Potential conflict of interest: Nothing to report.
Hepatic ischemia and reperfusion (IR) injury is a major clinical problem that leads to frequent extrahepatic complications including intestinal dysfunction and acute kidney injury (AKI). In this study we aimed to determine the mechanisms of hepatic IR-induced extrahepatic organ dysfunction. Mice subjected to 60 minutes of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased interleukin-17A (IL-17A) levels in portal vein plasma and small intestine. We also detected increased levels of IL-17A messenger RNA (mRNA) and protein in Paneth cells after hepatic IR with laser capture dissection. IL-17A-neutralizing antibody treatment or genetic deletion of either IL-17A or IL-17A receptors significantly protected against hepatic IR-induced acute liver, kidney, and intestinal injury. Leukocyte IL-17A does not contribute to organ injury, as infusion of wildtype splenocytes failed to exacerbate liver and kidney injury in IL-17A-deficient mice after hepatic IR. Depletion of Paneth cell numbers by pharmacological (with dithizone) or genetic intervention (SOX9 flox/flox Villin cre+/− mice) significantly attenuated intestinal, hepatic, and renal injury following liver IR. Finally, depletion of Paneth cell numbers significantly decreased small intestinal IL-17A release and plasma IL-17A levels after liver IR. Conclusion: Taken together, the results show that Paneth cell-derived IL-17A plays a critical role in hepatic IR injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR. (HEPATOLOGY 2011;)
Hepatic ischemia and reperfusion (IR) complicates liver transplantation and major liver resection.1 Furthermore, hepatic IR frequently leads to extrahepatic organ injury including the kidney, intestine, and lung.2 In particular, acute kidney injury (AKI) after major liver IR is extremely common (40-85% incidence) and the development of AKI after liver injury greatly increases patient mortality and morbidity during the perioperative period.2 Furthermore, extrahepatic manifestations of liver IR not only contribute significantly to remote organ (e.g., kidney, intestine) injury but also exacerbate hepatic IR injury. Unfortunately, the detailed mechanisms involved in extrahepatic organ dysfunction due to hepatic IR remain obscure.
Interleukin-17A (IL-17A) is a proinflammatory cytokine released by T cells as well as by innate immune cells and plays a critical role in both innate and adaptive immunity.3–6 Not surprisingly, IL-17A dysregulation has been implicated in several autoimmune diseases, with heightened inflammatory responses in humans and in mice.3 In our previous studies we showed that AKI leads to increased small intestinal IL-17A release and plasma IL-17A levels.7 Takahashi et al.4 recently demonstrated that small intestinal Paneth cells produce and release IL-17A to mediate tumor necrosis factor alpha (TNF-α)-induced shock. Therefore, small intestinal Paneth cells may function as a reservoir of proinflammatory IL-17A and Paneth cell-derived IL-17A may potentiate liver injury, systemic inflammation, and extrahepatic organ dysfunction.
In this study we tested the hypothesis that hepatic IR induces Paneth cell dysregulation and increased IL-17A production and release. A combination of pharmacological and genetic depletion approaches were used to determine the role of small intestinal Paneth cells as a source of IL-17A generation after hepatic IR resulting in exacerbation of liver injury and extrahepatic (kidney and intestine) organ dysfunction.
AKI, acute kidney injury; ALT, alanine aminotransferase; IL, interleukin; IR, ischemia and reperfusion; LCM, laser capture microdissection; TNF-α, tumor necrosis factor alpha.
Materials and Methods
Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO). Anti-(6C/A)-Crp1 antibody reactive against mouse alpha-defensin was a kind gift of Dr. Andre J. Ouellette (Keck School of Medicine of the University of Southern California, Los Angeles, CA).
All mice strains were bred or purchased on a C57BL/6 background. Male C57BL/6 mice (20-25 g) were obtained from Harlan (Indianapolis, IN). IL-17A-deficient mice (IL-17A−/−) were obtained as a gift from Yoichiro Iwakura (University of Tokyo, Tokyo, Japan) and IL-17A receptor-deficient mice (IL-17R−/−) were provided by Amgen. Both IL-17A−/− and IL-17R−/− mice were congenic with C57BL/6 mice.8 Paneth cell-deficient mice (SOX9 flox/flox Villin Cre+/−) were generated as described and obtained from Yuko Mori-Akiyama (Baylor College of Medicine, Houston, TX).9 SOX9 transcription factor is required for the differentiation of Paneth cells, as intestinal inactivation of SOX9 resulted in mice with Paneth cell deficiency without affecting differentiation of other intestinal epithelial cell types.9 SOX9 flox/flox Villin Cre−/− mice were used as wildtype controls.
Induction of Hepatic IR.
All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Male mice (20-25 g) were subjected to liver IR injury as described.10 This method of partial hepatic ischemia for 60 minutes results in a segmental (≈70%) hepatic ischemia but spares the right lobe of the liver and prevents mesenteric venous congestion by allowing portal decompression through the right and caudate lobes of the liver. Sham-operated mice were subjected to laparotomy and identical liver manipulations without vascular occlusion. Five to 24 hours after reperfusion, plasma, liver, kidney, and small intestine tissues were collected for analysis of tissue injury, inflammation, cytokine up-regulation, and apoptosis. We also collected systemic plasma 0.1, 1, 3, 5, 12, and 24 hours after reperfusion to measure IL-17A levels with enzyme-linked immunosorbent assay (ELISA). To deplete Paneth cell granules, mice were treated with dithizone (100 mg/kg, intravenously) 6 hours prior to hepatic ischemia as described.11, 12 Dithizone (10 mg/mL) was dissolved in saturated lithium carbonate (1 g/100 mL). To neutralize IL-17A, mice were treated intravenously with 100 or 200 μg of antimouse IL-17A antibody (eBioscience, San Diego, CA) immediately before reperfusion. In order to determine whether leukocyte IL-17A contributes to hepatic IR injury and extrahepatic organ dysfunction, spleens from wildtype (C57BL/6) mice were crushed and splenocytes were passed through a nylon cell strainer (BD Biosciences, San Jose, CA) and collected in phosphate-buffered saline (PBS). Red blood cells were lysed and single-cell splenocyte suspensions were transferred intravenously (6 × 106 to 1 × 107 splenocytes per transfer, 200 μL) to IL-17A−/− mice 24 hours before liver ischemia.
Measurement of Plasma Alanine Aminotransferase (ALT) Activity and Creatinine Level.
The plasma ALT activities were measured using the Infinity ALT assay kit according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA). Plasma creatinine was measured by an enzymatic creatinine reagent kit according to the manufacturer's instructions (Thermo Fisher Scientific). This method of creatinine measurement largely eliminates the interferences from mouse plasma chromagens well known to the Jaffe method.13
Histological Analysis of Hepatic, Small Intestinal, and Renal Injury.
For histological preparations, liver, small intestine, or kidney tissues were fixed in 10% formalin solution overnight. After automated dehydration through a graded alcohol series, tissues were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin-eosin (H&E). All H&E sections were evaluated for injury by a pathologist (V.D'A.) who was blinded to the treatment each animal had received. Liver H&E sections were graded for IR injury using the system devised by Suzuki et al.14 and as described.10 In this classification, three liver injury indices, sinusoidal congestion (score: 0-4), hepatocyte necrosis (score: 0-4), and ballooning degeneration (score: 0-4), are graded for a total score of 0-12. We also quantified percent liver necrotic area as well as degree of hepatocyte apoptosis after liver IR. Hepatic apoptosis was quantified by counting the number of apoptotic hepatocytes per high-power field (400×) in necrotic and in nonnecrotic (viable) zones. Total apoptosis score per liver section was estimated by multiplying the number of apoptotic cells in necrotic area by percent liver necrotic area. Intestine H&E sections were also blindly evaluated for intestinal epithelial cell necrosis, development of a necrotic pannus over the mucosal surface, villous endothelial cell apoptosis, and swelling and blunting of villi because of villous mononuclear cell mucosal inflammation and edema. Renal H&E sections were evaluated for the severity (score: 0-3) of renal cortical vacuolization, peritubular/proximal tubule leukocyte infiltration, proximal tubule simplification, and proximal tubule hypereosinophilia.
ELISA for IL-17A.
Twenty-four hours after liver reperfusion, plasma, small intestine, and isolated crypt IL-17A levels were measured with mouse specific IL-17A ELISA kit according to the manufacturer's instructions (eBiosciences). Tissues were homogenized in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, and 1% Triton-X [pH 7.4]) and samples processed for mouse-specific ELISA kits.
Small intestine tissues from SOX9 flox/flox Villin Cre+/− (Paneth cell-deficient) or SOX9 flox/flox Villin Cre−/− (wildtype control) mice were homogenized in ice-cold RIPA buffer and processed for cryptdin-1 immunoblotting with Anti-(6C/A)-Crp1 antibody as described.10, 15
Assessment of Liver,
Small Intestine, and Kidney Inflammation. Liver, kidney, and small intestine inflammation after hepatic ischemia was determined with detection of neutrophil infiltration by immunohistochemistry 24 hours after hepatic IR as described16 and by measuring messenger RNA (mRNA) encoding markers of inflammation, including keratinocyte-derived cytokine (KC), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractive protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) 5 hours after liver IR (Supporting Table 1). Semiquantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed as described.10
Detection of Liver, Small Intestine, and Kidney Apoptosis.
We used two additional independent assays to assess the degree of liver and intestine apoptosis 24 hours after sham surgery or liver IR: in situ transferase-mediated dUTP nick-end labeling (TUNEL) assay and the detection of DNA laddering. For the TUNEL assay, formalin-fixed sections were deparaffinized in xylene and rehydrated through graded ethanol to water. In situ TUNEL staining was used for detecting DNA fragmentation in apoptosis using a commercially available in situ cell death detection kit (Roche, Nutley, NJ) according to the manufacturer's instructions. For DNA laddering, apoptotic DNA fragments were extracted according to the methods of Herrmann et al.17 and electrophoresed at 70 V in a 2.0% agarose gel in Tris-acetate-EDTA buffer. This method of DNA extraction selectively isolates apoptotic, fragmented DNA and leaves behind the intact chromatin. The gel was stained with ethidium bromide and photographed under ultraviolet (UV) illumination. DNA ladder markers (100 basepairs) were added to a lane of each gel as a reference for the analysis of internucleosomal DNA fragmentation.
Isolation of Intestinal Crypts.
Intact small intestinal crypts were isolated with the distended intestinal sac method as described by Traber et al.18 with slight modifications. Small intestine (jejunum and ileum) was removed and rinsed thoroughly with intestinal wash solution (0.15 M NaCl, 1 mM dithiothreitol [DTT], and 40 pg/mL phenylmethylsulfonyl fluoride [PMSF]) and then filled with buffer A (in mM): 96 NaCl, 27 sodium citrate, 1.5 KCl, 8 KH2P04, 5.6 Na2HP04, and 40 pg/mL PMSF (pH 7.4). The ends were clamped with microclips and the intestine was filled to a pressure of 50 cm H20. The filled intestine was submerged in oxygenated 0.15 M NaCl at 37°C for 40 minutes, drained, and the solution was discarded. The intestine was then filled with buffer B (in mM): 109 NaCl, 2.4 KCl, 1.5 KH2PO4, 4.3 Na2HPO4, 1.5 EDTA, 10 glucose, 5 glutamine, 0.5 DTT, and 40 pg/mL PMSF (pH 7.4), incubated at 37°C for another 20 minutes, and the intestinal contents were drained and collected. The cells from 40-60 minutes fraction containing intact and isolated crypts were collected by pelleting at 100g for 5 minutes at 4°C and washed once with PBS.
Laser Capture Microdissection (LCM) of Paneth Cells.
LCM of individual Paneth cells was performed with the PixCell I LCM System (Arcturus Engineering, Mountain View, CA) as described.19 Small intestine tissues were excised and embedded in Optimum Cutting Temperature (OCT) compound (Sakura, Torrance, CA), sectioned at a thickness of 10 μm, and mounted on 1.0 PEN Membrane Slides (Carl Zeiss, Thornwood, NY). The sections were then prepared for microdissection using an LCM staining kit (Ambion, Austin, TX) through a graded alcohol series (95%, 75%, 50%) followed by cresyl violet staining. After destaining by way of second graded alcohol series (50%, 75%, 95%), they were dehydrated in 100% ethanol followed by xylene. LCM was performed on a Zeiss Axiovert 200M microscope equipped with PALM RoboSoftware and the total area of tissue collected per slide was tracked and recorded. RNA was isolated from the dissected tissue by following the protocol provided by the RNAqueous-Micro kit (Ambion) by way of column purification.
Small intestines were fixed in 4% paraformaldehyde / 3% glutaraldehyde in 10 mM sodium phosphate buffer (pH 7.4) for 48 hours. All samples were postfixed with 1% osmium tetroxide in 100 mM cacodylate buffer (pH 7.4) on ice for 1 hour. Samples were then treated with 0.5% aqueous uranyl acetate, dehydrated in graded alcohol, treated with propylene oxide, and embedded in Embed 812 (Electron Microscopy Sciences). The resin was polymerized in a 60°C oven for 2-3 days. Sections were cut with a Dupont diamond knife in Reichert-Jung UltraCut E ultramicrotome, collected on copper grids, and doubly stained with saturated aqueous uranyl acetate and lead citrate. Ultrathin sections were imaged for Paneth cells using a JEM-1200EX electron microscope manufactured by JEOL.
Protein contents were determined with a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL), using bovine serum albumin as a standard.
All data are reported as mean ± standard error. The overall significance of the results was examined using one-way analysis of variance and the significant differences between the groups were considered at P < 0.05 with the appropriate Tukey's post hoc test made for multiple comparisons. The ordinal values of the liver and kidney injury scores were analyzed by the Mann-Whitney nonparametric test.
Paneth Cells Degranulate After Ischemic AKI or Bilateral Nephrectomy.
Histological examination of small intestines from sham-operated mice showed Paneth cells containing densely packed eosinophilic secretory granules (Fig. 1A). In contrast, after hepatic IR rapid and extensive degranulation of Paneth cells was observed (magnification 400×, representative of five experiments, arrows and magnified insert) compared to sham-operated animals. Further evidence of Paneth cell degranulation was apparent by electron microscopy of small intestines following hepatic IR (Fig. 1B). The crypt lumen from sham-operated mice was devoid of Paneth cell granules, whereas the crypt lumen from mice subjected to hepatic IR showed granules being released into the lumen.
Increased IL-17A mRNA and Protein in Small Intestinal Crypt Paneth Cells After Hepatic IR.
With LCM we selectively isolated Paneth cells to determine whether Paneth cells produce increased IL-17A mRNA 5 hours after liver IR. mRNA recoveries were sufficient for performance of semiquantitative RT-PCR for GAPDH and IL-17A, which demonstrated increased IL-17A mRNA after bilateral nephrectomy (11 ± 1-fold over sham, n = 4, P < 0.01, Fig. 2). We also isolated intact small intestinal crypts containing Paneth cells 24 hours after sham-operation or liver IR. Small intestinal crypts isolated with the distended sac method and stained with eosin-Y showed red staining characteristic of Paneth cells (Supporting Fig. 1). IL-17A ELISA performed in these isolated crypts showed that IL-17A protein levels were significantly increased (59 ± 4 pg/mg protein, n = 4) compared to sham-operated mice (9 ± 3 pg/mg protein, n = 4).
Hepatic IR Increases Plasma and Tissue IL-17A Production in Mice.
Wildtype (C57BL/6) mice subjected to 60 minutes liver IR increased both systemic (Fig. 3A) and portal venous (Fig. 3B) IL-17A levels compared with the sham-operated mice (undetectable levels). The rise in systemic plasma was very rapid, occurring within 1 hour after reperfusion. Moreover, the rise in portal venous levels of IL-17A was significantly greater (P < 0.05) than the level detected in the systemic circulation. In addition, hepatic, renal, and small intestine (jejunum) tissue IL-17A levels all increased (P < 0.01) after hepatic IR compared to IL-17A levels detected in sham-operated mice liver, kidney, and small intestine (Fig. 3C). Consistent with higher portal venous IL-17A levels, small intestine IL-17A levels after liver IR were greater than the levels determined from the liver or kidney (P < 0.01).
IL-17A Plays a Critical Role in Hepatic, Renal, and Intestine Injury After Hepatic IR in Mice.
Mice subjected to liver IR not only developed severe liver dysfunction with significantly higher plasma ALT levels (P < 0.0001 compared to sham-operated mice) but also developed AKI with significant rises in plasma Cr (P < 0.01 versus sham-operated mice) 24 hours after hepatic ischemic injury (Fig. 3D). Induction of IL-17A plays a critical role in generating hepatic and renal injury after liver IR as mice treated with IL-17A neutralizing antibody were significantly and dose-dependently protected against hepatic and renal injury after liver IR. In addition, mice deficient in IL-17A receptor or IL-17A were protected against hepatic and renal injury after liver IR. Furthermore, transfusion of IL-17A wildtype splenocytes failed to exacerbate hepatic or renal injury in IL-17A-deficient mice after liver IR, suggesting that IL-17A from a nonleukocyte source contributes to hepatic and renal injury. Plasma (systemic and portal vein) and tissue (liver, kidney and jejunum) levels of IL-17A were significantly reduced in mice treated with IL-17A antibody, in IL-17A receptor, or IL-17A-deficient mice (Fig. 3A-C) after liver IR. We determined in pilot experiments that transfusion of IL-17A wildtype splenocytes to IL-17A wildtype mice did increase plasma and tissue IL-17A levels or alter renal or hepatic injury after liver IR (data not shown). Importantly, we were able to detect IL-17A protein expression plasma (Fig. 3A,B) and tissues (Fig. 3C) of IL-17A-deficient mice transfused with IL-17A wildtype splenocytes 24 hours after hepatic IR. We also detected IL-17A mRNA expression in the kidney, liver, and intestine of IL-17A-deficient mice transfused with IL-17A wildtype splenocytes (data not shown).
Representative H&E slides (magnification, 40×) of liver tissues from mice subjected to 60 minutes ischemia and 24 hours reperfusion or to sham-operation are shown in Fig. 4A. Sixty minutes of partial hepatic IR in wildtype mice produced large necrotic areas after reperfusion (average percent necrotic area = 92 ± 2%, n = 6). Livers were also analyzed for the degree of hepatocellular damage using the Suzuki et al.'s criteria.14 The ischemic lobes in the control group showed severe hepatocyte vacuolization, necrosis and sinusoidal congestion (Suzuki score = 9.4 ± 0.3, n = 6, Fig. 4B). Neutralization of IL-17A (200 μg antibody), deficiency in IL-17A receptor or IL-17A significantly reduced liver necrosis and lowered Suzuki liver injury scores (Fig. 4B). Moreover, transfusion of IL-17A wildtype splenocytes failed to exacerbate liver necrosis in IL-17A-deficient mice after liver IR.
Representative H&E slides from kidneys from mice subjected to 60 minutes liver ischemia and 24 hours reperfusion are shown in Fig. 5A (magnification 200×). Kidneys from the wildtype mice subjected to liver IR demonstrated multifocal acute tubular injury including S3 segment proximal tubule necrosis, cortical tubular simplification, cytoplasmic vacuolization, and dilated lumina as well as focal granular bile/heme casts (Fig. 5A). The summary of renal injury scores for percent renal tubular hypereosinophilia, percent peritubular leukocyte margination, and percent cortical vacuolization are shown in Fig. 5B. Neutralization of IL-17A (200 μg antibody), deficiency in IL-17A receptor or IL-17A significantly reduced kidney injury. Consistent with plasma creatinine, IL-17A-deficient mice transfused with IL-17A wildtype splenocytes were still protected against kidney injury after liver IR.
Hepatic IR injury also caused severe small intestinal injury (Fig. 6). Small intestine histology assessed 24 hours after 60 minutes hepatic IR in H&E-stained sections demonstrated villous endothelial cell apoptosis (Fig. 6B, magnified insert), villous epithelial cell necrosis, and the development of a necrotic epithelial pannus over the mucosal surface. Neutralization of IL-17A (200 μg antibody, Fig. 6C), deficiency in IL-17A (Fig. 6D) or IL-17A receptor (Fig. 6E) significantly reduced small intestine injury 24 hours after 60 minutes hepatic IR. In addition, infusion of wildtype splenocytes into IL-17A-deficient mice did not reverse the intestinal protection in these mice (Fig. 6F).
IL-17A Blockade or Deficiency Reduces Hepatic, Renal, and Intestine Inflammation and Apoptosis After Liver IR.
We assessed tissue inflammation by detecting neutrophil infiltration and by measuring proinflammatory mRNA up-regulation. Sixty minutes of hepatic ischemia resulted in significant recruitment of neutrophils into the liver, kidney, and intestine in IL-17A wildtype mice (Supporting Fig. 2A-C). Neutrophil infiltration coincided with areas of liver necrosis. Neutralization of IL-17A, deficiency in IL-17A receptor or IL-17A significantly reduced neutrophil infiltration in all three organs.
We also measured the expression of proinflammatory cytokine mRNAs in the liver, kidney, and intestine 24 hours after liver IR with semiquantitative RT-PCR. Hepatic IR significantly increased proinflammatory mRNA expression (ICAM-1, KC, MCP-1, and MIP-2) in all three organs compared to the sham-operated mice (Supporting Fig. 3A-C). However, neutralization of IL-17A, deficiency in IL-17A receptor or IL-17A significantly reduced proinflammatory mRNA expression in all three organs. We were able to detect IL-17A mRNA expression in all tissues (data not shown) of IL-17A-deficient mice transfused with IL-17A wildtype splenocytes. Furthermore, wildtype IL-17A splenocyte transfused IL-17A-deficient mice showed significantly attenuated proinflammatory mRNA expression in the liver, kidney, and small intestine (data not shown).
We used three separate indices to detect apoptosis: (1) TUNEL staining (Supporting Fig. 4), (2) DNA laddering (Supporting Fig. 5), and (3) counting the number of apoptotic hepatocytes in high-power field (400×) H&E images (Supporting Fig. 6). Hepatic IR caused massive hepatocyte apoptosis. Moreover, we determined that apoptotic hepatocytes can be detected in both necrotic and nonnecrotic areas after IR with significantly higher number of apoptotic cells in the necrotic zones of the liver. After hepatic IR, kidney and small intestine also showed severe capillary endothelial apoptosis (insert expanded in Supporting Fig. 4B,C). Neutralization of IL-17A, deficiency in IL-17A receptor, or IL-17A significantly reduced apoptosis in all three organs (Supporting Figs. 4–6).
Pharmacological Depletion of Paneth Cells Attenuates Hepatic, Renal, and Intestine Injury After Hepatic IR.
Zinc depletion with dithizone treatment selectively and rapidly (within 1 hour) results in the loss of Paneth cell secretory granules in mice.11, 12 Accordingly, we treated mice with dithizone to deplete Paneth cell granules to test the effect of this pharmacological ablation on the response to hepatic IR injury. Secretory granules are evident and abundant in ileal Paneth cells from vehicle (lithium carbonate)-treated mice (Fig. 7A, left panel, arrows). In contrast, dithizone administration to mice almost completely depleted ileal Paneth cells of their granules within 6 hours of dithizone exposure (Fig. 7A, right panel, asterisk).
We also stained small intestine crypts with lysozyme specific antibody as a marker of Paneth cell depletion after dithizone treatment. We demonstrate that Paneth cell granule depletion with dithizone treatment reduced lysozyme staining in small intestinal crypts after bilateral nephrectomy (Fig. 7B). Note that lysozyme staining was heavy in Paneth cells (arrows) of small intestinal crypts of mice treated with vehicle (Li2CO3). Paneth cell depletion with dithizone treatment eliminated lysozyme staining in Paneth cells (asterisk).
Treatment of Paneth cells with dithizone resulted in an approximately 64% reduction in plasma IL-17A levels 24 hours after liver IR (Fig. 7C). Furthermore, dithizone granule depletion drastically reduced IL-17A protein levels in the liver (76%), kidney (51%), and small intestine (67%) 24 hours after liver IR (Fig. 7C). Notably, Paneth cell depletion with dithizone caused the greatest reduction in IL-17A levels in isolated crypts after liver IR to near sham-operated values (Fig. 7C). Dithizone alone did not significantly affect IL-17A levels in sham-operated mice (data not shown).
Depletion of Paneth cell granules with dithizone improved liver and kidney function after 60 minutes of liver ischemia and 24 hours of reperfusion (Fig. 7C). We also determined that Paneth cell granule depletion with dithizone significantly attenuated renal, hepatic and intestinal apoptosis (Supporting Figs. 5-7) and neutrophil infiltration (Supporting Fig. 8) after liver IR. In small intestine, we show that apoptotic cells are localized primarily to the tops of the villi and that dithizone treatment reduced intestinal apoptosis. Dithizone treatment did not induce Paneth cell or intestinal crypt apoptosis (Supporting Fig. 7C).
Mice Genetically Deficient for Paneth Cells Are Protected Against Hepatic, Renal, and Intestine Injury After Hepatic IR.
To confirm that Paneth cell secretory products are required for hepatic, renal, and intestinal injury induced by liver IR, we investigated the responses in mice genetically deficient in the Paneth cell lineage. We first confirmed that intestine-specific SOX9-null (SOX9 flox/flox Villin Cre+/−) mice were deficient in Paneth cells by performing RT-PCR and immunoblotting for detection of the mouse Paneth cell α-defensin cryptdin-1, a Paneth cell-specific marker. Intestine-specific SOX9-null mice have significantly reduced cryptdin-1 mRNA and cryptdin-1 protein (Fig. 8A), and H&E staining confirmed absent Paneth cell secretory granules in these intestine-specific SOX9-null mice (Fig. 8B), confirming stable genetic ablation of the lineage.
Intestine specific SOX9-null mice subjected to liver IR had significantly reduced IL-17A protein levels in plasma (≈40%) and in the liver (≈34%), kidney (≈52%), and small intestine (≈33%) 24 hours after liver IR (Fig. 8C). However, we demonstrate that Paneth cell deficiency in intestine-specific SOX9-null mice reduced IL-17A protein levels in isolated crypts to near sham levels when compared to the wildtype mice after liver IR (Fig. 8C). Furthermore, Paneth cell-deficient intestine specific SOX9-null mice were protected against hepatic and renal injury after 24 hours after liver IR (Fig. 8D) as measured by reduced plasma ALT and creatinine.
We hypothesized that small intestinal Paneth cell-derived IL-17A plays a critical role in generating liver, kidney, and intestine injury after hepatic IR. Our results support this hypothesis, as (1) small intestinal Paneth cells degranulate and increase IL-17A production after liver IR; (2) plasma and tissue levels of IL-17A increase significantly with the highest IL-17A levels detected in portal vein plasma and in the small intestine; (3) depletion of IL-17A with neutralizing antibody or genetic deletion of either IL-17A or the IL-17A receptor protected against liver IR injury and extrahepatic organ dysfunction; (4) pharmacological (with dithizone treatment) or genetic depletion (with intestine specific SOX9 deletion) of Paneth cells attenuated hepatic, renal, and intestinal injury following hepatic IR; and (5) depletion of Paneth cell granules markedly decreased small intestinal IL-17A release and significantly attenuated plasma and tissue IL-17A levels after hepatic IR.
Hepatic IR injury is a common and unavoidable clinical complication in many major surgical procedures involving prolonged occlusion of the portal vein, inferior vena cava, or aorta. Furthermore, hepatic IR injury frequently leads to extrahepatic multiorgan dysfunction, making therapeutic interventions extremely difficult.10, 20 For example, patients subjected to hepatic IR frequently suffer from renal, respiratory, and intestinal failure which drastically increases mortality, morbidity, and prolongs intensive care unit care. Initiation of remote organ injury in patients after liver IR further exacerbates liver injury creating a vicious cascade of multiorgan derangements. Therefore, a better understanding of the mechanisms of hepatic IR injury and extrahepatic organ dysfunction would lead to improved therapy for patients subjected to unavoidable hepatic IR during the perioperative period. However, the detailed mechanisms involved in extrahepatic organ dysfunction due to hepatic IR are not fully elucidated. Studies to date implicate a complex orchestration of necrosis, apoptosis, and inflammation mediated by hepatic (hepatocytes, Kupffer cells) and extrahepatic (leukocytes, circulating cytokines) components.1, 21
We show that hepatic IR resulted in severe small intestinal injury as evidenced by villous endothelial apoptosis and villous epithelial necrosis (Fig. 6). Small intestine has been implicated as a source of systemic inflammation, bacterial translocation, and infection contributing significantly to multiorgan failure of critically ill patients.22, 23 Furthermore, small intestine has been implicated in generating hepatocellular dysfunction in trauma or hemorrhagic shock, as the injurious factors derived from the intestine attacks the liver first.22 Our results show that the concentration of IL-17A was highest in small intestine and in portal vein plasma (Fig. 3).
We propose that hepatic IR up-regulates small intestinal Paneth cell IL-17A production and Paneth cell-derived IL-17A plays an important role in propagating multiorgan injury after hepatic IR. We demonstrate rapid degranulation of small intestinal Paneth cells with induction of IL-17A after liver IR. Small intestinal Paneth cells are crucial for both mucosal as well as innate immunity against pathogens and can actively secrete several antimicrobial peptides (e.g., lysozyme, α-defensins/cryptdins) as well as proinflammatory molecules (e.g., inducible NO synthase, phospholipase A2, IL-17A).4, 12, 24-27 Therefore, although the Paneth cells (with the ability to kill bacteria and release proinflammatory mediators) are essential barriers providing mucosal and innate immunity,28, 29 their dysregulation and overproduction of IL-17A after hepatic IR may lead to a systemic inflammatory syndrome and exacerbation of hepatic, intestinal, and renal injury. It is likely that Paneth cell-derived IL-17A resulted in small intestinal inflammation and the influx of proinflammatory leukocytes with subsequent small intestinal tissue destruction and barrier disruption. Draining of proinflammatory mediators to the liver would then lead to exacerbation of hepatic IR injury.
Because freshly isolated individual crypts are free of leukocytes as well as cells of myeloid origin, we can rule out the contribution of leukocyte and myeloid source of increased IL-17A mRNA and protein after liver IR. However, because isolated crypts also contain stem cells and transit amplifying cells in addition to Paneth cells, we also performed LCM to specifically capture Paneth cells. We again confirmed increased expression of IL-17A mRNA in these Paneth cells captured by LCM (Fig. 2). Furthermore, we demonstrate in this study that IL-17A generated from leukocytes do not contribute to hepatic IR injury and AKI, as IL-17A-deficient mice transfused with wildtype splenocytes were still protected against liver and kidney injury. Collectively, these data suggest that Paneth cell-derived IL-17A is responsible for generating intestinal, renal, and hepatic injury after liver IR.
IL-17A is an important regulator of both innate and adaptive immunity and plays a critical role in host immune defense and inflammation.3, 4 IL-17A production was originally characterized from Th17 cells of the CD4+ T-cell subset distinct from Th1 or Th2 cells.5, 6, 30, 31 Subsequent studies showed that other cell types including CD3+ natural killer T cells, myeloid cells, neutrophils, as well as Paneth cells can produce IL-17A in response to various inflammatory and pathogenic stimuli.3, 4 Therefore, it is not surprising that IL-17A acts on various cell types, including neutrophils, endothelial cells, and renal proximal tubule epithelial cells, inducing the expression of proinflammatory mediators such as IL-8, IL-6, and CXC chemokines.32 Interestingly, the intestinal lamina propria was shown to be a unique site for detectable IL-17A levels in naive animals.8 Atarashi et al.33 confirmed these findings and demonstrated high amounts of IL-17A-producing Th17 cells in the intestinal lamina propria but not in the spleen, mesenteric lymph nodes, or Peyer's patches of a healthy mouse. Recently, Takahashi et al.4 showed that IL-17A produced by intestinal Paneth cells drive TNF-α-induced inflammation and shock. These previous and our current studies suggest that Paneth cell dysregulation and IL-17A release plays a major role in multiorgan dysfunction and inflammation.
Pharmacological or genetic Paneth cell granule depletion attenuated hepatic, intestinal, and renal injury and reduced tissue and plasma IL-17A levels after liver IR. We depleted Paneth cell granules with dithizone, a zinc chelator, as Paneth cell granule formation requires zinc.11, 12 Although our TUNEL data (Supporting Fig. 7C) demonstrate that dithizone did not induce small intestinal Paneth cell apoptosis, use of dithizone may be limited by systemic side effects (e.g., pulmonary toxicity) at high doses and Paneth cell depletion is transient (with complete repopulation of Paneth cells at 12-24 hours after injection). Therefore, we complemented the dithizone studies with studies in intestine-specific SOX9-null mice. Wnt, the Wnt Frizzled-5 receptor, Math1, Gfi1, and SOX9 are required for the development of Paneth cells.9, 34 SOX9/Villin Cre+/− mice lack SOX9 transcription factor in intestinal epithelia and as a result show absent or significantly reduced numbers of mature Paneth cells in adult mice.9 These two approaches of Paneth cell depletion allowed us to conclude that Paneth cells are critical in generating extrahepatic intestinal and renal injury after liver IR. However, Paneth cell-depleted mice still had a significant degree of hepatocyte necrosis (and elevated plasma ALT) due to prolonged liver ischemia (Figs. 7D, 8D). These findings suggest that both Paneth cell independent (hepatocyte necrosis) and Paneth cell-dependent extrahepatic injury contribute to hepatic IR injury in vivo.
With pharmacological or genetic Paneth cell granule depletion, we observed a striking reduction in IL-17A up-regulation in isolated crypts with profound hepatic, intestinal, and renal protection after liver IR. Although significantly attenuated, plasma and tissue IL-17A levels in Paneth cell-depleted mice (Figs. 7, 8) subjected to liver IR were still elevated compared to sham-operated mice. It is likely that several cell types including leukocytes and epithelial cells can generate IL-17A in response to liver IR and oxidant stress during reperfusion.3, 6
The mechanisms leading to Paneth cell degranulation and increased Paneth cell-derived IL-17A after hepatic IR remain to be determined. Our model of hepatic IR with partial portal vein and artery occlusion avoids total intestinal outflow obstruction. However, intestinal venous congestion with resultant partial intestinal ischemia may occur, as 100% of intestinal blood flow is diverted to ≈30% of hepatic mass. Partial intestinal IR may have contributed to Paneth cell degranulation and dysregulation. In addition, hepatic IR releases endogenous damage-associated molecular pattern molecules (DAMPs including endotoxin, HMGB-1, mitochondrial DNA, urinic acid) that can activate several Toll-like receptors (TLRs).35 TLR-mediated Paneth cell degranulation has been described.36, 37
In summary, we show that neutralization or genetic deletion of IL-17A provides powerful multiorgan protection after liver IR. In addition, we demonstrated that small intestinal Paneth cells degranulate to play a critical role in hepatic, intestinal, and renal injury after liver IR. In addition, Paneth cells are a major initial source of IL-17A production after hepatic IR. We propose that the small intestinal Paneth cell generation of IL-17A leads to hepatic injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have important therapeutic implications in reducing systemic complications arising from hepatic IR injury.
We thank Dr. Andre J. Ouellette (Keck School of Medicine of the University of Southern California, Los Angeles, CA) for providing mouse alpha-defensin antibody and Dr. Yuko Mori-Akiyama (Baylor College of Medicine, Houston, TX) for sending breeder pairs of intestine-specific SOX9 null mice.