Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection (1). In the United States, sepsis results in between 270,000 to 380,000 deaths annually (2). Despite increasing knowledge of sepsis, if antimicrobial treatment is unsuccessful, management relies upon supportive care, without proven benefit of any adjunctive therapy (3).
The gut is frequently referred to as the “motor” of critical illness (4–6) due to a number of abnormalities that can cause and/or propagate sepsis. Under basal conditions, cellular proliferation is balanced by cell loss by apoptosis and exfoliation of live cells into the gut lumen. However, this balance is altered in critical illness. Sepsis induces intestinal epithelial apoptosis in both preclinical models as well as in human autopsy studies (7). Notably, overexpression of the anti-apoptotic protein Bcl-2 prevents sepsis-induced gut epithelial apoptosis, and this confers a survival advantage in murine models of cecal ligation and puncture (CLP) and Pseudomonas aeruginosa pneumonia (8, 9), although no difference in survival is seen with Bcl-2 overexpression following acute lung injury (10). Although numerous theories have been proposed, the etiology through which prevention of gut apoptosis by Bcl-2 overexpression improves survival in sepsis (as opposed to noninfectious inflammation) is unclear.
Barrier function is maintained by allowing paracellular movement of water, solutes, and immune modulating factors while preventing diffusion of potentially harmful components of the microbiome (11). Sepsis induces intestinal hyperpermeability in preclinical models of sepsis and in critically ill patients which can potentially allow components of the pathobiome to escape the gut lumen and cause or worsen systemic illness (12, 13). Sepsis-induced gut barrier dysfunction is mediated via alterations in the apical tight junction (TJ) which alters molecular via two distinct pathways—a high-capacity, size, and charge-selective route (pore) and a low-capacity, nonselective route (leak).
The relationship between apoptosis and permeability is complex. While agents that increase apoptosis have been demonstrated to be associated with increased permeability in the small intestine (14), the majority of studies linking these two processes have been performed in cell culture (15–18) and conflicting data exists regarding the relationship between apoptosis and permeability. To determine whether prevention of sepsis-induced gut apoptosis by the anti-apoptotic protein Bcl-2 prevents intestinal hyperpermeability in sepsis in vivo, mice that overexpress Bcl-2 in their gut epithelium (Fabpl-Bcl-2 mice) (8, 9, 19, 20) were assayed for permeability and TJ following CLP.
MATERIALS AND METHODS
Experiments were performed on 6 to 12-week-old, gender-matched WT FVB/N and Fabpl-Bcl-2 mice, which are on the same genetic background (21). All experiments were performed in the morning to minimize diurnal variation. Mice were maintained on a 12 h light–dark schedule in a specific pathogen-free environment and received standard laboratory mouse chow and water ad libitum both before and after sepsis. All studies were performed in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Emory University School of Medicine (Protocol 201800033).
CLP or sham laparotomy was performed according to the method of Baker et al. (22). Under isoflurane anesthesia, a midline abdominal incision was made, and the cecum was exteriorized and ligated below the ileocecal valve to avoid intestinal obstruction. The cecum was then punctured twice with a 25-gauge needle and gently squeezed to extrude a small amount of stool. After replacing the cecum in the abdomen, the abdominal wall was closed in layers. All mice received 1 mL of normal saline via subcutaneous injection to account for insensible fluid losses (23). To mimic clinical management, mice were also given subcutaneous injections of ceftriaxone (50 mg/kg, Sigma-Aldrich, St. Louis, Mo) and metronidazole (30 mg/kg, Apotex Corp, Weston, Fla) after CLP and every 12 h thereafter (3). Mice were sacrificed at 6, 12, 24, 48, or 96 h following CLP. All mice received buprenorphine (0.1 mg/kg, McKesson Medical, San Francisco, Calif) immediately prior to laparotomy to minimize animal suffering and were redosed with buprenorphine postoperatively when deemed appropriate by the staff of the Division of Animal Resources at Emory University.
Intestinal permeability was measured in vivo by measuring the concentration of FD-4 (22 mg/mL, molecular mass 4 KDa, Sigma-Aldrich) in the blood (24, 25). Mice were orally gavaged with 0.5 mL of FD-4 5 h prior to sacrifice (i.e., 1, 7, 19, 43, or 91 h following induction of CLP or sham laparotomy). Blood was collected at the time of sacrifice and centrifuged at 10,000 rpm at 4°C for 10 min. A total of 50 μL of plasma was then diluted with an equal amount of sterile phosphate-buffered saline (pH 7.4), and the concentration of FD-4 was determined using fluorospectrometry (Synergy HT, BioTek, Winooski, Vt) using an excitation wavelength of 485 nm and an emission wavelength of 528 nm with serially diluted samples as standards. All samples and standards were run in triplicate.
Quantification of mRNA expression
Segments of jejunum were treated with RNAlater Stabilization Solution (Thermo-Fisher Scientific, Waltham, Mass) for 24 h at 4oC and stored at −80oC. Frozen segments of jejunum were thawed and lysed using QIAshredder homogenizer spin columns (QIAGEN, Venlo, the Netherlands). Total RNA was extracted using the QIAGEN RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. Complementary DNA was synthesized from 0.5 ug of total RNA using the iScript cDNA Synthesis kit (Bio-rad, Hercules, Calif) following the manufacturer's instruction. Messenger RNA quantification was measured using TaqMan Gene Expression Assays (Thermo-Fisher Scientific, Waltham, Mass) using comparative Ct method and calculated in the open-source software package R (26).
Western blot analysis
For Western Blots examining TJ, segments of jejunum were snap-frozen in liquid nitrogen and stored at −80oC. These were subsequently weighed and homogenized in 4× volume of ice-cold lysis buffer (50 mM Tris HCl; 10 mM EDTA; 150 mM NaCl; 1% Triton X-100, 1% NaDOC, 1 mM EDTA; 0.1% SDS) with protease inhibitor cocktail mix (cOmplete Mini EDTA-free, Roche, Indianapolis, Ind) using BulletBlender Storm homogenizer (BBY24 M, Next Advance, Inc, Averill Park, NY) for 2 min. Samples were then centrifuged at 10,000 rpm at 4oC for 5 min. The supernatant was collected, and total protein concentration was determined using the Pierce 660 nm protein assay (Thermo-Fisher Scientific). Protein samples of 30 μg and an equal volume of 2× Laemmli buffer (Bio-Rad) were then heated at 95oC for 5 min. Samples were loaded on Mini-PROTEAN TGX Stain-Free polyacrylamide gels (Bio-Rad) for 2 h at 70 V. The gel was activated for 5 min, and the protein was transferred to polyvinylidene difluoride membrane for 7 min using the Trans-blot Turbo system (Bio-Rad). Membranes were then blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich) at room temperature for 60 min and incubated overnight with primary antibody at 4oC. Primary antibodies used were rabbit anti-β-actin (1:2,000, 4967S, Cell Signaling Technology, Danvers, Mass), 51–9100, Life Technologies), rabbit anti-claudin 3 (1:500, 34–1,700, Life Technologies, Carlsbad, Calif), rabbit anti-claudin 4 (1:2,000, PA5–34437, Life Technologies), rabbit anti-claudin 5 (1:2,000, 34–1,600, Life Technologies), and rabbit anti-occludin (1:250, 710192, Life Technologies). The following day, membranes were washed and incubated for 60 min at room temperature with goat antirabbit secondary antibody linked to horseradish peroxidase (1:1,000, Cell Signaling Technology). Membranes were then stripped and probed for actin.
A modification of this technique was performed for Western Blots examining pro- and anti-apoptotic proteins. Samples were loaded on polyacrylamide gels (Bio-Rad) for 45 min at 180 V. Proteins were detected with a chemiluminescent system (ChemiDoc Touch, Bio-Rad). Resulting bands were analyzed using intensity quantification software (ImageLib 6.0, BioRad). Linear dynamic detection range with stain-free technology was used for lane protein normalization and comparisons (27). Membranes were incubated overnight with primary antibody at 4oC with rabbit anti-Bax (1:1,000, Cell Signaling Technology 2772) or rabbit anti-Bcl-xL (1:10,000, Cell Signaling Technology, 2762). Protein bands were detected with a chemiluminescent system (BioRad) and visualized with a charged coupled device (ChemiDoc Touch, BioRad). Densitometric analysis was performed by image software (ImageLib 6.0, BioRad). Data are presented as relative protein expression compared with WT mice for all Western blots.
Slides were deparaffinized, rehydrated and washed with phosphate-buffered saline (PBS). Samples were incubated with 20% goat serum albumin for 30 min and then stained with rabbit anti-claudin-3, anti-claudin-4, anti-claudin-5, or anti-occludin (1:200, Thermo Fisher Scientific) overnight at 4oC. The next day, samples were washed with PBS, then incubated with 1:500 biotinylated anti-rabbit antibody for 1 h at room temperature. After washing in PBS, the samples were incubated with 1:500 horseradish peroxidase streptavidin for 1 h at room temperature. After washing in PBS, the samples were counterstained with 3,3’-diaminobenzidine solution.
Intestinal epithelial apoptosis
Crypt apoptotic cells were quantified in the jejunum by two complementary methods: H&E-staining (morphological analysis) and active caspase-3 staining (functional analysis). Apoptotic cells were identified on H&E-stained sections by characteristic nuclear condensation and fragmentation. For active caspase-3 staining, immunohistochemistry was performed as above and sections were stained for rabbit-anti cleaved caspase 3 (1:100 Cell Signaling Technology 9664). Apoptotic cells were quantified in 100 contiguous well-oriented crypt-villus units per animal by an examiner (TO) blinded to sample identity.
Statistical analyses were performed using Prism (version 7.0, GraphPad Software, San Diego, Calif). Data are presented as mean ± SEM. Data were tested for normality by the D’Agostino-Pearson omnibus normality test. Data with a normal distribution were compared using Student t test. Data that did not have a normal distribution were compared using the Mann–Whitney U test. A P value of ≤0.05 was considered to be statistically significant.
Effect of Bcl-2 overexpression in the intestinal epithelium on jejunal apoptosis
Jejunal apoptosis was decreased in Fabpl-Bcl-2 mice compared with WT mice 24 h after CLP when measured by morphologic criteria (Fig. 1,A–C) or by active caspase 3 staining (Fig. 1, D–F). This decrease in cell death was not associated with differences in levels of the apoptotic mediators Bax or Bcl-XL in WT and transgenic mice (Supplemental Figure 1, Supplemental Digital Content 1, https://links.lww.com/SHK/A959).
Effect of intestinal epithelial apoptosis prevention on sepsis-induced hyperpermeability
Under sham conditions, permeability was similar between WT and Fabpl-Bcl-2 mice (Fig. 2). Sepsis induced intestinal hyperpermeability in WT mice, with increasing FD-4 appearance in the blood detectable at 6 h persisting out to 48 h. In contrast, permeability was not significantly different between Fabpl-Bcl-2 mice at any timepoint following CLP compared with sham Fabpl-Bcl-2 mice. In addition, permeability was lower in Fabpl-Bcl-2 mice than WT mice at all timepoints between 6 and 48 h.
Effect of intestinal epithelial apoptosis prevention on TJ mRNA expression
Since permeability was similar in WT mice at multiple timepoints after CLP but apoptosis peaks 24 h after the onset of sepsis (19), mechanistic studies were performed at 24 h. Messenger RNA levels were assayed in the jejunum of TJs previously determined to be present in the intestine. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice than septic WT mice (Fig. 3). In contrast, claudin 4 mRNA expression was significantly higher in septic Fabpl-Bcl-2 mice. No difference was detected in expression of mRNA expression of claudins 2, 7, 15, JAM-A, and ZO-1 between septic WT and transgenic mice.
Effect of intestinal epithelial apoptosis prevention on TJ protein expression and immunohistochemistry
To determine whether transcriptional changes in intestinal TJ protein were associated with changes at the protein level, Western Blots were performed on TJ that were different between WT and transgenic mice at the mRNA level (Fig. 4). Similar trends were noted in claudins 3 and 5 (decreased) and 4 (increased) in Fabpl-Bcl-2 mice 24 h after CLP. In contrast, occludin showed the opposite expression pattern. While mRNA levels of occludin were lower in transgenic mice following CLP, protein levels were nearly 100 times higher. To confirm the differential expression patterns of intestinal TJ in vivo, immunohistochemistry was performed on WT and transgenic mice 24 h after CLP (Fig. 5). Patterns identified were generally consistent with that seen on Western Blotting, albeit with differential cellular and geographic specificity along the crypt villus axis depending on TJ assayed.
This study demonstrates that while gut barrier function is similar in sham conditions between WT mice and those that overexpress Bcl-2 in their intestinal epithelium, sepsis-induced intestinal hyperpermeability is prevented in transgenic mice. This is associated with alterations in numerous TJ proteins that may mechanistically prevent induction of intestinal hyperpermeability in Fabpl-Bcl-2 mice.
While often thought of as independent processes, the relationship between apoptosis and permeability is complex. Knocking out the TJ protein occludin in normal cells in vitro not only leads to hyperpermeability, it also leads to increased apoptosis via the extrinsic (receptor-mediated) pathway (28). In theory, elimination of dead cells via apoptosis could also cause a gap in an epithelial layer, leading to hyperpermeability. However, as a programmed activity that is evolutionarily conserved from simple multicellular organisms up to humans, tissues have developed methods to eliminate apoptotic cell without altering host homeostasis (29). Sepsis is well known to induce both augmented apoptosis and permeability, in both patients and in animal models. Previous experiments examining a potential link between increased apoptosis and permeability in the intestine have dominantly been performed in cell culture, often with conflicting results. Both Fas and LPS individually induce apoptosis and increase permeability in intestinal epithelial cell monolayers, and permeability is restored when apoptosis is blocked (17, 18). Similarly, apoptosis induced by Giardia labmlia infection in enterocyte monolayers is prevented by pretreatment with caspase 3 inhibitors (17). In contrast, TNF and IFN-γ-induced gut barrier dysfunction is independent of apoptosis in an epithelial cell line since apoptosis inhibition by the caspase inhibitor z-VAD does not alter paracellular permeability (16). Further, while both TNF and IFN-γ- induce hyperpermeability in a three-dimensional cell culture of intestinal epithelial cells, TNF-induced apoptosis contributes to worsened barrier function while IFN-γ's role does not involve apoptosis (15). The results presented herein expand our understanding of this relationship in a number of ways. First, the results are in vivo. It is essentially impossible to model the complexities of sepsis in a cell culture setting, and the finding that permeability is normalized by Bcl-2 overexpression shows a link between apoptosis and permeability in a setting that is more clinically relevant than in vitro experiments. Next, the findings examine the intrinsic (mitochondrial) pathway of apoptosis. This is mechanistically distinct from the majority of in vitro findings examining the extrinsic (receptor-mediated) pathway. In addition, to the best of our knowledge, our findings represent the most comprehensive analysis of intestinal tight junctions in the setting of gut apoptosis prevention.
The relationship between intestinal TJ proteins and permeability in sepsis is also complex. We have previously demonstrated that CLP induces jejunal claudin 2 and JAM-A while decreasing claudin 5 and occludin (25). Pseudomonas pneumonia impacts the same TJ while also decreasing ZO-1. In contrast, subcellular localization of colonic claudins 1, 3, 4, 5, and 8 all altered and claudin 2 is increased following CLP (30). Intestine-specific overexpression of epidermal growth factor improves gut barrier dysfunction and normalizes sepsis-induced increases in claudin 2 (31). Further, systemic epidermal growth factor decreases intestinal permeability in septic mice with pre-existing chronic alcohol ingestion, associated with increases in claudin 5 and JAM-A (32). In a complementary fashion, this study finds that normalization of intestinal permeability by Bcl-2 overexpression is associated with alterations in claudin 3, claudin 4, claudin 5, and occludin. Paracellular flux through the TJ occurs through two distinct routes—the leak and pore pathways. (11, 33). The leak and pore pathways are modulated by different TJ and TJ-associated proteins—occludin, MLCK, ZO-1 for leak and the claudin family for pore—and are activated by different cytokines (34, 35). Notably, claudins can be either pore forming or barrier enhancing depending on their structure. Additionally, the different sizes of these pathways are potentially mechanistically important since intact bacteria are too large to translocate through the pore pathway, suggesting intestinal hyperpermeability may play a role in mediating mortality through mechanisms independent of bacterial translocation (although this does not rule out a role for translocation through larger pathways). Putting our results into context suggests that sepsis impacts both the pore and leak pathways, and strategies that improve the gut barrier alter TJ in both pathways. Further, while numerous TJ are present in the intestine, only a subset are altered in sepsis and are thus more likely to play a mechanistic role in gut hyperpermeability, which suggests a targeted approach may be beneficial in efforts to improve the gut barrier in sepsis.
This study has a number of limitations. First, we have used Bcl-2 overexpression as a proxy for apoptosis prevention. Mechanistically, cell death in the intrinsic pathway of apoptosis is modulated by the balance of pro- and anti-apoptotic Bcl-2 family members, and there is extensive mechanistic data for the role of Bcl-2 in modulating apoptosis (36). Despite there being no known role for Bcl-2 in mediating permeability, we cannot absolutely rule out an unknown role of Bcl-2. Further, while our data convincingly demonstrate a role for intestinal Bcl-2 overexpression in modulating gut permeability, this does not mean that all apoptosis mediators impact the gut barrier. In addition, all studies of TJ proteins were carried out only at 24 h. This experimental design was used because this is the timepoint where maximal apoptosis occurs and also because there was a significant difference in permeability between WT and Fabpl-Bcl-2 mice at this timepoint. Nonetheless, it is possible that we missed important changes in the TJ at earlier or later timepoints by assaying mice only at 24 h. Further, we cannot conclude which TJs are mechanistically responsible for normalization of permeability in Fabpl-Bcl-2 mice. While numerous TJ proteins were altered in Fabl-Bcl-2 mice compared with WT mice, additional studies either knocking out or augmenting each protein would be required to distinguish which (if any) are causative for the alteration in permeability. It is also unclear why occludin mRNA levels were decreased in Fabpl-Bcl-2 mice whereas protein levels were nearly 100 times higher although we speculate that the differences between mRNA and protein levels were due to post-transcriptional modification. It should be noted that mechanistically increased occludin levels would be expected to be associated with improved permeability, as was seen in transgenic mice. Next, claudins 3, 4, and 5 are all sealing claudins, and, each would be expected to increase if they were mechanistically responsible for improved permeability. However, only claudin 4 was increased while claudins 3 and 5 were decreased in transgenic mice. FD4 has a diameter of 28 Å and measures permeability via the leak pathways (altered by occludin) but not the pore pathway (altered by the claudin family). Identifying the importance (if any) of each of these four TJ proteins in mediating decreased permeability after Bcl-2 overexpression would require future experiments where each TJ was either knocked out or ovreexpressed in transgenic mice. Finally, the cellular and/or subcellular mechanisms through which altering apoptotic machinery leads to changes in permeability require further study.
Despite these limitations, this study demonstrates that apoptosis prevention in the intestine normalizes sepsis-induced hyperpermeability associated with numerous TJ changes. Further mechanistic studies are required to determine what role this plays in the survival benefit conferred by intestine-specific Bcl-2 overexpression in sepsis.
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