Multiple sclerosis (MS) is a common neurological disorder of putative autoimmune origin. Clinical and experimental studies delineate abnormal expression of specific cytokines over the course of the disease. One major cytokine that has been shown to play a pivotal role in MS is tumor necrosis factor (TNF). TNF is a pleiotropic cytokine regulating many physiological and pathological functions of both the immune system and the central nervous system (CNS). Convincing evidence from studies in human and experimental MS have demonstrated the involvement of TNF in various pathological hallmarks of MS, including immune dysregulation, demyelination, synaptopathy and neuroinflammation. However, due to the complexity of TNF signaling, which includes two-ligands (soluble and transmembrane TNF) and two receptors, namely TNF receptor type-1 (TNFR1) and type-2 (TNFR2), and due to its cell- and context-differential expression, targeting the TNF system in MS is an ongoing challenge. This review summarizes the evidence on the pathophysiological role of TNF in MS and in different MS animal models, with a special focus on pharmacological treatment aimed at controlling the dysregulated TNF signaling in this neurological disorder. The natogenesis . Autoreaogenesis . The conogenesis . Of noteogenesis .TNF is a pleiotropic cytokine increasingly recognized to regulate important physiological processes not only in the immune system, but also in the brain ,10,11. EHere, we review the pleiotropic functions of TNF in both the immune system and the CNS, with a focus on those processes that are dysregulated in MS, such as Treg function, neuronal transmission, BBB permeability and myelination. Moreover, we provide an updated overview of data from different animal models of MS and from MS patients about the role of TNF and its receptors in MS pathology, proposing a re-examination of therapies targeting TNF signaling in MS.TNF is the prototypical cytokine of the so-called TNF superfamily, which includes 19 ligands, while the TNF receptor (TNFR) superfamily (TNFRSF) consists of 29 related receptors . Newly sTNF family members exert their cellular effect through two distinct surface receptors, the TNF Receptor Superfamily Member 1A (TNFRSF1A-TNFR1), also known as p55 or p60, and TNF Receptor Superfamily Member 1B (TNFRSF1B-TNFR2), also known as p75 or p80 . ThroughThe TNF-TNFR signaling pathways are complex and wide-ranging in different cell types and precise circumstances, thereby accounting for TNF pleiotropic nature of action . DespiteTNF has long been regarded as the key regulator of the inflammatory response, as shown in sepsis . NowadayBelow, we revise main findings regarding the involvement of TNF in both immune system and brain functioning.Cytokines provide essential communication signals to the highly motile cells of the immune system. Upon cellular activation, TNF is mostly produced by cells of the innate immune system like mononuclear phagocytes but it is also expressed by neutrophils and lymphocytes ,36. ThisTNF regulates many aspects of T cell biology, like proliferation, priming and survival and/or apoptotic fate . A relevTNF is known to play an important role in the conclusion of lymphocyte responses, as indicated by the ability of this molecule to promote activation-induced cell death in both CD4+ and CD8+ T cells, mainly through TNFR1 . HoweverIt has recently become apparent that TNF can exert a strong activity on CD4+ Tregs, which are a subset of CD4+ T cells that help to prevent or treat autoimmunity by maintaining self-tolerance, immune homeostasis, and suppression of cytotoxic T cells . MountinWhile most of the studies on human and murine cells have addressed the effects of TNF on T cells, a poorly explored and clarified issue regards the role of T cell-derived TNF in pathophysiological conditions . T cellsTNF is constitutively expressed at low levels in the normal adult brain  and its Glial cells, astrocytes and microglia, are the main source of TNF in the brain in both physiological and pathological conditions ,68. NoteTNF has been reported to control glutamate transmission also by modulating NMDA currents. The use of TNF KO mice has revealed the permissive role of constitutive TNF in the control of glutamate release from astrocytes in response to purinergic signaling, with the consequent enhancement of the excitatory synapses in the dentate gyrus of the hippocampus through the activation of presynaptic NMDAR . ApplicaTNF can potentiate glutamate excitotoxicity even in conditions of subthreshold glutamate levels, directly through activation of glutamate-NMDA receptors  and locaIn addition to excitotoxicity, in the injured CNS the increased concentration of TNF released by astroglia and microglia boost neuroinflammatory responses . As elseOverall data from murine studies of brain functioning implicate TNF in several CNS activities and suggest that, under specific conditions of elevated TNF levels, TNFR1 is more likely implicated in glutamatergic transmission potentiation and demyelination, while TNFR2 signaling may be involved in reparative processes.Data reported in the previous chapter highlight the crucial involvement of TNF in several immune and CNS functions that are particularly relevant for MS pathogenesis. However, the centrality of TNF in the above biological functions is a double-edged sword, because, on the one hand, it clearly links TNF to MS pathology, but, on the other hand, it makes hard to dissect the contribution of TNF to each pathological hallmark of MS, namely immune dysregulation, demyelination, neuroinflammation and neurodegeneration. In the following paragraph we revise the literature addressing the role of TNF in animal models of MS and in human studies.Due to the complexity of disease manifestations and the underpinning mechanisms, no single animal model can fully recapitulate the broad spectrum of MS symptoms and pathological hallmarks. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model in MS research to study the autoimmune component of MS pathology . Though EAE disease course is significantly influenced by several factors, including species, strain and the autoantigen used. In this model, autoreactive T cells are actively induced by peripheral immunization with a myelin antigen. Such T cells collect in the spleen before migrating to the CNS, where they recognize their cognate antigen on local APCs and activates, thus starting an inflammatory cascade leading to tissue injury ,102.In EAE, TNF has been shown to be expressed by local microglia and infiltrating macrophages  and by eThe induction of EAE in transgenic mice for TNF or TNFRs has significantly contributed to understanding TNF role in MS pathophysiology . SeveralTNF KO mice were also used to investigate TNF involvement in BBB leakage in the EAE model. BBB permeability changes were significantly reduced in TNF KO mice with EAE compared with WT congenic animals, suggesting that TNF plays a role in promoting BBB damage induced by EAE, possibly through its known effect on the induction of adhesion molecules and chemokines ,116. MorEvidence of TNF pathogenic role was further provided by anti-TNF treatment. The administration of a recombinant TNFRp55 protein constructed by fusing TNFRp55 extracellular domain cDNA to a human IgG (TNFRp55-IgG1), showed a beneficial effect in both preventive and therapeutic schedule . IntrapeTo better understand the role of TNF in MS pathology, several studies addressed whether individual TNFRs were involved in different processes of disease progression. Findings in transgenic mice indicate that TNFR1 plays a detrimental role in MS whereas TNFR2 has a protective one. In particular, TNFR1/TNFR2 double-KO and TNFR1 KO mice were completely protected against EAE showing a reduction in maximal disease severity compared to WT mice, whereas TNFR2 KO mice showed exacerbated disease symptoms, demyelination, enhanced Th1 cytokine production, and enhanced CD4+ T cell infiltration in the CNS ,122,123.The TNFR2-mediated protective action was also supported by evidence in conditional KO mice that demonstrated the crucial role of TNFR2 in oligodendrocyte differentiation  and suppRecent studies indicated an important role for TNF and TNFR2 in the activation of Treg cells. It was shown that Treg-TNFR2-deficient mice developed exacerbated EAE disease, indicating that intrinsic TNFR2 signaling in Tregs provides protection in CNS autoimmunity . HoweverThe individual roles of the two TNF-TNFR signaling pathways have also been investigated by pharmacological treatments in the EAE model, providing solid and convincing evidence that the specific inhibition of solTNF or TNFR1 is beneficial in EAE and may represent a valuable anti-TNF therapy for MS. The selective inhibition of solTNF through Xpro1595, which does not alter tmTNF signaling, has been studied by different groups and has proven a promising therapeutic strategy . TreatmeRegarding TNFR targeting, the first compound used in EAE was an antagonist to TNFR1, PEGylated R1antTNF (PEG-R1antTNF) . PEG-R1aIn line with the idea of a beneficial role of TNFR2 signaling, exogenous activation of TNFR2 has been shown to ameliorate EAE disease. Treatment with solTNFR:Fc/p80 fusion protein reduced the clinical deficit of the first attack of relapsing-remitting EAE and decreased chemokine expression . MoreoveAll together, these results of pharmacological manipulation of TNF signaling mirror data obtained in conditional KO mice and clearly indicate divergent roles for the individual TNFRs in CNS autoimmunity; TNFR1 seems to promote CNS inflammation and demyelination and TNFR2 apparently acts limiting pathology, by eliminating autoreactive CD4+ T cells and macrophages, by maintaining Treg functionality, and by promoting remyelination.Finally, TNF has been shown to promote the synaptic alterations typically affecting the brain of EAE mice and consisting in a potentiation of the glutamatergic transmission that has been proposed to contribute to neurodegeneration in EAE/MS brains . TNF relCPZ model provides an ideal system to study neuroinflammation, demyelination and remyelination in the absence of autoimmune response ,140,141.Like in EAE, in CPZ mice TNF was demonstrated to act in a bivalent way . In theiIn line with these findings, a recent paper has demonstrated that local CNS production of solTNF inhibits the onset of remyelination and CNS repair in CPZ mice . In partThese results show that TNF plays a crucial role in pathological processes of CPZ model and, in agreement with data in EAE, reveal that the selective inhibition of solTNF prevents neuroinflammation and demyelination, by increasing the number of OPCs in the lesion sites and by promoting the clearance of myelin debris by phagocytic CNS macrophages.TMEV is a picornavirus which, when injected intracranially into susceptible mice, causes a chronic, inflammatory demyelinating disease. The histopathology of the virally-induced demyelination consists of mononuclear cell infiltration and myelin sheath damage limited to the white matter . The iniThese data suggest that TNF is most likely involved in the pathogenesis rather than protection against virally-induced inflammatory demyelinating disease and further studies should be addressed to evaluate the potential therapeutic role of specific TNFR antagonists in this animal model.First evidence of the involvement of TNF in the neuroinflammatory process occurring in MS were provided by post mortem studies in MS patients, showing high cytokine levels in close proximity to active CNS lesions ,150,151.Post mortem studies represent a worthwhile tool to deepen the pathophysiology of neurological disorders, but they are intrinsically devoid of prognostic value. Thus, different groups have turned their attention to detectable fluid TNF-signaling biomarkers in MS patients, showing differential expression of TNF depending on disease stage and providing evidence that TNF levels correlate with MS progression ,156 Tab. With soThe detection of TNF in easily accessible fluids, like serum or plasma, is appealing in terms of clinical monitoring of patients, but it cannot entirely explain disease development. Recent technical progress in terms of test sensitivity has made possible the detection of the levels of cytokines and other molecules in the CSF, which is more informative of the brain activity than the blood . Thus, iElegant studies of combined tissue characterization and CSF analysis, have provided compelling evidence that the released inflammatory/cytotoxic mediators into the CSF by immune cells in the subarachnoid space create an intracerebral milieu that sustains chronic compartmentalized inflammation and, at the same time, directly mediates and/or exacerbates cortical pathology and disease progression ,175,176.Collectively these data indicate that TNF is more associated to PMS than RRMS and suggest that further investigations are needed.In MS, the detrimental effect of glutamate signaling in the CNS is widely recognized to contribute to neurodegeneration ,177,178 Chimeric experiments have been carried out to address the causal link between TNF and excitotoxic damage in MS. TNF-enriched CSF from PMS subjects was incubated with corticostriatal slices to study the synaptic effect of inflammatory milieu on synaptic transmission . GlutamaOverall, findings in human MS suggest the involvement of TNF in MS pathology. Moreover, though not fully exploring the whole TNF signaling and its receptors, they indicate that TNF is more associated to PMS than RRMS and suggest that further investigations are needed.Based on the recognized role of TNF in autoimmune disorders, since 1988 different drugs targeting TNF signaling have been successfully introduced for the treatment of inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, Crohn’s disease and ankylosing spondylitis, psoriasis ,187,188.Despite promising results in other autoimmune diseases, anti-TNF therapy has proven unsuccessful in MS. Following the encouraging results gained in rheumatoid arthritis  two femaBased on current knowledge of TNF biology, a possible explanation for these results, which at that time were quite unexpected, could be linked to the pleiotropic actions of TNF, including both pro- and anti-inflammatory functions as well as the almost ubiquitous expression and action of such cytokine. Moreover, the relationship between nonselective TNF inhibitors and MS is further supported by several cases of MS onset observed in patients with other diseases ,193. PatIn order to clarify the effect of anti-TNF therapy on MS patients and non-MS autoimmune diseases, genome-wide association studies (GWAS) may help in identifying a link between the specific response to anti-TNF therapy and the presence of the single nucleotide polymorphism in TNF-related genes that may affect TNF signaling. In particular, two polymorphisms of the TNFR1-encoding gene, TNFRSF1A, the rs1800693 with high frequency and the rs4149584 with low frequency, have been identified and associated with an increased risk for MS and not for other autoimmune disorders . The resAs reviewed, TNF is an important mediator of disease in MS playing a key role in the pathogenesis of the main hallmarks characterizing MS and its related preclinical models. Although clinical trial with nonselective anti-TNF antibodies completely failed and discouraged neurologists from using anti-TNF agents for MS patients, in the light of the recent advances in the knowledge of TNF signaling, the anti-TNF therapy for MS treatment should be re-examined. On one hand, data from MS models in TNF KO mice or in mice treated with pan-anti-TNF drugs, agree with human studies. On the other hand, recent studies in experimental MS provide clear evidence of the complexity of the TNF/TNFRs signaling in MS, clarifying some crucial aspects of the disease and opening the possibility to test novel and more selective compounds. In particular, the opposite immunoregulatory effects of TNFRs suggest that the low efficacy of nonselective TNF blockers (like etanercept) in the EAE model is probably due to the abrogation of TNFR2 neuroprotective action and leads to the speculation that selective regulation of TNFR1 and TNFR2 may be a new molecular target for the development of therapeutic agents in MS. Testing anti-TNF signaling drugs in animal models of MS in combination with genetic ablation of TNFRs in specific cellular population, could be of great value to distinguish the role of each TNFRs in a specific cellular context. Indeed, the use of selective conditional KO mice has highlighted subtle but relevant differences in the function of TNFR1 and TNFR2 in a cell- and context-dependent manner ,126. MorBased on the literature reviewed here, a dichotomic, albeit simplistic, vision of TNF signaling and targeting in MS can be proposed. Considering that TNFR1 exerts inflammatory and proapoptotic functions, whereas TNFR2 has a neuroprotective function, the combination of inhibitors of TNFR1 signaling and TNFR2 agonists represents promising a therapeutic approach for the treatment of MS . The pro


Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2−∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders . Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.Around a 20–30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders ( Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is an autoimmune disorder whose pathogenesis genetics and environmental factors play a key role ,2. ArounBiological therapy, mainly anti-TNF agents, has revolutionized the treatment of IBD and other inflammatory diseases. The phenotype of pIBD usually leads to earlier use of biological therapy in children . HoweverMucosal healing is the best outcome measure in pIBD and it is used to compare the efficacy of different biological therapies . HoweverSerological or fecal biomarkers, such as trough serum anti-TNF levels and antidrug antibodies and fecal calprotectin, have been related to the response to anti-TNF treatment and are usually monitored in clinical practice ,9,10,11.The identification of genomic biomarkers could help us to identify groups of children who are less likely to respond to anti-TNFs before the beginning of treatment. The expression profile of several genes in inflamed tissues have been associated with a response to anti-TNF agents in adults diagnosed with IBD or other autoimmune disorders ,21,22,23SMAD7, TNF, TLR2, TNFRSF1B, TBX21, DEFA5, IL11, TREM1, and OSM (previously studied as related to the anti-TNF response or IBD in adults) [In the present study, we measured the expression of the genes  adults) ,29,30,31Forty-three patients met the inclusion criteria and were included in the study . Twenty-The anti-TNF treatment failure rate was 18.2%, which was much lower than expected. The characteristics of both groups of patients are summarized in p value = 0.013)  .TLR2, TNF, TNFRSF1B, IL11, TBX21, SMAD7, TREM1, OSM and DEFA5 in responders and non-responders immediately prior to the first administration of the anti-TNF agents. The analysis was performed using responders (R-T0) as the biological reference group. The SMAD7 mRNA expression was decreased in non-responders compared to responders . In addition, a decrease in expression of DEFA5 (–2.0-fold) and TLR2 (–1.8-fold) were observed in non-responders compared with responders  in non-responders compared with responders . The results showed a lower expression of sponders .TLR2 was lower (–1.6-fold) in non-responders, although this difference was not statistically significant.These results and inter-sample variability were observed more clearly when the relative expression values of each sample were represented graphically . The expDEFA5 decreased 1.75-fold after the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders, although this difference was not statistically significant. These results and inter-sample variability were observed more clearly when the relative expression values of each sample were represented graphically  and before initiation of treatment (t0) (t2/t0). The expression of phically .SMAD7 mRNA expression at t0 was decreased in non-responders compared to responders  was 3.75 (95% CI 1.70–8.27), and the negative LR (–LR) was 0.21 (95% CI 0.03–1.32).The sponders . The posNOD2/CAR15, ATG16L1, IL23R, and IL10R have been involved in susceptibility to pIBD or to very-early-onset ulcerative colitis [ATG16L1, CDKAL1, ICOSLG, BRWD1, and HLA-DQA1 have been associated with an anti-TNF response specifically in children [TNF, SMAD7, and DEFA5 to be potential pharmacogenomic markers for early response to anti-TNF drugs in pediatric patients with IBD. This discovery is a first step for what in the future may constitute a minimally invasive routine test in daily clinical practice that could help to select the best treatment options for these patients. Several authors have included these three genes in their gene expression studies in the search for a biomarker that would enable us to predict the response to anti-TNF agents in patients with various diseases, mainly rheumatoid arthritis [Although the anti-TNF agents infliximab and adalimumab have revolutionized the treatment of autoimmune diseases, a significant percentage of patients do not respond to this therapy . Knowing colitis ,33. Diff colitis . Concernchildren . Howeverchildren ,20,23. Trthritis ,26,36,37SMAD7 mRNA was downregulated in the peripheral blood cells of non-responders after 2 weeks of anti-TNF treatment. SMAD7 negatively regulates phosphorylation of the SMAD2/SMAD3 complex, which is necessary for TGF-ß signaling [SMAD7 in the affected regions of the intestine [SMAD7 increases values for proinflammatory cytokines, such as IL-2, thus driving inflammation and enhancing the proinflammatory Th17 immune response [SMAD7 also decreases in the peripheral blood mononuclear cells of patients with multiple sclerosis [SMAD7 cells are enriched at inflammation sites and, in parallel, poorly expressed SMAD7 cells are enriched in peripheral blood. TGF-β is a cytokine with important immunomodulatory properties that control the activation status of all immune system cells and contributes to peripheral differentiation of regulatory cells (anti-inflammatory cells) and Th17 T cells (pro-inflammatory cells) [SMAD7 is a valuable biomarker of early response to anti-TNF response in children with IBD, although this should be validated in a larger cohort.Our results showed that expression of ignaling ,39. Seveignaling  and IBD.ntestine . Howeverresponse . Expressclerosis ,44. Receclerosis . We suggy cells) . This duDEFA5 mRNA levels tend to decrease between week 0 and 2 in responders, but not in non-responders. DEFA5 is a cytotoxic peptide that is produced in large quantities by Paneth cells. It is involved in the control of intestinal microbiota and plays a crucial role in its homeostasis [DEFA5 mRNA expression in blood during the first 2 weeks of anti-TNF treatment in pediatric responders. As for SMAD7, expression in inflamed tissues and in blood may be increased in one and decreased in the other.In addition, our results showed that eostasis . DEFA5 aeostasis . Recentleostasis  demonstrTREM1 and TNF genes have been associated with a response to anti-TNF drugs in adults [n adults ,49. Furtn adults . In our The main limitation of the study is its sample size, which is insufficiently large to take account of the wide interindividual variability in gene expression observed in blood ,26. GeneIn addition, comparison with similar studies is difficult for two reasons. First, there are no data on children. Second, response criteria differ with studies. Thus, for example, the response may be based on various parameters, such as endoscopic and histological healing , or the More studies involving larger populations are necessary to confirm these findings. Nevertheless, the various blood biomarkers of response to anti-TNF agents identified to date could help to improve the therapy in pIBD.SMAD7 expression prior and after 2 weeks of anti-TNF treatment were associated with a lack of response in children diagnosed with IBD. Moreover, TLR2 and DEFA5 were also differentially expressed in responder patients to anti-TNF drugs when compared to non-responders, but these trends need to be validated in larger cohorts to achieve significant results.In summary, we verified that low SMAD7 mRNA in blood as pharmacogenomic markers of early response to anti-TNF drugs in pIBD.In conclusion, to the best of our knowledge, this is the first study to assess levels of Between March 2017 and March 2019, 43 IBD patients aged under 18 years were prospectively recruited from the Pediatric Gastroenterology Units of 14 Spanish hospitals. The groups were matched for age and gender. The inclusion criteria were as follows: (1) diagnosis of IBD established based on clinical manifestations, radiological findings, and endoscopic and histological criteria; (2) age 1 to 18 years old; and (3) start of treatment with infliximab  or adalimumab . Demographic and clinical information were also collected. Disease activity was assessed using the Pediatric Crohn’s Disease Activity Index (PCDAI) and Pediatric Ulcerative Colitis Activity Index (PUCAI). Patients were classified as responders and non-responders to anti-TNF therapy based on changes in the PCDAI or PUCAI. Response was defined as a decrease higher than 15 points in any of these indexes from week 0 to 10 for infliximab or from week 0 to 26 for adalimumab. Response was defined as reaching a value of 0 in patients with a value lower than 15 points prior to treatment in any of these indexes.The present study was approved by the Ethic Committee of the Hospital General Universitario Gregorio Marañón and the other participating hospitals . All patients from 6 to 18 years old assented and parents or legal representatives provided their written informed consent to participate, in accordance with relevant guidelines and regulations.A blood sample was collected in PAXgene Blood RNA tubes  before the administration of the anti-TNF agent (week 0) and after 2 weeks of treatment (week 2). Total whole blood RNA was extracted using PAXgene Blood RNA kits  according to the manufacturer’s instructions. RNA concentrations were measured using the Quawell Q5000 Spectrophotometer . RNA integrity was verified using the Agilent RNA 6000 Nano Kit in a 2100 Bioanalyzer . Extracted RNA was stored at −80 °C until further processing. All RNA samples fulfilled both of the following criteria: RNA integrity ˃ 7 and an optical density ratio of absorbance at 260/280 of between 1.8 and 2.−∆∆Ct method. GAPDH and HPRT1 were used for normalization. Real-time PCR was performed in triplicate using 2 μL/well of the cDNA dilution, and 0.04 μM SMAD7, TNF, TLR2, TNFRSF1B, TBX21, DEFA5, IL11, GAPDH, and HPRT1  following the manufacturer’s instructions. A 1:20 dilution of each cDNA synthesized from 1 µg of total RNA in a volume of 20 µL was used. Expression of the selected genes was quantitated using qPCRBIO SyGreen™ Mix  and a StepOnePlus real-time PCR system (Applied Biosystems). Gene expression was calculated using the 2t-test was used to compare qualitative and quantitative variables, respectively. Continuous clinical and demographic variables were expressed as the mean and standard deviation (SD) or as the median and interquartile range (IQR); qualitative variables were presented as absolute and relative frequencies. The Fisher exact test or t-test was applied to compare gene expression between groups, with a confidence level of 95% and maximum Ct of 35. Efficiency for each primer pair probe was calculated and used for correction. A p value < 0.05 was considered statistically significant. The statistical analyses were performed using ExpressionSuite v1.1  and GraphPad Prism v5.1 .Gene expression was quantified using ExpressionSuite v1.1  and data were reported as the mean relative quantification and maximum and minimum relative quantification; all the assays were run in triplicate. The unpaired SMAD7 relative expression were calculated as previously described [The PPV, NPV, sensitivity, specificity and diagnostic odds ratios for escribed . Likelih


Thiothrix, ‘Candidatus Thiothrix moscowensis’ and ‘Candidatus Thiothrix singaporensis’. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin–Benson–Bassam cycle; sulfide oxidation , sulfur oxidation (rDsr complex), direct (soeABC) and indirect  sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus  Thiothrix remain poorly understood [T. nivea, T. fructosivorans, T. unzii, T. caldifontis and T. lacustris. Despite the fact that the genomes of T. lacustris (GCF_000621325.1), T. caldifontis (GCF_900107695.1), and T. nivea (GCF_000260135.1) are available from databases, comprehensive analysis of the main metabolic pathways of sulfur, carbon, and nitrogen has not been reported. In addition, 25 metagenome-assembled genomes (MAGs) obtained from metagenomes of natural hydrogen sulfide biotopes are currently available. Two of these genomes were attributed to certain species, T. nivea DOLJORAL78_51_81 [T. lacustris A8 [Thiothrix sp. or uncultured Thiothrix sp. [Filamentous colorless sulfur bacteria (FCSB) have been a classic object of microbiology since the time of S.N. Winogradsky, who used them in the research that culminated in the discovery of chemosynthesis. Nevertheless, the genomics and metabolic potential of the FCSB from the genus derstood ,2,3. Thederstood  include s GCF_00021325.1, GCF_90010695.1, anThiothrix, where filamentous bacteria often form massive fouling. There are reports of intense growth of Thiothrix in wastewater rich in reduced sulfur compounds [Thiothrix in wastewater treatment plants leads to the formation of fouling that can disrupt the operation of the entire system, degrading the quality of the wastewater and increasing the treatment time [Thiothrix from activated sludge to accumulate polyphosphates and participate in the removal of phosphorus from wastewater was found along with other polyphosphate accumulating organisms (PAOs) [Candidatus Accumulibacter phosphatis’, Tetrasphaera [Halomonas [Due to the feature of energy metabolism, where hydrogen sulfide is the main donor of electrons during chemosynthesis, sulfur springs were considered to be a traditional habitat for ompounds ,12,13,14ent time ,16. On ts (PAOs) ,18,19, sasphaera ,21, and alomonas .Thiothrix genomes—Thiothrix sp. RT and Thiothrix sp. SSD2, recovered from metagenomes of laboratory-scale enhanced biological phosphorus removal bioreactors (EBPR). Based on the phylogenetic analysis of MAGs, we propose two new species, ‘Candidatus Thiothrix moscovensis’ sp.nov. and ‘Candidatus Thiothrix singaporensis’ sp.nov.The present study aims to analyse of genomic and metabolic potential of two recently obtained 3COONa·3H2O, 50 mg/L NH4Cl, 54.3 mg/L KH2PO4, 56.7 mg/L Na2HPO4, and 8.6 mg/L yeast extract. The bioreactor was operated at room temperature in a cyclic mode. A 6 h cycle of cultivation included the stages of filling the bioreactor with fresh medium (30 min), anaerobic growth (110 min), aerobic growth (180 min), precipitation of activated sludge (30 min), and discharge of the spent medium (10 min). Under these conditions, the PAO bioreactor stably removed 75–80% of phosphorus from the medium [Enrichment and cultivation of the PAO microbial community in a lab-scale bioreactor operated at Research Center of Biotechnology  was reported earlier . Brieflye medium .® Ultra™ II DNA Library Prep Kit  and sequenced on the Illumina HiSeq2500 instrument (250 nt single-end reads). Adapter sequences and low-quality reads were removed using Cutadapt [https://github.com/najoshi/sickle accessed on 10 November 2020), respectively. A total of about 50 million high-quality reads were obtained. The reads were de novo assembled into contigs using MetaSPAdes v. 3.7.1 [The total DNA was extracted from 1 g of sludge sample using a DNeasy PowerSoil DNA isolation kit . A total of about 1.5 μg of DNA was obtained. A metagenomic DNA sample was sequenced using the Illumina  platform. The TruSeq DNA library was prepared using NEBNextCutadapt  and Sickv. 3.7.1 . Completv. 3.7.1 . The taxv. 3.7.1  and Genov. 3.7.1 .Gene search and annotation were performed using the RAST server 2.0  followedThiothrix sp. SSD2 was obtained from sludge from a full-scale wastewater treatment plant  and assembled in a single contig with a total length of 4,541,730 [Thiothrix sp. SSD2 genome was obtained from NCBI GenBank (CP059265).MAG of ,541,730 . The inf,541,730 . Genome ,541,730 . For thehttps://www.ezbiocloud.net/tools/ani) based on the OrthoANIu algorithm, using USEARCH [https://ggdc.dsmz.de/ggdc.php# accessed on 10 November 2020).Average nucleotide identity (ANI) was calculated using an online resource  using Illumina technique. Analysis of the taxonomic affiliation of the obtained MAGs showed that one of them, designated d sheath .Thiothrix sp. RT MAG consisted of 78 contigs  with a total length of 3,693,042 bp; the CheckM-estimated completeness of this MAG as 99.8% with 0.92% possible contamination. Genome annotation revealed 16S rRNA gene, 38 tRNA genes, and 3645 potential protein-coding genes. The Thiothrix sp. RT 16S rRNA gene shares 97.9% sequence identity with Thiothrix nivea DSM 5205 and more than 99.5% identity with several environmental 16S rRNA gene sequences detected in activated sludge [The assembled d sludge .Thiothrix sp. RT, only one genome of Thiothrix sp. from EBPR bioreactors, Thiothrix sp. SSD2, is available [Thiothrix, which have all been isolated from natural sulfidic environments.Besides MAG of vailable . Here weThiothrix sp. RT and Thiothrix sp. SSD2 with genes of other members of the genus Thiothrix varies from 93.0 to 97.9%, while the 16S rRNAs of all described representatives of the genus are 93.3-98.9% identical to each other . ‘Ca. Thiothrix singaporensis’ sp.nov. .Based on results of phylogenomic analysis, we propose classifying strains RT and SSD2 as ‘Thiothrix are known representatives of sulfur-oxidizing prokaryotes. Dissimilatory sulfur metabolism of the genus Thiothrix is represented by various sulfur-oxidizing complexes involved in the oxidation of hydrogen sulfide, thiosulfate, elemental sulfur, and sulfite.Members of the genus Thiothrix sp. RT and Thiothrix sp. SSD2.Genes that encode enzymes involved in the oxidation of sulfide to elemental sulfur, sulfide:quinone oxidoreductase (SQR) and flavocytochrome c-sulfide dehydrogenase (FCSD) were identified in sqrA, sqrF, and fccAB genes were found in the genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. The sqrD gene, phylogenetically close to sqrF, was additionally revealed in Thiothrix sp. SSD2  and 12 genes (dsrABCEFHLMKJOP), respectively  and the sat gene encoding ATP sulfurylase (Sat)) were also found. Oxidation of sulfite to sulfate occurs via the APS intermediate.Genomes of Thiothrix sp. RT and Thiothrix sp. SSD2, like the well-known pure cultures of the genus Thiothrix, have pathways for the oxidation of reduced sulfur compounds, which provide the oxidation of hydrogen sulfide, elemental sulfur, sulfite, and thiosulfate.Thus, it can be assumed that 2 through the Calvin–Benson–Bassham cycle  were found in all members of the genus Thiothrix, as well as in Thiothrix sp. RT and Thiothrix sp. SSD2 , the main enzyme that catalyzes the transfer of ATP (ppk1) or GTP (ppk2) to the active site of the protein during the synthesis of the poly-P chain [ppk1 gene encoding PPK1 was found in the genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. Since this reaction is reversible, in the case of PPK1, it could be used to make ATP from poly-P [epp), which catalyzes the hydrolysis of inorganic phosphorus from the polyphosphate chain, was also found in the genomes of Thiothrix sp. RT and Thiothrix sp. SSD2. Genes for PPK and EPP are also present in other members of the genus Thiothrix.The accumulation of polyphosphate (poly-P) granules is known for members of the genus hiothrix ,43. Both-P chain . The ppkm poly-P . The genphoURB, pstSACB which encodes systems for the regulation and transfer of inorganic phosphorus into the cell were also found in the genomes of Thiothrix sp. SSD2 and Thiothrix sp. RT. Along with participation in biosynthetic processes, polyphosphates are probably used for temporal ATP storage, which is realized through the activity of polyphosphate kinase PPK1 and exopolyphosphatase EPP in members of the genus Thiothrix.Thus, both enzymes, PPK1 and EPP, can regulate the accumulation and degradation of polyphosphates in the cell. Genes Thiothrix sp. RT and Thiothrix sp. SSD2 revealed the presence of a complete set of genes for the Embden–Meyerhof (EM) pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle -dependent malate dehydrogenase, which is found in the vast majority of prokaryotes. Instead, the mqo gene encoding membrane-bound FAD-dependent malate:quinone oxidoreductase was found in the genomes of representatives of the genus Thiothrix, including Thiothrix sp. RT and Thiothrix sp. SSD2.TCA cycle has a remarkable feature in the known species of the genus Thiothrix sp. RT and Thiothrix sp. SSD2 include all the genes of the EM pathway. The gene pckA for ATP-dependent phosphoenolpyruvate (PEP)-carboxykinase, which decarboxylates oxaloacetate to form pyruvate, was also revealed in the genomes of Thiothrix sp. SSD2 and Thiothrix sp. RT. The genomes contain genes for pyruvate carboxylase (pyc) and decarboxylating NADP-dependent malate dehydrogenase (maeB). The two latter enzymes likely shunt the reaction of malate conversion to oxaloacetate in the TCA cycle and glyoxylate cycle in order to ensure the production of sufficient oxaloacetate for gluconeogenesis and amino acid biosynthesis.Genomes of aspB), while the conversion of 2-oxoglutarate is carried out by glutamate synthase (glt) to glutamate.The release to constructive metabolism from the TCA cycle occurs at the level of oxaloacetate and 2-oxoglutarate. Oxaloacetate is involved in aspartate synthesis with the participation of aspartate aminotransferase , an iron-sulfur protein (sdhB), a flavoprotein subunit (sdhA), a hydrophobic membrane anchor protein (sdhD). The genes form a single sdhABCD cluster in the genome of Thiothrix sp. RT, and are located in two separate loci, sdhBCD and sdhA, in Thiothrix sp. SSD2.The succinate dehydrogenase II complex includes a cytochrome bc reductase, consists of an iron-sulfur subunit (encoded by the rpi1 gene), a cytochrome b subunit (encoded by the cytB gene), and a cytochrome c 1 subunit (encoded by the cyc1 gene).Complex III, ubiquinol-cytochrome 3 type subunits (encoded by ccoNOPGQS), six cox subunits , and four cytochrome d subunits of ubiquinol oxidase (encoded by cydABCDX). Conversion of ADP to ATP occurs via F-type ATP synthetase, encoded at the atpABCDEGH cluster, with a separate gene for the F-type ATP synthetase B subunit, atpF. The phosphates required for ATP synthetase to function are supplied by inorganic pyrophosphatase, encoded by the ppa gene, and polyphosphate kinase, encoded by ppk.Complex IV is represented by five types of oxidases: six cbbThiothrix sp. SSD2 and Thiothrix sp. RT did not reveal any important differences in the composition of genes for metabolic pathways from other representatives of the genus Thiothrix. The scheme of potential metabolic pathways of representatives of the genus Thiothrix, based on our genome-based reconstructions, is shown in Overall, genome analysis of Thiothrix sp. RT and Thiothrix sp. SSD2 and other members of the genus Thiothrix supported classification of the two new strains as new species ‘Candidatus Thiothrix moscovensis’ sp.nov. and ‘Candidatus Thiothrix singaporensis’ sp.nov., respectively.Phylogenetic analysis of Thiothrix sp. RT and Thiothrix sp. SSD2 genomes show that the studied organisms carry out the respiratory type of metabolism, and that they are facultative aerobes and facultative heterotrophs. Complete oxidation of organic substrates can occur through the TCA pathway with the formation of a transmembrane proton gradient, which is further used by ATP synthase for the synthesis of ATP. Thiothrix sp. RT and Thiothrix sp. SSD2 are potentially capable of assimilation of molecular nitrogen, anaerobic respiration on nitrates and incomplete denitrification.The results of the analysis of Thiothrix, including Thiothrix sp. RT and Thiothrix sp. SSD2, which ensure the lithotrophic growth of these organisms. A feature of thiosulfate oxidation is the presence of a branched Sox pathway (the SoxAXBYZ enzymatic complex) and the absence of soxCD genes, which leads to the oxidation of thiosulfate to sulfates and elemental sulfur.Autotrophic growth in the presence of reduced sulfur compounds  with the participation of enzymatic systems of dissimilatory sulfur metabolism is possible for representatives of the genus Thiothrix sp. RT and Thiothrix sp. SSD2, as well as other representatives of the genus Thiothrix, is the absence of the classical cytoplasmic NAD-dependent malate dehydrogenase, which is found in the vast majority of prokaryotes. Instead, the mqo gene encoding a FAD-dependent malate:quinone oxidoreductase was found in the genomes of two MAGs, as well as in the genomes of other known representatives of the genus Thiothrix. The presence of only MQO, as an enzyme capable of directly oxidizing malate to oxaloacetate in TCA and in the glyoxylate cycle, is quite rare in prokaryotes [A notable feature of karyotes . In termkaryotes ,46.Thiothrix may also be due to the fact that the samples were taken from EBPR systems. During organoheterotrophic growth, the presence of MQO in these organisms gave an advantage when growing on acetate relative to organisms with classical MDH. This capacity probably facilitated enrichment of Thiothrix species in the reactors, as well as their ability to accumulate and degrade polyphosphates as typical phosphate-accumulating microorganisms. It should be noted that the growth of a number of members of the genus Thiothrix  is preferable under chemolithoheterotrophic conditions (cell biomass increases by 2–3 times). With batch cultivation under organoheterotrophic conditions other than the EBPR system, growth is ineffective and is accompanied by a slight increase in biomass, by 20–30% (our unpublished data).The two EBPR systems analyzed used a batch reactor with alternating aerobic and anaerobic conditions with acetate as a carbon and energy source. The fact that we managed to assemble MAGs representing new species of the genus Thiothrix usually grows in nature in organic-poor hydrogen sulfide biotopes or in organic-rich wastewater, but in the presence of sulfide. In this case, bacteria use a lithoautotrophic or lithoheterotrophic type of metabolism.Thiothrix species that were not previously identified in natural biotopes and wastewater. There is now sufficient evidence that Thiothrix can participate in the removal of inorganic phosphorus [Thiothrix species, but also for creating optimal conditions for the growth of Thiothrix as phosphate accumulating organisms in the EBPR system. In particular, the addition of nitrate could probably stimulate the growth of Thiothrix in the EBPR bioreactors, since they are able to survive anaerobic conditions by switching to anaerobic respiration on nitrates. The addition of acetate to the culture medium promotes the induction of MQO activity involved in the conversion of malate to oxaloacetate. This process is vital because oxaloacetate is an important precursor of gluconeogenesis and amino acid synthesis of the aspartate branch.Periodic changes in the growth conditions realized in the EBPR system probably provided an advantage for the enrichment of new osphorus ,18,19. A


Malocclusion features differ across various populations and ethnicities. At this time, no data are available regarding the dentofacial differences between Syrian and European adolescents with Class II division 1 malocclusion, which is one of the most frequently treated pathologies in orthodontic practice. The present combined cephalometric and tooth-size study aimed to compare the dentoskeletal and tooth-size characteristics of Syrian and Hungarian adolescents with Class II division 1 malocclusion.(p < 0.01). Hungarian boys had significantly more protruded maxillae (p < 0.001) and less retruded mandibles (p < 0.01) when compared with Syrian boys, while Hungarian girls had significantly shorter mandibles relative to those of Syrian girls (p < 0.01). Syrian girls had significantly more protrusive lower incisors (p < 0.001), accompanied by significantly larger anterior tooth-size ratios when compared to Hungarian girls (p < 0.001). In conclusion, these findings underscore the importance of considering ethnic differences during orthodontic diagnosis and may have implications for optimizing orthodontic treatments in Syrian and Hungarian adolescents with Class II division 1 malocclusion.Class II division 1 malocclusion in Hungarian adolescents was a sagittal discrepancy, while in Syrian adolescents, it was a result of excessive vertical development. Syrian adolescents had a significantly excessive vertical development when compared with Hungarian adolescents, regardless of sex  Although several studies have reported ethnic differences in dentoskeletal structure and tooth-size characteristics –8, limitVarious factors cause Class II/1 malocclusion. However, conflicting results, possibly due to ethnic variations, have prevented their general characterization –11. LikeThe influence of skeletal age variations on results’ reliability has been neglected, since most Class II/1 malocclusion studies grouped patients by their chronological age , 19–23.While most studies selected patients with Class II/1 malocclusion based on their dental relationships (molar and/or incisor relationships) , 23–25, The dentoskeletal structure of individuals with Class II/1 malocclusion was analyzed during childhood , 11, aduLimited studies have included cephalometric and tooth-size measurements in the same investigation . This coDespite the evaluation of Class II/1 malocclusion in multiple studies, the following factors limit their results:This combined cephalometric and tooth-size study aimed to compare the skeletal morphology, dental position, and tooth size of skeletal age- and sex-matched Syrian and Hungarian adolescents with skeletal and dental Class II/1 malocclusion.t test for assessment. According to previous similar dentoskeletal and tooth-size studies [The sample size assumed a bilateral two-sample  studies , 27, eff studies , respect studies ; lateral studies . Age andAge, 12–17 years.Syrian ethnicity for group 1; Hungarian ethnicity for group 2.Overjet > 4 mm without upper incisor retroclination (U1/NA angle ≥ 22°).Half-unit or greater bilateral distal occlusion with permanent dentition.ANB angle > 4° with a convex facial profile.No extractions, interproximal restorations, or conditions affecting the mesio-distal teeth diameter.The inclusion criteria were as follows:The exclusion criteria were patients with history of orthodontic treatment, trauma, or craniofacial syndromes. The Human Investigation Review Board at the University of Szeged (151/2018-SZTE) approved this retrospective cross-sectional study. The participants or their parent/legal guardian provided written informed consent to participate in this study.Pretreatment lateral cephalograms were obtained for each patient in both ethnic groups using the same protocol . Magnification was corrected to 1:1 since the cephalograms were acquired with two different machines. The cephalometric measurements used herein were derived from the analyses developed by Jarabak, Steiner, and the University of Bonn –32. DefiThe teeth in both arches—from the right first permanent molar to the left first permanent molar—were measured at the largest mesio-distal dimension , to the t-tests showed no systematic error (p > 0.05). Intraclass correlation coefficients were > 0.95.To establish intra-examiner reliability, measurements of 20 randomly selected cephalograms and casts were replicated several weeks later by the same investigator. Dahlberg’s formula  showed rTo establish inter-examiner reliability, measurements of 20 randomly selected cephalograms and casts were replicated again by another investigator. Random errors were ≤ 0.46 mm and ≤ 0.48° for linear and angular cephalometric variables, respectively, and ≤ 0.33 mm for tooth-size measurements. Systematic error was absent (p > 0.05). Intraclass correlation coefficients were > 0.93.t- or Mann–Whitney U-tests, depending on data normality (according to Shapiro–Wilk’s test). For normally-distributed data, two-sample or Welch’s t-tests were used depending on equality of variance (according to F-test). The level of statistical significance was set at 0.05.Descriptive statistics of each variable were calculated using SPSS software 24.0 . Intergroup comparisons were performed using Results for sex-based comparisons between the two ethnic groups are presented in Table Results for overall comparisons between the two ethnic groups are presented in Additional file p < 0.001), while Syrian boys had significantly more retruded mandibles (SNB) (p < 0.01). Hungarian girls had significantly smaller mandibular lengths (Go-Gn) than their Syrian counterparts (p < 0.01)  than their Syrian counterparts p < 0.00, while Sp < 0.01) between Syrian and Hungarian adolescents, regardless of sex, indicating a hyperdivergent facial pattern in Syrian ethnicity  showed significant differences (p < 0.001) than Hungarian girls. These observations did not extend to the male populations  (ns Table .p < 0.001). Syrian and Hungarian boys showed no such difference , and ethnicity , and mosSkeletal comparisons showed that sagittal measurements significantly differed between the two groups—Hungarian boys had more protruded maxillae compared to Syrian boys, while Hungarian girls had significantly shorter mandibles than Syrian girls Fig. . These oMany authors have recognized the importance of excessive vertical development in the determination of mandibular position , 20, 26.The position of dentition, relative to the skeletal structure, is another important aspect of Class II/1 malocclusion. The only significant difference in the dental position found in this study was related to the lower incisors and was evident among girls. Syrian girls had more protrusive lower incisors than Hungarian girls. Status on the position of lower incisors in patients with Class II/1 malocclusion remains unclear. Most non-Caucasian studies have reported protruded lower incisors –22, 26, First, because Class II/1 malocclusion in Hungarian adolescents represents sagittal discrepancy , treatment strategies should aim to inhibit maxillary growth using extra-oral forces in Hungarian boys and enhance mandibular growth using functional appliances in Hungarian girls. Second, since Syrian adolescents had considerable vertical tendencies and most orthodontic treatment mechanics tend to open the bite, greater care should be exercised to control the vertical dimension when treating Syrian adolescents compared with Hungarian adolescents. Treatment strategies for Class II/1 Syrian adolescents should aim to alter the extent and direction of vertical facial growth and prevent posterior mandibular rotation. Finally, Class II/1 Syrian girls exhibited more protrusive lower incisors with relative tooth-size excess in the lower anterior segment, which affects treatment objectives regarding the optimal final position of these teeth. Therefore, a more interproximal reduction might be a better indication for Syrian girls than Hungarian girls, if the treatment decision is to decrease protrusion. Contrarily, protruded lower incisors, more than the standard levels, might be acceptable outcomes in Syrian girls.Class II/1 malocclusion reflects sagittal discrepancy in Hungarian adolescents , while it was caused by excessive vertical growth among Syrian adolescents, regardless of sex.Class II/1 Syrian girls have more protrusive lower incisors with a relative tooth-size excess in the lower anterior region compared with Class II/1 Hungarian girls.Class II/1 treatment strategies for Hungarian adolescents are not applicable to Syrian adolescents, because.Thus, even among patients with the same malocclusion type, dentoskeletal and tooth-size characteristics can vary with ethnicity. Hence, orthodontists should be aware of this variation to optimize their differential diagnosis and treatment planning.Although sample size estimation showed sufficient sample sizes, they were relatively small. This was the major drawback of the present study; therefore, the results should be interpreted with caution, and additional studies with larger sample sizes are warranted.Additional file 1: Table S1. Definitions of the cephalometric measurements used in this study.Additional file 2: Figure S1. Landmarks and reference lines used in this study.Additional file 3: Table S2. Overall comparison of cephalometric measurements and tooth-size ratios between the two ethnic groups.


ΔARE/+) that develops rheumatoid arthritis and Crohn’s disease. 5XFAD/TNFΔARE/+ mice showed significantly decreased amyloid deposition. Interestingly, microglia but not astrocytes were activated in 5XFAD/ TNFΔARE/+ brains. This microglial activation was associated with increased infiltrating peripheral leukocytes and perivascular macrophages and synaptic degeneration. APP levels and APP processing enzymes involved in Aβ production remained unchanged, suggesting that the reduced amyloid burden can be attributed to the increased microglial and perivascular macrophage activation caused by TNF-α. Peripheral TNF-α levels were increased while brain TNF-α remained the same. These data provide further evidence for peripheral TNF-α as a mediator of inflammation between the periphery and the brain.Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, involved in Alzheimer’s disease pathogenesis. Anti-TNF-α therapeutic approaches currently used in autoimmune diseases have been proposed as a therapeutic strategy in AD. We have previously examined the role of TNF-α and anti-TNF-α drugs in AD, using 5XFAD mice, and we have found a significant role for peripheral TNF-α in brain inflammation. Here we investigated the role of mouse TNF-α on the AD-like phenotype of 5XFAD mice using a knock-in mouse with deletion of the 3’UTR of the endogenous TNF-α (TNF Although beta-amyloid (Αβ) is traditionally accepted as the main initiator of the disease, numerous failed clinical trials targeting Αβ2 have changed research focus on other factors involved in the disease as brain inflammation, commonly called neuroinflammation4. Moreover, clinical and experimental studies have provided significant evidence that peripheral inflammation may be associated with increased neuroinflammation and accelerated AD progression. A number of pro-inflammatory molecules including TNF-α, have been implicated in AD pathogenetic process5. TNF-α has been shown to colocalize with amyloid plaques in AD human brains and animal models7 and elevated TNF-α levels have been detected in the serum and cerebrospinal fluid (CSF) of AD patients9. TNF-α is a pleiotropic pro-inflammatory cytokine that mediates its effect through two different receptors, TNF-α receptor I (p55) and TNF-α receptor II (p75)10. TNF-α central role in the pathogenesis of chronic autoimmune diseases like rheumatoid arthritis (RA), psoriasis and Crohn’s disease14 has led to the development of anti-TNF-α therapeutics widely used in the treatment of autoimmune diseases16. Case studies have reported that treatment of AD patients with the anti-TNF-α agents currently used in RA or Crohn’s disease, was beneficial and improved cognitive impairment19 while epidemiological studies showed that AD risk is reduced in RA patients receiving anti-TNF-α agents20.Alzheimer’s disease (AD) is the most common cause of dementia among the elderly characterized by severe memory loss and cognitive impairment22. 5XFAD mice, carrying a 3’-UTR modified human TNF-α (hu TNF-α) transgene23 (5XFAD/Tg197) that results in increased peripheral expression of huTNF-α protein, develop an AD-like phenotype along with TNF-induced inflammatory arthritis, which can be suppressed by peripheral treatment with infliximab, a monoclonal anti-huTNF-α antibody widely used for RA treatment in patients24. 5XFAD/Tg197 mice exhibit robust brain inflammation, with extensive microglial and astrocytic activation and increased meningeal and perivascular macrophage activation that compromises neuronal integrity and synaptic health, despite reduced amyloid deposition. Interestingly, these alterations in the 5XFAD/Tg197 brains, correlated with increased levels of peripheral human TNF-α, suggesting that peripheral TNF-α alone is able to modulate brain inflammation through a TNF-α dependent periphery-to-brain communication pathway.We have previously shown that peripheral human TNF-α and anti-TNF-α therapy, currently used in RA treatment, play a significant role in modulating neuroinflammation, amyloid deposition and neuronal degeneration in AD miceΔARE/+ mouse model and generated 5XFAD/TNFΔARE/+ mice. In TNFΔARE/+ mice deletion of the 3’-UTR of the endogenous mouse gene (muTNF-α) results in increased peripheral expression of the muTNF-α and the development of autoimmune inflammatory disease25. Analysis of 5XFAD/TNFΔARE/+ mice revealed increased peripheral muTNF-α levels, reduced amyloid deposition, activated microglia and increased perivascular macrophages and infiltrating leukocytes. 5XFAD/TNFΔARE/+ brains showed increased synaptic degeneration. Interestingly astrocytes were not activated, while APP processing enzymes remained unchanged. Our results provide further evidence for an association between systemic peripheral inflammation and AD pathogenesis and suggest peripheral TNF-α as a central link.To further elucidate the role of the TNF signaling pathway and evaluate the effect of the modulation of endogenous mouse TNF-α gene (muTNF-α), in the current study, we employed the TNFΔARE/+ mice has been shown to significantly increase muTNF-α protein levels in the periphery25. To examine the effect of the modification of the endogenous muTNF-α gene and upregulation of muTNF-α levels in the 5XFAD/TNFΔARE/+ mice, we measured muTNF-α protein levels in the serum and the brain of the mice using an anti-muTNF-α specific ELISA. Our analysis detected significantly elevated muTNF-α protein levels in the sera of 5XFAD/TNFΔARE/+ mice (>200 pg/mL) while muTNF-α was not detected in 5XFAD sera 25 to generate 5XFAD/TNFΔARE/+ mice. In this study we used heterozygous TNFΔARE/+ mice, as homozygous (TNFΔARE/ΔARE) develop a wasting phenotype and die early27. 5XFAD littermates were used as controls. We used only female mice for analysis, as male 5XFAD transgenic mice develop the amyloid phenotype with a two-month delay compared to females28.To evaluate the effects on the amyloid phenotype of AD mice, from the genetic modification of the endogenous muTNF-α gene, we used the 5XFAD transgenic miceΔARE/+ and 5XFAD 4-month-old mice. Confocal microscope image analysis revealed a significant reduction in Thioflavine-S positive amyloid plaque burden in the hippocampus and the cortex of 4-month-old 5XFAD/TNFΔARE/+ mice compared to the 5XFAD , Nicastrin and Aph1. 5ΧFAD/TNFΔARE/+ and 5XFAD total brain protein extracts were analyzed by Western blot analysis using specific antibodies to detect the above APP processing proteins. Since TNF-α is the main substrate of TACE, we examined whether muTNF-α gene modification can affect TACE protein levels in 5XFAD/TNFΔARE/+ mice. Our analysis confirmed that TACE protein levels remained the same between the two groups  PS1 between the two groups  and synapsin in brain sections from 5ΧFAD/TNFΔARE/+ and 5XFAD control mice. Confocal analysis confirmed extensive loss of MAP2 and synapsin in 5ΧFAD/TNFΔARE/+ mice compared 5XFAD control mice  that results in elevated mouse TNF-α levels in the blood and development of RA and Crohn’s disease25. Analysis of the 5XFAD/TNFΔARE/+ mice showed that the genetic modification of the TNF-α endogenous gene protects against the amyloid phenotype in the mouse brain. 5XFAD/TNFΔARE/+ brains exhibit significantly decreased amyloid deposition and increased microglial activation. Perivascular macrophages and infiltrating leukocytes are also increased. Astrocytic activation in the brain is not affected and APP processing enzymes remain in the same levels compared to 5XFAD mice. Interestingly, peripheral TNF-α levels are increased in the 5XFAD/TNFΔARE/+ mice compared to 5XFAD, while brain TNF-α levels are not affected.In the present study, we investigated the role of TNF-α in the development of the amyloid phenotype in an AD mouse model by modulating the 3′UTR of the endogenous mouse gene. We employed a widely used AD mouse model, the 5XFAD6. RA is a chronic autoimmune disease driven by TNF-α13. RA patients receiving anti-TNF-α drugs show reduced risk of developing AD20, raising questions about the role of RA and/or anti-TNF-α therapy in AD. Despite numerous studies focusing on the therapeutic role of TNF-α in AD and the potential protective effects of anti-TNF drugs, the underlying mechanisms are still unknown46. In this study, analysis of 4-month-old 5XFAD/TNFΔARE/+ mice that develop an AD-like phenotype, revealed a significant decrease of Thioflavine-S positive plaques and Αβ deposition in mouse brains compared to the 5XFAD controls, suggesting that the modification of the 3′UTR of the muTNF-α endogenous gene, exerts a protective effect against amyloid deposition in the brain. This result is similar to our previous study22, where 5XFAD mice carrying a human TNF-α transgene with a modified 3′UTR23 developed reduced amyloid deposits and robust brain inflammation. In both studies, alteration of the 3′UTR of the TNF-α gene, resulted in decreased amyloid plaque formation in the brain. Interestingly the same result, is observed by blocking the TNF-α signaling pathway either by genetically eliminating TNF-α or the p55 TNF-α receptor in AD mice48. Genetic deletion of TNF-α is protective against amyloid plaque formation and Αβ production in AD mice, by decreasing amyloidogenic APP enzymatic processing and Aβ production47. Deletion of the p55 receptor in AD mice also reduces amyloid plaque deposition by decreasing Aβ production48, whereas genetic deletion of the p75 receptor exacerbates amyloid deposition49 suggesting a complex role for TNF-α signaling pathway in AD pathogenesis. To elucidate the mechanism involved in the reduction of amyloid deposits in 5XFAD/TNFΔARE/+ mice, we first looked for differences in APP protein levels and the APP processing enzymes involved in Aβ production between 5XFAD/TNFΔARE/+ and control 5XFAD brains. Our analysis did not show any differences in APP protein levels or the APP processing enzymes between the two groups. BACE1, which is the β-secretase involved in Aβ generation50 as well as TACE, and ADAM10, two of the α-secretases that cleave APP54, did not show any significant changes in their protein levels in the mouse brains. Previous studies, including ours48, have shown that genetic deletion of TNF-α or the TNF-α receptors’, p55 and p75, can modulate α- and β-secretase protein levels in AD mice, affecting Aβ production and amyloid plaque deposition51. Since TNF-α has been implicated in APP cleavage by modulating PS1, a component of the γ-secretase complex, through the JNK-dependent pathway30 we also looked for differences in protein levels of PS1, as well as Nicastrin and Aph1, proteins of the γ-secretase complex, in the brains of 5XFAD/TNFΔARE/+ mice. Our analysis for PS1, Nicastrin and Aph1 showed no difference in protein levels between 5XFAD/TNFΔARE/+ and control brains. Overall, our analysis of the APP processing enzymes in the 5XFAD/TNFΔARE/+ mice provide evidence that modification of the 3’UTR of the TNF-α gene does not affect APP processing or Aβ production in the brain.TNF-α is a major pro-inflammatory cytokine that has been identified as a key molecule involved in autoimmune diseases as RA and neurodegenerative diseases as AD26, we examined the glial response in the 5XFAD/ TNFΔARE/+ brains. Our analysis revealed that modification of the 3′UTR of the TNF-α mouse gene results in a robust increase in microglial activation with a small increase in phagocytic and autophagy markers as CD68 and LC3 in the 5XFAD/TNFΔARE/+ brains. To further analyze this result, we examined brains of TNFΔARE/+ mice where we also found a robust increase of microglial activation, which suggests that modification of the 3′UTR of the muTNF-α gene is responsible for the microglial activation in the 5XFAD/TNFΔARE/+ brains. This finding is similar to our previous study21 where transgenic mice expressing a human TNF-α transgene with a modified 3′UTR also showed highly activated microglia with increased phagocytic capacity. Our data, together with other studies where genetic elimination of the TNF-α gene or the p55 receptor gene, or pharmacological inhibition of TNF-α suppresses microglial activation48, suggest a key role for TNF-α in modulating microglial response. Next, we analyzed astrocytic activation in the 5XFAD/TNFΔARE/+ and 5XFAD control brains and, we found no differences between the two groups. Further analysis between TNFΔARE/+ and control brains confirmed this finding suggesting that modification of the 3′UTR of the muTNF-α gene does not affect the activation status of the astrocytes in the physiological or the AD brain context. Similarly, to this finding, previous studies in mice using LPS induction and inhibition of the TNF-α signaling with Etanercept in the CNS, have confirm specific activation of microglia and not astrocytes55. These findings suggest that increased levels of muTNF-α either from acute (LPS)55 or chronic induction (expression of muTNF-α in TNFΔARE mice) might not be sufficient or capable to activate astrocytes. Interestingly, this is in contrast to our previous study where modification of the 3′UTR of a human TNF-α transgene resulted in robust astrocytic response in the mouse brain21. The increased astrocytic activation observed in our previous study, might be related to the activation level of microglia56 as in the huTNF-α expressing brains21 microglia is highly activated throughout the brain, in comparison to the muTNF-α expressing TNFΔARE brains, where microglial activation was milder. These data imply for a possible differential activation of the two glial cell types, microglia and astrocytes, by TNF-α and further studies are needed to investigate this effect. Overall, our results suggest for a central role of microglia in restricting the amyloid deposits in the mouse brains.Microglia and astrocytes are the immune cell populations of the brain involved in Aβ clearance. Since 5XFAD mice display increased microglial and astrocytic activation associated with amyloid depositionΔARE/+ mutant mice25. To evaluate this effect in 5XFAD/TNFΔARE/+ mice we measured TNF-α levels in the serum of mice as well as in brain protein extracts. Our analysis revealed a significant increase in TNF-α levels only in the periphery of 5XFAD/TNFΔARE/+ mice compared to 5XFAD controls, in similar levels to our previous study21 (~200 pg/ml) while brain TNF-α remained the same. This result suggests that, similarly to our previous study21, modification of the 3′UTR of the TNF-α gene, can result in significantly increased TNF-α levels in the periphery, while brain levels remains unchanged. To further evaluate whether this increase in peripheral TNF-α can affect bone marrow derived cell populations involved in Αβ clearance41, as brain infiltrating leukocytes and perivascular and meningeal macrophages, we looked for CD45 and CD206 positive cells in the 5XFAD/TNFΔARE/+ brains. Our analysis revealed a significant increase of CD45 positive leukocytes in the brain as well as CD206-positive meningeal/perivascular macrophages in the 5XFAD/TNFΔARE/+ mouse brains compared to the 5XFAD. A previous study by D′ Mello et al., has supported the central role of TNF-α during peripheral inflammation in orchestrating the periphery to brain communication pathway32. CD45 positive leukocytes that infiltrate the brain, have been identified as a key cell population involved in this pathway together with activated microglia. The abovementioned study together with our previous21 and current study support CD45 leukocytes as a mediator and potent regulator, between the peripheral immune system and the brain’s innate immune cell type, microglia. Furthermore, CD206 perivascular macrophages, that are known to participate in Aβ clearance41 are also activated in the mouse brains. Our analysis support CD45 and CD206 positive cells as the mediators of the effects of peripheral TNF-a into the 5XFAD/TNFΔARE/+ mouse brains.Modification of the 3′UTR of the TNF-α gene results in significant increase in the levels of peripheral TNF-α in the TNFΔARE/+ mice display a clear reduction for both. Several lines of evidence have suggested that TNF-α regulates neuronal and synaptic health in the brain42. Neuronal TNF-α expression in AD mice results in neuronal death and extensive microglial activation57 and TNF-induced neuronal and synaptic loss is mediated by microglial phagocytosis59. These findings suggest for microglial activation resulting from peripheral TNF-α as a significant modulator of neuronal and synaptic integrity.Our analysis for MAP2 and synapsin, cell markers that reveal neuronal and synaptic health, shows that 5XFAD/TNF60. Transgenic mice that carry modified TNF-α or TNF-α receptors genes have helped to better comprehend the role of TNF-α in the AD pathogenetic mechanisms. These studies have been sometimes conflicting and not easy to interpret, suggesting a complex effect of TNF-α on the parameters involved in AD pathogenesis. We and others have found that inactivating the TNF-α signaling pathway by genetically deleting the TNF-α gene or the TNF-α receptor p55 gene results in decreased amyloid deposition in the mouse brain48. On the contrary genetic deletion of the TNF-α receptor p75 gene or both receptors p55 and p75 results in increased amyloid deposition suggesting a complex mechanism involving the TNF-α signaling pathway60. In our previous study overexpression of a huTNF-α modified transgene in AD/arthritic mice (5XFAD/Tg197) has resulted in a brain phenotype with exacerbated neuroinflammation and reduced amyloid deposition21. Surprisingly this brain phenotype could be modified by regulating peripheral huTNF-α levels with the use of an anti-TNF-α therapeutic antibody for RA, administered at the periphery, thus confirming the key role of peripheral TNF-α. To further investigate the role of muTNF-α in the AD pathogenetic mechanism in the 5XFAD mice, in the current study, we employed a TNF-α knock-in mouse, the TNFΔARE/+, where the effects observed on the AD-like phenotype are mediated by muTNF-α. These effects mediated by the mouse TNF-α differ from the previously characterized effects of a human TNF-α transgene21. Specifically, the brain inflammatory phenotype in 5XFAD/TNFΔARE/+ mice is milder with less activated microglial cells and lacks astrocytic activation compared to the 5XFAD/Tg197. Yet, in both studies a significant reduction in amyloid plaque formation is observed that coincides with activated microglia and synaptic degeneration, suggesting a central role for microglia in modulating the AD-like phenotype. A possible explanation for this difference in the glial activation between the two studies could be that in the 5XFAD/Tg197 brains, where mice express the human TNF-a, the mouse p75 receptor is unable to bind the human TNF-α61. Therefore, in 5XFAD/Tg197 brains, huTNF-α binds only p55 and mediates its effects through this receptor only, while in 5XFAD/TNFΔARE/+ brains, muTNF-α binds both p55 and p75 receptors. Previous studies involving traumatic brain injury, have suggested for a protective role for p75 in brain health, while the role p55 has been detrimental62. Further supporting the protective role of p75 in the brain, Etanercept, a fusion protein of p75, used in RA treatment, has shown promising results in AD treatment20. Finally in agreement with our previous study21, in 5XFAD/TNFΔARE/+ brains, CD45 and CD206 positive cell activation remains strong supporting the role of these bone-marrow derived cell populations in mediating the effect of peripheral TNF-α.We and others have shown that interfering with the TNF-α signaling pathway can modify the AD-like phenotype in the brain of AD mouse modelsOur current and previous studies dissociate the human and the mouse TNF, in modulating neuroinflammation, in an AD/TNF transgenic mouse system. Our findings suggest for a potent protective role of the p75 TNF receptor in modulating brain inflammation and point out the TNF pathway as a major therapeutic target in AD therapeutics. More elaborate studies that involve genetic or pharmaceutical manipulation of the p75 TNF receptor in AD/TNF transgenic mice will help to further elucidate the underlying mechanism.26 were purchased from The Jackson Laboratory  and were on a C57Bl6/J genetic background. TNFΔARE/+ mice27 were kindly provided by Dr. G.Kollias  and were on a C57BL/6 J genetic background. Both 5XFAD and TNFΔARE/+ transgenic lines were maintained as hemizygotes. For the purposes of this study, 5XFAD hemizygous mice were crossed with TNFΔARE/+ mice to generate 5XFAD/TNFΔARE/+ double transgenic mice along with 5XFAD, TNFΔARE/+ and wild-type non-transgenic littermates used as controls. Only female mice were used in the analysis, as 5XFAD females develop the amyloid phenotype 2 months earlier than 5XFAD males28. Mouse genotyping was performed with polymerase chain reaction. All mice were housed in the SPF facility of the Biomedical Research Foundation and maintained on a standard chow diet containing 5% fat . All animal procedures were approved by the Bioethical Committee of the Biomedical Research Foundation and were in agreement with ethical recommendations of the European Communities Council Directive (86/609/EEC). The approved procedures for animal care and treatment were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the recommendations of Federation of European Laboratory Animal Science Association (FELASA).5XFAD mice63 After euthanasia, the brains were removed and cut along the sagittal midline. Left hemibrains were snap-frozen for protein analysis. Right hemibrains were immersion-fixed in PBS-buffered 4% paraformaldehyde (Sigma Aldrich) for 48 hours and then cryoprotected in 20% sucrose in PBS for histological analysis.Mice were anesthetized and transcardially perfused with ice-cold PBS as previously described63. Tissues were homogenized in ice-cold PBS, containing protease inhibitors  with a Tissue homogenizer (Wheaton). The homogenate was centrifuged at 12500 rpm  for 45 min at 4 °C. The supernatant (PBS fraction) was removed and used to evaluate Iba1 and CD206 protein levels. The pellets were resuspended in ice-cold lysis buffer  and centrifuged at 9000 rpm for 10 min at 4 °C. The supernatant (lysis fraction) was removed and used to evaluate APP/CTFs, BACE1, ADAM10, TACE, PS1/CTF, Nicastrin, Aph1, synapsin and GFAP protein levels.Protein extraction from hemibrains was carried out in sequential steps as previously described63. Specifically, protein concentrations of all samples were determined using the BCA Protein Assay Kit  according to the manufacturer’s protocol. Equal amounts of total protein from hemibrains were separated on SDS-PAGE 8–16% Tris-Glycine gels (or 12% Tris-Tricine gels for APP/CTFs and Iba1) and electrophoretically transferred to nitrocellulose membrane . For the detection of PS1/CTF and APP/CTFs, proteins were transferred to PVDF membrane . Membranes were cut in two pieces, one piece, containing protein extracts of molecular weight approximately around 50 kDa, was processed separately for tubulin, and the other half was incubated separately with each of the below mentioned primary antibodies. Only for the detection of GFAP that has a molecular weight close to tubulin (50 kDa GFAP-55kDa tubulin), membranes were first processed for detection of GFAP and then stripped  and re-incubated for tubulin. Specifically, membranes were blocked with 3–5% non-fat milk in TBS/Tween-20 0.05–0,1% and then incubated with specific primary antibodies: mouse monoclonal anti-BACE1 , anti-PS1 C-terminal , anti-GFAP  and anti-tubulin , rat anti-CD206 , goat polyclonal anti-TACE  and anti-Nicastrin  and rabbit polyclonal anti-APP C-terminal , anti-ADAM10 , anti-Aph-1 , anti-synapsin , anti-Iba1  and anti-LC3  Membranes were then incubated with the corresponding HRP-conjugated secondary antibody  and developed using enhanced chemiluminescence. Densitometric analysis was performed using the NIH Image J software.Analysis was performed as previously described64. Optical densities at 450 nm of each well were read on a microplate reader , and sample TNF-α concentrations were determined by comparison with the respective standard curves using the Gen5 software (BioTek Instruments). Brain sample values were normalized to total brain protein concentrations determined using the BCA Protein Assay Kit. The results were calculated as mean ± SEM for each genotype.For muTNF-α quantitation by ELISA, serum and PBS brain homogenates were diluted with the ELISA sample buffer and sample duplicates were run on specific sandwich colorimetric ELISAs (Biolegend) according to the manufacturer’s protocol as previously described63. Specifically, fixed hemibrains were cut in 40μm sagittal free-floating sections from the genu of the corpus callosum to the most caudal hippocampus using a vibratome . For the quantitation of amyloid plaque load, sections were incubated for 9 minutes in 1% w/v Thioflavine-S (Sigma Aldrich) aqueous solution and then differentiated 2 times with 80% ethanol for 3 min each, followed by another 3 min wash with 95% ethanol. Sections were rinsed three times with ddH2O and coverslipped with mounting medium for fluorescence . Imaging for Thioflavine-S was performed on a Leica TCS SP5 confocal microscope and image captivation was performed using the Leica LAS AF Suite. Images for Thioflavine-S load quantitation were captured using HCImage software (Hamamatsu) in a Leica DMRA 2 microscope.Analysis was performed as previously described63. Specifically, 40μm sagittal free-floating sections were permealized with TBS/Triton X-100 0.1% 3 times for 10 min each, incubated for 30 min with 0.6% H2O2 in TBS and washed 3 times 5 min each with TBS. Antigen retrieval was performed with 98% formic acid (AppliChem) for 5 min, followed by 3 washes with TBS. Sections were blocked in 15% normal goat serum (Vector Laboratories) in TBS/Triton X-100 0.1% for 1 hour and incubated overnight at 4οC with the mouse monoclonal biotinylated 6E10 antibody  in 5% normal goat serum (Vector Laboratories) in TBS/Triton X-100 0.1%. Sections were then washed 4 times with TBS plus one final wash with PBS, and incubation in avidin-biotinylated horseradish peroxidase complex  followed for 120 min at room temperature. Peroxidase labeling was visualized with DAB/Ni . After a 1–2 min incubation period, sections were washed with ddH2O, mounted on polylysine-coated slides (Thermo Fisher Scientific), dehydrated in increasing ethanol concentrations from 50 to 100% followed by xylene (Sigma Aldrich) and coverslipped with DPX mounting medium (AppliChem). Imaging was performed with a DM LS2 Leica microscope, and image capture was carried out using the Leica Application Suite (version V4.6).Analysis was performed as previously described63. For immunofluorescent labeling, 40μm sagittal free-floating sections were first subjected to three 5 min washes with PBS and antigen retrieval with 10 mM sodium citrate buffer pH 6, for 30 min at 80 °C. The sections were washed again with PBS, then blocked for 1 h in 10% FBS (Thermo Fisher Scientific), 1% BSA (Sigma Aldrich) in PBS/Triton X-100 0.3% and incubated overnight at 4 °C with rat anti-CD206 , rabbit anti-Iba1 , rabbit anti-smooth muscle actin (α-SMA) , rabbit anti-synapsin , rat anti-CD45  and rat anti-CD68  antibodies in 1% FBS, 1% BSA in PBS/Triton X-100 0.3%. Sections were washed with PBS and developed with anti-rabbit Cy3-conjugated , anti-rat Alexa-633-conjugated  and anti-mouse Alexa-546-conjugated  secondary antibodies for 1 h at room temperature. Where needed, sections were treated with 1% Thioflavine-S aqueous solution for 5 min, differentiated twice in 70% ethanol and washed in PBS. For GFAP and Thioflavine-S double labeling, sections were blocked for 1 h in TBS/Triton X-100 0.4% supplemented with 5% normal goat serum and immunolabeled with anti-GFAP  antibody in the blocking solution overnight at 4 °C. Sections were washed with TBS, developed with an anti-mouse Alexa-546-conjugated secondary antibody  for 1 h at room temperature and then processed for Thioflavine-S staining. For MAP-2 immunofluorescent detection, sections were permeabilized for 1 h in PBS/Triton X-100 0.3%, blocked for 1 h in 10% FBS, 1% BSA in PBS/Triton X-100 0.3%, and incubated overnight at 4 °C with a rabbit anti-MAP-2  antibody in 1% FBS, 1% BSA in PBS/Triton X-100 0.3%. Sections were washed with PBS and processed with an anti-rabbit Cy3-conjugated  secondary antibody for 1 h at room temperature. All sections were finally mounted on polylysine-coated slides and covered with mounting medium (Vectashield). Imaging was performed on a Leica TCS SP5 confocal microscope and images were captured using the Leica LAS AF Suite.Analysis was performed as previously described63. For Thioflavine-S load quantitation, six 40μm sagittal hippocampal sections 240μm were chosen. Images were analyzed with the NIH Image J software in an 8-bit grayscale format. Figures were generated using adobe. photoshop. elements 12 Version 12.1 (20140303.12.1.49334) software. Hippocampal or cortical area was outlined manually, and the surrounding area was cleared. Thioflavine-S load was expressed as the percent area covered by Thioflavine-S positive plaques (% Thioflavine-S)63. For the quantitation of cortical CD45high leukocytes, Three 40μm sagittal sections 240μm apart from each other per brain (n = 3) were immunostained for CD45 and 20x magnification image stacks were captured on a Leica TCS SP5 confocal microscope. Cell counting was performed using the Cell Counter Image J plugin as previously described21. For the quantitation of microtubule-associated protein 2 (MAP2) immunoreactivity, six 40 μm sagittal sections 240 μm apart from each other per mouse brain were immunostained and 40x magnification image stacks were captured on a Leica TCS SP5 confocal microscope. The percentage area covered by MAP2-positive dendrites was measured (% of dendritic MAP2)21. All the obtained measurements per section were calculated as the mean value for each mouse brain and used for statistical analysis. Statistical analyses were performed using GraphPad Prism  as previously described63. All data were analyzed using two-tailed unpaired t-test and expressed as mean ± SEM. Statistical significance was defined as P < 0.05.All confocal microscopy images were taken using the Leica Application Suite, Advanced Fluorescence Lite, 263 build 8173 software (Leica Microsystems). All images were analyzed using the Image J software (NIH) as previously describedSupplementary information.