Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1.TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of DOI:http://dx.doi.org/10.7554/eLife.10592.001 Psoriasis is an inflammatory disorder that causes red, flaky patches of skin. The disease affects around 2% of the world’s population, and is most common in people of northern European descent. TNF is one of the key proteins in the development of psoriasis and drugs that inhibit TNF have been very successful in the treatment of this disease. However, these drugs are expensive and for unknown reasons at least 10% of patients do not respond to them.et al. set out to obtain a more detailed molecular understanding of this disease by developing new mouse models of the condition. Mice were genetically engineered such that a key gene was deleted specifically from the skin cells that form the main barrier to the environment. These mice demonstrated that defects in skin cells called keratinocytes, rather than defects in the immune response, could lead to a psoriasis-like disease.Attempts to develop better drugs for psoriasis would be assisted by an improved understanding of this disease in terms of the genes and proteins involved. Etemadi et al. also showed that the skin cells with this genetic defect die in the presence of TNF and this cell death in mice caused a rapidly-appearing form of psoriasis. However, in the absence of TNF the mice still developed psoriasis, albeit more slowly. In this case, the condition was due to an excessive activation of a protein called NF-κB, which is known to play a role in maintaining balance in the immune system and in psoriasis.Etemadi These findings reveal how keratinocytes, cell death and inflammation can directly contribute to psoriasis-like conditions in mice. The next challenge will be to determine whether these findings can be used to help patients with this condition.DOI:http://dx.doi.org/10.7554/eLife.10592.002 TNF Receptor Associated Factor 2 (TRAF2) is an adaptor protein that transduces signals following ligation of certain cytokine receptors including those binding TNF. It was first identified together with TRAF1 as a component of TNF receptor-2 and then TNF receptor-1 (TNFR1) signalling complexes . TRAF2, RING domains are nearly always associated with ubiquitin E3 ligase activity  and TRAF-/- Traf2mice while only JNK signalling was affected in lymphocytes from transgenic mice that express a dominant negative (DN) form of TRAF2 that lacks the RING domain (Traf2-/-Traf5-/- mouse embryonic fibroblasts (MEFs) have a pronounced defect in activation of NF-κB by TNF, suggesting that absence of TRAF2 can be compensated by TRAF5  is a pleiotropic sphingolipid mediator that regulates proliferation, differentiation, cell trafficking and vascular development . S1P is 1-5 . However1-5  and the 1-5 .et al. Alvarez and colleagues, observed that in the absence of SPHK1, TNF-induced NF-κB activation was completely abolished.Recently, it was suggested that the RING domain of TRAF2 requires S1P as a co-factor for its E3 ligase activity . Alvarez-/- Traf2and -/-Sphk1 cells to TNF were not compared. Therefore, we undertook an analysis of TRAF2 and SPHK1 function in TNF signalling in a number of different tissues.Although we know a lot about TRAF2, there are still important gaps particularly with regard to cell type specificity and in vivo function of TRAF2. Moreover, despite the claims that SPHK1 and its product, S1P, are required for TRAF2 to function as a ubiquitin ligase, the responses of cpdm mutant  and keratinocytes required TRAF2 but not SPHK1 for full strength TNF signalling. In these cell types, absence of TRAF2 caused a delay in TNF-induced activation of NF-κB and MAPK, and sensitivity to killing by TNF was increased. Absence of TRAF2 in keratinocytes in vivo resulted in psoriasis-like epidermal hyperplasia and skin inflammation. Unlike TNF-dependent genetic inflammatory skin conditions, such as IKK2 epidermal knock-out  and the m mutant , the onslox/loxLyz2-Cre (Traf2LCTraf2) mice. The Lyz2-Cre recombinase transgene is expressed in all cells of the myeloid lineage including macrophages , from wild type and rophages . TNF stirophages . In Trafpe BMDMs .10.7554/Traf2-deficient and wild-type BMDMs with TNF for 24 hr and assayed their viability using Propidium Iodide (PI) staining and flow cytometry. Taking into account the slightly higher background death of -/- Traf2BMDMs compared with wild type BMDMs, TNF was not cytotoxic to either wild-type or -/- Traf2macrophages  by crossing lox/loxTraf2 mice with transgenic mice constitutively expressing Cre recombinase under the control of the keratin-14 promoter (K14-Cre). Primary keratinocytes isolated from wild-type, EKOTraf2 and -/- Sphk1mice were tested and, similarly to MEFs and MDFs, TNF signalling was delayed and weakened in Traf2-deficient  beads to precipitate ubiquitylated proteins. Within 5 to 15 min of TNF stimulation RIPK1 became strongly ubiquitylated in both wild-type and -/- Sphk1MDFs, and this decreased over 60 min , but occurred after 60 min with a different pattern  mice. EKOTraf2 mice were born at the expected Mendelian ratios but developed an inflamed skin phenotype from 6 weeks of age. This phenotype became worse over time and the mice were sacrificed usually before 15 weeks of age  and cleaved caspase-3 (CC3), respectively  mouse, loss of one allele of Tnf significantly reduced the systemic inflammatory disorder , the skin of most of the EKOTnfTraf2-/- mice started to become inflamed. Lesions became severe by 30 weeks, and these necessitated sacrifice of the affected animals . To molecularly define the type of TNF-induced cell death in -/- Traf2keratinocytes, we looked at the necroptotic and apoptotic markers upon TNF stimulation in detail  or -/-Ifng mice. However, both wild type and -/-Ifng chimeras developed skin inflammation to the same extent, indicating that hematopoietic cell-derived IFNγ is not a major contributor to the psoriasis-like skin inflammation  expressed by keratinocytes that attracted these inflammatory cells independently of TNF. TRAF2 suppresses non-canonical NF-κB activation in many different cell types and loss of TRAF2 induces constitutive activation of non-canonical NF-κB . However, no difference was observed in the proliferation of -/-Traf2 versus wild-type keratinocytes  and without any stimulation , cells were lysed and their RNA were extracted and analysed by the qPCR array. Out of more than 600 inflammatory genes analysed, about 300 genes were constitutively expressed in the keratinocytes under these conditions  mice, that bear an inactive point mutant of NIK, or Nfkb2 (p100)-/- mice. Contrary to our hypothesis, deficiency in Map3k14 or Nfkb2 did not prevent the psoriasis-like skin disease in EKOTraf2 mice . However, depletion of both TNF and NF-κB2 completely prevent the psoriasis-like inflammation in the EKOTraf2 mice . Thus, total lack of SPHK isoenzymes did not phenocopy the skin defects seen in the absence of TRAF2, again arguing against an essential role for S1P in TRAF2's function.Studies on TRAF2 deficient B cells revealed a crucial role for TRAF2 in B cell development and examined the consequences of gnalling . Aside fithelium . In anotone week . However reports , no signcpdm/cpdmSharpin mice can be driven by TNF-induced apoptotic death, because deletion of just one allele of Casp8 or epidermal Fadd deletion markedly ameliorated or prevented the TNFR1-dependent inflammatory phenotype (cFlip (cflar) knock-out causes excessive apoptotic death and an initial wave of very rapid hyperplasia in the epidermis that is rescued by TNF inhibition  and cytotoxic T cells have also been reported to have essential roles in initiation and development of psoriasis . This failure might be due to low levels of secretion of these factors by keratinocytes maintained in serum-free media, so we investigated the transcription of inflammatory genes. At the mRNA level, several inflammatory genes were highly elevated in EKO/EKOTnfTraf2-/- compared to EKO/+TnfTraf2-/- keratinocytes, and many of these, such as M-CSF and IL-23, have been implicated in the development of inflammatory diseases  under the approval of WEHI ethics committee. Animal technicians performed unbiased mouse monitoring and informed us if mice developed skin inflammation and needed to be sacrificed according to the WEHI animal ethic guidelines.2O2PO7). Cell lysates were loaded on NuPAGE Bis-Tris gels  and transferred on to Immobilon-P PVDF membranes  or Hybond-C Extra . Membranes were blocked and antibodies diluted in 5% skim milk powder or Bovine Serum Albumin (BSA) in 0.1% Tween20 in PBS or TBS. Antibodies used for Western blot were: Cleaved caspase-3 (9661) and -8 (8592), phospho-JNK1/2 (4668P), phospho-p38 (4511), p38 (9212), caspase-8 (4927), JNK1/2 (9252), IκBα (CN: 9242) and phospho-p65 (3033) from Cell Signaling Technology , β-actin , cFLIP , RIPK1  and VDAC1 . Monoclonal antibody for TRAF2 was made in-house and raised against the RING domain of TRAF2 (Clone: 6/12-2D1-22-1). cIAP1 antibody was made in house and is distributed by ENZO Life Sciences . MLKL antibody was made in-house as described  in the absence or presence of Q-VD-OPh  and Necrostatin  for the times indicated and lysed in ice-cold DISC buffer. The lysates were incubated with TUBE beads for 2 hr at 4°C before washing 3x in ice-cold PBS-Tween. Washed beads were resuspended in 1x SDS sample buffer, separated on SDS-PAGE gels and subjected to Western blotting and probed with the indicated antibodies.Tandem Ubiquitin Binding Entities  beads were used to purify ubiquitin conjugates from MDFs according to the manufacturer’s recommendations . Wild–tySkin samples were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned for routine histology staining (H&E). For skin immunofluorescence, parafin sections were de-waxed, subjected to heat-induced epitope retrieval (HIER) with citrate buffer, blocked in 3% goat serum, then permeabilised with 0.3% Triton X-100, and stained with keratin 6 (Covance) or keratin 14, and goat anti-rabbit Alexa 594  antibodies. Nuclei were visualized using Hoechst (Invitrogen). For tissue immunohistochemistry, sections were dewaxed and subjected to antigen retrieval in trypsin buffer or HIER with citrate buffer and stained with anti-CC3 , anti-CD45  and goat anti-rabbit biotinylated antibodies. Images were taken using a DP72 microscope and cellSens Standard software .Skin was separated from the tail of an adult mouse and incubated in dispase II (20 mg/ml) at 4°C for 24 hr and keratinocytes and MDFs were isolated from the epidermal and dermal layers respectively as described before . TNF ELIMEFs were generated from E15 embryos by removing their head and liver and passing through a cell strainer. MEFs and MDFs were immortalised by infecting these cells with a lentivirus expressing SV40 Large T antigen.Cultured keratinocytes in one well of a 6-well plate were lysed in 500 μl TRIzol (Invitrogen) and the RNA was purified using PureLink RNA purification kit . cDNA was made with SuperScript VILO cDNA Synthesis kit (Invitrogen) and qPCR was performed using TaqMan Open Array mouse inflammation panel  on a QuantStudio 12K Flex machine (Life Technologies) following the manufacturer protocols.-/-Tnf-/-Traf2 and +/-Tnf-/-Traf2 keratinocytes using empirical Bayes t-statistics  and Smac-mimetic (TetraLogic), and cell death was measured by Propidium Iodide (PI) staining and flow cytometry performed on a FACSCalibur flow cytometer  and analysed using Weasel (WEHI). Keratinocyte viability was assayed using CellTiter 96  according to the manufacturer’s instructions.Epidermal and dermal layers of ears were separated after 30 min incubation with 2.4 U/ml Dispase  at 37°C, followed by cutting into fine pieces and digested with 50 U/ml collagenase for 45 min at 37°C. Single-cell suspension was made in FACS buffer  and blocked with Fc block (eBioscience) and stained with the following antibodies in FACS buffer at 4°C as described before . AntibodDermal T cells were isolated from the skin and resuspended in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-Glutamine (GIBCO), 50 μM 2-mercaptoethanol  100 U/ml penicillin/streptomycin (GIBCO). Cells were stimulated for 4 hr in presence of PMA (20 ng/ml) and Ionomycin (1 μg/ml) in presence of the GolgiPlug (BD Biosciences).EKOTraf2. Mice were maintained on 2 mg/ml neomycin in drinking water for three weeks post irradiation.Mice were irradiated with 550 rads twice with 3 hr minimum between irradiation. Approximately 5 million cells from the bone marrow of C57/BL6 or IFNγ mice were injected by tail i.v. to Stimulated keratinocytes were permeabilised in buffer containing 0.025% digitonin. Cytosolic and crude membrane fractions were further solubilized in 1% digitonin, resolved by Bis·Tris Native PAGE as described before , and imm In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been favorably evaluated by Tadatsugu Taniguchi (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of SPHK1" for peer review at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Manolis Pasparakis and Hao Wu (peer reviewers).The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.The authors used genetic, cellular, immunological, and histological tools to investigate the roles of TRAF2 in inflammation and cell death. TRAF2 knockout does not affect TNF-induced canonical NF-κB or MAPK signaling, or cell death in macrophages. The authors show that TRAF2 is required for TNF signaling in fibroblasts and keratinocytes, but SPHK1 (sphingosine kinase 1) knockout cells did not show altered canonical NF-κB signaling, MAPK, or apoptotic cell death when compared to wild-type cells. Loss of TRAF2 leads to epidermal hyperplasia and psoriasis-like inflammation in mice, which can be delayed but not fully reversed by deletion of TNF. Deletion of TRAF2 activates the non-canonical NF-κB signaling pathway through NIK, and double deletion of NF-κB2 and TNF fully rescued inflammation induced by TRAF2 deletion. In addition, they conclude that in contrast to its previously suggested essential in regulating TRAF2 E3 ligase activity dependent TNF signalling, SPHK1 is not required for TNF-induced activation of NF-κB or MAPK and for resistance to TNF-induced cell death.This is a very nice manuscript reporting clear and well-controlled experiments that reveal an important function of TRAF2 in the maintenance of skin homeostasis. Also, the data on the (lack of) function of SPHK1 in TNF signalling is very convincing. The authors should discuss more carefully the skin pathology developing in their mice comparing with human psoriasis in terms of histological findings, the immune infiltrates, gene expression as well as the genetic studies.Reviewer #1 (Minor Comments):1) The Materials and methods is missing information about how TUBE experiment was conducted, and source of RIPK1 and cIAP antibodies.2) -/-Traf2 and -/-Sphk1 cells.3) The authors should be more consistent in all blots regarding time points when they compare effects of WT, -/-Sphk1 cells upon TNF stimulation, since cIAPs and TRAF2  should be active? The authors may consider discussing this issue.4) In -/-Traf2 which would confirm their hypothesis about delayed NF-kB activation.5) EKOTnf-/-Traf2 as they did for other cell suspensions, and compare the data with results for CD4+ T cells.6) 7) The authors should focus in Discussion on experiments with IL-17 and discuss potential other signals for activation of non-canonical NF-kB independent of TNF.EKOTnf-/-Nfkb2-/-Traf2 mice for up to 36 weeks. Instead, it is showing only survival percentage for other 6 genotypes. Since this survival curve is one of the most crucial results, the authors should add it.8) Traf2 mostly induced lesions in anterior parts of the body, while animals lacking active TRAF2 and functional non-canonical NF-kB signaling were more susceptible to develop lesions in ventral and posterior parts. They should discuss possible reasons for these differences.9) The authors describe that loss of EKOTraf2/+-/-Nfkb2-/-Tnf which isn't presented nowhere on figures. Please, provide figures showing these results or correct that paragraph.10) In the last Results paragraph, the authors twice talk about the phenotype of Reviewer #2 (Minor Comments):In Reviewer #3 (Minor Comments):-/-, Nfkb2-/-Tnf) 'occasionally' developed skin lesions but do not indicate how many mice have been analysed and how many developed lesions. Please clearly state the number of mice analysed for each genotype. It would also be helpful to clearly state the age of mice shown in all figures.The authors state that the triple mutant mice ." The citation Pasparakis, Schmidt-Supprian, and Rajewsky, 2002 is not the right one, the correct one is Pasparakis, Courtois et al., 2002.EKOTraf2 mice is characterised by skin infiltration of neutrophils and IFNγ producing CD4+ T cells in” referring to the data on IFNγ+ T cells should be eLife. Your revised article has been favorably evaluated by Tadatsugu Taniguchi (Senior Editor), a Reviewing Editor, and one reviewer. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.Thank you for resubmitting your work entitled "TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of Sphingosine Kinase 1" for further consideration at The inflammatory skin lesions developing in mice with keratinocyte-specific TRAF2 knockout can be described as psoriasis-like, but it is not correct to write that these mice develop psoriasis, as in the following instances in the manuscript:EKOTraf2 mice is characterised by skin infiltration of neutrophils and IFNγ producing CD4+ T cells in”: "However, both wild type and -/-Ifngchimeras developed skin inflammation to the same extent indicating that IFNγ is not a major contributor to the psoriasis  function of SPHK1 in TNF signalling is very convincing. The authors should discuss more carefully the skin pathology developing in their mice comparing with human psoriasis in terms of histological findings, the immune infiltrates, gene expression as well as the genetic studies.Reviewer #1 (Minor Comments):1) The Materials and methods is missing information about how TUBE experiment was conducted, and source of RIPK1 and cIAP antibodies.Thanks for picking this up, we have now included the full information for the method and the source of the antibodies (subsection “Immuno precipitation of ubiquitin-conjugates (TUBE IP)”).2) Sphk1-/- and wild type MEFs but incomplete in -/-Traf2MEFs . Likewise, JNK phosphorylation is maximal in wild type and Sphk1-/- MEFs and is reduced and delayed in -/-Traf2MEFs (n=3). In the previous figure we had included phospho-p38 and phospho-ERK blots, however, in repeating experiments with different sources of MEFs for the revision, we observed variations in these responses. Therefore we cannot draw conclusions based on this data and have removed them. This fits with our poor experience with different biological MEF lines and is the reason that we prefer to use the far more reliable dermal fibroblasts (e.g. We repeated this experiment using the same time points as before and tested with a full panel of antibodies and the appropriate loading controls new . As befosts e.g. .10.7554/-/- and Sphk1-/- cells.3) The authors should be more consistent in all blots regarding time points when they compare effects of WT, Traf2In -/-Traf2cells we extended the analysis to 60 minutes  should be active? The authors may consider discussing this issue.4) In Bax-/- cells to examine ubiquitylation of caspase-8, which they observed most strongly 1 hr post stimulation. In Sphk1-/- cells. However we think the completely different treatments and cell types used in these two works preclude us from drawing any conclusion or adding anything productive to the Discussion.In Gonzalvez et althe authors use TRAIL or the Fas activating antibody Jo2 in HCT116 -/- which would confirm their hypothesis about delayed NF-kB activation.5) -/-Traf2MDFs compared to wild type or Sphk1-/- cells.As requested we have repeated this experiment new , which rEKOTnf-/- as they did for other cell suspensions, and compare the data with results for CD4+ T cells.6) EKOTnfTraf2-/- mice (new panel in EKOTraf2and EKOTnfTraf2-/- mice compared with wild type dermis (New We have added the requested data quantifying CD11b Ly6G neutrophils from the epidermis of rmis New .7) The authors should focus in Discussion on experiments with IL-17 and discuss potential other signals for activation of non-canonical NF-kB independent of TNF.In the Discussion section, we have added discussion on the clinical results with secukinumab and ixekizumab and the implications and limitations of our in vitroexperiments.Traf2EKO/EKOTnf-/- keratinocyte proliferation compared to Traf2EKO/+Tnf-/- cells in vitroand loss of TRAF2 had no impact on IL-17 mediated activation of NF-κB and p38 in keratinocytes. While this does not exclude a role for IL-17 in the psoriasis observed in EKOTraf2or EKOTnfTraf2-/- mice we hypothesized that Traf2EKOTnf-/- keratinocytes produced other factors to recruit inflammatory cells to the skin.""IL-17 plays an important role in inflammatory diseases and monoclonal antibodies targeting IL-17 such as secukinumab and ixekizumab are performing well in psoriasis patients . […] However, in our experiments IL-17 did not increase Likewise we have included a discussion on the implications of our results regarding non-canonical NF-κB signalling and psoriasis:"This observation might be relevant to human psoriasis because it has been reported that the levels of TWEAK and its receptor Fn-14 are elevated in human psoriatic lesions  and it is well established that TWEAK/Fn14 signalling induces degradation of TRAF2 and activation of non-canonical NF-κB signalling . Thus, our results suggest that inhibition of non-canonical NF-κB signalling might be beneficial in chronic inflammatory disease together with TNF inhibitions."EKOTnf-/-Nfkb2-/- mice for up to 36 weeks. Instead, it is showing only survival percentage for other 6 genotypes. Since this survival curve is one of the most crucial results, the authors should add it.8) -/-TnfTraf2-/-Nfkb2-/- (from 4 to 13) and -/-TnfTraf2-/-Nfkb2-/+ (from 9 to 15). These new data also incorporate three -/-TnfTraf2-/-Nfkb2-/- mice that die of other causes than psoriasis. These causes are most likely due to their immune deficiency and also occur in Tnf-/-Nfkb2-/- and EKO/EKONik(Map3K14)aly/alyTraf2mice (Our apologies. The data was there but it was incorrectly colour coded (pink instead of blue) and was in an unfortunate position so that it gave the impression of demarking the graph boundary. We have now changed the format of this graph so that it is clear which survival line corresponds to which mouse strain. We have also substantially increased the numbers of the strains that are most directly relevant, namely /alymice  and 10C.9) The authors describe that loss of Traf2 mostly induced lesions in anterior parts of the body, while animals lacking active TRAF2 and functional non-canonical NF-kB signaling were more susceptible to develop lesions in ventral and posterior parts. They should discuss possible reasons for these differences.EKO/EKONfkb2Traf2-/-Tnf-/- and Tnf-/-Nfkb2-/- mice  that we have cited.We note that we also see the same lesions developing in -/- mice . NIK andEKO/+Tnf-/-Nfkb2-/- which isn't presented nowhere on figures. Please, provide figures showing these results or correct that paragraph.10) In the last Results paragraph, the authors twice talk about the phenotype of Traf2EKO/EKOTnf-/-Nfkb2-/-Traf2and we have now fixed this error.These mice were shown in Reviewer #2 (Minor Comments):In In We repeated the experiment in Reviewer #3 (Minor Comments):-/-, Nfkb2-/-) 'occasionally' developed skin lesions but do not indicate how many mice have been analysed and how many developed lesions. Please clearly state the number of mice analysed for each genotype. It would also be helpful to clearly state the age of mice shown in all figures.The authors state that the triple mutant mice ." The citation Pasparakis, Schmidt-Supprian, and Rajewsky, 2002 is not the right one, the correct one is Pasparakis, Courtois et al., 2002.Thank you for this correction.EKO mice is characterised by skin infiltration of neutrophils and IFNγ producing CD4+ T cells in” referring to the data on IFNγ+ T cells should be The whole figure has changed and we have endeavoured to cite the new figure correctly.The inflammatory skin lesions developing in mice with keratinocyte-specific TRAF2 knockout can be described as psoriasis-like, but it is not correct to write that these mice develop psoriasis, as in the following instances in the manuscript:EKO mice is characterised by skin infiltration of neutrophils and IFNγ producing CD4+ T cells in”: "However, both wild type and Ifng-/- chimeras developed skin inflammation to the same extent indicating that IFNγ is not a major contributor to the psoriasis (In the subsection “Psoriasis-like inflammation in Traf2soriasis ."EKO mice (In the subsection “Blocking non-canonical NF-κB signalling and depletion of TNF together prevent inflammation caused by TRAF2 deficiency in keratinocytes”: "Contrary to our hypothesis, deficiency in Map3k14 or Nfkb2 did not prevent the psoriasis in Traf2EKO mice  and 10."-/-Casp8-/- on the late onset psoriasis."Discussion: "While this does not exclude a role for IL-17 in the psoriasis observed in Traf2EKO or Traf2EKOTnf-/- mice we hypothesized that Traf2EKOTnf-/- keratinocytes produced other factors to recruit inflammatory cells to the skin."Discussion: "Unfortunately, we were unable to age these mice more than 15 weeks as they developed lymphadenopathy and could therefore not examine the effect of MlklEKOTraf2mice, which are consistent with the title and rest of the manuscript.Thanks for pointing out this. We have changed all the above items to the appropriate and relevant terms to the phenotype of In Now we have changed and improved


Among targets of biological agents, tumor necrosis factor remains in the exclusive group that has retained its historical name - or at least one of them. Tumor necrosis factor is usually known by the three-letter abbreviation TNF and somehow has escaped a more bland and nondescript moniker such as IL-something. Endogenous pyrogen is now IL-1, B-cell stimulatory factor-2 is now IL-6, and T-cell growth factor is IL-2. But TNF is still TNF. The other name for TNF, cachectin, seems long gone, although arguably the metabolic actions of TNF are more relevant to the setting of inflammatory and autoimmune disease than a capacity to kill tumors. Although the name TNF has withstood the test of time, it depicts few of its myriad activities and little of its profound impact on the function of the brain, liver, and heart among other organs and tissues.The designation TNF or tumor necrosis factor reflects the original discovery in the 1970s of a cytotoxic substance produced by immune cells stimulated by endotoxin. Since in animal models TNF kills tumors, investigators tried to exploit this action therapeutically in patients with cancer, but the administration of this cytokine made recipients too systemically sick. Though limiting TNF’s development in oncology, these studies revealed the powerful role of TNF in immunity. In addition to killing tumors, TNF can cause the metabolic derangements of cachexia as shown initially in studies on a novel factor produced by macrophages stimulated with bacterial or protozoal products. These studies called this factor cachectin. After studies showed that TNF and cachectin are the same molecule, only one name was possible and TNF became it.With the recognition of TNF’s role in inflammation, studies focused on its potential as a target in the treatment of sepsis. Even though studies pointed to the efficacy of TNF blockade in animal models of sepsis, trials in humans were unsuccessful. Fortunately, these trials provided valuable reagents that led to the striking success of TNF blockade in the treatment of rheumatoid arthritis, ankylosing spondylitis, and Crohn’s diseases among many other inflammatory diseases. In the context of treating inflammation, the effects of TNF on cancer cells remain a concern as a side effect, with worries that a malignancy could emerge if TNF blockade impairs immune surveillance.Trying to change TNF’s name at this point would be foolhardy and destined to fail if it eliminated the three-letter abbreviation. Knowing the power of name recognition, I would like to suggest a modest revision that would be more accurate while retaining the TNF nomenclature. Rather than having the N in TNF stand for necrosis, I think that it should stand for necroptosis. The brand identity thus remains intact.Motivating consideration of this change is exciting research showing that the death form induced by TNF is better conceptualized as necroptosis than necrosis. For those readers who are not aficionados of death, necroptosis is a form of programmed cell death sometimes called programmed necrosis. Unlike classic necrosis , necroptosis is highly regulated and depends on the action of enzymes that internally deliver the death blow to a cell. As recent studies show, necroptosis is closely allied with apoptosis in the role of enzymes in cellular demise. Whereas the caspase enzymes mediate apoptosis, two enzymes called receptor-interacting protein (RIP) kinases 1 and 3 play a central role in killing cells by necroptosis by binding to form a necrosome.The outcome of death via the caspases and RIP kinases is vastly different even though they can emerge from the same starting or check point and have cross-talk.  In simple form, apoptosis renders a cell immunologically impotent. In contrast, necroptosis creates a cell that can induce inflammation. Indeed, recent studies suggest that the elaborate cleavage reactions and cellular rearrangements that characterize apoptosis have evolved not to kill cells but to strip away its immunological potential. In this sense, apoptosis may be a ‘good death’.in vitro killing of the L929 fibrosarcoma line - is now one of the standard models for necroptosis, and caspase inhibition as well as treatment with TNF leads to this form of cell death.Reflecting the key role of cell injury and destruction in a host of diseases, the literature of cell death is now one of the liveliest in all of biology (pun intended) and is providing new insights into diverse processes that span the spectrum of medicine. Importantly, as these studies show, necroptosis, not necrosis, is truly the counterpart to apoptosis, and these two death forms are possible outcomes of stressors that include cytotoxic drugs, toxins, ischemia, or infection. With caspases inhibited, the cell can die by necroptosis and become more immunologically active. Interestingly, the venerable bioassay for TNF - the A focus on necroptosis is not just a matter of semantics or intellectual disputation: it has important therapeutic implications. In oncology, an important goal in enhancing therapy is to boost killing by cytotoxic agents - whether chemotherapeutics, biologicals, or radiation - making them more lethal and rendering the dead cell more immunogenic. In contrast, in cardiovascular or cerebrovascular disease, the goal is the opposite: to prevent cell death from dangers such as ischemia and curtail the subsequent inflammation that can intensify injury and inflict bystander damage. Controlling, channeling, and commandeering death processes are therefore literally a matter of life and death in medicine.Whereas necrosis, especially if caused by a harsh physical or chemical agent, is an explosion beyond the reach of therapy, necroptosis is a process and therefore can be contained. An agent named necrostatin 1 can block necroptosis by inhibiting the action of the RIP kinase 1 and its downstream effects. This agent and related analogues have shown impressive activity in models such as solid organ transplantation, ischemia-reperfusion injury, and acetaminophen toxicity. Acetaminophen toxicity has long been a model of necrosis. Guess what. The death form is more likely necroptosis than necrosis; as such, this important model of drug-induced liver injury can be inhibited by necrostatin 1 and other strategies to reduce RIP kinases.The place of necroptosis in autoimmune and inflammatory disease is less certain since few studies have directly explored the role of this death form in immunopathogenesis. Right now, NETosis is hot but that form of death involves mostly neutrophils and other immune cells. Death is central to the pathogenesis of many rheumatological diseases, however. Muscle cells die in myositis, endothelial cells die in vasculitis, and glomerular cells die in nephritis. In general, these events have been called necrosis but, if they are more properly categorized as necroptosis, then the use of agents like necrostatin 1 could rescue and preserve tissue until glucocorticoids or immunosuppressives can more decisively halt the inflammation. In conditions such as rapidly progressing glomerulonephritis, an agent that stops injury could be the difference between renal failure and recovery. The time is now to make necroptosis a new watchword in rheumatology.Had necroptosis been recognized when TNF was discovered, it is likely that this powerful cytokine would have been called tumor necroptosis factor. Although such a term is not as catchy as tumor necrosis factor (necroptosis’s four syllables are too bulky and jarring to come trippingly from the tongue), it provides a more clear and precise picture of the pathway induced and its consequences. By incorporating necroptosis into our thinking about inflammatory and autoimmune disease, new insights into the effects of TNF blockade should be possible and certainly could lead to the development of therapy directed at end-organ damage. In this case, death is not the end but the beginning, and its study gives new life to a critical domain of modern rheumatology.The author declares that he has no competing interests.RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation.PLoS One 2011, 6:e23209.Cho YS, McQuade T, Zhang H, Zhang J, Chan FK-M: RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.Immunity 2011, 35:908–918.Duprez L, Takashi N, Van Hauwermeiren F, Vandendriessche B, Goossens V, Berghe TV, Declercq W, Libert C, Cauwels A, Vandenabeele P: Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α.Cell 2009, 137:1100–1111.He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X: Necroptosis: the release of damage-associated molecular patterns and its physiological relevance.Immunity 2013, 38:209–223.Kaczmarek A, Vandenabeele P, Krysko DV: RIPK3-mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival.Am J Transplant 2013, 13:2805–2818.Lau A, Wang S, Jiang J, Haig A, Pavlosky A, Linkermann A, Zhang Z-X, Jevnikar AM: Necroptosis in immunity and ischemia-reperfusion injury.Am J Transplant 2013, 13:2797–2804.Linkermann A, Hackl MJ, Kunzendorf U, Walczak H, Krautwald S, Jevnikar AM: Necroptosis.N Engl J Med 2014, 370:455–465.Linkermann A, Green DR: Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.Hepatology 2013, 58:2099–2108.Ramachandran A, McGill MR, Xie Y, Ni H-M, Ding W-X, Jaeschke H: Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.Cell Death Dis 2012, 3:e437.Takashi N, Duprez L, Grootjans S, Cauwels A, Nerinckx W, DuHadaway JB, Goossens V, Roelandt R, Van Hauwermeiren F, Libert C, Declercq W, Callewaert N, Prendergast GC, Degterev A, Yuan J, Vandenabeele P: zVAD-induced necroptosis in L929 cells depends on autocrine production of TNFα mediated by the PKC-MAPKS-AP-1 pathway.Cell Death Differ 2011, 18:26–37.Wu Y-T, Tan H-L, Huang Q, Sun X-J, Shen H-M:


Neuroinflammation and mitochondrial dysfunction are common features of chronic neurodegenerative diseases of the central nervous system. Both conditions can lead to increased oxidative stress by excessive release of harmful reactive oxygen and nitrogen species (ROS and RNS), which further promote neuronal damage and subsequent inflammation resulting in a feed-forward loop of neurodegeneration. The cytokine tumor necrosis factor (TNF), a master regulator of the immune system, plays an important role in the propagation of inflammation due to the activation and recruitment of immune cells via its receptor TNF receptor 1 (TNFR1). Moreover, TNFR1 can directly induce oxidative stress by the activation of ROS and RNS producing enzymes. Both TNF-induced oxidative stress and inflammation interact and cooperate to promote neurodegeneration. However, TNF plays a dual role in neurodegenerative disease, since stimulation via its second receptor, TNFR2, is neuroprotective and promotes tissue regeneration. Here we review the interrelation of oxidative stress and inflammation in the two major chronic neurodegenerative diseases, Alzheimer's and Parkinson's disease, and discuss the dual role of TNF in promoting neurodegeneration and tissue regeneration via its two receptors. The adult human central nervous system (CNS) consists of approximately 100 billion neurons and a similar amount of glia cells, namely, astrocytes, oligodendrocytes, and microglia , 3. MoreAll types of glia cells are of relevance to maintain the homeostasis of the CNS. Of particular importance are astrocytes, which are essential for the trophic support of neurons and oligodendrocytes, and microglia, the immune cells of the CNS. Under physiological conditions the so-called resting microglia, which are kept quiescent by interaction with neuronal proteins such as CX3CL1  and CD200 , constanDuring transient injuries microglia activation is usually not detrimental to the CNS. Although the release of proinflammatory cytokines and ROS may be harmful to neurons and oligodendrocytes, microglia are essential to remove the cellular debris and secrete neurotrophic factors after resolution of the injury and are thus essential for tissue regeneration . If, howHere we review the relationship between oxidative stress and inflammation in chronic neurodegenerative disease. In particular we will focus on the role of the tumor necrosis factor (TNF), which is released by activated astrocytes and microglia and which can exacerbate inflammation and promote the release of ROS from microglia thereby promoting neurodegeneration. Interestingly, TNF not only elicits detrimental and degenerative responses. Particularly in the nervous tissue TNF can also ameliorate immune responses and promote regeneration and neuroprotection , 14.We will restrict our discussion on the interrelation of oxidative stress and inflammation in the two major chronic neurodegenerative diseases, namely, Alzheimer's disease (AD) and Parkinson's disease (PD). Where applicable we will correlate certain aspects to multiple sclerosis (MS), the predominant inflammatory disease of the CNS.β), which are generated by proteolytic processing of the C-terminus of the amyloid precursor protein (APP) by specific proteases, β-secretase and γ-secretase. Moreover, intracellular protein aggregates, the so-called neurofibrillary tangles, which consist predominantly of hyperphosphorylated and misfolded tau protein, are characteristically found in the neurons of AD patients. Since tau is important for stability of microtubules its deregulation may result in impaired axonal transport. Although more than 90% of AD cases are sporadic, there are also familial forms of this disease, which are caused by mutations in APP or presenilin, the catalytic subunit of γ-secretase [AD is characterized by memory impairment and cognitive decline due to neuronal loss mainly in the neocortex and the hippocampus. The main histopathological hallmark is the formation of extracellular plaques consisting predominantly of aggregates formed by amyloid beta .2−∙), hydroxyl radical (∙OH), and hydrogen peroxide (H2O2). H2O2, in particular, has been implicated in signaling pathways important for cell growth, proliferation, and survival [ROS have important physiological functions, for example, by oxidation of cysteines in proteins necessary for the formation of disulfide bonds , 11. Exasurvival . Howeversurvival , cells hsurvival   located in the mitochondrial matrix or SOD1 located in the cytosol [2O2 can act as an oxidant and, moreover, in the presence of reduced metal ions such as ferrous iron (Fe2+), can be converted by the Fenton reaction into the highly reactive hydroxyl radical, the most harmful species of all ROS [2O2 is rapidly converted to water by mitochondrial glutathione (GSH) with the participation of GSH reductase and peroxiredoxins [During mitochondrial activity superoxide is produced in the electron transport chain (ETC). Since superoxide can inactivate proteins containing iron-sulfur clusters in the mitochondrion, it is immediately converted to H cytosol , 28. H2O all ROS , 24. Theredoxins . The GSHredoxins .A second source of free radical superoxides are NADPH oxidases, multisubunit enzyme complexes located in the cell membrane, which are classified by their catalytic subunit, NOX. Several NOX proteins are expressed in cells of the CNS including neurons, astrocytes, and microglia, with NOX2 as the predominant form in microglia and astrocytes , 31. In −) due to interaction of nitric oxide (NO) with superoxide. ONOO− can react with CO2 to form the highly reactive radicals ∙NO2 (nitrogen dioxide) and CO3−∙. NO2 and NO can then react further to form N2O3 (dinitrogen trioxide). NO itself is produced by NO synthase (NOS), which has three isoforms in the CNS, endothelial NOS (eNOS), neuronal NOS (nNOS) identified in neurons, and inducible NOS (iNOS) identified in glial cells [Next to ROS, also reactive nitrogen species (RNS) can contribute to oxidative stress . One exaal cells , 33. Undal cells .At the cellular level ROS and RNS can cause DNA and protein oxidation as well as lipid peroxidation . The latTwo main aspects contribute to the vulnerability of the CNS to oxidative stress mediated neurodegeneration: high metabolism and restricted cell renewal. First, the CNS is a metabolically highly active organ, requiring approximately 20% of the total energy consumption of the body. Therefore the CNS contains high amounts of mitochondria, which are particularly active, resulting in high amounts of ROS . AccordiSecond, in postmitotic cells such as neurons and oligodendrocytes, nonfunctional proteins and organelles have to be degraded to prevent their cellular accumulation which may eventually cause cell death. Nonfunctional proteins are predominantly degraded in an ubiquitination-dependent manner by the proteasome . ImportaThere is strong support for the role of ROS and mitochondrial dysfunction in AD. For example, neurons of AD patients have a high percentage of damaged mitochondria, which may be due to the increased presence of mutations in the mitochondrial DNA . Moreoveβ peptide, derived from APP, as well as misfolded tau protein and both have been implicated in mitochondrial dysfunction. In particular, mitochondrial damage may be caused by intracellular APP and/or Aβ, since these proteins can directly bind to the protein import machinery of mitochondria thereby impairing import of mitochondrial proteins [β and tau can deregulate the mitochondrial ETC at distinct sites resulting in mitochondrial dysfunction and oxidative stress [β-secretase through activation of c-jun N-terminal kinase (JNK) and p38 MAP kinase thereby promoting the generation of Aβ [As described in proteins . This ree stress . On the on of Aβ . Similaron of Aβ , 54.β can activate NADPH oxidase in primary cultures of cortical neurons thereby causing the generation of ROS within the cells [Interestingly, Ahe cells . Furtherhe cells .+). MPP+ is a substrate for the dopamine transporter and is taken up selectively into dopaminergic neurons where it inhibits complex I of the mitochondrial electron transport chain ultimately resulting in PD-like symptoms [Dopaminergic neurons are particularly vulnerable to oxidative stress since dopamine metabolism and transport can contribute to ROS production , 57, 58.symptoms , 60. Thesymptoms –63. Moresymptoms , 65.α-synuclein, parkin, PINK1, and DJ-1, affect mitochondrial function resulting in increased ROS production [α-synuclein, the major component of Lewy bodies, since α-synuclein can directly impair the mitochondrial complex I in dopaminergic neurons [α-synuclein are implicated in increased ROS production while oxidative stress promotes α-synuclein aggregation thus creating a vicious cycle promoting neurodegeneration [Importantly, mutations in most genes that have been implicated in hereditary forms of PD, such as oduction –68. This neurons . Moreoveneration –73.So far we have focused on the role of cell intrinsic oxidative stress resulting in neuronal damage. However, during infection or injury, factors are released, the so-called pathogen associated and danger associated molecular patterns (PAMPs and DAMPs), respectively, that are recognized by pattern recognition receptors. Important pattern recognition receptors in the CNS are, among others, the toll-like receptors (TLRs) which are broadly expressed by microglia and astrocytes , 74. UpoIn general, inflammation is a protective response to various cell and tissue injuries to destroy and remove the detrimental agents and injured tissues, thereby promoting tissue repair. However, when inflammation is uncontrolled, it can cause excessive cell and tissue damage ultimately leading to destruction of normal tissue and chronic inflammation . This isβ deposition in AD and in the substantia nigra in PD patients [β and α-synuclein can bind and activate TLRs, in particular TLR2, in the membrane of astrocytes and microglia [Important roles of microglia and astrocytes in chronic neurodegeneration have first been suggested due to the increased presence of both cell types around sites of Apatients –82. Bothicroglia , 83, 84.icroglia , which microglia , whereasicroglia , 86.Upon chronic activation microglia and astrocytes can also generate ROS and RNS . Particuβ and α-synuclein can directly activate NOX2 in microglia causing a burst of superoxide [β and α-synuclein has been implicated in the chronic neurodegeneration in AD and PD patients [β-plaques in AD brains and in microglia in the substantia nigra of PD patients [Both Aperoxide , 63. It patients –97. Simipatients , 63. Imppatients .α-synuclein in PD. As mentioned in α-synuclein amplify its oxidization and aggregation [α-synuclein subsequently promotes activation of NADPH oxidase in microglia and thus enhances the generation of free ROS thus boosting the oxidization of α-synuclein in intact neurons [α-synuclein is altered by oxidative stress and the oxidized α-synuclein is promoting chronic activation of microglia, creates a feed-forward state for the progressive neuronal death seen in PD [Particular support for the link of ROS production and inflammation in neurodegeneration is again obtained from the role of mutated regation , 72. Aft neurons , 99–101.en in PD , 102.κB as well as induction of the stress-responsive protein kinases JNK and p38 MAP kinase [κB, in particular, has been recognized as a major player in governing cellular responses to oxidative stress.Although oxidative stress is implicated as a causative factor in neurodegenerative disorders, the signaling pathways linking ROS production with neuronal cell death are not well characterized. However, certain ROS-mediated signaling pathways relevant for the induction of inflammatory responses have been elucidated . The actP kinase , 104. NFκB consists of five members that are characterized by a Rel homology domain, which is required for dimerization and DNA binding. RelA (p65), RelB, and cRel contain a transactivation domain required to activate transcription of NFκB target genes. The other two members, p100 and p105, are formed as precursor proteins, which are processed by the proteasome to p52 and p50, respectively. Although the NFκB family members can form several homo- and heterodimers, two main signaling pathways have been described, the canonical and the noncanonical NFκB pathways, which are usually mediated by p65/p50 and RelB/p52 dimers, respectively. NFκB-activity is inhibited by IκB proteins which prevent translocation of NFκB into the nucleus [κB activation can have various physiological consequences and has been implicated in cell proliferation, differentiation, and survival by the induction of antiapoptotic proteins. However, NFκB is best known for its role in the immune system where it acts as an important positive regulator of the inflammatory response, predominantly, but not exclusively, by activation of the canonical NFκB signaling pathway [β and α-synuclein to TLR2 results in the activation of the canonical NFκB-signaling pathway [κB pathway is predominantly activated by various proinflammatory cytokines such as TNF and IL-1 [The family of NF nucleus . NFκB ac pathway . Similarand IL-1 , 110 andκB activation can be highly diverse. Of relevance for the development of chronic inflammatory and neurodegenerative diseases is the NFκB-mediated induction of inflammatory mediators, in particular cytokines such as IL-6, IL-8, and TNF, and adhesion molecules thus promoting recruitment of immune cells to inflamed tissues [κB is a potent inducer of NOX2 and iNOS and thus contributes to the generation of ROS/RNS under proinflammatory conditions [κB can induce the expression of COX-2 and cPLA2, which are involved in the generation of prostaglandins thus further promoting recruitment of inflammatory cells [κB demonstrates the strong interrelationship between ROS/RNS production and the induction of proinflammatory cytokines, which results in enhanced cell damage and thus aggravates neurodegeneration  , 118. TNα converting enzyme (TACE) results in the release of soluble TNF (sTNF). TNF can bind to two membrane receptors, TNF receptor (TNFR) 1 and TNFR2. Whereas TNFR1 is constitutively expressed on virtually all tissue cells, the expression pattern of TNFR2 is restricted to cells of the immune systems, especially regulatory T cells, endothelial, and neuronal tissues and its expression can be highly regulated by the cellular activation status. Both receptors are typical type I transmembrane proteins with extracellular and intracellular domains of about equal sizes and single transmembrane domains [TNF is synthesized as a type II transmembrane protein that self-assembles into noncovalently linked homotrimers. Proteolytic cleavage of the ectodomain of this initial membrane-bound form of TNF (memTNF) by the TNF- domains .The extracellular domains of both receptors are quite similar comprising four cysteine rich domains, which are required for ligand binding but also carry a homophilic interaction motif. Interestingly, whereas TNFR1 can be equally well activated by both memTNF and sTNF, the activation of TNFR2 is dependent on the presence of memTNF . A furthβ-activating kinase1 (TAK1) and its associated proteins TAK1-binding protein2 (TAB2) and TAB3  complex, by linear ubiquitination, while IKKβ, one of the catalytic subunits of the IKK-complex, associates with ubiquitinated RIP1 resulting ultimately in activation of the complete IKK-complex [κB proteins resulting in their ubiquitination and proteasomal degradation, which enables nuclear translocation and DNA binding of NFκB dimers , the activation of p38 MAP kinase and JNK, two important pathways which are associated with the induction of apoptosis via mitochondrial dysfunction  containing receptors (TNFR1) and the TRAF-interacting receptors (TNFR2), respectively . The DD and TAB3 . This me-complex . SubsequB dimers . As menttly, TNF , 126 thufunction  and the After complex I internalization TRAF2 and cIAPs dissociate from TNFR1 and a secondary proapoptotic signaling complex II is formed by recruitment of the adaptor protein Fas-associated death domain protein (FADD) and the procaspase 8 to the receptor complex resulting in the formation of the death inducing signaling complex (DISC) , 130. AfκB signaling pathways. Moreover, by an as yet not fully elucidated mechanism TNFR2 can activate the phosphatidyl inositol (PI) 3-kinase/Akt pathway thus promoting cell survival and proliferation [In contrast to TNFR1, TNFR2 does not contain a DD and therefore cannot directly induce cell death. However, TNFR2 can directly interact with TRAF-proteins, namely, TRAF 1, 2, and 3, as well as cIAPs and can thus influence TNFR1 signaling –139. Sigferation –144. In feration . in vivo, although many other cell types are also capable of producing TNF under certain circumstances. TNF acts as a costimulator for natural killer cells, activated B and T lymphocytes, and it enhances the pathogen-directed cytotoxicity of monocytes, neutrophils, and eosinophils.TNF is the prototypic member of a large family of cytokines that play an important role in the regulation of the innate and adaptive immune system . TNF itsOf great importance are the stimulatory effects on endothelial cells resulting in enhanced surface expression of adhesion molecules, a central step for the recruitment of immune cells, for example, neutrophils, lymphocytes, and monocytes, which in the case of the CNS might be followed by transendothelial cell migration into neuronal tissues , 148. ThThe pleiotropic functions of TNF in regulation of the adaptive and innate immune response explain why deregulated TNF production due to overreaction of the host or expression in an inappropriate location can lead to major pathogenic consequences. Indeed, persistently elevated levels of TNF have been implicated in chronic inflammation and have been associated with a variety of diseases including autoimmune diseases and neurodegenerative diseases. Blocking of TNF signaling has therefore been evaluated in various inflammatory diseases and is successfully used for treatment of autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis , 152.Interestingly, increased serum TNF levels due to systemic inflammation have been associated with an increased cognitive decline in AD and PD suggesting a link between systemic inflammation and neurodegeneration , 153. SiAlthough other members of the TNF superfamily, for example, Fas ligand and TRAIL, have been linked to neuroinflammation , TNF cleInterestingly, distinct effects of TNF in the CNS have also been revealed by using TNFR knockout mice in MPTP-induced neurotoxicity. In this model, deficiency for both TNFRs protects against MPTP-induced striatal dopaminergic neurotoxicity, suggesting a role for TNF in neurodegeneration in PD. In the hippocampus, however, TNFR-deficient mice showed exacerbated neuronal damage after MPTP treatment. Taken together, the results are indicative of a region-specific and dual role for TNF in the brain: a promoter of neurodegeneration in the striatum and a protector against neurodegeneration in the hippocampus .A direct role of TNF in PD is also suggested by genetic studies, which identified polymorphisms in the TNF gene, which are associated with an increased risk to develop sporadic PD. In contrast the role of genetic variations of TNF is less clear for the development of AD . ImportaThere is increasing evidence that the detrimental effects of TNF in chronic neurodegenerative diseases are largely mediated by sTNF. For example, transgenic mice expressing a mutant TNF that cannot be cleaved, thus preventing the formation of sTNF, are largely protected against the induction of autoimmune diseases , 161. Si35-55 induced EAE [35-55 induced EAE [Importantly, polymorphisms in the TNFR1 gene have been associated with the risk to develop PD or MS , 164. Mouced EAE –169. Induced EAE , 171. Fuuced EAE –174. TheκB. First, as mentioned in κB signaling pathway in endothelial cells can induce the expression of cell adhesion molecules thereby promoting the transmigration of immune cells into the CNS parenchyma [κB signaling pathway can induce the production of proinflammatory cytokines such as IL-6, IL-8, and TNF itself. Enhanced TNFR1 signaling will thus greatly enhance the inflammatory response thereby promoting neurodegeneration. Indeed, astrocyte-specific inhibition of NFκB signaling in transgenic mice strongly suppresses the inflammatory response of the CNS in various animal disease models, such as spinal cord injury, EAE, and optic neuritis, thereby reducing neurodegeneration and improving functional recovery [TNFR1 can exacerbate neurodegeneration at several routes, which are mainly mediated by activation of NFrenchyma . Moreoverecovery –183.γ), increase the expression of iNOS in microglia leading to the secretion of NO [κB-dependent manner again demonstrating the relevance of this signaling pathway for the role of TNF in neurodegeneration. Of relevance for AD, the intracerebroventricular injection of Aβ in mice caused marked deficits of learning and memory, which were greatly reduced by TNF and iNOS inhibitors. Importantly, a similar reduction of AD symptoms was observed in TNFR1-deficient mice demonstrating the role of TNFR1 in this animal model of AD [Next to the induction of proinflammatory signaling pathways, TNF can contribute to chronic neurodegeneration by promoting the generation and release of ROS and RNS. Indeed, the high systemic toxicity of TNF has been related to excessive induction of NO resulting in vasodilatory shock . In the on of NO . Moreoveon of NO . In thisel of AD .κB-dependent manner [Moreover, TNF can also directly exacerbate the formation of ROS by activating NADPH oxidases thus inducing the production of superoxide . For exat manner . Importat manner . A specit manner .κB target gene. Indeed, it has been shown that TNFR1 is required for the efficient induction of COX-2 in activated macrophages [β-induced activation of COX-2, which is correlated to the cognitive decline of the animals [Another mechanism by which TNF, via TNFR1, can promote the generation of ROS is the activation of COX-2, another NFrophages . Importa animals .Although sustained activation of JNK, in particular JNK3, has been implicated in neurodegeneration, the evidence for a role of TNF-mediated JNK-activation in chronic neurodegeneration is scarce. One example is the role of TNFR1-induced JNK-activation in the death of retinal ganglion cells following optic nerve crush injury . Of rele in vitro studies have shown that TNF, via TNFR1, can also mediate direct apoptosis of neurons by activation of caspase 8 [ in vivo [Next to the indirect neurodegenerative effects of TNF by promoting neuroinflammation and oxidative stress mediated by glial cells,aspase 8 . Elevate in vivo . Whether in vivo .κB activation. Indeed, whereas induction of NFκB in astrocytes and microglia promotes inflammation and neurodegeneration  and remyelination are significantly delayed in TNF and TNFR2 knockout mice, demonstrating that tissue regeneration is dependent on the signaling of TNF via TNFR2  Table 1Table 1. tion see , neuronae in EAE . In thistoxicity . This peκB pathway, also the PI 3-kinase/Akt survival pathway has been implicated in TNFR2 mediated neuroprotection [ In vitro studies have revealed that neuroprotective effects of TNFR2 indeed require the activation of the PI 3-kinase/Akt pathway [2O2 or 6-OHDA induced cell death by selective activation of TNFR2 after the toxic insult [Besides the NFotection , 143. Mootection , thereby pathway , 177. Imc insult . Althougc insult –200. Nexc insult , thereby2O2-induced cell death [On a mechanistic level, TNFR2 activation promotes the release of anti-inflammatory and neurotrophic factors from astrocytes and microglia , 202, whll death . These rll death , 206.The two most common features of neurodegenerative diseases are sustained oxidative stress and inflammation. Here, we have provided an overview on the interrelation between these two hallmarks of neurodegeneration. Of particular importance is the excessive generation of ROS, for example, due to mitochondrial dysfunction, which causes neuronal damage and thus the release of cytosolic factors that activate neighboring microglia and astrocytes. These cells respond by the release of proinflammatory cytokines as well as ROS and RNS thus further promoting the inflammatory response and exacerbating the neuronal damage. Accordingly, persistent activation of glia cells can ultimately result in an amplification loop resulting in chronic neurodegeneration.κB pathway in glia cells is a key mediator of these detrimental effects of TNF leading on one hand to the elevated production of proinflammatory cytokines and on the other hand to the production of iNOS, COX-2, and NOX subunits, thereby activating NADPH oxidases, ultimately leading to the production of ROS. TNF itself is a response gene of the NFκB pathway and, moreover, ROS can activate the NFκB pathway thereby amplifying the TNF/ROS/NFκB responses. In consequence, this aggravates the neuronal damage, which further promotes neuroinflammation ultimately resulting in a feed-forward loop that causes chronic neurodegeneration. This central role of NFκB and the resulting strong cross talk between proinflammatory cytokines, mainly TNF, and ROS/RNS emphasize the interrelation between inflammation and oxidative stress in neurodegeneration (TNF is a key cytokine of the immune system that initiates and promotes inflammation, which under uncontrolled conditions may lead to the development of neurodegenerative diseases. The activation of the NFneration .Translating these findings into the production of novel therapies to attenuate neurodegeneration is, however, challenging due to the distinct effects of TNF via its two receptors, which is exemplified by the failure of TNF-inhibitors in MS treatment . Since t


The goal of therapy of rheumatoid arthritis is to achieve a remission or at least low disease activity. TNF inhibitors induce high remission rates only in combination with methotrexate, whereas the efficacy of tocilizumab is optimal even as a monotherapy. In this article, the differing dependence of the biological drugs on methotrexate is explained from the viewpoint of an immunologist. A selective search and evaluation of the literature was performed with regard to the mode of action of TNF inhibitors, tocilizumab and methotrexate in rheumatoid arthritis. Methotrexate primarily inhibits the activation and proliferation of lymphocytes. TNF inhibitors suppress monocytes and myeloid dendritic cells, and tocilizumab has a broader activity and is directed against both the lymphoid as well as the myeloid compartment. In view of the broad mode of action of tocilizumab, it can be explained why this drug, in contrast to TNF inhibitors, is acting optimally even in monotherapy. Adalimumab Actemra (ADACTA) trial, adalimumab and tocilizumab were compared in RA patients intolerant of methotrexate [Since the introduction of biologics into the treatment of rheumatoid arthritis (RA), patients’ symptoms have been significantly reduced and the process of joint destruction markedly decelerated. At present, about 15 % of RA patients in Germany are treated with biologics. Five TNF inhibitors, together with tocilizumab and abatacept, are approved for use as first-line biologics following failure of at least one or two conventional disease-modifying anti-rheumatic drugs (DMARDs). Earlier studies, e.g. the TEMPO trial for etanercept , showed The initiation of RA is facilitated by a genetic predisposition. In addition, the probability of developing RA is influenced by environmental factors such as smoking, alcohol and nutrition. The autoimmune aspect of the disease begins many years before overt arthritis occurs. In this ‘pre-arthritis’ phase, the autoantibodies rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA) can usually be detected ; howeverThe joint inflammation, which usually starts after a long period of pre-arthritis, has three main phases:At the initiation of arthritis, cells of the immune system move into the joints. Arthritis is probably triggered by an autoantigen in the joint. This autoantigen has not yet been identified, and triggers are likely to vary from patient to patient. Initially, antigen-presenting cells in the joint loaded with an autoantigen probably move to the central lymphatic organs and activate T cells there. They then migrate back to the joints, along with circulating immune cells.+ T-helper cells that are mainly part of the Th1 and proinflammatory Th17 subsets [Most of the lymphocytes in the synovial membrane comprise CD4 subsets , 9. B ceThe cells of the immune system interact in the inflamed joint and activate each other by cell-cell contact as well as by the production of cytokines. The formation of cytokines such as TNF-α and IL-6 at the site of inflammation leads to activation of the endothelium cells in newly formed vessels and to an increase in adhesion receptors such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) . This fu2 in the synovial fluid is always markedly below that in the blood. Angioneogenesis therefore is essential for the perpetuation of the inflammation [Fibroblast-like synovial cells are activated and produce collagenases , which attack the cartilage . Then, aammation .Insights into the pathophysiology of RA have led to the development and use of biologics. The mode of action of classical DMARDs, and specifically that of methotrexate, can now be at least partially explained.Methotrexate (MTX) was first used in the treatment of RA in 1951 , but hasIn patients with RA, methotrexate inhibits the proliferation of cells and induces apoptosis (cell death) of activated cells. Both mechanisms mainly affect the lymphocytes and also, to a lesser degree, monocytes . MethotrMethotrexate probably reduces the interaction of T lymphocytes and synovial fibroblasts by reduced cytokine production, e.g. of IL-6, IL-15 and IL-17 . On monoThe production of RANK-L by synovial fibroblasts is decreased and that of osteoprotegerin is increased . Both meAs described above, TNF-α is one of the dominant cytokines in the pathogenesis of RA. TNF-α is predominantly produced by monocytes/macrophages, as well as by T cells, and activates, for example, macrophages and endothelial cells, after binding to the TNF receptor (CD120), which is expressed on multiple cells. As a consequence, there is an increase in the production of IL-1, IL-6 and other proinflammatory cytokines. Due to the important role of TNF-α in the pathophysiology of RA, TNF inhibitors are used in therapy. Infliximab is a chimaeric antibody targeted at TNF-α. Today, complete human antibodies against TNF-α are available in the form of adalimumab and golimumab, while certolizumab is a pegylated human anti-TNF-α antibody (which does not bind to Fc receptors); etanercept is a soluble TNF receptor. Everyday clinical experience shows that some RA patients respond to a second, but not the first, TNF blocker. Therefore, there may be subtle differences in the modes of action of the individual TNF inhibitors, which will not be discussed here in general, however.TNF inhibitors influence the pathophysiology of RA on several levels.At least in vitro, TNF inhibitors reduce the expression of VCAM on endothelial cells from the microvasculature of the intestine  and therThe degree of activation of the monocytes is markedly reduced , 25. In The influence on lymphocytes is less pronounced. In vitro studies have shown that infliximab is able to induce apoptosis (programmed cell death) at least in some T lymphocytes. In vivo, however, both infliximab and etanercept were able to trigger apoptosis only in monocytes, but not in lymphocytes . The actThe change in cell populations affects the production of cytokines in the blood. Specifically, the concentration of proinflammatory cytokines produced by monocytes, such as IL-6, MIP-1 and CCL20 , is reduTNF inhibitors decelerate joint destruction even if the inflammation does not improve. TNF inhibitors reduce the production of MMPs  that contribute to the chondroclast activation and cartilage destruction . Furthertrans-signalling). IL-6 therefore has a widespread effect on many cells and functions. In the inflammatory process, IL-6 increases the production of chemokines and expression of adhesion molecules on lymphocytes [Tocilizumab binds both to the membrane-bound and soluble IL-6 receptor and can thus completely interrupt the effect of IL-6. IL-6 is a proinflammatory cytokine that binds to the IL-6 receptor and then to gp130, a signal transduction molecule. The membrane-bound IL-6 receptor is only expressed on few cell types. However, the soluble IL-6 receptor circulates in the blood and binds to IL-6. This complex can bind to gp130, which is expressed on the surface of most cells , tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and “C-X-C motif chemokine 10” (CXCL10) was reduced in responders . MIF is According to current insights, tocilizumab therefore has a very broad effect and regulates B and T lymphocytes, as well as the monocytes and dendritic cells.TNF inhibitors, whose mode of action (MoA) primarily targets monocytes and dendritic cells, and MTX, which inhibits lymphocytes, exert a synergistic therapeutic effect in RA.Tocilizumab has a broader effect than TNF inhibitors and mainly targets T and B lymphocytes, but also dendritic cells and monocytes.The combination of TNF inhibitors with methotrexate fills the gap of each substance with regard to the MoA, while tocilizumab alone has a very broad effect.The observation from studies, which show that tocilizumab monotherapy is as effective as tocilizumab in combination with methotrexate, is therefore not surprising.


Evidence suggests that activation of κ-opioid receptor (KOR) by U50,488H exhibits potential cardiovascular protective properties. However, the effects of U50,488H on vascular dysfunction in diabetes mellitus (DM) are still not clear. The present study was designed to investigate the effects of U50,488H on vascular dysfunction in diabetic rats and explore the underlying mechanisms involved.Rats were randomly divided into control, DM, DM + vehicle, DM + U50,488H and DM + nor-binaltorphimine (nor-BNI) groups. Streptozotocin injection was used to induce DM. Weight, blood glucose, blood pressure and plasma insulin for each group were measured. Arterial functions were assessed with isolated vessels mounted for isometric tension recordings. Angiotensin II (ANG II), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin (IL)-6 and IL-8 levels were measured by ELISA, and endothelial nitric oxide synthase (eNOS) phosphorylation and NF-κB p65 translocation were measured by Western blot.Activation of KOR by U50,488H reduced the enhanced contractility of aortas to KCl and noradrenaline and increased acetylcholine-induced vascular relaxation, which could also protect the aortal ultrastructure in DM. U50,488H treatment resulted in reduction in ANG II, sICAM-1, IL-6 and IL-8 levels and elevation in NO levels, while these effects were abolished by nor-BNI treatment. Further more, eNOS phosphorylation was increased, and NF-κB p65 translocation was decreased after U50,488H treatment.Our study demonstrated that U50,488H may have therapeutic effects on diabetic vascular dysfunction by improving endothelial dysfunction and attenuating chronic inflammation, which may be dependent on phosphorylation of eNOS and downstream inhibition of NF-кB. Diabetes mellitus (DM) has become an increasing burden on human health worldwide . It is rOpioid receptors, discovered by Solomon H. Snyder in 1973, belong to the G protein-coupled receptor superfamily . There aIt has been shown that KOR is the predominant type of opioid receptors in the cardiovascular system , and hasRecent data have suggested that U50,488H is a highly selective KOR agonist with lower abuse potential and fewer side effects, such as respiratory depression, convulsions and gastrointestinal intolerance ,15. UponIt is well known that endothelial dysfunction and low-grade inflammation play important roles in vascular dysfunction -22. EndoIn this study, we investigated whether KOR activation was involved in attenuating diabetic vascular dysfunction by U50,488H and explored the downstream molecular mechanisms.In order to establish diabetic model, streptozotocin  injection was applied. Male Sprague–Dawley (SD) rats (weight 200–240 g) received intraperitoneal injection of STZ (35 mg/kg) for 3 days . Blood gBlood pressure was monitored with a tail cuff system  as described previously . BrieflyTo detect the KOR expression in thoracic aorta, immunohistochemical staining was performed. Briefly, the thoracic aortas were isolated, fixed by 4% paraformaldehyde and paraffin embedded. Serial tissue sections (5 μm) were mounted on slides, then xylene and ethanol were used to deparaffinize and dehydrate the tissue respectively. Non-specific antigens were blocked by 20% normal bovine serum for 30 minutes, which was followed by treatment with 0.3% hydrogen peroxide. Then the sections were incubated overnight at 4°C with KOR antibody . After three washes in PBS, the sections were incubated with a biotinylated secondary antibody  for 1 hour at 37°C and washed again in PBS. Then horseradish peroxidase was developed with 3,3-diaminobenzadine (DAB)  as the chromogen substrate. After rinsing, the slides were mounted with cover slips, and the positive samples displayed a brown color under a light microscope.Transmission electron microscope (TEM) was performed to identify the ultrastructural changes of thoracic aortas. After anesthetized, the thoracic aortas from the rats were immediately isolated and cut into 1–2 mm wide, and then kept in glutaraldehyde and osmium tetroxide in order. Subsequently, the TEM specimens were dehydrated using graded ethanol and embedded in resin. The ultrastructure of thoracic aortas were visualized by ultra-thin sections under transmission electron microscope .Enzyme-linked immunosorbent assay  was performed to determine the serum levels of ANG II, sICAM-1, IL-6 and IL-8, as previously described . StandarThe arterial functions were assessed with endothelium-intact isolated rats vessels mounted for isometric tension recordings in organ chambers, as described previously . Rats we2 + 5% CO2) Krebs solution . The vessels were allowed to equilibrate for 60 minutes before the onset of experiments, with Krebs solution changed every 15 minutes. After a washout period, vascular rings were challenged with a cumulative concentration of KCl (10–90 mmol/L) or noradrenaline  to test vasoconstrictive activity. In addition, arterial contractions were induced by phenylephrine , and then were relaxed by acetylcholine  or sodium nitroprusside  to test vasodilative activity. Myograph data were recorded by PowerLab and Chart software , and cumulative concentration response curves were then generated.Vascular rings were mounted on a myograph  under their optimal resting tension (1.0 g) in 15 ml organ baths containing warmed (37°C), oxygenated (95% OWestern blot was performed as previously described . BrieflyThe following primary antibodies were used: KOR (1:1 000), endothelial nitric oxide synthase , phospho-eNOS (at Ser-1177) (1:1 000), NF-κB p65 (1:1 000), histone 3 (1:1 000) and β-actin (1:1 000). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG at 1:5,000 dilution. All above antibodies were purchased from Santa Cruz Biotechnology.All statistical analysis were performed by GraphPad Prism software version 5.0 . Values were presented as mean ± SD and analyzed using ANOVA with Bonferroni corrected t-test. Western blot results were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Differences were considered statistically significant if P < 0.05.vs. 8.2 ± 0.94 mmol/L, P < 0.05), decreased body weight gain  and reduced insulin levels  compared with CON group. There was no significant difference in blood pressure  between CON and DM groups. Neither U50,488H nor nor-BNI administration influenced body weight, blood glucose levels, blood pressure and insulin levels in diabetic rats.Administration of U50,488H or nor-BNI was initiated 4 weeks following the onset of experimental DM. The metabolic characteristics of rats in different groups are shown in Table As shown in Figure −9-10−4 mol/L) are shown in Figure To determine the effects of KOR activation on vasoconstrictive function of thoracic aortas in DM rats, isometric tension recordings were performed. The concentration-response curves for KCl (0–100 mmol/L) and NE (10−9-10−4 mol/L) or SNP (10−9-10−4). Concentration-response curves  staining showed intact endothelium in the CON group, that the endothelium was falling in the DM group, and only part of the endothelium was falling in the DM + U50,488H group Figure A. TransmSerum NO concentrations in the DM group were significantly decreased compared with the CON group Figure A. TreatmTo determine whether U50,488H inhibited the DM-induced inflammatory response in rats, pro-inflammatory cytokines and adhesion molecule levels were measured. As shown in Figure It is reported that inhibition of the transcription factor NF-кB is important in mediating anti-inflammatory activity. Therefore, Western blot was performed to investigate NF-кB nuclear translocation. The results showed that nuclear translocation of p65 protein induced by DM was markedly attenuated by U50,488H treatment, while nor-BNI treatment increased nuclear translocation of p65 protein  or autoimmunity (type 1 DM) [There are two main types of diabetes: insulin-dependent DM (type 1) and non-insulin-dependent DM (type 2). Type 1 DM is strongly associated with autoimmunity, while type 2 DM is strongly associated with obesity and insulin resistance. STZ-induced DM offers a very cost effective and expeditious technique for DM research. STZ also offers the additional benefit of being able to select specific traits of interest, which can be important for specific experimental design . In thispe 1 DM) ,39.Previous research has indicated that opioid receptors are widely distributed in the cardiovascular system . Opioid We further found that KOR activation could improve vasoconstrictive function in DM, evidenced by a reduction in vascular contractility to NE and KCl. We also observed that the impaired endothelium-dependent vasorelaxation in response to ACh in DM was ameliorated significantly by KOR activation, which suggests that the attenuation of endothelial dysfunction by KOR activation may, at least in part, account for the improvement in vasorelaxation.Endothelial dysfunction has been recognized as one of the major causes involved in the development of cardiovascular diseases . ClinicaEndothelium can maintain vascular homeostasis through multiple regulatory pathways that involve the release of vasoactive factors such as NO and ANG II ,46. NO iet al. reported that ANG II contributed to impairment of insulin-stimulated vasodilation in vascular endothelium in DM rats [We further found that KOR activation by U50,488H reduced serum levels of ANG II. As we known, ANG II exerts an important action in mediating endothelial dysfunction, vascular inflammation, hypertrophy and remodeling. Park  DM rats ,49.To explore the mechanism by which U50,488H improved DM-induced endothelial dysfunction, we further investigated eNOS phosphorylation. Evidence showed that eNOS phosphorylation was associated with the increase in NO production, as well as the decrease in ANG II production . Consistet al. reported that U50,488H administration could inhibit neutrophil accumulation and TNF-α induction in myocardium subjected to ischemia/reperfusion [et al. reported that U50,488H administration could inhibit TLR4/NF-κB signaling induced by ischemia/reperfusion in rats hearts [Chronic inflammation is another important cause accounting for vascular dysfunction. Accumulating evidence suggests that U50,488H has therapeutic potential in cardiovascular protection through an anti-inflammatory response, which exhibits a broad inhibitory influence on cytokines, chemokines and chemokine receptor expression. Wu erfusion . Lin et s hearts . In thiss hearts .We further investigated the mechanisms responsible for U50,488H-associated vascular protection in DM. It is reported that NF-κB plays a central role in the development of inflammation through regulation of genes encoding not only pro-inflammatory cytokines, but also adhesion molecules . Our resin vitro experiments. Therefore, further study is needed to evaluate the effects of KOR activation on vascular function in patients with DM.This study provides evidence supporting the protective effects of KOR activation by U50,488H against DM-induced vascular dysfunction. These findings indicate that KOR activation may represent a promising therapeutic strategy to maintain vascular function in patients with DM. Nonetheless, it is noteworthy that our findings were mainly based on rodent models and KOR activation may have therapeutic effects on diabetic vascular dysfunction by attenuating chronic inflammation and improving endothelial function, which may be dependent on eNOS phosphorylation and downstream inhibition of NF-кB.


Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.The spread of  Many plant and animal viruses induce the formation of insoluble aggregates/inclusion bodies inside infected cells. These structures usually contain viral and host proteins, and can vary in location, size, content and biological function1131Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement78The interaction with cytoskeleton elements was studied mostly, but not only, with RNA viruses. The involvement of cytoskeletal elements in virus inter- and intra-cellular movement and/or in the formation of VFs was shown for many plant viruses1Tobacco mosaic virus (TMV) and facilitate the transport of the viral MP and MP-containing viral replication complexes (VRCs) along the ER to the plasmodesmata (PD)3410Actin filaments and associated myosin motor proteins are also important for Potato leafroll virus (PLRV) MP17 localizes to PD of infected source cells in an actin filaments/ER-Golgi-dependent manner, while in sink tissues, it is degraded in a microtubule/proteasome-dependent manner. Inhibition of the proteasome in sink leaves leads to the formation of a single large aggregate per cell, resembling the mammalian aggresomeThe movement protein of Cauliflower mosaic virus (CaMV)15Tomato yellow leaf curl virus (TYLCV) coat protein (CP) in tomato (Solanum lycopersicum) leaf cellsBegomovirus, family Geminiviridae) transmitted in nature by the whitefly Bemisia tabaciMicrotubules play an important role also in the formation of electron lucent inclusion bodies (ELIBs) from electron dense inclusion bodies (EDIBs) during infection with the double stranded (ds) DNA virus Nicotiana tabacum protoplasts showed V2 to be localized mainly in the cytoplasm, around the nucleus and at the cell periphery, as well as in punctate bodies distributed throughout the cytoplasm2123In the current study, we investigated the subcellular accumulation pattern and properties of V2, a protein encoded by the same ssDNA genome as CP and transcribed from the same promoter as CPHere, we show that TYLCV V2 forms aggregates in tomato tissues and is able to bind TYLCV genomic ssDNA (but not dsDNA replication form). We also demonstrate that V2 associates with the cytoskeleton; furthermore intact actin filaments and microtubules are needed to deliver V2 to the 26S proteasome for degradation. We propose that the cytoskeleton is required for the development of V2 aggregates, a parameter of the successful TYLCV invasion of plant cells. Moreover, our data indicate that V2 aggregates are targeted for degradation by the 26S proteasome.B. tabaci B biotype) were reared on cotton plants grown in insect-proof cages at 26 °C, as described. Nicotiana benthamiana plants, four weeks after sowing, were used for infiltration experiments. All plants were grown in a greenhouse kept at 24 °C  to 18 °C .TYLCV from Israel (GenBank X15656.1) was maintained in tomato plants (cv. Daniella) by whitefly-mediated inoculationNdeI and BamHI sites of pET-14B (Novagen). The recombinant plasmid pET-V2 was used to transform E. coli cells, strain BL21 (DE3). pBIN19-V2:GFP was a gift from Prof. Y. Gafni, The PCR-amplified V2 ORF (nucleotides 314-664) was cloned into the Western blotting was performed according to standard procedures. The source of antibodies was as follows: anti-TYLCV-V2 primary antibody was prepared similarly to the anti-TYLCV-CP2, 0.2 mM EDTA, 1 mM dithiothreitol and Complete Protease Inhibitor Mixture - Roche, Mannheim, Germany). Stem pellet after spin down was re-suspended in buffer H and drill-homogenized. The leaf and stem homogenates were filtered through a cellulose membrane  to separate debris and insoluble cell wall fraction from the filtrate. Proteins in cell debris and cell wall fraction were not gradient-separated further; the whole fraction was named Pellet (P). The filtrate was subjected to centrifugation at 3000 g for 20 min to obtain a pellet (P3) and a supernatant (S3). Native total proteins were isolated as follows: leaves and stem spin down pellet (pooled from three plants) were drill-homogenized in buffer H supplemented with 0.5% Nonidet P40. Homogenates were incubated on ice for 45 min, vortexed and centrifuged at 1200 g for 10 min at 4 °C. The supernatant containing native proteins was further analyzedLeaves (pooled from three plants) were collected and drill-homogenized in detergent-free buffer H  leaf and stem samples were embedded in wax (PEG 400 distearate and 1-hexadecanol – both from Sigma – mixed at a ratio of 9:1). Fifteen micrometer-thick wax-embedded tissues were sectioned with a microtome , rehydrated and blocked for 1 h at 25 °C in 2% BSA/MTSB prior to incubation for 18 h at 4 °C with anti-TYLCV-V2 primary antibody diluted 1:100 and/ or with anti-TYLCV-CP diluted 1:100 in 2% BSA/MTSB. After washing with MTSB the samples were incubated for 1.5 h at 25 °C with a Cy3- and Cy2-conjugated anti-rabbit secondary antibody  diluted 1:200. The samples were inspected using a stereoscopic fluorescence zoom microscope  and fluorescence microscope ; V2 was detected as red fluorescent signal, CP was detected as green fluorescent signal. Plant nuclei were stained with DAPI , at 1 μg/ml for 20 min at 25 °C, and detected as blue fluorescent signal.For histological analyses, cross sections of tomato leaves and stems (cut into 0.5 cm squares) were processed as describedN. benthamiana leaves were infiltrated with Agrobacterium tumefaciens transformed with the expression plasmids. The bacteria were grown for 48 h at 28 °C in LB medium containing antibiotics. The culture was centrifuged at 4,000 × g for 8 min, and the pellet was re-suspended in water to a final optical density (OD 600) of 0.1-0.6. The suspension was used to infiltrate the underside of young leaves of 5-weeks-old plants using a syringe. The GFP/RFP-fusion proteins were visible under the fluorescence microscope 48 h after infiltration. For confocal imaging, we used an Olympus IX 81 inverted laser scanning confocal microscope (Fluoview 500) equipped with an argon ion laser and a 60 × 1.0 N.A. PlanApo water-immersion objective. Excitation/emission wavelengths were 488 nm/505 to 545 nm for GFP and 543 nm/585 to 615 nm for RFP. Plant nuclei were stained with DAPI infiltrated into leaves 30 min prior to detection at excitation/emission wavelengths of 350 to 470.http://imagej.en.softonic.com/). The number of aggregates per image-area and their size were measured using the “threshold” and “particle analyzer” functions of the program.To determinate the number and cross-sectional areas of aggregates, images were imported into ImageJ  were placed in micro-tubes containing 20 μM oryzalin, 10 μM latB and/ or 50 μM MG132 . Water with the same final concentration of DMSO but without the drugs was used as control. The solutions were replaced daily for 4 days. Proteins were extracted, separated on sucrose gradients and immuno-detected as described.Oryzalin, latrunculin B (latB)  and MG132  were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions of 100 mM. For Interaction between V2 and total DNA extracted from leaves of TYLCV-infected tomato plants was detected by immuno-capture-PCR (IC-PCR)TYLCV V2 was immuno-detected in protein extracts of tomato leaves and stems using a specific antibody, 28 and 49 days post whitefly-mediated inoculation (dpi) . The reaTYLCV CP was shown previously to be more abundant in insoluble than in soluble protein fractions of tomato leaf and stem5Following this observation, we determined the state of V2 aggregation by ultracentrifugation of leaf and stem protein extracts in non-denaturing 10-50% sucrose gradients18in situ immuno-fluorescent visualization of V2 in leaf and stem sections of symptomatic tomato plants at 28 and 49 dpi. The first appearance of V2 aggregates was detected at 28 dpi. At later stages (49 dpi), the abundance of V2 aggregates increased , the PCR amplicon was not obtained , we tested whether V2:GFP aggregation was dependent on the integrity of microtubules and actin filaments. The potential involvement of microtubules in V2 aggregation was investigated by labeling microtubules with the microtubule marker MAP4:RFP , a toxin that inhibits the polymerization of actin microfilamentsThe potential involvement of actin filaments in V2 aggregation was investigated by treating N. benthamiana leaves expressing V2:GFP were treated with the proteasome inhibitor MG132Several plant viral proteins were shown to be degraded by the 26S proteasome14To determine whether the proteasome and cytoskeleton inhibitors have similar effects on V2 aggregation during virus infection, leaves of TYLCV-infected tomato plants were treated with oryzalin, latB, and MG132, separately or in combination . Proteins were extracted after 4 days and separated by ultracentrifugation in sucrose gradients, and the viral V2 was immuno-detected on western blots. In samples from tissues with disrupted cytoskeleton, V2 was observed in fractions reflecting aggregates of smaller sizes than in untreated leaves . TreatmeViral proteins form conspicuous aggregates in the cytoplasm and nucleus of plant cells infected by viruses. Although the role of aggregation was studied extensively in mammalian cells, less is known about the role of aggregation of viral proteins during infection in plant cells. Following the dynamics of aggregation of viral proteins in plant host cells provides new insights into virus-host interactions, especially with respect to the cellular structures involved in the defense against viruses or exploited by viruses (reviewed in 2). In general, aggregates may reflect the presence of local scaffolds for anchoring and accumulation of protein complexes. Such anchored accumulation sites can support virus infection by increasing the local concentration of viral and host components required for replication and assembly, and by shielding the process of replication from host defense. Aggregation of proteins with support of cellular scaffolds may also facilitate innate cellular responses that recognize virus components and target them for storage and degradationin vitro and in vivo .