intestinal inflammation is a common occurrence for premature infants . after a relatively protective intrauterine environment , the newborn
is abruptly exposed to nonmaternal antigens and abnormal bacterial flora in the intensive care setting .
exposure to formula has been shown to increase proinflammatory cytokines in the immature intestine , and breast milk from mothers exposed to a western diet has been shown to contain increased amounts of long chain and saturated fatty acids that induce inflammation in the immature intestine .
while these data indicate that diets of premature infants put them at risk for chronic gut inflammation , the effects of chronic inflammation on the developing intestine are not fully understood .
notably , enteral feeding is one of the strongest risk factors for the development of necrotizing enterocolitis ( nec ) , leading to questions regarding the role that feeding - induced chronic intestinal inflammation may play in the induction and pathophysiology of the disease .
nec is predominantly a disease of premature infants , primarily affecting infants born at the shortest gestations [ 4 , 5 ] .
the incidence of nec is the highest between the ages of 28 and 32 weeks relative gestation regardless of when an infant is born , suggesting that changes in gut development regulate susceptibility [ 6 , 7 ] .
a key component of nec is exaggerated inflammation [ 810 ] mediated by potent inflammatory cytokines such as tumor necrosis factor ( tnf ) and platelet activating factor ( paf ) . in human studies ,
newborn infants who developed nec expressed significantly higher serum tnf levels compared to controls , and in animal models of nec , animals treated with anti - tnf antibodies have lower incidence and severity of disease .
our lab recently reported that significant developmental - stage dependent changes can be seen in intestinal goblet cell mucus secretion in the murine ileum eight hours following tnf exposure .
furthermore , acute tnf exposure mediates the early stages of tissue damage seen in salmonella infection of the intestinal tract   and can induce decreases in villus height in burn patients .
however , the specific role of tnf in the pathophysiology of nec is not completely known and the mechanisms behind the initiation of inflammatory signaling represent a major gap in our understanding . considering that ( 1 ) susceptibility to nec appears to be developmentally - dependent , ( 2 ) premature infants have frequent exposure to intestinal inflammation , and ( 3 ) tnf induced inflammation can have developmental dependent effects on the intestinal epithelium , we tested whether tnf induces other developmentally dependent changes in the small intestine . to better understand how different exposures of inflammation would affect the developing intestinal tract , we examined components of intestinal architecture following several different types of exposure to tnf . in the following studies ,
we show that both acute and chronic exposure to tnf have developmental - stage dependent effects on the developing small intestine .
c57bl/6j , tnfr1 , tnfr2 , and tnfr12 mice were purchased from the jackson laboratory .
all genetically modified mice were maintained on a c57bl/6j background and c57bl/6j was used as wild type comparisons . for acute tnf experiments , c57bl/6j or cd-1 mice
were injected intraperitoneally at the indicated ages with 0.5  g / gbw tnf ( peprotech ) and maintained for 8 hours separated from mothers and without feeds at 33c in a humidified incubator ( petiatric.com ) .
tissues were fixed by snap freezing or carnoy solution ; tissues prepared for immunohistochemistry were paraffin - embedded and sectioned at 5  m . for chronic tnf experiments ,
, mice were housed under normal conditions at the animal care facility at the university of iowa . on the final day of experimentation
all animal experiments were performed according to protocols approved by the institutional animal care and use committee at the university of iowa .
ileal sections were stained with h&e ( sigma - aldrich ) . in an attempt to minimize sectioning variability ,
all sections were obtained from the center of the intestinal sample and only areas with full villi were included . in each sample used for measurement , at least 3 areas were counted to minimize sectioning variances .
measurements were obtained at each timepoint from at least five separate animals . a total of at least 20 consecutive villi from 3 separate areas per animal were analyzed in each experimental group .
villus length and surface area measurements were taken with a 10x objective ( 100x total magnification ) from the tip of the villus to the entrance of the crypt opening .
villus length was measured by drawing a bisecting line through the center of the villi .
villus epithelial cells were counted with a 60x objective ( 600x total magnification ) from the entrance of the crypt opening to the beginning of the curve at the villus tip .
intestinal sections from at least five animals for a total of at least 70 villi per animal were analyzed for each experimental group . to detect paneth cells ,
slides were stained with alcian blue / periodic acid schiff stain ( sigma - aldrich ) .
cells were quantified with a 60x objective ( 600x total magnification ) by a single blinded investigator .
intestinal sections from at least five animals were analyzed for each experimental group and at least 100 crypts were counted per animal .
all data were obtained using a nikon niu microscope using nikon elements software ( nikon ) .
ileal samples were homogenized using a tissuelyser lt ( qiagen ) , then cleared , and boiled as previously described .
membranes were blocked for 1  h in tris - buffered saline with 0.05% tween 20 ( tbst ) and 5%
nonfat dry milk , incubated with anti - tnfr1 primary antibody ( santa cruz biotechnology ) overnight at 4c , and incubated with secondary antibody ( cell signaling ) for 45 minutes .
horseradish peroxidase was detected with the western lightning enhanced chemiluminescence kit ( perkinelmer life sciences ) .
for mrna quantification , ileal samples were homogenized as above and rna was isolated using rneasy plus mini kit ( qiagen ) according to manufacturer 's directions .
rna concentration and quality were determined using a nanodrop 1000 spectrophotometer ( thermo fisher scientific ) .
first - strand cdna was synthesized using high - capacity cdna reverse transcription kit with rnase inhibitor ( life technologies ) .
quantitative real - time reverse transcription - polymerase chain reaction ( qrt - pcr ) was performed using taqman fast universal pcr master mix ( 2x ) ( life technologies ) and taqman gene expression assays for tnfr1 primers ( life technologies ) .
qrt - pcr reactions were run in a c1000 thermal cycler ( eppendorf ) and using the cfx96 real - time pcr detection system ( bio - rad ) .
the threshold cycle ( ct ) value for each well was obtained by using the instrument 's software .
fold change in gene expression was determined by normalizing gene expression to -actin in each sample .
rates of apoptosis on histological specimens were determined by in situ oligo ligation dna fragmentation assay ( isol ; millipore ) , as we have previously reported .
. statistical significance of differences was assessed with one - way analysis of variance ( anova ) .
post - test comparisons between groups were made using holm - sidak 's multiple comparisons test .
minimum level of significance was set at < 0.05 and error bars designate standard error of the mean .
we have previously shown that goblet cell numbers in the murine ileum increase in a developmental - dependent manner during the first four weeks of life . to determine if ileal height developed in a similar pattern , we measured the length of ileal villi at various stages of development throughout the first four weeks of life of the mouse .
birth did not affect villus length , as there was no difference between e19 and p0 mice .
we have previously shown that tnf has an age - dependent effect on intestinal mucus production and secretion ; however , the effects of tnf on the developing intestinal morphology in the immature intestine remain unknown . to test these effects , we examined the ileum eight hours after an intraperitoneal injection with tnf .
this time point was based on our earlier studies of tnf exposure on goblet cell numbers in immature intestine [ 13 , 16 ] .
tnf induced significant villus blunting at p7 , p14 , p21 , and p28 ( figure 1(b ) ) .
in contrast , p0 pups were protected from this effect , suggesting either the presence of a protective signaling pathway or decreased responsiveness to tnf at this stage of development . to determine if tnf - induced villus blunting was specific to the c57bl/6j strain , we repeated this experiment in p14 cd-1 mice and compared these data to the results seen in c57bl/6j mice from figure 1(b ) .
tnf significantly blunted ileal villi in cd-1 mice as well ( figure 1(c ) ) .
our data are thus consistent with a strain - independent effect , though it should be noted that showing true strain independence would require experiments with many more lines , which is beyond the scope of this study .
to further characterize tnf - induced changes in intestinal morphology , we quantified the epithelial cells lining villus cross - sections .
similar to the effects seen on villus length , the number of villus epithelial cells was not affected by tnf at birth . however , in the subsequent 4 weeks , tnf induced a significant decrease in total epithelial cells compared to controls ( figure 2(b ) ) .
we next measured the average villus area of mice treated with tnf compared to controls . in control animals ,
villus area significantly decreased in the first week of life , followed by significant increases during the second , third , and fourth weeks of life .
tnf treatment again had no significant effect at birth but induced significant reductions in villus surface area at later ages ( p < 0.0001 ) ( figure 2(a ) ) . to test
if tnf - induced loss of villus height , surface area , and epithelial cell mass could be due to induction of apoptosis , histological samples from p14 mice treated with tnf were stained by in situ oligo ligation ( isol ) to detect dna fragmentation and compared to controls .
mice exposed to acute tnf had significantly more apoptosis than age - matched controls ( p = 0.03 ) ( figure 3 ) .
we have previously shown that tnf can affect both intestinal epithelial cells and ileal goblet cells through tnf receptor-1 ( tnfr1 ) signaling [ 13 , 16 ] . to determine if the tnf - dependent effects on villus length , area , and epithelial cell mass were also dependent on tnfr1 , p14 tnfr1 , tnfr2 , and tnfr12 mice were given intraperitoneal injections of tnf and their ileal villus measurements were compared to knockout controls .
tnf induced villus blunting in wild type and tnfr2 mice , but not mice lacking tnfr1 .
similarly , tnf treatment induced loss of villus area only in wild type and tnfr2 mice , again demonstrating a requirement for the presence of tnfr1 .
however , only wild type mice showed a significant loss in epithelial cell number ( figure 4 ) .
our findings indicate that the degree of tnf - induced villus blunting varies depending on the stage of development .
to test whether these effects were due to differences in ileal maturity , rather than ontogenic differences in tnfr1 expression , ileal tissue was harvested from wild type mice at various ages and examined by western blot analysis for expression of tnfr1 .
this finding is important , as it demonstrates a developmental stage - dependent change in downstream tnfr1 effectors that alter response without changing receptor expression . since our data demonstrate a developmentally dependent effect of acute tnf on the architecture of the ileum , we investigated the effects of chronic exposure . to accomplish this ,
mice were treated with tnf once a week beginning on p7 until one week prior to euthanasia . in this fashion
, we developed three groups of mice : control mice , mice exposed to a single dose of tnf one week prior to examination ( brief exposure , or b - tnf ) , and mice exposed to weekly tnf treatments beginning on p7 , and ending with the last dose occurring one week prior to euthanasia ( chronic exposure , or c - tnf ) . using this methodology
similar to the acute exposure , mice exposed to chronic tnf showed a significant loss in villus length .
mice treated with brief exposure ( b - tnf ) had significantly shorter villi than controls , and mice treated with chronic exposure ( c - tnf ) had significantly shorter villi than those with brief exposure ( figure 6(a ) ) . to determine if chronic exposure to tnf would impact the levels of tnfr1 , we measured levels of tnfr1 mrna in tissues of p21 mice treated with b - tnf and c - tnf compared to controls .
we chose p21 as a time point because at that age the chronic mice had received more than one tnf dose , but the intestine was still immature ( as opposed to the p28 intestine which is considered to be matured ) .
there was no significant difference in tnfr1 levels in either b- or c - tnf groups compared to controls ( figure 6(d ) ) .
as chronic tnf ( c - tnf ) caused developmentally dependent villus blunting , we next investigated its effects on villus surface area and epithelial cell counts . while acute tnf treatment induced decreases in villus area and epithelial counts at p14 and 21 ( figure 2 ) , brief ( b - tnf ) and chronic ( c - tnf ) treatments induced significant increases in both area and epithelial cell counts at these ages , suggesting a change in villus architecture from long thin villi to shorter , wider villi in the face of chronic inflammation ( equal area with less length ) .
exposure to b - tnf induced a significant increase in villus area but a decrease in epithelial cell numbers , and exposure to c - tnf induced no change in villus area and a decrease in epithelial cell counts .
these effects demonstrate a developmental stage - dependent effect of chronic exposure to tnf on the small intestinal architecture ( figures 6(b ) and 6(c ) ) ( p < 0.0001 ) . our lab and others have described a key protective role for paneth cells in the immature intestinal tract [ 8 , 1820 ] .
tnf has been shown to be important in paneth cell granule release in adult intestine [ 21 , 22 ] ; however , the effect of either acute or chronic tnf on paneth cells from immature intestine is less well described . to examine this we treated mice with acute , brief , or chronic tnf exposures as above and quantified the number of granule containing paneth cells in the ileum compared to controls .
mice were only examined from p14 to p28 as mature paneth cells do not appear in mice until between p7 and p10 [ 23 , 24 ] .
acute tnf induced significant loss of granulated paneth cells in both p14 and p28 mice ( figure 7(a ) ) ( p = 0.002 ) .
however , there was no difference in paneth cell numbers in mice treated with brief or chronic treatments ( figure 7(b ) ) . to determine if acute tnf - induced paneth cell loss was tnfr dependent , we examined the effects of tnf on p14 mice lacking tnfr1 , tnfr2 , or both .
tnf induced significant loss of granulated paneth cells only in tnfr2 mice demonstrating a requirement for tnfr1 ( figure 7(c ) ) ( p = 0.035 ) .
premature infants are routinely subject to intestinal inflammation , both as a result of normal exposure to new environmental factors and through pathologic situations such as intestinal perforations and necrotizing enterocolitis .
however , the ramifications of inflammation during development of the intestinal tract are not fully understood . in this study
we next provide evidence that acute tnf exposure can blunt normal villus height during development .
since tnfr1 expression remains constant through development of the small intestine , tnf - induced villus blunting may be dependent on downstream tnf signaling targets that are differentially expressed during development .
lastly , we show that acute tnfr1-mediated signaling also induces a significant decrease in the number of granule containing paneth cells . taken together , these morphologic changes caused by tnf exposure may help to explain why this cytokine plays a prominent role in the development of nec in the immature intestine .
our data demonstrate that both acute and chronic tnf exposures induce significant changes in the immature ileum .
tnf mediates distinct physiological effects through two separate transmembrane receptors , tnfr1 , and tnfr2 [ 25 , 26 ] .
physiological levels of tnf result in a preferential ligation to tnfr2 , which promotes cellular migration , proliferation , and wound healing , while higher concentrations of tnf lead to ligation of tnfr1 and subsequent activation of inflammatory responses .
our lab has recently shown that tnf causes tnfr1-dependent , developmental stage - dependent changes in intestinal goblet cell secretion of mucus .
similarly , we demonstrate in these experiments that tnf - induced villus blunting through a tnfr1-dependent , tnfr-2 independent mechanism .
the developmental - stage dependent differential effects of tnfr1 are intriguing as protein levels of tnfr1 remain constant from birth through four weeks of life , when the ileum reaches maturation .
it was also interesting to note that tnf had little effect on the most immature intestine ( p0 ) despite having similar levels of tnfr1 .
our initial hypothesis was that the most immature intestine would show the greatest effects of tnf exposure . however , tnf had no effect on villus length , villus area , or epithelial cell counts .
this was similar to our data regarding tnf effects on goblet cells   and correlates clinically with a lack of development of nec during extremely premature ages .
these data imply that either an unidentified downstream target of tnfr1 has developmental - stage dependent expression or that another developmental stage - dependent influence acts as a repressor of tnfr1 signaling . either way
, this alteration of tnfr1 signaling may be important , as intestinal inflammation is a common occurrence in premature infants .
the embryogenesis of the gastrointestinal tract is nearly identical in all mammals ; however , the stage of development when birth occurs can vary dramatically between species . while human infants have mature intestinal tracts at term birth ,
mice are born much earlier in the developmental process [ 29 , 30 ] and do not reach a maturity level equivalent to term human infants until four weeks after birth .
thus , the first four weeks of mouse life reasonably approximate the last half of human fetal development , the stage of development when premature infants are most susceptible to developing nec .
thus it is important to note that villus length increased during the first four weeks of life as a function of age ( figure 1(a ) ) . in our experiments to study the effects of chronic inflammation on the intestine , we modeled two types of chronic tnf treatments . brief dosing examined mice one week following a single tnf treatment , and chronic dosing examined mice one week following the last of multiple weekly tnf treatments .
our data show that both types of chronic exposure to tnf induced villus blunting at p14 , p21 , and p28 . however , it was interesting to note that while chronic exposure to tnf induced loss of villus height at all ages , p14 and p21 mice had significantly increased villus area and epithelial cell counts with this exposure .
this suggests a conformation change from thin long villi to shorter , wider villi in the face of chronic inflammation .
this may be a compensation mechanism to place the villus epithelial layer closer in proximity to the lamina propria where the majority of adaptive immune cells reside .
interestingly , this trend was not seen in p28 mice , again demonstrating developmental stage - dependent effects of tnf on the small intestinal architecture .
we and others have recently reported a loss of paneth cells in infants who developed nec [ 18 , 31 ] , and we have recently developed a novel two - hit model of nec in mice that utilizes paneth cell loss to develop injury that is consistent with human disease . in this model , significant injury only occurs following ablation of paneth cells , suggesting that the injury to , or loss of , the paneth cell is required to develop nec .
tnf has been shown to induce paneth cell degranulation in adult mice , thus we desired to determine if tnf would have similar effects in the developing intestine .
our data show that acute tnf significantly reduces granule containing paneth cells in both p14 and p28 mice , and that this reduction is due to a tnfr1-dependent mechanism .
interestingly , chronic exposure to tnf had no effect on the paneth cell population , suggesting that while paneth cells are responsive to tnf exposure , the ileum naturally compensates to preserve the paneth cells in the face of chronic inflammation .
these data correspond to additional results from our laboratory that shows decreased expression of lysozyme , a key granular secretion of paneth cells , in ileal enteroid cultures exposed to tnf . in summary
, we have shown that chronic exposure of the immature ileum to tnf induces not only functional changes but architectural changes as well .
these findings may have important implications for elucidating the pathophysiology of nec . our recently proposed 
model of nec hypothesizes that bacteria invade the intestinal tissue principally through the crypts , rather than through the villus tips as previously thought . in this model ,
tnf - induced blunting caused by feeding - induced or other chronic inflammation could subsequently decrease the distance between the luminal contents of the intestine and the lamina propria .
this shortening of distance would greatly increase the ability of bacteria to reach the crypt and infiltrate the intestine .
this , along with other effects of tnf such as depletion of the mucous layer and degranulation of paneth cells , may allow for easier bacterial penetration into the intestinal lamina propria , leading to the inflammatory response and coagulation necrosis characteristic of nec .
 
 background . 
 premature infants are commonly subject to intestinal inflammation . since the human small intestine does not reach maturity until term gestation , premature infants have a unique challenge , as either acute or chronic inflammation may alter the normal development of the intestinal tract . tumor necrosis factor ( tnf ) has been shown to acutely alter goblet cell numbers and villus length in adult mice . in this study we tested the effects of tnf on villus architecture and epithelial cells at different stages of development of the immature small intestine . 
 methods . to examine the effects of tnf - induced inflammation 
 , we injected acute , brief , or chronic exposures of tnf in neonatal and juvenile mice . 
 results . 
 tnf induced significant villus blunting through a tnf receptor-1 ( tnfr1 ) mediated mechanism , leading to loss of villus area . 
 this response to tnfr1 signaling was altered during intestinal development , despite constant tnfr1 protein expression . 
 acute tnf - mediated signaling also significantly decreased paneth cells . conclusions . taken together 
 , the morphologic changes caused by tnf provide insight as to the effects of inflammation on the developing intestinal tract . 
 additionally , they suggest a mechanism which , coupled with an immature immune system , may help to explain the unique susceptibility of the immature intestine to inflammatory diseases such as nec . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion