Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Exposure to Microbial Metabolite Butyrate Prolongs the Survival Time and Changes the Growth Pattern of Human Papillomavirus 16 E6/E7-Immortalized Keratinocytes in Vivo

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. Although cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells'' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure may increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. These results suggest that fermentation products of tonsillar bacteria may play an important role in the long-term persistence of high-risk HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.

Human papillomavirus (HPV) is a small nonenveloped circular double-stranded DNA virus that infects keratinocytes. Among the five genera identified so far, α-papillomavirus is the most commonly studied group because of its association with cancer/precancerous lesions,¹ and is subdivided into low-risk (eg, HPV6 and HPV11) and high-risk (eg, HPV16 and HPV18) genotypes. High-risk HPV encodes two major oncoproteins, E6 and E7, that play a pivotal role in driving the infected keratinocytes toward malignancy. The E6 oncoprotein inactivates the tumor suppressor and cell cycle checkpoint protein TP53, whereas the E7 oncoprotein inactivates the retinoblastoma (RB1) tumor suppressor protein.^2,3 Together, E6/E7 oncoproteins target diverse cellular pathways involving cell cycle, apoptosis, and cell polarity, resulting in cell cycle deregulation and genome instability.^4,5 Knockdown of E6/E7 induces cell senescence or apoptosis, highlighting their crucial roles in the persistence of HPV-mediated cancers.^6,7 Infection with high-risk HPV is responsible for ≈5% of all human cancers and accounts for 70% of oropharyngeal malignancies.⁸HPV infection has caused an epidemic increase in the incidence of oropharyngeal cancers worldwide.⁹ HPV-related head and neck squamous cell carcinomas (SCCs) occur almost exclusively in the oropharynx.^10,11 HPV-positive oropharyngeal SCC (OPSCC) often exhibits a high-grade morphology, nonkeratinized and basaloid, deviating from the conventional well-differentiated, keratinized SCC in the oral cavity.¹² HPV-positive OPSCC appears to be molecularly and pathologically distinctive from its HPV-negative counterpart. Clinically, HPV-positive OPSCC has a unique pattern of metastasis, a longer disease-free interval, and a better prognosis.¹³ A new cancer staging system for HPV-positive OPSCC was established to guide treatment,¹⁴ partly because de-escalation of treatment does not compromise the cure rate but decreases morbidity caused by adverse effects of the therapy. However, treatment de-escalation for HPV-positive OPSCC has been recently discouraged after the publication of RTOG 1016 and De-ESCALaTE studies, both showing that outcomes of HPV-positive OPSCC patients strongly depend on the type of treatment received.^15,16HPV-related premalignant progression is traditionally studied on uterine cervical premalignant lesions. The derived cell lines have been the major tools for researchers to understand the biological process of HPV-associated malignant transformation.¹⁷ After prolonged passaging, the cell lines are able to undergo phenotypic progression from low-grade through high-grade dysplasia to invasiveness when differentiating into stratified squamous epithelium in organotypic raft epithelial tissue cultures.^18,19 Unlike cervical cancers, the process involving HPV-positive oropharyngeal malignancy is less well understood, and at least two major issues remain to be elucidated. First, premalignant lesions (dysplasia) have rarely, if ever, been identified in clinical oropharyngeal specimens in the absence of an invasive component.²⁰ Because premalignant lesions are present in HPV-related SCCs involving other anatomic sites, including the adjacent oral cavity,²¹ their absence in the oropharynx remains an enigma. Second, HPV-related head and neck cancers have a strong site preference for the oropharynx.^10,22The unusually high incidence of HPV-positive SCC in the oropharynx suggests the existence of unique local factors that promote HPV-induced oncogenesis. The oropharynx contains palatine tonsils and accessory lymphoid tissues of the Waldeyer ring that form deep crypts lined by patches of nonkeratinized stratified squamous epithelium and reticulated spongiotic epithelium with a gapped, noncontinuous basement membrane.²³ The crypt epithelium is constantly infiltrated by lymphoid cells and forms a loose network abutting the subjacent lymphoid tissue. The deep crypts provide a low-oxygen habitat for anaerobic bacteria,^24,25 which produce a variety of fermentation products, including butyrate, which contributes to halitosis.²⁶ Butyrate is a histone deacetylase (HDAC) inhibitor and has been shown to function as an epigenetic modulator to condition intestinal immunity and improve the health of intestinal epithelium.^27,28 However, butyrate has both protumor and anti-tumor properties that have been observed on cultured HPV-immortalized epithelial cells.^29,30Although HPV is a ubiquitous human pathogen, host immunity is capable of clearing the infection in most cases, including those caused by the high-risk group. However, a small percentage of the patients develop persistent infection, which is associated with an increased risk of cancer development.31, 32, 33 Although humans are the natural host of HPV, various studies have shown that HPV16 oncoproteins can target mouse TP53 and RB1 pathways similar to those in the human cells.³⁴ Therefore, mice provide a valuable animal model to investigate HPV precancer-host or cancer-host interaction.The current study showed that butyrate treatment changed the in vitro and in vivo properties of HPV16 E6/E7-expressing keratinocytes. The treatment prolonged the survival time and changed the growth pattern of the cells after injection into syngeneic, immunocompetent mice. The results suggest a link between the byproducts produced by tonsillar bacteria and chronic HPV infection.     

Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.Visceral fat necrosis has been noted to occur with pancreatitis for over 100 years.^1,2 This fat is typically located in or around the pancreas^3,4 and is a major component of necrotizing pancreatitis and peripancreatic necrosis.^5,6 Despite being a part of the criteria for staging the severity of acute pancreatitis (AP) in humans^7–9 and being included as a separate entity in the recently revised Atlanta criteria,⁶ the pathogenesis and role of fat necrosis, that is, whether it is a marker or mediator of severe AP (SAP), remains unclear.Clinical correlates of the relevance of fat necrosis include the several epidemiological studies that show individuals with increased intra-abdominal fat^10–13 or obese patients being at an increased risk for SAP.^11,14–20 Peripancreatic fat necrosis may occur independent of pancreatic necrosis⁵ and is associated with an increased risk for severe attacks.^21,22 Although there is basolateral leakage of pancreatic enzymes during pancreatitis,^23–26 and lipases have been noted in fat necrosis in human disease,^27,28 it is unclear whether these lipases cause fat necrosis or are a remnant of pancreatic damage.Triglycerides compose 80% to 90% of the volume of adipocytes^29–31 and can be hydrolyzed by lipases released basolaterally during pancreatitis.^23–26 Although in vitro studies done more than 2 decades ago showed lipase inhibition to reduce pancreatic acinar injury on co-incubation with pancreatic homogenates and triglycerides,³² in vivo lipase inhibition in SAP models showed no benefit³³ until recently, when this was studied in the context of increased visceral fat.^34,35Previous studies have shown that long-chain unsaturated fatty acids (UFAs) form the majority of the nonesterified fatty acids in necrotic collections^36,37 and are more toxic than are saturated fatty acids.^32,34,35,38 Since obese (ob/ob) mice and obese humans may have a similar visceral fat composition,^3,35 we chose to study whether a classically mild model of pancreatitis, that is, cerulein pancreatitis, would result in different outcomes in these mice versus lean mice and explore the role of fat necrosis in these outcomes. Our previous work has shown a benefit of pharmacological inhibition of pancreatic lipases in biliary, IL-12–induced, and IL-18–induced pancreatitis^34,35; we therefore used this as a tool to influence outcomes. Since obesity is also known to be associated with an exaggerated baseline inflammatory state, we went on to study whether the inflammatory response was different between the groups that had different outcomes. Additionally, since SAP may occur along with severe pancreatic necrosis or with multisystem organ failure (MSOF) with insignificant necrosis,^39–41 we also compared pancreatic necrosis between groups with different outcomes.Reasons for preferring a pharmacological approach^34,35 over a genetic one include: i) previous validation in mechanistically distinct models,^34,35 ii) the redundant roles of pancreatic lipases⁴² (which are supported by this study), iii) the deletion of the two lipases of interest being embryonically lethal,⁴³ and iv) the inability to make mice with a genetic deletion of lipases obese on a high-fat diet (unpublished data). Our results, in concert with those from previous studies,^34,35 show that pancreatic lipase–dependent visceral fat lipolysis worsens outcomes of AP, unrelated to the initiator of the disease, and suggest alternate ways to interpret the parameters of severity in AP, along with suggesting a potential approach to improving SAP outcomes.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Immunogenicity of Decellularized Porcine Liver for Bioengineered Hepatic Tissue

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham–operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.Within the United States alone, tens of thousands of patients are awaiting a liver transplant, with only a few thousand donor organs available annually.¹ This widening mismatch has led physicians and researchers to pursue alternative therapies for chronic liver disease, including in situ cell-based therapies or xenotransplantation of organs.^2–4 The field of regenerative medicine offers another approach, in which elements of both would be combined for the bioengineering of neo-organs for transplantation.^5,6The concept of whole liver tissue engineering aims to combine patient-specific autologous hepatocytes or hepatic progenitor cells and a carrying platform, or scaffold, to allow for three-dimensional tissue growth and permit the complex cellularity of hepatic tissue. Use of decellularized organ matrices preserves the natural extracellular matrix (ECM) proteins and growth factors that guide cell attachment and proliferation in an organ-specific manner.⁷ Proper processing of the matrix scaffolds removes all cytotoxic chemicals from the decellularization process and performs complete degradation of donor nucleic acids to prevent an adverse host immune response.⁸ These bioengineered livers have the ultimate potential to surpass the current allograft gold standard.The process begins by removing the native cellular components from a donor tissue using detergents and enzymes and leaving behind an ECM scaffold with preserved vasculature and essential biological factors. The concept has been applied to many tissues, including the heart,^9,10 lungs,^11–14 bladder,¹⁵ blood vessels,^16,17 muscle,¹⁸ intestines,^19,20 trachea,^21–23 kidney,^7,24,25 and liver.^26–30 Each detergent, enzyme, washing buffer, and sterilization technique used to decellularize a tissue can have a direct influence on the host remodeling response and functional outcome.³¹ In a previous study, decellularized matrix scaffolds were immunologically favorable up until cells were added to the scaffolding material, where proinflammatory macrophages were activated.³² To evaluate whether a decellularized tissue represents a viable scaffold option, the generated matrices need to be implanted and evaluated over time, without cells, to allow a host’s immune cells to infiltrate and respond to the material.³³ The initial response can begin as early as 2 days and last for months.³⁴ During that time, the environment from both the host itself and the degrading matrix material can influence the phenotype of the host immune cells switching between activation states that will determine the future clinical viability of the biological matrix material.^35–37 Triggering of proinflammatory macrophage activation results in the release of cytokines, growth factors, proteolytic enzymes, and reactive oxygen and nitrogen intermediates that will greatly inhibit the integration of the biomaterial with the host tissue.³⁸ Studies on the immunogenicity of decellularized whole tissue are limited, and a key criterion of transplantation viability will be evaluating the activation of host macrophages toward the classically proinflammatory phenotype (M1) or the regenerative and repair phenotype (M2).The objectives of the current study were to generate a decellularized porcine liver by scaling up our previously established rodent perfusion protocol,²⁸ characterize the resultant scaffold, and compare the in vivo immunological response of a rodent host between allograft and xenograft decellularized liver matrices. We hypothesized that both tissues would elicit a similar host response as the result of the high levels of preservation the ECM protein structures share between species.³⁹ The generation of large-scale hepatic tissue platforms, and an understanding of the inherent immune response by a host species, will be vital in producing implantable bioengineered livers.     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

VEGFA Activates Erythropoietin Receptor and Enhances VEGFR2-Mediated Pathological Angiogenesis

Clinical and animal studies implicate erythropoietin (EPO) and EPO receptor (EPOR) signaling in angiogenesis. In the eye, EPO is involved in both physiological and pathological angiogenesis in the retina. We hypothesized that EPOR signaling is important in pathological angiogenesis and tested this hypothesis using a rat model of oxygen-induced retinopathy that is representative of human retinopathy of prematurity. We first determined that EPOR expression and activation were increased and that activated EPOR was localized to retinal vascular endothelial cells (ECs) in retinas at postnatal day 18 (p18), when pathological angiogenesis in the form of intravitreal neovascularization occurred. In human retinal microvascular ECs, EPOR was up-regulated and activated by VEGF. Lentiviral-delivered shRNAs that knocked down Müller cell–expressed VEGF in the retinopathy of prematurity model also reduced phosphorylated EPOR (p-EPOR) and VEGFR2 (p-VEGFR2) in retinal ECs. In human retinal microvascular ECs, VEGFR2-activated EPOR caused an interaction between p-EPOR and p-VEGFR2; knockdown of EPOR by siRNA transfection reduced VEGF-induced EC proliferation in association with reduced p-VEGFR2 and p-STAT3; however, inhibition of VEGFR2 activation by siRNA transfection or semaxanib (SU5416) abolished VEGFA-induced proliferation of ECs and phosphorylation of VEGFR2, EPOR, and STAT3. Our results show that VEGFA-induced p-VEGFR2 activates EPOR and causes an interaction between p-EPOR and p-VEGFR2 to enhance VEGFA-induced EC proliferation by exacerbating STAT3 activation, leading to pathological angiogenesis.Retinopathy of prematurity (ROP) is an important cause of vision loss and blindness in infants worldwide.¹ Because of the limited ability to study human preterm infant eyes, models have been established in which newborn animals that normally vascularize their retinas after birth are exposed to oxygen stresses that lead to retinal features similar to human ROP.² Based on such models of oxygen-induced retinopathy (OIR) and on observations in human infants, ROP has been described as having two phases.^3,4 In phase I, infants experience delayed physiological retinal vascular development and sometimes vasoattenuation from high oxygen.⁵ Phase II is characterized by aberrant disordered developmental angiogenesis in the form of vasoproliferative intravitreal neovascularization (IVNV).² Several angiogenic agonists and inhibitors have been recognized as potentially involved in human ROP. Of these, the most studied is vascular endothelial growth factor A (VEGFA).^6–9Besides being involved in human pathological angiogenic eye disease,¹⁰ VEGFA is also important in retinal vascular development.^11,12 Inhibition of the bioactivity of VEGFA in preterm infants with severe ROP reduced the IVNV of phase II,¹³ but reports of persistent avascular retina and recurrent pathological angiogenesis raised concern.¹⁴ Furthermore, neutralizing VEGFA with an antibody similar to that used in human preterm infants with severe ROP initially reduced IVNV in the rat ROP model, but caused recurrent pathological angiogenesis in association with up-regulation of several angiogenic agonists, including erythropoietin (EPO).¹⁵EPO is known mainly for hematopoiesis, and it has been used to treat anemia.¹⁶ However, a growing body of evidence indicates that EPO has other biological effects, including neuroprotective,^17–19 antiapoptotic,^19,20 antioxidative,^20,21 and angiogenic^22–24 properties. Evidence supporting the role of EPO in angiogenesis comes from clinical and animal studies. In clinical studies, proliferative diabetic retinopathy and severe ROP have been associated with increased EPO. In proliferative diabetic retinopathy, vitreous EPO was increased,²⁵ and a promoter polymorphism in the EPO gene resulting in increased production of EPO was associated with severe diabetic retinopathy in a largely European-American population.²⁶ In ROP, greater risk of severe ROP was associated with EPO treatment for anemia of prematurity.^27,28 In a mouse OIR model, hyperoxia down-regulated EPO expression in the retina and decreased vascular stability in association with vaso-obliterated retina,²⁹ and, after relative hypoxia, retinal EPO was increased and contributed to IVNV.³⁰ EPO was also identified as a target in OIR in a study using a transgenic mouse in which hypoxia inducible factor 2α (HIF-2α; EPAS1, alias HLF) was knocked down,³¹ and EPO synergistically increased VEGFA-induced human retinal microvascular endothelial cell (hRMVEC) proliferation.¹⁵ However, EPO also promoted physiological retinal vascularization in a rat OIR model.³² Thus, the evidence is mixed, in that EPO has been associated with both physiological and pathological retinal angiogenesis. EPO is now being considered as a neuroprotective agent to promote cognitive development in preterm infants.³³ Greater understanding is needed regarding EPO and EPO receptor (EPOR) signaling in ROP and developmental angiogenesis.In the present study, we used a rat ROP model³⁴ in which VEGFA is overexpressed by postnatal day 12 (p12) and causes IVNV at p18.³⁵ Our research group previously found that the VEGFA signal is detected in Müller cells and developed a method using a lentiviral vector that targets Müller cells and knocks down VEGFA in vivo, thereby inhibiting IVNV without interfering with pup growth or serum VEGFA.³⁶ The present investigation of the role of EPOR adapted lentiviral vector rat model. In phase I, EPOR activation was lower than in phase II, when VEGFA expression and VEGFR2 expression and activation were increased.³⁷ In hRMVECs, VEGFA up-regulated and activated EPOR and (through crosstalk between activated VEGFR2 and EPOR) increased the activation of STAT3 to enhance angiogenesis. Our present findings support the hypothesis that VEGFA activates and causes an interaction between EPOR and VEGFR2 to contribute to pathological angiogenesis.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Short Hairpin RNA-Mediated Knockdown of VEGFA in Müller Cells Reduces Intravitreal Neovascularization in a Rat Model of Retinopathy of Prematurity

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30–embedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNA–targeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.Retinopathy of prematurity (ROP) remains a leading cause of childhood blindness and is increasing in frequency in developing countries. The hypothetical proposed pathophysiological characteristics of ROP have been recently refined to be that stresses in prematurity cause delayed physiological retinal vascular development and potentially some high oxygen-induced capillary constriction that results in avascular retina.^1–4 Once supplemental oxygen is removed from the preterm infant, the retina becomes hypoxic, and hypoxia stimulates the release of angiogenic factors with growth of new blood vessels into the vitreous as intravitreous neovascularization (IVNV). Many angiogenic factors can result in pathological IVNV in animal models, such as insulin-like growth factor-1,^5,6 hepatocyte growth factor,⁷ erythropoietin,^8–10 platelet-derived growth factor,¹¹ and angiopoietins,^12,13 but vascular endothelial cell growth factor A (VEGFA) has become one of the most studied factors leading to IVNV. VEGFA mRNA was found in the retina of a preterm infant eye with severe ROP,¹⁴ and VEGFA protein was increased in vitreous from preterm infants who underwent surgery for stage 4 ROP compared with controls.¹⁵ VEGFA inhibitors reduce pathological angiogenesis in adult retinal diseases, including diabetic retinopathy^16,17 and age-related macular degeneration.^18–20 Therefore, there is reason to consider VEGFA in the pathological characteristics of human ROP. However, in the preterm infant retina, VEGFA is also important in the development of retinal blood vessels^21–23 and other organs.^24,25 After a recent clinical trial testing intravitreal delivery of a broad anti-VEGFA antibody in infants with severe ROP, there have been reports of persistent avascular retina and reactivation of IVNV with subsequent total retinal detachment, even 1 year after treatment.²⁶ In addition, by using a relevant ROP model, we found that inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area without adversely affecting physiological retinal vascular development 6 days after antibody injection, but significantly reduced body weight gain in the pups, suggesting an adverse effect.²⁷ Therefore, safer ways to inhibit pathological IVNV while preserving physiological retinal vascularization are needed.One way to target pathological IVNV is to determine the cells within the retina that overproduce VEGFA during pathological stress. In preterm infant eyes, it is not possible to safely localize where VEGFA is produced. Therefore, we used a relevant model of ROP today, the rat 50/10 oxygen-induced retinopathy (OIR) model, to localize the VEGFA signal within the retina and determine its role in pathological IVNV in ROP. This model causes features of severe ROP and produces extrauterine growth restriction, a risk for ROP in human preterm infants.²⁸ The oxygen exposure recreates arterial oxygen fluctuations similar to those experienced by infants with severe ROP.²⁹ Previously, we found that VEGFA and VEGFR2 were both increased as early as at postnatal day 8 (p8) in whole retinas from eyes of pups in the 50/10 OIR model compared with room air–raised counterparts.³⁰In the retina, several cells have been shown to produce VEGFA to support retinal development and physiological functioning. These include ganglion cells,³¹ astrocytes,³² Müller cells, and retinal pigment epithelium.³³ In pathological IVNV, the VEGFA signal has been localized to many of the same cells: Müller cells,^34,35 astrocytes,³⁶ and, possibly, ganglion cells. However, there has been disagreement as to the cell type that overproduced VEGFA to cause IVNV,^34,36 and these articles also used the mouse model of OIR that exposes pups to constant and higher oxygen levels than currently used in the management of ROP.³⁷In the current study, we generated shRNAs to VEGFA and used a lentiviral miRNA-based system to target Müller cells, where the VEGFA message was localized in the 50/10 OIR model. We tested the hypothesis that knocking down VEGFA to physiological levels in Müller cells would inhibit IVNV without adversely affecting physiological retinal vascular development.     

FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration

Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain–containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain–containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal–regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration.CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.The asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor 1α (FIH-1; official symbol HIF1AN) was originally identified as a protein that interacts with and inhibits the activity of hypoxia-inducible factor 1α (HIF-1α) in the C-terminal transactivation domain^1,2 by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α.³ Significantly, proteins containing the ankyrin repeat domain, such as Notch, are other substrates for FIH-1.³ Only recently has FIH-1 been recognized to have pleiotropic roles in maintaining epithelial homeostasis.^4,5 For example, FIH-1 negatively regulates glycogen metabolism in corneal epithelium in a HIF-1α–independent manner via the direct involvement of the Akt/glycogen synthase kinase 3β signaling pathway.⁴ Furthermore, in epidermal and corneal epithelial keratinocytes, FIH-1 was shown to act as a negative regulator of differentiation via a coordinate decrease in Notch signaling.⁵ What is not clear in these studies is whether FIH-1 affects other signaling pathways known to influence keratinocyte growth, differentiation, and migration. For example, the regulation of Notch 1 activity by FIH-1⁵ raises the possibility of cross-talk with the epidermal growth factor receptor (EGFR)-signaling pathway, since EGFR signaling has been shown to be a negative regulator of Notch 1 gene expression and activity in keratinocytes.⁶Once EGF binds to the EGFR, numerous signaling pathways are activated that impact on cell proliferation, migration, differentiation, and survival.^7–9 With respect to the skin, EGFR impacts on epidermal and hair follicle development, keratinocyte proliferation, survival, cancer, and immune homeostasis.¹⁰ EGFR signaling also plays a prominent role in epidermal and corneal epithelial migration and wound repair. For example, in the epidermis, EGFR signaling has been shown to promote keratinocyte migration and wound repair.¹¹ Likewise, corneal perturbations activate the EGFR and downstream Ras-Raf-Mek-Erk1/2 (Ras, Raf, mitogen-activated protein kinase kinase, extracellular signal–regulated kinase 1/2) and phosphoinositide 3 kinase–Akt signaling cascades, which are required for efficient wound healing and are attenuated in patients with diabetic keratopathies.^12–14The activation of EGFR also commences endocytic trafficking, whereby the receptor is either packaged in lysosomes for degradation or recycled to the cell surface.^15,16 Endosomal trafficking is essential for establishing the extensiveness of the EGF-mediated signal, and thus much attention has been directed toward understanding the steps involved in the movement of the EGFR from the cell surface to cytoplasmic vesicles, such as the endosome, multivesicular body, and lysosome.^16–18 Recently, leucine-rich repeat kinase 1 (LRRK1) was recognized as a key regulator of EGFR endosomal trafficking.^19,20 Specifically, it is believed that LRRK1 forms a complex with activated EGFR through an interaction with growth factor receptor–bound protein 2 and that this complex is internalized in early endosomes.¹⁹ The mechanism by which LRRK1 regulates EGFR transport is from early to late endosomes.¹⁹LRRK1 protein kinase is one of the ROCO proteins, which contain a GTPase-like domain [Ras of complex proteins (Roc)] and a C-terminal of Roc (COR) domain.²¹ ROCO proteins have a series of leucine-rich repeats and/or ankyrin repeats, with LRRK1 containing six N-terminal ankyrin repeats.²² This latter aspect of LRRK1 is noteworthy since, as mentioned above, proteins with ankyrin repeat domains are potential substrates for FIH-1.³ Thus, FIH-1 has the potential to directly interact with LRRK1, which could impact on EGFR signaling.Here we show that ectopic expression of FIH-1 in keratinocytes increases phosphorylation of the EGFR, which positively affects keratinocyte migration via stimulation of the mitogen-activated protein kinase pathway, resulting in an increase in phosphorylated ERK1/2. Such enhanced migration is independent of Notch signaling. Moreover, our studies reveal that FIH-1 interacts with LRRK1 and prevents the formation of an EGFR/LRRK1 complex necessary for proper EGFR turnover.¹⁹ The identification of LRRK1 as a substrate for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. Thus, the breadth of FIH-1 epithelial biology is considerably larger than previously realized.     

Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A^−/− mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A^−/− mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.To support efficient gas exchange, the lung must maintain a barrier between the atmosphere and fluid-filled tissues. Without this crucial barrier, the air spaces would flood, and gas exchange would be severely limited.^{1, 2} In acute lung injury and acute respiratory distress syndrome, fluid leakage into the lung air space is associated with increased patient mortality and morbidity.^{3, 4} Lung fluid clearance is maintained, in part, by tight junctions that regulate paracellular flux between cells.^{5, 6, 7}Tight junctions are multiprotein complexes located at sites of cell-cell contact and are composed of transmembrane, cytosolic, and cytoskeletal proteins that together produce a selective barrier to water, ions, and soluble molecules. Among the transmembrane proteins required for epithelial barrier function is the Ig superfamily protein junctional adhesion molecule A (JAM-A).^{8, 9, 10, 11} JAM-A is ubiquitously expressed and regulates several processes related to cell-cell and cell-matrix interactions, including cell migration and proliferation in addition to barrier function regulation. Specific mechanistic roles for JAM-A in regulating tight junctions continue to be elucidated.JAM-A signaling is stimulated by cis-dimerization, which provides a platform for multiple proteins to cluster in close apposition.¹² In particular, JAM-A has been shown to recruit scaffold proteins, such as zonula occludens 1 (ZO-1), ZO-2, and Par3, to tight junctions, where these proteins enhance the assembly of multiprotein junctional complexes.^{13, 14} More recently, it was demonstrated that JAM-A directly interacts with ZO-2, which then recruits other scaffold proteins, including ZO-1.¹⁵ This nucleates a core complex that includes afadin, PDZ-GEF1, and Rap2c and that stabilizes filamentous actin by repressing rhoA.¹⁵ Together, all of these activities of JAM-A promote tight junction formation and barrier function.Although JAM-A is part of the tight junction complex, the main structural determinants of the paracellular barrier are proteins known as claudins. Claudins are a family of transmembrane proteins that interact to form paracellular channels that either promote or limit paracellular ion and water flux.^{16, 17, 18} Claudins that promote flux are known collectively as pore-forming claudins, whereas claudins that limit flux are known as sealing claudins.¹⁹ In fact, there is a link between JAM-A and claudin expression because it was demonstrated that JAM-A–deficient intestinal epithelium has increased expression of two pore-forming claudins, claudin-10 and claudin-15.²⁰ Critically, increased claudin-10 and claudin-15 leads to a compromised intestinal barrier, as demonstrated by an enhanced susceptibility of JAM-A^−/− mice to dextran sulfate sodium–induced colitis.²⁰ However, it is not known whether this relationship between JAM-A and claudin expression occurs in other classes of epithelia.Several claudins are expressed by the alveolar epithelium. The most prominent alveolar claudins are claudin-3, claudin-4, and claudin-18; several additional claudins are expressed by alveolar epithelium and throughout the lung as well.^{21, 22} A central role for claudin-18 in regulating lung barrier function was demonstrated in two independently derived strains of claudin-18–deficient mice that showed altered alveolar tight junction morphologic features and increased paracellular permeability.^{23, 24} Claudin-4 also is an important part of the lung response to acute lung injury because it improves barrier function by limiting alveolar epithelial permeability and promoting lung fluid clearance.^{25, 26} Although claudin-4–deficient mice show a relatively mild baseline phenotype, these mice have impaired fluid clearance in response to ventilator-induced lung injury.²⁷ An analysis of ex vivo perfused human donor lungs revealed that increased claudin-4 was linked to increased rates of alveolar fluid clearance and decreased physiologic respiratory impairment,²⁸ further underscoring the importance of claudin regulation in promoting efficient barrier function in response to injury.Although JAM-A has a clear role in regulating gut permeability,²⁰ a recent report that wild-type and JAM-A^−/− mice show comparable levels of pulmonary edema in response to intratracheal endotoxin challenge²⁹ raises questions about potential roles for JAM-A in lung barrier function. Herein we used a combination of in vivo and in vitro approaches to assess the contributions of JAM-A to alveolar barrier function. Using a model of mild systemic endotoxemia induced by i.p. injection of Escherichia coli–derived lipopolysaccharide (LPS), we found that JAM-A^−/− mice showed greater lung edema than comparably treated wild-type mice. Greater sensitivity to injury was due to aberrant regulation of tight junction protein expression, which was recapitulated by JAM-A–depleted alveolar epithelial cells. JAM-A depletion also resulted in decreased β1 integrin protein levels and disrupted cytoskeletal assembly. Together, these effects indicated that the loss of JAM-A impaired tight junction formation, thus rendering the lung more susceptible to edema and injury.