Moreover, weinvestigated the effect of TNF-α inhibition onhuman endothelial cells (HUVECs) incubated with serumfrom RHTN and normotensive subjects. HUVECs containingserum obtained from normotensive (n = 8) and hypertensive (n = 8) individuals were treated withTNF-α inhibitor (infliximab). Cellsuspensions were used for measurement of DNAfragmentation and reactive oxygen species (ROS)content. RHTN patients showed higher levels ofTNF-α compared to normotensive subjects, aswell as higher PWV. Positive correlation was foundbetween TNF-α levels and PWV measures in thewhole group. HUVECs incubated with serum from RHTNshowed increased cell apoptosis and higher ROScontent compared to normotensive subjects.Infliximab attenuated the apoptosis of HUVECsincubated with serum from RHTN, but no effect inROS production was observed. Our findings suggestthat TNF-α might mediate, at least in part,vascular damage in resistant hypertension.Inflammatory cytokines have been associated withthe pathophysiology of hypertension and target organdamage (TOD). Resistant hypertensive patients (RHTN)are characterized by poor blood pressure control andhigher prevalence of TOD. This study evaluated therelationship between plasma levels of TNF- In these animals, the inhibition of TNF-α prevented increased vascular superoxide production and hypertension mediated by angiotensin II [α inhibition decreases blood pressure and prevents target organ damage in animal studies [α in humans showed BP reduction [Several studies have demonstrated the participation of inflammatory cytokines in the genesis of hypertension in humans , 2 and astration , 5. Moreensin II . Althoug studies , 6. Alsoα, and the expression of cell adhesion molecules including VCAM-1 and MCP-1 [α, C-reactive protein, and interleukin-6 are correlated with arterial stiffness in hypertensive subjects [−/−) or interleukin-17a (IL-17a−/−) reduces collagen deposition in the aorta and superoxide production and preserved endothelium-dependent vasodilatation compared to wild type animals [α showed increased expression of several genes related to vascular dysfunction and inflammation [Resistant hypertensive patients (RHTN) represent extreme phenotype of hypertension, characterized by poor blood pressure control and higher prevalence of target organ damage, which explain the unfavorable prognosis associated with this condition . Vasculand MCP-1 , 15. In subjects . In fact animals . Interesammation . Taken tα inhibition with infliximab reduced systolic BP, left ventricular hypertrophy, and vascular inflammation in spontaneously hypertensive rats (SHR) [α compared with normotensive subjects [α inhibition has been discussed as potential strategy to improve vascular function [α improves vascular endothelial function and decreases arterial stiffness in postmenopausal women [We recently found that TNF-ts (SHR) . Moreovesubjects . TNF-α ifunction , 21. On al women  and rheual women .α as a potential marker of vascular inflammation, the causal role of this cytokine in the pathogenesis of hypertension is underexplored. The association of TNF-α has been extensively reported in hypertension, but no previous study evaluated the effects of TNF-α on human endothelial cells. Thus, this study was designed to evaluate the relationship between plasma levels of TNF-α and arterial stiffness in RHTN and normotensive subjects. Moreover, we investigated the effect of TNF-α inhibition on human endothelial cells incubated with serum from RHTN and normotensive subjects.Despite some studies suggesting TNF-This cross-sectional study was performed in the Outpatient Resistant Hypertension Clinic at the University of Campinas Hospital. Thirty-two patients classified as RHTN and 19 normotensive subjects were included in this study. Resistant hypertension (RHTN) was defined as blood pressure (BP) that remained above goals despite the concurrent use of 3 antihypertensive agents of different classes, including a diuretic, at optimal dose amounts. Also, patients whose blood pressure was controlled but required 4 or more medications were also considered resistant . Hyperteα were determined by ELISA  according to manufacturer's instructions. Biochemical assessments, including serum cholesterol, LDL, HDL, triglycerides, glucose, aldosterone, and creatinine, were performed by central laboratory at the University of Campinas Hospital.Blood samples were collected at 8:00 a.m. after overnight fasting. The plasma levels of TNF-Pulse wave velocity was measured using the SphygmoCor System  with the patient in the supine position. Pulse wave of the carotid and femoral arteries was determined by estimating the delay with respect to the electrocardiogram wave. A measuring tape was used to measure the distance from the sternal notch to the carotid-femoral recording site. Carotid-femoral PWV was calculated by dividing traveled distance by transit time (PWV = distance/time). At least two measurements were performed in each patient. The PWV value was reported as the mean and the values were corrected for mean arterial pressure.At the study visit, blood was collected by antecubital vein puncture using serum-separating tubes (BD Vacutainer System). After 30 minutes of resting at room temperature, whole blood samples were centrifuged for 10 minutes at 4,000 rpm. Serum was stored at −80°C in 1 mL aliquots until the cytokine measurements.2 in Dulbecco's Modified Eagle's Medium , supplemented with glucose 4,500 mg/L and 10% (v/v) fetal bovine serum (FBS). The cells were used at passage 3 for the experiments. HUVECs were plated in 6-well plates at a density of 2 × 105 cells/well (for flow cytometry assays) and in 25 cm2 tissue culture flasks at a density of 4 × 105 cells/flask (for gene expression experiments). After 24 hours, the medium was replaced by FBS-free medium containing 10% of serum obtained from normotensive (n = 8) and hypertensive (n = 8) individuals. The cells were incubated for additional 24 hours with the patients' serum. The experiments were performed in duplicate. When used, infliximab (400 μg/mL) was added to the cells concomitantly with human serum.HUVEC cell lines  were cultured at 37°C in 5% COThe medium was discarded and adherent cells were washed with Krebs buffer. The cells were detached using a cell scraper. Cell suspensions were divided into two aliquots used for measurement of DNA fragmentation and ROS content.μL of DNA fragmentation buffer  and incubated overnight at 4°C. After the incubation, samples were analyzed using a FACSCalibur flow cytometer . Fluorescence of 10,000 events was acquired in the FL2 channel and analyzed using the CellQuest software. The cells with fragmented DNA emitted lower fluorescence signal, compared to cells with intact diploid and tetraploid DNA, which emitted characteristic two-peaked high-intensity fluorescence. The percentage of apoptotic cells was calculated using the number of low fluorescence events.For DNA fragmentation measurements, cells were centrifuged  and the pellets were resuspended in 250 For ROS content measurements, cells were incubated with 1 mL of Krebs buffer containing 5 mM 2,7-dichlorodihydrofluorescein diacetate (DCFH)  for 30 min at room temperature. After incubation, samples were analyzed using a FACSCalibur flow cytometer. Fluorescence of 10,000 events was acquired in the FL1 channel and analyzed using the CellQuest Pro software. Mean fluorescence was obtained from a M1 population defined in the histograms. The limits for the M1 population were set based on the fluorescence of the unstained cells.2 tissue culture flasks using QIAzolLysis Reagent  according to manufacturer's instructions. The concentration and purity of the isolated RNA were determined using UV spectrophotometry . The integrity of the RNA was verified using agarose gel electrophoresis stained with ethidium bromide. Reverse transcription was performed using TaqMan Reverse Transcription Reagents  using 1 μg of total RNA. Real-time RT-PCR was performed using SYBR Green . Specific sequences of the primers for endothelial NO synthase (ENOS), inducible NO synthase (INOS), catalase (CAT), arginase II (ARGII), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  as a housekeeping gene control are shown in The total RNA was extracted from 25 cm t-test was used to compare apoptosis and ROS content between 2 groups. Comparisons among more than 2 groups were performed using 1-way ANOVA with Tukey's post hoc test. Categorical data were presented in percentages and compared by Fisher's exact test. Spearman correlation was performed between TNF-α and PWV. The level of significance (α) accepted was less than 0.05.Continuous variables were expressed as mean and standard deviation (SD). Normality of distribution was assessed by Shapiro Wilk test. Mann-Whitney test was used to compare clinical data in 2 groups, while 2-tailed unpairedα compared to normotensive subjects (3.3 versus 2.1 pg/mL), as well as higher PWV (10.5 versus 7.2 m/s), represented in α levels and PWV measures in the whole group  and higher reactive oxygen species (ROS) content , compared to normotensive subjects (p = 0.04) but exerted no effect on ROS production. Although we observed a tendency pointing to an increase, infliximab did not change the apoptosis of HUVECs incubated with normotensive serum   . ENOS, INOS, CAT, and ARGII was shown in  ENOS expression were found between cells incubated with NT or RHTN serum, we found increased ENOS expression after treatment with infliximab only in the normotensive group  expression was lower in HUVECs incubated with serum of resistant hypertensive patients compared to normotensive serum, but no effect of infliximab was found in both groups.Normalized expression of the genesve group . No diffα plasma levels compared to normotensive subjects. Moreover, TNF-α levels were positively correlated with carotid-femoral PWV. We also found that endothelial cells incubated with RHTN serum showed higher apoptosis rate than cells incubated with serum from normotensive subjects. The treatment with TNF-α inhibitor reduced apoptosis induced by RHTN serum. The inhibition of TNF-α increased the expression of ENOS in the cells incubated with normotensive serum, but no changes in INOS, CAT, and ARGII expression were observed. In addition, the expression of ARGII decreased in cells incubated with RHTN compared to NT serum.The present study demonstrated that resistant hypertensive subjects have higher arterial stiffness and increased TNF-α inhibition reduced systolic BP and left ventricular hypertrophy and activated AKT/eNOS pathway, improving vascular function in hypertensive rats [α and increased arterial stiffness were found in hypertensive subjects compared with normotensive subjects [α were positively correlated in hypertensive patients [α plasma levels are associated with elevated blood pressure in apparently healthy subjects [The contributions of immune system to cardiovascular damage have been largely investigated. The lack of immune cells prevents vascular damage and the development of hypertension on several animal models of hypertension as angiotensin II infusion and DOCA-salt , 25. Takive rats . Indeed,subjects . Also, apatients . Previousubjects , 2.α levels, we investigated the effects of RHTN serum on endothelial cells in culture, by measuring apoptosis percentage and ROS content and whether those effects were mediated by TNF-α. We found that serum from RHTN promotes endothelial cells apoptosis and increases ROS content. We found reduction in cell apoptosis after the treatment with TNF-α inhibitor, but no changes in ROS content. Corroborating with these findings, we did not find changes in catalase gene expression after TNF-α inhibition. Our findings suggest that TNF-α may participate in apoptosis of endothelial cells but seems to not affect oxygen species.Since RHTN patients have increased arterial stiffness and positive correlation with TNF-α was also shown to stimulate endothelial microparticles (EMPs) releasing, which is a marker of endothelial dysfunction, and reactive oxygen species (ROS) production, which suggest that ROS are important mediators of TNF pathway [α inhibition, other factors present in RHTN serum may be stimulating ROS formation.TNF- pathway . Both hi pathway . Howeverα upregulates the expression of arginase in ECs, which decreases L-arginine availability to eNOS and leads to O−2 production. ROS production impairs vasodilatation mediated by NO [ in vitro system. Therefore, we may speculate that serum from RHTN has substances able to stimulate NO production in our in vitro system  but endothelial cells of the patients may not respond to these stimuli in vivo, explaining the presence of endothelial dysfunction in these patients [α might impair NO synthesis in ECs. Indeed, animal study with estrogen-deficient rats treated with anti-TNF-α had increased expression of tissue eNOS [α has a crucial role in expression of eNOS suppressing eNOS mRNA and protein levels by decreasing mRNA stability [The endothelium represents the main regulator of wall homeostasis through releasing of several molecules such as nitric oxide (NO) that is continuously produced by healthy endothelial cells. L-arginine is converted into NO by endothelial NO synthase. Also, NO can be produced in response to immunological stimuli through inducible NO synthase (iNOS). Interestingly, HUVECs incubated with RHTN serum had tendency of higher expression of eNOS compared to cells treated with NT serum, irrespective of the increased endothelial dysfunction found in those patients , 27. Mored by NO , 28. Surpatients . We alsosue eNOS . TNF-α htability .α may modulate other NO synthases, as inducible NO synthase (iNOS), promoting nitrosative stress and endothelial dysfunction [On the other hand, we do not discard the possibility that TNF-function ; howeverα inhibitors have an emerging role in prevention of vascular dysfunction. Furthermore, arterial stiffness was improved in patients with rheumatoid arthritis treated with TNF-α inhibitors [α on arterial stiffness. In conclusion, serum TNF-α in resistant hypertensive subjects induces apoptosis in human endothelial cells. These results suggest that TNF-α might mediate, at least in part, vascular injury in resistant hypertension. Finally, future clinical trials with TNF-α inhibitors in these patients may represent a field of interest.The treatment of inflammatory disease (anti-neutrophil cytoplasmic antibody-associated systemic vasculitis) with infliximab improved endothelial function ; thus, Thibitors . Further


The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation.+), TNF+PAD4+/+, and TNF+PAD4-/- mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF+PAD4+/+ and TNF+PAD4-/- mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis.Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically  contains supplementary material, which is available to authorized users. Over the past decade, our understanding of rheumatoid arthritis has grown with a potential role for citrullinated proteins and the peptidylarginine deiminases (PADs) in disease development. Rheumatoid arthritis may start with an environmental trigger leading to protein citrullination. Genetically susceptible individuals form anti-citrullinated protein antibodies (ACPAs) as part of a loss of tolerance to citrullinated and other posttranslationally modified proteins. As inflammation increases secondary to the loss of tolerance, the levels of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) rise and ultimately inflammatory arthritis develops.Citrullination is a posttranslational modification catalyzed by the PAD enzymes whereby arginines are converted to citrullines. PAD4 is particularly interesting in rheumatoid arthritis for several reasons. It is expressed in immune cells and is increased in the rheumatoid joint , 2. AlsoLung inflammation is tightly linked to rheumatoid arthritis with clinical interstitial lung disease occurring in about 10 % of rheumatoid arthritis patients and subclinical lung disease in up to 80 % of rheumatoid arthritis patients . Also, pTo test causality, it is often necessary to use animal models. One good model of rheumatoid arthritis is the TNF-α transgenic mouse, in which human TNF-α is overexpressed systemically causing a severe inflammatory and destructive arthritis that starts at 4–8 weeks of age depending on the number of copies of the TNF-α transgene , 20. TheIn this study, we employ mice that overexpress TNF-α to determine if TNF-α can induce lung citrullination, if PAD4 is required for lung citrullination and/or lung inflammation downstream of TNF-α, and if TNF-α-induced lung inflammation is sufficient to induce arthritis.+ [+PAD4+/+ and TNF+PAD4-/- mice. Littermates were used for all experiments. Mice that overexpress TNF-α only in the lungs under the control of the surfactant promoter [Mice that overexpress systemically one copy of the TNF-α transgene (Tg3647 transgenic mice referred to here as TNF+ ) were cr+  to generpromoter  and wildLung tissue was harvested from mice, flash frozen and homogenized using a Bullet Blender  in RIPA buffer supplemented with protease inhibitor cocktail . Lysates were sonicated briefly and total protein was quantified.Equal amounts of protein were diluted with trichloroacetic acid and incubated with Rh-PG as previously described . The reaEqual amounts of protein were subjected to gel electrophoresis using two polyacrylamide gels. One gel was stained with brilliant blue and the other was transferred to a polyvinylidene difluoride membrane  using a semi-dry blotting system . The membrane was blocked with milk, incubated overnight at 4 °C in 1:1000 mouse monoclonal anti-peptidyl citrulline (F95) antibody  in milk,Lungs and hind legs were fixed in 10 % neutral-buffered formalin, processed with a VIP processor, embedded in paraffin , sectioned and stained with hematoxylin and eosin (H&E). Hind legs were decalcified for 30 hours with Surgipath Decalcifier 1  prior to processing and embedding. Lung disease was scored in a blinded manner by a board-certified pathologist on a scale of 0–5  for the following characteristics: lymphocytic parabronchiolar foci, perivascular lymphoid infiltrates, destructive vasculitis, interstitial inflammatory infiltrates, and alveolar infiltrates. Scores were not limited to integer values. Ankle joints were evaluated for synovitis and erosions in a blinded manner by a board-certified pathologist.t test with the results considered significant if the two-tailed p value was less than 0.05.All data were analyzed using a + mice) and wild-type littermates. As shown in Fig. + compared to wild-type mice. No significant increase in protein citrullination is seen in TNF+ compared to wild-type mice at 2 and 3.5 months of age (data not shown).To quantify protein citrullination, we allowed a citrulline-specific, fluorescently labeled chemical probe, Rh-PG , to bind+ and control mice by western blot. As shown in Fig. + mice have increased lung protein citrullination compared to wild-type littermates at 5 months of age. No significant increase in citrullination was seen at 2 and 3.5 months of age in TNF+ mice using F95 (data not shown). Taken together, our data suggest that TNF-α induces citrullination in murine lungs.Although normal lungs have some baseline PAD activity  and nati+ mice, we wanted to determine if the citrullination seen might require PAD4. Therefore, we used Rh-PG as above to quantify protein citrullination in lung protein lysates from 5-month-old TNF+PAD4+/+ and TNF+PAD4-/- mice. We did not detect a reduction in total protein citrullination in the lung . Also, we did not detect a significant reduction in citrullination of specific protein bands in TNF+PAD4-/- compared to TNF+PAD4+/+ mice (data not shown). These data suggest that PAD4 is not critical for gross protein citrullination in TNF+ mice.After demonstrating that lung citrullination is increased in TNFung Fig.  and b inice Fig. . Similar+ mice, we hypothesized that PAD4 might be important for TNF-α-induced lung inflammation since it contributes to TNF-α-induced joint inflammation [+PAD4+/+ and TNF+PAD4-/- mice were scored for the severity of lung disease in a blinded manner. At 3.5 months of age, TNF+PAD4-/- mice have reduced perivascular lymphoid infiltrates and interstitial infiltrates compared to TNF+PAD4+/+ mice  show no inflammatory infiltrates or destructive vasculitis . Taken together, these data suggest that PAD4 contributes to interstitial and perivascular inflammation as well as destructive vasculitis downstream of TNF-α in murine lungs.Despite our observation that PAD4 is not critical for total lung citrullination in TNFammation . To testice Fig.  and b. Ftis Fig. . There wSince TNF-α induces protein citrullination in the lung, which is thought to be a potential trigger for rheumatoid arthritis, we hypothesized that TNF-α-induced lung inflammation might induce arthritis development. Therefore, we harvested the joints from almost 2-year-old mice that overexpress TNF-α only in the lungs under the control of the surfactant promoter . We prepIn the present study, we show that overexpression of TNF-α, a major pathologic cytokine in rheumatoid arthritis, induces murine lung citrullination providing evidence for a pathway by which lung citrullination may be induced in human disease. In human rheumatoid arthritis, high levels of TNF-α may be a trigger for the observed citrullination in the rheumatoid lung . The preDespite our observation that TNF-α induces lung citrullination, we found that TNF-α-driven lung inflammation is not sufficient to induce arthritis. Our work builds on previous studies, which found that chronic exposure to tobacco smoke or intratracheal bleomycin is not sufficient to drive arthritis in SKG mice , a murinThe same mechanism may be true in humans. Once tolerance is lost, increasing levels of inflammatory cytokines like TNF-α in the preclinical phase of rheumatoid arthritis may drive both joint and lung inflammation. It is known that cytokines rise prior to the onset of arthritis , but les+ mice. This result is surprising given previous studies that show a relationship between TNF-α, nuclear localization of PAD4, citrullination, and rheumatoid arthritis [+ mice as detected by a colorimetric assay [+ mice, which is similarly seen in the rheumatoid lung [We have also found that PAD4 does not play a major role in lung citrullination in TNFrthritis , 35–37. ic assay . Thus, aic assay , but grooid lung , is not oid lung , 38. Furoid lung , 40 and oid lung . PAD2 isoid lung . FurtherWe have also shown that PAD4 is important for TNF-α-driven lung inflammation in agreement with previous findings that PAD4 contributes to TNF-α-driven joint inflammation . Initial+PAD4-/- lungs points to a role for PAD4 in the lymphoid compartment, although we cannot conclude if PAD4 is required in lymphocytes or in cells that regulate lymphocytes. PAD4 may have large effects on immune cells while citrullinating only a few total arginines. Such a small amount of citrullination would likely not be detected with our methods. Nonetheless, PAD4 appears to contribute to inflammation independently of gross citrullination. Additional studies are needed to better characterize the role of PAD4 in immune cells, clarify the role of PAD2 in citrullination and arthritis, and determine how both PADs contribute to human rheumatoid arthritis.Our finding that PAD4 significantly contributes to lung inflammation, but not total protein citrullination in the lung, supports a role for PAD4 apart from mass antigen citrullination. This role is likely multifactorial since, in addition to a role for PAD4 in neutrophils, PAD4 is expressed in monocytes and macrophages  and it cWe have shown that PAD4 contributes to lung inflammation, but is not critical for gross protein citrullination in the inflamed lung. These findings suggest that PAD4 exacerbates inflammation apart from antigen citrullination and that PADs other than PAD4 are responsible for the majority of inflammation-dependent protein citrullination in the lung. Further, we have shown that TNF-α-induced lung inflammation is not sufficient to cause murine arthritis, suggesting that lung and joint inflammation are two separate outcomes of a single underlying pathologic process instead of lung inflammation driving murine inflammatory arthritis. Further work is needed to extrapolate these findings to human rheumatoid arthritis.ACPA, anti-citrullinated protein antibody; MHC, major histocompatibility complex; NET, neutrophil extracellular trap; PAD, peptidylarginine deiminase; TNF-α, tumor necrosis factor alpha


Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.Tumor necrosis factor- For example, increased level of TNF-α is detected in the brain and plasma in AD patients and mouse models of AD. In CNS, TNF-α is mainly produced by activated microglia and astrocytes in response to various stimuli including infection and injury. Genetic deletion of TNFR1 has been shown to attenuate the production of the amyloid-β  (Aβ) and to improve impairments in mice with AD [α or TNF receptor protects against dopaminergic neurotoxicity [α is strongly linked with neuronal damage, and blockage of TNF-α-mediated neurotoxic pathway emerges as an attractive strategy for the treatment of degenerative diseases such as AD and PD. Although TNF-α has been shown to be neurotoxic to cultured neurons by promoting glutamate production [α-initiated neurotoxicity are not yet understood.Massive loss of a particular subset of neurons is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Cytokine-driven neuroinflammation and neurotoxicity have been implicated in the initiation and progression of these devastating diseases . Ample eem (CNS) . TNF-α i with AD , 4. Moretoxicity , 6. Theroduction , the sigα is involved in diverse cellular responses including apoptosis and necrosis. TNF family of cytokines, such as TNF-α, TRAIL, and FasL, triggers apoptosis by recruiting and activating caspase-8 through the adaptor protein FADD. In some cell types, suppression of caspase-8 or FADD sensitizes cells to programmed necrosis (termed necroptosis) in response to these cytokines as well as ligands of Toll-like receptors (TLRs) [α-induced necroptosis, deubiquitination of RIP1 by cylindromatosis (CYLD) is a critical process for necrosome formation and activation [As a pleiotropic factor, TNF-s (TLRs) . Necropts (TLRs) , receptos (TLRs) –13, and s (TLRs) , 15. In tivation , 17. Alttivation  and calctivation  have beeα-induced neurotoxicity in vivo. Here, we demonstrated that deficiency of RIP3 alleviates the loss of hippocampal neurons in the mouse hippocampus after intracerebroventricular injection of TNF-α. Using an in vitro hippocampal neuronal model, we provided a detailed molecular characterization of TNF-α-induced death of hippocampal neurons.The connection between necroptosis and neuronal damage has been suggested by studies demonstrating a protective effect of necroptosis inhibitor on brain injury in experimental stroke and trauma models , 21. We α. In brief, 2.5 μg or 5 μg TNF-α was dissolved in PBS to make a total volume of 20 μL and then injected into each lateral ventricle. The control group mice received 20 μL PBS. After 3 days, mice were scarified and the proteins were extracted from hippocampus and subjected to western blot analysis. Morphology of hippocampal neurons was analyzed by Nissl staining of brain sections. All animal experiments were performed in accordance with protocols by the Institutional Animal Care and Use Committee at Soochow University.RIP3 knockout mice were generated as described previously  and cros3·7H2O) were from Sigma. Recombinant TNF-α was purified as described previously [β-actin (Sigma).Dulbecco's modified Eagle's medium (DMEM) was from Thermo. Penicillin/streptomycin, L-Glutamine, and fetal bovine serum (FBS) were from GIBCO. BHA, NAC, phosphate buffered saline (PBS), and Lanthanum(III) chloride heptahydrate  cells were a gift from the Lab of Dr. Zhenghong Qin (Soochow University). Mouse embryonic fibroblasts (MEFs) were isolated from day 14.5–15.5 embryos. These cells were grown in DMEM supplemented with 10% fetal bovine serum.Lentiviral expression construct containing mouse RIP3 was amplified from RIP3 plasmid with primers containing an N-terminal Flag epitope and then cloned into pCAG-MCS-IRES vector that was a gift from the Lab of Dr. Yun Zhao (Soochow University). Lentiviral expression construct containing RIP3-RHIM domain mutant (RIP3-RHIM-Mut) or RIP3 kinase mutant (RIP3 K51A) was generated by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Mouse RIP3, RIP1, MLKL, and CYLD siRNAs were synthesized by GenePharma: RIP3-1 (cccgacgaugucuucugucaa), RIP3-2 (cuccuuaaagucaauaaacau), RIP1-1 (ccacuagucugacugauga), RIP1-2 (ucaccaauguugcaggaua), CYLD-1 (uccauugaggauguaaauaaa), CYLD-2 (aaggguugaaccauuguuaaa), MLKL-1 (gagauccaguucaacgaua), and MLKL-2 (uaccaucaaaguauucaacaa).α. Brains were dissected out of the skull and put in 4% paraformaldehyde to fix the tissue for 24 hours at room temperature and then stored in 30% sucrose phosphate buffer overnight until the tissue sank to the bottom of the solution. 20 μm sections were cut in the coronal plane using a freezing microtome (Leica CM19500) and mounted on gelatin coated slides. The sections were further stained in 0.1% cresyl violet solution (Sigma-Aldrich) at 37°C for several minutes. Rinse quickly in distilled water followed by differentiation in 95% ethyl alcohol and check microscopically for best result. Dehydrate in 100% alcohol and clear in xylene. Finally, the sections were mounted using a neutral balsam and photos were taken under microscope.The mice were sacrificed 72 h after intracerebroventricular injection of TNFβ-glycerol-phosphate, and 1 mM PMSF supplemented with protease inhibitor cocktail (Roche). The mouse tissue was grinded and resuspended in lysis buffer with 0.1% SDS. The resuspended cell pellet or tissue was vortexed for 10 seconds, then incubated on ice for 20 min, and then centrifuged at 20,000 g for 20 min. Protein concentration was determined by Quick Start Bradford 1x Dye Reagent (Bio-Rad). The protein samples were prepared for western blot analysis.Cell pellets were lysed in lysis buffer containing 20 mM Tris-Hcl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1% Glycerol, 0.5 mM DTT, 1 mM Na3VO4, 25 mM 293T cells werecotransfected with lentiviral expression construct and packaging plasmids mix, and viral particles were collected after 48 hours and 72 hours. HT-22 cells were infected with lentivirus containing RIP3, RIP3K51A, and RIP3-RHIM-Mut, respectively. 72 hours later cells were selected with GFP by fluorescence-activated cell sorting.HT-22 cells were transfected with siRNAs by Lipofectamine RNAiMAX Reagent (Invitrogen) for 60 h and then treated with the indicated drug for about 20 h. Cell survival was determined by Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega).α-induced neurotoxicity, we challenged wild-type and RIP3-deficient mice with intracerebroventricular injection of TNF-α. Histological analysis with Nissl staining of neurons was performed to evaluate TNF-α-induced damage of neurons. We observed that administration of TNF-α to wild-type mice caused a reduction in neuronal density in the hippocampus especially CA3 region in a dose-dependent manner as compared with control-treated mice . The kinase activity and RHIM domain of RIP3 are critical for its function in mediating necroptosis . We exam Figures , which w Figures , indicatα-induced necrosis of HT-22 cells. As shown in Figures α-induced necrosis. In addition, knockdown of CYLD, a deubiquitinase of RIP1, blocked TNF-α-induced necrosis of HT-22 cells  and N-acetylcysteine (NAC). MEF cells are known to undergo necroptosis in response to TNF-α, Smac mimetic and z-VAD. In the presence of BHA at 100 μM, the survival rate of HT-22 cells was increased by 13% and around 30% cells still underwent necrosis in response to TNF-α plus z-VAD, while TNF-α-induced necrosis in MEF cells was entirely prevented by BHA  , 18, so  Figures . NAC had Figures . Recentl and NAC . These rα is a key mediator of neuroinflammation. Elevated levels of TNF-α are associated with various neurodegenerative conditions and contribute to neurotoxicity. The mechanisms underlying TNF-α-initiated neurotoxicity are largely unknown. Our present work revealed an important role of necroptosis in TNF-α-induced toxicity of hippocampal neurons.TNF-α. Using RNAi approach, we demonstrated that TNF-α-induced neuronal necrosis is mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. Our present study suggests that the core components of necrosome have conserved roles in neuronal necrosis initiated by TNF-α.Necroptosis is a form of programmed necrosis which is regulated by RIP1 and RIP3 kinases. Apoptosis is known to negatively regulate necroptosis via active caspase-8, which is able to cleave necrosis regulators including RIP1, RIP3, and CYLD . CYLD waα-induced necroptosis of HT-22 cells mouse hippocampal neurons is largely independent of ROS accumulation. Recently, TRPM7 has been identified to mediate calcium influx through acting downstream of MLKL membrane localization in TNF-α-induced necroptosis [α-induced necroptosis of HT-22 cells largely proceeds even in the presence of calcium channel inhibitor and ROS scavenger. The data indicate that TNF-α-induced necroptosis of HT-22 cells largely bypasses ROS accumulation and calcium influx. Therefore, it is tempting to speculate that different downstream responses activated by necrosome depend on cell type. Future studies are required to clarify crucial downstream events for the execution of necroptosis in hippocampal neurons.Necroptotic cell death is characterized by disrupted plasma membrane; however, the downstream events of necrosome leading to collapse of membrane are largely unknown. ROS production has been shown to be required for necroptosis in several mouse cell lines including L929, MEF, NIH3T3, and macrophages, whereas it is not involved in necroptosis in HT-29 human colon cancer cells . NADPH oroptosis . In our α. Importantly, genetic deletion of RIP3 reduced the loss of hippocampal neurons after intracerebroventricular injection of TNF-α. To our knowledge, the data represent the first in vivo evidence for a role of RIP3 in TNF-α-induced neurotoxicity of hippocampal neurons. Blockage of necroptosis by necrostatin-1, a chemical inhibitor targeting RIP1 kinase, has provided protective effects on neuronal damage in animal models of brain injury [α-initiated damage of hippocampal neurons. Given the elevated levels of TNF-α in the brains during various neurodegenerative diseases, neuronal cells may be susceptible to necroptosis upon stimulation of TNF-α, therefore contributing to the pathogenesis of neurodegenerative diseases. Targeted prevention of neuronal necroptosis may provide a novel therapeutic approach for the treatment of the related neurodegenerative diseases.Necroptosis is emerging as an important process involved in various pathological conditions including ischemic injury, acute pancreatitis, inflammatory bowel disease, and neuronal damage. Recent studies have demonstrated that increased level of RIP3 expression in the damage tissue correlates with the induction of necroptosis in the mouse models of acute pancreatitis and inflammatory bowel disease (IBD)  and patin injury , stroke n injury , and amyn injury . Our stuα-induced neurotoxicity. Necroptosis can be activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. HT-22 hippocampal cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. A detailed molecular characterization demonstrates that TNF-α-induced necrosis in HT-22 cells is mediated by CYLD-RIP1-RIP3-MLKL necroptotic signaling pathway and largely independent of both ROS accumulation and calcium influx.Taken together, our study revealed an important role of necroptosis in TNF-


TNF conditional knock out mice (LysMcreTNFfl/fl) in which the TNF gene was deleted in cells of the myeloid lineage, including microglia. The deletion reduced secreted TNF levels in lipopolysaccharide-stimulated cultured primary microglia by ~93%. Furthermore, phosphorylated-ERK/ERK ratios were significantly decreased in naïve LysMcreTNFfl/fl mice demonstrating altered ERK signal transduction. Micro-PET using 18[F]-fluorodeoxyglucose immediately after focal cerebral ischemia showed increased glucose uptake in LysMcreTNFfl/fl mice, representing significant metabolic changes, that translated into increased infarct volumes at 24 hours and 5 days compared to littermates (TNFfl/fl). In naïve LysMcreTNFfl/fl mice cytokine levels were low and comparable to littermates. At 6 hours, TNF producing microglia were reduced by 56% in the ischemic cortex in LysMcreTNFfl/fl mice compared to littermate mice, whereas no TNF+ leukocytes were detected. At 24 hours, pro-inflammatory cytokine  levels were significantly lower in LysMcreTNFfl/fl mice, despite comparable infiltrating leukocyte populations. Our results identify microglial TNF as beneficial and neuroprotective in the acute phase and as a modulator of neuroinflammation at later time points after experimental ischemia, which may contribute to regenerative recovery.Microglia are activated following cerebral ischemia and increase their production of the neuro- and immunomodulatory cytokine tumor necrosis factor (TNF). To address the function of TNF from this cellular source in focal cerebral ischemia we used  Microglial activation constitutes a major defense mechanism against ischemic brain injury, with dual effects linking microglial activation to both neurotoxic and neuroprotective effects in the brain12345678The effect of TNF is mediated by two cell surface receptors, TNF-R1 and TNF-R2. These receptors may feed into diverse signaling pathways according to differences in their intracellular domains. A death domain in TNF-R1, not present in TNF-R2, can lead to apoptotic cell death, although most cell types are protected due to expression of anti-apoptotic proteins. Alternative pathways of TNF-R1 signaling involve activation of the ERK1/2 MAP kinases and NF-κB activation106912−/−) mice display increased infarct volumes and worse functional outcome after experimental stroke414In experimental stroke, genetic ablation of solTNF, with sustained mTNF expression, is associated with reduced infarct volume, improved functional outcome and an altered pro-inflammatory environmentfl/fl mice lacking TNF in myeloid cells display phenotypical alterations in microglia, including altered ERK signaling. In addition, FDG uptake in the ischemic brain during the acute phase after ischemia (1–3 hours) is increased in LysMcreTNFfl/fl mice resulting in increased infarct volumes 24 hours and 5 days after focal cerebral ischemia. The relevance of the present study was underlined by investigating glial activation, TNF and TNF receptor expression in human autopsy material from stroke patients. This may have significant implications for the development of future neuroprotective treatment strategies in the acute phase after stroke, as modulating the microglial TNF response acutely can provide a viable option to boost neuroprotection.Given the contribution of microglia/macrophages to the pathophysiology of stroke, in the present study, we investigated the effect of TNF ablation specifically in myeloid cells on neuroprotection and functional outcome after focal cerebral ischemia. We demonstrate that LysMcreTNFfl/fl mice under naïve conditions. LysMcreTNFfl/fl mice were comparable to TNFfl/fl mice displaying normal cerebrovascular anatomy at the level of the circle of Willis  revealed higher CD11b . Since microglia increase LysM expression under ischemic and inflammatory conditions1718fl/fl and LysMcreTNFfl/fl mice    and at 5rs (30%)  after pMrs (30%) .fl/fl and LysMcreTNFfl/fl mice 2 and 5 days after pMCAO , we performed  imaging . Dynamic imaging . Ten to controls , demonst18[F]-FDG-PET imaging comparing the normalized rate constants  revealed significant differences between TNFfl/fl and LysMcreTNFfl/fl mice   after pMCAO. Despite tendencies, pERK1/ERK1 and pERK2/Erk2 ratios were not different between TNFfl/fl and LysMcreTNFfl/fl mice 6 hours after pMCAO  , suggester pMCAO . No differ pMCAO , or p-p3er pMCAO  ratios ifl/fl and LysMcreTNFfl/fl mice, we assessed cytokine, chemokine, and TNFR protein expression in naïve conditions and after pMCAO by multiplex analysis fl/fl and LysMcreTNFfl/fl mice 6 and 24 hours after pMCAO by the use of flow cytometry . The infarcted area, identified by HE staining .To address TNF signaling in the context of human stroke pathophysiology, we analyzed TNF, TNF-R1, and TNF-R2 expression in the staining , was chastaining . Iba1+ c infarct . GFAP+ a infarct . TNF+ cet tissue . On the issue  and then by flow cytometry 6 hours after pMCAO demonstating a 56% reduction in TNF+ microglia in the ischemic brain. On the contrary, release of TNF was not affected in vitro in astrocytes, further validating efficient and selective ablation of TNF from microglial cells.Eventhough in LysMcreTNFfl/fl mice to have significantly increased FDG uptake acutely (>3 hours) and larger infarct volumes at 24 hours and 5 days after pMCAO, supporting our previous findings that constitutively expressed or early induced, microglial-derived TNF is neuroprotective in experimental strokefl/fl and LysMcreTNFfl/fl mice, we found early microglial-derived TNF to affect FDG uptake in the ischemic tissue after pMCAO. Though assumptions of a steady state system is hampered in the acute phase after pMCAO and care should be taken not to use the kinetic model data as absolute true numbers, the findings of hyper-FDG in the ispsilateral cortex compared with the contralateral cortex within the first 2 hours after pMCAO in both strains of mice are consistent with previous findings of increased accumulation of glucose around the ischemic border29293010fl/fl mice compared to TNFfl/fl mice during acute ischemia supports the findings of increased infarct volumes 24 hours and 5 days after pMCAO and demonstrate that conditional ablation of TNF in microglia modifies neuronal sensitivity to ischemia. The fact that we in the present study were not able to differentiate between FDG uptake in infarct and peri-infarct areas is due to the limited resolution (1 mm) of the PET scanner as imaging spatial resolution is essential to delineate these areasWhen we tested the effect of myeloid TNF deletion in experimental stroke, we found LysMcreTNFfl/fl mice, with a tendency still present 6 hours after pMCAO. These findings suggest that ERK signal transduction is altered in LysMcreTNFfl/fl mice. TNF is known to activate all three MAPKs in neurons, with ERK1/2 activation mediated via MEK1/2−/− and WT mice after pMCAOfl/fl mice. In support of this, Rousseau et al.in vitro LPS stimulation of macrophages results in sequential activation of the TPL2-MKK1/MKK2-ERK1/2 pathway followed by secretion of TNF and that inhibition of this pathway resulted in loss of TNF production. Alternatively, we speculate that conditional ablation of TNF in microglia may have affected microglial expression of P2X7 receptors, as previously shown7 receptors on microglia and thereby activate MAPK , which results in the release of neuroprotective TNFIn some studies JNK/SAPK and p38 MAPK were shown to promote cell death, whereas ERK1/2 opposed cell death32fl/fl and TNFfl/fl mice after pMCAO. We found that numbers and percentages of CD11b+CD45+Gr1− monocytes and CD45+CD3+ T cells were significantly increased in LysMcreTNFfl/fl mice 24 hours after pMCAO suggesting that ablation of mesodermally-derived TNF results in an increase in the number of newly produced, bone marrow-derived monocytes and T cells. The signals that trigger leukocyte mobilization from the bone marrow after stroke are unknown and may represent potential targets for future therapies40fl/fl mice, which also display significantly increased infarcts. We and others have shown that following cerebral ischemia cellularity decreases in the spleen343+ T cells in LysMcreTNFfl/fl mice compared to TNFfl/fl mice can be due to decreased ischemia-induced apoptosis of spleen lymphocytes. On the other hand, early splenic responses after stroke involve increased production of pro-inflammatory mediatorsAs a consequence of ischemia, microglial numbers decrease in the ipsilateral hemisphere after experimental stroke whereas leukocyte infiltration increases at the same time438−/− compared to WT mice 24 hours after strokefl/fl mice at this time point, nor at 6 hours after pMCAO. Anti-TNF therapies decrease the number of granulocytes infiltrating the ischemic infarct 24 hours after pMCAOfl/fl micefl/fl mice, given that we previously demonstrated reduced infiltration of macrophages into the brain of mTNFΔ/Δ mice 24 hours after pMCAO+ leukocytes 6 hours after pMCAO.We have previously shown that the number of infiltrating leukocytes is significantly increased in TNFAll together, the present study supports microglial-derived TNF as beneficial and neuroprotective in the acute phase after experimental stroke and as a strong modulator of the neuroinflammatory response at later time points after experimental ischemia.cre/x TNFflox/flox (LysMcreTNFfl/fl) breeding couples, with a specific deletion of TNF in myeloid cellsflox/flox (TNFfl/fl) mice, with normal TNF epxressioncre/micetdTomato miceMLys1fl/fl mice were genotyped by a two-oligonucleotide primer PCR assay and Cre-mediated recombination and completion of deletion were established using a three-oligonucleotide primer PCR assayTNFfl/fl and TNFfl/fl mice essentially as previously describedThe circle of Willis and its branches were examined in LysMcreTNF2), bone mineral content  and bone mineral density  were measured in naïve LysMcreTNFfl/fl and TNFfl/fl using dual-energy X-ray absorptiometry (DXA) using a PIXImus2  as previously described5Total tissue mass (g), fat mass (g), fat-%, lean tissue mass (g), lean-%, bone area  under Hypnorm/Stesolid anesthesia 2218[F]-FDG via a tail vein catheter. The animals were anesthetized throughout the entire imaging procedure using a mixture of 1.5–2% isoflurane and 100% oxygen.Micro PET imaging was performed on a Siemens Inveon PET scanner. The animals were scanned twice. The first scan was performed in the acute phase, 1–2 hours after surgery, and the second scan 24 hours later. Animals were placed in prone position on a heated scanner bed and injected with 18[F]-FDG was injected and data was acquired for 90 min. Data was binned in 20 frames (4 × 30 s + 10 × 1 min + 1 × 3 min + 5 × 15 min), beginning with short frames to capture the fast vascular phase moving to a slower frame rate appropriate for the dynamics beyond 15 min. At day 1 (24 hours after pMCAo) 10.7 ± 1.2 MBq 18[F]-FDG was injected. After a 30 min delay a 15 min static image was acquired.At day 0 (1–2 hours after pMCAo) 10.9 ± 2.4 MBq All images were reconstructed using OSEM3D/MAP matrix 128 × 128, 4 OSEM3D iterations and 18 MAP iterations with requested resolution of 0.8 mm. A zoom factor of 2 was applied yielding a voxel size of approximately 0.4 mm × 0.4 mm in the XY-plane and a slice thickness of 0.8 mm. The reconstruction was carried out with the build in algorithm of Siemens Inveon Acquisition Workplace software version 1.5.1 (transport rate constant from blood to brain tissue), k2 (rate constant from brain tissue back to blood), k3 (binding in the cells (phosphorylation))i was calculated as Ki = K1k3/(k2 + k3). The glucose uptake (metabolism) is proportional to Ki, glucose uptake = Ki (blood glucose/LC), where LC is the lumped constant. The FDG uptake is a very sensitive measure and is influenced by many factors including dietary condition (insulin level), ambient temperature and mode of anesthesiaA computer tomography (CT) image of a single animal was acquired and used as an anatomical reference. All images were analyzed and the kinetic modeling done within the Siemens Inveon Research Workplace software version 4.2. The reference CT and the PET images were manually co-registered. The images acquired at day 0 were analyzed using a 2-tissue-compartment irreversible FDG model. Image derived blood input functionsVolumes of interest were semi-automatically made by first drawing a volume covering the increased FDG uptake in the ipsilateral region. The final VOI was calculated by setting a threshold of 70% of maximum within the last image of the dynamic series. This VOI was mirrored onto the contralateral brain hemisphere as a reference VOI for the unaffected brain tissue. The blood input VOI was manually created in the first or second frame where the vena cava was clearly visualized. The blood input VOI was manually drawn as the part of vena cava with highest activity.fl/fl and TNFfl/fl mice as previously describedLocomotor activity was investigated using the open field test in a group of naïve LysMcreTNFfl/fl and TNFfl/fl the spontaneous alternation test was performed using a Y-maze as previously describedIn order to test spatial leaning and memory in naïve LysMcreTNFIn order to evaluate motor coordination/performance and balanceIn order to test stepping, inter-limb coordination and balance, the rung walk test was performed 2 days after pMCAO as previously described6The grip strength measuring device  was used to study neuromuscular function in mice subjected to pMCAO. The peak amount of force was recorded in 5 sequential trials and the highest grip value was recorded as the score2 and cut coronally in six parallel series of 30 μm and stored at −80 °C until further processingMice were killed by cervical dislocation, and brains quickly removed, frozen in COtdTomato mice were anesthetized with Ketamine/Xylazine (100 mg/10 mg/kg) and transcardially perfused with 4% PFA. For cell counting, tissue was processed as previously describedfl/fl and TNFfl/fl mice were overdosed using Pentobarbital (200 mg/ml)/Lidocainhydrochlorid (20 mg/ml) and transcardially perfused with 4% PFA. Brains were fixed in 4% PFA overnight, changed to 1% PFA and finally 0.1% PFA before they were horizontally vibratome-cut into six 60 μm-thick parallel series of free floating sections and cryoprotected in de Olmo’s solution2 snow and cut into 10 series of parallel 20 μm-thick coronal sections on a cryostat. Sections were stored at −20 °C until further processing.LysMEvery sixth section (1 series) from mice with 24 hours and 5 days survival after pMCAO was stained with TB for estimation of infarct volume using the Cavalieri principle28fl/fl and LysMcreTNFfl/fl mice using Cavalieri’s principle as previously decribed in detailThe volume of the neocortex was estimated in naïve TNFIn fresh-frozen sections, TNF was detected using a rabbit anti-TNF antibody  and AP-conjugated anti-rabbit antibody as previously describedet al.b  and mouse IgG-488 (Invitrogen) were used as isotype controls. Substitution of the primary antibody with isotype control or rabbit IgG gave no signal. Digital images were captured using an Olympus BX51 microscope connected to a PC containing DP manager software (version 2.1.1.158). Figures were composed using Photoshop CS5 software.Immunofluorescent staining for TNF co-locatization was performed essentially as described in Clausen LysMcre co-localization in the adult brain was investigated using rat anti-CD11b , rat anti-GFAP , mouse anti-NeuN , rabbit anti-red fluorescent protein (RFP) , and rabbit anti-Olig2 . Donkey anti-mouse Alexa Fluor-488, donkey anti-rat Alexa Fluor-488, donkey anti-rabbit Alexa Fluor-488, and goat anti-rabbit Alexa Fluor-568 (1:500) were used as secondary antibodies. DAPI  was used as a nuclear marker. Sections were coverslipped using Vectashield (Vectorlabs). Digital images of the cortex were captured using a Nikon Eclipse Ti epifluorescent microscope with NIS Elements software.+ cells was based on co-localization of the tdTomato reporter signal, which was enhanced using a RFP antibody with the various cell identification markers . Co-localization with oligodendroglial Olig2 was performed with the endogenous tdTomato signal. In total, 151–171 LysM+ (tdTomato) cells spread across at least three non-consecutive tissue sections were counted per animal (n = 3). All cells had a clearly identifiable DAPI+ nucleus.Quantification of LysMfl/fl and TNFfl/fl pups were decapitated, the brains isolated, and the meninges removed. Tail biopsies were obtained for genotyping. Brains were then placed in pre-warmed DMEM-GlutaMAX culture media (Gibco) containing 20% FCS and 1% penicillin/streptomycin (Gibco), mechanically homogenized by pipetting and filtered through a 70 μm cell strainer . Cells corresponding to the number obtained from 1 brain were seeded into poly L-ornithine pre-coated 24-well culture dishes (Costar) and grown at 36 °C (20% O2 and 5% CO2) for up to 21 days. Fifty percent of the culture medium was changed every 3–4 days. After 7 days, the medium was changed to culture medium containing 10% FCS and 1% penicillin/streptomycin and cells grown for an additional 7 days. After 14 days in vitro, microglia were removed from the layer of confluent astrocytes by gently shaking at 100 rpm for 2 hours at room temperatur. Microglia were harvested from the same dish twice with 7 days intervals. Microglia and astrocytes were separated and centrifuged separately at 1,000× g for 5 min at room temperature. The supernatant was removed and cells resuspended in medium before plated into 24-well plates . Microglia were then stimulated with 10 ng/mL lipopolysaccharide (LPS)  and astrocytes with either 10 ng/ml recombinant TNF (Sigma) or 10 ng/ml recombinant interleukin (IL)-1β (Sigma) for 24 hours. Unstimulated cultures were used as controls. After 24 hours, the media was removed and stored at −80 °C until further analysis. Cells were then prepared for immunohistochemistry by 3 x rinses in 0.15 M Sorensen Buffer (SB), fixation for 20 min i 4% PFA, 3 x rinses in 0.15 M SB and stored in 0.15 M SB for evaluation of purity (described above), which was found to be >95% (data not shown).For preparation of primary microglia and astrocyte cultures, 0–2 day old LysMcreTNFTM Deluxe Sets (Biolegend) according to the manufacturer’s instructions. The obtained data was processed and analyzed by the interface software program SoftMax®Pro. Each standard and test was determined in duplicate.The amount of TNF present in the media from LPS-stimulated and unstimulated microglia and TNF- or IL-1β-stimulated or unstimulated astrocyte cultures was examined using the Mouse TNF ELISA MAXtdTomato mice were isolated as previously describedFor flow cytometry, cells from LysM+CD45dim or CD11b+CD45dimLy6ClowLy6G−], macrophages [CD11b+CD45highGr1− or CD11b+CD45highLy6ChighLy6G−], granulocytes [CD11b+CD45highGr1+ or CD11b+CD45highLy6C+Ly6G+], T cells [CD45highCD3+], TNF+ microglia and TNF+ macrophages were identified as previously described3Flow cytometry was performed as previously described using the FACSVerse (BD Biosciences) and data analyzed using the FACSuite software3in vitro with the protein transport inhibitor GolgiPlug (BD Biosciences) and processed as previously described3Prior to fixation, cells were stained for live/dead cells using a Fixable Viability Dye eFluoro 506 (eBioscience) diluted in PBSPositive staining was determined based on fluorescence levels of the respective isotype and fluorescence minus one (FMO) controls. Antibodies were directly conjugated with fluorochromes: PerCP Cy5.5 anti-CD45 , PE anti-CD11b , PE-Cy7 anti-Ly6G/Ly6C (Gr1) , PE-Cy7 anti-Ly6C , BV421 anti-Ly6G , APC anti-TNF , and APC anti-CD3 . Isotype controls used were hamster IgG1κ , rat IgG2b , rat IgG1κ , rat IgMκ , Lewis IgG2aκ , and mouse IgG1 . The mean fluorescence intensity (MFI) was calculated as the geometric mean of each population in the TNF, CD45 and CD11b positive gates, respectivelyOne series of brain tissue was lysed in 1x Tris Lysis buffer  containing phosphatase inhibitor (Sigma) and cOmplete mini, EDTA-free proteinase inhibitor cocktail (Roche). Protein estimations were performed using the Bradford method.Plasma and brain samples were measured in the MSD Mouse Proinflammatory V-Plex Plus Kit  and in brain samples the mouse TNF-RI and TNF-RII Ultra-Sensitive Kits  using a MSD QuickPlex (SQ120) Plate Reader  according to the manufacturer’s instructionsfl/fl mice or as ratios (for MAPK).Western blot analysis for TNF  was performed using 20 μg protein extract separated on 4–12% SDS-PAGE gels  essentially as previously described36post mortem brain tissue from two stroke cases obtained from the Department of Pathology, Odense University Hospital and the use of human brains was approved by the Danish Biomedical Research Ethical committee for the Region of Southern Denmark (permission number S-20080042). Sex, age, infarcted brain area and age of infarct are given in The study was performed on 2O2 in Tris-buffered saline (TBS) and demasked in T-EG buffer , whereafter they were loaded onto a Dako autostainer 423Tissue blocks containing infarcted human brain tissue were imbedded in paraffin and 2 μm thick, serial sections were cut on a microtome, deparaffinized, blocked in 1.5% HFor multiple comparisons, one-way or two-way ANOVA was applied. When appropriate, repeated measures were used. Each test was followed by the relevant post-hoc test. For single comparisons unpaired and paired student’s t-tests were applied. P values ≤ 0.05 were considered statistically significant. Data are presented as mean ± SEM.How to cite this article: Clausen, B. H. et al. Conditional ablation of myeloid TNF increases lesion volume after experimental stroke in mice, possibly via altered ERK1/2 signaling. Sci. Rep.6, 29291; doi: 10.1038/srep29291 (2016).


Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast  Plasmodium falciparum, tumor epitopes [Viral structural proteins produced in heterologous expression systems and self-assembled to virus-like particles (VLPs) represent promising tools for modern vaccinology and diagnostics. Since the beginning of the 1980s, over 100 recombinant VLPs that originated from microbial, plant, insect, and mammalian viruses, belonging to 35 different virus families, have been constructed and characterized. Recombinant VLPs are extensively used for various applications, from basic virus assembly and structure studies to the production of human and animal vaccines . Therefoepitopes . Anotherepitopes ,14,15,16epitopes . Howeverepitopes ,14, as wepitopes ,17. MoreSaccharomyces cerevisiae and shown to self-assemble to VLPs [Major capsid proteins VP1 of hamster, murine and human polyomaviruses  have been generated previously in yeast expression system ,19. Very to VLPs . Purifie to VLPs ,20. TherThe preS1 epitope DPAFR spanning 31–35 aa residues of HBV preS1 protein has been identified as a recognition site of a neutralizing monoclonal antibody (MAb) MA18/7 ,22. The + cytotoxic T cells [The PADRE epitope AKFVAAWTLKAAA is a 13 aa-long universal helper T cell peptide that is non-natural but is designed to have a high affinity for multiple DR alleles in humans and mice . PADRE i T cells . The epi T cells . Recent  T cells . The broIn the current study, the preS1 and PADRE epitopes were inserted into either TSVyP VP1 protein or HaPyV VP1 protein at different positions and the resulted chimeric proteins were evaluated in terms of their capability to self-assemble to VLPs and immunogenicity in mice.Search of templates for structure modeling of recombinant polyomavirus VP1 proteins/pentamers, as well as of optimal sequence alignments of query sequence to template, were carried out using a sensitive profile-profile comparison method HHPRED . ObtaineE. coli K12 DH5a strain. Cloning and expression in yeast of the entire TSPyV VP1-encoding sequence (GenBank: KP293746) optimized for yeast was described recently [Construction of plasmids and all DNA manipulations were performed according to standard procedures . Recombirecently . The TSPTSV-VP1-1Bg-D, 5′-tcagatctaggttcttctggtcaagataaaccaacttctggt-3′,TSVP1-1Bg-R, 5′-ctagatctgaaccagaagaaccattagcaacagtaactttttcag-3′,TSVP1-4Bg-D, 5′-ggagatctaggttctggtgatatgcaatatagaggtttc-3′,TSVP1-4Bg-R, 5′- ctagatctccagaaccaaccaaaaaaccaacaatatc-3′.The nucleotide sequences of the mutated VP1-encoding variants: TSVP1-1Bg and TSVP1-4Bg were verified by DNA sequencing. The mutated TSVP1-1Bg and TSVP1-4Bg were then cloned into the XbaI site of the yeast expression vector pFX7 . HaPyV VThe four mutated VP1 gene variants  inserted into pFX7 plasmid were digested with BglII, and the preS1 epitope-encoding and PADRE epitope-encoding oligonucleotide duplexes were introduced into each construct. The oligonucleotide duplexes were synthesized by Metabion . The insertion sites of the resulting recombinant plasmids were verified by DNA sequencing.Saccharomyces cerevisiae strain AH22-214p . Purification of chimeric proteins was performed as described previously [2, 0.25 M L-arginine, 0.001% Triton X-100, 10 mM Tris/HCl, pH 7.2), lysed by vortexing with glass beads, mixed with the SDS-PAGE sample buffer , boiled for 5 min and then subjected to SDS-PAGE. For purification of chimeric proteins, yeast cells producing recombinant proteins were suspended in DB450 buffer  with 2 mM PMSF and EDTA-free Complete Protease Inhibitor Cocktail tablets  and then homogenized with glass beads using Bead-Beater GB26 . The chimeric proteins were purified from the supernatant of yeast cell lysate by ultracentrifugation overnight at 4 °C in 20%–60% sucrose gradient . Fractions containing chimeric proteins were diluted in DB150 buffer and subjected to further ultracentrifugation at 100,000 × g for 18 h on CsCl gradient (1.23–1.38 g/mL). Purified chimeric proteins were collected, pooled and after dilution in DB150 buffer precipitated by ultracentrifugation for 4 h at 100,000 × g. Pellets containing recombinant chimeric proteins were dissolved in phosphate-buffered saline , dialyzed against PBS, aliquoted  and then lyophilized or stored in PBS with 50% glycerol (at concentrations 1–2 mg/mL) at −20 °C until use. Concentrations of purified VLPs were determined using Bradford reagent  and by the densitometric scanning of the Coomassie brilliant blue-stained protein bands in SDS-PAGE using the ImageScanner III  device. Protein concentrations in each preparation of chimeric VLP were determined using the ImageQuantTL software based on the comparison with known quantities of the purified unmodified VP1 proteins and bovine serum albumin  run in the same gel. The unmodified TSPyV VP1 and HaPyV VP1 proteins were prepared as described previously [The chimeric VP1 proteins of TSPyV and HaPyV were generated in yeast eviously ,36. BrieTo verify the VLP assembly of VP1-derived chimeric proteins, samples of each purified chimeric protein were placed on 300-mesh carbon-coated palladium grid. VP1-derived VLPs adhering to the grid were negatively stained with 2% aqueous uranyl acetate solution and examined with a Morgagni 268 electron microscope .The immunogenicity of chimeric VLPs was investigated by immunization of 8-week old female BALB/c mice obtained from the breeding colony at the Center for Innovative Medicine, Dept. of Immunology . Each group of mice (n = 3 per group) was immunized 2 times with different chimeric VLPs with or without adjuvant at 21-day intervals. For a primary immunization, mice were injected subcutaneously with 50 μg/mouse of chimeric VLPs either dissolved in PBS or emulsified in complete Freund’s adjuvant . On day 21, the same groups of mice were immunized with chimeric VLPs (50 μg/mouse) either in PBS, or in incomplete Freund’s adjuvant , respectively. Blood serum specimens were collected from a tail vein on day 21 after the first immunization and 14 days after the 2nd and the 3rd immunizations. Spleen cells for VLP stimulation were collected on day 21 after the 2nd immunization.The work with experimental mice was performed by FELASA-certified personnel in accordance with the Lithuanian and European legislation. The Center for Innovative Medicine  has permissions for breeding experimental mice  and their use for generation of polyclonal and monoclonal antibodies (permission No. 209).Hybridomas producing monoclonal antibodies (MAbs) against TSPyV VP1 VLPs were generated essentially as described by Kohler and Milstein (1975) . BALB/c 2SO4. The optical density (OD) was measured at 450 nm in a microtiter plate reader .The levels of specific antibodies in blood sera of immunized mice were determined by an indirect ELISA using microtiter plates  coated with the respective antigens: chimeric VLPs, non-modified VLPs, synthetic biotinylated preS1 peptide  and synthetic biotinylated PADRE peptide (Pepscan). Chimeric VLPs and non-modified VLPs were immobilized by adding 100 μL of the antigen solution (2 μg/mL) in coating buffer  and incubation overnight at +4–8 °C. The plates were blocked for 30 min at room temperature (RT) with 1% BSA in PBS. For peptide immobilization, the plates were incubated overnight with streptavidin  added at concentration 5 μg/mL in coating buffer, then blocked as above and incubated with the respective biotinylated peptide added at concentration 5 μg/mL in PBS with 0.1% Tween-20 (PBST). After antigen immobilization and blocking, the plates were rinsed with PBST and incubated for 1 h at RT with mouse antiserum specimens. Individual sera from mice were serially diluted in two-fold steps starting at 1:100 in PBST. Following additional rinsing, the plates were incubated with horse-radish peroxidase (HRP)-labeled anti-mouse IgG  for 1 h at RT. After washing, the enzymatic reaction was detected with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) and stopped by adding 1 M H450 values greater than three times the background. The background was calculated as the mean OD450 value plus 3 standard deviations (SD) for OD450 values of a preimmune mouse serum that was diluted 1:100 and added to the antigen-coated wells in triplicates. The preimmune serum was included into each ELISA plate.Titers of antigen-specific IgG antibodies were determined as the reciprocal of the highest antiserum dilutions giving ODRecombinant proteins were fractionated by SDS-PAGE in 12% mini-gels and transferred to polyvinyldifluoride (PVDF) membranes (Merck-Millipore) under semidry conditions. Membranes were blocked with 1% gelatine in PBS for 1 h at RT, rinsed with PBST and incubated either with monoclonal antibody 3D10  for dete6 cells/mL and stimulated either with VLPs  at a concentration 5 μg/mL or polyclonal activators Concavalin A (ConA) (Sigma-Aldrich) and lipopolysaccharide (LPS) (Sigma-Aldrich) added at concentrations 3 μg/mL and 1 μg/mL, respectively, to the growth medium: RPMI-1640 containing 10% FBS (Merck-Millipore) and antibiotics. After 24 h and 48 h of incubation, culture supernatants were collected and stored at −80 °C until analysis. The suspension of spleen cells from immunized mice was prepared by mechanical disruption. Ammonium chloride lysing buffer was used for the lysis of red blood cells. The prepared cells were seeded into 96-well cell culture plates at a density of 1 × 10Interleukin 12 (IL-12), interleukin 2 (IL-2) and interferon gamma (IFN-γ) concentrations in mouse spleen cell cultures were determined by sandwich ELISA using Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go, Mouse IFN gamma ELISA Ready-Set-Go  and Mouse IL-2 ELISA set  according to the recommendations of the suppliers.6 cells/well in duplicate. After 24 h of incubation at 37 °C in 5% CO2, spleen cells were removed and the plates were incubated with detection antibody  for 8 h at +4–8 °C, followed by 2 h incubation with streptavidin-AP . The BCIP/NBT substrate  was used to visualize the enzymatic reaction. The immunostained spots were counted with CTL-ImmunoSpot® S6 FluoroSpot Line counter  and analyzed using Immuno Capture 6.4 software (C.T.L.). For each mouse and stimuli, the ELISPOT count  was reported as the average number of spots of replicates. The results were expressed as the number of responding cells per one million of spleen cells.The frequency of IFN-γ-producing cells in the spleen cell cultures of immunized mice was determined by an enzyme-linked immunospot (ELISPOT) assay. Briefly, 100 μL of spleen cell suspension from the immunized mice were suspended in RPMI-1640 medium containing 10% FBS (Merck-Millipore) and antibiotics. The cells were seeded into sterile 96-well plates for ELISPOT assays  pre-coated overnight with anti-IFN-γ capture antibody  in 100 μL of growth medium containing either ConA (3 μg/mL) or VLPs  at a concentration 5 μg/mL. Spleen cell cultures were diluted in two-fold steps starting from a density 0.5 × 10in vitro  flow cytometer and analyzed using FlowJo, Version 10 software .A three color staining flow cytometry analysis of the murine spleen T cells was carried on the 28th day after the 3rd immunization with VLPs and re-stimulation t-test was used to compare the titers of insert-specific IgG antibodies between the groups of immunized mice and to compare cytokine levels between the untreated and VLP-treated murine spleen cell cultures. The differences were accepted as statistically significant if p < 0.05.Statistical analysis of experimental data was performed using OriginPro 8 software . The The potential insertion sites for foreign sequences within the TSPyV VP1 protein were determined based on the known three-dimensional fold structures of other polyomavirus VP1 proteins and successful exploring of HaPyV VP1 insertion sites located at the BC loop  and HI loop  ,13. Structure modeling of recombinant polyomavirus VP1 proteins/pentamers was performed using a sensitive profile-profile comparison method HHPRED. It has been found that the best structural template for modeling both native and recombinant versions of TSPyV VP1 protein is PDB:1sva–SV40 capsid . Though VP1 hexamer has been shown to be an asymmetric unit of the capsid, we have used the pentamer (a compact part) as a template. For modeling both native and recombinant versions of HaPyV VP1 protein the best template was PDB:1sid–murine polyomavirus (MPyV) capsid .The generated three-dimensional molecular model of TSPyV VP1 pentamer with indicated surface-exposed sites #1 and #4 is presented in Furthermore, molecular models of TSPyV VP1 pentamers with inserted 5 aa-long HBV PreS1 epitope (short name PS1) or a universal 13 aa-long T cell epitope PADRE either at site #1 A or siteBased on the molecular model of TSPyV VP1 protein, potential surface-exposed positions for inserting peptides PS1 and PADRE were selected. The peptides were inserted into the TSPyV VP1 protein at either position #1 (aa 76–80) or position #4 (aa 278–281). The same peptides PS1 and PADRE were inserted into HaPyV VP1 protein at previously selected positions  - eitherS. cerevisiae strain AH22-214p was analyzed by SDS-PAGE and Western blot using VP1-specific MAbs . All eight chimeric proteins formed VLPs E. HaVP1-To analyze whether the selected insert positions are located on the surface of TSPyV VP1 protein, a collection of monoclonal antibodies (MAbs) against an intact (non-modified) TSPyV VP1 protein was generated. To generate hybridomas, spleen cells of the BALB/c mouse immunized with TSPyV VP1 VLPs were fused with myeloma cells. Hybrid clones were screened in ELISA for their reactivity with TSPyV VP1 VLPs. In total, 17 positive hybrid clones secreting TSPyV VP1-specific MAbs of IgG isotype were identified that were further stabilized by cloning and propagated in culture . The reaThe immunogenicity of chimeric VLPs harboring either HBV preS1 (PS1) or PADRE epitopes at different positions was evaluated. For this purpose, the development of IgG antibodies against the inserted target peptides as well as against VP1 carrier was analyzed by an indirect ELISA in different groups of BALB/c mice immunized with chimeric VLPs. On day 21st after the primary immunization, IgG antibodies against the inserted peptides and the chimeric VLPs were detected in all groups of mice . High tiThus, the immunogenicity study showed that all chimeric VLPs were immunogenic in mice and induced activation of insert-specific B cells. E. coli lipopolysaccharide (LPS), a potent stimulator of dendritic cells used as a positive control (mean concentration 102 pg/mL). In contrast, the levels of IL-12 in spleen cell cultures incubated for 48 h without adding VLPs (negative control) were in the range of 7–12 pg/mL  secretion levels in spleen cell cultures of BALB/c mice were analyzed by ELISA. Both chimeric VLPs and intact VLPs added to murine spleen cultures induced high IL-12 secretion after 48 h of incubation  indicati12 pg/mL . Comparein vitro was analyzed. Activation of T cells was analyzed by ELISA, ELISPOT and flow cytometry assays. Furthermore, the ability of spleen T cells derived from BALB/c mice immunized with chimeric VLPs harboring T cell-specific PADRE epitope to respond to restimulation with recombinant VLPs The levels of T-cell specific cytokines IL-2 and IFN-γ were measured in spleen cell cultures treated either with chimeric VLPs or unmodified VLPs. Significant amounts of IL-2 (in the range of 3–23 pg/mL) and IFN-γ (in the range of 0.2–2.3 ng/mL) were determined in spleen cell cultures after 24 h of incubation with VLPs A–D. In cin vitro stimulation with recombinant VLPs, three-color staining (CD4+ CD69+ and CD8+ CD69+) and subsequent flow cytometry analysis of spleen cell cultures was performed. The percentages of CD4+ CD69+ and CD8+ CD69+ cells were higher in VLP-treated spleen cell cultures as compared to untreated spleen cell cultures indicating activation of antigen-specific memory T cells . Similar tendency was observed in CD8+ T cell population: higher percentage of CD69+ T cells (0.74%–2.52%) was determined within the CD8+ T cell population as compared to the untreated spleen cell cultures (0.64%–0.68%). However, the difference between the VLP-stimulated and the untreated CD8+ T cell population was less pronounced as compared to the CD4+ T cell population. More efficient activation of memory CD8+ T cells was observed in a group of mice immunized with HaVP1-PADRE-1 VLPs , while only marginal increase of CD8+CD69+ T cells after VLP stimulation was determined in other groups of immunized mice (An increase of a percentage of CD69+ T cells (0.50%–1.13%) was determined within the CD4zed mice . Summarizing, the analysis of VLP-treated murine spleen cell cultures by different assays demonstrate that T cell-specific epitopes derived from recombinant VLPs are efficiently presented to T cells and induce their activation.E. coli that allowed the subsequent in vitro assembly of VLPs under certain conditions [S. cerevisiae. The first polyomavirus-derived VLPs produced in yeast were HaPyV VP1 VLPs [S. cerevisiae-based expression system for the production of VLPs originating from MPyV, SV40, BKPyV and JCPyV [Recombinant polyomavirus-derived VLPs that resemble native virions in size and shape are extensively used for targeted epitope presentation and show a great potential for vaccine development, gene delivery and diagnostics ,38,39,40nditions ,42. AnotVP1 VLPs . Furthernd JCPyV . Recombind JCPyV . The mulnd JCPyV ,42,44. Tnd JCPyV . Therefond JCPyV ,47. Amonnd JCPyV ,13,14,15nd JCPyV ,48,49,50S. cerevisiae in a form of VLPs [During the last decade, 11 novel human polyomaviruses have been identified ,52,53,54 of VLPs . Serolog of VLPs . No specOne of the newly discovered human polyomaviruses is trichodysplasia spinulosa-associated polyomavirus (TSPyV) that causes a rare skin disease trichdysplasia spinulosa in immunocompromised patients . TSPyV VIn line with computer modeling and EM data, immunogenicity study revealed lower levels of PADRE-specific IgG antibodies in mice immunized with TSVP1-PADRE-4 chimeric VLPs as compared to TSVP1-PADRE-1 chimeric VLPs. PADRE peptide presented on HaPyV VP1-derived chimeric VLPs was more immunogenic, which may be explained by a more efficient VLP assembly. In addition, the immunogenicity of HaVP1-PADRE-4 correlates with lower structural flexibility of the inserts at site #4 (HI loop) of HaPyV VP1 modeled pentamer, thus suggesting that the flexibility of inserts plays a significant role in peptide presentation and activation of B cells. In contrast to chimeric VLPs with inserted PADRE epitope, the TSPyV VP1-derived chimeric VLPs with inserted 5 aa-long HBV preS1 epitope were more efficient in inducing insert-specific antibodies as compared to HaPyV VP1-derived chimeric VLPs. The immunogenicity data demonstrate the importance of the surrounding aa residues for the efficiency of peptide presentation to B cells.All types of chimeric VLPs efficiently activated memory B cells as demonstrated in a long-term immunogenicity study that revealed a significant increase of insert-specific IgG titers after repeated injections, even when the VLPs were applied without any adjuvant. As an activation of T helper cells is necessary for inducing memory B cell response and immunoglobulin class switch, the long-term immunogenicity data indicate an efficient T helper activation by chimeric VLPs. Moreover, the ability of all chimeric VLPs to activate dendritic cells and T cells was demonstrated by enhanced expression of cytokines IL-12, IL-2 and IFN-γ, as well as T cell activation-related surface marker (CD69) in the cultures of murine spleen cells treated with chimeric VLPs. Summarizing, the TSPyV VP1-derived recombinant VLPs are immunogenic and represent an efficient carrier for foreign epitopes. To our knowledge, this is the first study that describes chimeric VLPs derived from the capsid protein of the newly discovered human polyomaviruses. TSPyV VP1 protein represents a new carrier for insertion of foreign epitopes at surface-exposed positions within the HI or BC loop. The yield and stability of chimeric TSPyV VP1-derived VLPs depends on the position and hydrophobicity of the insert.A large collection of novel MAbs against TSPyV VP1 protein has been generated. The selective reactivity of the MAbs with TSPyV VP1-derived chimeric VLPs confirms the surface exposure of the inserts.All chimeric VLPs are immunogenic and induce formation of insert-specific IgG antibodies in immunized mice. An increase of the titers of insert-specific IgG antibodies after repeated immunizations indicates immune memory, even when the VLPs are applied without any adjuvant.All chimeric VLPs induce activation of dendritic cells and T cells in murine spleen cells cultures as demonstrated by the analysis of cytokine production profiles and T cell surface marker CD69.The immunogenicity of TSPyV VP1-derived chimeric VLPs is similar to that observed with HaPyV VP1-derived chimeric VLPs. However, highly hydrophobic PADRE epitope more disturbs VLP assembly and induces lower antibody response when inserted into TSPyV VP1 protein as compared to HaPyV VP1 protein. In contrast, the HBV preS1 epitope is more immunogenic when presented on TSPyV VP1-derived chimeric VLPs.The current study provides the first example of chimeric VLPs constructed on the basis of the capsid protein of the newly discovered human polyomaviruses.