and overexpression of asb degrades mll to reduce its transactivation activity. zinc finger protein, a member of the tristetraprolin family of tandem ccch finger proteins that bind to au-rich elements in the 3’-untranslated region of mrnas, is a critical modulator of definitive hematopoiesis. zinc finger protein, a member of the c2h2-zinc finger family of proteins, appears to function as a transcriptional and cell cycle/apoptosis regulator and play a role in the establishment and maintenance of pluripotency. although these proteins play roles in several important cellular events such as hematopoiesis, these functions do not appear to be relevant to the pathogenesis of caws-induced vasculitis. scatter plot analysis indicated that mrna levels of irf cebpb, stat nurp irf and brd were high enough to conduct physiologically significant comparison between b and dba/ mice according to their heatmaps at both w and w following caws administration, suggesting that their superficially augmented expression is physiologically significant. by contrast, mrna levels of asb zfp zfp zfp zfp and tbx were too low to conclude that heatmap comparison of these genes between b and dba/ mice is physiologically significant. finally, caws administration resulted in a slight, but comparable increase in dectin- mrna levels in both b and dba/ mice. moreover, scatter plot analysis indicated that the mrna levels of dectin- mrna were lower than those of dectin- a similar c-type lectin molecule. discussion in the present study, we used dna microarray analysis to identify genes whose mrna levels in pbmcs were upregulated in dba/ mice but not in b mice at w, w, w, and w following caws administration. we found that the mrna levels of c c cfb, and cfh, which regulate the alternative pathway of the complement system, are conspicuously increased in dba/ mice at w, w, and w following caws administration, whereas b mice showed decreased mrna levels of these proteins, and this notable difference was confirmed by qrt-pcr. we also observed that mrna levels of ifng and interferon-related genes were augmented in the pbmcs of dba/ mice compared with those of b mice. ifng expression is induced upon infection by pathogens such as viruses, bacteria, or parasites, suggesting that some components of caws, possibly the polysaccharide chains of α and β mannoproteins in the cell wall extract, are recognized as pathogens after injection. ifng enhanced c mrna and protein expression levels in a human astroglioma cell line. ifng increased steady state mrna levels of both c and c in three different human cell types. ifng stimulation also increased c and c protein synthesis. moreover, ifng induced biosynthesis of complement components c c and cfh at the transcriptional level in human proximal tubular epithelial cells. in mice, cfb mrna expression was synergistically upregulated in murine macrophages, and ifng directly stimulated transcription or stabilized complement c and c mrnas. transgenic mice over-expressing ifng in the brain exhibited neuroinflammation induced by upregulation of the early components of the complement cascade, including c and c4a. thus, increased c and c mrna expression following caws administration appears to be due to increased ifng expression, which may occur due to recognition of caws as a pathogen. interferon regulatory factors, including irf, irf, and irf, are ifng-induced genes encoding transcription regulators that induce the expression of other immune-related genes, including genes that mediate the antiviral activity of ifng and play pivotal roles in the regulation of cellular responses, such as inflammation in host defense. indeed, ifng enhanced the expression of itgam, icam1, and il-12rb1. in particular, irf which regulates the expression of ifn-induced genes and type i interferon, is an early signal that promotes inflammation; irf1-deficient mice are protected from autoimmune brain inflammation through regulation of th2-type cytokines. moreover, expression levels of irf were significantly higher in the synovium of joints with rheumatoid arthritis than in that of those with osteoarthritis. irf gene activation by reactive oxygen species is an early signal that promotes inflammation after ischemic renal injury. notably, ifng increases the expression of irf protein in human pbmcs, and cebpb, which plays a fundamental role in regulating activated macrophage functions, is necessary for ifng-induced upregulation of irf mrna in cultured mouse bone marrow-derived macrophages. cebpb is also involved in expression of ccl mrna induced by ifng and lipopolysaccharide. the low but similar expression levels of dectin- in b and dba/ mice suggest that dectin- plays similar roles in caws-vasculitis in these two mice. the lethality of caws-vasculitis in dba/ mice, but not in b mice, may be due to the increased expression and activity of components in the ifng-complement axis. dectin- a β-glucan specific receptor, may not be involved in caws-vasculitis, because caws is a mannoprotein and lacks the dectin- ligand, beta- -glucan, which results in no signal being transmitted via the dectin- route. a recent report suggests that the mannan-dectin-2/fceric pathway promotes phosphorylation of a protein called linker for activation of b cells, which induces natural killer and t cell-mediated ifng production following c. albicans infection. based on these observations, we propose the following molecular model to explain how caws administration causes lethal vasculitis in dba/ mice but not in b mice. following injection into the mouse body, the α-mannan component of caws binds to the α-mannan receptor dectin- and is recognized as a pathogen, putatively leading to induced ifng expression. one downstream pathway, shown by dotted green arrows, may lead to mild inflammation and non-lethal vasculitis in both b and dba/ mice. the other pathway, shown by dotted black arrows, results in enhanced expression of ifng. ifng then enhances c and c expression, which leads to neutrophil activation and causes a cytokine/chemokine storm, inducing damage and necrosis of vascular endothelial cells, severe inflammation and finally, a lethal vasculitis. ifng also enhances expression of irf irf and irf transcription factors that regulate the expression of immune-related genes that may also lead to severe inflammation of the arteries and subsequent lethal vasculitis. cebpb expression induced by ifng may play a role in enhanced expression of ifg and ccl which also leads to arterial inflammation, and itgam, icam and il-12rb induced by ifng may also participate in inducing severe inflammation. it was recently shown that caws-treated dba/ mice developed left ventricular dilution and dysfunction with macroscopic cardiomegaly and concentric left ventricular hypertrophy. we propose here that this chronic mortality of caws-treated dba/ mice is due to abnormal activation of the complement system induced by ifng upregulation. this proposal is supported by recent reports showing that c5a elicits anti-inflammatory action through c5l and that inflammation in dba/ mice deficient in c cannot be inhibited following caws treatnment. the proposal is also supported by the observation that inflammation cannot be inhibited in a mutant mouse deficient in c expression with acute pancreatitis and associated lung injury. caws also induced vasculitis in other mouse strains, such as c3h/hen, c3h/hej, dba/ a/j, cba/n, c57bl, akr, and balb/c mice. it would be interesting to test whether the ifng-complement axis is also activated following caws administration in these mouse models in the future. we and others have reported that complement activation is involved in the pathogenesis of systemic autoimmune diseases. in active anti-neutrophil cytoplasmic antibody-associated vasculitis with renal lesions, significantly higher levels of plasma c3a, c5a, soluble c5b- and bb, a component of the alternative complement pathway, was observed during active development of the disease. ficolin- mrna level showed a four-fold increase in the pbmcs of ta patients, and that ficolin- protein expression was observed in the inflamed regions of surgical aorta specimens, in particular in cd68-positive cells, which are involved in the induction of inflammation. ficolin- mrna level was also higher in pbmcs isolated from microscopic polyangiitis patients than in pbmcs isolated from healthy volunteers. moreover, increased expression of ficolin- protein was also detected by immunostaining in renal specimens taken from mpa patients, and many of the ficolin- positive cells coincided with cd68-positive cells in the glomeruli of mpa patients. these results suggest that enhanced expression of ficolin- in monocytes and macrophages is also involved in the pathogenesis of human vasculitis.  our results suggest that caws administration induces non-lethal caws-vasculitis in both b and dba/ mice via a dectin- pathway, but induces enhanced expression of ifng only in dba/ mice, which leads to augmented expression of c c cfb, cfh, and fcna, thereby causing lethal inflammation of the arteries and severe vasculitis. thus, the ifng-complement axis may play an important role in the pathogenesis of lethal caws-vasculitis, and suggest that this axis may also be important in the pathogenesis of human vasculitis. 
candida albicans water-soluble fraction, a mannoprotein-β-glucan complex obtained from the culture supernatant of c. albicans nbrc causes caws-mediated vasculitis in b and dba/ mice with mild and lethal symptoms, respectively. we found that the mrna levels of interferon-γ and several genes that regulate the complement system, such as c c cfb, cfh, and fcna, were increased dramatically only in dba/ mice at and weeks after caws administration. moreover, mrna levels of immune-related genes, such as irf irf irf cebpb, ccl itgam, icam and il-12rb whose expression levels are known to be increased by ifng, were also increased, but only in dba/ mice. by contrast, the mrna level of dectin- the critical receptor for the α-mannans of caws, was increased slightly and similarly in both b and dba/ mice after caws administration. taken together, our results suggest that caws administration induces dectin- mediated caws-vasculitis in both b and dba/ mice and the expression of ifng, but only in dba/ mice, which led to increased expression of c c cfb, cfh, and fcna and an associated increase in lethality in these mice.
 candida albicans water-soluble fraction, a mannoprotein-β-glucan complex obtained from the culture supernatant of c. albicans nbrc causes caws-mediated vasculitis in b and dba/ mice with mild and lethal symptoms, respectively. why caws is lethal only in dba/ mice remains unknown.  we performed dna microarray analyses using mrna obtained from peripheral blood mononuclear cells of b and dba/ mice and compared their respective transcriptomes. we found that the mrna levels of interferon-γ and several genes that regulate the complement system, such as c c cfb, cfh, and fcna, were increased dramatically only in dba/ mice at and weeks after caws administration. the dramatic increase was confirmed by quantitative real-time polymerase chain reactions. moreover, mrna levels of immune-related genes, such as irf irf irf cebpb, ccl itgam, icam and il-12rb whose expression levels are known to be increased by ifng, were also increased, but only in dba/ mice. by contrast, the mrna level of dectin- the critical receptor for the α-mannans of caws, was increased slightly and similarly in both b and dba/ mice after caws administration.  taken together, our results suggest that caws administration induces dectin- mediated caws-vasculitis in both b and dba/ mice and the expression of ifng, but only in dba/ mice, which led to increased expression of c c cfb, cfh, and fcna and an associated increase in lethality in these mice. this model may contribute to our understanding of the pathogenesis of severe human vasculitis. cawsdna microarraydba/2kawasaki diseasecomplement factorsifng 