Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9

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ORIGINAL RESEARCH Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9 Q10

Godhev Kumar Manakkat Vijay,1,2 Changyun Hu,2 Jian Peng,2 Irma Garcia Martinez,3 Rafaz Hoque,3 Rejina Mariam Verghis,4 Yun Ma,1 Wajahat Mehal,3 Debbie Lindsay Shawcross,1,* and Li Wen2,* 1

Liver Sciences Department, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom; Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; 3 Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; and 4Welcome Wolfson Institute of Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queens University, Belfast, United Kingdom 2

Wild type (WT)

NH4-Ac stimulation (4mM)

DNA release

DNA activates TLR9 in neutrophils and macrophages

Tlr9-/- and LysmCre Tlr9fl/fl

DNA release

Neutrophils

Macrophages

Production of pro-inflammatory Cytokine IFN-γ, TNFand IL-6 in macrophages and T cells

Increase in brain water

ODN2088 disrupts colocalization of DNA with TLR9 in innate immune cells abrogating cytokine production No increase in brain water

WT + TLR9 antagonist (ODN2088)

SUMMARY

and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50mg/mouse) was evaluated.

Utilizing a TLR9-deficient mouse model and the TLR9 antagonist ODN2088, we have shown that ammonia-induced brain edema requires macrophage and T cell expression of TLR9 supporting exploration of TLR9 antagonism in acute liver failure.

RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (p<0.0001) in WT but not Tlr9-/- mice (p<0.001). ODN2088 inhibited macrophage and T cell cytokine production (p<0.05) and prevented an increase in BW (p<0.0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (p<0.05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (p<0.05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells.

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4þ and CD8þ T cells. Brain water (BW)

CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH. (Cell Mol Gastroenterol Hepatol 2019;-:-–-; https://doi.org/10.1016/j.jcmgh.2019.08.002) Keywords: Ammonia; Brain edema; Immune dysfunction; Tolllike receptor 9.

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mmonia plays a pivotal role in the development of hepatic encephalopathy and brain edema in acute liver failure (ALF).1–3 A robust systemic inflammatory response and susceptibility to developing infection are common in ALF exacerbate the development of ammoniainduced brain edema and are major prognosticators.4–10 Experimental models have unequivocally associated ammonia exposure with astrocyte swelling and brain edema, potentiated by proinflammatory cytokines.11–14 Toll-like receptor 9 (TLR9) is an innate pattern recognition receptor that binds to the CpG motif of bacterial and mammalian DNA.15 TLR9 plays an important role in acetaminophen-induced liver inflammation,16 and we recently reported that ammonia along with DNA induces neutrophil TLR9 expression in patients with acetaminophen-induced ALF and advanced hepatic encephalopathy.17 Although the evidence base supporting the relationship between ammonia, inflammation and brain edema is robust in ALF, there is a paucity of data characterizing the specific pathogenic mechanisms entailed. We hypothesized that ammonia-induced brain edema and immune dysfunction are mediated by TLR9. As TLR9 is necessary for the development of acetaminophen-induced acute liver injury in murine models,16 the hypothesis could only be tested in a murine model exposed to ammonium acetate (NH4-Ac) without liver injury. Using an acute hyperammonemic mouse model, we demonstrated that ammonia-induced brain edema and immune dysfunction, as measured by increased brain water (BW) content and intracellular cytokine production of macrophages and T cells are mediated through TLR9. In mice, hyperammonemia resulted in DNA release and activation of TLR9 inducing downstream inflammatory cytokine production. TLR9 in lysozyme expressing cells was critical for the development of brain edema and immune dysfunction. Administration of a TLR9 antagonist abrogated inflammation and prevented brain edema.

Results Ammonia-Induced Brain Edema and Changes in the Liver Were Dependent on TLR9 To determine whether TLR9 plays a role in ammoniainduced brain edema, we evaluated the BW content of wild-type (WT) and Tlr9–/– mice 6 hours after a single dose of NH4-Ac (4 mM) injection (intraperitoneal). Following NH4-Ac stimulation, there was a significant increase in the BW content in WT mice compared with control mice; which was significantly decreased in Tlr9–/– mice compared with WT mice (Figure 1A) indicating that TLR9 plays an important role in the development of brain edema. To determine whether the increased BW content was associated with any changes in the liver, we assessed the liver-to-body weight ratio and liver histopathology after NH4-Ac stimulation. In WT mice, there was a significant increase in liver-to-body weight ratio with evidence of hepatocyte swelling but not necrosis (there was no rise in serum aspartate and alanine transaminases), a finding which was abrogated in Tlr9–/– mice (Figure 1B and C). We hypothesized that DNA released

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following NH4-Ac stimulation can bind to TLR9 resulting in activation of the innate immune system. To test our hypothesis, we measured plasma DNA. Total plasma dsDNA levels were significantly increased in WT mice and Tlr9–/– mice following NH4-Ac stimulation compared with control mice, but there was no difference in Tlr9–/– mice compared with WT mice following NH4-Ac stimulation (Figure 1D).

Ammonia Altered the Function of Macrophages and T Cells in a TLR9-Dependent Manner To determine whether systemic inflammation contributed to the ammonia-induced increase in BW content through a TLR9-mediated pathway, we measured intracellular cytokine (interferon gamma [IFNg], tumor necrosis factor alpha [TNFa], and interleukin [IL]-6) production in T cells and macrophages isolated from spleen and liver in WT mice and Tlr9–/– mice following NH4-Ac stimulation. There was a significant increase in the intracellular cytokines produced by macrophages (Figure 2A–D) and CD4þ and CD8þ T cells (Figure 3A–F) isolated from the spleen in WT mice following NH4-Ac stimulation, compared with control mice which was abrogated in Tlr9–/– mice. A similar trend was observed in the immune T cells isolated from the liver (Figure 4). Neutrophil phagocytic activity was unaltered following NH4-Ac stimulation (Figure 5).

Ammonia-Induced Brain Edema and Inflammation Were Independent of Acetate or pH Acetate has been reported to influence inflammation in acute alcoholic hepatitis18 and the increased toxicity of ammonium salts promotes ammonium gas transfer across the blood brain barrier due to the rise in blood pH and the direct effect of alkalinization.19 Therefore, to confirm whether the TLR9-mediated brain edema and inflammation were solely induced by ammonia and not by the acetate or changes in pH, an alternate salt of acetate, sodium acetate (NaCH3CO2) (Na-Ac) was injected in WT mice after adjusting for pH (same as NH4-Ac). Na-Ac (4 mM) did not alter the BW content, liver-to-body weight ratio, or intracellular cytokine production of various immune cell subsets in WT mice compared with control mice, unlike NH4-Ac (Figure 6A–F). These results confirm that the observed ammonia-induced cytokine production and brain edema were induced by ammonia per se, and not influenced by acetate or pH changes.

*Authors share co-senior authorship. Abbreviations used in this paper: ALF, acute liver failure; BW, brain water; IFNg, interferon gamma; IL, interleukin; mAbs, monocloncal antibodies; Na-Ac, sodium acetate; NH4-Ac, ammonium acetate; TNFa, tumor necrosis factor alpha; TLR9, Toll-like receptor 9; WT, wild-type.

© 2019 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2352-345X https://doi.org/10.1016/j.jcmgh.2019.08.002

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Figure 1. TLR9 stimulates ammonia-induced brain edema and liver-to-body weight ratio through DNA. (A) Following NH4-Ac stimulation, there was a significant increase in the BW content in WT mice (n ¼ 9) compared with control mice (n ¼ 12) (P < .0001) (mean difference: 1.35; 95% confidence interval [CI], 0.9 to 1.8), which was ameliorated in Tlr9–/– mice (n ¼ 10) (P ¼ .006) (mean difference: –0.76; 95% CI, –1.3 to –0.25). (B) Following NH4-Ac stimulation, there was a significant increase in the liver-to-body weight ratio in WT mice (n ¼ 16) compared with control mice (n ¼ 13) (P < .0001) (mean difference: 0.019; 95% CI, 0.013 to 0.024), which was ameliorated in Tlr9–/– mice (n ¼ 11) (P < .0001) (mean difference: –0.014; 95% CI, –0.019 to –0.009). (C) Following NH4-Ac stimulation, there was a significant increase in the hepatocyte swelling in the liver histology (hematoxylin and eosin stain, 200 magnification) in WT mice compared with control mice, where the cytoplasm of cells remained intact. Following NH4-Ac stimulation, there was no difference in the hepatocyte morphology in Tlr9–/– mice compared with control mice but the hepatocyte swelling was reduced compared with WT mice. The images in the black box inset are the representative images at 400 magnification. (D) Following NH4-Ac stimulation, there was a significant increase in the total plasma DNA level in WT mice (n ¼ 12) compared with control mice (n ¼ 12) (P ¼ .007) (mean difference: 2.97; 95% CI, 0.88 to 5) and in Tlr9–/– mice (n ¼ 17) compared with control mice (n ¼ 14) (P ¼ .004) (mean difference: 1.76; 95% CI, 0.6 to 2.9).

Ammonia-Induced Brain Edema and Inflammation Were Mediated by TLR9 Expressed Within Lysozyme-Expressing Cells Our results showed that deletion of TLR9 tempered the proinflammatory state and abrogated the development of brain edema following ammonia stimulation. To examine the role of TLR9 in macrophages, we used Lysm-Cre Tlr9fl/fl mice that have TLR9 specifically deleted in lysozymeexpressing cells, namely macrophages and neutrophils.20 NH4-Ac (4 mM) was therefore injected into Lysm-Cre Tlr9fl/fl mice. Interestingly, we found that BW content, liverto-body weight ratio, hepatocyte swelling, and cytokines produced by macrophages were significantly ameliorated in Lysm-Cre Tlr9fl/fl mice compared with WT mice following NH4-Ac stimulation but with no difference compared with Tlr9–/– mice (Figure 7A–G). There was no difference in the total plasma dsDNA levels in Tlr9–/– mice and Lysm-Cre Tlr9fl/fl mice compared with WT mice following NH4-Ac stimulation (Figure 7H). We also tested another control mouse strain, Tlr9fl/fl mice (TLR9 is sufficient in all the cell types). As expected, Tlr9fl/fl mice were not protected from NH4-Ac induced inflammation compared with Lysm-Cre Tlr9fl/fl mice (Figure 8A–E).

TLR9 Antagonism Abrogates Ammonia-Induced Brain Edema and Inflammation

As Tlr9–/– mice were protected against NH4-Ac stimulation, we tested whether an antagonist of TLR9 (ODN2088) could inhibit the ammonia-induced proinflammatory changes and brain edema observed in the WT mice. Administration of ODN2088 (50 mg/mouse) with NH4-Ac (4 mM) significantly decreased the BW content and liver-tobody weight ratio and ameliorated the hepatocyte swelling (Figure 9A–C). Administration of ODN2088 did not alter the total plasma DNA levels in WT mice following NH4-Ac stimulation but they were increased compared with control mice (Figure 9D). There was also a significant reduction in the cytokines produced by macrophages (Figures 9E–G) and T cells (Figure 10A–F).

Discussion In this study, we have demonstrated a novel link between ammonia-induced inflammation and the subsequent development of brain edema mediated by TLR9 using different mouse models. In addition, we showed that TLR9 expressed by lysozyme-producing cells in mice was critical

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Figure 2. Ammonia-induced intracellular cytokine production by macrophages is mediated by TLR9. (A) Following NH4Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by splenic macrophages in WT mice (n ¼ 8) compared with control mice (n ¼ 7) (P < .0001) (mean difference: 2.3; 95% CI, 1.5 to 3), which was ameliorated in Tlr9–/– mice (n ¼ 8) (P ¼ .0006) (mean difference: –1.6; 95% CI, –2.4 to –0.83). (B) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by splenic macrophages in WT mice (n ¼ 8) compared with control mice (n ¼ 7) (P < .0001) (mean difference: 25; 95% CI, 22 to 28.5), which was ameliorated in Tlr9–/– mice (n ¼ 8) (P < .0001) (mean difference: –14; 95% CI, –17.2 to –10.8). (C) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IL-6 produced by splenic macrophages in WT mice (n ¼ 8) compared with control mice (n ¼ 7) (P < .0001) (mean difference: 4; 95% CI, 3.5 to 4.5), which was ameliorated in Tlr9–/– mice (n ¼ 8) (P < .0001) (mean difference: –4; 95% CI, –4.7 to –3.5). (D) Representative FACS plots of intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic macrophages in WT mice and Tlr9–/– control mice, and following NH4-Ac stimulation.

for ammonia-induced brain edema and inflammation to develop. Importantly, we also showed that administration of a TLR9 antagonist abrogated the inflammation and prevented brain edema supporting therapeutic exploration of TLR9 antagonism in early ALF. Ammonia stimulation upregulated the production of the cytokines IFNg, TNFa, and IL-6 by macrophages and T cells. The observation of increased cytokine production from the hepaticinfiltrated T cells indicates that the immune response originates from the liver. The cytokines IFNg, TNFa, and IL-6 produced by T cells are predominantly responsible for mediating inflammation within the liver.21–23 In this study ammonia activated cytokine production in macrophages indicated that those cells, along with CD4þ and CD8þ T cells, are responsible for promoting the proinflammatory environment. It is possible that ammonia and systemic inflammation induce brain edema by inducing astrocyte swelling. This is supported by the previously published observation that astrocyte swelling can be induced when astrocytes are exposed to proinflammatory cytokines after being exposed to ammonia.11 Though deficiency of TLR9

exacerbates cognitive impairment and severity of seizures in the brain,24 it has been demonstrated that activation of TLR9 in microglia and astrocytes induces the production of various proinflammatory cytokines in response to CpG DNA25 and activation of TLR9 in astrocytes leads to synaptic protein loss in the brain in chronic hyperglycemia.26 Activation of Tlr9 results in the upregulation of cytokine production and increase in the BW content, and the critical role of TLR9 in this mechanism has been demonstrated by using genetically modified mice. TLR9 is essential for macrophage production of TNFa and IL-6 in response to CpG DNA and IFNg produced by CD4þ T cells in response to CpG DNA and mycobacteria.15,27 The observation that ammonia-induced TLR9 activation induces systemic inflammation and brain edema is well supported by recently published studies that show TLR9 induces inflammation in acetaminophen-induced ALF and nonalcoholic steatohepatitis.16,20 Ammonia stimulation increased the total plasma DNA levels in all the different mouse models, thereby suggesting that ammonia activates TLR9 through DNA release. In

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Figure 3. Ammonia-induced intracellular cytokine production by T cells is mediated by TLR9. (A) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by splenic CD4þ T cells in WT mice compared with control mice (P < .0001) (median difference: 2.7; 95% CI, 1.5 to 3), which was ameliorated in Tlr9–/– mice compared with WT mice (P ¼ .0007) (median difference: –1.9; 95% CI, –2.8 to –0.8). (B) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by splenic CD4þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 49.5; 95% CI, 47 to 52), which was ameliorated in Tlr9–/– mice compared with WT mice (P < .0001) (mean difference: –27.5; 95% CI, –30 to –25). (C) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IL-6 produced by splenic CD4þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 2.2; 95% CI, 1.4 to 3), which was ameliorated in Tlr9–/– mice compared with WT mice (P ¼ .0052) (mean difference: –1.6; 95% CI, –2.7 to –0.5). (D) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by splenic CD8þ T cells in WT mice compared with control mice (P ¼ .0002) (mean difference: 3.9; 95% CI, 2.1 to 5.8), which was ameliorated in Tlr9–/– mice compared with WT mice (P ¼ .0003) (median difference: –3.5; 95% CI, –6.4 to –2.1). (E) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by splenic CD8þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 49; 95% CI, 46 to 52), which was ameliorated in Tlr9–/– mice compared with WT mice (P < .0001) (mean difference: –48; 95% CI, –51 to –45). (F) Representative FACS plots of the intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic T cells in WT mice and Tlr9–/– control mice and following NH4-Ac stimulation. WT control mice (n ¼ 11) and NH4-Ac-treated mice (n ¼ 13); Tlr9–/– control mice (n ¼ 10) and NH4-Ac-treated mice (n ¼ 10).

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Figure 4. Ammonia-induced intracellular cytokine production by liver-infiltrated T cells. (A) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by liver-infiltrated CD4þ T cells in WT mice compared with control mice (P < .0001) (median difference: 18.3; 95% CI, 15 to 21.6), which was ameliorated in Tlr9–/– mice compared with WT mice (P < .0001) (median difference: –14.5; 95% CI, –18.6 to –10.3). (B) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by liver-infiltrated CD4þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 21.4; 95% CI, 14.5 to 28.2), which was ameliorated in Tlr9–/– mice compared with WT mice (P < .0001) (mean difference: –20; 95% CI, –27 to –12.7). (C) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IL-6 produced by liver-infiltrated CD4þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 11.9; 95% CI, 8.1 to 15.6), which was ameliorated in Tlr9–/– mice compared with WT mice (P ¼ .0082) (mean difference: –7.3; 95% CI, –12.4 to –2.2). (D) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by liver-infiltrated CD8þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 18.3; 95% CI, 15.8 to 20.9), which was ameliorated in Tlr9–/– mice compared with WT mice (P < .0001) (median difference: –12.4; 95% CI, –16.7 to –8.2). (E) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by liver-infiltrated CD8þ T cells in WT mice compared with control mice (P < .0001) (mean difference: 47.3; 95% CI, 39 to 56), which was ameliorated in Tlr9–/– mice compared with WT- mice (P < .0001) (mean difference: –44; 95% CI, –56 to –32). WT control mice (n ¼ 10) and NH4-Ac-treated mice (n ¼ 10); Tlr9–/– control mice (n ¼ 4) and NH4-Ac-treated mice (n ¼ 7).

acetaminophen-induced ALF, DNA fragments released by apoptotic hepatocytes have been shown to be responsible for the activation of TLR9 and induction of systemic inflammation.16,28 These findings support the results of our recent human study, which showed profound TLR9

activation in neutrophils in the presence of plasma DNA in patients with acetaminophen-induced ALF, systemic inflammation, and brain edema,17 suggesting that TLR9 mediates the ammonia-induced brain edema in a DNAdriven manner. In this study, it is not possible to identify

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Figure 5. Neutrophil phagocytosis measured by dextran fluorescein isothiocyanate (FITC) using flow cytometry in WT and Tlr9–/– mice after NH4-Ac stimulation. Following NH4Ac stimulation, there was no difference in the mean fluorescence intensity of dextran FITC in neutrophils isolated from whole blood in WT mice or Tlr9–/– mice compared with control mice.

the source of the DNA, and we can only speculate that nonimmune cells are the source of DNA. TLR9 expressed in the lysozyme-producing cells played an important role in the ammonia-induced cytokine production of macrophages and brain edema. Marques et al29 recently demonstrated that neutrophils are the predominant innate immune cells that sense DNA through the TLR9/ nuclear factor-kappa B pathway and induce inflammation in the context of acetaminophen toxicity. These data are also well supported by the strong correlation demonstrated between neutrophil TLR9 expression, ammonia and IL-8 in acetaminophen-induced ALF and the abrogation of neutrophil TLR9 upregulation and cytokine production in acetaminophen-induced ALF plasma by DNAse-I.17 Administration of the TLR9 antagonist, ODN2088 abrogated the cytokine production and prevented the increase in BW content induced by stimulation of ammonia. These data are well supported by the findings of Imaeda et al,16 who in an acetaminophen-induced hepatotoxicity model, established that inhibition of TLR9 using ODN2088 and IRS954, a TLR7/9 antagonist, downregulated proinflammatory cytokine release and reduced mortality. The amelioration of brain edema and cytokine production by ODN2088 supports exploration of TLR9 antagonism as a therapeutic modality in early ALF to prevent the development of brain edema and intracranial hypertension. There are pros and cons of using an ammonia-induced murine model of cerebral edema without acute liver injury. Some of the best-characterized animal models of cerebral edema in ALF include the hepatic devascularized rat30 and pig,31 and the rat with thioacetamide-induced toxic liver injury.30 While these models develop cerebral edema and exhibit intracranial hypertension, the impact of hepatic devascularization and necrosis bring other sequelae, including systemic inflammation, bacterial and fungal sepsis, and coagulopathy. This makes it hard to tease out the specific mechanisms that underpin the development of ammoniainduced brain edema per se and how this relates to innate immune dysfunction. Furthermore, we know that murine acetaminophen-induced ALF is mediated by TLR9 and can be abrogated by a TLR9 antagonist.16 Therefore, we could not utilize an acetaminophen-induced model of ALF to assess TLR9-

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766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 Materials and Methods 783 Animals Used for This Study 784 All the procedures and protocols used in the studies with 785 animals were approved by the Institutional Animal Care and 786 Use Committee at Yale University. Four strains of mice on 787 C57BL/6 genetic background were used for the experi788 ments. C57BL/6 (WT) were originally obtained from the 789 –/– Jackson Laboratory (Bar Harbor, ME). The original Tlr9 790 fl/fl and Tlr9 breeders were kindly provided by Professor 791 15 and Professor Mark Shlomchik Shizuo Akira (Japan) 792 20 (University of Pittsburgh), respectively. We bred Lysm-Cre 793 fl/fl fl/fl mice to generate Lysm-Cre Tlr9 mice mice with Tlr9 794 with TLR9 specific deletion in lysozyme expressing cells.20 795 All the mice used in the study were bred and kept at the 796 Yale animal facility in specific pathogen–free conditions 797 with autoclaved food, bedding, and filtered cage. The mice 798 were fed on a regular chow diet and on a 12-hour light/dark 799 cycle. Male mice (7–9 weeks old) were used in the study. 800 801 Stimulation With Ammonium Acetate 802 To determine whether TLR9 plays a role in brain edema 803 in the mice, NH4-Ac (NH4CH3CO2) (4 mmol/kg of body- 804 weight) was injected intraperitoneally in WT mice, Tlr9–/– 805 mice, Lysm-Cre Tlr9fl/fl mice, and Tlr9fl/fl mice that were 806 sacrificed 6 hours after the injection. The concentration and 807 duration of NH4-Ac used for this experiment was optimized 808 in a pilot experiment. Na-Ac (NaCH3CO2) (J.T. Baker) Q7809 (4 mmol/kg of bodyweight) was injected intraperitoneally 810 in WT mice to demonstrate that any effect was due to 811 ammonia and not the acetate moiety. 812 813 Blood Collection and Tissue Harvesting 814 Six hours after the NH4-Ac stimulation, blood was 815 collected from the mice and liver, spleen and brain were 816 harvested. Plasma was collected and stored in –80 C. Spleen 817 was homogenized using rough sides of 2 grinding slides and 818 red blood cells were lysed by hypotonic solution and quickly 819 restored in isotonic phosphate-buffered saline. The single 820 suspension of splenocytes (106) was stained with mono- 821 cloncal antibodies (mAbs) conjugated with different fluo- 822 rochromes before flow cytometry analysis. Liver tissue was 823 homogenized using a plunge through a wire mesh and 824 mediated cerebral edema in this study and thus chose to examine this in a model of ammonia-induced cerebral edema. In summary, this study demonstrates that the development of ammonia-induced brain edema requires macrophage and T cell expression of TLR9, which may be stimulated by DNA release. The importance of TLR9-expressing neutrophils or Kupffer cells in the induction of brain edema and inflammation was confirmed by the deletion of the TLR9 gene only in lysozyme-expressing cells. We have therefore demonstrated that TLR9 is necessary for the development of ammoniainduced brain edema.16,29 The observation that systemic inflammation and brain edema can be prevented following administration of a TLR9 antagonist supports the exploration of TLR9 antagonism as a therapeutic modality in early ALF to prevent the progression to intracranial hypertension.

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Figure 6. Unaltered brain edema and intracellular cytokine production by macrophages after Na-Ac stimulation. (A) Following Na-Ac stimulation, BW content remained unaltered in WT mice (n ¼ 7) compared with control mice (n ¼ 12), but was significantly reduced compared with the NH4-Ac–stimulated WT mice (n ¼ 9) (P < .001) (mean difference: –1.1; 95% CI, –1.7 to –0.47). (B) Following Na-Ac stimulation, liver-to-body weight ratio remained unaltered in WT mice (n ¼ 7) compared with control mice (n ¼ 13), but was significantly reduced compared with the NH4-Ac–stimulated WT mice (n ¼ 16) (P < .001) (mean difference: –0.02; 95% CI, –0.028 to –0.012). Following Na-Ac stimulation, the intracellular cytokine (C) IFNg (P < .0001) (mean difference: –2.2; 95% CI, –3 to –1.4), (D) TNFa (P < .0001) (mean difference: –22.3; 95% CI, –28.3 to –16.3), and (E) IL-6 (P < .0001) (mean difference: –4.5; 95% CI, –5.2 to –3.9) produced by splenic macrophages were significantly reduced in WT mice (n ¼ 7) compared with the NH4-Ac–stimulated WT mice (n ¼ 8) but remained unaltered compared with control mice (n ¼ 7). (F) Representative FACS plots of the intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic macrophages in WT control mice, NH4-Ac–stimulated WT mice and Na-Ac–stimulated WT mice.

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digested using collagenase-I and DNAse-I (Sigma-Aldrich, St Louis, MO). Liver-infiltrated immune cells were isolated from the homogenized tissue using the density-gradient Polymorphprep solution (Axis Shield, Oslo, Norway), stained with mAbs and analyzed using flow cytometry. The entire brain was weighed immediately after sacrifice using an electronic balance to determine the wet weight. The brain was then dried in an oven at 100 C for 24 hours to obtain the dry weight. The BW content was then calculated according to the formula32: BW content ð%Þ ¼

Wet weight  Dry weight  100 Wet weight

Preservation of Tissues for Histopathological Examination For histopathological examination, tissues were harvested and stored in 10% formalin at room temperature. Specimens were then embedded in paraffin. Then, 6-mM tissue sections were stained with hematoxylin and eosin and examined under a light microscope.

Total DNA Estimation Total DNA was measured in the plasma samples stored at –80 C using the Quant-iT PicoGreen dsDNA quantitation kit (Life Technologies) as detailed previously.20

TLR9 Antagonist Injection To determine whether an inhibitor of TLR9 offers protection against NH4-Ac stimulation, the TLR9 antagonist (ODN2088) (InvivoGen, San Diego, CA) (50 mg/mouse) was injected intraperitoneally in WT mice immediately following NH4-Ac injection. Six hours later, blood was collected and organs were harvested as mentioned above. The time and concentration of ODN2088 were chosen based on a recently established mouse model of acetaminophen hepatotoxicity.16

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Stimulation of Intracellular Cytokine Production To determine the intracellular cytokine production of mononuclear cells from spleen and liver, up to 5.0  106 cells per mL were stimulated with phorbol 12-myristate 13acetate (50 ng) and ionomycin (InvivoGen) (500 pg) in complete media with Golgi plug (BD Biosciences, San Jose, CA) (1 mL) and incubated at 37 C for 5 hours in the presence of 5% CO2. At the end of 5 hours, stimulated cells were washed, stained with fluorochrome conjugated mAbs and analyzed using flow cytometry.

Cell Staining and Flow Cytometry Up to 1 million (1  106) cells were resuspended in 100 mL of staining buffer and stained with different fluorochrome conjugated mAbs in a tube followed by incubation at room temperature in darkness for 30 minutes and the stained cells were washed with phosphate-buffered saline. For intracellular cytokine staining, 100 mL of cytofix/cytoperm solution (BD Biosciences) was added to the cell pellet after staining with surface markers and kept at room temperature for 20 minutes. The cells were washed with 1 mL of permeabilization wash buffer (BD Biosciences) followed by resuspension in 300 mL of phosphate-buffered saline and acquired in a LSRII flow cytometry (BD Biosciences) using BD FACS DIVA software V6.0 (BD Biosciences).

Neutrophil Phagocytosis The phagocytic ability of neutrophils was determined by incubating the whole blood with Dextran FITC (1 mg/mL) at 37 C for 20 minutes in a water bath and measuring the mean fluorescence intensity of the neutrophils in a flow cytometer. Neutrophils were identified using Lys6-G (1A8) and CD11b (M1/70) antibodies.

Identification and Characterization of Various Immune Cell Subsets The different subsets of lymphocytes were identified using their specific markers. T cells were identified using CD3 (17A2); CD4 (GK1.5) and CD8 (53-6.7) markers were

Figure 7. (See previous page). TLR9 in lysosomal expressed cells stimulates ammonia-induced brain edema and cytokine production by macrophages. (A) Following NH4-Ac stimulation, BW content was significantly ameliorated in Tlr9–/– mice (n ¼ 10) (P < .05) (mean difference: –0.76; 95% CI, –1.5 to –0.06) and Lysm-Cre Tlr9fl/fl mice (n ¼ 9) (P < .001) (mean difference: –1.4; 95% CI, –2 to –0.7) compared with WT mice (n ¼ 9). (B) Following NH4-Ac stimulation, liver-to-body weight ratio was significantly ameliorated in Tlr9–/– mice (n ¼ 11) (P < .0001) (mean difference: –0.015; 95% CI, –0.021 to –0.008) and Lysm-Cre Tlr9fl/fl mice (n ¼ 10) (P < .0001) (mean difference: –0.019; 95% CI, –0.026 to –0.013) compared with WT mice (n ¼ 16). (C) Following NH4-Ac stimulation, there was a significant increase in the hepatocyte swelling in the liver histology (hematoxylin and eosin stain, 200 magnification) in WT mice compared with Tlr9–/– mice and Lysm-Cre Tlr9fl/fl mice, where the cytoplasm of cells remained intact. The images in the black box inset are the representative images at 400 magnification. (D) Following NH4-Ac stimulation, the intracellular cytokine IFNg produced by splenic macrophages was significantly ameliorated in Tlr9–/– mice (n ¼ 8) (P < .0001) (mean difference: –1.6; 95% CI, –2.4 to –0.84) and Lysm-Cre Tlr9fl/fl mice (n ¼ 9) (P < .01) (mean difference: –1; 95% CI, –1.75 to –0.25) compared with WT mice (n ¼ 8). (E) Following NH4-Ac stimulation, the intracellular cytokine TNFa produced by splenic macrophages was significantly ameliorated in Tlr9–/– mice (n ¼ 8) (P < .0001) (mean difference: –14; 95% CI, –17.4 to –10.6) and Lysm-Cre Tlr9fl/fl mice (n ¼ 9) (P < .0001) (mean difference: –11.7; 95% CI, –15 to –8.4) compared with WT mice (n ¼ 8). (F) Following NH4-Ac stimulation, the intracellular cytokine IL-6 produced by splenic macrophages was significantly ameliorated in Tlr9–/– mice (n ¼ 8) (P < .0001) (mean difference: –4.1; 95% CI, –4.7 to –3.4) and Lysm-Cre Tlr9fl/fl mice (n ¼ 9) (P < .0001) (mean difference: –3.6; 95% CI, –4.2 to –3) compared with WT mice (n ¼ 8). (G) Representative FACS plots of the intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic macrophages in WT mice, Tlr9–/– mice and Lysm-Cre Tlr9fl/fl mice following NH4-Ac stimulation. (H) There was no difference in the total plasma DNA level between WT mice (n ¼ 12), Tlr9–/– mice (n ¼ 17) and Lysm-Cre Tlr9fl/fl mice (n ¼ 12).

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Figure 8. Ammonia-induced brain edema and cytokine production in Lysm-Cre Tlr9fl/fl mice compared with Tlr9fl/fl mice. (A) Following NH4-Ac stimulation, there was a significant increase in the BW content in Tlr9fl/fl mice (n ¼ 11) compared with control mice (n ¼ 8) (P ¼ .0009) (mean difference: 1.05; 95% CI, 0.5 to 1.6), which was ameliorated in Lysm-Cre Tlr9fl/fl mice (n ¼ 9) (P ¼ .014) (mean difference: –0.7; 95% CI, –1.2 to –0.16). (B) Following NH4-Ac stimulation, there was a significant increase in the liver-to-body weight ratio in Tlr9fl/fl mice (n ¼ 12) compared with control mice (n ¼ 8) (P ¼ .002) (mean difference: 0.007; 95% CI, 0.003 to 0.015), which was ameliorated in Lysm-Cre Tlr9fl/fl mice (n ¼ 10) (P ¼ .007) (mean difference: –0.008; 95% CI, –0.017 to –0.002). (C) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IFNg produced by splenic macrophages in Tlr9fl/fl mice (n ¼ 10) compared with control mice (n ¼ 8) (P < .0001) (mean difference: 3.5; 95% CI, 2.5 to 4.5), which was ameliorated in Lysm-Cre Tlr9fl/fl mice (n ¼ 8) (P < .0001) (mean difference: –3.6; 95% CI, –4.6 to –2.7). (D) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine TNFa produced by splenic macrophages in Tlr9fl/fl mice (n ¼ 10) compared with control mice (n ¼ 8) (P < .0001) (mean difference: 14.3; 95% CI, 10.2 to 18.3), which was ameliorated in Lysm-Cre Tlr9fl/fl mice (n ¼ 8) (P < .0001) (mean difference: –13; 95% CI, –16.3 to –9.7). (E) Following NH4-Ac stimulation, there was a significant increase in the intracellular cytokine IL-6 produced by splenic macrophages in Tlr9fl/fl mice (n ¼ 10) compared with control mice (n ¼ 8) (P < .0001) (mean difference: 3; 95% CI, 2.5 to 3.5), which was ameliorated in Lysm-Cre Tlr9fl/fl mice (n ¼ 8) (P < .0001) (mean difference: –2.7; 95% CI, –3.2 to –2.2).

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used to characterize the different subsets of T cells and macrophages were identified using F4/80 (BM8) and CD11b (M1/70) markers. Intracellular cytokine production (IL-6 [MP5-20F3], IFNg [XMG1.2], and TNFa [MP6-XT22]) was determined in the CD4þ and CD8þ T cell subsets and F4/ 80þ and CD11bþ macrophages. Flow cytometry antibodies were purchased from BioLegend (San Diego, CA).

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Statistics For comparisons between 2 groups, Student’s t test (parametric data) or Mann-Whitney U test (nonparametric data) were used; for comparisons among 3 or more groups, 1-way analysis of variance with Tukey’s multiple comparison tests (parametric data) or Kruskal Wallis with Dunn’s multiple comparison tests (nonparametric data) were used based on the normal distribution of the data. All the results are presented as mean or median differences with 95% confidence intervals. Hypothesis testing was 2 tailed at an alpha level of .05. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA); P < .05 was considered as statistically significant.

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References 1. Bernal W, Hall C, Karvellas CJ, Auzinger G, Sizer E, Wendon J. Arterial ammonia and clinical risk factors for encephalopathy and intracranial hypertension in acute liver failure. Hepatology 2007;46:1844–1852. 2. Phear E, Sherlock S, Summerskill W. Blood ammonia levels in liver disease and hepatic coma. Lancet 1955; 7:836–840. 3. Butterworth RF. Pathogenesis of Hepatic Encephalopathy and Brain Edema in Acute Liver Failure. Journal of Clinical and Experimental Hepatology 2015;5:S96–S103. 4. Vaquero J, Polson J, Chung C, Helenowski I, Schiodt FV, Reisch J, Lee WM, Blei AT. Infection and the progression

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of hepatic encephalopathy in acute liver failure. Gastroenterology 2003;125:755–764. Rolando N, Wade J, Davalos M, Wendon J, PhilpottHoward J, Williams R. The systemic inflammatory response syndrome in acute liver failure. Hepatology 2000;32:734–739. Wright G, Shawcross D, Olde Damink SWM, Jalan R. Brain cytokine flux in acute liver failure and its relationship with intracranial hypertension. Metab Brain Dis 2007;22:375–388. Jalan R, Damink SWO, Hayes PC, Deutz NE, Lee A. Pathogenesis of intracranial hypertension in acute liver failure: inflammation, ammonia and cerebral blood flow. J Hepatol 2004;41:613–620. Blazka ME, Wilmer JL, Holladay SD, Wilson RE, Luster MI. Role of Proinflammatory Cytokines in Acetaminophen Hepatotoxicity. Toxicol Appl Pharmacol 1995;133:43–52. Ishida Y, Kondo T, Ohshima T, Fujiwara H, Iwakura Y, Mukaida N. A pivotal involvement of IFNg in the pathogenesis of acetaminophen-induced acute liver injury. FASEB J 2002;16:1227–1236. Dambach DM, Watson LM, Gray KR, Durham SK, Laskin DL. Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse. Hepatology 2002;35:1093–1103. Rama Rao KV, Jayakumar AR, Tong X, Alvarez VM, Norenberg MD. Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes. J Neuroinflammation 2010;7:66. Kato M, Hughes RD, Keays RT, Williams R. Electron microscopic study of brain capillaries in cerebral edema from fulminant hepatic failure. Hepatology 1992; 15:1060–1066. Shawcross DL, Jalan R. The pathophysiologic basis of hepatic encephalopathy: central role for ammonia and inflammation. Cell Mol Life Sci 2005;62:2295–2304.

Figure 9. (See previous page). ODN2088 inhibits the ammonia-induced brain edema and cytokine production by macrophages in WT mice. (A) Administration of the TLR9 antagonist, ODN2088, along with NH4-Ac prevented the rise in BW content (n ¼ 10) (P < .001) (mean difference: –1.07; 95% CI, –1.7 to –0.48) compared with the NH4-Ac alone–stimulated mice (n ¼ 9) and there was no difference compared with control mice (n ¼ 12). (B) Administration of ODN2088, along with NH4-Ac prevented the rise in the liver-to-body weight ratio in WT mice (n ¼ 15) (P < .0001) (mean difference: –0.02; 95% CI, –0.026 to –0.014) compared with the NH4-Ac alone–stimulated mice (n ¼ 16) and there was no difference compared with control mice (n ¼ 13). (C) Following NH4-Ac stimulation, there was a significant increase in the hepatocyte swelling in the liver histology (hematoxylin and eosin stain, 200 magnifications) in WT mice compared with control mice, where the cytoplasm of cells remained intact. Administration of ODN2088, ameliorated the hepatocyte swelling in WT mice. The images in the black box inset are the representative images at 400 magnification. (D) Following NH4-Ac stimulation, there was a significant increase in the total plasma DNA level in WT mice (n ¼ 12) (P ¼ .02) (mean difference: 12.58; 95% CI, 0.5 to 5.4) and in WT mice administered with ODN2088 (n ¼ 16) (mean difference: 11.4; 95% CI, 0.6 to 5.2) compared with control mice (n ¼ 12). There was no difference in the total plasma DNA level in NH4-Ac alone WT mice stimulated compared with ODN2088 treated group. (E) Administration of ODN2088, along with NH4-Ac prevented the rise in the intracellular cytokine IFNg produced by splenic macrophages in WT mice (n ¼ 11) (P < .0001) (mean difference: –2.5; 95% CI, –3.1 to –1.9) compared with the NH4-Ac alone–stimulated mice (n ¼ 8) and there was no difference compared with control mice (n ¼ 7). (F) Administration of ODN2088, along with NH4-Ac prevented the rise in the intracellular cytokine TNFa produced by splenic macrophages in WT mice (n ¼ 11) (P < .0001) (mean difference: –28.9; 95% CI, –32.1 to –25.6) compared with the NH4-Ac alone–stimulated mice (n ¼ 8) and there was no difference compared with control mice (n ¼ 7). (G) Administration of ODN2088, along with NH4-Ac prevented the rise in the intracellular cytokine IL-6 produced by splenic macrophages in WT mice (n ¼ 11) (P < .0001) (mean difference: –5; 95% CI, –5.5 to –4.5) compared with the NH4-Ac alone–stimulated mice (n ¼ 8) and there was no difference compared with control mice (n ¼ 7). (H) Representative FACS plots of the intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic macrophages in WT control mice and NH4-Ac–stimulated WT mice with and without ODN2088.

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Figure 10. ODN2088 inhibits the ammonia-induced cytokine production by T cells in WT mice. Administration of the TLR9 antagonist, ODN2088, along with NH4-Ac significantly inhibited (A) the intracellular cytokine IFNg (P < .01) (median difference: –1.8; 95% CI, –2.7 to –0.9), (B) the intracellular cytokine TNFa (P < .05) (median difference: –26; 95% CI, –29.5 to –22), and (C) the intracellular cytokine IL-6 (P < .001) (mean difference: –2; 95% CI, –2.8 to –1.1) produced by splenic CD4þ T cells in WT mice (n ¼ 11) compared with the NH4-Ac alone–stimulated WT mice (n ¼ 11). ODN2088 along with NH4-Ac also significantly inhibited (D) the intracellular cytokine IFNg (P < .0001) (mean difference: –3.3; 95% CI, –5.4 to –1.3) and (E) the intracellular cytokine TNFa (P < .0001) (mean difference: –45.5; 95% CI, –49 to –42) produced by splenic CD8þ T cells in WT mice (n ¼ 11) compared with the NH4-Ac alone–stimulated WT mice (n ¼ 11). (F) Representative FACS plots of the intracellular cytokines IFNg, TNFa, and IL-6 produced by splenic T cells in WT control mice and NH4-Ac–stimulated WT mice with and without ODN2088.

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14. Ratnakumari L, Qureshi IA, Butterworth RF. Effects of congenital hyperammonemia on the cerebral and hepatic levels of the intermediates of energy metabolism in spf mice. Biochem Biophys Res Commun 1992;184:746–751. 15. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740–745. 16. Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, Flavell RA, Mehal WZ. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009;119:305–314. 17. Manakkat Vijay GK, Ryan JM, Abeles RD, Ramage S, Patel V, Bernsmeier C, Riva A, McPhail MJ, Tranah TH, Markwick LJ, Taylor NJ, Bernal W, Auzinger G, Willars C, Chokshi S, Wendon JA, Ma Y, Shawcross DL. Neutrophil Toll-like receptor 9 expression and the systemic inflammatory response in acetaminophen-induced acute liver failure. Crit Care Med 2016;44:43–53. 18. Kendrick SF, O’Boyle G, Mann J, Zeybel M, Palmer J, Jones DE, Day CP. Acetate, the key modulator of inflammatory responses in acute alcoholic hepatitis. Hepatology 2010;51:1988–1997. 19. Warren KS, Nathan DG. The Passage of Ammonia Across the Blood-Brain-Barrier and its Relation to Blood pH. J Clin Invest 1958;37:1724–1728. 20. Garcia-Martinez I, Santoro N, Chen Y, Hoque R, Ouyang X, Caprio S, Shlomchik MJ, Coffman RL, Candia A, Mehal WZ. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of Q8 TLR9. J Clin Invest 2016;126:859–864. 21. Lin F, Taylor NJ, Su H, Huang X, Hussain MJ, Abeles RD, Blackmore L, Zhou Y, Ikbal MM, Heaton N, Jassem W, Shawcross DL, Vergani D, Ma Y. Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcoholrelated cirrhosis. Hepatology 2013;58:314–324. 22. Blackmore LJ, Ryan JM, Huang X, Hussain M, Triantafyllou E, Vergis N, Vijay GM, Antoniades CG, Thursz MR, Jassem W, Vergani D, Shawcross DL, Ma Y. Acute alcoholic hepatitis and cellular Th1 immune responses to alcohol dehydrogenase. Lancet 2015;385(Suppl 1):S22. 23. Yuksel M, Wang Y, Tai N, Peng J, Guo J, Beland K, Lapierre P, David C, Alvarez F, Colle I, Yan H, MieliVergani G, Vergani D, Ma Y, Wen L. A novel "humanized mouse" model for autoimmune hepatitis and the association of gut microbiota with liver inflammation. Hepatology 2015;62:1536–1550. 24. Matsuda T, Murao N, Katano Y, Juliandi B, Kohyama J, Akira S, Kawai T, Nakashima K. TLR9 signalling in microglia attenuates seizure-induced aberrant neurogenesis in the adult hippocampus. Nat Commun 2015;6:6514. 25. Butchi NB, Du M, Peterson KE. Interactions between TLR7 and TLR9 agonists and receptors regulate innate immune responses by astrocytes and microglia. Glia 2010;58:650–664. 26. Zhao Y, Pu D, Sun Y, Chen J, Luo C, Wang M, Zhou J, Lv A, Zhu S, Liao Z, Zhao K, Xiao Q. High glucose-

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1710 1711 1712 27. 1713 1714 1715 1716 1717 28. 1718 1719 1720 1721 1722 1723 1724 29. 1725 1726 1727 1728 1729 1730 1731 1732 30. 1733 1734 1735 1736 31. 1737 1738 1739 1740 32. 1741 1742 1743 1744 1745 1746 Correspondence 1747 Address correspondence to: Debbie Lindsay Shawcross, BSc, MBBS, PhD, Liver Sciences Department, Faculty of Life Sciences and Medicine, King’s 1748 College London, King’s College Hospital Campus, Denmark Hill, London, 1749 SE5 9RS United Kingdom; fax: þ44 (0)20 3299 3167. e-mail: Q1 1750 [email protected]; or Li Wen, MD, PhD, Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, PO Q31751 Box 208056, 333 Cedar Street, New Haven, CT 06520. e-mail: Q21752 [email protected]; fax: 1-203-737–5558. 1753 Acknowledgments 1754 The authors thank all the Wen Lab members for their technical help and scientific input during the study and are grateful to Professor Mark Jay 1755 Shlomchik from the University of Pittsburgh for providing the Tlr9fl/fl mice. 1756 1757 Conflicts of interest The authors disclose no conflicts. 1758 1759 Funding This study was funded by the UK Institute of Liver Studies Charitable Fund and Q41760 National Institutes of Health. GKMV was supported by a King’s College London 1761 International PhD studentship. The infrastructure to support this study was provided by National Institutes of Health Grant Nos. RO1 DK-092882 and 1762 DK-100500, Mouse Genetic Core Grant No. P30 DK-405735 (to Li Wen), the 1763 Medical Research Council Centre for Transplantation, King’s College London Medical Research Council Grant No. MR/J006742/1, and the National Q91764 Institute for Health Research Biomedical Research Centre based at Guy’s 1765 and St Thomas’ NHS Foundation Trust and King’s College London. The 1766 views expressed are those of the author(s) and not necessarily those of the National Institutes of Health, National Health Service, the National Institute 1767 for Health Research, or the Department of Health. 1768 induced defective thrombospondin–1 release from astrocytes via TLR9 activation contributes to the synaptic protein loss. Exp Cell Res 2018;363:171–178. Bafica A, Scanga CA, Feng CG, Leifer C, Cheever A, Sher A. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J Exp Med 2005; 202:1715–1724. Marques PE, Amaral SS, Pires DA, Nogueira LL, Soriani FM, Lima BHF, Lopes GAO, Russo RC, Ávila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 2012;56:1971–1982. Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, Pires DA, Novaes JT, Patricio DO, Cisalpino D, Menezes-Garcia Z, Leevy WM, Chapman SE, Mahecha G, Marques RE, Guabiraba R, Martins VP, Souza DG, Mansur DS, Teixeira MM, Leite MF, Menezes GB. Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. Hepatology 2015;61:348–360. Butterworth RF, Norenberg MD, Felipo V, Ferenci P, Albrecht J, Blei AT. Experimental models of hepatic encephalopathy: ISHEN guidelines. Liver Int 2009; 29:783–788. Ytrebo LM, Nedredal GI, Langbakk B, Revhaug A. An experimental large animal model for the assessment of bioartificial liver support systems in fulminant hepatic failure. Scand J Gastroenterol 2002;37:1077–1088. Tang J, Liu J, Zhou C, Alexander JS, Nanda A, Granger DN, Zhang JH. Mmp-9 deficiency enhances collagenase-induced intracerebral hemorrhage and brain injury in mutant mice. J Cereb Blood Flow Metab 2004; 24:1133–1145.

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