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.     

Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl⁻ currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel by 4,4''-diisothiocya-natostilbene-2,2''-disulfonic acid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl⁻ channel blockers against ER stress-associated cardiac anomalies.The endoplasmic reticulum (ER) is characterized as an organelle that participates in the folding of membrane and secretory proteins.^1,2 Efficient functioning of the endoplasmic reticulum is important for cell function and survival. Perturbations of ER homeostasis by energy deprivation and glucose,³ viral infections⁴ and accumulation of misfolded and/or unfolded proteins² interfere with ER function, leading to a state of ER stress.^{5, 6, 7} A cohort of chemicals, for example, tunicamycin and thapsigargin, also trigger ER stress.^{8, 9, 10} Thapsigargin disrupts the calcium storage of ER by blocking calcium reuptake into the ER lumen, thus by depleting calcium from the organelle.¹¹ In particular, tunicamycin is a highly specific ER stress inducer by inhibiting N-linked glycosylation of protein, representing a well-documented method to artificially elicit unfolded protein response.⁸ In response to ER stress, ER chaperones such as glucose-regulated protein 78 kDa (GRP78) and glucose-regulated protein 94 kDa (GRP94) are upregulated to facilitate the recovery of unfolded or misfolded proteins.¹² ER stress may act as a defense mechanism against external insults; however, prolonged and/or severe ER stress may ultimately trigger apoptosis.⁸ The C/EBP homologous protein (CHOP) has been defined as a pivotal mediator of cell death signaling in ER stress.^{13, 14} Accumulating evidence has demonstrated that ER stress-induced cell death is an essential step in the pathogenesis of a wide variety of cardiovascular diseases such as ischemia reperfusion heart diseases,¹⁵ atherosclerosis,^{5, 16, 17, 18} myocardial infarction,¹⁹ hypertension^{20, 21} and heart failure.^{8, 22, 23} Inhibiting ER stress has great therapeutic values for cardiac anomalies. However, the precise mechanism involved in ER stress-induced cardiovascular diseases has not been well identified, which impedes the translation of our understanding of ER stress-induced cardiovascular anomalies into effective therapeutic strategies. Apoptosis induction requires persistent cell shrinkage, named apoptotic volume decrease (AVD).^{24, 25, 26, 27} It is an early prerequisite for the activation of caspases.²⁴ In various types of cells including cardiomyocytes, AVD process is accomplished by the activation of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel and is concomitant with the egress of water from the cells undergoing mitochondrion-initiated or death receptor-induced apoptosis.^{25, 28, 29, 30} Although inhibition of VSOR Cl⁻ channel by DIDS (4,4''-diisothiocyanatostilbene-2,2''-disulphonic acid) and DCPIB (4-(2-butyl-6,7- dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid) blocked AVD and rescued cardiomyocytes from mitochondrial and death receptor pathway-induced apoptosis,^{31, 32} it remains largely unknown concerning the role of VSOR Cl⁻ channel and how it is regulated in ER stress-induced apoptotic cardiomyocyte death.Emerging evidence indicates that Wnt signal pathways are found to be anti-apoptotic in the cardiovascular diseases,^{33, 34, 35} regulating crucial aspects of cardiovascular biology. However, up to now, its activity in ER stress-induced apoptosis and in the process of AVD in cardiomyocytes remains elusive.In the present study, we probed the role of VSOR Cl⁻ channel in ER stress-induced apoptosis of cardiomyocytes, which intimately correlates with cardiac contractile dysfunction (CCD). We hypothesized that VSOR Cl⁻ channel controls the process of AVD occurring concomitantly with ER stress-induced apoptosis of cardiomyocytes. To test this hypothesis, we investigated VSOR Cl⁻ currents in cardiomyocytes treated with the ER stress inducer tunicamycin. The pathophysiological role of VSOR Cl⁻ channel and the potential signaling mechanisms in the development of ER stress-induced apoptosis in CCD were also dissected.     

Suppression of epithelial–mesenchymal transition and apoptotic pathways by miR-294/302 family synergistically blocks let-7-induced silencing of self-renewal in embryonic stem cells

The embryonic stem cell (ESC)-enriched miR-294/302 family and the somatic cell-enriched let-7 family stabilizes the self-renewing and differentiated cell fates, respectively. The mechanisms underlying these processes remain unknown. Here we show that among many pathways regulated by miR-294/302, the combinatorial suppression of epithelial–mesenchymal transition (EMT) and apoptotic pathways is sufficient in maintaining the self-renewal of ESCs. The silencing of ESC self-renewal by let-7 was accompanied by the upregulation of several EMT regulators and the induction of apoptosis. The ectopic activation of either EMT or apoptotic program is sufficient in silencing ESC self-renewal. However, only combined but not separate suppression of the two programs inhibited the silencing of ESC self-renewal by let-7 and several other differentiation-inducing miRNAs. These findings demonstrate that combined repression of the EMT and apoptotic pathways by miR-294/302 imposes a synergistic barrier to the silencing of ESC self-renewal, supporting a model whereby miRNAs regulate complicated cellular processes through synergistic repression of multiple targets or pathways.Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into any cell type.¹ Therefore, they hold great potential for clinical applications in regenerative medicine. However, the molecular mechanisms regulating the self-renewal and differentiation of ESCs are still not fully understood. miRNAs are an important class of short non-coding RNAs that regulate ESC self-renewal and differentiation.² miRNA-deficient ESCs proliferate at a slower rate with a slight accumulation of cells in the G1 phase, and they cannot silence the self-renewal program when induced to differentiate.^{3, 4, 5} Introducing individual members from an miRNA family highly expressed in ESCs partially rescues the proliferation defect and reverses the G1 accumulation.⁶ The family shares a seed sequence (5′-AAGUGCU-3′) and has eight members, including miR-294 and miR-302a-d. Because of their role in influencing the ESC Cell Cycle, they have been called the ESCC family of miRNAs. In addition, ESC cell cycle regulating miRNAs (ESCC miRNAs) suppress the G1 restriction point by inhibiting retinoblastoma (Rb) family proteins, preventing ESCs from exiting the cell cycle during serum starvation or contact inhibition.⁷ In contrast to ESCC miRNAs, the introduction of let-7 family miRNAs that are enriched in somatic cells as well as several other lineage-specific miRNAs such as miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218 silences self-renewal in Dgcr8−/− (DiGeorge syndrome critical region gene 8−/−) ESCs but not wild-type ESCs.^{7, 8} Interestingly, the ESCC miRNAs prevent these miRNAs from silencing ESC self-renewal. Consistent with their roles in promoting self-renewal, ESCC miRNAs dramatically enhance the de-differentiation of human and mouse fibroblasts to induced pluripotent stem cells (iPSCs).^{9, 10, 11, 12, 13}How ESCC miRNAs maintain self-renewal in the presence of differentiation-inducing miRNAs is not clearly understood. Genomic studies have shown that these miRNAs target hundreds of mRNAs enriched in many biological processes.^{8, 14, 15, 16} Functional analysis of a small number of targets chosen based on their known roles has begun to give some insights into their functions in reprogramming somatic cells to iPSCs.^{10, 11, 17} However, due to the inherent differences between the maintenance and establishment of pluripotency,¹⁸ what targets or pathways underlie the antagonism between the two opposing families of miRNAs in regulating ESC self-renewal remains unknown. Recent work showed that while the miR-294/302 family suppresses and let-7 induces the G1/S restriction point, this cell cycle function cannot explain their antagonistic roles in maintaining pluripotency.⁷ Therefore, we set out to search for additional functions of the two miRNA families that directly underlie their opposing roles in regulating pluripotency. In this study, we found that combined repression of epithelial–mesenchymal transition (EMT) and apoptotic pathways by miR-294/302 forms a synergistic barrier to block the silencing of ESC self-renewal by let-7 and other differentiation-inducing miRNAs.     

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.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

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.     

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.     

Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl⁻ transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases.Neurodegeneration restricts neuron numbers during development but can become a serious issue in disease conditions such as temporal lobe epilepsy (TLE).¹ GABA-activated Cl⁻ channels contribute to activity-dependent refinement of neural networks by triggering the so-called giant depolarizing potentials providing developing neurons with a sense of activity essential for neuronal survival and co-regulation of excitatory glutamatergic and (inhibitory) GABAergic synapses.² By regulating transmembrane Cl⁻ gradients KCC2 plays a vital role in development and disease.³ In addition, KCC2 plays a protein structural role in spine formation through its C-terminal protein domain (CTD).^{4, 5} Hence, regulation of KCC2 expression and function is relevant for development and disease-specific plasticity of neural networks.^{6, 7, 8, 9}GlyR α3K RNA editing leads to proline-to-leucine substitution (P185L) in the ligand-binding domain and generates gain-of-function neurotransmitter receptors.^{10, 11, 12, 13} GlyR RNA editing is upregulated in the hippocampus of patients with TLE and leads to GlyR α3K^185L-dependent tonic inhibition of neuronal excitability associated with neurodegeneration.¹⁴ KCC2 expression promotes neuroprotection^{14, 15} but whether this involves regulation of transmembrane Cl⁻ gradient or protein structural role is a matter of debate.^{14, 15}Here, we assessed neuroprotection through several KCC2 variants in two different models of neurodegeneration including chronic neuronal silencing (α3K^185L model) and acute neuronal overexcitation (NMDA model).^{14, 15} The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased Cl⁻ transport activity, but they also demonstrate that the N-terminal KCC2 protein domain (NTD) is sufficient for neuroprotection.     

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.