interferon tau ( ifnt ) is produced by trophectoderm cells of conceptuses of ruminant species and is the maternal recognition of the pregnancy signal .
besides its critical roles in implantation and establishment of pregnancy in ruminants [ 1 , 2 ] , it has a plethora of physiological functions in various cell types such as macrophages , lymphocytes , and epithelial cells in humans and mice [ 35 ] .
it is a type i interferon ( ifn ) , which includes ifn alpha ( ifna ) , ifn beta ( ifnb ) , ifn delta ( ifnd ) , and ifn omega ( ifnw ) . after binding to a common receptor , ifna receptor 1 ( ifnar1 ) , and ifnar2 , type
i ifns affect the production of inflammatory cytokines such as interleukin- ( il- ) 1 and tumor necrosis factor  ( tnf- ) [ 6 , 7 ] .
thus , type i ifns have widely recognized roles in inflammatory diseases , such as experimental allergic encephalomyelitis , multiple sclerosis , and spontaneous autoimmune diabetes [ 5 , 810 ] .
notably , unlike other members of type i ifn family , ifnt has few adverse effects and low cytotoxicity even at high dosages [ 11 , 12 ] , suggesting its therapeutic potential as an alternative to other type i ifns due to its anti - inflammatory effects .
recent compelling findings about the anti - inflammatory effects of ifnt include lower nlrp3 ( nucleotide - binding oligomerization domain - like receptor , pyrin domain - containing 3 ) inflammasome - driven il-1 secretion by human macrophages , mitigation of obesity - associated systemic tissue inflammation in mice , and promotion of th2 biased immune response in mice .
the intestinal microbiota provides important benefits for the development of immune responses ; however , the disturbances in the intestinal microbiota are associated with numerous chronic inflammatory diseases [ 13 , 14 ] . also , the effect of ifnt on expression of il-17 in the intestine is not known .
the potential effect of ifnt on expression of il-17 is important as il-17 promotes local chemokine production to recruit monocytes and neutrophils to sites of inflammation that leads to development and pathogenesis of various autoimmune diseases , including rheumatoid arthritis , psoriasis vulgaris , multiple sclerosis , and inflammatory bowel diseases [ 15 , 16 ] . in this study ,
the intestinal microbiota and expression of il-17 in the intestine were explored after two weeks of ifnt supplementation in a mouse model .
the hypothesis is that ifnt supplementation alters intestinal microbiota and intestinal innate immunity in mouse model .
this study used the escherichia coli f4-producing strain w25k ( hereafter referred as etec ; o149:k91 , k88ac ; lt , stb , east ) , which was originally isolated from a piglet with diarrhea .
this study was conducted according to the guidelines of the laboratory animal ethical commission of the chinese academy of sciences .
icr ( institute for cancer research ) mice ( six weeks of age ) were purchased from slac laboratory animal central ( changsha , china ) .
the mice were housed individually in a pathogen - free animal vivarium ( temperature , 25c ; relative humidity , 53% ; 12  h dark/12  h light ) and had free access to a standard rodent diet   and drinking water .
after three days of accommodation , mice were assigned randomly into two groups ( ifnt and control ; n = 10/group ) .
mice in the control group were fed the basal diet   and normal water , while mice in ifnt group were fed the basal diet and water containing recombinant ifnt ( 40  g / l ) for two weeks .
the effective supplemental dosage of ifnt was established in previous study [ 5 , 19 ] . at the end of the two weeks of experimental period
, mice were sacrificed to collect contents of the lumens of the jejunum , ileum , and colon , as well as feces .
after three days of accommodation to the conditions of the vivarium , icr mice were assigned randomly into two groups ( etec and ifnt+etec ; n = 10/group ) .
mice in ifnt+etec group were fed the basal diet and recombinant ifnt - supplemented water ( 40 
g / l ) for two weeks , while mice in etec group were fed the basal diet and had normal water .
after two weeks of feeding , mice in both groups were inoculated with 10  cfus of etec w25k by oral gavage . at 6 hours after infection , all active mice were sacrificed to collect the jejunum , and the samples were stored at 80c until processed .
dna was extracted from the luminal contents of the jejunum , ileum and colon , and feces using the qiagen qiaamp dna stool mini kit according to the protocol for isolation of dna .
equal amounts of dna from six different mice were pooled to generate one common sample for each type of sample ( i.e. , control versus ifnt , intestinal source , and feces ) .
the v4-v5 region of the bacterial 16s ribosomal rna gene was amplified by pcr using primers 515f 5-barcode - gtgccagcmgccgcgg-3 and 907r 5-ccgtcaattcmtttragttt-3 , where barcode is an eight - base sequence unique to each sample .
illumina miseq sequencing and general data analyses were performed by a commercial company ( biotree , shanghai , china ) .
miseq pe libraries , miseq sequencing , and further analyses were based on previous work .
total rna was isolated from liquid nitrogen frozen and ground jejunum , ileum , and colon using trizol regent ( invitrogen , usa ) and then treated with dnase i ( invitrogen , usa ) according to the manufacturer 's instructions .
synthesis of the first strand ( cdna ) was performed using oligo ( dt ) 20 and superscript ii reverse transcriptase ( invitrogen , usa ) .
the rt - pcr experiment was conducted according to previous studies [ 18 , 21 ] .
data shown are the means  the standard error of the mean ( sem ) .
all statistical analyses for data were performed using spss 16.0 software ( chicago , il , usa ) .
to investigate the effect of ifnt supplementation on mouse growth performance , feed intake , water intake , and body weight were monitored in ifnt - supplemented mice and control mice . with two weeks of ifnt supplementation ,
the averages for feed intake and water intake for ifnt - supplemented mice were significant ( p < 0.05 ) higher than for control mice ( figures 1(a ) and 1(b ) ) . however , ifnt supplementation had no significant effect on body weight of mice ( figure 1(c ) ) . to explore the influence of ifnt supplementation on the intestinal microbiota
, we analyzed the intestinal microbiota at end of two weeks of ifnt supplementation with 16s rdna sequencing ( table 1 ) .
for microbiota in the jejunum , both shannon and simpson indices demonstrated that the diversity of microbiota in mice with ifnt supplementation was higher than the control mice , while the richness indices ( ace and chao ) suggested that the community richness in ifnt - supplemented and control mice was similar ( table 1 ) .
for the microbiota in the ileum , the diversity of microbiota ( shannon and simpson ) and richness indices ( ace ) for mice with ifnt supplementation were higher than that of control mice ( table 1 ) .
for the microbiota in the colon , the diversity of microbiota ( shannon ) and richness indices ( ace and chao ) were similar for ifnt - supplemented and control mice ( table 1 ) .
for the fecal microbiota , microbial diversity ( shannon and simpson ) in mice with ifnt supplementation was lower than for control mice , while the community richness ( ace and chao ) for ifnt - supplemented mice was similar to that for control mice ( table 1 ) .
collectively , ifnt supplementation increases the diversity of microbiota in small intestine , while decreasing the diversity of microbiota in the feces . the taxonomy of the intestinal microbiota was assessed using a taxon - dependent analysis and the rdp classifier .
seven phyla , including one candidate division ( tm7 ) , were found in the microbiota of the jejunum for all samples , including six phyla in the control mice and seven phyla in mice with ifnt supplementation .
eight phyla were found in the microbiota of the ileum of all samples , including six phyla in control mice and seven phyla in mice with ifnt supplementation .
ten phyla were found in the microbiota of the colon of all samples , including ten phyla in control mice and eight phyla in mice with ifnt supplementation .
ten phyla were found in the microbiota of the feces of all samples , including nine phyla in control mice and ten phyla in mice with ifnt supplementation .
for the jejunum , the two most abundant phyla in ifnt - supplemented mice , accounting for approximately 99% of all assigned sequence readings , were firmicutes ( 94.5% ) and bacteroidetes ( 4.4% ) ( figure 2(a ) ) . in control mice ,
most abundant phyla were firmicutes ( 97.6% ) and bacteroidetes ( 1.2% ) ( figure 2(a ) ) .
for the ileum , the three most abundant phyla in ifnt - supplemented mice were firmicutes ( 95.3% ) , candidate_division_tm7 ( 2.3% ) , and proteobacteria ( 1.1% ) ( figure 2(b ) ) , while in control mice , they were firmicutes ( 97.7% ) , proteobacteria ( 1.5% ) , and bacteroidetes ( 0.5% ) ( figure 2(b ) ) .
for the microbiota in the colon , the three most abundant phyla in ifnt - supplemented mice were bacteroidetes ( 72.2% ) , firmicutes ( 23.0% ) , and proteobacteria ( 4.1% ) ( figure 2(c ) ) , while they were bacteroidetes ( 75.8% ) , firmicutes ( 18.8% ) , and proteobacteria ( 4.3% ) in control mice ( figure 2(c ) ) .
for feces , the three most abundant phyla in ifnt - supplemented mice were bacteroidetes ( 43.6% ) , firmicutes ( 48.1% ) , and proteobacteria ( 5.9% ) ( figure 2(d ) ) , while bacteroidetes ( 53.2% ) , firmicutes ( 39.2% ) , and proteobacteria ( 5.3% ) were most abundant for control mice ( figure 2(d ) ) .
for the microbiota of the jejunum , the two most abundant genera in ifnt - supplemented mice , accounting for approximately 99% of all assigned sequence readings , were lactobacillus ( 94.3% ) and s24 - 7_norank ( 4.9% ) ( figure 3(a ) ) . in control mice ,
they were lactobacillus ( 97.3% ) and s24 - 7_norank ( 1.2% ) ( figure 3(a ) ) .
for the ileum , the five most abundant genera in ifnt - supplemented mice were lactobacillus ( 93.3% ) , candidatus - saccharimonas ( 2.3% ) , allobaculum ( 1.2% ) , desulfovibrio ( 1.1% ) , and enterorhabdus ( 0.6% ) , while they were lactobacillus ( 97.3% ) , candidatus - saccharimonas ( 0.3% ) , allobaculum ( 0.1% ) , desulfovibrio ( 1.5% ) , and enterorhabdus ( 0.07% ) in control mice ( figure 3(b ) ) .
for the microbiota in the colon , ifnt supplementation increased the percentages of blautia ( 7.0% versus 5.1% ) , bacteroides ( 6.4% versus 3.7% ) , alloprevotella ( 5.2% versus 1.3% ) , and lactobacillus ( 4.0% versus 2.5% ) , compared with control mice ( figure 3(c ) ) .
for the fecal microbiota , ifnt supplementation increased the percentages of lactobacillus ( 30.0% versus 16.7% ) , bacteroides ( 3.3% versus 1.8% ) , and allobaculum ( 4.5% versus 0.4% ) , while decreasing the blautia ( 2.7% versus 6.5% ) compared with control mice ( figure 3(d ) ) .
collectively , ifnt supplementation affects the composition of intestinal microbiota in mice , especially those for the colon and feces .
the effect of ifnt supplementation on activation of intestinal innate immunity in mice was further explored , focusing on the expression of polymeric immunoglobulin receptor ( pigr ) , mucin-4 , cryptidin-1 , cryptidin-4 , cryptidin-5 , il-17 , interferon gamma ( ifn- ) , lysozyme ( lyz ) , and j - chain in the jejunum , ileum , and colon [ 18 , 21 ] . in the jejunum ,
ifnt supplementation significantly decreased the expression of cryptidin-5 , il-17 , ifn- , and lyz , while it had little effect on the expression of the other transcripts ( figure 4(a ) ) .
ifnt supplementation had no significant effect on the expression of those transcripts in the ileum of mice ( figure 4(b ) ) .
in the colon , ifnt supplementation significantly lowered the expression of cryptidin-1 and il-17 but had little effect on the expression of the other transcripts ( figure 4(c ) ) .
as ifnt supplementation decreased the expression of il-17 in the jejunum and colon , we further validated the effect of ifnt to decrease expression of il-17 in etec infected mouse model .
we found that etec infection promotes the il-17 expression in the mouse jejunum at 6 hours after infection ( w. ren and y. yin , unpublished results ) .
after two weeks of ifnt supplementation , expression of il-17 in the jejunum was significantly lower in ifnt - supplemented mice , compared to that of nonsupplemented mice during etec infection ( figure 4(d ) ) .
thus , ifnt supplementation reduces the expression of the inflammatory cytokine , il-17 , in the intestine of mice .
in this study , although two weeks of ifnt supplementation increases the mouse feed and water intake but has little effect on body weight of mice . results of a previous study revealed that ifnt supplementation ( 8  g / kg bw / day ) reduces body weight beginning at 3 weeks after ifnt supplementation in zucker diabetic fatty rats , while lower dose of ifnt supplementation ( 4  g / kg bw / day ) has no significant effect on body weight during 8 weeks of ifnt treatment , indicating that the effect of ifnt on body weight depends on dosage and duration of ifnt treatment .
however , in a mouse model with high - fat or low - fat diet , 12 weeks of ifnt treatment does not significantly affect body weight
. however , ifnt supplementation has little effect on feed intake and water intake in those investigations [ 4 , 5 ] . in the present study ,
ifnt supplementation increases the microbial diversity in the jejunum and ileum , while decreasing the microbial diversity in the feces of mice .
the gut microbiota affects numerous biological functions [ 22 , 23 ] and is linked to the pathogenesis of various diseases , such as obesity , cancer , and liver cirrhosis .
the influence of the gut microbiome on host physiological functions and the pathogenesis of disease in hosts may result from the activities of the microbiome and its metabolic products .
it is widely accepted that body weight is associated with the composition of intestinal microbiome and its metabolic capacity . an increase in the relative proportion of firmicutes
is linked to obesity as firmicutes ferments plant polysaccharides to produce short - chain fatty acids ( scfa ) , which provides additional energy for the host . in phyla ,
ifnt supplementation decreases the percentage of firmicutes , while increasing the bacteroidetes in the jejunum and ileum
. however , ifnt supplementation increases the percentage of firmicutes , while decreasing the bacteroidetes in the colon and feces .
thus , ifnt supplementation may regulate body weight and metabolism through effects on the intestinal microbiota . at the genus level ,
ifnt supplementation decreases the lactobacillus in the jejunum and ileum but increases the percentage of lactobacillus and bacteroides in the colon and feces .
lactobacillus has critical roles in the intestine to combat gastrointestinal bacterial pathogens and rotaviruses through competitive metabolic interactions and the production of antimicrobial molecules .
bacteroides are known for their capacity to metabolize a wide variety of oligosaccharides from the intestinal luminal , such as xylan , starch , and host - derived glycans .
thus , results of the present study suggest that ifnt supplementation affects those functions of the intestinal microbiome in mice .
ifnt supplementation inhibits intestinal expression of il-17 , which suggests that ifnt reduces intestinal inflammation .
il-17 is produced by inducible th17 ( ith17 ) cells and natural th17 ( nth17 ) cells and regarded as an intestinal proinflammatory cytokine .
il-17 can activate nuclear factor b ( nf-b ) transcription factors , extracellular signal - regulated protein kinase ( erk1 and erk2 ) , c - jun n - terminal kinases ( jnk-1 and jnk-2 ) , and mitogen - activated protein kinases ( p38 mapks ) pathways , leading to upregulation of expression of inflammatory cytokines , such as il-6 and il-1 .
recent investigations have revealed that mammalian target of rapamycin ( mtor ) is a critical signaling pathway for th17 responses and il-17 expression [ 3337 ] .
the mtor signaling regulates il-17 expression through hypoxia - inducible factor 1  ( hif-1 ) and ribosomal protein s6 kinase ( s6k : s6k1 and s6k2 ) [ 3740 ] .
mtor signaling activates hif-1 , which promotes il-17 expression by activating rort ( a key transcriptional regulator of th17 cells ) and mediating degradation of foxp3 ( a key transcriptional regulator of treg cells ) .
s6k1 promotes the expression of early growth response protein 2 ( egr2 ) , which then inhibits growth factor independent 1 transcription repressor ( gfi1 ) , which can negatively regulate expression of il-17 without affecting rorc expression [ 37 , 38 ] .
s6k2 ( the nuclear - localized counterpart of s6k1 ) binds to rort to promote nuclear translocation of rort , which can complex with hif- and p300 in the nucleus to promote expression of il-17 [ 3739 ] .
thus , the underlying mechanism by which ifnt supplementation reduces intestinal il-17 expression is of interest .
the effect of ifnt supplementation to decrease expression of il-17 in the intestine indicates a potential therapeutic application of ifnt to mitigate intestinal inflammatory diseases associated with expression of il-17 . in conclusion ,
ifnt supplementation affects the diversity and composition of the intestinal microbiota and decreases expression of il-17 in mice .
the findings from this study are significant in understanding the physiological and immunological functions of ifnt in treatment of inflammatory diseases .
 this study was conducted to explore the effects of interferon tau ( ifnt ) on the intestinal microbiota and expression of interleukin 17 ( il-17 ) in the intestine of mice . 
 ifnt supplementation increased microbial diversity in the jejunum and ileum but decreased microbial diversity in the feces . 
 ifnt supplementation influenced the composition of the intestinal microbiota as follows : ( 1 ) decreasing the percentage of firmicutes and increasing bacteroidetes in the jejunum and ileum ; ( 2 ) enhancing the percentage of firmicutes but decreasing bacteroidetes in the colon and feces ; ( 3 ) decreasing lactobacillus in the jejunum and ileum ; ( 4 ) increasing the percentage of blautia , bacteroides , alloprevotella , and lactobacillus in the colon ; and ( 5 ) increasing the percentage of lactobacillus , bacteroides , and allobaculum , while decreasing blautia in the feces . also , ifnt supplementation decreased the expression of il-17 in the intestines of normal mice and of an intestinal pathogen infected mice . in conclusion , 
 ifnt supplementation modulates the intestinal microbiota and intestinal il-17 expression , indicating the applicability of ifnt to treat the intestinal diseases involving il-17 expression and microbiota . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 