IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes).^{1, 2} Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.²Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.^{3, 4, 5} HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.^{6, 7, 8, 9} About 40–70% of surgeries and over 91% of burn injuries result in HS.¹⁰ A key feature of HS is a metabolic disorder of collagen-based ECM proteins.^{11, 12, 13} Autophagy has an important role in homeostasis of tissue structure and function.^{2, 14, 15} Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.^{16, 17, 18, 19, 20, 21, 22, 23}Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.^{24, 25, 26} Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.^{27, 28} IL10 has a pivotal role in wound healing^{29, 30} and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.^{9, 31, 32}Fibroblasts are one of the most important effector cells responsible for HS formation.^{12, 33, 34} Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs.     

Animal Models of Q Fever (Coxiella burnetii)

Q fever, caused by the pathogen Coxiella burnetii, is an acute disease that can progress to become a serious chronic illness. The organism leads an obligate, intracellular lifecycle, during which it multiplies in the phagolytic compartments of the phagocytic cells of the immune system of its hosts. This characteristic makes study of the organism particularly difficult and is perhaps one of the reasons why, more than 70 y after its discovery, much remains unknown about the organism and its pathogenesis. A variety of animal species have been used to study both the acute and chronic forms of the disease. Although none of the models perfectly mimics the disease process in humans, each opens a window onto an important aspect of the pathology of the disease. We have learned that immunosuppression, overexpression of IL10, or physical damage to the heart muscle in mice and guinea pigs can induce disease that is similar to the chronic disease seen in humans, suggesting that this aspect of disease may eventually be fully understood. Models using species from mice to nonhuman primates have been used to evaluate and characterize vaccines to protect against the disease and may ultimately yield safer, less expensive vaccines.Coxiella burnetii is the causative agent of human Q fever. Infection can take several forms and has been described as clinically polymorphic.⁶ In humans, presentation ranges from asymptomatic, through acute disease, to chronic illness. In the majority of cases, acute disease presents as a self-limiting febrile illness, with half of cases also having severe headaches.⁸⁸ In severe cases of acute disease, atypical pneumonia is often found.⁸⁸ A small proportion (2% to 4%) of subjects with symptomatic acute Q fever are admitted to hospital.^70,88 Chronic disease may develop in approximately 5% of those infected;¹⁶ the vast majority of these cases will present as a bacterial culture-negative endocarditis^16,22 often in those with predisposing heart-damage¹⁹ or immunosuppression.¹⁶ Without effective treatment, Q fever endocarditis is generally fatal, but early diagnosis coupled with novel treatment strategies has brought the death rate down to less than 5%.⁶⁹ The 2009 outbreak in the Netherlands involved 2357 human cases, of which more than 400 required hospitalization.⁹⁰ The animal cost in the Netherlands was far higher, with more than 50,000 pregnant goats culled in an attempt to control the epidemic.⁸²Two other clinical manifestations of Q fever are worthy of mention owing to their less-than-satisfactory outcomes with current treatment strategies. These are Q fever during pregnancy and Q fever fatigue syndrome. C. burnetii infection during pregnancy results in premature delivery in almost half of those affected and spontaneous abortion in more than a quarter.¹⁴ There have been few studies in this area, but there are indications that among those infected during the first trimester and treated suboptimally, the abortion rate is 100%.⁶⁸ This effect is compounded by the fact that the frontline bactericidal drugs for treatment (doxycycline and hydroxychloroquine) are contraindicated for use during pregnancy.⁶⁸ A bacteriostatic regimen (cotrimoxazole) has therefore been proposed for use⁶⁸ until delivery. Without satisfactory treatment during and after pregnancy, there is also a high probability for infection to lead to chronic Q fever: an incidence of 70% was reported in a group of pregnant women in France.⁶⁸Post-Q fever fatigue syndrome was first reported in 1996,⁵² but an association between Q fever and chronic fatigue had been observed as early as 1982.⁵² Between 10% and 15% of those who have had acute Q fever develop a chronic fatigue syndrome that can last between 5 and 10 y—and even longer in some cases.⁵³ Some of these patients have been found to have long-term persistence of C. burnetii cell components and LPS associated with traces of genomic DNA,⁵³ suggesting that Q fever fatigue syndrome may be immunologically mediated rather than caused by the organism directly.Q fever is a zoonosis that has been described worldwide,⁵⁶ and human outbreaks are often associated with contact with the birth products of farm animals.⁵⁶ However, outbreaks associated with the birth products of domestic cats have also been reported.⁵⁴ Human infection primarily occurs via the inhalation of infectious aerosols.⁵⁶ Over the past 10 y, outbreaks have been reported in the Netherlands,⁷¹ Slovenia,²⁶ the United Kingdom,^91,97,99 Israel,² Iraq,¹⁸ the United States,¹¹ Germany,²⁴ Bulgaria,⁶³ Croatia,⁵⁸ Spain,²³ Italy,⁸³ and France.⁸⁸A very small number of C. burnetii organisms can cause infection by inhalation. Infection has been predicted to be possible after exposure to only a single organism.³³ This low dosage, coupled with the organism''s ability to cause debilitating disease and high levels of resistance to various means of inactivation^67,77,78 have resulted in it being listed as a category B biologic warfare and bioterrorism agent by the Centers for Disease Control.⁴⁹Prevention of Q fever in man can be achieved by vaccination; the only vaccine available for general use is Q-Vax, which was licensed in Australia in 1989.⁵¹ This vaccine consists of formalin-inactivated C. burnetii whole cells, produced in chick embryos. Its use has been associated with severe local reactions in those with preexisting immunity. As a precaution, prevaccination screening (history, skin test, and serology) must therefore be performed prior to administration.³⁵ Despite this safeguard, severe local reactions to vaccination are reported.⁴⁴ The vaccine is also hazardous to produce, with the organism requiring culture in chick-embryos at biosafety level 3 prior to inactivation.⁵¹ There is, therefore, a need for a vaccine that is safer to produce and safer to use and that does not require prevaccination screening.The organism displays antigenic phase variation often paralleled with the rough-smooth variation seen in Enterobacteriaceae. In C. burnetii, phase variation has been demonstrated to be due to differences in LPS. Phase I has been shown to contain a unique disaccharide galactosaminuronyl glucosamine and 9 unidentified components in addition to the components of phase II LPS.¹ Organisms with the phase I phenotype are the infectious and virulent form found in the environment. Organisms with the phase II phenotype are observed only during repeated subculture in laboratory chick embryo or cell culture systems;²⁷ they have a chemically simpler LPS¹ and several deletions in the genome.^32,92 Phagocytosis of phase I, but not phase II, organisms by macrophages involves an interaction between the bacterial LPS and Toll-like receptor 4. This mechanism also stimulates F-actin reorganization of the host cells and stimulates the release of type 1 cytokines including IFNγ and TNF.³⁰ This interaction appears important in the initial priming of the immune response and could provide an explanation for the limited protection of vaccines based on potential virulence genes (omp1, HspB, Pmm, Fbp, Orf 410, Crc, CbMip, MucZ, P28) singly and in combinations but containing no LPS.^47,89,102In addition to its antigenic phase variation, C. burnetii occurs in 2 morphologic forms, a large-cell variant and a small-cell variant. These forms differ antigenically due to differences in the proteins expressed on their surface. It has been suggested that the resistance of C. burnetii to host defense mechanisms may be enhanced by antigenic differences between the different developmental forms.^57,94 The small-cell morphologic form is highly resistant to destruction by chemical and environmental factors and is likely the transmissible form of the pathogen.^15,67 After infection, which generally occurs by inhalation of the small-cell form, the organisms are taken up by host alveolar macrophages.⁸¹ Morphogenesis from the small-cell to large-cell form then occurs, the large-cell variant being the replicative form of the organism.¹⁵ These organisms then replicate within parasitophorous vacuoles.⁵⁰ As the organisms enter the stationary phase of their growth within the cell, they condense back into the small-cell form.¹⁵ During replication within the host cell, the organism subverts cellular processes though active mechanisms to avoid and modify the host immune response.⁵⁰ C. burnetii possesses a type IV secretion system, and the proteins that cause this subversion are likely delivered to the host cell by this machinery.^50,93Because C. burnetii is an obligate intracellular organism, it has only been possible to study the organism within living animal hosts. Host-cell–free growth of the organism has been reported recently,⁶² but the technique has yet to be exploited fully. Cell-culture–based in vitro systems remain limited in the study of C. burnetii, given that the organism soon reverts to the avirulent (at least in immunocompetent hosts) phase II form (characterized by the loss of the phase I LPS phenotype) in these systems.¹⁰ A key problem in comparing models of C. burnetii infection is related to the organism''s intracellular nature, which complicates attempts to count the organisms used for infection. The literature reflects this difficulty in the fact that there are many different methods used (including plaque assay in primary cell cultures, median infectious doses in chick eggs or mice, and median lethal dose in SCID mice) and no way to directly compare them.     

Effects of Helicobacter Infection on Research: The Case for Eradication of Helicobacter from Rodent Research Colonies

Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.The reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome. Infections of mouse colonies are important to the scientific community because they can introduce such harmful variables. Therefore, the ultimate goal of laboratory animal facilities is to maintain disease-free animals, to eliminate those unwanted variables.Numerous pathogenic microbes can interfere with animal research (reviewed in reference 57), and colonization of mouse colonies with members of the family Helicobacteriaceae is an increasing concern for the research community. Naturally acquired Helicobacter infections have been reported in all commonly used laboratory rodent species.^{3,10,36,44,45,49,82,124} A study of mice derived from 34 commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States showed that 88% of these institutions had mouse colonies infected with 1 or more Helicobacter spp.¹⁰⁹ Approximately 59% of these mice were infected with Helicobacter hepaticus ; however monoinfections with other species also were encountered. In another study, at least 1 of 5 Helicobacter spp. was detected in 88% of the 40 mouse strains tested.⁴Surveys such as these have established that a broad range of Helicobacter spp. may be present in mouse research colonies. Several of those Helicobacter species cause disease in laboratory mice. H. hepaticus first was identified as a pathogen when it was discovered to be the cause of chronic hepatitis and hepatocellular carcinoma in mice,^26,31,116 either alone or in combination with other Helicobacter spp.⁷⁸ In addition, H. typhlonius causes intestinal inflammation in mice with immunodeficiency or defects in immune regulation;^28,37 H. muridarum has been associated with gastritis,⁸⁶ and H. bilis has been associated with hepatitis^35,38 and colitis.^60,61 Although, H. rodentium appears to be relatively nonpathogenic in wild-type and SCID mice,⁷⁸ combined infection with H. rodentium and H. typhlonius results in a high incidence of inflammation-associated neoplasia in IL10^−/− mice.^9,46 Further, it is becoming increasingly clear that the effects of Helicobacter infections are not limited to the gastrointestinal system. Helicobacter infections have been documented to directly or indirectly affect responses as diverse as reproduction, development of breast cancer, and altered immune responses to vaccines.^65,95,99 In addition to effects on rodents, Helicobacter spp. can infect other laboratory animals^{2,5,27,29,33,36,107} and can colonize different anatomic regions of the gastrointestinal system.³⁵ This review focuses on the potential effect of these organisms on in vivo experiments and biomedical research. The results summarized here emphasize the importance of knowledge of colony infection status and prevention of unintentional infections to achieve the goal of providing a consistent and reliable environment for research studies.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Housing Conditions Modulate the Severity of Mycoplasma pulmonis Infection in Mice Deficient in Class A Scavenger Receptor

Mycoplasmosis is a frequent causative microbial agent of community-acquired pneumonia and has been linked to exacerbation of chronic obstructive pulmonary disease. The macrophage class A scavenger receptor (SRA) facilitates the clearance of noxious particles, oxidants, and infectious organisms by alveolar macrophages. We examined wildtype and SRA^−/− mice, housed in either individually ventilated or static filter-top cages that were cycled with fresh bedding every 14 d, as a model of gene–environment interaction on the outcome of pulmonary Mycoplasma pulmonis infection. Intracage NH₃ gas measurements were recorded daily prior to infection. Mice were intranasally infected with 1 × 10⁷ cfu M. pulmonis UAB CT and evaluated at 3, 7, and 14 d after inoculation. Wildtype mice cleared 99.5% of pulmonary M. pulmonis by 3 d after infection but remained chronically infected through the study. SRA^−/− mice were chronically infected with 40-fold higher mycoplasma numbers than were wildtype mice. M. pulmonis caused a chronic mixed inflammatory response that was accompanied with high levels of IL1β, KC, MCP1, and TNFα in SRA^−/− mice, whereas pulmonary inflammation in WT mice was represented by a monocytosis with elevation of IL1β. Housing had a prominent influence on the severity and persistence of mycoplasmosis in SRA^−/− mice. SRA^-/- mice housed in static cages had an improved recovery and significant changes in surfactant proteins SPA and SPD compared with baseline levels. These results indicate that SRA is required to prevent chronic mycoplasma infection of the lung. Furthermore, environmental conditions may exacerbate chronic inflammation in M. pulmonis-infected SRA^−/− mice.Abbreviations: BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; KC, keratinocyte-derived chemokine (CXCL1); MCP1, monocyte chemotactic protein 1; SPA, surfactant protein A (SFTPA1); SPB, surfactant protein B (SFTPB); SPD, surfactant protein D (SFTPD); SRA, class A scavenger receptor (MSR1); WT, wildtypeThere are numerous options for the housing and husbandry of rodents in the laboratory setting. Various available choices in caging, bedding material, and cage-change frequency have the potential to effect physiologic values and thus experimental outcomes.^20,108 In many facilities, current practices involve performing cage changes every 1 to 2 wk, with some facilities exploring the possibility of extending these practices to every 4 wk.⁹⁷ Cage-change frequency practices are established at various institutions after consideration of several variables that affect animal health, welfare, and cost. Ideally, an appropriate sanitation program provides clean and dry bedding, adequate air quality, and clean cage surfaces and accessories.⁴⁴ When establishing performance standards for a sanitation program that are different from those which are recommended in the Guide for the Care and Use of Animals in Research,⁴⁴ microenvironmental conditions, including intracage humidity, temperature, animal behavior and appearance, microbiologic loads, and levels of pollutants such as CO₂ and NH₃, should be evaluated and verified. Although there are currently no established NH₃ exposure limits for laboratory animals, the human occupational exposure limit of 25 ppm as an 8-h time-weighted average, established by the National Institute for Occupational Safety and Health, is often referenced as a guideline for animals.⁹⁵ Multiple factors, such as animal cage density, sex, age, bedding type, reusable compared with disposable caging, static caging compared with IVC, and cage-change frequency, influence intracage and ambient NH₃ levels.^82,83,97 Only limited information is available that addresses the effect of natural intracage NH₃ levels on respiratory function in experimental rodents and whether exposure to high NH₃ levels under current standard practices affects the results of respiratory disease research.Ammonia is an alkaline, corrosive, and irritant gas that is very water soluble. It reacts with the moisture of the mucous membranes of the eyes, mouth, and respiratory tract to form ammonium hydroxide in an exothermic reaction, resulting in thermal and chemical burns.⁶⁸ Clinical symptoms in humans exposed to high levels of NH₃ include eye irritation, headaches, and multiple acute and chronic respiratory symptoms, such as irritation of the nose, pharynx, and sinuses, and in severe cases, development of bronchitis and hyper-reactive airway disease.⁷⁹ Animals are similarly susceptible to NH₃-induced pulmonary disease.^23,31,48Mice exposed to naturally increasing levels of intracage NH₃ can develop lesions in the rostral nasal cavity, with decreasing severity of the lesions moving caudally into the nasopharynx, and no lesions in the lung.⁹⁷ However, dust is another common environmental pollutant that is often present in animal settings. Dust particles readily absorb NH₃, which then serve as a source of NH₃ deposition into the lower respiratory tract. Dust particulate can range from large (300 µm), minimally respirable particles to very fine (< 50 µm) particulate matter, which can settle deep within the alveoli.^10,102 The mucociliary system of the respiratory tract is the first line of defense against inspired noxious stimuli and pathogens. Exposure of the ciliated respiratory epithelium to the damaging effects of NH₃ are known to cause decreased mucociliary beating.⁵⁶ Disruption of the respiratory mucociliary escalator initiated by NH₃ exposure can then promote establishment of chronic infections and inflammation of the airway mucosa.^11,87 Therefore, NH₃ potentially can cause pathophysiologic changes of the lung in the absence of histopathologic lesions.Our primary goal was to analyze the effect of 2 housing modalities, which result in different intracage NH₃ concentrations, on mice that were challenged with a respiratory pathogen. Mycoplasma pulmonis was chosen as a model because it is a well-established model in rodents which causes chronic mycoplasmosis and reproduces the features of M. pneumoniae in humans.^22,41 M. pneumoniae infection is a frequent and contagious etiology of community-acquired pneumonia causing tracheobronchitis, sneezing, cough, and inflammation of the respiratory tract.^8,12,47,63 Moreover, atypical and difficult-to-detect respiratory pathogens such as Chlamydophila pneumoniae and Mycoplasma pneumoniae that can establish chronic asymptomatic infections may contribute to both the development and exacerbation of COPD^{26,45,57,58,62,63,66,72,96,103} and asthma.^8,51,65 Infection with M. pulmonis in rodents causes rhinitis, otitis media, tracheitis, and pneumonia, which can be exacerbated by housing conditions and genetic background.^14,32,85 The mechanism of pathogenicity of mycoplasmas continues to be an area of interest in the research.The innate host factors protecting against pulmonary mycoplasmosis include the secreted surfactant protein opsonins SPA and SPD, surfactant phospholipids, and the molecular pattern-recognition receptor TLR2.^15,16,54,74 Therefore, compared with their wildtype (WT) counterparts, SPA-deficient mice infected with either M. pulmonis or M. pneumoniae develop more severe inflammation and have decreased capacity to clear these infections from the lungs.⁴³ In addition, TLR2-deficient mice exhibit decreased clearance and increased inflammation in response to mycoplasma infection.^60,104Second, we wanted to study the effects of SRA deficiency in mycoplasmosis. The class A scavenger receptor (SRA) modulates inflammatory responses and mediates the clearance of airborne oxidants, particulates, and respiratory pathogens.^{3,17,18,49,88,101} Inhibition of SRA expression in alveolar macrophages in an elastase–LPS model of COPD was associated with decreased clearance of Haemophilus influenzae.³³ Lack of SRA similarly impaired alveolar macrophage-mediated clearance of Streptococcus pneumoniae,⁵ environmental particles,⁶ and ozone-oxidized lipids¹⁸ by alveolar macrophages. Absence of SRA also enhanced hyperoxia-induced lung injury⁴⁹ and exacerbated inflammation in response to Staphylococcus aureus infection.⁸⁸ SRA appears to have antiinflammatory properties with the capacity to modify macrophage phenotype and suppress polarization toward the M1 alternative macrophage activation state.¹³ The SRA gene (MSR1) is polymorphic in both mice and humans.^19,29,105 Genetic association studies in humans, however, showed that subjects with truncations or point mutations in MSR1 have significantly increased risk for the development of pulmonary diseases such as COPD^33,38,71,94 and asthma.⁵ Our understanding of the immune factors that contribute to mycoplasmosis is far from complete.In the present study, by investigating the role of SRA in mycoplasmosis jointly with the effects of housing, we demonstrated that genetic and environmental factors both serve as critical players in disease progression. We show that SRA-deficient mice are susceptible to chronic colonization with M. pulmonis and development of chronic mycoplasma-induced bronchopneumonia characterized by persistent multicellular inflammation. Furthermore, we show that housing conditions influence the effect of SRA deficiency on the severity of mycoplasmosis. Taken together, these results indicate that lack of SRA function impairs host protection against both infectious and environmental insults.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Serologic Evaluation of Clinical and Subclinical Secondary Hepatic Amyloidosis in Rhesus Macaques (Macaca mulatta)

Secondary hepatic amyloidosis in nonhuman primates carries a grave prognosis once animals become clinically ill. The purpose of this study was to establish serologic parameters that potentially could be used to identify rhesus macaques undergoing subclinical development of secondary hepatic amyloidosis. A retrospective analysis was completed by using serum biochemical profiles from 26 histologically diagnosed amyloidotic macaques evaluated at 2 stages of disease, clinical and subclinical (3 to 32 mo prior to clinical signs of disease). Standard serum biochemistry values for cases were compared with institutional age- and gender-specific references ranges by construction of 95% confidence intervals for the difference between means. In addition, 19 histologically diagnosed amyloidotic macaques and 19 age-matched controls were assayed for changes in various parameters by using routinely banked, frozen (–80 °C) sera available from clinical and subclinical time points. Clinically amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyltranspeptidase, and macrophage colony-stimulating factor and significantly decreased quantities of albumin and total cholesterol. Subclinical amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, and serum amyloid A and decreased concentrations of albumin and total cholesterol. The serologic parameters studied indicate a temporal relationship of these factors not previously described, show a clear pattern of disease progression, and could be useful in subclinical disease detection.Abbreviations: mCSF, macrophage colony stimulating factor; SAA, serum amyloid AAmyloid is an eosinophilic substance made of insoluble fibrillar protein.³² When deposited extracellularly, amyloid causes displacement of tissue form and disruption of organ function.³² Persistent accretion of amyloid can result in organ failure and ultimately animal death.²² Clinical signs of disease depend on the tissues affected and the degree of involvement.³² Amyloidosis has been well documented in humans, other mammals, birds, and reptiles.³⁸ In humans, amyloidosis plays a key role in many diseases, including Alzheimer disease, type II diabetes, rheumatoid arthritis, and Down syndrome.^15,20,35,38Amyloidosis generally is classified into 3 categories: primary, secondary, and hereditary. Primary amyloidosis consists of the immunoglobulin- and myeloma-associated types. Secondary (reactive) amyloidosis is associated with chronic inflammation.²⁴ Common causes of secondary amyloidosis in humans include rheumatoid arthritis, idiopathic colitis, infectious diseases, such as tuberculosis and leprosy, and malignant tumors, such as mesothelioma and Hodgkins disease.²⁸ Hereditary amyloid syndromes are rare and include Mediterranean fever, Muckle–Wells syndrome, and familial amyloid cardiomyopathy.^32,38Secondary amyloidosis is the most common form of amyloidosis in animals.³⁸ Amyloidosis occurs in many species of nonhuman primates including the common marmoset (Callithrix jacchus),²³ squirrel monkey (Saimiri sciureus),³⁴ rhesus macaque (Macaca mulatta),^9,10 pigtailed macaque (Macaca nemestrina),^18,27 crab-eating macaque (Macaca fascicularis),²⁷ barbary ape (Macaca sylvanus),⁶ baboon (Papio spp.),¹⁷ mandrill (Papio sphinx), and chimpanzee (Pan troglodytes).^16,39 Although a definitive cause of secondary amyloidosis has not been identified in nonhuman primates, this condition has been associated with chronic inflammation due to rheumatoid arthritis,⁶ viral infection,¹⁸ parasitism,¹ respiratory disease,^27,30 trauma,³⁰ and bacterial enterocolitis.^27,30,31 Shigella spp. have received particular attention as a common etiology linking enterocolitis with amyloidosis.^4,7,38Previous research on amyloidosis in nonhuman primates has yielded clinical and serologic profiles in end-stage amyloidotic animals, but little is known about the serologic status in the subclinical stages of disease. Amyloid can accumulate for as long as 3 y before severe organ disruption occurs¹⁴ and clinical signs of amyloidosis become evident.¹⁶ With appropriate analysis, detection of amyloidosis could occur much earlier than typically now achieved, thus allowing for targeted preventative therapy to potentially halt the progression of this insidious disease.     

Infection with Murine Norovirus 4 Does Not Alter Helicobacter-Induced Inflammatory Bowel Disease in Il10?/? Mice

Infection of laboratory mice with murine noroviruses (MNV) is widely prevalent. MNV alters various mouse models of disease, including the Helicobacter bilis-induced mouse model of inflammatory bowel disease (IBD) in Mdr1a^−/− mice. To further characterize the effect of MNV on IBD, we used mice deficient in the immunoregulatory cytokine IL10 (Il10^−/− mice). In vitro infection of Il10^−/− bone marrow-derived macrophages (BMDM) with MNV4 cocultured with H. bilis antigens increased the gene expression of the proinflammatory cytokines IL1β, IL6, and TNFα as compared with that of BMDM cultured with H. bilis antigens only. Therefore, to test the hypothesis that MNV4 infection increases inflammation and alters disease phenotype in H. bilis-infected Il10^−/− mice, we compared the amount and extent of inflammation in Il10^−/− mice coinfected with H. bilis and MNV4 with those of mice singly infected with H. bilis. IBD scores, incidence of IBD, or frequency of severe IBD did not differ between mice coinfected with H. bilis and MNV4 and those singly infected with H. bilis. Mice infected with MNV4 only had no appreciable IBD, comparable to uninfected mice. Our findings suggest that, unlike in Mdr1a^−/− mice, the presence of MNV4 in Il10^−/− mouse colonies is unlikely to affect the IBD phenotype in a Helicobacter-induced model. However, because MNV4 altered cytokine expression in vitro, our results highlight the importance of determining the potential influence of MNV on mouse models of inflammatory disease, given that MNV has a tropism for macrophages and dendritic cells and that infection is widely prevalent.Abbreviations: BMDM, bone marrow-derived macrophages; IBD, inflammatory bowel disease; MLN, mesenteric lymph node; MNV, murine norovirusInflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn disease, is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. In addition, patients with IBD may be at increased risk of developing colorectal cancer.^15,46 Although the exact mechanisms of disease are still not understood fully, the pathogenesis of disease is likely multifactorial, with components of the innate and adaptive immune systems, host genetics, and environmental factors (for example, the commensal gut microflora) all playing a role.^4,37,55Animal models of IBD have been used to advance our knowledge and understanding of IBD pathogenesis and treatment.^{16,20,37,38,52} One such model that has been widely used to elucidate the mechanisms of IBD is the interleukin10–deficient (Il10^−/−) mouse.^{3,5,6,20,21,29,33,57} The antiinflammatory cytokine IL10 modulates both innate and adaptive immune responses.⁴¹ Produced mainly by dendritic cells, monocytes, macrophages, and T regulatory cells, IL10 exerts its immunomodulatory effects by various mechanisms including decreasing secretion of proinflammatory cytokines (for example, interferon γ, IL1, IL2, IL6, IL12 and TNFα) and downregulating important components of innate immune responses and T-cell activation (for example, MHC class II, costimulatory molecules, and nitric oxide production) in antigen presenting cells.^14,41 As a consequence, Il10^−/− mice, which lack the suppressive effects of IL10, develop IBD in response to their commensal gut microflora or to certain microbial triggers such as Helicobacter infections.^{5,6,11,21,29,52,57}Antigen-presenting cells such as macrophages and dendritic cells play key roles in the inflammatory responses in IBD.^32,47,50 In 2003, a newly discovered murine norovirus (MNV) in laboratory mice was shown to infect macrophages and dendritic cells.^27,53 Subsequent studies indicated widespread MNV infection in laboratory mice used for biomedical research, with a serologic prevalence as high as 32%.^25,43 Members of the genus Norovirus are regarded as gastrointestinal pathogens in humans and animals, eliciting both innate and adaptive immune responses.¹⁹ Therefore, in light of the cellular (macrophages and dendritic cells) and tissue (gastrointestinal) tropisms of MNV as well as the high prevalence of MNV infection in laboratory mice, we hypothesized that MNV infection could be a potential confounder in mouse models of inflammatory diseases including IBD. In support of this idea, our laboratory recently reported that MNV infection in Mdr1a^−/− mice (FVB.129P2-Abcb1a^tm1Bor) accelerated weight loss and exacerbated IBD progression initiated by H. bilis infection.³¹ This effect potentially was mediated in part through modulating dendritic cell and cytokine responses. In addition, others have reported gastrointestinal abnormalities as a result of MNV infection in some strains of mice,^7,26,36 whereas others have described the importance of both innate and adaptive immune responses during MNV infection.^{8,9,10,28,34,36,48} Collectively, these data indicate that MNV could alter inflammatory responses in laboratory mice.Here we extended our studies of MNV beyond Mdr1a^−/− mice to Il10^−/− mice, another common animal model of IBD, to further examine the potential effect of MNV on IBD research. Disease was initiated in Il10^−/− mice with H. bilis, and we determined whether coinfection with MNV altered disease development, incidence, and severity and the production of cytokines. We demonstrated that although MNV stimulates a Th1 skewing of cytokines in Il10^−/− bone marrow-derived macrophages (BMDM) in vitro, MNV does not alter the development, incidence, or severity of disease in vivo. Therefore, although MNV may not affect disease in Il10^−/− mouse models, the virus may influence in vitro cytokine phenotypes and thus complicate interpretation of such data. To our knowledge, this report is the first to describe the evaluation of MNV infection in the Helicobacter-induced Il10^−/− mouse model of IBD.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.