Raymond Kwan

An Indispensable Role for the Chemokine Receptor CCR10 in IgA Antibody-Secreting Cell Accumulation

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.

Panhematin provides a therapeutic benefit in experimental pancreatitis

Background and aim Acute pancreatitis (AP) can result in pancreatic necrosis and inflammation, with subsequent multi-organ failure. AP is associated with increased neutrophil recruitment and a rise in pro-inflammatory cytokines such as TNFα. Pretreatment with haemin, results in recruitment of haem-oxygenase-1 (HO-1)+ macrophages and protects against experimental pancreatitis. It is not clear whether modulation of HO-1 after onset of disease has a protective role. In this study, we tested the utility of Panhematin, a water-soluble haemin formulation, in activating and inducing pancreatic HO-1, and as a therapeutic agent in treating mouse acute pancreatitis. Methods We defined the distribution of radiolabelled haemin, then used in vivo HO-1–luciferase bioluminescence imaging and the CO-release assay to test Panhematin-induced upregulation of HO-1 transcription and activity, respectively. Using two well-defined AP murine models, we tested the therapeutic benefit of Panhematin, and quantified cytokine release using a luminex assay. Results Intravenously administered Panhematin induces rapid recruitment of HO-1+ cells to the pancreas within 2 h and de novo splenic HO-1 transcription by 12 h. Despite high baseline spleen HO-1 activity, the pancreas is particularly responsive to Panhematin-mediated HO-1 induction. Panhematin-treated mice, at various time points after AP induction had significant reduction in mortality, pancreatic injury, together with upregulation of HO-1 and downregulation of pro-inflammatory cytokines and CXCL1, a potent neutrophil chemoattractant. Conclusions Despite AP-associated mortality and morbidity, no effective treatment other than supportive care exists. We demonstrate that Panhematin leads to: (i) rapid induction and activation of pancreatic HO-1 with recruitment of HO-1+ cells to the pancreas, (ii) amelioration of AP even when given late during the course of disease, and (iii) a decrease in leucocyte infiltration and pro-inflammatory cytokines including CXCL1. The utility of Panhematin at modest doses as a therapeutic in experimental pancreatitis, coupled with its current use and safety in humans, raises the potential of its applicability to human pancreatitis.

Heme oxygenase-1 is induced in peripheral blood mononuclear cells of patients with acute pancreatitis: a potential therapeutic target

Heme oxygenase-1 (HO-1) induction by hemin or Panhematin protects against experimental pancreatitis. As a preclinical first step toward determining whether HO-1 upregulation is a viable target in acute pancreatitis (AP) patients, we tested the hypothesis that HO-1 expression in peripheral blood mononuclear cell (PBMC) subsets of hospitalized patients with mild AP is upregulated then normalizes upon recovery and that cells from AP patients have the potential to upregulate their HO-1 ex vivo if exposed to Panhematin. PBMCs were isolated on days 1 and 3 of hospitalization from the blood of 18 AP patients, and PMBC HO-1 levels were compared with PMBCs of 15 hospitalized controls (HC) and 7 volunteer healthy controls (VC). On day 1 of hospitalization, AP patients compared with VCs had higher HO-1 expression in monocytes and neutrophils. Notably, AP monocyte HO-1 levels decreased significantly upon recovery. Panhematin induced HO-1 in ex vivo cultured AP PBMCs more readily than in HC or VC PBMCs. Furthermore, PBMCs from acutely ill AP patients on day 1 were more responsive to HO-1 induction compared with day 3 upon recovery. Similarly, mouse splenocytes had enhanced HO-1 inducibility as their pancreatitis progressed from mild to severe. In conclusion, AP leads to reversible PBMC HO-1 upregulation that is associated with clinical improvement and involves primarily monocytes. Leukocytes from AP patients or mice with AP are primed for HO-1 induction by Panhematin, which suggests that Panhematin could offer a therapeutic benefit.

Human keratin 8 variants promote mouse acetaminophen hepatotoxicity coupled with c‐jun amino‐terminal kinase activation and protein adduct formation

Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple‐type digestive epithelia and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and poor outcomes in acute acetaminophen (APAP)‐related liver failure. Given that K8 G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Mice that overexpress the human K8 variants, R341H or R341C, were generated and used together with previously described mice that overexpress wild‐type K8 or K8 G62C. Mice were injected with 600 mg/kg of APAP or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative real‐time polymerase chain reaction, immunoblotting, histological and immunological staining, and biochemical assays. Under basal conditions, the K8 G62C/R341H/R341C variant‐expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, whereas K8 G62C/R341C animals had aberrant disulphide cross‐linked keratins. Animals carrying the K8 variants displayed limited gene expression changes, but had lower nicotinamide N‐methyl transferase (NNMT) levels and were predisposed to APAP‐induced hepatotoxicity. NNMT represents a novel K8/K18‐associated protein that becomes up‐regulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced keratin solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione, or adenosine triphosphate levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. Conclusion: Expression of human K8 variants G62C, R341H, or R341C in mice predisposes to acute APAP hepatotoxicity, thereby providing direct evidence for the importance of these variants in human acute liver failure. (Hepatology 2015) Hepatology 2015;62:876–886)

Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice

Human mutations in keratin 8 (K8) and keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8‐null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2‐type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild‐type control, K8‐null, and K18‐null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81 % of K8‐null male mice 8 mo or older. Similar autoantibodies were detected in aging K18‐null male mice that had a related liver phenotype but normal colon compared with K8‐null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG‐CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase‐2 as additional autoantigens. Therefore, absence of the hepatocyte keratins results in production of anti‐mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with keratin mutations that associate with liver and other diseases.—Toivola, D. M., Habtezion, A., Misiorek, J. O., Zhang, L., Nyström, J. H., Sharpe, O., Robinson, W. H., Kwan, R., Omary, M. B. Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice. FASEB J. 29, 5081–5089 (2015). www.fasebj.org

Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina‐associated proteins predispose to NAFLD and used a candidate gene‐sequencing approach to test for variants in 10 nuclear lamina‐related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina‐related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina‐associated polypeptide‐2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin–LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin‐binding protein p62/SQSTM1. Conclusion: Several variants in nuclear lamina‐related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710‐1725).

Su1799 Liver-Specific Nuclear Lamin a Deficiency Causes Male-Selective Hepatic Steatosis via Induction of Lipogenic Genes

Background: Total parenteral nutrition (TPN) is a life-saving therapy in critically ill patients. Unfortunately, it is associated with significant morbidity and mortality, including gut atrophy and Parenteral Nutrition Associated Liver Disease. A lack of enteral feeding during TPN, disrupts key signaling molecules and the enterohepatic circulation, which appears to be a major contributor to disease pathology. Our group previously demonstrated thatenterally administeredbile acid,oleanolic acid (OA)significantly improved gut density andmorphology. OAis a ligand for the G-protein coupled receptor TGR-5. TGR-5 is known to induce transcription of glucagon-like peptide (GLP)-1 and GLP-2. While GLP-1 regulates hepatic steatosis and inflammation, GLP2 is involved in gut growth and motility.Hypothesis: We hypothesize that TPN infusion disrupts the TGR5-GLP axis and exogenous GLP-1, GLP-2 infusion will ameliorate TPN induced injury. Methods: Seven to ten day old piglets were implanted with jugular venous (JVC) and duodenal catheters (DC). Animals were randomized to receive Enteral Feeding (EN) or TPN. The TPN group was subdivided into TPN plus GLP-1 and GLP-2 (n=3). Milk was delivered via the DC and TPN was infused via the JVC. GLP-1 and GLP-2 were dosed at3 mcg/kg/day and 30 mcg/kg/day respectively. Results: Mean daily weight gain (grams) for control Enteral Fed (EN) vs TPN animals and standard deviation (±SD) was 102.4±10.8, 91.03±12.1 respectively, p>0.05. Marked gut atrophy was noted with TPN.Mean gut density as measured in grams/cm of gut tissue and standard deviation (±SD) in EN vs TPN was 0.35±0.03, 0.185±0.05, p<0.05. GLP treatment caused a robust increase in gut density(0.28±0.07). Conclusion: GLP treatment improves gut atrophy in TPN infused animals. This result also provides mechanistic validation for effects of OA treatment. We believe that modulation of TPN associated pathology via bile acid regulation of signaling molecules is novel and could bring a paradigm change in current treatment strategies for unfortunatecomplication of a lifesaving therapy.

Mo1772 Kinase Inhibition Normalizes Mutation-Mediated Keratin Filament Disruption and Predisposition to Apoptosis and Liver Injury

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.