REDUCED EXPRESSION OF MIR-146A POTENTIATES INTESTINAL INFLAMMATION FOLLOWING ALCOHOL AND BURN INJURY

Abstract

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. Using a mouse model of acute ethanol intoxication and burn injury, we found that small intestinal epithelial cell expression of miR-146a is significantly decreased one day following injury. Using in vitro studies, we show that reduced miR-146a promotes intestinal epithelial cell inflammation by promoting p38 MAPK signaling via increased levels of its target TRAF6. Furthermore, we demonstrate that in vivo miR-146a overexpression significantly inhibits intestinal inflammation one day following combined injury and potentially supports intestinal barrier homeostasis. Overall, this study highlights the important impact that miRNA expression can have on intestinal homeostasis and the valuable potential of harnessing aberrant miRNA expression as a therapeutic target to control intestinal inflammation.

Introduction

Every year in the United States nearly 500,000 people receive medical treatment for burn injuries and 40,000 of these cases require some form of hospitalization(1). In addition, the prevalence of alcohol use at the time of burn injury can be as high as 50%(2). Alcohol intoxication is associated with poorer prognosis in burn patients, resulting in more adverse outcomes, longer hospitalization, and increased mortality(2–4). Disruption of the intestinal barrier is associated with a wide variety of gastrointestinal disorders and is a major contributing factor to mortality and morbidity following severe burn injury(5, 6). Alcohol intoxication at the time of burn injury promotes intestinal inflammation, which exacerbates disruption of the gut barrier and contributes to severe consequences of burn injury, including sepsis and multiple organ failure(6–9). Intestinal epithelial cells form a crucial component of the gut’s physical barrier, maintaining tight junctions that prevent translocation of bacteria and their pro-inflammatory pathogen associated molecular patterns (PAMPs) to the underlying immune compartment and systemic circulation(10). Previous studies have shown that following severe burn injury, toll-like receptor 4 (TLR4) activation by bacterial products can promote excessive small intestinal inflammation(11). This intestinal inflammatory response is characterized by elevated levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6), and neutrophil infiltration which can occur between hours to one day after injury(9, 12–14). Acute intestinal inflammation contributes to intestinal barrier disruption, which results in translocation of bacteria and PAMPs across the intestinal barrier, system inflammation, and distant organ injury(9–11, 15). Studies suggest that reducing inflammatory cytokines, including IL-6, can promote intestinal barrier function following alcohol and burn injury(16, 17). It has also been shown that reducing intestinal inflammation after alcohol and burn injury can prevent tissue damage and barrier disruption associated with neutrophil infiltration(12, 18, 19).

MicroRNAs (miRNAs) are small noncoding RNA first transcribed from the genome as pri-miRNA molecules. Following cleavage by Drosha into a pre-miRNA molecule and export from the nucleus, the miRNA is further cleaved into a miRNA duplex by Dicer. A functional mature miRNA is then generated by selection and loading of one strand into the RISC complex. The mature miRNA can then associate with the 3’ UTR of target mRNAs via complementary sequence binding, resulting in repression of target mRNA expression(20). Within the intestinal epithelium, miRNAs have been shown to play a key regulatory role in gut homeostasis(21–23). Due to the expansive nature of miRNA regulation, small changes in miRNA expression can have dramatic consequences for normal cellular process. Unsurprisingly, altered miRNA expression has been associated with a wide variety of diseases including cancer, neurodegenerative disorders, diabetes, and heart disease(24). As our understanding of miRNA associated pathophysiology expands, increased interest in targeting abnormal miRNA expression as a therapeutic intervention has developed. Several studies have shown the potential efficacy of different miRNA therapeutics and the therapeutic potential of miRNAs has become widely recognized. Although miRNAs heavily regulate inflammatory signaling and gut barrier homeostasis, the role of altered miRNA expression within the intestinal epithelium following burn injury, with or without prior alcohol exposure, is poorly understood.

In this study, we hypothesized that downregulation of key anti-inflammatory miRNAs would promote excessive intestinal inflammation and GI dysfunction following alcohol and burn injury. Using a mouse model of acute ethanol intoxication and burn injury, we found significantly reduced expression of miR-146a and miR-150 within small intestinal epithelial cells one day following combined injury, potentially contributing to excessive inflammation and intestinal barrier disruption. Several studies have shown that miR-146a plays a critical role in controlling inflammatory responses of immune cells, particularly macrophages(25–27). Furthermore, some studies indicate that miR-146a expression can reduce epithelial cell inflammation following cytokine or LPS stimulation(28, 29). Therefore, we sought to investigate the mechanism by which downregulation of miR-146a could contribute to intestinal inflammation and gut barrier disruption after alcohol and burn injury. Our findings suggest that downregulation of miR-146a following alcohol and burn injury promotes intestinal inflammation by preventing repression of its target, TRAF6, and elevating intestinal epithelial cells p38 MAPK signaling. Overall, this study highlights the important impact that miRNA expression can have on intestinal homeostasis and the valuable potential of harnessing aberrant miRNA expression as a therapeutic target to control intestinal inflammation.

Materials and Methods

Animals.

Male C57/BL6 mice (8 weeks old) were obtained from Charles River Laboratories and maintained in animal housing facilities at Loyola University Chicago Health Sciences Division, Maywood, Illinois, USA.

Mouse Model of Acute Ethanol Intoxication and Burn Injury.

Male C57/BL6 mice (9–10 weeks old, 22–26 g) were randomly assigned into one of four experimental groups: sham injury + vehicle treatment (sham vehicle, SV), sham injury + ethanol treatment (sham ethanol, SE), burn injury + vehicle treatment (burn vehicle, BV), or burn injury + ethanol treatment (ethanol burn, EB). For miR-146a mimic experiments, male C57/BL6 mice were randomly assigned into four experimental groups: Sham Vehicle + Scramble Mimic, Sham Vehicle + miR-146a Mimic, Burn Ethanol + Scramble Mimic, or Burn Ethanol + miR-146a Mimic. Ethanol intoxication and burn injury were carried out as previously described(8, 14, 17, 30–33). One day prior to injury, mice were given an intraperitoneal injection containing 50 ug of either scramble or miR-146a mirVana mimic (ThermoFisher Scientific, HPLC purified, in vivo ready) with 6 uL in vivo-jetPEI reagent in 250 μL 5% glucose (Polyplus Transfection). On the day of injury, ethanol mice were gavaged with 400 μL of 25% ethanol in water (2.9 g/kg), while vehicle animals were gavaged with 400 μL water. Three hours following the gavage, mice were given 1 mg/kg buprenorphine subcutaneously for pain management. Four hours following the gavage, mice were anesthetized with a ketamine hydrochloride/xylazine cocktail (~ 80 mg/kg and ~ 1.2 mg/kg respectively) via intraperitoneal injection. The dorsal surface of each mouse was shaved before placing the mice in a prefabricated template exposing ~ 12.5% total body surface area, calculated using Meeh’s formula(34). Burn group animals were immersed in ~85 °C water bath for ~7 seconds to induce a full-thickness scald burn injury. Sham animals were placed in a 37 °C, lukewarm water bath for an equal length of time. Following burn or sham injury, animals were dried gently and given 1.0 mL normal saline resuscitation by intraperitoneal injection. Animals were returned to their cages, which were placed on heating pads to help maintain their body temperature and observed to ensure recovery from anesthesia. Mice were then returned to their normal housing and allowed food and water ad libitum.

Small Intestinal Epithelial Cell Isolation.

One day after injury, mice were euthanized, and the abdominal cavity was exposed via midline incision. Approximately ~8 cm of the distal small intestine was harvested and opened longitudinally and washed twice in ice cold PBS + 100 U/mL penicillin + 100 μg/mL streptomycin. Small intestines were then incubated in HBSS buffer without phenol red supplemented with 10 mMol/L HEPES, 50 μg/mL gentamicin, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 mM EDTA and 1 mM DTT (pre-digestion solution) for 20 min at 37 °C with agitation at 250 rpm. Samples were vortexed to disrupt epithelial cells from the lamina propria and epithelial cells were collected through a 100 μm strainer. This process of pre-digestion solution incubation and epithelial cell collection was repeated a second time, pooling isolated epithelial cells(35, 36). Epithelial cells were then washed with PBS twice to remove pre-digestion solution and then used in downstream applications.

Total RNA Isolation.

Total RNA for miRNA or mRNA analysis was extracted from small intestinal epithelial cells using the mirVana miRNA Isolation Kit (Invitrogen) according to manufacturer’s instructions. If only mRNA analysis was being performed, then total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. RNA concentration and purity were assessed using a Nanodrop 2000 spectrophotometer (ThermoScientific).

qRT-PCR Analysis of microRNA Expression.

Using total RNA isolated via the mirVana miRNA Isolation Kit, reverse transcription and cDNA amplification was performed using the miRCURY LNA RT Kit (Qiagen). Relative expression of target miRNAs was then assessed via quantitative real time PCR (qPCR) using miRCURY LNA SYBR Green PCR Kit and miRCURY LNA PCR Assay primers (Qiagen) specific to each target miRNA. Each sample’s target miRNA Ct cycle values were normalized to SNORD68 housekeeping control and relative expression was calculated using the ΔΔCT method(37).

qRT-PCR Analysis of mRNA Expression.

Using total RNA isolated via either the mirVana miRNA Isolation Kit or RNeasy Mini Kit, reverse transcription and cDNA amplification was performed using the High Capacity Reverse Transcription Kit (Applied Biosystems). Relative expression of target genes was then assessed via quantitative real time PCR (qPCR) using TaqMan Fast Advanced Master Mix and FAM TaqMan primer probes (Life Technologies) specific to each target gene. Each well’s target gene Ct cycle values were normalized to either Beta Actin or GAPDH housekeeping control Ct values using VIC Taqman primer probes in the same reaction. Relative expression was calculated using the ΔΔCT method(14, 30, 37).

Murine Duodenal Epithelial Cell (MODE-K) Culture.

To investigate miRNA regulation of small intestinal epithelial inflammation in vitro, MODE-K cells were obtained from the laboratory of Dr. Jin Mo Park (Harvard Medical School, Mass General Research Institute, Charlestown, MA). MODE-K cells are small intestinal epithelial cells isolated from mouse duodenum tissue and immortalized with SV40 large T gene. They are widely used in literature and exhibit normal enterocyte characteristics, including the formation of intercellular tight junction and inflammatory cytokine responses to TLR ligands such as LPS(38–40). MODE-K cells were cultured in Dulbecco’s Modified Eagle Media (DMEM) containing 4.5 g/L glucose, 1 mM L-glutamine, 1 mM sodium pyruvate (Gibco, ThermoFisher Scientific) and supplemented with 1% penicillin-streptomyocin cocktail (ThermoFisher Scientific), 1 mM HEPES, and 5% fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO₂.

Overexpression or Inhibition of miR-146a and LPS Stimulation.

MODE-K cells were seeded into 6-well plates at 200,000 cells/well to obtain ~30–40% confluency the next day. One day after seeding, culture media was replaced, and cells were transfected with 10 pmol scramble or miR-146a specific miRCURY LNA mimics or 50 pmol scramble or miR-146a specific miRCURY LNA inhibitors (Qiagen) using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) according to manufacturer instructions. After 24 hours, culture media was replaced, and cells were treated with 1 ug/mL LPS. For pro-inflammatory cytokine expression, media supernatant and cells were collected 24 hours later for ELISA analysis or RNA isolation and RT-qPCR analysis. For western blot analysis of pro-inflammatory signaling, cells were collected for protein isolation after 30 minutes, 1 hour, 2 hours, 3 hours, or 4 hours of LPS stimulation.

ELISA.

Media was collected from MODE-K cells 24 hours after LPS stimulation and centrifuged at 10,000 × g for 10 min. Supernatants were then analyzed for IL-6 (R&D Systems) and KC (R&D Systems) using their respective ELISA kits according to manufacturer instructions. Cytokine levels were expressed per milliliter of supernatant.

Protein Isolation and Western Blot.

Cell pellets from MODE-K cell culture or small intestinal epithelial cell isolation were lysed in Cell Lysis Buffer (Cell Signaling Technology) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) added. Lysates were homogenized and centrifuged at 10,000 × g for 10 min. Protein concentration was measured by DC Protein Assay (BioRad). Equal amounts of protein were then loaded and run on an SDS-PAGE gel and transferred to a PVDF membrane for blotting. Membranes were blocked for 1 hour at room temperature with 5% blocking grade milk (BioRad) in TBS-T (0.1% Tween 20 in TBS). After washing membranes twice for 5 min in TBS-T to remove excess milk, membranes were incubated with desired primary antibody overnight at 4°C. Membranes were then washed three times for 10 min in TBS-T and incubated in the appropriate secondary antibody conjugate to horseradish peroxidase for one hour at room temperature. Triplicate washes were then repeated, and membranes were developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer) and exposed on a ChemiDoc (BioRad) for imaging. Densitometric analysis was performed using Image Lab software (BioRad). Bands were normalized to Beta Actin probed on the same membrane and expressed as densitometric units. Levels of phosphorylated protein were expressed relative to its total protein after normalization to Beta Actin.

Pharmacological Inhibition of Pro-inflammatory Signaling.

Inhibitors specific for p38 MAPK (SB 203580), NF-κB (CAPE), and STAT3 (Stattic) were obtained from R&D Systems. Pharmacological inhibitors were reconstituted in DMSO and stored at recommended stock concentrations at −20°C. Treatment concentrations were determined based on published active concentrations for each inhibitor and Alamar Blue Viability Assays were prerformed (not shown) to validate low cytotoxicity at chosen concentrations. 24 hours after transfection with scramble or miR-146a specific inhibitors, MODE-K cells were treated with either 10 μM SB 203580, 20 μg/mL CAPE, 10 μM Stattic, or DMSO control in culture media. Following one hour of pre-treatment, 1 ug/mL LPS was added, and cells were incubated for 24 hours. Media supernatant was collected for ELISA analysis and cells were lysed for RNA isolation and RT-qPCR analysis.

TRAF6 Knockdown.

Predesigned scramble and TRAF6 specific siRNAs (Ambion Silencer Select) were obtained from ThermoFisher Scientific. MODE-K cells were co-transfected with 10 pmol siRNA and 50 pmol miRNA inhibitors using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) according to manufacturer instructions. The following day, culture media was replaced, and cells were treated with 1 ug/mL LPS overnight. Media supernatant was collected for ELISA analysis and cells were lysed for either protein and western blot analysis or RNA and RT-qCPR analysis. Knockdown was confirmed via western blot analysis of TRAF6 protein level 24 hours after siRNA transfection.

Measurement of Intestinal Barrier Permeability.

One day following alcohol gavage and burn injury, mice were given a gavage of 0.4 ml Fluorescein Isothiocyanate (FITC)-dextran at 22 mg/ml in PBS. 3 hours later, mice were euthanized, and blood was collected by cardiac puncture into a heparin coated syringe. Blood was centrifuged at 8,000 rpm for 10 min at 4°C to collect plasma. Samples were assessed via fluorescent spectrophotometry (480 nm excitation and 520 nm emission wavelengths) against a standard curve to quantify plasma FITC-dextran levels as μg FITC-dextran per μl plasma.

Statistics

Data is presented as means ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7 as defined in figures legends. Briefly, experiments containing 2 groups were analyzed via student’s t test. Experiments containing more than two groups were analyzed via 2-way ANOVA with the p-values from t tests between two groups being adjusted for false discovery rate via two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. P-values or adjusted p-values < 0.05 were considered significant.

Study Approval.

All animal experiments were conducted in accordance with the guidelines set forth by the Animal Welfare Act and approved by the Institutional Animal Care and Use Committee (IACUC) at Loyola University Health Sciences Division.

Data Availability.

All underlying data is available upon request.

Results

Downregulation of Anti-Inflammatory miRNAs in Small Intestinal Epithelial Cells After Ethanol and Burn Injury

To identify miRNAs that could contribute to elevated intestinal inflammation, we sought to identify anti-inflammatory miRNAs with downregulated expression following ethanol and burn injury. Following literature review, a panel of anti-inflammatory miRNAs were selected for initial assessment via RT-qPCR analysis. To begin, intestinal epithelial cell miRNA expression was assessed across three separate two group experiment comparing sham vehicle to ethanol burn mice. As shown in Figure 1A, both miR-146a and miR-150 were significantly downregulated in intestinal epithelial cells one day following combined injury. Although miR-194 showed a minor trend towards downregulation, its expression was not significantly altered in ethanol burn mice. Intestinal epithelial expression of miR-381, miR-495, and miR-574 were also assessed (data not shown), but no significant changes were observed. To determine the impact of either ethanol or burn injury alone versus ethanol and burn combined on the expression miR-146a, miR-150, and miR-194, small intestinal epithelial cells were isolated one day after injury from mice belonging to four experimental groups: sham vehicle, sham ethanol, burn vehicle, and ethanol burn. We observed no significant changes in miR-194 expression with ethanol and burn injury alone or combined. Additionally, although miR-150 expression was slightly reduced following burn injury alone, the expression of miR-146a and miR-150 were only reduced with combined ethanol and burn injury (Figure 1B).

Figure 1. Profiling Expression of Anti-Inflammatory miRNAs in Small Intestinal Epithelial Cells One Day After Ethanol and Burn Injury.

Small intestinal epithelial cells were isolated one day after ethanol and burn injury in experiments with either (A) two treatment groups (across 3 independent experiments, n=11–15 per group) or (B) four treatment groups (one experiment, n=5–8 per group). Total RNA was extracted for RT-qPCR analysis of miRNA expression using primers specific for miR-146a-5p, miR-150–5p, and miR-194–5p. Expression depicted relative to sham vehicle control, Snord68 used as housekeeping. Statistical analysis via student’s t test between groups depicted as * p<0.05, ** p<0.01, *** p<0.001.

miR-146a Expression Modulates Intestinal Epithelial Cell Production of Inflammatory Cytokines

Several studies have shown that miR-146a is an important brake to control inflammation and innate immunity, while others suggest that miR-146a also plays a regulatory role in epithelial cell inflammatory responses(25–29). MODE-K cells are a mouse duodenal epithelial cell line commonly used in literature which exhibit normal enterocyte characteristics, including intercellular junction proteins and cytokine responses to TLR ligands such as LPS(38). To investigate the impact of miR146a expression on small intestinal epithelial cell inflammation after ethanol and burn injury, MODE-K cells were stimulated with LPS for 24 hours and then IL-6 and KC (also known as CXCL1) were measured to assess inflammation. Cells were transfected with miR-146a antagomir inhibitor molecules to imitate reduced miR-146a expression seen in our in vivo mouse model, or mimic molecules to study the impact of miR-146a overexpression on small intestinal epithelial cell inflammation. Figure 2 demonstrates that LPS stimulation of MODE-K cells significantly upregulates IL-6 and KC gene expression, measured via RT-qPCR, and protein secretion, measured via ELISA. Inhibition of miR-146a results in a significant enhancement in gene expression and secretion of IL-6 compared to LPS alone. Gene expression, but not overall secretion, of KC is also significantly enhanced by miR-146a inhibition after LPS stimulation. As shown in Figure 3, overexpression of miR-146a via miR-146a mimic transfection shows a dramatic and opposite effect. Overexpression of miR-146a significantly reduces baseline levels of IL-6 and KC and significantly inhibits IL-6 and KC gene expression and protein secretion in response to LPS.

Figure 2. Inhibition of miR-146a Promotes LPS Induced Small Intestinal Epithelial Cell Inflammation.

MODE-K cells were transfected with scramble or miR-146a specific antagomir inhibitor and the next day stimulated with 1 μg/mL LPS for 24 hours. RNA was extracted for RT-qPCR analysis of gene expression using primers specific for (A) IL-6 or (B) KC. Expression depicted relative to sham vehicle control, Beta Actin used as housekeeping. Media supernatant collected and (C) IL-6 or (D) KC quantified via ELISA. Concentrations in pg/mL depicted relative to scramble inhibitor control. Graphs are means ± SEM, n=6 (across three independent experiments). Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests with p values adjusted for false discovery rate, * padj<0.05, **** padj<0.0001 as compared to Scramble Inh or # padj<0.05, ### padj<0.001, #### padj<0.0001 as compared to Scramble Inh + LPS.

Figure 3. Overexpression of miR-146a Inhibits LPS Induced Small Intestinal Epithelial Cell Inflammation.

MODE-K cells were transfected with scramble or miR-146a specific mimic and the next day stimulated with 1 μg/mL LPS for 24 hours. RNA was extracted for RT-qPCR analysis of gene expression using primers specific for (A) IL-6 or (B) KC. Expression depicted relative to sham vehicle control, Beta Actin used as housekeeping. Media supernatant collected and (C) IL-6 or (D) KC quantified via ELISA. Concentrations in pg/mL depicted relative to scramble inhibitor control. Graphs are means ± SEM, n=5 (across two independent experiments). Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests with p values adjusted for false discovery rate, *** padj < 0.001, **** padj < 0.0001 as compared to Scramble Mimic or ### padj < 0.001, #### padj < 0.0001 as compared to Scramble Mimic + LPS.

Modulation of Intestinal Epithelial Cell Inflammatory Signaling via miR-146a Regulation of p38 MAPK/TRAF6 Signaling Axis

Our results clearly indicate miR-146a expression in small intestinal epithelial cells can regulate inflammatory responses, therefore we next sought to determine which signaling pathways are involved in miR-146a mediated regulation of IL-6 and KC production. There are several signaling pathways activated following LPS stimulation that could be regulated by miR-146a expression. Activation of p38 MAPK and NF-κB can occur directly downstream of LPS stimulation of TLR4 signaling(29, 41, 42). In addition, LPS stimulation can result in STAT3 activation following IL-6 production(43). Activation of these pathways can cooperatively or independently promote pro-inflammatory cytokine expression in small intestinal epithelial cells. To examine which pro-inflammatory signaling pathways are required for miR-146a mediated elevation of LPS induced cytokine expression, MODE-K cells were transfected with miR-146a inhibitor antagomirs and pretreated with 10 μM SB 203580 (p38 MAPK inhibitor), 20 μg/mL CAPE (NF-κB inhibitor), or 10 μM Stattic (STAT3 inhibitor) for one hour prior to LPS stimulation. Active concentrations of pharmacological inhibitors were identified following literature review and Alamar Blue viability assay (data not shown) to confirm low cytotoxicity. As previously established, Figure 4A demonstrates that LPS stimulation significantly induced IL-6 expression and the addition of miR-146a inhibition significantly enhanced LPS-induced IL-6 expression. Although only the p38 MAPK inhibitor significantly reduced LPS-induced IL-6 expression, both p38 MAPK and STAT3 inhibitors significantly lowered elevated IL-6 expression seen following miR-146a inhibition. The p38 MAPK inhibitor appeared to exhibit the greatest effect, bringing IL-6 expression back to levels seen with LPS stimulation alone. Inhibition of NF-κB did not impact IL-6 expression but instead dramatically elevated KC expression following LPS stimulation, as shown in Figure 4B. Although inhibition of p38 MAPK appeared to restrict elevated KC expression following miR-146a inhibition, none of the changes reached statistical significance.

Figure 4. Pharmacological Inhibition of p38 MAPK Suppresses Small Intestinal Epithelial Cell Inflammation Induced by miR-146a Inhibition.

MODE-K cells were transfected with scramble or miR-146a specific antagomir inhibitor and the next day pretreated with DMSO control (black), 10 μM SB 203580, 20 μg/mL CAPE, or 10 μM Stattic for 1 hour before stimulation with 1 μg/mL LPS for 24 hours. RNA was extracted for RT-qPCR analysis of gene expression using primers specific for IL-6 or KC. Expression depicted relative to scramble inhibitor DMSO control, GAPDH used as housekeeping. Graphs are means ± SEM, n=4 (across two independent experiments). Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests with p values adjusted for false discovery rate, * padj < 0.05, ** padj < 0.01, **** padj < 0.0001 as compared to DMSO control within the same group or for comparisons as indicated.

To further explore the role of p38 MAPK and STAT3 in miR-146a mediated regulation of pro-inflammatory cytokine expression, MODE-K cells were transfected with either miR-146a mimic or inhibitors following LPS stimulation for a time series ranging from 15 min to 4 hours. Western blot analysis of total protein was performed to assess activation of these pathways via p38 MAPK and STAT3 phosphorylation. As shown in Figure 5A, phosphorylation of p38 MAPK increases within 30 minutes of LPS stimulation while phosphorylation of STAT3 increases after approximately 4 hours. Overexpression of miR-146a inhibits LPS induced phosphorylation of both p38 MAPK and STAT3. On the other hand, Figure 5B demonstrates that inhibition of miR-146a primarily increases LPS induced phosphorylation of p38 MAPK but has less effect on STAT3 phosphorylation.

Figure 5. Impact of miR-146a Overexpression or Inhibition on Intestinal Epithelial Cell p38 MAPK and STAT3 Phosphorylation Following LPS Stimulation.

(A) MODE-K cells were transfected with scramble or miR-146a specific mimic and the next day stimulated with 1 μg/mL LPS for 24 hours. Total protein was isolated and western blot analysis performed to compare relative levels of p38 MAPK and STAT3 phosphorylation following LPS stimulation. (B) MODE-K cells were transfected with scramble or miR-146a specific inhibitor and the next day stimulated with 1 μg/mL LPS for 24 hours. Total protein was isolated and western blot analysis performed to compare relative levels of p38 MAPK and STAT3 phosphorylation following LPS stimulation. One representative blot shown for experiments repeated in triplicate.

As our results indicate that miR-146a likely controls small intestinal epithelial cell inflammation primarily by regulating a target upstream of p38 MAPK signaling, we next assessed protein levels of upstream signaling proteins to identify a potential target of miR-146a. Several studies have validated miR-146a targeting of both IRAK1 and TRAF6, which are downstream of LPS induced TLR4 stimulation and can lead to p38 MAPK and NF-κB activation (26, 28, 29). As shown in Figure 6A, miR-146a overexpression in MODE-K cells significantly decreases TRAF6 protein levels. Although IRAK1 is expressed at lower levels than TRAF6, we also see a slight decrease in IRAK1 protein following miR-146a overexpression. Other studies have validated that TRAF6 is a direct target of miR-146a mediated repression of inflammatory signaling(25–29). To determine if miR-146a downregulation promotes small intestinal epithelial cell inflammation via targeting of TRAF6, MODE-K cells were co-transfected with miR-146a inhibitor and TRAF6 siRNA and stimulated with LPS. Figure 6B demonstrates successful knockdown of TRAF6 in MODE-K cells transfected with as little as 5 pmol siRNA compared to scramble control transfected cells. As shown in Figure 6C, knockdown of TRAF6 on its own significantly inhibited the expression of both IL-6 and KC. Although, LPS-induced expression of KC was significantly impaired by TRAF6 knockdown, LPS-induced IL-6 expression was not affected by TRAF6 knockdown. However, further elevation of IL-6 expression by miR-146a inhibition was successfully blocked by TRAF6 knockdown.

Figure 6. TRAF6 Knockdown Inhibits Small Intestinal Epithelial Cell Inflammation Following miR-146a Inhibition and LPS Stimulation.

(A) MODE-K cells were transfected with scramble or miR-146a specific mimic and total protein was isolated 24 hours for western blot analysis of proteins upstream of p38 MAPK activation, including previously validated targets of miR-146a. (B) MODE-K cells were transfected with scramble or TRAF6 specific siRNA constructs. Total protein was isolated after 24 hours for western blot analysis of TRAF6 knockdown. (C) MODE-K cells were transfected with scramble siRNA or TRAF6 siRNA #1 in combination with scramble or miR-146a specific inhibitor and then stimulated with 1 μg/mL LPS for 24 hours. RNA was extracted for RT-qPCR analysis of IL-6 or KC gene expression, depicted relative to scramble siRNA scramble inhibitor, Beta Actin used as housekeeping. Graphs are means ± SEM, n=4 (across two independent experiments). Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests compared to sham vehicle with p values adjusted for false discovery rate, * padj < 0.05, ** padj < 0.01, *** padj < 0.001, **** padj < 0.0001 as compared to Scramble No Tx control, ### padj < 0.001, #### padj < 0.0001 as compared to Scramble + LPS control, and &&& padj < 0.001, &&&& padj < 0.0001 for indicated comparison.

miR-146a Expression Inhibits Intestinal Inflammation Following Ethanol and Burn Injury

As our results demonstrate that downregulation of miR-146a significantly elevates intestinal epithelial cell inflammation, we next sought to investigate the impact of in vivo miR-146a mimic administration on intestinal inflammation and subsequent gut barrier disruption after ethanol intoxication and burn injury. To validate miR-146a overexpression following in vivo mimic administration, miR-146a expression was analyzed in small intestinal tissue and isolated epithelial cells one day after ethanol and burn injury. As shown in Figure 7, mice receiving miR-146a mimic had significantly higher miR-146a expression in both total small intestinal tissue and isolated intestinal epithelial cells compared to mice receiving scramble mimic. This overexpression successfully elevated miR-146a expression following ethanol and burn injury.

Figure 7. In Vivo Administration of miR-146a Mimic Significantly Increases miR-146a Expression in Small Intestine.

(A) Small intestinal tissue or (B) small intestinal epithelial cells were isolated one day after ethanol and burn injury and total RNA extracted for RT-qPCR analysis of miRNA expression using primers specific for miR-146a-5p. Expression depicted relative to scramble sham vehicle control, Snord68 used as housekeeping (one experiment, n=4–7 animals per group). Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests compared to sham vehicle with p values adjusted for false discovery rate, * padj<0.05, ** padj<0.01.

Previous studies in our laboratory have demonstrated that elevated intestinal inflammation following ethanol and burn injury is characterized by increased levels of pro-inflammatory mediators, including pro-inflammatory cytokine IL-6(8, 17, 31, 44). To assess the overall impact of miR-146a overexpression on intestinal inflammation following ethanol and burn injury, gene expression of IL-6 was measured in small intestinal tissue. Figure 8A demonstrates significantly elevated IL-6 gene expression in small intestinal tissue one day following ethanol and burn injury. Overexpression of miR-146a almost entirely inhibited IL-6 expression, firmly supporting the anti-inflammatory capacity of miR-146a. We also found that IL-6 expression was significantly reduced by miR-146a overexpression in isolated small intestinal epithelial cells, indicating that epithelial cell expression of miR-146a likely contributes to the observed reduction in intestinal tissue inflammation. Expression of miR-146a has been previously studied for its regulation of inflammatory signaling, where its expression reduced inflammatory cytokine expression via inhibition of NF-κB signaling(25, 29, 45). In addition, our in vitro studies suggest that p38 MAPK signaling is required for miR-146a mediated regulation of pro-inflammatory cytokine expression in small intestinal epithelial cells. To further characterize the impact of in vivo miR-146a overexpression on intestinal inflammation following ethanol and burn injury, activation of pro-inflammatory signaling within small intestinal epithelial cells was assessed by western blot analysis of p38 MAPK and NF-κB p65 phosphorylation. As shown in Figure 8B, elevated phosphorylation of p38 MAPK and NF-κB p65 in small intestinal epithelial cells one day after ethanol and burn injury appears to be reduced by miR-146a overexpression.

Figure 8. In Vivo Overexpression of miR-146a Inhibits Intestinal Inflammation Following Ethanol and Burn Injury.

(A) Small intestinal tissue or small intestinal epithelial cells were isolated one day after ethanol and burn injury and total RNA extracted for RT-qPCR analysis of miRNA expression using primers specific for IL-6. Expression depicted relative to scramble sham vehicle control, Beta Actin used as housekeeping. n=4–7 animals per group. Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests compared to sham vehicle with p values adjusted for false discovery rate, * padj<0.05. (B) Small intestinal epithelial cells were isolated one day after ethanol and burn injury. Western blot analysis was performed to analyze activation of inflammatory signaling via quantification of phosphorylated p38 MAPK and NF-κB. Graphs depict density of phospho- relative to total protein bands normalized to beta actin and presented relative to average Scramble SV, with mean ± SEM.

Studies have shown that neutrophil accumulation, promoted by chemokines such as KC, contributes to intestinal tissue damage and inflammation following ethanol and burn injury(12, 14, 18). Although previous studies have demonstrated significantly increased KC expression in small intestinal tissue one day following ethanol and burn injury, changes in KC expression did not reach significance in our current study. Nevertheless, we observe a clear trend (Student’s t test p=0.06) indicating that miR-146a mimic administration reduces small intestinal KC expression after combined injury (Figure 9A). To further evaluate neutrophil infiltration of intestinal tissue, gene expression of neutrophil marker Ly6G was assessed via RT-qPCR. In addition, we assessed gene expression of the neutrophil effector enzyme Lipocalin-2 (Lcn2), which is stored in specific granules and is induced by inflammatory cytokines and bacterial products. Small intestinal expression of Ly6g and Lcn2, shown in Figure 9B, are significantly elevated following ethanol and burn injury. Overexpression of miR-146a dramatically decreases Ly6g and Lcn2 expression after ethanol and burn injury, indicating reduced neutrophil infiltration, which could contribute to reduced intestinal inflammation and tissue damage.

Figure 9. In Vivo Overexpression of miR-146a Reduces Small Intestinal Neutrophil Infiltration Following Ethanol and Burn Injury.

Small intestinal tissue was isolated one day after ethanol and burn injury and total RNA extracted for RT-qPCR analysis of gene expression using primers specific for neutrophil markers KC, Ly6g or Lcn2. Expression depicted relative to scramble sham vehicle control; beta actin used as housekeeping. n=4–7 per group. Statistical analysis via two-way ANOVA with on graph significance depicting individual t tests compared to sham vehicle with p values adjusted for false discovery rate, * padj<0.05, ** padj<0.01.

Impact of miR-146a Overexpression on Intestinal Barrier Integrity After Ethanol and Burn Injury

Intestinal inflammation and neutrophil infiltration can result in gut barrier disruption and promote consequences including systemic inflammation and remote organ injury(9, 12, 17, 19, 32, 33, 46). The intestinal barrier is maintained by a variety of mechanisms, including the formation of tight junctions holding intestinal epithelial cells closely together and the constant regeneration of the intestinal epithelial layer(47, 48). To analyze the proliferative state of intestinal epithelial cells following ethanol and burn injury, RT-qPCR and western blot analysis of cell cycling protein CyclinD1 was performed. Figure 10A demonstrates that CyclinD1 protein expression is reduced one day following ethanol and burn injury and is significantly elevated by miR-146a overexpression. CyclinD1 gene expression, shown in Figure 10B, is also significantly increased one day after combined injury following miR-146a overexpression. Additionally, our results indicate that miR-146a overexpression enhances intestinal epithelial cell tight junction protein expression. Western blot analysis, shown in Figure 10A, and RT-qPCR, shown in Figure 10B, demonstrate reduced expression of Occludin, a crucial component of tight junctions, one day after ethanol and burn injury. Occludin expression after ethanol and burn injury is enhanced by miR-146a overexpression. These results demonstrate that miR-146a overexpression may promote the intestinal barrier by increasing epithelial cell proliferation and tight junction protein expression.

Figure 10. Impact of miR-146a Overexpression on Intestinal Epithelial Barrier Following Ethanol and Burn Injury.

Small intestinal epithelial cells were isolated one day after ethanol and burn injury. (A) Western blot to assess CyclinD1 and Occludin protein levels. Graphs depict density normalized to beta actin and presented relative to average Scramble SV, with mean ± SEM (n = 2–4 per group). Significance of depicted comparisons analyzed via student’s t tests, *p<0.05. (B) Total RNA extracted for RT-qPCR using primers specific for CyclinD1 or Occludin. Expression depicted relative to scramble sham vehicle control. Beta Actin used as housekeeping, n=4–7 per group. (C) Mice were gavaged with FITC-Dextran one day after ethanol and burn injury. Three hours later, blood was collected for spectrophotometric analysis of serum FITC-dextran levels. Concentration of FITC-dextran shown as μg FITC-dextran per μL serum, with mean ± SEM (one experiment, n = 4–7 per group). Statistical analyses were performed via two-way ANOVA with p values of individual t tests adjusted for false discovery rate, * padj<0.05, ** padj<0.01, **** padj<0.0001.

Following ethanol and burn injury, intestinal inflammation and disruption of the epithelial barrier culminates in heightened intestinal permeability and gut leakiness. This allows for the translocation of bacteria and inflammatory products such as endotoxin, which has been shown to contribute to serious pathologies following ethanol and burn injury including sepsis and multiple organ failure. To measure intestinal permeability, FITC-dextran was introduced into the gastrointestinal tract via gavage. Blood was collected three hours later to quantify the amount of FITC-dextran that was able to leak from the intestinal tract into systemic circulation. As shown in Figure 10C, intestinal permeability is significantly increased one day following ethanol and burn injury. Overexpression of miR-146a may slightly impact intestinal permeability, however no significant reduction in serum FITC-dextran was found. Although our data suggests that miR-146a overexpression may significantly impact individual components of the intestinal barrier, such as inflammation or small intestinal epithelial cell proliferation, miR-146a overexpression alone is unable to fully restore gut barrier integrity after ethanol and burn injury.

Discussion

Although miRNAs have been shown to impact wound healing, insulin resistance, and cardiac dysfunction after severe burn, the contributions of miRNA expression to intestinal inflammation and barrier disruption following alcohol and burn injury have remained largely unexplored(49–52). Within the intestinal epithelium, miRNAs play a critical role in maintaining gut homeostasis(21–23). Changes in miRNA expression have been implicated in the pathogenesis of several disorders involving intestinal inflammation and barrier disruption(23, 53). While less is known regarding the impact of miRNAs on gut dysfunction following acute clinical conditions such as alcohol and burn injury, several miRNAs have been studied as candidate biomarkers for sepsis, which can result from intestinal barrier disruption after burn injury. Reduced levels of circulating miR-150, miR-381, miR-495, and miR-574 are associated with inflammation and sepsis severity(54–61). Additionally, previous studies have shown that toll-like receptor activation by bacteria products, such as LPS, can promote excessive intestinal inflammation following burn injury(11). Several anti-inflammatory miRNAs regulate toll-like receptor signaling to control inflammatory responses, particularly in immune cells. Studies have shown that expression of miR-146a, miR-150, miR-194, miR-495, and miR-574 can significantly reduce LPS induced inflammation(25, 28, 37, 60, 62–67). Due to these associations, we sought to determine if reduced expression of these anti-inflammatory miRNAs promotes excessive intestinal inflammation and gut dysfunction following alcohol and burn injury. Using a well characterized mouse model of acute ethanol intoxication and burn injury, we found significantly reduced expression of miR-146a and miR-150 in small intestinal epithelial cells one day following ethanol and burn injury. These results further support previous findings demonstrating reduced miR-150 intestinal epithelial cell expression after ethanol and burn injury, and that miR-150 expression can regulate intestinal epithelial cell inflammatory cytokine production in response to LPS(37). First identified as a LPS responsive gene in monocytes, miR-146a was shown to be an important brake for macrophage inflammation with important roles in autoimmunity and endotoxin-induced tolerance(25–27, 68). In monocytes, miR-146a expression is induced by inflammatory stimuli and then miR-146a targets MyD88 adaptor molecules, including TNF receptor associated factor 6 (TRAF6), to limit over-activation of inflammatory pathways(26). More recent studies indicate that miR-146a may be similarly important in intestinal epithelial cells. Using rat and human colonic epithelial cell lines, Anzola et al demonstrated that miR-146a expression inhibits LPS or IL-1β induced production of MCP-1 and IL-8(28). In addition, He et al revealed the protective effect of miR-146a expression on intestinal injury following ischemia-reperfusion and linked reduced injury to lower TRAF6 levels and reduced NF-κB activation(29). If miR-146a does indeed play an important role in regulating intestinal epithelial cell inflammatory signaling, then changes in miR-146a could significantly contribute to gut dysfunction in disease. Therefore, we sought to further elucidate the impact that miR-146a downregulation could have on intestinal inflammation following alcohol intoxication and burn injury.

Activation of toll-like receptor 4 (TLR4) signaling has been shown to mediate intestinal barrier disruption following burn injury(11, 69). Therefore, we recapitulated activation of this crucial inflammatory pathway in vitro by stimulating MODE-K small intestinal epithelial cells with LPS. Stimulation of MODE-K cells for 24 hours significantly induced the expression of the pro-inflammatory cytokine IL-6 and the neutrophil chemokine KC. These pro-inflammatory mediators promote intestinal inflammation, neutrophil recruitment, and barrier disruption following ethanol and burn injury(8, 9, 12, 17). Our results demonstrate that miR-146a inhibition in MODE-K cells significantly elevates LPS induced IL-6 and KC expression, while miR-146a overexpression significantly dampens this inflammatory response. Previous studies have established that miR-146a regulates inflammation by inhibiting NF-κB signaling via targeting of TRAF6 or other targets including IRAK1/4 or TAB1(26, 29, 45). However, we found that pharmacological inhibition of NF-κB in MODE-K cells had no significant impact on IL-6 expression while significantly enhancing KC expression induced by miR-146a inhibition and LPS stimulation. Although this may appear counterintuitive, NF-κB signaling has been shown to play different roles in epithelial and immune cells. While generally considered pro-inflammatory in immune cells, NF-κB signaling is critical for homeostasis of the intestinal epithelium and therefore loss of NF-κB within intestinal epithelial cells has been associated with increased inflammation(70, 71). Instead, we found that miR-146a inhibitor mediated increases in LPS induced IL-6 expression was significantly reduced by both p38 MAPK and STAT3 inhibition. Furthermore, western blot analysis of p38 MAPK and STAT3 phosphorylation following LPS stimulation of MODE-K cells demonstrates that while miR-146a overexpression inhibits phosphorylation of both p38 MAPK and STAT3, miR-146a inhibition only significantly promoted LPS induced phosphorylation of p38 MAPK. The difference observed between miR-146a overexpression and miR-146a inhibition is likely due to the overwhelming increase in miR-146a expression that occurs following miR-146a mimic treatment. Inhibition of miR-146a, instead, reduces endogenous miR-146a and is more physiologically relevant to the downregulation of miR-146a seen after alcohol and burn injury.

The most well characterized and validated target of miR-146a is TRAF6, which has generally been demonstrated as the primary target of miR-146a responsible for regulation of NF-κB signaling(25–29). Although our results indicate that miR-146a primarily modulates inflammation of MODE-K cells via the p38 MAPK pathway, TRAF6 is also upstream of p38 MAPK activation and therefore is still the most likely direct target of miR-146a in our model. We found that overexpression of miR-146a does indeed reduce TRAF6 protein levels. Furthermore, the results from our TRAF6 knockdown experiments support targeting of TRAF6 by miR-146a significantly contributes to miR-146a mediated regulation of LPS induced cytokine production in MODE-K cells. Overall, our results suggest that reduced expression of miR-146a in intestinal epithelial cells one day after alcohol and burn injury potentiates intestinal inflammation via the TRAF6/p38 MAPK pathway (Figure 11). Studies have shown that inhibition of p38 MAPK can protect intestinal barrier integrity and reduce intestinal permeability after burn injury(72, 73). In addition, activation of p38 MAPK in Kupffer cells has been linked to pulmonary and hepatic inflammation following alcohol and burn injury(74). Therefore, our studies also support the therapeutic potential of targeting this miR-146a/TRAF6/p38 MAPK pathway to promote intestinal barrier integrity after alcohol and burn injury, potentially reducing poor patient outcomes associated with sepsis and multiple organ failure.

Figure 11. miR-146a Expression Regulates Intestinal Epithelial Cell Inflammatory Gene Expression After Alcohol and Burn Injury.

Under normal conditions, miR-146a is an important brake mechanism which controls inflammatory signaling to promote resolution of inflammation. Following alcohol intoxication and burn injury, small intestinal epithelial cell expression of miR-146a is significantly impaired. Reduced expression of miR-146a promotes proinflammatory signaling and intestinal epithelial cell cytokine expression, which potentiates gut inflammation and barrier disruption following alcohol intoxication and burn injury.

As insight into miRNA associated pathophysiology expands, targeting of miRNA expression has emerged as a promising therapeutic strategy. Although miRNA-based therapies have yet to reach federal approval, several have demonstrated the efficacy of targeting miRNA expression in clinical trials. Studies have shown that miRNA-based therapies can be used to control excessive activation of inflammatory signaling, including in diseases associated with intestinal inflammation(24, 75, 76). Indeed, clinical trials are currently ongoing for the drug ABX464 which treats ulcerative colitis by inducing miR-124 expression(77–80). Therapeutic targeting of intestinal inflammation has the potential to reduce gut barrier disruption and prevent serious consequences following alcohol and burn injury, including sepsis and multiple organ failure(81). Although activation of toll-like receptor 4 (TLR4) signaling and subsequent pro-inflammatory cytokine production have been connected to intestinal barrier disruption following burn injury, therapeutic targeting of these pathways may have unintended consequences within the intestinal epithelium(11, 69). Studies have shown that loss of either Myd88, a signaling molecule immediately downstream of TLR4 activation, or subsequent NF-κB signaling are both associated with increased inflammation and intestinal barrier disruption(70, 71). Due to the more fine-tuned regulation that miRNAs provide, targeting of this pathway via modulation of key miRNAs that regulate its activity may provide a more promising therapeutic strategy to control intestinal inflammation and support barrier integrity following alcohol intoxication and burn injury.

As our in vitro studies suggest that downregulation of miR-146a within small intestinal epithelial cells promotes LPS induced inflammation via the TLR4/TRAF6/p38 MAPK signaling axis, we next sought to determine if restoration of miR-146a expression could reduce intestinal inflammation and promote barrier integrity following ethanol and burn injury. Our results demonstrate that in vivo administration of a miR-146a mimic significantly elevates miR-146a expression in both small intestinal tissue and epithelial cells one day after ethanol and burn injury. This in vivo overexpression significantly reduced intestinal inflammatory cytokine expression and lowered intestinal neutrophil marker levels, indicating that miR-146a administration successfully prevents intestinal inflammation following alcohol and burn injury. Additionally, we saw increased intestinal epithelial cell expression of CyclinD1 and Occludin after alcohol and burn injury in mice receiving miR-146a mimic compared the scramble control. Although these improvements were unable to fully prevent intestinal permeability following ethanol and burn injury, our findings suggest that miR-146a expression may impact intestinal epithelial cell barrier function either as a secondary result from reducing inflammatory signaling or via direct regulation of intestinal epithelial cell proliferation or tight junction protein expression. Further characterization, both in vitro and in vivo, is required to understand these mechanisms and the impact they may have on intestinal epithelial barrier integrity after alcohol intoxication and burn injury.

Although our mouse model does not use a large burn area (only ~12.5% TBSA) and does not exhibit intestinal pathology unless combined with alcohol intoxication, some cytokines, including IL-6, have been observed to be elevated by burn injury alone(8, 31). However, the addition of alcohol does significantly elevate inflammatory cytokine levels compared to burn alone, which leads to the recruitment of inflammatory immune cells to the intestine and results in increased intestinal permeability. Other studies have shown that inhibiting inflammation via anti-IL-6 antibody treatment prevents neutrophil accumulation, promotes tight junction organization, reduces intestinal damage, and prevents bacterial translocation after alcohol and burn injury(17). While our studies demonstrate that miR-146a expression significantly reduced intestinal inflammation, including reduced IL-6 and neutrophil marker expression, administration of miR-146a mimic alone was unable to fully restore the gut barrier and prevent significant intestinal permeability. Inflammation is just one of the many factors that impact intestinal barrier integrity following alcohol and burn injury. Other studies have shown that the loss of intestinal IL-22 after alcohol and burn injury contributes to reduced intestinal proliferation and barrier disruption(30, 35). Like miR-146a mimic treatment, however, recombinant IL-22 treatment alone was unable to significantly reduce intestinal permeability following alcohol and burn injury(30). Administration of miR-146a mimic reducing intestinal inflammation and recombinant IL-22 treatment promoting intestinal barrier integrity could work together to successfully prevent intestinal permeability after alcohol and burn injury and therefore is a promising option for a combination therapy.

It is important to consider a major limitation to the conclusions that can be drawn from this study regarding the role of intestinal epithelial cell miR-146a expression on intestinal inflammation and barrier integrity after alcohol and burn injury. Administration of miRNA mimics, including via intraperitoneal injection, can result in miRNA expression in a variety of organs and cell types. In the current study, we are unable to clarify the contribution that increased miR-146a expression in immune cells, present both in circulation and within intestinal tissue, versus epithelial cells of the intestinal epithelium might have on mitigating intestinal inflammation. Both inflammatory immune cell infiltration and inflammatory cytokine production from epithelial cells have been implicated in barrier disruption following alcohol and burn injury(12, 16, 17, 19). While our results support the conclusion that restoration of intestinal epithelial cell miR-146a expression contributes to reduced intestinal inflammation and prevents recruitment of neutrophils to the intestine, it is likely that miR-146a expression in immune cells also plays a role in the observed outcomes. Further studies evaluating restoration of miR-146a specifically in intestinal epithelial cells or the immune cell compartment are required to fully understand the contributions of miR-146a expression in different cell type. While our study demonstrates the impact of miR-146a downregulation on intestinal inflammation following alcohol and burn injury, there are limitations to the conclusions that can be drawn regarding miR-146a mimic administration as a viable treatment option. In our study, mice were injected with miR-146a mimic one day prior to burn injury. Although miR-146a expression is upregulated 24 hours after injury and significantly controls intestinal inflammation, we are unable to conclude that miR-146a treatment immediately following alcohol intoxication and burn injury would provide similar results. A more thorough study exploring miR-146a administration at several time points within the first 24 hours following burn injury would provide more concrete support of the therapeutic potential of targeting miR-146a expression and deeper understanding of its limitations.

Although we’ve only begun to lay the foundations, these studies address an important gap in our current understanding of the mechanisms underlying intestinal dysfunction in patients exhibiting alcohol intoxication at the time of burn injury. Our studies identify numerous miRNAs which are crucial regulators of intestinal homeostasis and highlight their potential contributions to intestinal dysfunction after alcohol intoxication and burn injury. In particular, we demonstrate significantly reduced expression of miR-146a in intestinal epithelial cells one day following the combined insult and elucidate the mechanism by which decreased intestinal epithelial cell miR-146a expression perpetuates intestinal inflammation and potentially impacts gut barrier integrity. Future studies will continue to illuminate the role of altered miRNA expression in gut dysfunction and the therapeutic potential of harnessing these important miRNAs to reduce intestinal inflammation and improve outcomes after alcohol intoxication and burn injury.

Key points.

• Alcohol and burn injury decrease miR-146a in small intestinal epithelial cells.

• Reduced miR-146a promotes intestinal inflammation after alcohol and burn injury.