Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

A hallmark of epithelial cells is polarization, which is achieved by the orchestration of external cues, such as cellular contact, extracellular matrix, signal transduction, growth factors, and spatial organization.¹ Hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell.² The apical poles of adjacent hepatocytes form a continuous network of bile canaliculi into which bile is secreted, whereas the basolateral membrane domain forms the sinusoidal pole, which secretes various components, such as proteins or drugs, into the blood circulation.³ Loss of hepatic polarity has been associated with several cholestatic and developmental disorders, including progressive familial intrahepatic cholestasis (PFIC) and primary sclerosing cholangitis (PSC).^4,5 Although the molecular mechanisms governing hepatocyte polarity have been extensively studied in the in vitro systems, there is still a significant gap in our understanding of how polarity is established within the context of tissue during development or maintained during homeostasis.^6,7 Similarly, the molecular pathways contributing to hepatic polarity are not entirely understood, and a better comprehension of hepatic polarity regulation is thus warranted.Previous studies have confirmed the role of hepatocellular junctions, such as tight and gap junctions, in the maintenance of hepatocyte polarity.^8,9 Studies done in vitro and in vivo have shown that loss of junctional proteins, such as zonula occludens protein (ZO)-1, junctional adhesion molecule-A, and claudins, lead to impairment of polarity and distorted bile canaliculi formation.10, 11, 12, 13 In addition, proteins involved in tight junction assembly, such as liver kinase B1, are also involved in polarity maintenance.¹⁴ Among adherens junction proteins, various in vitro cell culture models have confirmed the role of E-cadherin in the regulation of hepatocyte polarity, possibly through its interaction with β-catenin.^15,16 However, there is a lack of an in vivo model to study the role of adherens junction proteins in hepatocyte polarity and their misexpression contributing to various liver diseases.β-Catenin plays diverse functions in the liver during development, regeneration, zonation, and tumorigenesis.17, 18, 19 The relative contribution of β-catenin as part of the adherens junction is challenging to study because like in other tissues, γ-catenin compensates for the β-catenin loss in the liver.^20,21 To address this redundancy, we previously reported a hepatocyte-specific β-catenin and γ-catenin double-knockout (DKO) mouse model was reported.²² Simultaneous deletion of β-catenin and γ-catenin in mice livers led to cholestasis, partially through the breach of cell-cell junctions. However, more comprehensive understanding of the molecular underpinnings of the phenotype is needed.In the current study, prior preclinical findings of dual β-catenin and γ-catenin loss were extended to a subset of PFIC and PSC patients. In vivo studies using the murine model with hepatocyte-specific dual loss of β-catenin and γ-catenin showed complete loss of hepatocyte polarity compared to the wild-type controls (CONs). Loss of polarity in DKO liver was accompanied by epithelial-mesenchymal transition (EMT), activation of transforming growth factor (TGF)-β signaling, and reduced expression of hepatocyte nuclear factor 4α (HNF4α). Our findings suggest that β-catenin and γ-catenin and in turn adherens junction integrity, are critical for the maintenance of hepatocyte polarity, and any perturbations in this process can contribute to the pathogenesis of cholestatic liver disease.     

Integrin β1 Establishes Liver Microstructure and Modulates Transforming Growth Factor β during Liver Development and Regeneration

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin β1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin β1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell–cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor β (TGF-β) and widespread fibrosis. Targeted deletion of integrin β1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin β1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-β. Taken together, these findings identify integrin β1 as a key determinant of liver architecture with a critical role as a regulator of TGF-β secretion. These results suggest that disrupting the hepatocyte–extracellular matrix interaction is sufficient to drive fibrosis.

The homogeneous appearance of the liver belies a complex microstructure essential for proper function. A fenestrated endothelium provides minimal resistance to blood flow to maintain low portal pressure and allows filtered plasma to bathe hepatocytes, permitting nutrient exchange and release of synthetic products. At the apical hepatocyte surface, a specialized canalicular network conveys bile out of the liver for secretion into the intestine.Establishing precise microstructure is thus critical for liver function. The role of hepatocyte–extracellular matrix (ECM) interactions in the development of liver microstructure was therefore examined in the current study.Integrins play an essential role in hepatocyte–ECM interactions. They are single-pass transmembrane receptors that function as adhesion molecules on the cell surface and mediate cell–matrix and cell–cell interactions.¹ They exist on the hepatocyte surface as heterodimers of an α and β subunit and contain a short cytoplasmic tail responsible for chemical signaling and physical force transduction through links to the actin cytoskeleton.² Component subunits exhibit high selectivity in their interactions. In particular, integrin receptors on hepatocytes bind collagen I, laminin, and fibronectin.3, 4, 5Integrin signaling involves a multiprotein complex that is recruited to and becomes associated with the intracellular portion of the protein. Although heterogeneous, signaling often involves focal adhesion kinase (FAK) as a platform for various phosphorylation events, including autophosphorylation, as well as signaling involving Src-family kinase, integrin-linked kinases, and paxillin.6, 7, 8 Principal integrin pairs expressed by hepatocytes are α1β1, α5β1, and a9β1.^9,10 The α1β1 heterodimer predominantly binds collagen IV, the α5β1 receptor binds fibronectin, and the α9β1 receptor binds to a non-RGD (Arg-Gly-Asp) site on tenascin.^11,12 Data on specific roles for integrins in the liver are limited to a few reports. α5β1 integrins sense tauroursodeoxycholic acid and become active in response to cell swelling.¹³ Mice with transgene-mediated osteopontin expression in hepatocytes develop fibrotic livers.¹⁴ Disruption of integrin-linked kinase in hepatocytes leads to increased deposition of ECM.¹⁵ Integrin β1 is essential for liver regeneration, but its effect on fibrosis has not been determined.¹⁶ In humans, hepatitis C is associated with increased expression of various integrins, including β1, α1, α5, and α6, which reflect disease severity.¹⁷ Expression of integrin α6 occurs in a variety of chronic liver diseases.¹⁸A critical role for integrins after injury is also suggested by the known role of ECM during injury response. ECM remodeling via matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is critical in recreating normal liver architecture. MMP-2, MMP-3, and MMP-14 are expressed by stellate cells during activation.^19,20 MMP-9 is expressed in hepatocytes and Kupffer cells after setting of injury.²¹ TIMP-1 modulates liver MMPs, and both TIMP-1 and TIMP-2 are expressed in hepatocytes during injury.22, 23, 24To determine how hepatocyte integrins help establish liver microstructure and re-establish it after liver injury, models were created to assess the consequences of hepatocyte-specific deletion of integrin β1 from before birth and in the adult mice after injury. Hepatocyte size and shape, interactions between hepatocytes and other cells, and signaling pathways relevant to integrin signaling and fibrosis were investigated.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.     

Host Cxcr2-Dependent Regulation of Pancreatic Cancer Growth,Angiogenesis, and Metastasis

Pancreatic ductal adenocarcinoma (PDAC) manifests aggressive tumor growth and early metastasis. Crucial steps in tumor growth and metastasis are survival, angiogenesis, invasion, and immunosuppression. Our prior research showed that chemokine CXC- receptor-2 (CXCR2) is expressed on endothelial cells, innate immune cells, and fibroblasts, and regulates angiogenesis and immune responses. Here, we examined whether tumor angiogenesis, growth, and metastasis of CXCR2 ligands expressing PDAC cells are regulated in vivo by a host CXCR2-dependent mechanism. C57BL6 Cxcr2^−/− mice were generated following crosses between Cxcr2^−/+ female and Cxcr2^−/− male. Cxcr2 ligands expressing Kirsten rat sarcoma (KRAS-PDAC) cells were orthotopically implanted in the pancreas of wild-type or Cxcr2^−/− C57BL6 mice. No significant difference in PDAC tumor growth was observed. Host Cxcr2 loss led to an inhibition in microvessel density in PDAC tumors. Interestingly, an enhanced spontaneous and experimental liver metastasis was observed in Cxcr2^−/− mice compared with wild-type mice. Increased metastasis in Cxcr2^−/− mice was associated with an increase in extramedullary hematopoiesis and expansion of neutrophils and immature myeloid precursor cells in the spleen of tumor-bearing mice. These data suggest a dynamic role of host CXCR2 axis in regulating tumor immune suppression, tumor growth, and metastasis.

Pancreatic ductal adenocarcinoma cancer (PDAC) is the fourth leading cause of cancer-related death, for which treatment options are limited.¹ PDAC progression, metastasis, and therapy resistance involve complex interactions between tumor cells and dynamic tumor microenvironment (TME).² PDAC tumors are known to contain infiltrates of immune cells that help to suppress the anti-tumor responses.3, 4, 5 Such immune infiltrates, composed of both myeloid and lymphoid lineages, include neutrophils, myeloid-derived suppressor cells (MDSCs), macrophages, and T-regulatory (Treg) cells.⁵Chemokine CXC-receptor-2 (CXCR2) and its ligands regulate the growth of the tumor, angiogenesis, and metastasis in various cancers.6, 7, 8, 9 CXCR2 is known to orchestrate immune responses in multiple diseases, including cancer.10, 11, 12 Several studies have confirmed the presence of CXCR2 and its ligands in human PDAC tissues and cell lines.13, 14, 15, 16, 17 In addition to PDAC cells, CXCR2 is expressed on endothelial cells and neutrophils.18, 19, 20 The expression of CXCR2 has also been reported on PDAC fibroblasts²¹ and is a known mediator of tumor progression, angiogenesis, and metastasis in PDAC.^16,22,23 Studies from our group and others show that CXCR2 is expressed on endothelial cells.^19,24,25 Cxcr2⁺ neutrophils are one of the innate immune cell types that originate from the myeloid precursor,²⁶ and play a protumor role in cancer.^27,28 Deletion of host Cxcr2 in melanoma and breast cancer leads to inhibition of tumor growth, angiogenesis, and metastasis.^9,29,30 However, the role of host Cxcr2 expressed on stromal cells (neutrophils, fibroblasts, and endothelial cells) in PDAC growth and metastasis remains unclear.^12,31Here, we hypothesized that tumor growth, angiogenesis, and metastasis in CXCR2 ligands expressing PDAC cells are regulated by a host stromal Cxcr2-dependent mechanism in the PDAC TME. Syngeneic immunocompetent wild-type (WT) and whole-body Cxcr2 knockout (Cxcr2^−/−) mice showed no significant difference in PDAC tumor growth and enhanced spontaneous and experimental liver metastasis in Cxcr2^−/− mice compared with WT mice. The deletion of Cxcr2 led to inhibition in microvessel density, in situ cell proliferation, and increased apoptosis in PDAC tumors. Additionally, increased metastasis in Cxcr2^−/− mice was associated with an increase in extramedullary hematopoiesis (EMH) and expansion of neutrophils and immature myeloid precursor cells in the spleen of tumor-bearing mice. These data reveal a dynamic role of host CXCR2 axis in regulating pancreatic tumor growth, immune suppression, and metastasis.     

Unbound Corneocyte Lipid Envelopes in 12R-Lipoxygenase Deficiency Support a Specific Role in Lipid-Protein Cross-Linking

Loss-of-function mutations in arachidonate lipoxygenase 12B (ALOX12B) are an important cause of autosomal recessive congenital ichthyosis (ARCI). 12R-lipoxygenase (12R-LOX), the protein product of ALOX12B, has been proposed to covalently bind the corneocyte lipid envelope (CLE) to the proteinaceous corneocyte envelope, thereby providing a scaffold for the assembly of barrier-providing, mature lipid lamellae. To test this hypothesis, an in-depth ultrastructural examination of CLEs was performed in ALOX12B^−/− human and Alox12b^−/− mouse epidermis, extracting samples with pyridine to distinguish covalently attached CLEs from unbound (ie, noncovalently bound) CLEs. ALOX12B^−−/− stratum corneum contained abundant pyridine-extractable (ie, unbound) CLEs, compared with normal stratum corneum. These unbound CLEs were associated with defective post-secretory lipid processing, and were specific to 12R-LOX deficiency, because they were not observed with deficiency of the related ARCI-associated proteins, patatin-like phospholipase 1 (Pnpla1) or abhydrolase domain containing 5 (Abhd5). These results suggest that 12R-LOX contributes specifically to CLE–corneocyte envelope cross-linking, which appears to be a prerequisite for post-secretory lipid processing, and provide insights into the pathogenesis of 12R-LOX deficiency in this subtype of ARCI, as well as other conditions that display a defective CLE.

The corneocyte lipid envelope (CLE) (Figure 1D) is a lipid membrane composed of ω-hydroxy ceramides and ω-hydroxy fatty acids that are covalently bound to the extracellular surface of the proteinaceous corneocyte envelope (CE).^1,2 Investigation of autosomal recessive congenital ichthyosis (ARCI), caused by mutations in enzymes that contribute to CLE and CE synthesis, has revealed that the CLE-CE complex is required for skin barrier function.3, 4, 5, 6 Altered CLE-CE composition and structure are also associated with barrier dysfunction in many other skin conditions, including atopic dermatitis⁷ and psoriasis.⁸Open in a separate windowFigure 1Unbound corneocyte lipid envelopes (CLEs) in ALOX12B^−/− autosomal recessive congenital ichthyosis (ARCI). A: The current model of CLE biogenesis involves esterified ω-hydroxy-acyl sphingosine (EOS) synthesis catalyzed by patatin-like phospholipase 1 (PNPLA1) and abhydrolase domain containing 5 (ABHD5), transport to the plasma membrane (not shown), and EOS oxidation catalyzed sequentially by 12R-lipoxygenase, epidermal lipoxygenase 3, and short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7). A subsequent reaction between EOS–epoxy enone (EpO) and peptides of the corneocyte envelope (CE) is thought to occur nonenzymatically to generate protein-bound ω-hydroxy-acyl sphingosine (OS), which may subsequently be hydrolyzed by ceramidases (C''ase) to form bound ω-hydroxy fatty acids (OHFA). B: A 53-year–old male ARCI patient with homozygous deletion/in-frame mutations (c.786_788delCTT leading to amino acid loss p. Phe263del) in ALOX12B. Note the accentuated skin lines (black arrow) and erythroderma that are typical findings in retinoid-treated ARCI. C–F: Skin samples from an ARCI-unaffected human subject (C and D) or from the ALOX12B^−/− patient (E and F) were divided and processed for electron microscopy with or without pyridine (Pyr) extraction, as indicated before fixation to extract unbound lipids (osmium post-fix). D–F: Covalently bound CLEs were visualized in the Pyr-extracted, ARCI-unaffected sample (D), whereas CLEs in the ALOX12B^−/− sample seemed to be unbound because they were removed by Pyr extraction (E and F). G: Quantification of CLE abundance. Black arrowheads indicate CEs; white arrowheads, CLEs; white arrows, corneodesmosomes (shown to demonstrate that images are in focus). Data show the means ± SEM (G). n = 10 randomly acquired high-powered fields used for quantification in each determination (G). ∗P < 0.05, ∗∗∗P < 0.001. Scale bars = 100 nm (C–F). C, corneocyte; C18:2-TAG, linoleoyl triacylglycerol; EpOH, epoxy alcohol; OOH, hydroperoxide.The major unbound lipid precursor of the CLE is most likely esterified ω-hydroxy-acyl sphingosine (EOS; an abundant ω-O-acyl ceramide in mammalian epidermis). In the current model (Figure 1A), the final step of EOS synthesis is transfer of linoleate (C18:2) from triglyceride to ω-hydroxy-acyl sphingosine (the ceramide ω-hydroxy-acyl sphingosine), catalyzed by patatin-like phospholipase 1 (PNPLA1) and its coactivator abhydrolase domain containing 5 (ABHD5)/comparative gene identification-58 (CGI-58).^4,9, 10, 11 EOS and glucosylated EOS are then stored and transported in lamellar bodies,¹² whose limiting membranes fuse with the plasma membrane during lipid secretion in the outer stratum granulosum at the site of the developing CLE.⁵ Subsequently, oxidation reactions catalyzed sequentially by 12R-lipoxygenase (12R-LOX; encoded by the gene ALOX12B), epidermal lipoxygenase 3 (encoded by ALOXE3), and short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) are proposed to transform the pentadiene group in the ω-linoleate of EOS into an epoxy enone that can react nonenzymatically with peptides on the external face of the CE (especially involucrin, envoplakin, and periplakin¹³) to form protein-bound ω-hydroxy-acyl sphingosine.^14,15 The bound ω-hydroxy fatty acids could be formed either by a similar sequence starting with ω-O-acyl fatty acids instead of EOS¹⁶ or via hydrolysis of the amide bond of bound ω-hydroxy-acyl sphingosine by ceramidases.¹⁷This model explains why both CLEs and their ω-hydroxy ceramide and ω-hydroxy fatty acid constituents are diminished in ARCI with defects in EOS synthesis^4,5 or EOS oxidation. The phenotype associated with these two subgroups of ARCI is indistinguishable,¹⁸ consistent with the possibility that the disease features are a consequence of the shared CLE defect. However, one major difference is that unbound EOS is reduced in EOS synthesis defects, but normal or increased in EOS oxidation defects (the metabolic changes associated with these differences are illustrated) (Supplemental Figure S1).^19,20 Some of this excess EOS could be contained within abnormal CLEs that fail to be cross-linked to protein (referred to as unbound CLEs throughout this article) due to the defect in EOS oxidation, although these have not yet been reported. The identification of such unbound CLEs would support a specific role for 12R-LOX in CLE-CE cross-linking and could provide insight into CLE biogenesis and the potential treatment of CLE defects in ichthyosis and other conditions. Therefore, in the current study, a detailed ultrastructural examination was performed on epidermis from an ALOX12B^−/− ARCI patient and from Alox12b^−/− mice, using the amphipathic solvent pyridine to distinguish unbound from bound CLEs.     

PCTR1 Enhances Repair and Bacterial Clearance in Skin Wounds

Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.

Inflammation is a critical phase of the tissue repair program that aids in containment of pathogens and clearance of dead tissue and debris.^1,2 Neutrophils are among the first innate immune cells recruited to injured tissue, and they possess an armament of chemical mediators that facilitate pathogen eradication. Monocytes are subsequently recruited and differentiate into macrophages that assist in pathogen detection and killing, as well as efferocytosis (ie, clearance of apoptotic cells). Distinct monocyte/macrophage subsets are temporally reprogrammed to directly promote tissue matrix remodeling, vascularization, and re-establishment of protective epithelial barriers through the release of growth factors, cytokines, and lipid mediators.3, 4, 5 If the tightly coordinated temporal dynamics of inflammation and its subsequent resolution are altered, delayed tissue repair can manifest and lead to necrosis and susceptibility to ongoing infection with pathogenic microbes.^2,6 Indeed, altered tissue repair is associated with several chronic diseases and thus new approaches to stimulate tissue repair are urgently needed.6, 7, 8, 9Lipid mediators are enzymatically generated from polyunsaturated fatty acids in injured tissues and govern both the initial phases of inflammation (eg, leukocyte recruitment, activation), as well as the resolution phase.^10,11 A superfamily of structurally diverse specialized pro-resolving mediators (SPM), which include lipoxins, resolvins, protectins, and maresins, have emerged as key mediators of active resolution that engage immune cells via specific receptors to blunt excessive neutrophil recruitment and to expedite macrophage efferocytosis.¹¹ Several SPM, including lipoxins, E-series resolvins, and D-series resolvins, also actively participate in tissue repair and regeneration in distinct contexts, including the skin, intestine, skeletal muscle, eye, gut, and periodontium.12, 13, 14, 15, 16, 17 Importantly, SPM facilitate host defense by stimulating macrophages and neutrophils to phagocytose and kill bacterial pathogens.¹⁸A novel series of cysteinyl-SPM (cys-SPM) comprising peptide conjugates within the resolvin, protectin, and maresin families were discovered and were coined conjugates in tissue regeneration (CTRs) based on their roles in promoting tissue regeneration in planaria.18, 19, 20 The complete structures of these novel mediators were systematically elucidated, and stereochemical assignments were performed, which was facilitated by total organic synthesis.18, 19, 20 They are biosynthesized via conjugation of glutathione to epoxide intermediates involved in protectin (16S,17S-epoxy protectin), maresin (13S,14S-epoxy maresin), and resolvin (7S,8S-epoxy resolvin) biosynthesis, yielding protectin CTRs (PCTRs), maresin CTRs (MCTRs), and resolvin CTRs (RCTRs), respectively¹⁸ (Figure 1A). The glutathione-conjugated mediators are designated PCTR1, MCTR1, and RCTR1, whereas cleavage of the γ-glutamyl group yields PCTR2, MCTR2, and RCTR2. These products are subsequently converted to cysteine-containing PCTR3, MCTR3, and RCTR3. These cys-SPM have been identified in human and mouse tissues, including spleen, lymph nodes, and self-resolving inflammatory exudates, and actively promote resolution of acute inflammation in vivo.¹⁸ They have direct actions on human leukocytes and promote macrophage efferocytosis, as well as bacterial phagocytosis, to facilitate host defense.²⁰ These pro-resolving roles act in concert with their roles in promoting tissue regeneration.18, 19, 20 Recent studies confirm and extend the potent inflammation-resolving actions of cys-SPM in multiple injury contexts.21, 22, 23, 24, 25Open in a separate windowFigure 1Structure-activity analysis of cys-SPM in promoting human keratinocyte migration. A: Cys-SPM biosynthetic pathways and structures, depicting key epoxide intermediates involved in the biosynthesis of protectins (PD1 and PCTRs), D-series resolvins (RvD1, RvD2, and RCTRs), and maresins (MaR1, MaR2, and MCTRs). Right panel, assessment of closure in scratch-wounded monolayers of human primary keratinocytes stimulated with EGF (100 ng/mL) or structurally distinct synthetic cys-SPM (1 and 10 nmol/L; 24 hours). B: Representative images of control and PCTR1-treated keratinocytes at baseline and 24 hours after wounding, with the black dotted line indicating the cell border. C: Assessment of proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation in keratinocytes in the presence of full serum medium or indicated concentrations of PCTR1 for 24 hours. D: Measurement of cAMP accumulation in keratinocytes stimulated with PCTR1 (10 nmol/L). E: Assessment of closure in wounded keratinocytes stimulated with PCTR1 in the presence or absence of PKA inhibitor, H89 (5 μmol/L). Data are expressed as means ± SEM. n = 5 independent experiments (A); n = 10 to 13 replicates from 2 independent experiments (C); n = 4 replicates from 2 independent assays (D); n = 15 to 17 replicates from 3 independent experiments (E). ∗P < 0.05 by one-way analysis of variance followed by Dunnett''s post hoc tests (A, C, and E), or unpaired t-test (D).The actions of cys-SPM in mammalian tissue repair programs, including re-establishment of epithelial barriers, have yet to be addressed. Here, evidence is presented that PCTR1 directly stimulates migration of human keratinocytes in vitro which translates to accelerated closure of full-thickness skin wounds in vivo. Importantly, PCTR1 was produced in wounds infected with the common skin pathogen, Staphylococcus aureus, and PCTR1 accelerated bacterial clearance, suggesting novel roles of this SPM in both facilitating host defense, as well as engaging in tissue repair programs.     

Lysosomal Acid Lipase Deficiency Controls T- and B-Regulatory Cell Homeostasis in the Lymph Nodes of Mice with Human Cancer Xenotransplants

Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal^−/− mice. In the lal^−/− lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal^−/− lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal^−/− lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal^−/− Treg and Breg elevation and PD-L1 expression in lal^−/− Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal^−/− lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.

Transplantable animal models, in which the relationship and interplay between malignant cells and immune cells can be studied, play a fundamental role in the study of oncoimmunology and development of therapeutic approaches to treat human cancer. Utilization of proper preclinical models accelerates development of immunotherapies and understanding of underlying mechanisms.^1,2 The host immune system determines the fate of invading cancer cells.³ In searching for appropriate immunosuppressive mouse models to study human cancer-derived xenografts, a genetic ablation mouse model (lal^−/−) of lysosomal acid lipase (LAL) was evaluated.⁴ LAL is a lipid metabolic enzyme catalyzing the hydrolysis of cholesteryl esters and triglycerides in the lysosome to generate free fatty acids and cholesterol.⁵ The hydrolyzed products are transported to the cytoplasm for either storage or utilization in membrane biogenesis, steroid hormone synthesis, and energy production. Although lal^−/− mice survive into adulthood, the metabolic defect of LAL deficiency leads to severe immunodeficiency, in which the lymphocyte levels are extremely low because of impaired development, maturation, and proliferation.⁶ The ratio of T-regulatory cells (Tregs) and myeloid-derived suppressive cells (MDSCs) is significantly increased, which suppresses T-cell function and directly stimulates tumor growth and invasion.6, 7, 8, 9, 10 More importantly, the compromised immunity accelerates growth and invasion of various murine tumor cells not only in syngeneic, but also allogeneic, lal^−/− mice.⁹ In this report, we show that immunodeficiency delays and reduces immune rejection of human cancer cells in lal^−/− mice. Herein, functional roles of Tregs and B-regulatory cells (Bregs) in the lal^−/− lymph nodes during failure of immune rejection, as well as the abnormal expression and function of PD-L1 in lal^−/− Tregs and Bregs in association with the metabolic switch toward glycolysis and glutamine utilization are systematically evaluated. Furthermore, the functional roles of mammalian target of rapamycin (mTOR) and peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor are characterized in Tregs and Bregs of the lal^−/− lymph nodes during human cancer cell rejection. We conclude that Tregs and Bregs in the lal^−/− lymph nodes play critical roles in suppression of immune rejection of human cancer growth, together with other cell types [eg, MDSCs and endothelial cells (ECs)], in lal^−/− mice. Therefore, the immunodeficient lal^−/− mouse model serves as a potential preclinical model that can be used for clinical human cancer studies.     

Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist,SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza–infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

Influenza virus infections during pandemic outbreaks and yearly seasonal epidemics cause significant morbidity and mortality rates globally.¹ Seasonal influenza-associated deaths have increased in recent years, with an estimated of >600,000 fatalities annually.² A significant proportion of hospitalized patients with influenza develop complications of acute respiratory distress syndrome, characterized by widespread alveolar-capillary injury, inflammation, edema, and parenchymal hemorrhage.3, 4, 5, 6, 7, 8 These pathologic manifestations are driven by virus-inflicted cytopathic effects as well as exaggerated host immune responses.9, 10, 11 Vaccination is the logical choice for controlling the virus. However, because of unrelenting emergence of new strains and their mutative ability, vaccination presents a major challenge during influenza outbreaks.^12,13 In such cases, treatment primarily depends on antiviral therapy. Administration of antiviral drugs may not always be effective, as considerable lung pathology is mediated by exaggerated host-immune responses in addition to virus-inflicted cytotoxicity.14, 15, 16, 17Previously, we established that massive neutrophil influx, their induced neutrophil extracellular traps (NETs), and extracellular histones (ECHs) aggravate pulmonary pathology in severe influenza.18, 19, 20, 21, 22, 23 Aberrant neutrophil activity and accumulation of NETs are also documented in patients with severe influenza.^24,25 Neutrophils are recruited to the site of injury/infection via chemokine signaling, mediated through chemokine receptors. Among various chemokine receptors, CXC chemokine receptor 2 (CXCR2) plays a critical role in modulating neutrophil functionality during influenza.²⁶ Numerous clinical studies have also tested CXCR2 antagonists for their efficacy in reducing inflammation and organ injuries in acute and chronic diseases.27, 28, 29, 30, 31 Recently, human phase 2 trials evaluated the safety and efficacy of a CXCR2 antagonist, danirixin, alone or in combination with oseltamivir in influenza-infected patients.^27,28 Although administration of danirixin was found to be safe and well-tolerated, no differences in the clinical scores were observed between patients given oseltamivir alone and those given danirixin plus oseltamivir.²⁷ Furthermore, there was inconsistency in neutrophil numbers among different treatment groups. This inconsistency may be attributed to the absence of rational determination of the optimal timing and dosing of danirixin, to achieve the fine balance of suppressing excessive neutrophil influx, without compromising the beneficial host immunity by neutrophils. Moreover, the underlying mechanistic roles of targeting CXCR2 and its pathogenic association with influenza pneumonia have not been established.NETs are large extracellular web-like chromatin strands that were initially proposed to have a defense mechanism against invading pathogens.³² However, excessive release of NETs aggravates tissue injury and death, as reported in several disease conditions.33, 34, 35, 36 NETosis is regulated by various granule and nuclear proteins.³⁷ Myeloperoxidase (MPO) and neutrophil elastase (NEs) are released from azurophilic granules, anchor chromatin scaffolds in NETs, and mediate histone degradation during NETosis.³⁸ We reported earlier that blocking MPO decreases NETs, but signaling mechanisms in influenza-induced NETosis remain unclear.^17,18 Herein, we evaluated the therapeutic efficacy of a CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide) alone or in combination with antiviral agent, oseltamivir (which inhibits viral neuraminidase and prevents progeny virus release from infected cells), in models of lethal influenza-infected mice and sublethal swine influenza-infected piglets. SCH527123 plus oseltamivir significantly improved survival in lethal influenza-challenged mice, and attenuated lung pathology in swine influenza-infected piglets. Thus, SCH5277123 plus oseltamivir represents a promising combination treatment against influenza pneumonia.