Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a Vmax increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from  Cytochromes P450 are heme-thiolate monooxygenases found in all domains of life Coon,  able to They catalyze many chemical reactions such as hydroxylation of non-activated carbon atoms, epoxidation of double bonds and dealkylation on C-, N-, and S- atoms  and its flavin-containing reductase domain (BMR) that, unlike in other cytochromes P450, are all part of the same polypetide chain. For this reason we already reported the construction of different fusion human P450 enzymes, engineered by connecting the reductase domain BMR with human cytochromes P450 2E1 to transfer electrons in the redox chain from NADPH to the heme is evaluated by varying the length of the linker region between BMR and the N-terminally modified 3A4 heme domain. As shown in Figure via the short linker Pro-Ser-Arg, two new constructs are engineered to include 3- or 5-Gly inserted after the Pro-Ser-Arg loop leading to 3A4-3GLY-BMR and 3A4-5GLY-BMR chimeric proteins. This glycine rich region close to the C-terminal of P450 3A4 has been proposed to function as a flexible hinge that can provide the enzyme the degrees of freedom required to achieve a functional complex. The effect of the linker length on the electron transfer between the reductase and the catalytic domains in the presence of NADPH as electron supplier is studied for the three chimeras.Flexibility between protein domains is a critical parameter in modulating the uncoupling process via the formation of an active complex and by tuning the electron flow to the heme. The ability of the reductase of P450 BM3 from c as an artificial electron acceptor  linked via a Pro-Ser-Arg linker was available in our laboratory  and Promega . Horse heart cytochrome c, erythromycin, testosterone, 6β-hydroxytestosterone, horseradish peroxidase type X, superoxide dismutase from bovine erythrocytes, catalase, N,N-dimethylaniline, and 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one were purchased from Sigma . NADPH  was acquired from Calbiochem (Germany). Human CPR was purchased from Life Technologies. All other chemicals, biochemicals used in this study were purchased from Sigma  at the highest available grade and used as recommended by the manufacturer.The primers for PCR and oligonucleotides for the linker region between P450 3A4 and BMR reductase were prepared by MWG (Germany). DNA Ladder, restriction endonucleases enzymes The strategy used in the construction of the fusion proteins is based upon introduction of 3 or 5 glycines in the pre-existent Pro-Ser-Arg linker connecting P450 CYP 3A4 domain and BMR one domain via 3 amino acids (Pro-Ser-Arg) by using as a starting template the vector pCW-3A4-BMR available in our laboratory Figure . GlycineAll PCR reactions were performed in a DNA thermal cycler  and employed Vent polymerase under conditions recommended by the manufacturer .B. megaterium. The 3A4-5GLY-BMR chimera contains the N-terminally modified human P450 3A4 (aa 13-503), the linker Pro-Ser-Arg-Gly-Gly-Gly-Gly-Gly and the C-terminal reductase domain (aa 472-1049) of B. megaterium. The gene coding for the BMR reductase was amplified using the forward 5-AGTGGAGCCCCTAGGTCAGGCGGTGGCAAAAAGGCAGAAAACGCTCATAAT-3′ and reverse 5′-GCTCATGTTTGACAGCTTATCATCGATAAGCTTTTACCC-3′ primers for the chimera with 3 glycines. The forward 5′-AGTGGAGCCCCTAGGTCAGGCGGTGGCGGTGGCAAAAAGGCAGAAAACGCTCATAAT-3′ and reverse 5′-GCTCATGTTTGACAGCTTATCATCGATAAGCTTTTACCC-3′ primers were used for the 5-glycine chimera. The forward primers were synthesized with an overhanging Hind III restriction site upstream of the BMR start codon, whereas the reverse primers contained an Avr II site downstream of the stop codon. The underlined letters represent the endonuclease restriction sites. DNA was melted for 2 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min and 45 s at 60°C, 1 min at 72°C. The polymerase chain reaction products obtained were digested with Hind III and Avr II endonucleases and purified by the GeneClean II method as described by the manufacturer. The pCW-3A4-BMR vector was digested for 1 h at 37°C with Hind III and Avr II and genomic DNA corresponding to pCW-3A4 was isolated by NAGEL KIT extraction and then ligated using T4 DNA ligase with the sticky sites of the digest PCR products. The ligation mix was transformed into E. coli DH5α cells and plated on LB agar containing 100 μg/ml ampicillin. The positive clones were chosen and the plasmid DNA was purified using a Mini-prep plasmid extraction kit . The correct insertion of the glycine codons was verified by DNA sequencing.The 3A4-3GLY-BMR chimera was engineered to contain the N-terminally modified human P450 CYP 3A4 (aa 13-503), the linker Pro-Ser-Arg-Gly-Gly-Gly and the C-terminal reductase (aa 472-1049) of P450 BM3 from E. Coli DH5α cells with the corresponding plasmid vectors. The proteins were purified using diethylaminoethyl and hydroxylapatite columns as described before  and the fluorescence was determined again. The concentrations of FAD and FMN in the supernatant were determined from the fluorescence intensity compared to those obtained for standard solutions of flavins by using as extinction coefficients 11.3 and 12.2 mMThe stability of 3A4-BMR fusion proteins was compared with that of CYP 3A4 by following the loss of CO-binding at a temperature of 37°C. Recombinant CYP3A4 was expressed and purified as described by Dodhia et al. . CO-bind−1 cm−1) for 750 sunder anaerobic conditions. Proteins and reducing agent solutions (in the absence and presence of saturating amounts of substrate) were prepared in syringes in an anaerobic glove box and transferred to the sample handling unit of the stopped-flow instrument. The experiment was performed by mixing the NADPH  and the enzyme or enzyme plus substrate solutions . Analysis of the resulting kinetic data was performed with Kinetic studio V3 software . In order to calculate the NADPH consumption rate, the plot of absorbance at 340 nm vs. time was fitted to a single exponential decay function.Rates of NADPH oxidation were measured using a Hi-Tech scientific SF-61 single mixing stopped-flow system  at 25°C in a glovebox  with an oxygen concentration below 10 ppm. NADPH consumption was monitored at 340 nm (ε = 6.22 mMEm = −440 mV), benzyl viologen (Em = −359 mV), safranine O (Em = −280 mV), anthraquinone-2,6-di sulfonic acid (Em = −185 mV), phenazinemethosulfate (Em = +80 mV), methylene blue (Em = +11 mV). The modified cuvette was flushed with oxygen-free nitrogen before and during the measurements. After equilibration at each potential, the optical spectrum was recorded. Reduction of the heme in 3A4-BMR fusion proteins was followed by the increase in the absorption at 550 nm related to the merging of Q-bands, using a diode array spectrophotometer . The fraction of reduced heme, proportional to the measured spectral transition, was plotted against potential, and the data were fitted to the Nernst equation:ox] and [red] are the concentrations of oxidized and reduced heme, respectively, E is the solution reduction potential at equilibrium, n is the number of electrons, Em is the midpoint potential, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.Potentiometric titrations were performed in an anaerobic glovebox  with an oxygen concentration below 10 ppm using a cuvette equipped with a gold laminar working electrode and an Ag/AgCl reference electrode connected to a potentiostat controlled by GPES3 software . Ambient reduction potentials were adjusted by addition of aliquots (2–5 μL) of anaerobically freshly prepared sodium dithionite solution. Titrations were performed in 50 mM potassium phosphate, pH 8. Electrode-solution mediation was facilitated by adding the following mediators at 5 μM concentration: methyl viologen  as described before  equipped with a 4.6 × 250 mm, 5 μm C18 column. Testosterone and 6β-hydroxytestosterone were separated using a gradient elution programmed as follows: isocratic elution of 20% acetonitrile and 80% water, 0–2 min; linear gradient elution from 20 to 90% acetonitrile and from 80 to 10% water, 2–20 min; linear gradient elution from 90 to 20% acetonitrile and from 10 to 80% water, 20–22 min; isocratic elution of 20% acetonitrile and 80% water 22 min-end of the run. The flow rate was set at 1 mL/min and detection wavelength was 244 nm. Retention times were 9.2 min and 13.1 min for 6β-testosterone and testosterone, respectively. Coupling efficiency was calculated as the ratio of 6β-hydroxytestosterone produced and the total amount of NADPH consumed by the chimeras.c reduction activity spectrophotometric assay was performed as described by Guengerich et al. (c (in 0.1 M potassium phosphate buffer pH 8.0) were pipetted into a 0.5 mL quartz cuvette (1 cm path-length). Then, 0.1 μM of chimeric proteins were added and gently mixed. The assay was started by adding NADPH to a final concentration of 75 μM. Using the kinetic mode of the spectrophotometer, the absorbance increase at 550 nm upon cytochrome c reduction was monitored for 5 min. The rate of cytochrome c reduction was calculated using reduced cyt c extinction coefficient at 550 nm,7.04 mM−1cm−1, as nmol of reduced cyt c per min. The experiment was carried out both in the absence and in the presence of 20% glycerol.Cytochrome h et al.  with slipost hoc test using SigmaPlot software.All collected data were recorded at least in triplicate and expressed as mean ± standard deviation. The comparison of the results obtained was performed by one way ANOVA and two way ANOVA followed by Student-Newman-Keuls After expression and purification Figure , the UV-Full reduction of the chimeras was achieved by addition of excess sodium dithionite leading to the hydroquinone forms with a concomitant decrease in the intensity in the visible region spectra , both in the presence and in the absence of substrate. Moreover, the presence of both erythromycin and testosterone significantly enhances NADPH consumption rate in all the examined fusion proteins. This effect is probably due to the fact that the substrate-bound complex has a higher reduction potential and is capable to accept electrons from the redox partner more easily leading to a faster reduction of the heme-Fe3+ to heme-Fe2+  to the reduced (Fe2+) state upon addition of sodium dithionite reducing agent was monitored by observing the spectral transition related to the merging of the Q-bands in a single band at 550 nm. Reduction potential values were calculated by plotting the calculated 3A4-BMR reduced fraction vs. the recorded potential using the Nernst's equation. Midpoint potential values were calculated to be −367±3 mV, −327±8 mV, and −397±8 mV for 3A4-BMR, 3A4-3GLY-BMR and 3A4-5GLY-BMR, respectively.Reduction potentials of the heme were determined with anaerobic spectro-potentiometric titrations of the chimeras using the method described by Dutton . The chaThe differences observed in NADPH oxidation and reduction potentials indicate that the loops indeed have an impact on these parameters and therefore also different functional performace in catalysis are expected.−1, whereas 3A4-BMR  when compared to the 3A4 alone.In order to study if and how the length of the loop has an impact on the stability of the P450 catalytic module, the time dependent decrease of the absorbance at 450 nm upon reduction and CO binding was used to compare the relative stability of P450 proteins (Lin et al., R Figure , 3A4-3GLR Figure , and 3A4R Figure  show a hM-values of 3A4-BMR, 3A4-3GLY-BMR, and 3A4-5GLY-BMR were 20.8 ± 3.6, 27.4 ± 4.0, and 21.3 ± 4.4 μM respectively. In this case no statistically relevant differences were found. These results show that the glycine linker length does not influence 3A4-BMR affinity for erythromycin substrate, indicating that the binding pockets of the heme domain of the chimeras are not perturbed.Turnover of the different chimeras was studied in the presence of the substrates erythromycin and testosterone using NADPH as electron source, and the results are reported in Table max-values of 0.0515 ±.0004, 0.1177 ± 0.0006, and 0.1387 ± 0.0009 nmol min−1 for 3A4-BMR, 3A4-3GLY-BMR, and 3A4-5GLY-BMR (Figure max for 3A4-3GLY-BMR and 3A4-5GLY-BMR with respect to the 3A4-BMR construct was observed. This shows that the glycine linker increases the enzyme turnover rate in the presence of erythromycin substrate. Moreover, a significant increase in Vmax-value was found for 3A4-5GLY-BMR compared to 3A4-3GLY-BMR.Further analysis in terms of turnover rate led to VR Figure , respectM-values obtained from fitting the Michaelis-Menten curves of 3A4-BMR, 3A4-3GLY-BMR, and 3A4-5GLY-BMR were 19.6 ± 2.6, 21.3 ± 2.7, and 19.8 ± 2.2 μM respectively, indicating again no statistically relevant differences in the binding pocket. The Vmax-values were 0.0066 ± 0.0002, 0.0089 ± 0.0002, and 0.0113 ± 0.0003 nmol min−1 respectively for 3A4-BMR, 3A4-3GLY-BMR, and 3A4-5GLY-BMR enzymes (Figure max results, a statistically significant increase was found for 3A4-3GLY-BMR and 3A4-5GLY-BMR with respect to the 3A4-BMR construct. Also in this case the glycine linker increases the enzyme turnover rates, with a significant higher Vmax-value for 3A4-5GLY-BMR compared to 3A4-3GLY-BMR.Also the testosterone 6β-hydroxylation rates were calculated at increasing concentrations of the substrate in the presence of all the 3A4-BMR constructs, and the results were fitted to the Michaelis-Menten model in order to calculate kinetic parameters Figure . The KM-s Figure . As obseThe coupling efficiency percentage during erythromycin N-demethylation, calculated as ratio of HCHO formed and NADPH consumed, were found to be 9.4 ± 0.5, 10.0 ± 0.8, and 12.5 ± 0.9% for 3A4-BMR, 3A4-3GLY-BMR, and 3A4-5GLY-BMR, respectively Figure . When pec was therefore used as external probe to check its ability to intercept the electrons of NADPH delivered by the BMR when this is exposed and hence available to interact with proteins other than the 3A4 domain. The ability of cytochrome c to be reduced by NADPH via free cytochrome P450 reductase has already been described in the literature (Guengerich et al., The results from enzymatic activity, coupling and reduction potentials indicate that the different loops affect the interaction between the BMR and 3A4 domains of the chimeras, probably due to the different degrees of length and flexibility associated with them. Cytochrome c is strictly related to cytochrome c ability to interact directly with the BMR domain when this is exposed. In order to explore the effect of viscosity on the interaction of cytochrome c with the chimera BMR domain, we performed the experiments both in the absence and in the presence of 20% glycerol. The reduction rates were calculated as nmoles of reduced cytochrome c per min and the results are summarized in the Figure The capability of 3A4-BMR chimeras to use NADPH electrons to reduce cytochrome c reduction rate compared to the other two proteins. Moreover, the presence of 20% glycerol significantly impaired cytochrome c reduction ability of all the three constructs, with the 3A4-5GLY-BMR being the most affected one.The 3A4-5GLY-BMR chimera shows the highest cytochrome Cytochromes P450 have been extensively used in our laboratories to investigate their potential for applications in biocatalysis as electron transfer domains in chimeric constructs (Cunha et al., 3+ to heme-Fe2+ (Sligar et al., The stability of the P450 catalytic domain is significantly improved in the presence of the BMR domain since all the three fusion proteins showed a 1.5–2 fold lower rate of P450 loss compared to P450 3A4. In this case, the presence of an extra domain rather than the length of the loop is essential for stability as no significant differences are observed among the three proteins. However, the length of the loop has an impact on other parameters, such as NADPH oxidation rates and reduction potential. In particular, the chimeric proteins show a higer NADPH consumption rate compared to the CYP3A4-hCPR system at a 1:1 molar ratio. Moreover, the 3A4-3GLY-BMR protein shows a 2-folds faster rate in NADPH consumption with respect to the other two constructs. This effect suggests that the 3GLY construct is more prone to accept electrons from the redox partner with respect to no GLY and 5GLY leading to a faster reduction of the heme-FeThe fastest oxidation rate in the 3GLY protein is explained by its more positive reduction potential compared to the other two fusion proteins. It is known that in redox proteins, the interaction with the redox partner can influence the conformation, the reduction potential and in general, the dielectric environment of the redox cofactor Bendall, . Here, tM-values measured for both erythromycin and testosterone substrates are similar for the three proteins and within the range of previously published data (Riley and Howbrook, When investigating the catalytic domain ability to use reducing equivalents for erythromycin N-demethylation and testosterone 6β-hydroxylation during the catalytic event, the 5GLY construct resulted the best for both turnover rate and coupling efficiency. Thus, the increase in turnover number and coupling efficiency is neither directly related to an improvement in electron transfer rate nor to a more positive heme iron reduction potential. Differences in substrate binding can also be excluded as the Kc was used as external probe to check its ability to intercept the electrons of NADPH delivered by the BMR when this is exposed and hence available to interact with proteins other than the 3A4 domain. The 5GLY resulted in the fastest in cytochrome c reduction and this result can be explained by the highest flexibility between the reductase and catalytic domain of the chimera allowing a better interaction with cytochrome c and an easier electron delivery from BMR reductase domain (Figure c reduction rate in all the three constructs, with a more marked effect on the more flexible for 5GLY loop.In order to investigate if there is a correlation between the observed improved turnover/coupling efficiency and an increased inter-domain flexibility in the 5GLY protein, cytochrome n Figure . This inTaken together, the erythromycin N-demethylation and testosterone 6β-hydroxylation activity and the coupling results show that the 3A4-5GLY-BMR chimera is the more catalytically efficient and the more coupled. This result suggests that the higher linker flexibility allows a more proficient arrangement of catalytic and reductase domains and improves the tuning of the electron transfer to the active site leading to a more accurate electrons delivery from the NADPH electron source during the catalytic event Figure .The creation of chimeric proteins was shown before to improve the solubility of P450 3A4 and to generate catalytically self-sufficient enzymes (Degregorio et al., This molecular design approach can be successfully developed for the obtainment of performing enzymatic paradigms suitable for biotechnological applications. The next step is the introduction of such catalysts in bacterial cells for the large-scale production of drug metabolites using whole cells as a less expensive system.DD performed the catalysis experiments on Erythromycin. SD performed the catalysis experiments on Testosterone. SC performed the uncoupling experiments, redox potential determination and cyt c interaction. GDN and GC performed the modeling of the chimeras. SS and GG interpreted the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of  Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium’s resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates. Burkholderia pseudomallei and melioidosis, the disease that it causes, were traditionally documented mainly in northern Australia and Southeast Asia, it has now been established that the bacterium is endemic to many parts of the tropics, including the Americas, the Indian subcontinent, other parts of Asia, and Africa -sulfamethoxazole (SMX) combination co-trimoxazole (SXT) is considered the standard for melioidosis eradication-phase therapy and is also the drug of choice for postexposure therapy , encoded by folA, and dihydropteroate synthetase (DHPS), encoded by folP , FolM likely provides the essential tetrahydromonapterine cofactor for PhhA  that is annotated as ptr1, forms an operon with folE . Ma. MaB. psin vitro . This, hin vitro . FolM isro state . Leishma folates . In bactfor PhhA . In thesbacteria  17). B.. B.B. psith folE  18), an, anB. ps10.1128/mBio.01357-17.5FIG S4 Burkholderia pseudomallei folE encodes GTP cyclohydrolase I. Download FIG S4, PDF file, 0.1 MB.Copyright © 2017 Podnecky et al.2017Podnecky et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the r) bacteria (19–r in a range of Gram-positive and Gram-negative bacteria (r) in B. pseudomallei has been controversial, but recent studies indicate that primary resistance to this drug is uncommon (<1%)  , 24. To Pseudomonas aeruginosa that B. pseudomallei BpeEF-OprC, an efflux pump of the resistance nodulation cell division (RND) family, extrudes chloramphenicol and TMP , 31P. aeC operon . The neeB. pseudomallei infections, the focuses of this study were (i) to determine and characterize the mechanisms leading to decreased SXT susceptibility using attenuated excluded  select-agent strain Bp82  to provide a possible explanation as to why primary SXTr is uncommon.Because of the pivotal role that SXT plays in treatment and prophylaxis of ain Bp82 , (ii) tor cutoff for resistance (bpeF (>30-fold) and bpeT (~2-fold) mRNA expression in strains Bp82.102 and Bp82.103 , Bp82.102, Bp82.103, and Bp82.104, were selected for further testing and characterization  in bpeT resulting in amino acid substitutions in the carboxy-terminal domain of BpeT, namely, C310R in Bp82.102 and L265R in Bp82.103  and Bp82.269 (BpeTL265R), had bpeF mRNA expression profiles similar to those found in the original TMPr isolates  factors other than mutations in bpeT contribute to the increased TMP, SMX, and SXT resistance levels observed in the selected Tmpr strains.To determine if changes to Bp82.103 . Strain isolates . MIC tesisolates . Howeverisolates  that werr strain Bp82.104 did not contain any bpeT mutations and did not overexpress BpeEF-OprC. To identify other potential mechanisms of resistance, the folA gene encoding DHFR, the TMP drug target  and Bp82.184 (folAI99L). Both folA mutations caused TMPr in the engineered mutant strains  or strain Bp82 ΔbpeT. Twelve resulting TMPr isolates capable of growth on 16 µg/ml TMP were sequenced to identify folA mutations. All had the FolAI99L mutation that was identified in Bp82.104, possibly due to clonal expansion (data not shown). Together, these findings suggest that folA target mutations may be a common cause of or contributor to acquired TMPr in the presence or absence of BpeEF-OprC efflux pump expression or in other strongly selective environments.To assess the propensity for selection of bpeT in transcriptional control of this efflux pump is not well understood. The genetic organization and substrate specificity of the P. aeruginosa MexEF-OprN efflux pump, which is positively regulated by the LysR-type regulator MexT, and those of BpeEF-OprC are similar, which led us to suggest that they may be regulated in analogous fashions   . The SXTctively) . DeletioSXT MICs , and subisolates . A possi10.1128/mBio.01357-17.8TABLE S3 TABLE S3, PDF file, 0.1 MB.The BpeEF-OprC efflux pump is required for increased folate inhibitor resistance in laboratory strains and clinical isolates. Download Copyright © 2017 Podnecky et al.2017Podnecky et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the bpeT and bpeF mRNA in the six isolates (Bp82.191 to Bp82.207) with reduced SXT susceptibility both with and without the BpeT transcriptional regulator. Expression of bpeT mRNA was detected in all six isolates relative to Bp82, but intraisolate bpeT expression level differences were not statistically significant   , a gene otation) . This idBP1026B_II0040  gene, which is annotated as ptr1 (pteridine reductase 1). This gene likely corresponds to the folM gene found in other bacteria and is referred to as folM from this point on  strains Bp82.246 and Bp82.249; repair of folMV15G resulted in folM+ strains Bp82.247 and Bp82.250; and repair of both bpeSK267T and folMV15G resulted in bpeS+folM+ strains Bp82.248 and Bp82.251. MIC testing indicated that loss of either SNP differentially restored susceptibility to TMP, SMX, and SXT (bpeS gene (Bp82.246 and Bp82.249) caused a greater reduction in MIC, where the susceptibilities of the repaired mutants matched that of the parental strain, Bp82. Repair of the folM gene (Bp82.247 and Bp82.250) reduced the SMX MIC from the detection limit of 1,024 μg/ml to 32 μg/ml and the SXT MIC from 4 to 6 μg/ml to 0.75 μg/ml but did not affect the TMP MIC, which remained above the ≥32 μg/ml detection limit. For unknown reasons, repair of both SNPs (Bp82.248 and Bp82.251) resulted in MICs below those of the original Bp82 parent strain.To confirm the role of the identified mutations in  and SXT . Repair bpeSK267T or folMV15G or both. Introduction of bpeSK267T into bpeSWT strain Bp82 resulted in >100-fold increased expression of bpeF, and, as expected, there was no change in bpeF expression seen with the introduction of the folMV15G mutation (bpeF expression patterns were observed in Bp82.202 and Bp82.204 with neither mutation repaired and in their derivatives that contain only the folMV15G mutation (Bp82.247 and Bp82.250)  .llpE-bpeE-bpeF-oprC operon transcription. To this end, mini-Tn7 vectors were constructed where bpeS was constitutively expressed from the P1 promoter. The mini-Tn7-P1-bpeS construct was transposed into ΔbpeS strain Bp82.264 to create Bp82.289 or into ΔbpeS ΔbpeT strain Bp82.286 to create Bp82.288. RT-qPCR analyses indicated that bpeS mRNA levels in these strains were increased ~12-fold compared to the Bp82 results (data not shown). However, this BpeS overexpression affected neither the expression of bpeF nor the host strain’s susceptibility to TMP, SMX, or SXT  and a ΔbpeS ΔbpeT strain containing the same construct (Bp82.321) overexpressed BpeEF-OprC with bpeF mRNA levels >100-fold higher than those seen with Bp82 (bpeEF-oprC transcription independently of the presence of a coinducer), rather than an increase in bpeS transcription, is responsible for differential levels of expression of the llpE-bpeE-bpeF-oprC operon.Due to the high degree of similarity between BpeT and BpeS, we assessed the role of BpeS in BpeEF-OprC expression specifically via activation of , or SXT . In contith Bp82 . This ovith Bp82 . These rr strain Bp82 derivatives also contained mutations in SXTr clinical isolates, we analyzed genes of the few SXTr strains identified to date by targeted sequencing or genome sequencing or both. Strain pair 354b (SXTs) and 354e (SXTr) represents two sputum isolates obtained from the same patient 75 months apart (folM which frameshifts FolM after amino acid 20 (s) and MSHR663 (SXTr) are sequential isolates obtained from a relapse melioidosis patient in Darwin, Australia  or a Bp82 ΔbpeT ΔbpeS strain (Bp82.321) led to high-level bpeF mRNA expression  and DHPS (FolP) drug targets , 20B. psn Bp1651 , had preIn vitro exposure to either TMP or SXT selects for regulatory mutants that constitutively express BpeEF-OprC. However, the natures of the regulatory mutations and the levels of efflux pump expression selected by the two drugs are different and this differential regulation of expression determines the drug resistance profile. Exposure to TMP alone selected for mutations affecting the putative carboxy-terminal effector-binding domain of BpeT. These are likely activating mutations that relieve the protein’s dependence on interaction with an unknown coinducer, possibly a pump substrate. Constitutive BpeEF-OprC expression in the TMPr isolates was accompanied by differences in the SMX MICs, but this level of expression is insufficient to cause SMXr and thus to cause clinically significant SXTr. Exposure to increasing concentrations of SXT led to SXT resistance. This resistance is largely due to constitutive overexpression of BpeEF-OprC, likely by activated mutant BpeS. The underlying mutations affect either the carboxy-terminal domain or the amino-terminal helix-turn-helix DNA-binding domain of BpeS. The constitutive overexpression of the BpeEF-OprC efflux pump mainly confers TMP resistance but also confers resistance to SMX and thus to SXT.bpeEF-oprC gene expression, at present we do not fully understand the involvement of two LysR-type regulators in regulation of BpeEF-OprC expression. What we do know is that neither regulator is essential for control of bpeEF-oprC gene expression. If either bpeT or bpeS were an essential activator of pump gene expression, loss of one or both regulatory genes would result in abrogated pump expression, which is not what we observed in induction studies. These studies showed that bpeF expression was induced to similar levels in the wild type and in a bpeS bpeT double mutant by the BpeEF-OprC substrates chloramphenicol and doxycycline . Thus, both regulators and their mutant versions affect transcription of the llpE-bpeE-bpeF-oprC operon directly, but likely independently, which suggests that they might activate BpeEF-OprC expression in response to different stimuli. This idea is especially interesting when combined with the fact that RND efflux systems such as BpeEF-OprC are adaptation mechanisms needed for response to environmental stressors other than antimicrobials  with increased SXT resistance also contained mutations in the annotated ive DHFR , subsequive DHFR . The phyive DHFR  Fig. 1)ptr1 geneynthesis . Tetrahyynthesis . In thissistance . Our datr strains are corroborated by data obtained with clinical B. pseudomallei isolates. First, it has been postulated that the previously introduced B. pseudomallei SXTr clinical isolate 354e likely expresses BpeEF-OprC due to a truncation of the BpeT carboxy terminus, and this strain has been shown to exhibit an SXTr phenotype  containing 5 g/liter NaCl or cation-adjusted Mueller-Hinton II broth or agar media . Bacterial growth medium was supplemented with adenine  for the growth of Bp82 and its derivatives as follows. LB broth or agar was used with 80 μg/ml Ade and MH broth or agar with 40 μg/ml Ade. E. coli strains DH5α  following the guidelines provided by the Etest manufacturer . The MICs of other antibiotics were determined by the standard broth microdilution method, following Clinical and Laboratory Standards Institute (CLSI) guidelines  for expression of the bpeEF-oprC operon from the inducible Ptac promoter . Similarly, spontaneous mutants with reduced SXT susceptibility were selected by serial passage in LB with increasing concentrations of SXT. Briefly, Bp82 was grown overnight and then subcultured at a dilution of 1:100 into LB containing 0.064 μg/ml SXT (~1 times the MIC). The bacteria were successively subcultured into fresh LB with 4-fold increases in SXT, ending at 8 μg/ml SXT. Isolated colonies that grew on LBA with 8 μg/ml SXT were patched onto LBA with 16 μg/ml SXT to confirm the regrowth with SXT.TMPTaq DNA polymerase High Fidelity  and specifically designed primer sets  or B. pseudomallei K96243 (NC_006350.1 and NC_006351.1). SNP positions identified were required to have >10× coverage depth and >90% variant base calls.Whole-genome sequencing of Bp82 laboratory strains and clinical isolates was performed by paired-end sequencing using an Illumina GAIIx Genome Analyzer  and a Kapa Biosystems library preparation kit  protocol with an 8-bp index modification. Details of library preparation, sequencing, and data analysis are provided in E. coli RHO3 mobilizer strain as previously described  and 300 to 1,000 μg/ml kanamycin (Kan). Merodiploids were resolved by the use of sucrose  counterselection or, in some cases, both sucrose- and I-SceI-mediated counterselection (FRT)-flanked Kan or gentamicin resistance markers in deletion mutants were removed by Flp recombinase-mediated excision using the pFLPe2 plasmid or the pFLPe3 plasmid, as previously described  originating from strain 1026b or Bp82 utilizing the mini-Tn7 system, which allows stable and site-specific single-copy insertions into the B. pseudomallei genome at three possible glmS-associated attTn7 sites   .P1 promoter  (7T-Km (kanamycin resistance marker) (7 elements were transposed into the chromosomes of the ΔbpeT (Bp82.87) strain, the ΔbpeS (Bp82.264) strain, or the ΔbpeT ΔbpeS strain (Bp82.286).The promoter  was used marker)  or pUC18 marker)  as descr600 = 0.6 to 0.8) in LB. RNA was isolated using an RNeasy Protect Bacteria minikit  in biological triplicate, as previously described (P values of <0.05 were considered significant.Expression levels of mRNA of target genes were analyzed in bacteria grown to mid-log phase (ODescribed . Relativescribed  in techn10.1128/mBio.00356-17, doi:10.1128/genomeA.00254-15, doi:10.1371/journal.pone.0036507, and and NCBI BioProjects PRJNA397943 and PRJNA398084.Whole-genome sequencing data are available from NCBI and published sources, e.g., doi:


In this review, we discuss the role of TNF in graft-versus-host disease as an example of the ambivalence of this cytokine in the pathophysiology of an immunopathology, highlighting the therapeutic potential of triggering TNFR2 to boost Treg expansion. We also describe new targets in immunotherapy of cancer, emphasizing on the putative suppressive effect of TNF in antitumor immunity and of the interest of blocking TNFR2 to regulate the Treg compartment.Tumor necrosis factor α (TNF) is a potent pro-inflammatory cytokine that has deleterious effect in some autoimmune diseases, which led to the use of anti-TNF drugs in some of these diseases. However, some rare patients treated with these drugs paradoxically develop an aggravation of their disease or new onset autoimmunity, revealing an immunosuppressive facet of TNF. A possible mechanism of this observation is the direct and positive effect of TNF on regulatory T cells (Tregs) through its binding to the TNF receptor type 2 (TNFR2). Indeed, TNF is able to increase expansion, stability, and possibly function of Tregs  Tumor necrosis factor α is a pleiotropic cytokine produced by various cell types and involved in a wide range of pathological processes . It Tumor necrosis factor α binds to two receptors, namely, TNF receptor type 1 (TNFR1) and TNFR2 Figure . SeveralTNFR2 expression is upregulated in activated Tregs and can be detected in activated conventional T cells (Tconvs), although at lower levels than in activated Tregs. Some other members of the TNFR family, such as GITR, OX40, or 4-1BB, are also preferentially expressed by Tregs, and their expression is also upregulated upon activation . Remarkain vitro and has been reviewed elsewhere , a systemic and highly inflammatory complication that occurs after allogeneic hematopoietic stem cell transplantation  . TNF indThe key role of TNF in aGVHD pathophysiology logically led researchers and physicians to try to block this cytokine to decrease inflammation and consequently to prevent or treat aGVHD. Along this line, most of the clinical trials focused on two molecules: infliximab—a monoclonal antibody (mAb) that binds TNF—and etanercept—a soluble TNFR that competes with cellular receptors for TNF binding. The great hope risen by TNF targeting in aGVHD during the first decade of this century has unfortunately faded rapidly due to somewhat disappointing results of clinical studies. Indeed, clinical trials failed to prove any benefit in adding infliximab to standard treatment, both for aGVHD prophylaxis and treatment , 53. OnlRegarding etanercept, a single center prospective study showed a promising response rate when combining etanercept with standard high-dose corticosteroids for first-line treatment of aGVHD compared with a cohort of contemporaneous case-matched patients treated with high-dose corticosteroids alone . HoweverRegulatory T cells modulate alloreactivity during allo-SCT. Cell therapy using Tregs efficiently control GVHD , 68 wherIn a murine model of aGVHD prevention relying on Treg infusion, we have clearly shown using three different experimental approaches that the protective effect mediated by therapeutic Tregs was dependent on TNF produced by pathogenic Tconv and TNFR2 expressed by Tregs . Indeed,In vitro, STAR2 was able to stimulate expansion and activation of Tregs, an effect not observed with Tconvs. This selective Treg expansion and activation was also triggered in vivo, when mice were treated with STAR2 intraperitoneal injections. Most of all, in a murine model of aGVHD, pretransplant administration of STAR2 to recipient mice protected from aGVHD and significantly increased survival. The protective effect of STAR2 was associated with a preserved GVL effect and had no deleterious effect on posttransplant anti-cytomegalovirus immune reconstitution.The second study was published by Chopra and colleagues, who developed a TNFR2 agonist called STAR2 . In vitrin vitro with TNF (+IL-2) for a short period , 7675, 7in vivo (Most of the abovementioned treatments are thought to work mainly by (re)-activating the cytotoxic arm of the immune response –102, namin vivo .Foxp3 regulatory sequences, resulted in a partial regression of established melanoma that correlated with CD8+ T cell accumulation in the tumor. Furthermore, mouse studies point out that anti-CTLA-4 antibodies mainly act by eliminating or inhibiting the tumor-associated Tregs (which highly express this molecule) rather than by reinvigorating exhausted T cells  promote Treg accumulation and expansion, (ii) enhance Treg function, and even (iii) induce Treg conversion from conventional CD4+ T cells . Among tOn top of the abovementioned strategies designed to disarm Tregs for therapeutic aims, “cytokine starvation or cytokine deviation” represents an alternative promising approach. Namely, deprivation of Tregs from IL-2 and TNF—two key cytokines essential for their biology—should lead to Treg dysfunction or death. Clinical manipulation of IL-2 levels remains complex as IL-2 can act both as an immune stimulating or suppressive cytokine, depending on the dose. On one hand, low-doses of IL-2 favor Treg survival and suppressive function and lead to a better control of autoimmune and inflammatory diseases –131. On As suggested by its name, TNF was described initially as a killer of cancer cells. We now know that this cytokine plays a complex role in cancer and tumor immunity because of its pleiotropic effect and the fact that it has two receptors. Actually, TNF is even considered mostly as a pro-tumor cytokine. Numerous mouse studies have shown that anti-TNF drugs reduced tumor growth in different types of cancers. This deleterious effect of TNF was further supported in TNF knockout mice that display reduced tumor growth –146. TheThe pro-tumoral effect of TNF has been explained by different mechanisms Figure . TNF mayIn mouse models of cancers, reduced tumor growth in mice treated by anti-TNF drugs or in TNFR2 knockout mice was associated with decreased numbers of MDSCs suggesting that TNF increases survival, recruitment, or function of MDSCs that suppress antitumor immunity Figure  20, 140, 151. InIn the setting of hematologic tumor relapse after allo-SCT, a similar approach using an anti-TNFR2 blocking mAb or TNFR2 antagonist could be considered to inactivate the deleterious effect of Tregs Figure B. These in vitro or in vivo after xeno-transplantation in immunodeficient mice. Results indicated that blocking TNF may reduce tumor growth, which is associated with reduced tumor dissemination, angiogenesis, and infiltration with myeloid cells , the possibility that TNF inhibits antitumor immunity by boosting Tregs is a very attractive hypothesis that may play a major role in some cancer types.It has to be emphasized that these studies that provide possible mechanisms to explain the supratumoral effect of TNF were only based on correlations or Immunotherapy of cancers is a promising land but unfortunately only a minority of patients responds to these treatments. Among multiple targets that are being tested, TNFR2 is an attractive one. Indeed, TNF blockade may have different impacts by limiting cell transformation, neovascularization, or pro-tumoral inflammation and may boost antitumor immunity by acting on MDSC or Tregs. Recent works suggest that targeting TNFR2-expressing Tregs would be a safe and efficient way to stimulate antitumor immunity. Future experiments and clinical trials are required to validate this new therapy.BS, EP, and JC organized the plan and structure of the manuscript, and all the authors contributed to the redaction.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AHR) in asthma. Conversely, omega‐3 fatty acid derivatives have been shown to resolve inflammation in various tissues. Thus, the effects of docosapentaenoic acid monoacylglyceride (MAG‐DPA) were assessed on inflammatory markers and reactivity of human distal bronchi as well as in a cultured model of guinea pig tracheal rings. Human bronchi were dissected and cultured for 48 h with 10 ng/mL TNF‐α or IL‐13. Guinea pig tracheas were maintained in organ culture for 72 h which was previously shown to trigger spontaneous AHR. All tissues were treated with increasing concentrations of MAG‐DPA . Pharmacomechanical reactivity, Ca2+ sensitivity, and western blot analysis for specific phosphoproteins and transcription factors were performed to assess the effects of both cytokines, alone or in combination with MAG‐DPA, on human and guinea pig airway preparations. Although 0.1 μmol/L MAG‐DPA did not significantly reduce inflammatory biomarkers, the higher concentrations of MAG‐DPA (0.3 and 1 μmol/L) blunted the activation of the TNF‐α/NFκB pathway and abolished COX‐2 expression in human and guinea pig tissues. Moreover, 0.3 and 1 μmol/L MAG‐DPA consistently decreased the Ca2+ sensitivity and pharmacological reactivity of cultured bronchial explants. Furthermore, in human bronchi, IL‐13‐stimulated phosphorylation of CPI‐17 was reversed by 1 μmol/L MAG‐DPA. This effect was further amplified in the presence of 100 μmol/L aspirin. MAG‐DPA mediates antiphlogistic effects by increasing the resolution of inflammation, while resetting Ca2+ sensitivity and contractile reactivity.Bronchial inflammation contributes to a sustained elevation of airway hyperresponsiveness ( AHRairway hyperresponsivenessASMairway smooth muscleCPI‐17protein kinase C‐dependent phosphorylation of the 17 kDa myosin phosphatase inhibitor proteinHBhuman bronchiMAG‐DPAdocosapentaenoic acid monoacylglyceridePUFApolyunsaturated fatty acidαTNF‐tumor necrosis factor alphan−3 PUFA levels are decreased in airway illnesses which are characterized by excess airway inflammation and enhanced n6‐PUFA, including arachidonic acid derivatives, as in the case of asthma  can alter Ca2+ homeostasis in ASM and render cells nonspecifically overreactive to pharmacological agonists   are limited , eicosapentaenoic acid , and docosapentaenoic acid  have been shown to prevent autoimmune diseases  is an intermediary elongated fatty acid between EPA and DHA  , histamine, aspirin, and β‐actin antibodies were purchased from Sigma . U‐46619 as well as TNF‐α and COX‐2 antibodies were obtained from Cayman Chemical . PPARγ and P‐NFκB antibodies as well as IL‐13 cytokine were purchased from Cell Signaling Technology , anti‐CPI‐17 and anti‐phospho‐CPI‐17 antibodies were purchased from Cedarlane . GPR‐32 antibody was purchased from Abcam . DMEM/F12 and penicillin–streptomycin were obtained from Gibco Invitrogen Corp. .MAG‐DPA was synthesized and purified by SCF‐Pharma . TNF‐N = 15) hospitalized for lung carcinoma were recruited and human lung tissues were obtained from the operating room via the Department of Pathology. “Tumor‐free” tissue samples were transported to our laboratory in sterile Krebs' solution, pH 7.4. Distal bronchi samples were dissected under binocular control, and bronchial segments of 4 mm in length and 0.5–0.8 mm in diameter were cultured for 48 h in Dulbecco's modified Eagle's medium: nutrient mixture‐F12 (DMEM‐F12) containing 1% penicillin/streptomycin . Airways were either untreated (control) or treated with 10 ng/mL TNF‐α, TNF‐α + 0.1 μmol/L MAG‐DPA, TNF‐α + 0.3 μmol/L MAG‐DPA, or TNF‐α + 1 μmol/L MAG‐DPA. Other sets of human bronchi were pretreated with 10 ng/mL IL‐13 or IL‐13 combined with 1 μmol/L MAG‐DPA or 1 μmol/L MAG‐DPA + 100 μmol/L acetylsalicylic acid (ASA). All tissues were incubated at 37°C in 5% CO2. The n values for each experimental condition were reported in the figure legends.This study was approved by our Institutional Ethics Committee (Protocol No.: CRC05‐088‐S1‐R6). Consenting patients  were anesthetized by intraperitoneal injection of a lethal dose of sodium pentobarbital (40 mg/kg). All procedures involving animal tissues were performed according to Canadian Council for Animal Care (CCAC) guidelines (protocol number 018–12). The trachea was then sectioned into eight tubular segments of equal length and each main bronchus was dissected into two sections. The tissues were used directly after dissection (fresh) or placed in individual wells of a culture plate containing DMEM‐F12 culture medium  supplemented with 1% penicillin/streptomycin  or 80 mmol/L KCl for each tested explant  and permeabilized at 25°C with 50 μmol/L β‐escin. Tension developed by the permeabilized bronchial rings was measured at 37°C according to free Ca2+ concentrations expressed in terms of pCa (pCa = −log [Ca2+]). Step increases in free Ca2+ (from pCa = 9.0 to 5.3) induced reproducible concentration–response curves to free Ca2+ concentrations, indicating successful permeabilization under these conditions . The latter prevented the dephosphorylation of regulatory proteins. Tissues were homogenized at 4°C and centrifuged at 1500g in the same buffer. The nuclear, cytosolic, and microsomal fractions were prepared as described previously : 300 sucrose, 20K‐PIPES, 4K‐EGTA, pH 7.2, and a cocktail of protease  and phosphatase inhibitors . Differences were considered statistically significant when *P < 0.05. Data curve fittings were performed using Sigma Plot 10  to determine EC50 values.Results are expressed as means ± SEM with α pretreated human bronchi in the absence or presence of MAG‐DPA, cumulative concentration–response curves to pharmacological agonists such as methacholine (MCh) and histamine (His) were measured. Figure α‐treated human bronchi in the absence or presence of either 0.3 or 1 μmol/L MAG‐DPA. CCRC demonstrated that 48 h pretreatment with 10 ng/mL TNF‐α induced a hyperresponsiveness to MCh with an apparent EC50 value (μmol/L) and a mean maximal tension  of 0.09 μmol/L and 1.15 ± 0.07 g comparatively to 0.05 μmol/L and 0.23 ± 0.05 g in control conditions. Conversely, treatment with either 0.3 μmol/L or 1 μmol/L MAG‐DPA significantly reduced the mean amplitudes of 0.70 ± 0.03 g and 0.60 ± 0.03 g, respectively.To investigate the pharmacomechanical properties of TNF‐α significantly increased the responsiveness to this agonist and reduced the apparent EC50 value and MT to 0.5 μmol/L and 1.2 ± 0.07 g compared to control conditions   was able to reverse this overreactivity induced by TNF‐α. Hence, despite the presence of TNF‐α, the mean agonist‐induced tone in the presence of 1 μmol/L MAG‐DPA was close to the tone developed in control condition. Thus, μM concentrations of MAG‐DPA were able to blunt TNF‐α‐induced airway hyperresponsiveness to U‐46619.To assess the putative proresolving effect of MAG‐DPA on pre‐established inflammation, human bronchi were cultured either in the absence (control) or presence of 10 ng/mL TNF‐ist Fig. A as thisκB (P‐NFκB) and COX‐2 were analyzed by western blots in the nuclear fractions of TNF‐α‐treated human bronchi. TNF‐α treatment resulted in increased detection levels of P‐NFκB and COX‐2 protein when compared to mean levels obtained from control bronchi. MAG‐DPA treatments decreased the phosphorylation level of p65 NFκB and COX‐2 in lung tissues comparatively to the levels observed in TNF‐α‐treated tissues .To determine the molecular pathways involved in MAG‐DPA anti‐inflammatory effects, protein levels of phospho‐NF2+ sensitivity, specific measurements were performed using β‐escin permeabilized bronchial rings. Figure α treatment induced Ca2+ hypersensitivity when compared to Ca2+ sensitivity measured in control bronchi . MAG‐DPA  decreased the Ca2+ hypersensitivity triggered by TNF‐α, with calculated EC50 values of 0.15, 0.4, and 0.62 μmol/L, respectively. The Ca2+ sensitivity determined in the presence of TNF‐α + 1 μmol/L MAG‐DPA moreover reached that observed in control condition , thus suggesting that MAG‐DPA is able to completely reverse the effect induced by TNF‐α. However, no difference was observed in mean Ca2+ sensitivity between TNF‐α and TNF‐α combined with the lower MAG‐DPA concentration (0.1 μmol/L) in pretreated bronchi  when compared to control conditions  .The effect of MAG‐DPA on IL‐13 pretreated human distal bronchi was first assessed on mechanical reactivity. Results revealed that 10 ng/mL IL‐13 treatments induced an overreactivity to 30 nmol/L U‐46619 .Further experiments were performed to ascertain whether or not MAG‐DPA was able to downregulate the phosphorylation level of CPI‐17 protein Fig. B, under ent Fig. B. In ordα in microsomal fractions derived from 72‐h cultured guinea pig tracheas . In Figure κB and PPARγ are the main transcription factors associated with inflammatory status in several tissues, including airways  model of inflammation. Smooth muscle lysates were either untreated (control) or treated with TNF‐α (10 ng/mL) in the absence or presence of 0.1, 0.3, or 1 μmol/L MAG‐DPA. Relative detection levels calculated from western blots performed on these preparations revealed that PPARγ was present in all tested lysates, although TNF‐α treatment increased the ratio of detection of PPARγ/β‐actin  in an allergic model of asthma  in combination with omega‐3 fatty acids on the resolution of inflammatory signaling pathways (Blocker et al. α or IL‐13, while being upregulated by MAG‐DPA, MAG‐DPA combined to ASA, and RvD1. RvD1 is a putative DHA metabolite, which was described as a potent proresolving compound (Serhan et al. The ability of MAG‐DPA to generate new proresolving mediators that would act as agonists on specific receptors GPR32, ChemR23, and ALX/FPR2 (Serhan et al. α/NFκB and PPARγ signaling pathways to mediate resolving properties in human and rodent models of airway hyperresponsiveness. In addition, the present findings demonstrate that MAG‐DPA is able to decrease the activation of PKC/CPI‐17 pathway, leading to a rightward shift in Ca2+ sensitivity and a significant decrease in airway hyperresponsiveness. The clinical relevance of MAG‐DPA would be related to its capacity to generate lipid metabolites, such as Resolvin D1, which was associated with a concomitant increase in GPR‐32 expression. The ability of the Resolvin precursor to reduce both the inflammation and hyperresponsiveness of airway smooth muscle could be of valuable pathophysiological significance in the treatment of respiratory diseases such as asthma, COPD, and cystic fibrosis.In summary, to our knowledge, this is the first study demonstrating that MAG‐DPA interacts with TNF‐None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.Plate S1. Pharmacological effects of MAG‐DPA on GPR‐32 protein expression in TNF‐α‐pretreated human bronchi.Plate S2. Effects of MAG‐DPA on GPR‐32 protein expression in IL‐13‐pretreated human bronchi.Click here for additional data file.


Inhibitors of apoptosis (IAP) proteins contribute to cell death resistance in malignancies and emerged as promising targets in cancer therapy. Currently, small molecules mimicking the IAP-antagonizing activity of endogenous second mitochondria-derived activator of caspases (SMAC) are evaluated in phase 1/2 clinical trials. In cancer cells, SMAC mimetic (SM)-mediated IAP depletion induces tumor necrosis factor (TNF) secretion and simultaneously sensitizes for TNF-induced cell death. However, tumor cells lacking SM-induced autocrine TNF release survive and thus limit therapeutic efficacy. Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent induction of apoptosis and/or necroptosis. Hypertonicity allowed robust TNF-dependent killing in SM-treated human acute lymphoblastic leukemia cells, which under isotonic conditions resisted SM treatment due to poor SM-induced TNF secretion. Mechanistically, hypertonicity-triggered TNF release bypassed the dependency on SM-induced TNF production to execute SM cytotoxicity, effectively reducing the role of SM to TNF-sensitizing, but not necessarily TNF-inducing agents. Perspectively, these findings could extend the clinical application of SM. SM free caspases from XIAP-mediated inhibition and trigger degradation of cIAP1/2, which is death promoting in two interdependent ways: it initiates autocrine secretion of tumor necrosis factor (TNF) and concomitantly undermines the function of cIAP1/2 to counteract TNF-induced TNF-receptor 1 (TNFR1)-mediated cytotoxicity.7 Usually, TNFR1 activation first triggers formation of a receptor-associated signaling complex (termed ‘complex I’). cIAP1/2-mediated ubiquitination of complex I provides a scaffold for recruitment of kinases activating the survival-promoting canonical nuclear factor kappa B (NFκB)-pathway. SM abolish complex I ubiquitination and facilitate its transition to a secondary cytosolic complex (termed ’complex II’), which can induce apoptotic and necroptotic cell death via caspase-8 and RIPK1/RIPK3, respectively.8 How SM trigger TNF production is not fully understood, but SM-mediated loss of cIAP1/2 impairs constitutive ubiquitination and subsequent proteasomal degradation of NFκB-interacting kinase (NIK). Increasing NIK levels activate the non-canonical NFκB pathway and transcription of downstream target genes such as TNF.10Imbalances in pro- and anti-apoptotic proteins disturb cellular death pathways and allow tumor cells to escape from chemotherapy-induced apoptosis. Newer therapeutic approaches aim to reinstate the cell’s death machinery by targeting the pro-survival BCL-2 family members or the cellular inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2 and X-linked IAP (XIAP). Mechanistically, XIAP blocks apoptosis by direct caspase inhibition whereas cIAP1/2 shut down cell death promoting activities of receptor-interacting protein kinase-1 (RIPK1). Overexpression of IAP contributes to the onset of cancer and drug resistance,7 Cancer cells that do not release TNF upon SM treatment are consequently resistant even though SM-mediated IAP depletion sensitizes for TNF-induced cell death. Combining SM with exogenously added TNF could overcome this limitation, but is clinically challenging due to the severe cardio-vascular side effects of the cytokine. However, aside from SM-triggered activation of the non-canonical NFκB pathway, a plethora of stimuli can induce cellular TNF production.11 Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent initiation of apoptotic and/or necroptotic cell death. Human acute lymphoblastic leukemia (ALL) cells resisted SM treatment under isotonic conditions due to poor SM-induced TNF secretion. Under hypertonic conditions, however, SM treatment displayed a robust, TNF-dependent cytotoxicity. Mechanistically, hypertonicity-triggered TNF secretion bypassed the dependency on SM-triggered TNF production for SM cytotoxicity and reduced the role of SM to TNF-sensitizers, but not necessarily simultaneous TNF-inducers. Perspectively, our findings could extend the clinical application of SM.Efficient killing by SM alone requires both autocrine TNF secretion and TNFR1-mediated cytotoxicity.12 Synergism in terms of cell death induction was observed for both, BV6 and LCL161 with NaCl  expectedly abrogated cytotoxicity of BV6/NaCl  generates an osmotic pressure gradient between the intra- and extracellular space (hyperosmotic stress or hypertonicity). Hypertonicity generated by supplementing the cell culture medium with 75 mM NaCl or 150 mM mannitol (both increasing osmolarity by 150 mOsm/l) drastically increased cell death in L929 cells upon challenge with the SM BV6, LCL161 and birinapant compared with isotonic conditions . Althougith NaCl . This isBV6/NaCl . As the ng RIPK3 . Collect21 In human ALL cells (REH) and rhabdomyosarcoma cells (KYM-1), hypertonicity (established by addition of 50 mM NaCl or 100 mM mannitol to the cell culture medium) enhanced BV6-, LCL161- and birinapant-mediated cytotoxicity compared with isotonic controls  detection of annexin-V and 7-AAD in flow cytometry  incytometry  and (c) ytometry . In sum,κB pathway or itself triggered TNF production. Birinapant-induced non-canonical NFκB activation (as seen by decline of p100) was not enhanced under hypertonic conditions, potentially pointing to an additional, hypertonicity-activated TNF-producing pathway  NaC pathway  and (c)  pathway .24 Notably, NFAT5 also potently initiates TNF transcription under hyperosmotic stress.25 We observed hypertonicity-induced increase in TNF mRNA levels  along with its hypertonicity-induced activation through the mitogen-activated protein kinase (MAPK) p38.A levels . The notA levels  and 3a–cA levels  promptedA levels  and phosA levels  were dettoxicity  and  to treat early stages of bladder cancer triggered local inflammation and TNF release from recruited neutrophils. In combination with SM, neutrophil-derived TNF potently induced apoptosis.26 mediated through binding of the transcription factor NFAT5 to the TNF promoter.25 Hypertonicity-induced TNF release from cancer cells  could therefore substitute for lacking SM-induced TNF production, while still relying on the TNF-sensitizing activity of SM. Our study strengthened this concept by showing that hyperosmotic stress (a) initiated TNF release in human and murine cells (κB-mediated upregulation of pro-inflammatory cytokines (including TNF).31 However, artificially establishing (transient) hypertonic conditions in a narrowly defined area such as the tumor environment could attenuate systemic toxicity by spatially restricting TNF production and lowering SM concentrations necessary for cancer cell elimination  enhne cells , 4 and 7ne cells  and 7. Amination  and 3d–f32 Hyperosmotic stress activates p38,33 raising the possibility of reduced SM cytotoxicity (due to lower TNF production) under hypertonic conditions. Our results, however, indicate the opposite. Although p38 activation was clearly detectable under hypertonic conditions , TF-1 (#ACC334) and L929 (#ACC-2) cells were obtained from the German Collection of Microorganisms and Cell Cultures , KYM-1 cells were kindly provided by Harald Wajant (University of Wuerzburg). All cell lines were grown RPMI 1640 medium  supplemented with 10% (v/v) fetal calf serum . TF-1 cells were additionally supplemented with 5 ng/ml human granulocyte–macrophage colony-stimulating factor . Antibodies used in the study: TNF #3707, p38 #9212, phospho-p38 #9211, ERK #4695, phospho-ERK #4370 ; tubulin #MS-581: Dunnlab ; NFAT5 #PA1-023: Thermo Fisher Scientific . Chemicals: MTT: Biomol ; zVAD-fmk -fluoromethylketone): Bachem ; BIRB796, PH797804, SB203580, BV6, LCL161 and birinapant: Selleck Chemicals . Necrostatin-1 (Nec-1): Stress-Marq ; Etanercept was obtained from Pfizer .4 cells/well), REH cells (7 × 104 cells/well) and TF-1 cells (7 × 104 cells/well) were seeded in 96-well plates and challenged with the indicated concentrations of SM in triplicates . For co-culture assays, 1:1 mixtures of TF-1 and REH cells (5 × 104 cells of each cell line/well) were seeded in 96-well plates and stimulated as above. Cell viability was determined 18 h after stimulation using MTT staining (2 h at 37 °C). Staining intensity was measured at 595 nm and the mean was calculated from the technical replicates of each experiment. The mean value for untreated controls was set to 100%. For any other condition, the MTT staining intensity is given relative to the corresponding untreated group (% of control). Data points shown are mean values  of independent experiments (n≥3).L929  SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktails-I and -II (Sigma). Samples were sonicated and boiled for 5 min at 96 °C before proteins were separated by SDS-PAGE and transferred to PVDF membranes. To block non-specific binding sites, membranes were incubated in TBS containing 0.1% (v/v) Tween 20 and 5% (w/v) dry milk before primary antibodies of the specificity of interest were added. Antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies  and ECL technology .12 In this model, CI-values <1 are considered to be synergistic and CI >1 indicate antagonistic effects. Strength of synergism can be further graded: <0.1 very strong synergism, 0.1–0.3 strong synergism, 0.3–0.7 synergism, 0.7–0.85 moderate synergism, 0.85–0.9 slight synergism, 0.9–1.1 no synergism but nearly additive effects.CI-values were calculated with the freely available software CompuSyn version 1.0 using the median effect/combination index isobologram method.4/well) or REH cells (1 × 106/well) were seeded in 96-well tissue culture plates and cultured overnight. The next day, cells were challenged with SM in the presence and absence of NaCl. The supernatant was collected and TNF was quantified using an enzyme-linked immunosorbent assay . All groups were analyzed as triplicates .For measuring TNF secretion, L929 cells  according to the manufacturer’s instructions. Emitted fluorescence was quantified using a Victor3 Multilabel Reader .35 In brief, total RNA was isolated using the Qiagen RNeasy mini kit  according to the manufacturer’s instructions. Two micrograms of total RNA were transcribed into complementary DNA using the high-capacity cDNA reverse transcription kit . Tnf mRNA levels were quantified using the TaqMan human Tnf (Hs00174128_m1) gene expression assay (Applied Biosystems) and an ABI Prism 7900 sequence detector (Applied Biosystems). qRT-PCR reactions were performed in duplicates for each sample of an experiment and normalized to the housekeeping gene Hprt1 (Hs02800695_m1). mRNA levels were calculated using the SDS 2.1 software (Applied Biosystems).qPCR was essentially performed as described previously.μM LCL161 and birinapant for 16 h in the presence and absence of 50 mM NaCl. Afterwards, cells were stained with 7-AAD and Annexin-V (4 °C for 15 min in the dark) and analyzed immediately using a FACSCanto flow cytometer (BD Biosciences) following standard procedures.Cell death was assessed by Annexin-V and 7-AAD staining. In brief, REH cells were challenged with 2.5


Escherichia coli (STEC) O157 is an important foodborne pathogen that can be transmitted to humans both directly and indirectly from the feces of beef cattle, its primary reservoir. Numerous studies have investigated the shedding dynamics of E. coli O157 by beef cattle; however, the spatiotemporal trends of shedding are still not well understood. Molecular tools can increase the resolution through the use of strain typing to explore transmission dynamics within and between herds and identify strain-specific characteristics that may influence pathogenicity and spread. Previously, the shedding dynamics and molecular diversity, through the use of multilocus variable number of tandem repeat analysis (MLVA) of STEC O157, were separately investigated in an Australian beef herd over a 9-month study period. Variation in shedding was observed over time, and 33 MLVA types were identified. The study presented here combines the two datasets previously published with an aim to clarify the relationship between epidemiological variables and strain types. Three major genetic clusters (GCs) were identified that were significantly associated with the location of the cattle in different paddocks. No significant association between GCs and individual cow was observed. Results from this molecular epidemiological study provide evidence for herd-level clonal replacement over time that may have been triggered by movement to a new paddock. In conclusion, this study has provided further insight into STEC O157 shedding dynamics and pathogen transmission. Knowledge gaps remain regarding the relationship of strain types and the shedding dynamics of STEC O157 by beef cattle that could be further clarified through the use of whole-genome sequencing.Shiga toxin-producing  Escherichia coli (STEC) O157 (E. coli O157 is still being explored (Ruminants are the primary reservoir of Shiga toxin-producing EC) O157 . STEC inEC) O157 . An estiEC) O157 , and in EC) O157 , 7 and sEC) O157 , promptiEC) O157  and AustEC) O157 , 11. EnvEC) O157 . SheddinEC) O157 , with vaEC) O157 , 14, 15.EC) O157 , 17; howexplored .The molecular epidemiology of STEC O157 in cattle is complex, as multiple subtypes are often circulating simultaneously on individual farms . FurtherWith the increased application of whole-genome sequencing technology in outbreak investigations , progresA better understanding of the temporal variation of STEC O157 shedding can help target control efforts. Strain-specific characteristics, such as pathogenicity and adaptation mechanisms, can influence shedding  and ultiTo address the challenges described, the shedding dynamics and molecular diversity of STEC O157 in an Australian beef herd was recently investigated , 33. VarAs described in Lammers et al. , a beef d-glucuronide agar (Oxoid) (CT-SMAC + BCIG) containing cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (Oxoid). Plates were screened for non-sorbitol fermenting and β-glucuronidase-negative colonies and up to 10 presumptive colonies were tested using an E. coli O157 latex test (Oxoid). Samples negative after direct plating were subjected to immunomagnetic separation, plated onto CT-SMAC + BCIG, and incubated for 18–24 h at 37°C. Up to 10 individual colonies were confirmed as STEC O157 using a multiplex PCR, targeting the 1rfbE, stx, and 2stx genes  based on MLVA profiles was created using the software MSTgold v2.4 . DefaultPrevious WGS analysis by Jones et al.  revealedThe genetic dataset indicating STEC O157 GCs was merged with metadata from Lammers et al. . Fisher’Previously reported molecular typing data from Jones et al. , includiMT14, MT2, and MT3 were each the first shed MLVA type of GC1, GC2, and GC3, respectively, with MT2 and MT3 each found at a central node in the MST in relation to the remaining members of the cluster. MT14 was the first GC1 MT to be isolated (on 5th November 2012) and was predominantly found in the beginning of the study period, whereas MT20 was the most commonly isolated MLVA type and the only GC1 type found after movement to the final paddock on February 28, 21 weeks after the start of the study.n = 2) shedding isolates from a single GC, 61% (n = 14) shedding isolates from two different GCs, and 30% (n = 7) shedding isolates from three different GCs , but not within cows (P = 0.142), based on Fisher’s exact tests.All cows shed isolates from at least one of the GCs during the 9-month study period, with 9% n =  sheddingThe number of isolates identified from each GC at each sampling point is shown in Figure E. coli strain types is more strongly influenced by environmental exposure, than individual cow attributes . For example, starlings have been implicated in the transmission of STEC O157 between dairy farms , and freThe use of the distance method to generate an MST was found to be the most appropriate for the data available, as single-repeat changes were previously observed to occur most frequently ; howeverWhile the results generated by this study have provided additional insight into STEC O157 transmission dynamics on the study site, there are some shortcomings. In this study, paddock changes were convenience based and driven by herd management needs, such as pasture quality and nutritional requirements, and thus cows did not spend equal amounts of time in each location. As such, additional analysis of STEC O157 shedding patterns in other herds and environments are needed to substantiate our findings. Furthermore, as with any epidemiological study, unaccounted for selection bias could have occurred. The strong sampling design did, however, offer insight into the spatiotemporal nature of STEC O157 shedding of cattle in this pasture based environment.Mycobacterium avium subspecies paratuberculosis was found to be unreliable to detect related and unrelated isolates, as it both over- and underestimated relatedness in comparison with WGS  from an intensive sampling study performed by the same research group (Importantly, results from the current analysis could inform potential future typing studies that were out of scope for the investigation presented here and could provide a rationale for the prioritization of additional isolates for WGS using existing samples. For example, building onto these initial studies, STEC O157 isolates (ch group  have beeIn conclusion, this study has provided further insight into STEC O157 shedding dynamics in an Australian beef herd over time and identified environmental forces as a likely driver of pathogen transmission. The knowledge gained can be used as a stepping stone to design higher resolution studies to answer additional questions related to cow- and herd-level shedding synchronization of specific strains over time.PM, CA, and JH drafted the manuscript and made a lead contribution to the conception, design, analysis, and interpretation of the work. GL, MJ, SO, and RL critically reviewed the manuscript and made contributions to field, laboratory, analysis, and interpretation of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to investigate the effects of anti-tumor necrosis factor (TNF)-α antibody (Ab) on alteration of penile structure in the hyperprolactinemia (hyperPRL) rat model. HyperPRL was induced in 8-week-old male Sprague-Dawley rats by allografting anterior pituitary (AP) glands under the renal capsule (+AP rats). Rats implanted with cerebral cortex (CX) were used as sham control (+CX rats). At 6 weeks post implantation, rats received either a single intra-testicular dose of TNF-α Ab (12.5 μg/kg) or testosterone replacement , and they were sacrificed 1 week later. Blood and penile tissue was collected for analysis. Compared to +CX rats, the +AP group had lower serum testosterone concentration and neuronal nitric oxide synthase (nNOS) expression, but exhibited a higher ratio of collagen III/I in the corpus cavernosum. Smooth muscle content exhibited no significant changes. At 1 week post TNF-α Ab injection, the collagen III/I ratio in the +AP group was decreased, and the smooth muscle content and nNOS expression increased significantly. These findings were comparable to those observed in +AP rats receiving TE. Testicular TNF-α suppresses testosterone release, which in turn results in the erectile dysfunction (ED) seen in hyperPRL. Intra-testicular TNF-α Ab treatment is as effective as testosterone supplementation on penile structure normalization in the hyperPRL model. Prolactin (PRL), a 23 kDa peptide, is secreted from the lactotrophs of the anterior pituitary (AP) gland under the inhibitory control of hypothalamic dopamine. The main functions of PRL in females are inducing and maintaining lactation during the peripartum and postpartum phases. In males, the role of PRL is less significant. However, a PRL deficiency in childhood might interfere with development of the reproductive system , 2. OverIn clinical practice, antipsychotics and antidepressants used to treat psychiatric diseases, behavioral disorders, or depression usually result in lowering CNS dopamine levels and thus hyperPRL . The occStudies have shown that TNF-α can affect erectile function by reducing neuronal nitric oxide synthase (nNOS) expression, promoting inflammation and fibrosis . In addiGood penile erection determined by pressure maintenance in the cavernosal sinuses and it relies on competent functions of endothelium, collagen, smooth muscle and elastic fibers. Changes of the connective tissue and smooth muscle in rabbits with erectile dysfunction can be quantitatively assessed by collagen III/I and collagen/smooth muscle ratio . Based oad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University. Approval number IACUC 2012–074, IACUC 2013–143.Male Sprague-Dawley rats (8 weeks old) weighing 250 to 300 g were purchased from the Animal Center of National Yang-Ming University and housed at 22 ± 1°C, with a light cycle between 08:00h and 20:00h. Food and water were provided HyperPRL was induced by allografting AP glands under the left renal capsule as previously described . Brieflyn = 8 per group): (1) rats implanted with CX ; (2) rats implanted with AP (+AP); (3) and (4) +CX and +AP rats, respectively, with saline-dissolved anti-TNF-α antibody  injected 1× into the left testis at 1 week before experiment; and (5) and (6) +CX and +AP rats, respectively, with olive oil-dissolved testosterone enanthate  injected 2× intra-muscularly within 1 week, 3 days apart.Rats were randomly divided into the following six treatment groups . According to the manufacturer, the assay detection range was 0.39–50 ng/ml and the detection limit was 0.2 ng/ml.Serum (0.1 ml) was mixed with 0.4 ml diethyl ether, shaken for 1 h, and quickly frozen in an ethanol/dry ice mixture. The organic phase (supernatant) was collected, dried, and reconstituted in a buffer solution  before measuring testosterone concentration by radioimmunoassay . SensitiSerum TNF-α was measured with the Rat TNF-α/TNFSF1A ELISA kit (R&D Systems). According to the manufacturer, the assay detection range was 12.5–800 pg/ml. The detection limit was typically less than 5 pg/ml.Penile tissues were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 24 h. After PBS washing, tissues were dehydrated and embedded in paraffin at 60°C. Transverse tissue sections at 4-μm thickness were obtained.Picrosirius red stain  was used to distinguish collagen I (red/orange) from collagen III (green) under polarized microscopy. Eight randomly chosen cavernosal tissue areas were captured per slide, and areas of collagen I and III were measured with the Image J version 1.45s Threshold Color plugin . Data was expressed as the ratio of collagen III/I (%).Masson’s trichrome stain was used to differentiate areas of smooth muscle and collagen in the corpus cavernosum. Eight randomly chosen smooth muscle areas were quantified by the Image J Threshold Color plugin. The entire corpus cavernosum area was manually delineated, and quantitative results were expressed as percentage of smooth muscle within the corpus cavernosum.Penile dorsal nerve nNOS expression was analyzed by immunofluorescence microscopy using primary rabbit nNOS polyclonal Ab  and secondary antibody conjugated with rhodamine. Anti-β3-tubulin Ab  and secondary Ab conjugated with fluorescein were used to detect nerve fibers. Eight randomly chosen areas were quantified and the entire dorsal nerve area manually delineated as described above. Quantitative results were expressed as percentage of nNOS within the dorsal nerve area.To confirm whether macrophages or TNF-α are present in the penis in our animal model, we used immunohistochemistry to detect expression of TNF-α and the macrophage marker, ED1, in penis sections. Primary anti-TNF-α Ab (1:100 dilution) was obtained from R&D Systems. Anti-ED1 primary Ab (1:100 dilution) was obtained from Hycult Biotech . Splenic tissue was used as a negative (skipped the primary antibody step) and positive control.t-test, with P< 0.05 considered as statistically significant.All statistical analyses were performed in the SPSS software package . Quantitative data were expressed as the mean ± standard error of the mean (SEM). Analysis of variance with Fisher’s least significant difference post hoc test was used for group comparisons. Difference between two means was tested for significance by Student’s P<0.05).Serum PRL levels were examined to confirm the success of the hyperPRL rat model. As shown in P<0.05). One week after TNF-α Ab or TE injection, the collagen III/I ratio in +AP rats was significantly decreased compared to that in the untreated +AP group. Compared to that in the untreated +CX group, collagen III in the +CX group receiving TE was also significantly decreased. These findings suggested that intra-testicular TNF-α Ab injection and intra-muscular TE injection has similar effects on penile tissue.The differential distribution of collagen I and III in the corpus cavernosum is shown in The distribution of smooth muscle and collagen in the corpus cavernosum is shown in Dorsal nerve nNOS expression is shown in In penile sections of our animal model, only trace amounts of TNF-α could be detected in the corpora cavernosa of +AP and +CX rats, and no difference could be found between them . Regarding the serum TNF-α levels, there was no difference detected between +AP and +CX rats  . Since hRegarding the route of drug administration, intra-testicular way is definitely not practical in clinical setting. Some more less-invasive routes should be developed. Using intra-testicular injection is based on the rationale that the TNF-α in our model is released from the testicular interstitial macrophages . Our preOur study has some limitations that warrant discussion. First, we examined only morphological changes in penile structure without consideration of physiological or behavioral modifications. Second, we used polyclonal anti-TNF-α Ab, which is not identical to treatment agents used for inflammatory diseases. Additional studies using etanercept or infliximab in the hyperPRL model are therefore needed.Our study shows that testicular TNF-α decreases testosterone release and thus remotely induces subsequent penile structure disruption. To our knowledge, this study is the first to suggest a role of TNF-α in hyperPRL-related ED. Anti-TNF-α treatment rapidly recovers testosterone secretion and restores normal penile architecture. These findings imply that anti-TNF-α treatment may lead to beneficial effect on erectile function and that administration of TNF-α Abs may be a promising approach in managing ED in patients with psychotropic-induced hyperPRL.S1 TableS1A Table. Serum PRL (ng/ml) in +AP and +CX Rats. S1B Table. Collagen Distribution (%) in Rat Corpus Cavernosum. S1C Table. Smooth Muscle Content (%) in Rat Corpus Cavernosum. S1D Table. nNOS Expression (%) in Rat Penile Dorsal Nerve. S1E Table. Serum Testosterone Concentration (ng/ml) in Response to TNF-α Ab or TE Treatment.(DOCX)Click here for additional data file.S1 FigThe picture of corpus cavernosum in +CX and +AP rats both showed negative for TNF-α expression. Splenic tissue was used as a negative (skipped the primary antibody step) and positive control. Arrows indicate representative cells showing positive TNF-α. Scale bar = 100 μm.(TIF)Click here for additional data file.S2 FigBrown color represents macrophage by ED1 antibody immunohistochemistry. Macrophage in the blood of the penile dorsal vein was used as positive control. Arrows indicate representative cells showing positive ED-1. Scale bar = 100 μm.(TIF)Click here for additional data file.S3 FigEach column represents the mean ± SEM of 4 rats.(TIF)Click here for additional data file.