Surprisingly, TNF‐α prevented the differentiation of TRAcP+ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31 Excessive release of inflammatory cytokines is associated with chronic inflammation and bone destruction. Tumor necrosis factor α (TNF‐α) is one of the most prominent inflammatory cytokines, and therefore, has been targeted in therapies against bone diseases Bingham, .Osteoclasts, the multinucleated bone‐resorbing cells, are crucial in bone diseases with excessive bone loss. Severe bone destruction occurs when the equilibrium of osteoclast and osteoblast activity is disturbed. Osteoclasts arise from monocytic precursors under the influence of M‐CSF and RANKL. Next to these cytokines, TNF‐α has been shown to stimulate osteoclast generation and bone resorption both in vitro  and TNF‐R2 (p75) , myeloid blasts (CD31+ Ly‐6C+), and monocytes (CD31− Ly‐6Chi) . Femur and tibia from both legs were removed, followed by grinding in a mortar and cells were suspended in 10 ml culture medium. Culture medium was α‐MEM  supplemented with 5% Fetal Clone I Serum  and 1% penicillin‐streptomycin‐fungizone . The cell suspension was aspirated through a 21 gauge needle and filtered through 40 µm filter. A total of 4 × 102.2hi Ly‐6C−), myeloid blasts (CD31+ Ly‐6C+), and monocytes (CD31− Ly‐6Chi) were sorted with FACSAria (Becton Dickinson).Bone marrow cells were labeled with CD31 and Ly‐6C following the labeling procedure described previously , with or without 20 ng/ml RANKL , with 0, 10, or 100 ng/ml TNF‐α (R&D Systems), with or without 10 ng/ml IL‐1β (Sigma–Aldrich), with or without 10 ng/ml IL‐6 (Sigma–Aldrich). Cells were cultured on plastic or on 650 μm‐thick bone slices and culture media were refreshed every 3 days. After 5 days the cultures on plastic plates were stopped by fixation in 4% PBS‐buffered formaldehyde for TRAcP analysis, or lysed in RNA lysis buffer  at day 0, 3, and 5 for RNA isolation. Bone slices were stored in water after 6 days of culture and culture media was collected for CTX assay.The three sorted subsets, early blasts, myeloid blasts, and monocytes were seeded in 96‐well plastic plates  at a density of 1.5 × 102.4+ multinucleated cells by using a commercially available Leucocyte acid phosphatase kit (Sigma–Aldrich). The staining procedure was performed following the manufacturer's instructions. Nuclei were counterstained by 4′6‐diamidino‐2‐phenylindole (DAPI). The TRAcP+ multinucleated cells were categorized as cells containing 3–10, and >10 nuclei, and the number of cells in each category was counted using a combination of light and fluorescence microscopy .Osteoclasts were identified as TRAcP2.5In order to evaluate bone resorption, formation of resorption pits on bone slices was analyzed by Coomassie brilliant blue staining. After 6 days of culture, bone slices were washed with water and the cells were removed by sonication for 30 min in 10% ammonia  on ice. To reduce staining background the bone surface was incubated in saturated alum for 10 min and subsequently washed with water. Resorption pits were stained with Coomassie brilliant blue  and visualized by light microscopy (Leica DFC320). In addition, CTX concentrations of the culture supernatants were determined using CrossLaps® for culture ELISA . Culture supernatants were collected after 6 days and CTX assay was conducted following the manufacturer's instructions.2.6−ΔCt, ΔCt = C gene of interest  −Ct PBGD. The results were shown as fold increased, normalized by the same gene expression at day 0.The procedure of RT‐PCR was described in detail in a previous paper . To analyze the capacity of cells to take up RANKL, cells were incubated with TAMRA conjugated‐recombinant murine‐RANKL for 30 min. TAMRA labeled (rm)RANKL stock was prepared by mixing 5 mg 5(6)‐TAMRA, SE  with 10 μg (rm)RANKL (R&D Systems) and incubated for 2 hr at room temperature. Purification was conducted by ultimate 3000 HPLC (Thermo Scientific and RPC‐8‐Vaydac column [GRACE]), and authenticity was confirmed by mass spectrometry MALDI‐TOF Microflex . For RANK expression, nonspecific binding was blocked by incubation with a combination of TruStain fcXTM Antibody (Fc receptors block)  in PBS supplemented with 1% BSA for 10 min. Subsequently, staining was performed using 1:100 diluted PE‐conjugated anti‐mouse CD265 (RANK) antibody (BioLegend) and PE‐conjugated IgG2a, ĸ Isotype Ctrl antibody (BioLegend) in PBS supplemented with 1% BSA for 30 min on ice. Cells were subsequently fixed with 1% formaldehyde and analyzed by FACS (BD Accuri™ C6 Flow Cytometer). Data were analyzed by CFlow® Plus software (Version 1.0.202.1).2.8n = 6) and GraphPad Prism  was used for the statistical analysis. One‐way ANOVA followed by Tukey–Kramer's multiple comparison was used for multiple comparisons, and t‐test was used for two comparisons. Data are displayed as mean ± SD and p < 0.05 was considered as significant difference.All data were analyzed from six mice (33.1hi Ly‐6C−), myeloid blasts (CD31+ Ly‐6C+), and monocytes (CD31− Ly‐6Chi) were isolated according to the expression of CD31 and Ly‐6C from the whole population  and cultures in the presence of TNF‐α (condition 2) . This indicates that RANKL is essential for the TNF‐α induced osteoclast formation by monocytes.Next, we analyzed whether priming with M‐CSF alone (without RANKL) was sufficient to have such a TNF‐α induced effect on osteoclastogenesis. The three subsets were cultured with M‐CSF for 3 days before adding TNF‐α. Under these conditions only few TRAcP3.2+ cells were present in monocyte cultures . With these two cell lineages, no inhibitory effect was found in any of the conditions .Next to TNF‐α also two other inflammatory cytokines, IL‐1β and IL‐6, have been shown to stimulate osteoclastogenesis. We wondered whether IL‐1β or IL‐6 might influence the effect of TNF‐α as shown above. In the presence of IL‐1β or IL‐6, high numbers of TRAcPs Figure a. IL‐6 as Figure b. Monocys Figure . Howevers Figure . This wa3.3+ mononuclear and multinucleated cells . No significant differences were seen for any of the tested genes in early blast cultures . In myeloid blasts an up‐regulation of TNF‐αR2 was seen . At the onset of the culture period monocytes showed a significantly higher expression of TNF‐αR1 as well as TNF‐αR2 than the other two subsets . All three subsets expressed comparable RANK at the start of the culture period .3.6low) showed on the left hand side of Figure high), seen on the right hand side of the graph  than the RANKLlow sub‐population (34 ± 6%). In the group with TNF‐α, this pattern was reversed. The percentage of RANKLhigh sub‐population was much lower (37 ± 4%) than that of RANKLlow sub‐population (63 ± 4%). Thus, TNF‐α decreased the binding of fluorescently labeled RANKL; an effect particularly apparent in monocytes.In order to assess whether TNF‐α could interfere with binding of RANKL to RANK, FACS analysis was used to investigate the interaction of RANKL with the membrane‐bound receptor RANK. Monocytes and myeloid blasts were cultured for 3 days with M‐CSF and RANKL, with or without TNF‐α before labeling. The expression of RANK did not show a significant difference with or without TNF‐α in any of the cultures (data not shown). By adding TAMRA‐conjugated RANKL, we were able to analyze the binding of RANKL to its receptor RANK on the plasma membrane. TAMRA‐conjugated RANKL labeling of myeloid blasts was not influenced by the presence of TNF‐α Figure A. In monh Figure b. In the4− Ly‐6Chi). Our results indicate that TNF‐α interferes with the binding of fluorescently labeled RANKL in monocytes. This likely results in the impairment of the RANK‐induced signaling pathway for osteoclastogenesis. However, this inhibitory effect can be prevented in different ways. First, by the presence of M‐CSF and RANKL prior to the addition of TNF‐α. Second, by seeding monocytes on bone. These phenomena were not found with early blast cultures nor with myeloid blast cultures. Our findings indicate a multifunctional role of TNF‐α on osteoclastogenesis of a particular subset of osteoclast precursors, the monocytes.In the present study we found that TNF‐α had the capacity to prevent osteoclastogenesis of one specific precursor subset: bone marrow derived monocytes  condition we found a decreased mRNA level of RANK by the monocytes, and an increased population of monocytes with a lower level of fluorescently labeled RANKL. The protein level of RANK proved to be unaffected. Thus, it is likely that the RANKL/RANK signaling pathway was affected. Our findings indicate that TNF‐α can express both inhibitory and stimulatory effects on monocytes’ osteoclastogenesis. A hypothetical model that offers an explanation for our findings is shown in Figure When monocytes are cultured with M‐CSF and RANKL, RANKL will bind to RANK expressed on the membrane of monocytes, leading to up‐regulation of osteoclast‐related genes, such as NFATc1. This will result in osteoclast differentiation Figure a. Howeve4.2Clearly the relationship between TNF‐α and RANKL in osteoclastogenesis is complex. Kobayashi et al.  showed t+ multinucleated cells were formed . This indicates that M‐CSF alone is not sufficient; a RANKL exposure is needed for the TNF‐α‐ induced osteoclastogenesis. To assess if TNF‐α can substitute RANKL in osteoclastogenesis, we also cultured the three osteoclast precursor subsets with M‐CSF and TNF‐α . Interestingly, high number of TRAcP+ mononuclear cells were formed in early blast and myeloid blast cultures , but only a few of them were multinucleated . Almost no TRAcP+ cells were present in monocyte cultures . This shows that TNF‐α can partly substitute RANKL in early blasts’ and myeloid blasts’ osteoclastogenesis, but not with monocytes.In order to prove that TNF‐α is closely associated with RANK/RANKL interaction, we cultured the three osteoclast precursor subsets with only M‐CSF for 3 days before adding TNF‐α together with M‐CSF and RANKL. Under these conditions only very few TRAcP4.3The inhibitory effect of TNF‐α on osteoclastogenesis by monocytes was prevented not only with an M‐CSF and RANKL pre‐incubation, but also by seeding monocytes on bone slices. Under these conditions even higher numbers of osteoclasts were formed with TNF‐α. The data indicate that attachment of the monocytes to the bone surface changes the sensitivity of these cells to TNF‐α. Although we do not have an explanation for such an effect, several authors have shown that an interaction of osteoclast precursors with bone greatly affects release of compounds like IL‐1 . This is in line with our previous study in which we showed higher numbers of TRAcP4.4This study showed that from all the different bone marrow derived osteoclast precursors, only the monocyte‐derived osteoclast formation could be abolished by TNF‐α, a phenomenon that does not apply to early blast‐ or myeloid blast‐ derived osteoclast formation. A previous study already addressed the differences among the three subsets in osteoclast differentiation (De Vries et al., Is there a physiological explanation for a cell type that becomes osteoclastogenesis‐insensitive to a cytokine like TNF‐α? Under pathological conditions the cytokine is present throughout the body and this could in theory result in the formation of osteoclasts anywhere in the body. After all TNF‐α as well as the monocytes are present at many different sites, and RANKL is also present in serum (Findlay & Atkins, Additional Supporting Information may be found online in the supporting information tab for this article.Supporting Figures S1.Click here for additional data file.


Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT  and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR  analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. Summary: The study indicated that parecoxib may be a potential therapeutic approach to treat GBM by up-regulating miRNA-29c and subsequently inhibiting proliferation, migration and invasion of GBM. Glioblastoma (GBM) is a grade IV glioma classified by the World Health Organization (WHO), which is one of the most lethal and aggressive brain cancers, and accounts for 15% of brain malignancies . The typFrequently, overexpression of cyclooxygenase-2 (COX-2) had been found in several types of tumor, including breast cancer  and glioMicroRNAs (miRNAs) are a class of 21-25 nucleotide small noncoding RNAs that post-transcriptionally downregulate expression of various genes via binding to the 3′ untranslated region (UTR) of mRNA of the target gene, leading to translational suppression or mRNA cleavage . AccumulTo investigate the anticancer role of parecoxib in GBM, we treated GBM cells with parecoxib and detected cell proliferation, migration and invasion. The results suggested that treatment with parecoxib decreases the cell proliferation, migration and invasion ability of GBM cells in a dose-dependent manner. Further studies identified that miRNA-29c was significantly induced by parecoxib, and addition of a miRNA-29c inhibitor can significantly attenuate parecoxib's chemotherapeutic efficacy. These finding suggest that parecoxib treatment can inhibit GBM cell proliferation, migration and invasion via upregulating miRNA-29c.To investigate the effect of parecoxib on the cell growth of GBM cells, U251 and U343 cells were exposed to the increased parecoxib concentrations  for 24 and 48 h. The results of MTT assays showed that the growth rate of U251 and U343 cells was significantly decreased in a dose-dependent manner A,B. MoreTo investigate the effect of parecoxib on the migration ability of GBM cells, U343 cells were treated with parecoxib for 24 and 48 h, followed by the scratch wound healing motility assay. The results showed that U343 cells treated with 200 μM of parecoxib for 24 and 48 h migrated significantly slower than PBS-treated control cells A,B, suggThe effect of parecoxib on the invasive ability of U343 cells was further investigated by using a Transwell invasion assay. Compared with the control, the number of invasive GBM cells incubated with 100 μM and 200 μM parecoxib was decreased significantly A,B, indimiRNA-29c is an important tumor suppressor miRNA, which is downregulated in GBM  and can miRNA-29c expression was induced by parecoxib, indicating that miRNA-29c may mediate the chemotherapeutic efficacy of parecoxib. We thus investigated the effect of miRNA-29c on parecoxib-inhibited cell proliferation. The specific miRNA-29c mimics, miRNA-29c inhibitors or scramble miRNA were transiently transfected into U343 cells for 24 h, followed by treatment with parecoxib for another 24 h. RT-qPCR analysis showed that the expression of miRNA-29c was significantly increased and reduced after transfection of miRNA-29c mimics and inhibitors, respectively, in comparison with scramble miRNA A. The MTThe effects of miRNA-29c on parecoxib inhibited migration were also determined by using a scratch wound-healing motility assay in U343 cells. Although the migratory abilities of U343 cells in the miRNA-29c mimics+parecoxib group were almost the same as the NC miRNA+parecoxib group, the migratory abilities in the miRNA-29c inhibitors+parecoxib group were significantly lower than the NC miRNA+parecoxib group A,B.Fig.The effects of miRNA-29c on parecoxib-inhibited invasive ability of U343 cells were further assessed using a Transwell invasion assay. The invasion ability of U343 cells in the miRNA-29c inhibitors group was slightly increased by 21% compared to the NC miRNA group. Moreover, the invasion ability of GBM cells in the miRNA-29c inhibitors+parecoxib group was significantly increased by 26% compared to NC miRNA+parecoxib group A,B. ThesIn the present study, we found that parecoxib can inhibit cell proliferation, migration and invasion of GBM cells, and identified that miRNA-29c is an important miRNA that is potentially implicated in the inhibitory effect of parecoxib on GBM cell proliferation, migration and invasion.Cyclooxygenase-2 (COX-2) had been found to be involved in tumorigenesis, and COX-2 inhibitors exhibit a potent anticancer effect . As the miRNA-29c had been found to be significantly downregulated in GBM tissues and cells , and forIn conclusion, parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. Future studies are required to investigate the roles of parecoxib in GBM in animal models, and further elucidate the mechanisms underlying the anti-tumor effect of parecoxib, which will aid in developing therapeutic strategies for GBM.2 and 95% air. Parecoxib sodium  was diluted at concentration of 50 mM, and further diluted in DMEM to obtain the final concentration.Human GBM cell lines including U251 and U343 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences . The GBM cells were cultured in Dulbecco's Modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS) , 100 U/ml penicillin and 100 μg/ml streptomycin , and maintained in a humidified cell incubator at 37°C with 5% CO100 nM of the scramble miRNA (NC miRNA), miRNA-29c mimics and inhibitors  were transfected into U251 or U343 cells with 70% confluences by using Lipofectamin™ 2000  according to manufacturer's instructions. The oligonucleotide sequences of NC miRNA, miRNA-29c mimics and inhibitors are: NC miRNA: 5′-UCACAACCUCCUAGAAAGAGUAGA-3′ (sense), and 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′ (antisense); miRNA-29c mimics: 5′-UAGCACCAUUUGAAAUCGGUUA-3′ (sense) and 5′-UAACCGAUUUCAAAUGGUGCUA-3′ (antisense); miRNA-29c inhibitors: 5′-UAACCGAUUUCAAAUGGUGCUA-3′.After treatment, the cell growth of U251 and U343 cells plated in 96-well plates was detected by using 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the cells were treated with MTT and incubated at 37°C for 4 h. Supernatant was removed and the dye was dissolved with 100 μl of DMSO and shaken on an orbital shaker for 10 min in the dark. The optical density (OD) was recorded at 570 nm using a spectrophotometer . Each experiment was performed in triplicate.DNA synthesis in proliferating U251 and U343 cells was determined by using 5-Bromo-2-deoxyUridine (BrdU) incorporation assay. 10 μM of BrdU  was added to the cells for 1 h. Cells were washed with PBS and fixed for 20 min in 4% paraformaldehyde (PFA). After blocking with 10% goat serum for 1 h, cells were incubated with peroxidase-coupled anti-BrdU-antibody  overnight at 4°C, washed three times with PBS, subsequently incubated with peroxidase substrate (tetramethylbenzidine) for 1 min at room temperature. The absorbance at 450 nm was determined with a spectrophotometer. Each experiment was performed in triplicate.−ΔΔCt method. All primers were purchased from GeneCopoeia . All experiments were conducted in triplicate.Total RNA was extracted from U251 and U343 cells with TRIzol reagent , and 100 ng total RNA was subsequently reverse transcribed into cDNA by using TaqMan MicroRNA Reverse Transcription Kit  and mRNA Reverse Transcription Kit  according to the manufacturer's protocol. RT-qPCR analysis was performed in the 7500 RT-qPCR system (Applied Biosystems) using the SYBR Green PCR Master Mix (Applied Biosystems) under the following conditions: DNA denaturation at 95°C for 10 min, followed by 40 cycles of 95°C, 15 s for denaturation, 60°C, 60 s for annealing and extension. Small nuclear U6 snRNA (Applied Biosystems) was used as the endogenous control to miRNA-29c. The RNA levels were determined using the 22 for 24 and 48 h, and the same wound areas were observed and photographed under an inverted microscope . The distance of the scratch closure was examined at 0, 24 and 48 h.After treatment, U343 cells in six-well plates were scratched with a sterile 10 μl pipette tip three times to form parallel lines, subsequently washed by PBS for removal of non-adherent cells. The six-well plates were then incubated at 37°C, 5% CO4. Simultaneously, 400 μl of DMEM medium with 10% FBS was placed in the lower chamber in a 24-well cultural plate. After cells were cultured for 24 h at 37°C in 5% CO2, non-invading cells were removed with cotton swabs, and invaded cells on the bottom surface of filter were fixed in 100% methanol and stained with 0.1% Crystal Violet. Subsequently, the stained cells in the filter were photographed and counted under an inverted microscope at ×400 magnification from 10 random fields. Assays were performed in triplicate.The transwell assays were performed in 24-well chambers  to detect the effect of miRNA-29c mimics and inhibitors on the invasion of U343 cells. After transfection with miRNA-29c mimics or inhibitors, U343 cells were trypsinized, washed and resuspended in DMEM medium with 1% FBS, and seeded into the upper chamber of the wells with the density of 1×10t-test or one-way ANOVA analysis were used to compare differences between groups. P-values <0.05 were considered statistically significant.All statistical analyses were carried out by using SPSS 18.0 software (IBM). Results are expressed as mean±s.d. from the experiments that repeated at least three times, Student's


Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production.In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy.Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain.rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction . Furthermore, T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.Our data implicate TNF as a promoter of The online version of this article (doi:10.1186/s12974-017-0952-0) contains supplementary material, which is available to authorized users. Tnfr1 mRNA and nearly undetectable Tnfr2 mRNA, neither of which are upregulated after exposure to interferon gamma (IFNγ) or TNF [Trypanosoma brucei, and malaria, caused by Plasmodium sp. [Trypanosoma cruzi infection is unclear. This intracellular protozoan parasite is the etiologic agent of Chagas disease, a neglected tropical disease characterized by a systemic inflammatory response enriched in TNF in chronic patients [Astrocytes are heterogeneous cells that are involved in maintaining the homeostatic environment in the central nervous system (CNS) . They ar) or TNF . TNF and) or TNF , 12. Incdium sp. –15. Indedium sp. . Howeverpatients , 18 and patients .T. cruzi infection, particularly in infants and susceptible experimental mice, intense meningoencephalitis and accumulation of amastigote forms of the parasite inside glial cells are common, but CNS inflammation and parasitism are drastically reduced in the chronic phase [T. cruzi infection, neurological involvement is observed in 75–90% of human immunodeficiency virus (HIV)-coinfected patients and in individuals immunocompromised by cancer, suppressive treatment, organ transplantation, or malnourishment [T. cruzi-infected C3H/He mice undergoing immunosuppression, reactivation of infection with numerous and large parasite nests and inflammation is restricted to the CNS and occurs in the absence of parasitemia, supporting the existence of low-grade parasite persistence in CNS-resident cells [T. cruzi persistence in the CNS cells remain unclear. Recently, we showed that in chronically infected C3H/He mice, most T. cruzi-bearing astrocytes are located near interferon gamma (IFNγ)+ Iba1+ microglial cells, in the absence of neuroinflammation [T. cruzi infection is increased by IFNγ, and the anti-TNF antibody infliximab abolished this IFNγ-induced effect. Therefore, IFNγ-stimulated infected astrocytes are potential sources of TNF, which may facilitate astrocyte infection [T. cruzi infection triggers TNF production and to explore the biological effects of TNF on parasite/astrocyte interaction regarding the T. cruzi cycle  and cytokine profile. Furthermore, we treated T. cruzi-infected mice with anti-TNF antibody to investigate whether TNF indeed affects CNS parasitism.In the acute phase of ic phase –29. Neverishment , 31. In rishment , 24, 30.rishment –24. In crishment ; or incrnt cells . Neverthb) and C3H/He (H-2K) mice, as described previously [2 culture flask pre-coated with poly-ornithine  and maintained in DMEM/F-12 medium  containing 10% fetal bovine serum  at 37 °C in a 95% humid atmosphere with 5% CO2. Microglia and other non-adherent cells were removed by gently shaking the culture flasks. Astrocytes from 7 to 10 days of culture were detached with 0.25% trypsin +0.02% EDTA in phosphate-buffered saline (PBS), pH 7.2–7.4, and plated at a density of 105 cells per 13-mm-diameter poly-ornithine-coated coverslip in 24-well plates . Cells were cultured for 24 h to allow adhesion to coverslips. Details of each experiment are provided in the figure legends.For in vitro experiments, primary mouse astrocyte cell cultures were obtained from the cerebral cortex of 1-day-old C57BL/6   and FITC-conjugated anti-mouse immunoglobulin secondary antibody . Microglia were revealed by surface CD11b expression using PECy7-conjugated anti-mouse CD11b monoclonal antibody . After cultivation, cells that adhered to coverslips were washed with warm PBS and fixed with methanol. To block nonspecific binding, the cells were incubated for 30 min with normal goat serum diluted 1:50 in 0.1% sodium azide/PBS and then stained for GFAP and CD11b. Negative controls were performed by omitting the primary antibodies. RAW 264.7 mouse macrophage cells  cultured in parallel served as a positive control for CD11b expression . The cov2. Cells were detached with 0.25% trypsin +0.02% EDTA in PBS, washed, plated at a density of 5 × 104 cells per 13-mm-diameter coverslip in 24-well plates , and incubated for 24 h to allow adhesion. Cultures were then washed with warm PBS, and the medium was replaced. To assess the effects of TNF in cells other than astrocytes, the fibroblast cultures were either left untreated or were treated with TNF (1 ng/mL) for 2 h and then infected as described for astrocytes.The fibroblast cell line L-929 , originated from a C3H/An mouse, was kindly provided by Dr. Maria de Nazaré Correia Soeiro . Cells were cultivated in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin  and maintained at 37 °C in a 95% humid atmosphere with 5% CO2. Cell culture dissociation was performed by replacing medium by 0.25% trypsin +0.02% EDTA in PBS. Cells were plated at a density of 105 cells per 13-mm-diameter poly-ornithine-coated coverslips in 24-well plates . Details of each experiment are provided in the figure legends.Human U-87 MG glioblastoma cells were purchased from Banco de Células do Rio de Janeiro . Cells were cultivated in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin  and maintained at 37 °C in a 95% humid atmosphere with 5% COT. cruzi strain [2 cultivation bottles  using RPMI-1640/HEPES . After 5 to 7 days in culture, the trypomastigote-containing supernatants were centrifuged ; the parasites concentrated in the pellet were then resuspended, counted in a Neubauer chamber, and added to astrocyte or fibroblast cultures. Depending on the experimental design, we used a multiplicity of infection (MOI) of 1:1 or 10:1 (parasite/cell) and interaction periods of 4 or 24 h.Trypomastigote forms of the Colombian Type I i strain  were obtT. cruzi infection of astrocytes in vitro, astrocytes were plated at a density of 105 cells per 13-mm-diameter poly-ornithine-coated coverslip in 24-well plates  and allowed to adhere for 24 h. Cultures were then washed three times with warm PBS to remove non-adherent cells, and fresh culture medium supplemented with FBS was added. Cultures of U-87 MG human astrocytes or murine primary astrocytes were left untreated or were pre-treated with human or murine recombinant IFNγ  or TNF  for different time periods depending on the experimental design . The numbers of non-infected and T. cruzi-infected astrocytes (300 cells/coverslip) and of intracellular amastigote forms per infected cell were counted. Representative images were acquired on a light microscope coupled with a digital camera DS-L3 .To investigate the effects of IFNγ and TNF on the To evaluate the role of rIFNγ-induced TNF in astrocyte infection, cell cultures were treated with the anti-TNF chimeric monoclonal antibody infliximab  30 min before the addition of murine or human rIFNγ, as described in the experimental design , a phosphodiesterase inhibitor, has previously been shown to suppress n injury  and TNFRnfection . To explAstrocyte cell viability was observed by the reduction of MTT -2,5-diphenyltretrazolium bromide; Sigma-Aldrich, Steinheim, Germany). Viable cells convert the yellow, water-soluble tetrazolium salt to insoluble purple formazan. After 5 min, formazan was solubilized with dimethyl sulfoxide , and the optical density was evaluated at 490 nm using a microplate reader .TNF concentrations in cell culture supernatants and mouse sera were evaluated using an ELISA kit for the detection of mouse TNF  according to the manufacturer’s protocol, and the colorimetric reaction was assessed on an ELISA plate reader at 490 nm . Cytokines  were detected in astrocyte culture supernatants using the commercial cytometric bead array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit  and Human Inflammatory Cytokines Kit  according to the manufacturer’s instructions. The fluorescence produced by the beads was measured on a FACSCalibur flow cytometer  and analyzed using the kit’s FCAP Array software. In the ELISA and CBA tests, standard curves (1 pg/mL to 100 ng/mL) were generated in parallel. These methods consistently detected concentrations above 10 pg/mL.Tnf mRNA expression in brain tissue was assessed and analyzed as previously described [2, and 10 mM DTT. The mixtures were incubated for 5 min at 95 °C, 5 min on ice, and 5 min at 25 °C. At this step, 100 U of RT was added to each sample, and the reaction mixture was incubated for 60 min at 37 °C. The temperature was then elevated to 95 °C for 5 min, and the tubes were transferred to ice for 5 min. The cDNA products were diluted in 0.2 mL of sterile distilled water and used at 5 μL per reaction for PCR amplifications. The PCR was performed in 25-μL reactions with samples diluted in the following buffer: 250 mM dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 10 mM of each primer, and 0.5 U of Taq polymerase . After an initial incubation for 3 min at 95 °C, the cycling conditions were denaturation for 1 min at 94 °C, annealing for 1 min at 54 °C, and extension for 2 min at 72 °C. After the designated cycle numbers for each primer, the program executed a final extension of 7 min at 72 °C. The PCR products and molecular weight markers were separated on 6% polyacrylamide gels and stained with silver nitrate. Densitometry was carried out on a CS-9301PC Densitometer . The PCRs were standardized using hypoxanthine-guanine phosphoribosyl transferase (HPRT). The same primer sequences and PCR conditions indicated previously were here used for detection of Tnf and Hprt mRNA [escribed . Brieflyprt mRNA . Fold-in6 astrocytes were cultivated in a 6-well plate , infected with T. cruzi (MOI 1:1), and cultivated as described above. After 4 h of culture, astrocytes were washed with warm PBS to remove debris, harvested by gentle scraping with a rubber policeman, and frozen in RNAlater . Total RNA was extracted using TRI-Reagent , after the complete removal of RNAlater. All reverse transcriptase reactions were performed using the SuperScript VILO cDNA Synthesis Kit , and real-time RT-qPCR was performed on a Real-Time PCR System  using TaqMan gene expression kits  for Tnf (# Mm00443258_m1) and Tnfr1 (#Mm00441883_g1), as well as the endogenous housekeeping control genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (# Mm99999915_g1) and β actin (# Mm00607939_s1), purchased from Life Technologies . The reactions were performed and analyzed as described previously [For real-time quantitative RT-PCR (RT-qPCR), 10eviously .K) mice obtained from the animal facilities of the Oswaldo Cruz Foundation  were housed under specific pathogen-free conditions with a 12-h light/dark cycle and ad libitum access to food and water. Mice were intraperitoneally infected with 100 blood trypomastigotes of the Colombian Type I T. cruzi strain obtained by passages through C3H/He every 5 weeks. This infection model has been shown to develop self-resolving acute meningoencephalitis [T. cruzi-infected mice and three to five non-infected controls per experiment, in three independent experiments , when the first parasites were detected in peripheral blood, the mice were subcutaneously treated with injection-grade saline  containing 10 μg of anti-TNF antibody , at 48-h intervals over 14 days Fig. . Inflixited mice .T. cruzi antibody  and FITC-labeled goat anti-rabbit secondary antibody . Negative-control brain tissue sections were generated by omitting the primary antibody. Ten sections were analyzed for each studied encephalon. Images were acquired on a fluorescence microscope equipped with a digital camera Eclipse CI  and analyzed with the Nis-Elements BR 4.0 software . Areas containing parasites were identified and counted. Data are expressed as T. cruzi-positive areas per 100 microscopic fields.Groups of non-infected and infected mice, with or without anti-TNF treatment, were sacrificed under anesthesia , at 30 dpi. The brains were removed, embedded in tissue-freezing medium , and stored in liquid nitrogen. Sagittal brain tissue serial cryostat sections (8 μm thick) were fixed in cold acetone and subjected to immunohistochemical staining. Parasite antigens were revealed by staining with polyclonal rabbit anti-t test to compare two groups. Comparisons between groups were carried out by analysis of variance (ANOVA) followed by Bonferroni post hoc test. All statistical tests were performed with GraphPad Prism 5.0 . Differences were considered statistically significant when p < 0.05.Data are expressed as arithmetic means ± standard error (SE). For statistical analyses, we used Student’s + cells (≥ 99%), while CD11b+ cells (< 1%) were rarely detected  and TNF levels (p < 0.001) were detected after 4 h of T. cruzi/astrocyte interaction  and untreated T. cruzi-infected astrocytes (p < 0.05), TNF production was upregulated in rIFNγ-treated T. cruzi-infected astrocytes  than for untreated astrocytes  or 10:1 (p < 0.05) , but had no impact when the MOI was 10:1 (p = 0.339), when a high frequency (75–80%) of infected cells was already observed in untreated astrocytes  Fig. . TNF addtes Fig. .T. cruzi/astrocyte interaction was a common biological process in other cell types, L-929 mouse fibroblasts were pre-treated with TNF and then infected with T. cruzi , regardless of the MOI (1:1 or 10:1) and time (4 or 24 h) of interaction  and frequencies of T. cruzi-infected astrocytes (p < 0.05) were dependent on the initial MOI  the number of amastigote-like forms inside astrocytes  by the addition of the anti-TNF antibody infliximab prior to rIFNγ treatment  the mean number of parasites inside U-87MG astrocytes compared with that of untreated astrocytes . Comparing the mean number of parasites inside astrocytes at 8 and 24 h of interaction revealed that the proliferation ratio of amastigote forms inside rTNF-treated astrocytes (α = 1.8) was almost threefold higher than that inside untreated astrocytes (α = 0.62). At 8 and 24 h of infection, compared with their respective untreated control astrocytes, the frequencies of infected astrocytes were also augmented in rTNF-treated astrocytes (p < 0.001). At 48 h of infection, the frequency of infected astrocytes was drastically reduced when cells were pre-treated with rTNF, suggesting the rupture of astrocytes harboring a high number of parasites . At these time-points, higher parasite loads were also detected when rTNF was added prior to and sustained after infection than for the corresponding controls . However, when rTNF was added after T. cruzi infection, no effect  on the parasite load was seen compared with the corresponding untreated astrocytes  the frequency of astrocytes harboring a higher number of amastigote-like forms. At 48 h of interaction, compared with the corresponding untreated control astrocytes, both experimental conditions using rTNF prior to infection enhanced the frequency of astrocytes harboring > 6 amastigotes/astrocyte (p < 0.05); however, no change was detected when rTNF was added after infection . At 8 and 24 h of infection, no trypomastigotes were detected in any of the cell culture supernatants. At 48 h of infection, compared with the corresponding untreated control astrocytes, a significant increase in the egression of trypomastigote forms was detected when rTNF was added prior to and removed after infection or was added prior to and sustained after infection , but no significant effect (p = 0.431) was detected when rTNF was added after infection  compared with non-infected astrocytes. Treating non-infected astrocytes with rTNF also enhanced  IL-6 concentrations in the culture supernatants , rTNF-treated non-infected , and T. cruzi-infected astrocytes , although a synergistic effect of rTNF and T. cruzi infection was not detected. By contrast, under all experimental conditions, the regulatory cytokine IL-10 was barely detected and was comparable to non-infected controls , rTNF-treated (p < 0.05), or rTNF-primed and infected (p = 0.063) human U-87MG astrocytes. Under these conditions, IL-10 was not detected . Interestingly, a significant decay (p < 0.001) in TNF concentrations in the supernatants of rTNF-treated T. cruzi-infected astrocytes compared with rTNF-treated non-infected astrocytes was observed at 24 h, suggesting consumption of the added TNF  Tnfr1 mRNA expression . PTX treatment also partially inhibited  the rTNF-fueled augmentation of the frequency of T. cruzi-infected astrocytes  Colombian-infected C3H/He mice. At 28 dpi, increased brain Tnf mRNA expression (p < 0.01) and high serum TNF concentrations (p < 0.001) were detected in infected mice compared with those in non-infected controls  acutely infected C3H/He mice , at 30 dpi, ice Fig. . Initialice Fig. .T. cruzi-infected astrocytes and is upregulated by IFNγ stimulation prior to infection; (ii) TNF priming fuels astrocyte infection, accelerates amastigote multiplication and trypomastigote egression; (iii) TNF-primed infected astrocytes create a TNF- and IL-6-enriched inflammatory milieu; and (iv) TNF/TNFR1 signaling may fuel parasite infection. Furthermore, treating infected mice with TNF antibody revealed that TNF facilitates T. cruzi parasitism in the CNS, reinforcing the biological relevance of our in vitro findings.In the present study, we show that (i) TNF is produced by T. cruzi infection, corroborating previous data using astrocytes from C3H/He mice [T. cruzi infection in vitro is preserved in different mouse genetic backgrounds and among species. In Chagas disease patients and experimental models, astrocytes are preferred targets for T. cruzi invasion and proliferation in the CNS, regardless of the host species and parasite strain [We first showed that primary astrocyte cultures from C57BL/6 mice and the human U-87 MG lineage are targets of /He mice  and the /He mice . Therefoe strain , 29, 44.T. cruzi infection by producing TNF, which is upregulated by rIFN stimulation prior to T. cruzi infection. In experimental African trypanosomiasis (sleeping sickness), Tnf transcripts were detected concomitant with astrocyte activation, indicating that these glial cells may be sources of TNF [Tnf mRNA was detected by in situ PCR in astrocytes [T. cruzi/astrocyte interaction. Interestingly, the anti-TNF blocking antibody infliximab hampered the IFNγ-induced increase in human astrocyte susceptibility to T. cruzi infection, supporting the role of TNF in this process. Indeed, pre-treating murine and human astrocytes with TNF fuels T. cruzi invasion, revealing a conserved biological activity of TNF. However, this biological process is not common to all cell types because pre-treating fibroblasts with TNF did not facilitate T. cruzi infection.Mouse and human astrocytes respond to s of TNF . Additiotrocytes , indicattrocytes , 18, 47.trocytes , 48. TheT. cruzi depends on when astrocytes experience this cytokine. In fact, this outcome was restricted to treatment prior to infection, but not when TNF was added after infection. Interestingly, treating T. cruzi-infected astrocytes with TNF had no effect on parasite growth control. Previously, TNF has been shown to facilitate the invasion of non-professional phagocytic epithelial cell lines by T. cruzi via NF-kB activation [T. cruzi infection, transforming growth factor β has been shown to enhance T. cruzi invasion and replication in epithelial cells [T. cruzi to complete the cycle allowing egression of trypomastigote forms has not been previously studied. Our data showed that TNF increases intracellular parasite growth and accelerates trypomastigote egression when added to astrocytes prior to infection but not after infection. Therefore, depending on the timing of exposure, TNF favors the complete T. cruzi cycle in the primary CNS-resident cell. The possibility that TNF acts on the expression of cell surface molecules or other inflammatory mediators that drive the invasion of and egression from astrocytes by T. cruzi cannot be excluded. Indeed, we recently showed that nitric oxide may also contribute to IFNγ-driven astrocyte invasion by T. cruzi [The ability of TNF to fuel astrocyte invasion by tivation . Interestivation . Converstivation , 51. Regal cells  and primal cells . HoweverT. cruzi .Toxoplasma gondii create a regulatory milieu with high IL-10 concentrations that downmodulate the inflammatory profile of IFNγ-activated microglia and impair neuronal death [T. cruzi infection and were upregulated by TNF stimulation. Therefore, in the presence of T. cruzi infection, astrocytes likely generate an inflammatory milieu in the CNS. Nevertheless, whether the cytokines released by CNS-resident cells in response to T. cruzi infection are beneficial or detrimental remains unclear. In vivo, the production of pro-inflammatory cytokines such as IL-6 and TNF by CNS-resident cells may help clear debris, repair and regenerate nervous tissue. However, prolonged exposure to high levels of these cytokines may worsen or accelerate tissue dysfunction and degenerative processes [T. cruzi-infected mice present systemically elevated levels of IFNγ and TNF [+ Iba-1+ glial cells surrounding GFAP+T. cruzi-bearing astrocytes [T. cruzi infection, systemic TNF has emerged as a key cytokine in the pathogenesis of cardiomyopathy and the dysregulation of the immune response in the spleen and bone marrow [T. cruzi infection, systemically disturbed IFNγ and TNF production may create a TNF-enriched environment in the CNS that may fuel parasitism but also create a deleterious circuit, leading to behavioral alterations. In line with this idea, systemic treatment with anti-TNF antibody or with the TNFR1 modulator PTX ameliorated depressive-like behavior in chronically T. cruzi-infected mice [T. cruzi infection.Astrocytes infected by al death . By controcesses . IL-6 isrocesses . In experocesses . In chag and TNF , 40, 47 trocytes . In chroe marrow . In T. cted mice , suggestT. cruzi, we analyzed TNFR1 expression on these glial cells. TNF failed to upregulate Tnfr1 transcription, corroborating previous data [Tnfr1 mRNA expression was detected in the presence of both TNF and T. cruzi infection, accompanied by increased consumption of TNF. Thus, astrocytes may facilitate T. cruzi persistence in the CNS by inducing TNF production and, in conjunction, these signals may upregulate TNFR1 expression. Indeed, pre-treating astrocytes with PTX reduced the TNF-induced augmentation of astrocyte invasion by T. cruzi. PTX has previously been shown to reduce TNF [T. cruzi infection in astrocytes triggers TNF production, which upregulates TNF and TNFR1 expression, creating a feedback circuit that drives the inflammatory milieu and contributes to parasite persistence in the CNS.To shed light on the mechanism by which TNF fuels astrocyte infection by ous data . Howeverduce TNF –59 and Tduce TNF , 36 exprT. cruzi infection in C3H/He mice [Tnf mRNA expression and high serum TNF concentrations in acutely Colombian-infected C3H/He mice. Therefore, we tested the idea that in vivo TNF fuels T. cruzi parasitism in the CNS. Indeed, anti-TNF antibody treatment reduced the number of T. cruzi-positive areas in the brain. In chronically T. cruzi-infected mice, anti-TNF antibody treatment downregulated systemic TNF and TNFR1 expression [Tnf transcription in the CNS and produce cognitive dysfunction [T. cruzi-infected mice. High systemic TNF concentrations are also detected in chronically T. cruzi-infected mice [T. cruzi-infected individuals.Intense CNS parasitism and inflammation by blood-borne leukocytes are detected in the acute phase of /He mice , 27. Herpression  and abropression . Interesfunction . Neverthted mice , 40. ThuT. cruzi parasitism after anti-TNF treatment allowed an approximation with in vitro data, supporting a role for TNF in astrocyte invasion by T. cruzi, directly translating our findings to the pathophysiology of the CNS commitment in Chagas disease is undoubtedly the crucial limitation of our study.In vitro models are reductionist approaches and are therefore limited in their ability to reproduce the complexity of the network of interactions occurring in the CNS. Although our in vivo result showing reduced CNS T. cruzi infection of astrocytes may fuel parasite infection of these glial cells via TNF/TNFR1 signaling, thereby perpetuating the parasite cycle in the CNS.The TNF-enriched inflammatory milieu created by Additional File 1: Figure S1.5 cells per 13-mm-diameter coverslip and analyzed 24 h later.  Giemsa staining reveals cells resembling astrocytes with thin extensions, delicate branches, recognizable nucleoli and cytoplasm lightly stained.  Simultaneous immunohistochemical staining for GFAP and CD11b was used to determine the cell composition of primary astrocyte cultures. (e) Insert showing RAW 264.7 mouse macrophage lineage used as positive control for CD11b staining. (TIFF 1266 kb)Primary cell cultures of C57BL/6 cerebral cortex are enriched in astrocytes. CNS cells of seven to ten days of primary culture were seeded at a density of 10Additional File 2: Figure S2.Trypanosoma cruzi. (a) Monotypic astrocyte cell cultures were allowed to interact during 4 or 24 h with Vero cells-born trypomastigote forms of the Colombian T. cruzi strain, at a MOI of 1:1 or 10:1. (b-e) Astrocyte infected with T. cruzi at a MOI of 1:1 and analyzed after 4 h  or 24 h (e) of interaction. (f) Parasites per 100 cells and frequency of T. cruzi-infected astrocytes after 4 and 24 h of T. cruzi/astrocyte interaction at a MOI of 1:1 or 10:1 Data are presented as means ± SEM of triplicates. *, p < 0.05 and **, p < 0.01 MOI 1:1 vs. MOI 10:1 in each analyzed interval; #, p < 0.05 and ##, p < 0.01 4 h vs. 24 h of infection in each analyzed MOI. (TIFF 1349 kb)Astrocytes from neonatal C57BL/6 mice are susceptible to in vitro infection by Additional File 3: Figure S3.5 cells per 13-mm-diameter coverslip, 24 h later washed with warm PBS and left untreated (NT), treated with rTNF  or toxic agents (HCl pH 4.0 or DMSO). After 24 h of exposure, cells were submitted to MTT assay. Data are presented as means ± SEM of triplicates. ***, p < 0.001 untreated (NT) vs. astrocytes submitted to other experimental conditions. (TIFF 881 kb)Addition of rTNF to astrocyte cultures had no cytotoxic effects on astrocytes. C57BL/6 primary astrocyte cultures were seeded at a density of 10Additional File 4: Figure S4.Trypanosoma cruzi. (a) Experimental scheme showing that L-929 fibroblasts were pre-treated with TNF (1 ng/mL) and subsequently infected with trypomastigote forms of the Colombian T. cruzi strain. (b-c) The percentage of infected cells and the number of parasites per cells were analyzed at 4 h (b) and 24 h (c) of infection, at a MOI of 1:1 or 10:1. Data are presented as mean ± SEM of duplicates. (TIFF 1430 kb)Pre-treatment of fibroblast with rTNF does not affect infection by Additional File 5: Figure S5.Trypanosoma cruzi-infected astrocytes. C57BL/6 primary astrocyte cultures were seeded, exposed to rTNF and infected as described in Fig. *, p < 0.05, **, p < 0.01 untreated (NT) vs. rTNF-treated astrocytes. (TIFF 1045 kb)Pre-exposing astrocyte cultures to rTNF enhances parasite load in Additional File 6: Figure S6.Trypanosoma cruzi infection. Human astrocytes were submitted to treatment with rTNF and left non-infected (NI) or infected by T. cruzi (MOI 1:1). At 4 h of infection, supernatants were collected and submitted to detection of cytokines by CBA. Data are presented as means ± SEM of triplicates. *, p < 0.05, untreated (NT) vs. rTNF-treated astrocytes. (TIFF 1375 kb)rTNF amplifies the pro-inflammatory profile of human astrocytes exposed to Additional File 7: Figure S7.Trypanosoma cruzi/astrocyte interaction are conserved in primary astrocyte cultures of C3H/He mice. (a) Monotypic primary astrocyte cell cultures of C3H/He mice were seeded, left untreated or treated with rTNF and infected with trypomastigote forms of the Colombian T. cruzi strain at a MOI of 1:1 or 10:1. (b) After 4 h of interaction, the frequencies of infected cells and the number in untreated (NT), rTNF-treated astrocytes are shown. (c) Data show the frequencies of infected astrocytes stratified by classes according to the number of amastigote-like forms harbored in the cytoplasm. Data are presented as means ± SEM of triplicates. *, p < 0.05, ***, p < 0.001 untreated (NT) vs. rTNF-treated astrocytes. #, p < 0.05, ##, p < 0.01, ###, p < 0.001 MOI 1:1 vs. MOI 10:1. (TIFF 1337 kb)rTNF effects on


Coxiella burnetii is a globally distributed zoonotic bacterial pathogen that causes abortions in ruminant livestock. In humans, an influenza-like illness results with the potential for hospitalization, chronic infection, abortion, and fatal endocarditis. Ruminant livestock, particularly small ruminants, are hypothesized to be the primary transmission source to humans. A recent Netherlands outbreak from 2007–2010 traced to dairy goats resulted in over 4,100 human cases with estimated costs of more than 300 million euros. Smaller human Q fever outbreaks of small ruminant origin have occurred in the United States, and characterizing shedding is important to understand the risk of future outbreaks. In this study, we assessed bacterial shedding and seroprevalence in 100 sheep from an Idaho location associated with a 1984 human Q fever outbreak. We observed 5% seropositivity, which was not significantly different from the national average of 2.7% for the U.S. (P>0.05). Furthermore, C. burnetii was not detected by quantitative PCR from placentas, vaginal swabs, or fecal samples. Specifically, a three-target quantitative PCR of placenta identified 0.0% shedding . While presence of seropositive individuals demonstrates some historical C. burnetii exposure, the placental sample confidence interval suggests 2016 shedding events were rare or absent. The location maintained the flock with little or no depopulation in 1984 and without C. burnetii vaccination during or since 1984. It is not clear how a zero-shedding rate was achieved in these sheep beyond natural immunity, and more work is required to discover and assess possible factors that may contribute towards achieving zero-shedding status. We provide the first U.S. sheep placental C. burnetii shedding update in over 60 years and demonstrate potential for C. burnetii shedding to reach undetectable levels after an outbreak event even in the absence of targeted interventions, such as vaccination. Coxiella burnetii is an important pathogenic bacterium of humans and ruminant livestock that is transmitted by inhalation directly from animals or from a contaminated environment [C. burnetii is accompanied by conversion from the metabolically active large cell variant (LCV) form to the environmentally stable small cell variant (SCV) form, which is believed to contribute to its survival [C. burnetii can persist in the environment for extended periods [C. burnetii outbreaks within the United States [C. burnetii infection can result in abortions or moribund lambs or kids [ironment –3. Ruminironment . Environsurvival  during lsurvival . Since C periods , 8 and s periods –11, tracd States , 13. Thed States –16. In pd States –19, and d States –22. In s or kids , and cos or kids .C. burnetii, termed Q fever, occurs in approximately half of infected individuals [C burnetii particles, and the organism is recoverable from the air of cattle [Disease in humans from ividuals  and is cividuals , 26. In ividuals , and serividuals . Treatmeividuals . The estividuals . Human if cattle , sheep [f cattle , and goaf cattle  pens. Fuf cattle –35. Undef cattle –11.C. burnetii-associated abortion storms in 2005–2007 which were followed in 2007–2011 by more than 4,100 human Q fever cases [C. burnetii in small ruminants for decades, and there have historically been long periods between recorded Q fever outbreaks [C. burnetii outbreak were estimated at €307 million, with substantial costs to both small ruminant industry (€85 million) and to broader society (€222 million) [C. burnetii is endemic in most of the world , there is concern about large outbreaks that could occur potentially at any site throughout its distribution [C. burnetii shedding outside of current or recent outbreaks has been minimally addressed in field studies.In a recent outbreak in the Netherlands, a region with small ruminant dairies experienced er cases , 37. Maner cases . The Netutbreaks . Total cmillion) . Since Cribution . In the ribution . HoweverC. burnetii in sheep has been estimated at 2.7% as of 2011 in the U.S. by the National Animal Health Monitoring Service (NAHMS)  during the spring 2016 lambing season at the same sheep research station in Dubois, ID as previously described . The avePedigree and lambing records of sheep at the Idaho location since 1984, the year of the Q fever outbreak, were examined to determine the number of generations that had elapsed between ewes that lambed in 1984 and the 100 selected ewes of the present study. A tail-female pedigree was obtained for each ewe to calculate the average number of generations elapsed in the tail-female line since those that lambed in the 1984 outbreak. In cases where multiple female ancestors lambed in 1984, a simple average of the number of generations back was calculated for each present ewe prior to calculation of the overall population average.Coxiella burnetii by real-time quantitative PCR (qPCR) at the Veterinary Diagnostic Laboratory of Colorado State University. DNA was purified from placental tissues and vaginal swabs using the QIAamp DNA Blood Mini Kit (Qiagen). Samples were processed according to manufacturer’s instructions, with one exception–an increased 56°C incubation from 10 minutes to 15 minutes. Placental swab samples were mechanically homogenized prior to the extraction process using a Mini-Beadbeater (Biospec Products). DNA was purified from fecal samples using the ZR Fecal DNA MiniPrep kit (Zymo Research), according to manufacturer’s instructions.Placental tissue, vaginal swab, and fecal samples were tested for C. burnetii. This qPCR contains three primer/probe sets targeting three different regions of the genome, including both multi-copy and single-copy genes, which provide data to estimate C. burnetii genome copy number. DNA from the vaginal swab and fecal samples was assayed by a qPCR with a single primer/probe set targeting the multi-copy IS1111 transposon, using TaqMan Universal PCR master mix (Applied Biosystems) [DNA from the placental tissues was assayed by qPCR using the Laboratory Response Network (CDC) protocol for systems) , 47. BotSerum samples were analyzed using a CHEKIT Q Fever Antibody ELISA Test Kit (IDEXX) at the Washington Animal Disease Diagnostic Laboratory using the manufacturer-recommended protocol including Phase I and Phase II purified antigens. The sample/positive control (S/P) ratio of optical densities was recorded with a spectrophotometer, and according to manufacturer’s guidelines, each sample was considered ELISA positive if the ratio was 40% or more, indeterminate if between 30% and 40%, and negative if below 30%.C. burnetii positive fractions from different diagnostic tests or different populations with underlying positive count data were performed with Fisher’s exact test. Results with P<0.05 were considered significant.The Clopper-Pearson exact method was used for calculation of 95% confidence intervals because it has been shown to be consistently conservative, even for proportions near zero or 100% . CompariNo abortion storms or recent human Q fever diagnoses were observed at this location in 2016 or in records available from the preceding ten years. Of the 100 ewes selected for testing, serum was available from all ewes, placentas from 97 ewes, vaginal swabs from 99 ewes, and fecal samples from 96 ewes. Twin placentas were available from 25 ewes, and triplet placentas were available from one ewe. A minimum of two sample types were available for shedding analysis by PCR from each of the 100 selected ewes. Of the 100 selected ewes, at least one member of the tail-female line from 98 ewes had lambed during 1984, the year of the outbreak. Between 6–15 generations, with an average of 9.21 generations, had elapsed between each ewe and ancestors in the tail-female line that had lambed during 1984.C. burnetii exposure .In this study, we assessed seroprevalence and shedding of  in 1984 . In the burnetii , ongoingC. burnetii among sheep in large operations (>500 sheep) from sheep production facilities over the United States was estimated at 2.8% by the National Animal Health Monitoring System (NAHMS) in 2011 . The 5% seroprevalence observed in the current study was not significantly different from this estimated national average (P>0.05). In addition, more localized individual studies of C. burnetii exposure in domestic sheep within the U.S. between 1951 and 1985 have reported 5.7% [Seroprevalence to ted 5.7% , 21.2% [rations >0 sheep fted 5.7% . The latC. burnetii DNA was detected in all placental, vaginal, or fecal samples in spite of the previous outbreak of Q fever epidemiologically attributed to this facility [C. burnetii was significantly greater than prevalence of placental shedding (P = 0.017). This finding is in agreement with previous studies demonstrating seroprevalence as an overestimate of shedding, since seropositive ruminants may not shed detectable C. burnetii [C. burnetii infection in either animals or humans was reported on the premises in records available from the ten years preceding this study. While shedding prevalence of C. burnetii has been previously investigated, no studies of placental shedding in sheep have been conducted within the U.S. in half a century [No facility . While ffacility , the lacfacility . The preburnetii , 53, 54.g P = 0.0. This fiC. burnetii have been demonstrated to shed for two parturitions following initial abortion, but factors behind progression to lack of C. burnetii shedding remain mostly unknown. Elimination of large-scale shedding from primarily dairy goat farms affected by the Netherlands outbreak is attributed to a combination of strict hygiene protocols, surveillance, and vaccination [C. burnetii vaccine is licensed in the U.S. Short-term elimination of shedding has been documented in a German flock after six years [C. burnetii, the affected sheep are no longer infective to other sheep, implying a possible loss of shedding, again with unknown contributing factors [Sheep and goats experimentally infected with cination . Two stucination  or complcination , followiix years  with unk factors . In the  factors , 56, 57. factors , 20, wit factors . StudiedC. burnetii in the lambing area. The facility currently houses approximately 1,428 mature breeding ewes, decreased from 5,270 in 1984, with more than 20,000 acres of land plus. The land use consists of a seasonal schedule of pasture grazing, housing in feedlots for breeding, and winter lots for holding and lambing that have remained invariant or changed only mildly since the outbreak. Changes in husbandry may have reduced infection from the environment over time, and future work on the impact of management conditions on C. burnetii shedding may clarify which interventions are most helpful. In terms of fetal infection, elimination over subsequent generations may be possible. DNA from C. burnetii can be detected in the fetuses from infected small ruminant dams [Specific changes made at the outbreak location may also have contributed to the lack of demonstrable shedding in the current study. While no antibiotics were used after the outbreak, the demolition of the majority of the original lambing facilities and construction of a new facility, along with subsequent changes made in facility function over the ensuing decades, may have had an effect on the amount of residual ant dams , but thiant dams , and it C. burnetii prior to sample collection, we demonstrated lack of C. burnetii shedding decades after a small Q fever outbreak on the premises. Further characterization of long-term shedding in healthy flocks within the U.S. will better contextualize the lack of shedding observed in this study. Owing to the potential for a zoonotic event, studies are warranted assessing the ability of premises with current C. burnetii shedding to progress to a zero-shedding flock and the mechanisms by which this can occur.While there were a small number of seropositive sheep identified in this study (5 of 100), implying exposure to S1 TableA list of each ewe’s study ID with the results of the serum S/P ratio and qPCR results for vaginal swabs, feces, and placentas.(XLSX)Click here for additional data file.


Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program.Injury-induced  axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NADDOI:http://dx.doi.org/10.7554/eLife.22540.001 Axon loss is a hallmark of many neurological disorders. Developing methods to maintain axons and preserve neural circuit integrity following injury or disease will require a mechanistic understanding of the axonal degeneration program . Injury-+ biosynthetic enzyme, is an essential axon survival factor whose loss is sufficient to trigger axon degeneration  and its downstream MAPK JNK were the first axon degeneration factors identified . This MA+ synthesis, but rather by blocking SARM1-dependent NAD+ depletion , and the MAPKs JNK1-3. Within this pathway, the MAPKKs MKK4 and MKK7 represent a targetable bottleneck in which loss of these kinases abrogates the MAPK signaling necessary for axon degeneration . Lentivi+ depletion and MAPK activation  acting downstream of SARM1 as an intermediate between SARM1 activation and NAD+ and ATP loss, or (b) functioning upstream of SARM1 to promote injury-dependent SARM1 activation. To distinguish between these possibilities, we assessed whether MKK4/7 activity is necessary for NAD+ depletion induced by a constitutively active form of SARM1 that bypasses the need for injury-dependent SARM1 activation. To generate an activated form of SARM1, we used a pharmacologically controlled system in which the SARM1-TIR domain fused to FKBP is homodimerized upon application of the small molecule AP20187 . Hence, MAPK signaling is not an intermediate between activated SARM1 and NAD+ depletion. Taken together with the finding that MKK4/7 are required for injury-dependent NAD+ and ATP depletion, these results are consistent with the model that MAPK signaling functions upstream of SARM1 to promote injury-dependent SARM1 activation.We previously demonstrated that SARM1 is required for NAD axotomy . Hence,  AP20187 . Dimeriz axotomy . Surprisrization . ATP is rization . We find+ depletion following injury but not direct dimerization of SARM1-TIR. While we cannot replicate the finding that the MAPK pathway is required for SARM1-TIR induced axon degeneration, we do observe activation of MKK4 following SARM1-TIR dimerization  promotes axon degeneration of motoneurons in h injury . Thus, w+ depletion , mouse anti-β3 tubulin  Cat# 801202 RRID:AB_10063408), rabbit anti-MKK4  Cat# 9152 RRID:AB_330905), rabbit anti-MKK7 , rabbit HSP90 , rabbit Phospho-SEK1/MKK4(Ser257/Thr261) , mouse anti-Myc-Tag , rabbit anti-SCG10  Cat# T2200 RRID:ti-SCG10 , rabbit ti-SCG10 , Cy3 andet al. , Map2k4 (MKK4) gRNAs (TTGTTTTACAGGGCGACTGT and TTTGTAAAACTTATCGAACG), Map2k7 (MKK7) gRNAs (TTGCAGCAAATGCGGCGCT and CCAAAGCACTGAACGATGTA), Nmnat2 gRNA (GGAGCCCACCTGTTTTCCGT). gRNAs were designed with help from the Genome Engineering and iPSC center. gRNA sequences were cloned into LentiGuide-puro plasmid (Addgene # 52963).For CRISPR/gRNA studies, Cas9 knock-in mice from Jackson Laboratory (Strain# 024857)  were breGapdh as an internal control. Primers include: Nmnat2 Fwd: 5’ GACCGAGACCACAAAGACCC, NMNAT2 Rev: 5’ CCCTGGCTCTCTCGAACATC, Stmn2 (SCG10) Fwd: 5’ TGCGTGCACATCCCTACAAT, Stmn2 (SCG10) Rev: 5’ CTGCTTCACCTCCATGTCGT, Gapdh Fwd: 5’ TGTGAACGGATTTGGCCGTA, and Gapdh Rev: 5’ ACTGTGCCGTTGAATTTGCC.Total RNA was purified from DRG cultures using phenol-chloroform extraction using Trizol (Invitrogen). RNA was retro-transcribed using qScript cDNA supermix . Quantitative real-time PCR was performed using a SYBR green-based assay on a 7900 HT sequence detection system (Applied Biosystems) and quantified using delta-CT, with Drosophila melanogaster were raised on standard fly food at 25°C. The following strains were used in this study: Canton S (wild-type), ∆Nhiw , hiw∆N , UAS-bskN  on ice for five minutes. After spinning for five minutes, the supernatant was transferred to a new tube, 20 µL 3M potassium carbonate (K2CO3) was added. The supernatant was loaded for HPLC after a five minute spin as described . For quantification of axon degeneration, images were analyzed using ImageJ software with the Degeneration Index algorithm, which computes an unbiased degeneration score ranging from 0–1.0, with 1.0 being total fragmentation . In thisFor axon-only western blot, dense spot cultures were plated using 4 embyros per 24-well plate. A microscalpel was used to remove cell bodies. Axon-only lysate was collected from 4–6 wells. Samples were lysed on ice using Laemmli buffer with protease inhibitor cocktail (Roche #11836170001). A standard western blot procedure was followed. For quantification, bands were normalized to HSP90 or Tuj1 as loading controls. For half-life quantification, data were fit to a curve.Drosophila samples were mounted and imaged in 70% glycerol containing Vectashield. Samples were imaged on a Leica DMI 4000B Confocal microscope using 40x or 60x oil objectives. Images are maximal Z-projections of confocal stacks. Samples for each experiment were processed simultaneously with identical confocal gain settings. Intensity of NMNAT2-myc and SCG10 in DRGs was quantified using ImageJ by determining the mean grey value within the total axonal area as defined by Tuj1 staining with background subtracted. A similar strategy was employed for intensity of HA-dNMNAT, with HRP labeling total axonal area.DRGs were plated in spot cultures in 8-well Permanox slides . Neurons were fixed in 4% paraformaldehyde, washed, and blocked as described . NMNAT2-One-way ANOVA comparison with Tukey post-hoc was used to test for statistical significance. All data are presented as mean ± SEM. Data analysis was performed in GraphPad Prism. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "MAPK Signaling Promotes Axonal Degeneration by Speeding the Turnover of the Axonal Maintenance Factor NMNAT2" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in + level alterations constitutes "a significant data set that will contribute to the eventual full understanding of degenerative pathways".Overall, the reviewers were positive both in their reviews and in their online discussion. As one reviewer commented in our discussion, your study on the relationship between injury, MAPK signaling, NMNAT2, and NADThe reviewers recognize that your results differ from those in Yang et al., from the Tessier-Lavigne group but in no way dismiss your results. Rather, they offer suggestions, albeit requiring additional experiments, to parse out MKK4/7 control of SARM activation that in turn regulates the neuroprotective elements NMNAT2 and SCG10:Although only Reviewer 1 noted this in the reviews below, all three agreed in our discussion that the Bcl-XL overexpression, to enhance neuron viability after transfection with shRNA constructs, is one aspect that could contribute to these differences. Bcl-XL overexpression should be explained and discussed in the text and, ideally, tested.The epistasis experiments are welcome, but the reviewers called for analysis of activation of individual components. They also agree that it would be important to test the effects of reducing JNK activity.All three reviewers were concerned about the conclusion that the MAPK pathway regulates degradation: They ask for clarification on the degradation and turnover analyses that lead to the conclusion that MKK4/7 promote axonal turnover of NMNAT2 and SCG10 post-injury. Reviewer 2 indicates that the lower rate of protein degradation could result from the degradation components being saturated with the high levels of NMNAT2 and SC10 seen in the MKK4/7/ depleted condition. Reviewer 3 asks whether this is due to axonal turnover depending on concurrent MAP kinase activity, or increased axonal loading overwhelming the normal course of degradation. A suggested experiment would involve increasing the levels of NMNAT2 and SCG10 in cells (to levels comparable to those seen with MKK4/7/ depletion) and show that the rate of degradation is unaltered.Reviewer 3 asked for the FRET experiment to directly readout SARM, but believes that this would be challenging, and so we will not hold you to perform this experiment.eLife at this time. We would be happy to receive a new submission if you were to address the reviewers' concerns.Thus, based on consultation among the three reviewers with the Reviewing Editor on these aspects and the individual reviews below, several asking for additional analyses, we regret to inform you that your work as it stands will not be considered for publication in Reviewer #1:The mechanism of axon degeneration is an area of great interest and intense investigation. Several highly conserved components involved in axotomy induced injury have been identified, including a critical prodegenerative component SARM, a distinctive MAPK pathway comprised of DLK/MEKK4/JNKs, and an anti-degenerative metabolic enzyme NMNAT. Together these molecules alter the metabolic state of axons, and regulate axonal degeneration. Additional components, including ubiquitinating enzymes, Bcl2 family members, caspases, and calpain have all been tied to regulation of axon degeneration. Here Walker et al. try to develop a cohesive view of these multiple components, and how they work together. The findings presented contradict a recent paper in the field. While that study concluded that SARM is upstream of the MAPK cascade, the present study reaches the opposite conclusion, namely that MAPK regulates degradation of NMNAT2 (and other components). Thus, in reviewing the paper, I tried to consider both how the current study differs from Yang et al., and also how well the current model is explored. A major problem (with both studies) is the push to place all these components in a single linear pathway. The impetus to place all the components in a linear pathway may have oversimplified this complex process.1) A major methodologic difference with the Yang et al. report is that the current study expresses Bcl-XL in all cultures in which shRNA knock down is performed. . The authors note that this enhances the viability of neurons transfected with shRNA constructs. However, the Tessier-Lavigne group has reported that bclxl can promote axonal survival and prevent degeneration. Therefore, this overexpression could definitely alter the results throughout the study  The results suggest that MAPK is needed for activation of SARM1, but once activated SARM functions independently of MAPK. IF this is true, then genetic epistasis experiments showing that MAPK is not needed for the activity of a constitutively active SARM, do not indicate that MAPK must be downstream of SARM. I believe there could indeed be a feedback loop. The authors considered, and rejected this possibility. The rejection of this model was based on evidence that MAPK regulates NMNAT2 in the absence of SARM. Again, this result is only problematic if the authors feel that there is a single input into this MAPK cascade.3) In 4) I think that genetic epistatis experiments are very useful, but it is also important to examine directly activation of individual components. Does activated SARM stimulate the DLK/Mkk4/JNK pathway? To address this question, the authors should look at phosphorylation or catalytic activity of pathway components as an indicator that the MAPK cascade is activated. This is particularly important since the genetic epistasis experiments are not all consistent, and it is likely that many of the pathway components have multiple functions.Reviewer #2:+ in axons in response to axotomy and find that MKK4/7 depletion does not prevent the loss of NAD+ in neurons where Sarm1 is directly activated. These findings are interesting because MKK4/7 was previously shown to function after the point of Sarm1 activation to mediate axon degeneration. However, a major point of this manuscript is MKK4/7 functions before the point of Sarm1 activation in this pathway.This manuscript by Walker and colleagues provides interesting mechanistic insights into the pathway of Wallerian degeneration, which has made significant advances in recent years. Specifically, this manuscript elucidates the order of events that have been recently shown to be key mediators of this pathway, namely-MAPK, NMNAT, and Sarm1. The authors here show that MKK4/7 are important for the loss of NADThe authors follow up this finding by identifying the role of MKK4/7 in regulating the levels of NMNAT2 and SCG10, both previously implicated in protecting against Wallerian degeneration. Loss of MKK4/7 leads to increase levels of NMNAT2 and SCG10, which maintain axon survival.Overall, these are interesting findings. However, additional experiments are needed to strengthen the conclusions. Also, the current title is not fully supported with the data shown. The data mainly show that MKK4/7 acts upstream of Sarm1 activation and mediates axon degeneration by regulating levels of NMNAT2 and SCG10.1) The differences in results between this manuscript and the Yang 2015 paper are significant and there does not seem to be any straightforward explanation for this. The Yang 2015 paper shows that both MKK4/7 depletion and JNK1/2/3 depletion prevented active Sarm1-induced axon degeneration. Since the results in his manuscript show that MKK4/7 depletion did not block axon degeneration in the context of direct Sarm activation, the authors should also examine whether similar results are obtained with JNK depletion (or inhibition).2) In 3) In 4) Have the authors considered increased transcription or translation as factors that could result in the increased accumulation of NMNAT2 or SCG10 in MKK4/7 depleted conditions? Since the title of this manuscript seems to imply turnover as the main mechanism, they need to be more rigorous to not only show convincing degradation data but also to eliminate the other possibilities.+ loss and axon degeneration after axotomy but not in response to constitutively active SARM1”, the authors conclude that MKK4/7 are "required" for NAD depletion after axotomy. Perhaps it is more accurate to state that MKK4/7 is important for NAD depletion after axotomy  In the third paragraph of the subsection “MKK4/7 are necessary for NADReviewer #3:+. These are interesting and significant findings given the prominent role of these proteins in axon degeneration. I have a few suggestions before this manuscript would be appropriate for eLife.This paper revises the current understanding of the relationship between injury, MAPK signaling, NMNAT2, and NADa) Although the epistasis is convincing, in general a direct readout of SARM would be very powerful. This would allow testing whether activating or inactivating MAP kinase signaling is sufficient to activate SARM, independently of axon injury. Perhaps this could be done with a FRET reporter for SARM conformation.b) The experiments in cultured cells and flies suggest that MAP kinase signaling is active even in uninjured neurons, and acts to limit levels of axon survival factors. This is a surprising result that needs more discussion. Can MAP kinase signaling be blocked acutely? How quick is the resulting accumulation of NMNAT2 and SCG10?c) An important conclusion is that MKK4/7 promote axonal turnover of NMNAT2 and SCG10 post-injury. However, in these experiments the initial amount of NMNAT2 and SCG10 is greatly increased. Thus, it is impossible to know whether axonal turnover really depends on concurrent MAP kinase activity, or whether increased axonal loading overwhelms the normal course of degradation. Perhaps these proteins could be overexpressed to mimic what happens in the MKK4/7 knockdown, and then turnover after injury could be measured.d) In the final experiment, NMNAT2 is shown to be downstream of MKK4/7, but not SARM. I have concerns about the interpretation. For NMNAT2, the data do show that "NMNAT2 is required for the axonal protection afforded by MKK4/7 knockdown", as stated, but not that "the protective effect of inhibiting MAPK signaling is conferred by elevated levels of NMNAT2" . In fact, the proposed model also includes SGC10, and there may be other protective effectors as well.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "MAPK Signaling Promotes Axonal Degeneration by Speeding the Turnover of the Axonal Maintenance Factor NMNAT2" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:All three reviewers are satisfied with the amendments and agree that the manuscript is strengthened. They have two remaining sets of recommendations.Essential revisions:Reviewer 3 asks that some of the key data be included in the revised manuscript, either in the main or figure supplements. This reviewer believes that including the following data in figures is important, especially as some of the data directly address and sometimes contradict the published data from Yang et al.a) You repeated the SARM1-TIR experiment using JNK inhibitor and found that, like MKK4/7 depletion, JNKi did not block axon degeneration induced by direct SARM1 activation. In particular, these data should be in the main figures.b) You report that NMNAT2 and SCG10 transcripts were not elevated upon depletion of MKK4/7 by RT-PCR. Based on this result, and the turnover data, you conclude that MAPKs influence the turnover rate of NMNAT2 and SCG10. Since this result is important for the overall conclusion of the manuscript, it would be important to also show it in a figure. [Editors’ note: the author responses to the first round of peer review follow.]The reviewers recognize that your results differ from those in Yang et al., from the Tessier-Lavigne group but in no way dismiss your results. Rather, they offer suggestions, albeit requiring additional experiments, to parse out MKK4/7 control of SARM activation that in turn regulates the neuroprotective elements NMNAT2 and SCG10:Although only Reviewer 1 noted this in the reviews below, all three agreed in our discussion that the Bcl-XL overexpression, to enhance neuron viability after transfection with shRNA constructs, is one aspect that could contribute to these differences. Bcl-XL overexpression should be explained and discussed in the text and, ideally, tested.does not influence the SARM1 Wallerian degeneration program. Indeed, the one point of experimental  disagreement between our work and the work of Yang et al. relates to the degeneration induced by dimerizing the SARM1-TIR domain. Importantly, Yang et al. state that this degeneration is not influenced by overexpression of Bcl-XL. In the Yang et al. paper, the Tessier-Lavigne group finds that “Treatment of sensory neurons expressing FKBP(F36V)-TIR with AP20187 induced degeneration of distal axons (overexpression of Bcl-xl(data not shown)” . We also previously demonstrated that Bcl-XL overexpression does not affect axon degeneration induced by activated SARM 1 , we show that this function is not downstream of SARM1 because it occurs in SARM1 KO neurons, and demonstrate that this function is necessary for the protection afforded by loss of MAPK signaling in our epistasis experiments. We do now expand our model to acknowledge this additional MAPK activation, and while we have no evidence that it is functionally relevant, we do discuss how this activation could serve to promote axon degeneration.We have addressed these issues via a series of experiments. We asked about MAPK activation by testing whether dimerization of the SARM1-TIR domain is sufficient to activate the MAPK pathway as described by Yang et al. We now show in We also perform additional experiments with JNK inhibition as requested. We now show that treatment of DRGs with JNK inhibitor is sufficient to increase the levels of NMNAT2 and SCG10 within two hours new  and demoAll three reviewers were concerned about the conclusion that the MAPK pathway regulates degradation: They ask for clarification on the degradation and turnover analyses, that lead to the conclusion that MKK4/7 promote axonal turnover of NMNAT2 and SCG10 post-injury. Reviewer 2 indicates that the lower rate of protein degradation could result from the degradation components being saturated with the high levels of NMNAT2 and SC10 seen in the MKK4/7/ depleted condition. Reviewer 3 asks whether this is due to axonal turnover depending on concurrent MAP kinase activity, or increased axonal loading overwhelming the normal course of degradation. A suggested experiment would involve increasing the levels of NMNAT2 and SCG10 in cells (to levels comparable to those seen with MKK4/7/ depletion) and show that the rate of degradation is unaltered.We thank the reviewers for the suggestions. First, we demonstrate that acute treatment with JNK inhibitor slows the turnover rate of survival factors before there is time for a significant increase in the levels of NMNAT2 and SCG10 . This isSecond, we performed the experiment requested by the reviewers. We find that when we simultaneously overexpress NMNAT2 and SCG10 (to mimic the effects of MAPK inhibition) their turnover rate is indistinguishable from their turnover rate when wild type levels of protein are present . Hence, Reviewer 3 asked for the FRET experiment to directly readout SARM, but believes that this would be challenging, and so we will not hold you to perform this experiment.We appreciate that the reviewers recognize that this would be beyond the scope of this proposal.Reviewer #1:[…] 1) A major methodologic difference with the Yang et al. report is that the current study expresses bclxl in all cultures in which shRNA knock down is performed. . The authors note that this enhances the viability of neurons transfected with shRNA constructs. However, the Tessier-Lavigne group has reported that bclxl can promote axonal survival and prevent degeneration. Therefore, this overexpression could definitely alter the results throughout the study  The results suggest that MAPK is needed for activation of SARM1, but once activated SARM functions independently of MAPK. IF this is true, then genetic epistasis experiments showing that MAPK is not needed for the activity of a constitutively active SARM, do not indicate that MAPK must be downstream of SARM. I believe there could indeed be a feedback loop. The authors considered, and rejected this possibility. The rejection of this model was based on evidence that MAPK regulates NMNAT2 in the absence of SARM. Again, this result is only problematic if the authors feel that there is a single input into this MAPK cascade.+ depletion. Because this simple model can explain all of the functional data, this is our preferred model. However, we certainly acknowledge that biology is complicated, and that additional regulatory mechanisms likely exist. We now discuss that SARM1-dependent activation of the MAPK pathway, although not necessary for the regulation of NMNAT2 and SCG10, could play an additional regulatory role in fine-tuning the process of axon degeneration.All of our functional data are consistent with a linear model in which the MAPK pathway negatively regulates NMNAT2 levels to control SARM1 activation and subsequent NAD3) In neuronal NAD+ following SARM1-TIR dimerization. We now demonstrate that MKK4/7 do not influence local axonal NAD+ following SARM1- TIR dimerization. This finding is fully consistent with our prior result.We appreciate the suggestion and performed the suggested experiment. In our original submission we showed that MKK4/7 does not influence total 4) I think that genetic epistatis experiments are very useful, but it is also important to examine directly activation of individual components. Does activated SARM stimulate the DLK/Mkk4/JNK pathway? To address this question, the authors should look at phosphorylation or catalytic activity of pathway components as an indicator that the MAPK cascade is activated. This is particularly important since the genetic epistasis experiments are not all consistent, and it is likely that many of the pathway components have multiple functions.We have discussed this above. We now confirm the finding of Yang et al. that activation SARM1 can activate the MAPK stress pathway. We now incorporate this finding into our Discussion.Reviewer #2:[…] 1) The differences in results between this manuscript and the Yang 2015 paper are significant and there does not seem to be any straightforward explanation for this. The Yang 2015 paper shows that both MKK4/7 depletion and JNK1/2/3 depletion prevented active Sarm1-induced axon degeneration. Since the results in his manuscript show that MKK4/7 depletion did not block axon degeneration in the context of direct Sarm activation, the authors should also examine whether similar results are obtained with JNK depletion (or inhibition).We repeated the SARM1-TIR experiment using JNK inhibitor and find that, like MKK4/7 depletion, JNKi does not block axon degeneration induced by direct SARM1 activation. This result has been incorporated into the Results section and fully supports our model.2) In We thank the reviewer for this observation. After quantifying 6 blots, we find that there is no significant change in SARM1 levels upon depletion of MKK4/7.3) In We appreciate the experimental suggestion. To address this concern, we have taken two approaches. First, as the reviewer suggested, we have overexpressed NMNAT2 and SCG10 to levels approximately 5 to 8-fold above endogenous levels . We find4) Have the authors considered increased transcription or translation as factors that could result in the increased accumulation of NMNAT2 or SCG10 in MKK4/7 depleted conditions? Since the title of this manuscript seems to imply turnover as the main mechanism, they need to be more rigorous to not only show convincing degradation data but also to eliminate the other possibilities.We find that NMNAT2 and SCG10 transcripts are not elevated upon depletion of MKK4/7 by rt-PCR (measured transcripts from 3 independent experiments). Thus, taken together with the additional turnover data from point 3 above, we are confident to conclude that MAPKs influence the turnover rate of NMNAT2 and SCG10. As such, we think it would be inappropriate to remove ‘turnover’ from the title.+ loss and axon degeneration after axotomy but not in response to constitutively active SARM1”, the authors conclude that MKK4/7 are "required" for NAD depletion after axotomy. Perhaps it is more accurate to state that MKK4/7 is important for NAD depletion after axotomy  In the third paragraph of the subsection “MKK4/7 are necessary for NADWe have changed the text to reflect this change.Reviewer #3:[…] a) Although the epistasis is convincing, in general a direct readout of SARM would be very powerful. This would allow testing whether activating or inactivating MAP kinase signaling is sufficient to activate SARM, independently of axon injury. Perhaps this could be done with a FRET reporter for SARM conformation.We agree that a FRET reporter for SARM1 activation would have numerous experimental applications. We appreciate that the reviewers find this to be beyond the scope of this study.b) The experiments in cultured cells and flies suggest that MAP kinase signaling is active even in uninjured neurons, and acts to limit levels of axon survival factors. This is a surprising result that needs more discussion. Can MAP kinase signaling be blocked acutely? How quick is the resulting accumulation of NMNAT2 and SCG10?We include a time course of NMNAT2 and SCG10 levels after treatment with JNK inhibitor in a new c) An important conclusion is that MKK4/7 promote axonal turnover of NMNAT2 and SCG10 post-injury. However, in these experiments the initial amount of NMNAT2 and SCG10 is greatly increased. Thus, it is impossible to know whether axonal turnover really depends on concurrent MAP kinase activity, or whether increased axonal loading overwhelms the normal course of degradation. Perhaps these proteins could be overexpressed to mimic what happens in the MKK4/7 knockdown, and then turnover after injury could be measured.This was the same suggestion made by reviewer 2 and is addressed above.d) In the final experiment, NMNAT2 is shown to be downstream of MKK4/7, but not SARM. I have concerns about the interpretation. For NMNAT2, the data do show that "NMNAT2 is required for the axonal protection afforded by MKK4/7 knockdown", as stated, but not that "the protective effect of inhibiting MAPK signaling is conferred by elevated levels of NMNAT2" . In fact, the proposed model also includes SGC10, and there may be other protective effectors as well.While our data demonstrate that NMNAT2 is required for the protective effect afforded by MKK4/7 knockdown, we have modified the text to note that SCG10 and other protective factors may also play a role in MAPK-mediate axon degeneration.[Editors' note: the author responses to the re-review follow.]Essential revisions:Reviewer 3 asks that some of the key data be included in the revised manuscript, either in the main or figure supplements. This reviewer believes that including the following data in figures is important, especially as some of the data directly address and sometimes contradict the published data from Yang et al.a) You repeated the SARM1-TIR experiment using JNK inhibitor and found that, like MKK4/7 depletion, JNKi did not block axon degeneration induced by direct SARM1 activation. In particular, these data should be in the main figures.This is now included as b) You report that NMNAT2 and SCG10 transcripts were not elevated upon depletion of MKK4/7 by RT-PCR. Based on this result, and the turnover data, you conclude that MAPKs influence the turnover rate of NMNAT2 and SCG10. Since this result is important for the overall conclusion of the manuscript, it would be important to also show it in a figure.This is now included as