Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

Levels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Il-1β protein levels are increased in neonatal and juvenile SP-A −/− mice compared to wild type mice. We next sought to determine if these changes are reflected at the level of mRNA. Using the primers listed in Table 1, we performed real-time quantitative RT-PCR analysis of TLR4, IL-1β, IL-6, CXCL1 and TNF-α mRNA expression in the ileum of SP-A −/− and wild type mice. As shown in Fig. 2A, expression of these mRNAs is increased in 1 week old SP-A −/− mice relative to wild type mice, although only TLR4, IL1-β and CXCL1 expression are significantly increased. On the other hand, expression of all assayed mRNAs is significantly increased in 2 weeks old SP-A −/− mice relative to wild type mice (shown in Fig. 2B) and the difference is greater than that seen in 1 week old mice, reflecting the results of Fig. 1. These results indicate that increases in ileal TLR4 and proinflammatory cytokines occur at the level of mRNA expression in the absence of SP-A.
Orogastric gavage of SP-A-deficient neonatal mice with purified SP-A reduces ileal TLR4 protein levels. Because we show that the absence of SP-A results in increased ileal TLR4 and proinflammatory cytokine levels, we then determined if the presence of exogenous SP-A in the ileum could reduce TLR4 protein levels. While our previous investigations demonstrated that its orogastric gavage could alter ileal physiology of neonatal rat pups in an experimental model of NEC, we did not determine the presence of gavaged SP-A in the ileum 37 . Using SP-A-deficient mice to mitigate any interference from endogenous SP-A, we determined if SP-A could be delivered to the ileum via orogastric lavage. One wk and 4 wk old wild type and SP-A −/− mice were orogastrically gavaged with 50 µg purified human SP-A in 100 µl PBS containing trypan blue as a marker. When mice were sacrificed 2 h later, the ileum had been stained with trypan blue indicating transit to that location.   Human   TLR4  5′-TTT GGA CAG TTT CCC ACA TTGA  5′-AAG CAT TCC CAC CTT TGT TGG  75 bp   IL-1β  5′-ACA GAT GAA GTG CTC CTT CCA  5′-GTC GGA GAT TCG TAG CTG GAT  72 bp   IL-6  5′-AAT TCG GTA CAT CCT CGA CGG  5′-TTG GAA GGT TCA GGT TGT TTTCT  112 bp   IL-8  5′-ATG ACT TCC AAG CTG GCC GTG GCT 5′-TCT CAG CCC TCT TCA AAA ACT TCT C 291 bp   Mouse   TLR4  5′-TTT ATT CAG AGC CGT TGG TG  5′-CAG AGG ATT GTC CTC CCA TT  185 bp   IL-1β  5′-GCC ACC TTT TGA CAG TGA TGAG  5′-AAG GTC CAC GGG AAA GAC AC  218 bp   IL-6  5′-TAG TCC TTC CTA CCC CAA TTTCC  5′-TTG GTC CTT AGC CAC TCC TTC  75 bp   CXCL1  5′-CTG CAC CCA AAC CGA AGT C  5′-AGC TTC AGG GTC AAG GCA AG  66 bp   TNF-α  5′-CAG CCT CTT CTC ATT CCT GC  5′-GGT CTG GGC CAT AGA ACT GA  132    www.nature.com/scientificreports/ Western blot analysis was performed to detect SP-A in the ileum. As seen in Fig. 3A, western analysis demonstrated that SP-A can be detected in the ileum of 1 week old SP-A −/− gavaged with SP-A, but not in similarly aged wild type and SP-A −/− mice receiving only PBS. Interestingly, SP-A was not detectable in 4 week old SP-A −/− gavaged with SP-A, suggesting that while the immature 1 week old stomach could not digest SP-A, the older 4 week old stomach could. These results indicate that orogastric gavage of mice 1 week and younger can deliver SP-A to the ileum. It should be noted that this analysis did not detect the presence of endogenous SP-A protein in wild type mouse ileum. We then determined if exogenous SP-A could reduce TLR4 protein levels in 1 week old SP-A −/− mouse ileum. Neonatal SP-A −/− mouse pups were gavaged with 5 µg purified human SP-A in PBS or PBS alone at 4 and 6 days of age and sacrificed on day 7 of life. Pups were maintained with dams throughout the experimental period. Western blot analysis shown in Fig. 3B indicates the levels of ileal TLR4 in mice that were either untreated, gavaged with PBS or gavaged with human SP-A in PBS. Orogastric gavage with PBS had little effect on TLR4 levels in the ileum while pups gavaged with SP-A decreased levels of TLR in the ileum.

SP-A reduces levels of TLR4 and proinflammatory cytokine mRNA in intestinal epithelial cells in cell culture. These investigations have demonstrated an extrapulmonary effect of SP-A to reduce TLR4
and proinflammatory cytokine levels in the ileum of the gastrointestinal tract. While it is known that SP-A can directly modulate TLR4 expression and activity in pulmonary alveolar macrophages 24,28 , it is unclear if SP-A can directly modulate TLR4 expression in intestinal epithelial cells. FHs74int cells are non-transformed epithelial cells derived from the small intestine of a normal human fetus that have been maintained in cell culture. FHs74int cells were incubated in the absence or presence of purified human SP-A (25 µg/ml for 24 h) and steady state levels of TLR4 and cytokine mRNA determined by real-time quantitative RT-PCR analysis. As shown in Fig. 4, SP-A significantly reduced TLR4 mRNA relative to cells not exposed to SP-A, suggesting a direct effect of SP-A on TLR4 expression in the intestinal epithelium. In addition, expression of IL-1β and IL-6 mRNA was significantly reduced. While expression of IL-8 mRNA in FHs74int cells was not impacted by the presence of SP-A, we have observed that expression of IL-8 mRNA and protein in FHs74int cells are greater than other cytokines by several orders of magnitude (unpublished results). CXCL1 is mouse homologue of human IL-8, and CXCL1 mRNA levels were impacted by SP-A in mice. Perhaps any effect of SP-A on IL-8 in FHs74int cells is masked by the high levels of IL-8 in these cells. In any case, these results indicate that the reduction of TLR4 and proinflammatory cytokine expression in the ileum by SP-A can be explained as a direct effect of SP-A on the intestinal epithelium.

SP-A does not reduce TLR4 protein levels in intestinal epithelial cells post-translationally.
We have shown that SP-A can reduce steady-state levels of TLR4 protein and mRNA in the intestinal epithelium, but it is unclear if the mechanism by which SP-A reduces TLR4 protein occurs by impacting TLR4 mRNA levels, protein levels or both. HEK293 cells do not express TLR4 38 , but an HEK293 cell line that has been stably transfected with an expression cassette that expresses hemagglutinin (HA)-tagged TLR4 (293/hTLR4-HA) from the relatively unregulated CMV1 promoter is commercially available. We assessed the ability of SP-A to specifically reduce TLR4 protein levels in 293/hTLR4-HA cells inasmuch as any potential effects of SP-A on transcription or  www.nature.com/scientificreports/ stability of endogenous TLR4 mRNA is absent in these cells. 293/hTLR4-HA cells were incubated in the absence of presence of SP-A (25 µg/ml) for 24 h and the steady-state levels of expressed TLR4 mRNA and protein were determined by real-time quantitative RT-PCR analysis and western analysis, respectively. As can be seen in Fig. 5A, expression of TLR4 mRNA is essentially undetectable in HEK293 cells. TLR4 mRNA expressed from the expression cassette is detectable in 293/hTLR4-HA cells and the presence of SP-A does not alter expression of the TLR4 mRNA. In Fig. 5B, it can be seen that the presence of SP-A does not alter expression of the HA-tagged TLR4 protein compared to untreated cells, suggesting that SP-A does not reduce TLR4 protein levels by activating protein degradation. It is known that levels of TLR4 can modulated through ubiquitination by the E3 ubiquitin-protein ligase RNF216, resulting in degradation via the ubiquitin-proteasome pathway 39 . If SP-A reduces TLR4 via proteasomal degradation, we reasoned that treatment of intestinal epithelial cells with proteasome inhibitors would abrogate the ability of SP-A to reduced TLR4 in the cells. HT-29 and IEC-6 ells were cultured in the absence or presence of SP-A (25 µg/ml) for 5 h. Various well-characterized inhibitors of proteasomal activity (MG132, carfilzomib and bortezomib) were added and incubation continued for 1 h until harvest. The levels of TLR4 protein were determined by western analysis. Shown in Fig. 6 are the results plotted as levels of TLR4 protein relative to β-actin protein of each sample normalized to TLR4 levels of untreated cells. As can be seen, the presence of SP-A significantly reduced TLR4 protein levels in all cases, but in no case did the presence of the proteasome inhibitor prevent the inhibitory activity of SP-A on TLR4 levels. These results suggest that while the presence of SP-A reduces steady-state levels of TLR4 mRNA and protein in intestinal epithelial cells, the mechanism of action does not appear to include enhanced proteasomal degradation of TLR4 protein.

SP-A-deficient neonatal mice are more susceptible to stress-induced intestinal injury than wild type neonatal mice in an experimental model of NEC.
This study has shown that levels of TLR4 protein and IL-1β inflammatory cytokines are increased in the ileum of SP-A-deficient mice, suggesting that the intestine of SP-A-deficient mice may be more susceptible to risk of inflammatory pathology in the neonatal intestine. We used an established model of experimental NEC involving neonatal mouse pups (8-10 days old) 40 that requires exposure to a variety of stresses (formula feeding, hypoxia, hypothermia) to determine the susceptibility of SP-A-deficient mice to intestinal stress. Wild type and SP-A −/− mice were divided into 4 groups; (1) dam-fed, (2) formula-fed, (3) formula-fed with hypoxia and hypothermia and (4) formula-fed with hypoxia and hypothermia with daily gavage of purified human SP-A (5 µg) to deliver SP-A to the intestine as shown in Fig. 3. Formula-feeding alone is a known risk factor for NEC in premature neonates and episodic hypoxia and hypothermia is meant to reproduce gastrointestinal reperfusion-like damage [1][2][3] . After the course of exposure in the model was completed, tissues were harvested for histochemical analysis. The incidence and severity of intestinal damage resembling NEC in the sections was determined by a scoring system (0-4) developed for use in neonatal rodents where the severity of damage was graded by three separate masked evaluators, with histological scores of ≥ 2 defined as having NEC 41 . As shown in Fig. 7A, there was no significant difference in NEC-like intestinal damage comparing dam-fed wild type and SP-A −/− neonatal pups. However, under the stress of formula feeding alone, 59% of SP-A −/− neonatal pups had damage resembling NEC, which was significantly higher than the wild type neonatal pups (23%). Upon exposure to the combined stresses of formula feeding, hypoxia and hypothermia, the amount of damage resembling NEC was high in both wild type (65%) and SP-A −/− (71%) neonatal pups, but there was no significant difference. As shown in Fig. 7A, and as expected from previous inves- www.nature.com/scientificreports/ tigations using neonatal rat pups, orogastric gavage of purified SP-A (5 µg/day) to SP-A-deficient pups treated with all stressors was sufficient to significantly reduce damage resembling NEC (p = 0.036). We then assessed the impact of orogastric gavage of purified SP-A on ileal proinflammatory cytokine expression in this model of experimental NEC. In Fig. 7B are shown the results of real-time quantitative RT-PCR analysis of ileal TLR4 and proinflammatory cytokine mRNA expression in neonatal wild type mice. As can be seen, formula feeding with hypoxia + hypothermia (FH) significantly increased TLR4 and IL-1β mRNA levels compared to formula feeding (FF) alone. Administration of purified SP-A (FHS) significantly reduced expression of TLR4, IL-1β and IL-6-compared to the FH group. Similar results are seen in Fig. 7C which shows that expression of ileal TLR4, IL-1β, and TNF-α mRNA in neonatal SP-A -/mice subjected to experimental NEC is significantly reduced upon orogastric gavage with purified SP-A.

Discussion
Traditionally, SP-A activity is thought to be limited to the lung, but numerous reports of extra-pulmonary SP-A expression 42 suggest that the function of this highly conserved component of the innate immune system may be important beyond the lung. SPA-deficient (SP-A −/− ) mice have no obvious phenotype other than enhanced susceptibility to pulmonary infections 43,44 . However, it was reported that neonatal and juvenile SP-A −/− mice housed in a non-hygienic environment have significant mortality that was associated with significant gastrointestinal tract pathology rather than the expected lung pathology 45 . Furthermore, that study showed that gavage of SP-A −/− pups with purified human SP-A improved newborn survival. We previously reported that orogastric gavage of purified human SP-A to neonatal rat pups significantly reduced intestinal damage and levels of intestinal TLR4 levels in a rat pup model of neonatal NEC 35 . Here, we furthered those investigations using mice deficient in the SP-A gene. We found that intestinal levels of TLR4 and inflammatory cytokines are increased in SP-A −/− mice compared to wild type mice. In addition, the presence of exogenous SP-A via orogastric gavage reduces ileal TLR4 protein levels. SP-A does not appear to increase degradation of TLR4 through the proteasomal pathway. We also show that neonatal SP-A −/− mice are more susceptible to stress that leads to intestinal damage resembling NEC and that exposure to exogenous SP-A can reduce ileal damage, TLR4 levels and inflammatory www.nature.com/scientificreports/ cytokine levels. SP-A is known to modulate TLR4 activity and inflammation in the lung and we now report a novel activity of SP-A to modulate TLR4 levels and inflammation in neonatal mouse intestine. While the results described here indicate that SP-A has a role in modulating TLR4 and inflammatory cytokine expression in the ileum, the source of SP-A and mechanism of action is still unclear. The results using cell lines suggest that SP-A can have a direct effect to reduce TLR4 and cytokine mRNA expression in the ileal epithelium, but the impact of gavaged SP-A in neonatal mice could be direct, indirect or both. Gastrointestinal expression of SP-A has been reported 32-34 , but the evidence is not very strong. We find that expression of SP-A mRNA in In these investigations we found that endogenous mouse SP-A was not detected in 1 week and 4 week old mouse pups by western analysis (Fig. 3A). Another complicating factor in these studies is the fact that SP-A has been reported to influence the immunomodulatory properties of breast milk 46,47 as well as impacting levels of IgA found in breast milk 48 . Realizing that the pups used in these investigations are being nursed, the complexities of the interaction of breast milk with inflammatory character of the gastrointestinal tract are not addressed here and could be contributing to the unexpected findings shown in Fig. 1C. These results can only definitely state that increased TLR4 and proinflammatory cytokine expression in the ileum occurs in the complete absence of SP-A expression throughout the body. www.nature.com/scientificreports/ SP-A has been reported to suppress pro-inflammatory cytokine production in alveolar macrophages through upregulation of IRAK-M expression 24 , a negative regulator of TLR-mediated NF-κB activation of cytokine transcription 31 , which suppresses the TLR4 signaling cascade. However, SP-A did not alter TLR4 expression in alveolar macrophages in those studies. SP-A has been shown to directly interact with TLR4 to reduce inflammation 49 , and a 20 amino acid-long fragment of SP-A has been shown to reduce inflammation in colonic epithelial SW480 cells 50 . We have observed that TLR4 levels protein are increased in lung alveolar epithelial cells of SP-A −/− mice compared to wild type mice, and TLR4 expression is markedly reduced in human alveolar epithelial A549 cells cultured in the presence of SP-A compared to cells cultured in the absence of SP-A (unpublished data). We suggest that SP-A reduces TLR4-mediated inflammatory responses in epithelial cells by reducing levels of TLR4 mRNA, a mechanism that is distinctly different from the ability of SP-A to reduce TLR4-mediated inflammatory responses in macrophages by suppressing the signal cascade initiated by activation of TLR4.
NEC is responsible for ~ 7% of all neonatal intensive care unit admissions, exacting huge healthcare costs (up to $1 billion annually) 1 . As complications occur before diagnosis of NEC and treatment is primarily supportive, surgical removal of affected bowel is frequent. While prematurity is the greatest risk factor for developing NEC 3 , the rate of feeding and type of feeding (formula > breast milk) is also associated with NEC. In two studies, 90% of infants with NEC were fed with infant formula (instead of breast milk) 51,52 . Hypoxia and damage to the intestinal mucosa are also considered risk factors, as NEC is thought to result from a reperfusion injury that stimulates an inflammatory cascade 51,52 . The presence of abnormal microbial flora along with additional risk factors of bowel ischemia/perforation, immaturity of host mucosal defense and enteral feedings is thought to contribute to the initiation and pathogenesis of NEC, likely due to an excessive inflammatory response 13,53,54 . Although the precise mechanisms responsible for initiation of NEC remain unclear, intestinal inflammation is key in its pathophysiology. While the innate immune system is essential in defense of pathogenic bacteria in the intestine, when over-stimulated or left unchecked, it may also contribute to inflammatory injury. Immature host defenses in premature intestine may lead excessive inflammation, consistent with the finding that human fetal intestinal epithelial cells produce higher levels of inflammatory cytokines in response to bacteria compared with adult intestinal epithelial cells 7 .
In large part, TLR4-mediated inflammation plays an important role in inflammatory diseases in the intestine 55 . One of the first histopathologic events in experimental models of NEC is caused by TLR4 activation in immature small intestinal tissue, but not in adult tissues 56,57 . Feeding with cow-based infant formula in both humans and animals is associated with increased activation of intestinal TLR4, and expression of TLRs and intestinal inflammatory cytokines precede observable injury in animal models of NEC 13 . Moreover, physiological stressors associated with the development of NEC (LPS and hypoxia) sensitize the intestinal epithelium through up-regulation of TLR4 12 . Taken together, these studies suggest that interactions of LPS from bacteria and TLR4, resulting in excess proinflammatory cytokines in premature intestinal epithelium, lead to intestinal damage and increase susceptibility of the neonate to intestinal diseases. A critical role TLR4 in these processes was demonstrated in TLR4 mutant mice which have reduced intestinal TNF-α levels resulting from formula-feeding and stress compared to wild type mice 58 , and in TLR4 null transgenic mice which are protected from the development of intestinal damage in experimental NEC models 14,15 .
Protection of the relatively immature intestine of the neonate comes from a variety of sources, such as epidermal growth factor (EGF) found in amniotic fluid (AF). SP-A can also provide protection to the immature intestine of the neonate, but it is unclear as to how sufficient levels of SP-A are delivered to the intestine. Expression of SP-A in the lungs is developmentally regulated; SP-A is detected in the lungs at 19 wks gestation in humans and expression increases dramatically after 32 wks gestation 59,60 , but very little is known concerning the ontogeny of SP-A in the intestine. Importantly for this study, the levels of SP-A in AF in humans has been shown to increase from < 3 µg/ml at 30 weeks gestation to > 24 µg/ml at term [60][61][62][63] . The changes in amniotic SP-A levels mirror that in the fetal lung, suggesting that SP-A in AF is from the lungs where secreted SP-A is transmitted to the AF through in utero fetal breathing movements. Thus, the most plausible source of SP-A for the intestine is the lung, and a mechanism to deliver SP-A from the lung to the gastrointestinal tract during fetal development is through AF. The fetus at term swallows up to a liter of AF (and SP-A) per day 61 , providing a rich source of SP-A to the immature intestinal mucosa at term. Interestingly, it has been reported that AF reduces inflammation and TLR4 activity in premature mouse intestine through the action of EGF, but in that study, the gestational age of the AF was unclear, as was the presence of SP-A 46 . We propose a model in which the presence of SP-A reduces TLR4 levels and inflammation in neonatal intestine, which lessens the possibility of intestinal inflammation when exposed to environmental insults (Fig. 7). SP-A is secreted from the developing fetal lung late in gestation and accumulates in AF. AF is swallowed by the fetus, providing a rich source of SP-A throughout the gastrointestinal tissue at term that reduces intestinal levels of TLR4 and inflammation. Prematurity is the greatest risk factor for the development of NEC in infants. Because expression of SP-A in the lungs is not initiated until the third trimester of gestation, premature delivery of infants (< 32 weeks gestation) increases the immature nature of neonatal intestine and will interrupt the natural in utero delivery of SP-A to the intestine, resulting in a situation where the immature intestine is predisposed to excessive inflammation and damage when exposed to the extrauterine environment, facilitating the development of intestinal diseases such as NEC. While this theory is not addressed by the findings here, we do show that SP-A does have a role in immunomodulation of the gastrointestinal tract.
Given that current technology cannot diagnose NEC in its early stages and that there are no treatments for the condition, development of prophylactic/treatment strategies to reduce the inflammatory environment in the premature GI tract is a worthy research goal. We have shown that orogastric gavage can deliver SP-A to neonatal intestine resulting in blunting of intestinal inflammation. Understanding the physiological role of SP-A in modulating excessive inflammation in neonatal intestine and the molecular mechanism by which SP-A reduces TLR4 in epithelial cells can potentially lead to the use of SP-A as a biomarker for and prophylactic agent against the onset of excessive inflammation that can lead to intestinal diseases such as NEC in premature infants.  64,65 , and were originally generated in the lab of Dr. Samuel Hawgood, who backcrossed the SP-A −/− locus to a C57B/6 background 66 . To reduce genetic and intestinal microbiome differences in our wild type and SP-A −/− lines used in the investigations, we crossed our SP-A −/− line with wild type C57BL/6 mice purchased from the Jackson Laboratory and the resulting heterozygotes from different breeding pairs were backcrossed to regenerate the lines. We repeat the backcross process on a yearly basis and house all lines in the location in our animal facility so they receive the same food, bedding and water. Studies were approved by the Animal Welfare Committee of the University of Texas Health Science Center at House (HSC-AWC-17-028) and thus, all methods were carried out in accordance with relevant guidelines and regulations.
NEC procedure. Induction of NEC in mouse pups was performed using the technique developed by Liu et al. 40 in which formula feeding combined with exposure to stress (hypoxia and hypothermia) induces NEC, but is not associated with excessive early mortality. Seven to 10-day old mouse pups were separated from dams, housed in an incubator and starved for 12 h before initiation of orogastric Tissue harvest and NEC evaluation. Following incision of the abdomen, the gastrointestinal tract was carefully removed. The small intestine was evaluated visually for typical gross signs of NEC, such as intestinal distension, wall hemorrhage, or necrosis. The last 5 cm of terminal ileum was excised. Parts of ileum for each animal were placed immediately in liquid nitrogen for RNA (2 cm) and protein (2 cm) isolation. The terminal 1 cm of ileum was fixed in 10% formalin, processed by the Cellular and Molecular Morphology Core Laboratory (the Texas Medical Center Digestive Disease Center, Houston, TX) and stained with hematoxylin and eosin for histological evaluation. Pathological changes in intestinal architecture were quantified using a modified NEC scoring system 67,69,70 . Histological changes in the ileum were scored by 3 blinded evaluators on a scale of 0 (normal), 1 (mild, separation of the villous core, without other abnormalities), 2 (moderate, villous core separation, submucosal edema, and epithelial sloughing), and 3 (severe, denudation of epithelium with loss of villi, fullthickness necrosis). Animals with histological scores 2 were defined as having NEC.   Tissue or cells were treated with Trizol (#15596026, Ther-moFisher Scientific, Waltham, MA), as per the manufacturer's instructions, and homogenized at full speed using a rotor-stator homogenizer. mRNA was then isolated, treated with Turbo DNA-free™ kit (#100789371; Ther-moFisher Scientific, Waltham, MA), and purified using a RNeasy ® mini kit (#160040219; Qiagen, Germantown, MD) according to the manufacturer's instructions. cDNA was synthesized from of total RNA (1 µg) using an iScript cDNA Synthesis Kit (#170-8891; Bio-Rad Laboratories, Hercules, CA). For qPCR analysis, primers listed in Table 1 were used. qPCR was performed using a LightCycler 480II System (Roche Diagnostics, Indianapolis, IN) and the iTaq Universal SYBR Green Supermix (#1725124; Bio-Rad Laboratories, Hercules, CA) as per the instructions of the manufacturers. Data were calculated by the comparative C T method (C T , threshold cycle) 73,74 .

Real-time quantitative RT-PCR of mRNA.
Data are plotted as fold increases in amplicon CT values normalized 18S RNA.
Cytokine measurement. Tissue was homogenized in RIPA buffer containing proteinase inhibitors and levels of IL-1β in the homogenate were determined by ELISA as per the manufacturer's instructions (#MLB00C; R&D Systems, Inc, Minneapolis, MN). Levels are expressed as pg per mg tissue.
Statistical analysis. Animal and cell data represent means ± SE with a vertical scatter plot of the individual data used to generate these values. Statistical analysis of protein and mRNA levels was performed using the student's t-test with Sigmaplot software, version 12. Statistical analysis comparing assessment of NEC between the groups was performed by Chi-square analysis. A value of P < 0.05 determined by either method was considered significant.

Data availability
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