CHMP2B, encoding a protein in the endosomal sorting complexes required for transport (ESCRT) machinery, causes frontotemporal dementia linked to chromosome 3 (FTD3). FTD, the second most common form of pre-senile dementia, can also be caused by genetic mutations in other genes, including TANK-binding kinase 1 (TBK1). How FTD-causing disease genes interact is largely unknown. We found that partial loss function of Ik2, the fly homologue of TBK1 also known as I-kappaB kinase ε (IKKε), enhanced the toxicity of mutant CHMP2B in the fly eye and that Ik2 overexpression suppressed the effect of mutant CHMP2B in neurons. Partial loss of function of Spn-F, a downstream phosphorylation target of Ik2, greatly enhanced the mutant CHMP2B phenotype. An interactome analysis to understand cellular processes regulated by Spn-F identified a network of interacting proteins including Spn-F, Ik2, dynein light chain, and Hook, an adaptor protein in early endosome transport. Partial loss of function of dynein light chain or Hook also enhanced mutant CHMP2B toxicity. These findings identify several evolutionarily conserved genes, including ik2/TBK1, cut up (encoding dynein light chain) and hook, as genetic modifiers of FTD3-associated mutant CHMP2B toxicity and implicate early endosome transport as a potential contributing pathway in FTD.Mutations in  Identified causative loci, collectively representing ~ 40% of all FTD cases, reveal a genetic, pathological and mechanistic overlap with amyotrophic lateral sclerosis (ALS)3. Extensive cell biological studies of these loci suggest RNA metabolism (TARDBP and FUS) and autophagy/endosomal-lysosomal function  as major contributors to neuronal pathology4. However, how different disease genes interact with each other in ALS/FTD pathogenesis remains poorly understood.Frontotemporal dementia (FTD) is an early onset dementia associated with frontotemporal lobar degeneration (FTLD)CHMP2B encodes a subunit of the endosomal sorting complex required for transport III (ESCRT-III) complex that is recruited to the surface of early endosomes to participate in the final step in membrane scission during the formation of multivesicular bodies (MVBs)5. MVBs are the endosomal and autophagosomal entry point to the late endosome and eventual lysosomal degradation. A splicing site mutation in CHMP2B resulting in a C-terminal truncation of the protein was identified in a Danish FTD patient cohort6 and other mis-sense mutations have since been identified in FTD and ALS pedigrees12. It is proposed that the truncation of the C-terminal in the CHMP2BIntron5 mutant protein promotes an ‘open’ configuration, locking the protein into an association with its binding partner Snf7-2/CHMP4B13. This blockage in ESCRT-III disassembly results in endosomal accumulation and deficient cellular trafficking15.Drosophila model expressing CHMP2BIntron5 post-mitotically in the fly eye16. Genetic screening for enhancers and suppressors of neurodegeneration in this model have so far identified activated innate immune signaling16, autophagosomal dysfunction17, and endosomal signaling disruption18 leading to JNK/AP-1 mediated pro-apoptotic signaling19. Here, using our fly eye model of CHMP2BIntron5 mediated neurodegeneration, we attempt to identify additional genetic modifiers of mutant CHMP2B toxicity and effectors of endosomal dysfunction. In particular, we focus on some other genes known to be involved in ALS/FTD pathogenesis.Why neurons are particularly susceptible to mutant CHMP2B induced endosomal dysfunction is not currently understood. To address this question, we established a Intron5 but not CHMP2BWT driven by GMR-Gal4 produced a retinal degeneration phenotype characterized by a few small melanin deposits in the fly eye16. Here we first confirmed this eye phenotype in flies expressing CHMP2BIntron5  or ik2 gene were enhancers of mutant CHMP2B toxicity  in Flybase. A point mutation named Alice (ik2Alice) compromises the normal function of ik220. Ik2Alice heterozygous flies did not show any eye degeneration phenotype . However, mutant CHMP2B toxicity was significantly enhanced in the ik2Alice heterozygous background  family. The Ird5KG08072 allele did not modify the eye phenotype of mutant CHMP2B  as the experimental system. Previously we reported that pan-neuronal expression of mutant CHMP2B resulted in a significant overgrowth at the Drosophila third instar larval NMJ19. Here we demonstrate that this synaptic overgrowth phenotype was also observed when CHMP2BIntron5 is expressed specifically in Drosophila motor-neurons, under the control of the OK6-Gal4 motorneuron driver. Motor-neuronal expression of CHMP2BIntron5 resulted in a significant increase in bouton number and NMJ length coupled with a significant decrease in muscle surface area  than with other human kinases such as IKK-epsilon and IKK-beta . Human TBK1 shares 35.5% and 18.5% identity with Ik2 and IRD5, respectively. Thus, Ik2 appears to be the Drosophila26. Therefore, we examined whether Spn-F is also a genetic modifier of mutant CHM2B toxicity. Indeed, the toxicity of mutant CHMP2B in the eye was greatly enhanced in the Spn-F2 heterozygous background  is a major phosphorylation target of Ik2, and these two proteins form a complex that regulates several developmental processes in und Fig. a,c; thisund Fig. b,d, whilUAS-EGFP-Spn-F using actin-Gal4 and used GFP antibody to pull down Spn-F from fly head lysates. We identified several proteins that bind to Spn-F (Table cut up (ctp). Although the functional homolog of Spn-F in mammals is unknown, both Hook and Dlc1 are highly conserved evolutionarily28. Spn-F directly interacts with Dlc1 in flies29 and Hook family proteins activate dynein adaptors in mammalian early endosome transport30. Thus, our interactome analysis identified Hook as another protein that interacts with Spn-F.To determine which cellular pathways regulated by the Ik2-Spn-F complex are involved in mutant CHMP2B toxicity, we sought to identify other interacting proteins by immunoprecipitation of Spn-F followed by mass spectrometry. We expressed -F Table . The mos30, we sought to determine whether this cellular pathway contributes to the toxicity of mutant CHMP2B. For this analysis, we used two hook mutant alleles (hook7 and hook11) and two hook-specific RNAi lines , and UAS-ctp RNAi (43115 and 104084) were from the Vienna Drosophila RNAi Center. Spn-F2 and UAS-GFP-Spn-F fly lines33 were kindly provided by Dr. Hsiu-Hsiang Lee, and hook7 and hook11 fly lines34 were from Dr. Helmut Krämer. The genetic aberrations of these alleles are summarized in Supplementary GMR-Gal4, UAS-CHMP2BIntron5/CyO, was crossed with individual classic mutants or RNAi lines. To quantify the CHMP2BIntron5 eye phenotype, we arbitrarily classified the eye phenotype with or without enhancers into three groups based on the relative abundance of black spots on the surface of the eye.Flies were raised at 25 °C on a standard diet. Actin-Gal4/UAS-GFP and Actin-Gal4/UAS-GFP-Spn-F flies were frozen with dry ice and vortexed to remove the heads. Heads from each genotype were homogenized in lysis buffer . Homogenates were centrifuged at 4 °C for 20 min at 12,000g. Protein concentrations were determined with the Bradford assay (Bio-Rad). For co-immunoprecipitation experiments, supernatants of GFP and GFP-Spn-F with the same amount of total proteins were incubated with GFP magnetic beads (Chromoteck) overnight at 4 °C. The beads were incubated, washed three times for 15 min each with washing buffer , and then suspended in the gel loading buffer and boiled for 5 min. The co-immunoprecipitation samples were then run on a 10% polyacrylamide-SDS gel for a short time, and stained with a silver staining kit (Sigma) for subsequent digestion and downstream LC–MS/MS analysis (Proteomics and Mass Spectrometry Facility at UMass).Adult Drosophila were raised on standard cornmeal medium at 18 °C on a 12-h light:dark cycle. Immunohistochemistry was performed described20. Motor-neuronal expression was under the control of the OK6-Gal4 driver. NMJs were imaged at 40× with a Hamamatsu ORCA-R2 C10600-10B digital camera on a Leica DM6000B microscope fitted with Qioptiq OptiGrid Structured-Light system using Leica MM AF software. Muscles were imaged with the same system at 10x, without the OptiGrid. NMJ analysis was done as described18. Prism 7 (GraphPad Software) was used for statistical analysis.For NMJ analysis, 36.This analysis was performed by UMass Proteomics Core Facility and Dr. John Leszyk provided the method description that was previously publishedSilver-stained gel bands were destained with a 1:1 ratio of potassium ferricyanide (30 mM) and sodium thiosulfate (100 mM). Gels were washed extensively with water to remove and destain the yellow color. Gel slices were cut into 1 × 1-mm pieces and placed in 1.5-ml eppendorf tubes with 1 ml of water for 30 min. The water was removed, and 200 µl of 250 mM ammonium bicarbonate was added. For reduction, 25 µl of a 45-mM solution of 1,4 dithiothreitol was added, and the samples were incubated at 50 °C for 30 min. After cooling to room temperature, the samples were alkylated by adding 25 µl of a 100-mM iodoacetamide solution for 30 min. The gel slices were washed twice with 1-ml aliquots of water. The water was removed, and 1 ml of a 50:50 mixture of 50-mM ammonium bicarbonate and acetonitrile was placed in each tube. After incubation at room temperature for 1 h, the solution was removed, and 200 µl of acetonitrile was added to each tube, turning the gel slices opaque white. The acetonitrile was removed, and the gel slices were further dried in a Speed Vac and rehydrated in 1,000 µl of 2 ng/µl trypsin (Sigma) in 0.01% ProteaseMAX Surfactant (Promega):50-mM ammonium bicarbonate. Samples were incubated at 37 °C for 21 h. The supernatant of each sample was removed and placed in a separate 1.5-ml eppendorf tube. Gel slices were further dehydrated with 100 µl of an 80:20 mixture of acetonitrile and 1% formic acid. The extract was combined with the supernatants of each sample. The samples were then dried in a Speed Vac.18 Magic 5-µm particle trap column. Peptides were eluted and sprayed from a custom-packed emitter (75 µm × 25 cm C18 Magic 3-µm particle) with a linear gradient from 95% solvent A (0.1% formic acid in water) to 35% solvent B (0.1% formic acid in acetonitrile) for 90 min at a flow rate of 300 nanoliters per minute on a Waters Nano Acquity UPLC system. Data-dependent acquisitions were done on a Q Exactive mass spectrometer (Thermo Scientific) according to an experiment in which full MS scans from 300 to 1,750 m/z were acquired at a resolution of 70,000 followed by 10 MS/MS scans acquired under higher-energy collisional dissociation (HCD) fragmentation at a resolution of 17,500 with an isolation width of 1.6 Da. Raw data files were processed with Proteome Discoverer  and then searched with Mascot Server  against the human index of Uniprot. The search parameters used were fully tryptic with 2 missed cleavages, parent mass tolerances of 10 ppm, and fragment mass tolerances of 0.05 Da. Variable modifications of acetyl (protein N-term), pyro glutamic for N-term glutamine, oxidation of methionine, and carboxymethyl cysteine were considered.After reconstitution in 25 µl of 0.1% trifluoroacetic acid in 5% acetonitrile, a 3-µl aliquot of each sample was directly injected onto a custom-packed 2 cm × 100 µm CSignificant difference between control and experimental groups were determined by using Chi-square tests to calculate P values for categorical data.Detailed information is also provided regarding genotypes of flies (Supplementary Supplementary Information


Treatment of hepatocellular carcinoma (HCC) using antibody-based targeted therapies, such as antibody conjugates and chimeric antigen receptor T (CAR-T) cell therapy, shows potent antitumor efficacy. Glypican-3 (GPC3) is an emerging HCC therapeutic target; therefore, antibodies against GPC3 would be useful tools for developing immunotherapies for HCC.We isolated a novel human monoclonal antibody, 32A9, by phage display technology. We determined specificity, affinity, epitope and anti-tumor activity of 32A9, and developed 32A9-based immunotherapy technologies for evaluating the potency of HCC treatment in vitro or in vivo.32A9 recognized human GPC3 with potent affinity and specificity. The epitope of 32A9 was located in the region of the GPC3 protein core close to the modification sites of the HS chain and outside of the Wnt-binding site of GPC3. The 32A9 antibody significantly inhibited HCC xenograft tumor growth in vivo. We then pursued two 32A9-based immunotherapeutic strategies by constructing an immunotoxin and CAR-T cells. The 32A9 immunotoxin exhibited specific cytotoxicity to GPC3-positive cancer cells, while 32A9 CAR-T cells efficiently eliminated GPC3-positive HCC cells in vitro and caused HCC xenograft tumor regressions in vivo.Our study provides a rationale for 32A9 as a promising GPC3-specific antibody candidate for HCC immunotherapy. Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases and is the sixth most common and the second most lethal cancer worldwide , 2. The Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is anchored on the cell surface by glycophosphatidylinositol (GPI) –7. GPC3 The mature form of human GPC3 has a protein core consisting of seven disulfide bonds that are highly conserved in the glypican family. In the flexible C-terminal region, GPC3 contains two binding sites (S495 and S509) for heparan sulfate (HS) chains . Our preIn the present study, we were interested in developing a novel anti-GPC3 monoclonal antibody that would recognize an epitope outside of the Wnt-binding motif or known epitope of other GPC3 antibodies and would be used to construct more effective antibody-based immunotherapies, such as immunotoxins , 25 and 2 at 37 °C. A431 cells were engineered to highly express GPC3 by transfection with a plasmid encoding full-length GPC3. PBMCs were isolated by Ficoll density centrifugation of healthy donors’ blood, obtained under the ethically protocol approved by Nanjing Jinling Hospital . The isolated PBMCs cultured in RPMI  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 0.1 mg/ml streptomycin and were incubated in 5% CO2 at 37 °C. All cell lines were evaluated and validated by their morphology and growth rate. All the cell lines were confirmed to be free of mycoplasma contamination by PCR using specific primers.The normal liver tissue and HCC tumor tissue specimens used in the current study were obtained from the patients recruited from the Nanjing Drum Tower Hospital  between November 2018 and March 2019. This study was approved by the Ethical Committee of Nanjing Drum Tower Hospital, and every patient provided written informed consent. Huh-7 cells were a kind gift from Dr. Xin-Wei Wang at the National Cancer Institute . A431, SK-hep-1, Hep3B, HepG2 and HEK293T cells were purchased from American Type Culture Collection . All cells were cultured in DMEM  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 0.1 mg/ml streptomycin  and were incubated in 5% COThe sequence of full-length GPC3 was cloned into the pLVX vector. For protein purification, truncated GPC3 (Q25-S550) was fused to a human Fc tag and cloned into the pFUSE vector. All point mutants of GPC3 and the GPC3 mutant lacking the HS chains (GPC3ΔHS) were generated by introducing point mutations using the overlapping PCR method. The mFrizzled8 ECD-hFc plasmid was purchased from Addgene . The 32A9 scFv sequence was fused to the human Fc tag and cloned into the pFUSE vector as well. The 32A9 heavy chain variable region and light chain variable region sequences were amplified by adding IL-2 signal peptide and were inserted into the expression vectors pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk , respectively. All plasmids were identified by sequencing and then transfected into 293T cells. After collecting the supernatant, protein purification was accomplished with a protein A affinity column . GPC3-his and GPC5-his proteins were purchased from R&D .YP7, a control mouse monoclonal antibody against GPC3 , was use11 ~ 1012 pFU/ml for the next round of panning.The TG1 clone was picked and cultured overnight in 2YT medium at 37 °C. GPC3-Fc protein in PBS buffer was used to coat an ELISA plate, which was incubated overnight at 4 °C. The next day, the phage library (Tomlison I library) and the coated wells were blocked with PBST with 3% milk at room temperature for 1 h. Then, the blocked phages were added to the coated wells and incubated at room temperature for 1 h. After washing 20 times with PBST, the GPC3-binding phages were eluted with 100 mM triethylamine . All eluted phages were collected and used to infect TG1 cells. After incubation with helper phages, the eluted phages were rescued with a titer of approximately 10The human GPC3 (NCBI Gene ID: 2719), mouse GPC3 (NCBI Gene ID: 14734), and human GPC5 (NCBI Gene ID: 2262) sequences were submitted to the protein structure modeling web server SWISS-MODEL . The C-t2SO4 were added to detect the OD450nm value.GPC3-hFc protein (5 μg/ml) was used to coat ELISA wells at 4 °C overnight. The wells were blocked with PBS containing 3% milk for 0.5 h at 37 °C. The antibodies or phage were added to the wells and incubated at 37 °C for 0.5 h. After washing with PBST 3 times, goat anti-human kappa chain HRP antibody (for 32A9) , goat anti-mouse HRP antibody (for YP7)  or rabbit anti-M13 HRP antibody (for phage)  was added to the wells and incubated at 37 °C for 0.5 h. TMB and H-cell suspension was incubated with 5 µg/ml of the indicated antibodies for 1 h on ice and then incubated with a 1:200 dilution of anti-human PE antibody (for 32A9)  or anti-mouse PE antibody (for YP7)  for 1 h on ice. The cells were analyzed using FACS Calibur .A singleThe sequence of 32A9 scFv was fused to a truncated and deimmunized Pseudomonas exotoxin (mPE24) fragment –37 and t4 cells were seeded into 96-well plates and cultured overnight at 80–90% density, and then different concentrations of immunotoxin were added to the wells. After 72 h, the OD450 nm values were detected by using a CCK-8 kit  to analyze the cytotoxicity of immunotoxin.A total of 10A 32A9-based second generation 4-1BB CAR fragment was synthesized . To detect the expression of CAR, an EGFP coding sequence was added upstream of the 32A9-CAR fragment separated by an F2A sequence. The whole sequence was subcloned into the pLVX lentiviral vector. The same version of FMC63 4-1BB CAR targeting CD19 was used as a negative control . To prodCAR-T cells were cocultured with GPC3-expressing cancer cells at the indicated ratios for 18 h. Then, the supernatant was collected to measure cytotoxicity by an LDH release assay , and IL-2 release was measured by an IL-2 detection kit .3, the mice were treated with PBS or 10 mg/kg 32A9 antibody by i.v. injection. Tumor dimensions were determined using calipers, and tumor volume was calculated by the formula V = 1/2 ab2, where a and b represent tumor length and width, respectively. To evaluate the antitumor activity of 32A9 CAR-T cells, five million of Huh-7 cells or luciferase-labeled Hep3B cells were subcutaneously inoculated into five-week-old NYG mice. When the average tumor size was ~ 50 mm3, 10 million PBMCs or 32A9 CAR-T cells were infused to mice for Huh-7 groups, PBS or 10 million FMC63-CAR-T cells or 32A9 CAR-T cells were infuse into mice for Hep3B groups by i.v. injection. The tumor size was measured every other day or imaged after the mice were injected with 100 µl D-luciferin  and imaged 10 min later. Living Image software was used to analyze the bioluminescence signal flux for each mouse as photons per second per square centimeter per steradian (photons/s/cm2/sr). Mice were killed when they showed any sign of sickness or when the tumor burden reached 1500 mm3 or the bioluminescence signal reached 1 × 1011 photons/s/cm2/sr.NYG mice which were established by knocking out Prkdc and Il2rg in NOD mice by Crispr-Cas9 technology. Four- to six-week-old female NYG mice were purchased from the Animal Facility of Nanjing Medical University. All mice were treated under the protocol approved by the Ethics Committee of the Animal Core Facility of Nanjing Medical University. To evaluate the antitumor activity of the 32A9 antibody, five million Huh-7 cells were subcutaneously inoculated into five-week-old NYG mice. When the average tumor size reached ~ 50 mmt test of the means and two-way ANOVA were used for statistical analysis, with P* < 0.05 defined as significant.Representative results were obtained with at least three independent experiments. All group data (except those indicated) are expressed as the mean ± standard deviation (SD) of a representative experiment performed at least in triplicate, and similar results were obtained in at least three independent experiments. All statistical analyses were conducted using GraphPad Prism 5.0. Two-tailed Student’s To evaluate the expression of GPC3 in HCC patients, we performed immunohistochemistry staining of HCC tumors and normal liver tissue Table . We foun450 nm value at least ten-fold higher than that of the negative control for sequencing  data . The 32A50 value of 0.68 nM but had no obvious effect on GPC3-negative cells   was used3 CAR  waThe HCC-specific expression and important function of GPC3 as a Wnt coreceptor make GPC3 an attractive therapeutic target in HCC. Here, we reported that 32A9, a novel human monoclonal antibody against GPC3, would satisfy the requirements for anti-GPC3 immunotherapy.Many GPC3-specific antibodies have been developed for HCC diagnosis or therapy; however, clear epitope information is available for few of them . Except Because the epitope of 32A9 identified in our study is quite close to the HS binding sites (residues S495 and S509), it seems that the HS chains of GPC3 contribute to the binding of 32A9 (Fig. In the present study, we constructed a human IgG, an immunotoxin and CAR-T cells as representative formats to verify the multiple potential applications of 32A9. The 32A9 immunotoxin showed specific cytotoxicity on GPC3-positive cells, which indicates that 32A9 could also be used to design other internalization-dependent antibody drugs, such as antibody drug conjugates . 32A9 CAOverall, we developed a novel human monoclonal anti-GPC3 antibody, 32A9, which targets the middle region of the GPC3 protein core. 32A9 IgG and 32A9-based immunotherapies showed potent antitumor activity. Our findings provide a rationale for 32A9 as a promising candidate antibody for GPC3 biological function investigation, HCC diagnosis and therapeutic applications.


C; n = 160) or non-conditioned ; source: auction market  or ranch direct ; and rest: 0 h  or 8 h . Means of non-esterified fatty acids (NEFA), serum amyloid-A, haptoglobin, creatine kinase (CK), and percentage of time standing from N calves were greater than C calves . Means of percentage of time standing and CK of R calves were greater than A calves (both p ≤ 0.05). The mean of NEFA of 0 h calves was greater than the 8 h calves (p < 0.01), while the percentage of time standing of 0 h calves was less than 8 h calves (p < 0.01). Statistically significant differences between means of NEFA and standing percentage, were observed between 0 h and 8h calves. Few and inconsistent indicators of reduced welfare were observed between auction market and ranch direct calves, while non-conditioning was associated with greater physiological and behavioural indicators of reduced welfare. Based on these results, conditioning should be implemented as a management practice to improve the welfare of transported calves.Consumers are increasingly concerned about the welfare of farm animals, especially during transportation. The Canadian transport regulations state that weaned cattle require an 8 h rest after 36 h of transport. There are, however, only a few studies that assess the effect of rest on the welfare of beef cattle. The aim of this study was to assess the effect of conditioning, source and rest on indicators of welfare in 7-8-month-old beef calves during long-distance transport. Treatments consisted of a 2 × 2 × 2 factorial design where the main factors were conditioning: conditioned ( Consumers are increasingly concerned about the welfare of farm animals being raised for food consumption . A studyTransport and weaning are stressful management procedures, which have been found to affect the immune response . PrecondReducing stressors associated with the marketing of feeder calves is known to reduce the risk of disease in beef cattle , 15. CalCurrently, there is a lack of literature on the effect of a rest stop on the welfare of beef cattle purchased from different sources and different management strategies. Therefore, the aim of this study was to assess the effect of conditioning, source, and rest, on welfare indicators in 7-8-month-old beef cattle transported by road.This protocol was approved by the Animal Care Committee of Lethbridge Research and Development Centre (LeRDC) (ACC number 1918). Animals were cared for in accordance with the Canadian Council of Animal Care [C; n = 160) or non-conditioned ; source: auction market  or ranch direct ; and rest: 0 h  or 8 h . Calves were divided into two groups (group 1 and 2) and each group was transported by road for 36 h, rested, and transported for an additional 4 h, 8 d apart  of body weight (BW)—were sourced from two different locations in southern Alberta, Canada. Treatments consisted of a 2 × 2 × 2 factorial design where the main factors included conditioning: conditioned , two groups of eighty calves were weaned and transported for approximately 1 h from the ranch of origin to the LeRDC. Upon arrival calves were processed, which included receiving a 7-way bovine clostridial vaccine ; a 5-way bovine viral diarrhea, rhinotracheitis, parainfluenza and bovine respiratory syncytial virus vaccine ; an antibiotic ; an anti-parasitic agent ; and an ear tag and a half duplex RFID tag. During the conditioning period (20 to 21 d) calves were housed in 4 pens (36.7 m × 22.2 m) with a central water trough, with 40 animals per pen. Calves received an ad libitum diet consisting of 35% corn silage, 20% alfalfa hay, 12% barley grain and 3% supplement with vitamins and minerals the first 3 d after arrival to the LeRDC feedlot and ad libitum feed consisting of 85% corn silage, 12% barley grain and 3% supplement with vitamins and minerals for the rest of the conditioning and experimental period.Twenty to twenty one d prior to LO1 , four groups of forty calves were separated from their dams and put on a truck and transported for approximately 1 h from the ranch of origin to the LeRDC. Non-conditioned calves received an ear tag and an RFID tag during LO1 sampling for identification. Processing  was postponed until UN2 to simulate industry practices, where calves are processed once they arrived at the feedlot.Prior to LO1  prior to LO1 sampling. After LO1 and LO2 sampling, calves were sorted into 5 pens, in order for treatments to be equally distributed into one of the 5 compartments within the trailer.n = 80) and non-conditioned (n = 80) calves from the groups described above were sampled (LO1) and transported for approximately 20 min  to a local auction market 7.9 km from the LeRDC, off-loaded and sorted into pens with access to hay and water. Within the 24 h that calves spent at the auction market, they were moved through the sale ring to mimic auction market conditions. Although it is common for calves to be mixed at the auction market, calves in the present study were not comingled with calves from other farms due to time and labour limitations.Conditioned (n = 80) calves and conditioned (n = 80) calves were mixed and sampled at LO1 on  prior to the 36 h transport.The remaining non-conditioned  at 1800 h. Calves did not have access to feed and water for 2.5 h prior to LO1, and for 1 h prior to LO2.ad libitum diet consisting of 35% corn silage, 20% alfalfa hay, 12% barley grain, and 3% supplement with vitamins and minerals for the first 3 d after arrival to the LeRDC feedlot. For the remainder of the conditioning and the experimental period, calves received ad libitum feed consisting of 85% corn silage, 12% barley grain and 3% supplement with vitamins and minerals to meet beef cattle nutrition requirements [ad libitum through a central water trough in the conditioning pens, and through a fence line water trough in the experimental pens.Calves were housed in 32 pens (21 × 27 m) with a fence line water through; 10 animals per pen. Calves received an irements . In addith and October 2nd, 2019), and both trailers arrived to the LeRDC at 0600 h . During both transports, calves were placed in the nose (n = 5), the deck (n = 26), the belly (n = 26), the back (n = 13), and the doghouse (n = 10). Loading densities  differed between compartments in order to have an equal representation of treatments per compartment. The two trailers leaving the auction market and the LeRDC on the same day travelled together to ensure similar road and environmental conditions. Drivers monitored the calves when they stopped for rest and made sure that all calves were standing to avoid injuries. The same experienced drivers transported both group 1 and group 2 calves.Two model 379 Peterbilt trucks and 2018 Merritt feeder cattle tri-axle trailers bedded with wood shavings were used to transport calves for 36 h and for an additional 4 h after rest. Calves transported for 36 h left the auction market and the LeRDC at 1800 h . Loggers were zip tied to the front of the ear tags of a subset of 128 calves, which were equally distributed by compartment. Relative humidity  and temperature  data were collected every 2 minutes during the 36 and 4 h transport and were used to calculate the temperature humidity index (THI)  using thn = 320) and a subset of 12 calves/treatment  were sampled for physiological and behavioural indicators of welfare. Calves were sampled prior to (LO1) and after (UN1) the 36 h transport, and prior to (LO2) and after (UN2) the additional 4 h transport. In addition, calves were sampled on d 1, 2, 3, 5, 14 and 28 after UN2.Weight and rectal temperature were recorded from all experimental calves  attached to the right hind leg of the calves using Vet Wrap . At LO1, accelerometers were placed, each in a vertical position on the hind right leg of the respective calf with the X-axis pointing up towards the backbone of the calf, and set to record data at 1-min intervals. Data from the days when the accelerometers were placed (d -2 and/or -1) and removed (d 5) from the calves were excluded from the analysis due to incomplete data. Standing and lying percentage per day, and standing and lying mean bout duration per day were summarized by day for further analysis.Calf attitude and gait were assessed after UN1 and UN2 as described previously by Meléndez et al. . An expeAttitude was evaluated using a 4-point scale . Normal,Gait score was evaluated using a 5-point scale . Cattle Dry matter intake , was determined by pen feed refusals recorded daily for d 0, 1 and 2 and 3 and weekly  until d 28 after transport for 24 pens. Feed samples were collected on feed refusal days to determine feed dry matter intake (DMI).Individual feeding behaviour was monitored in the remaining 8 pens using the GrowSafe feed bunk monitoring system  as previously described by Melendez et al . Calves The velocity at which animals exited the chute was collected at LO1, UN1, LO2, UN2, 1, 2, 3, 5, 14, and 28 d. The time it took an animal to travel a predetermined distance (2 m) was electronically recorded using two sets of light beams as described previously by Burrow et al. . Flight Calves were weighed while standing in a hydraulic squeeze chute  equipped with a weigh scale. Average daily gain (ADG) was calculated for the first week after transport by subtracting the d 5 BW from the initial (LO1) BW and dividing by the number of days on trial (A = 8 d and R = 7 d). The ADG of the second week was calculated by subtracting the d 14 BW from the d 5 BW and dividing by the number of days between sampling points (9 d). The ADG of the third and fourth week was calculated by subtracting the d 28 BW from d 14 BW and dividing by the number of days between samples (14 d). Shrink percentage was calculated for the 36 h transport (shrink 1) and the additional 4 h transport (shrink 2), using the formula: shrink = (1 - (BW after transport / BW before transport)) ×100.Morbidity and mortality were recorded for the experimental calves over a 28-d experimental period.g at 4°C. Serum was decanted and frozen at -80°C for further analysis, with the exception of osmolality, which was analyzed immediately after centrifugation.Blood samples were collected from a subset of 12 calves/treatment through jugular venipuncture at LO1, UN1, LO2, UN2, 1, 2, 3, 5, 14, and 28 d after UN2. Blood samples were collected into 3, 10-mL non-additive tubes  for osmolality, cortisol, non-esterified fatty acids (NEFA), haptoglobin (HP), serum amyloid-A (SAA), and creatine kinase (CK) analysis. Blood samples were also collected into a 7-mL sodium fluoride tube  for L-lactate analysis, and into a 6-mL EDTA tube  for complete blood cell count (CBC) analysis. Samples collected into the non-additive tubes and the sodium fluoride tube were left at room temperature for 1 h prior to centrifugation for 15 min at 2.5 × NEFA were collected as an indicator of fat mobilization due to feed deprivation. NEFA concentrations were quantified using a colorimetric assay (HR Series NEFA-HR (2), FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The intra-assay CV was 3.3% and the inter-assay CV was 3.6%. L-lactate was measured as an indicator of muscle damage using an L-lactate colorimetric assay  to quantify L-lactate concentrations in serum. The intra-assay CV was 3.7% and the inter-assay CV was 1.9%. CK concentrations were quantified as an indicator of muscle damage using a colorimetric assay . The intra-assay CV was 6.0% and the inter-assay CV was 2.3%. HP and SAA were collected as indicators of stress, inflammation, infection and trauma. HP concentrations were quantified using a colorimetric assay , while SAA concentrations were quantified using an enzyme linked immunosorbent assay . The haptoglobin intra-assay CV was 6.2% and the inter-assay CV was 5.2%. The SAA intra-assay CV was 5.6% and the inter-assay CV was 5.0%. Complete blood cell count (CBC) was measured as an indicator of immune function using a HemaTrueHematology Analyzer . Serum cortisol concentrations were collected as an indicator of acute stress and concentrations were quantified using an immunoassay kit . The intra-assay CV was 6.8% and the inter-assay CV was 11.3%. Osmolality (mOsm/kg) was collected as an indicator of dehydration and values were determined by freezing point depression .t) for each variable were tested and selected based on the Bayesian information criterion (BIC). After selecting the distribution, covariance structures: compound symmetry (CS), heterogeneous compound symmetry (CSH), variance component structure (VC), first-order autoregressive (AR1), and heterogeneous first-order autoregressive (ARH1) were tested and selected based on the BIC value  including the upper (u) and lower (l) limits at a 95% confidence. SAS PROC GLIMMIX iterated 1000 times at multiple levels of iterations . Bonferroni’s correction for multiple comparisons was used.Covariates in the model varied depending upon the variable assessed. Group, time of day, breed, and flight speed were included as covariates in the analysis of NEFA, L-lactate, haptoglobin, SAA, cortisol, CK, osmolality, and CBC. Group and breed were included as covariates for the analysis of BW, ADG, rectal temperature, shrink 1 and shrink 2. Feed intake and breed were included as covariates for the analysis of a subset of animals for shrink 2. Breed was included as a covariate for the analysis of GrowSafe data and group was included as a covariate for the analysis of DMI and accelerometer data. GrowSafe and accelerometer data collected on d 0 were adjusted to the proportion of time animals were in the pen, as this varied between the 0 h and 8 h groups. Results are reported as least squares-means , source (A and R) and rest stop (0 h and 8 h). For example, comparisons of interest included comparisons between treatments with the same source and rest effect, but differing conditioning effect (e.g. C-R-0h vs N-R-0h); the same conditioning and rest effect, but differing source effect (e.g. C-R-0h vs C-A-0h), or the same conditioning and source effect, but differing rest effect (e.g. C-R-0h vs C-R-8h) at a particular sampling point.Statistical significance was Data from 3 accelerometers  were excluded from the analysis due to technical issues and data from the 8h group was not collected on d 3 due to a severe snowstorm.p = 0.01) was observed for standing during the 36 h transport  was greater than the C-R-0h group μ = 40%  at hour 30 of transport. The mean standing percentage of the N-R-0h group μ = 62%  was greater than the C-R-0h group μ = 28%  at hour 32 of transport. The mean standing percentage of the C-A-8h group μ = 44%  was greater than the C-R-8h group μ = 16%  at 34 h of transport, while the mean standing percentage of the C-R-8h group μ = 71%  was greater than the C-A-8h group μ = 28%  at hour 35 of transport. Rest (nested in time) affected (p < 0.01) standing: the mean standing percentage where the 8h rested group was greater (p = 0.04) standing % than the 0 h unrested group at hour 3 during the additional 4 h transport (The three-way interaction of conditioning × source × rest (nested in time) effect  was observed for standing percentage after transport. The 8 h calves stood more (p < 0.01) than 0 h calves on d 0 .A rest (nested in time) effect (s on d 0 . This map < 0.01) were also observed for standing percentage. Mean standing percentage of the N calves was greater than the C calves, and mean standing percentage of the R calves was greater than the A calves (p < 0.01). The N calves μ = 45%  may have stood more than C calves μ = 41% because newly weaned calves typically walk more after weaning, in an attempt to reunite with their dam [μ = 44%  likely were characterized by greater standing percentages than A calves μ = 41%  because they may have been unsettled following weaning, transportation, and sampling (LO1) prior to the 36 h transport, while the A calves had the opportunity to rest for 24 h after sampling (LO1) and prior to the 36 h transport. Contrary to our results, we expected R calves to stand less due to greater fatigue than the A calves that were rested before the 36 h journey. Although differences in standing percentage were statistically significant, they were relatively small (< 4%) and may lack biological relevance. A limitation to our approach is that ‘true’ auction market calves would have been sourced from different ranches and could have displayed different behaviours compared to calves in the present study.A conditioning and a source effect (heir dam . The R cp = 0.04) was observed for mean lying bout duration. The N-0h calves were characterized by lower (p < 0.05) mean lying bout durations than N-8h calves on d 3 (A conditioning × rest (nested in time) effect (s on d 3 . A possip < 0.01) was observed for attitude score, where 8 h calves were characterized by a greater mean attitude score at UN2 μ = 0.30  than UN1 μ = 0.07 . We would have expected 8 h calves to exhibit signs of fatigue after the 36 h transport, rather than after the additional 4 h transport because the transport duration was much shorter and calves were given the opportunity to rest. A conditioning × source (p < 0.05) effect was observed for attitude score, where N-R calves μ = 0.3  were characterized by a greater attitude score than C-R calves μ1 = 0.1 . These results were expected because C calves are more likely to withstand transportation related stress than N calves [A rest (nested in time) effect  was observed for the DMI. Both C-0h and C-8h calves were characterized by greater (p ≤ 0.05) DMI than N-0h and N-8h calves on d 0 and 1 (p < 0.01) DMI than N-8h calves on d 2. These results are in agreement with the feeding behaviour data where overall, the C calves were characterized by greater mean feeding durations, intake, feeding rate, meal duration and meal size than N calves on d 0, 1, and 2. Results were inconsistent between treatments from d 3 to 28 (A conditioning × rest (nested in time) effect ( 0 and 1 . The C-8 3 to 28 . This isp < 0.01) was observed for mean BW, where C-0h was characterized by greater BW than the N-0h calves and the C-8h calves were characterized by greater BW than the N-8h calves BW at each sampling point (p < 0.01) (A conditioning × rest (nested in time) effect ( < 0.01) . This wap < 0.01) was observed for shrink after 36 h of transport, where C calves μ = 10%  characterized by greater shrink than N calves μ = 8% . Conditioned calves likely had more gut fill to lose during transport than N calves that had been recently weaned and transported from their ranch of origin. These results are contrary to a previous study were greater shrink was observed in newly weaned calves compared to calves weaned 15, 30, 45, and 60 days prior to transport [A conditioning effect  were observed between treatment groups for shrink after the additional 4 h transport. Data from a subset of 1 pen  housed in feed monitoring pens were analyzed to account for feed intake during the rest period. These results are similar to a previous study were no differences were observed in shrink after the additional 4 h of transportation between conditioned calves that received 0 h and 8 h of rest [No differences ( of rest . There a of rest  found no of rest  found dip < 0.01) were observed for ADG during the first week after transportation, where N μ = -0.2 kg  were characterized by greater ADG than C μ = -0.3 kg , and R μ = -0.2 kg  calves were characterized by greater ADG than A μ = -0.3 kg  calves. Greater ADG in N calves was likely due to a weight gain caused by a diet change, from an all forage low energy diet to a higher energy backgrounding (85% forage: 12% grain) diet [Conditioning and a source effects (in) diet . Similarin) diet , and conin) diet  during tin) diet . Greaterp = 0.03) and a rest effect (p <0.01) were observed for ADG during the second week. The C-A μ = 0.5 kg  and N-R μ = 0.5 kg  calves were characterized by greater (p <0.01) ADG than the C-R calves μ = 0.4 kg . The 8 h calves μ = 0.5 kg  were characterized by greater (p <0.01) ADG than the 0 h calves μ = 0.4 kg . Differences between N and C calves could be due to the observed weight gain in N calves once they started to eat a higher energy diet containing some grain, similar to the results observed in week 1. Differences observed between rest treatments could be because of the opportunity (or lack of) to rest and eat for 8 h prior to the additional 4 h transport. Calves that did not receive a rest may have been more fatigued and consequently preferred lying down rather than eating after the additional 4h transport, which could have affected ADG, however, no differences were observed in feed intake.A source × conditioning effect (p < 0.01) was observed for ADG between d 14 and d 28, where C-8h μ = 0.6 kg  and N-0h calves μ = 0.6 kg  were characterized by greater ADG than N-8h calves μ = 0.5 kg . Contrary to the first two weeks after transport, C were characterized by greater ADG than N calves and 0 h were characterized by greater ADG than the 8 h calves. This could be because during the first 2 weeks after transportation the rate of weight gain in N calves would be greater due to the change in energy (added grain) in their diet. The combined stress of transport and lack of rest may have affected the ADG of 0 h calves during the first two weeks after transport, while 8 h calves benefited from the rest which resulted in greater ADG during the first 2 weeks after transport. In a previous study, heifers were reported to take 2 weeks to reach a normal DMI after a period of feed restriction [A conditioning × rest effect , and one due to bloat (C-R-0h). Similar to a previous study , low morp < 0.01) was observed for mean NEFA concentrations. At LO1, N-0h were characterized by greater mean NEFA concentrations than C-0h calves, and N-8h calves were characterized by greater mean NEFA concentrations than C-8h calves (p < 0.01) (A conditioning × rest (nested in time) effect ( < 0.01) . Increasp > 0.10) were observed between A and the R calves at LO1. These results were expected because A calves had access to feed and water at the auction market and so would not be more energy depleted than calves transported directly from the ranch. Although the provision of feed is a common practice at the auction market used in this study it is not a common practice at most other auctions within Canada as typically only water is provided. We speculate that under these typical conditions (no feed provided) NEFA concentrations could be greater in A compared to R calves.No differences (p < 0.01) mean NEFA concentrations than C-8h calves. These differences were likely due to N calves not eating or eating less during the rest stop. To test this theory, data from a subset of 10 animals per treatment were summarized by hour and analyzed to assess feeding behaviour during the 8 h rest period. No treatment differences were observed for feeding time (p > 0.10), meaning that all calves spent an equal amount of time at the feed bunk, however a conditioning × rest (nested in time) effect (p = 0.03) was observed for feed intake, where C-8h calves μ = 4.1, 1.9, 1.9, 1.5 kg/h  were characterized by greater (p ≤ 0.02) feed intake than N-8h calves μ = 1.0, 0.6, 0.9, 0.6 kg/h  at hour 1, 2, 3, and 5 of the rest period. Although no differences were observed for feed intake at hours 4, 6, 7, and 8 the C-8h calves μ = 1.4, 1.4, 1.8, 2.0 kg/h  were characterized by numerically greater feed intake than N-8h calves μ = 0.8, 0.8, 1.3, 1.4 kg/h . These results indicate that N calves were eating during the rest stop, however, the amount of feed consumed was lower than in C calves, possibly due to a lack of feed and feed bunk adaptation.At LO2, N-8h calves were characterized by greater (p < 0.01) mean NEFA concentrations than C-8h calves. This is similar to the results observed for LO2, where N calves were characterized by lower feed intake likely due to a lack of adaptation to the feed and the feed bunk. In contrast, no treatment differences (p > 0.10) were observed between N-0h and C-0h calves, likely because calves did not receive a rest and therefore had no opportunity to eat.At UN2, N-8h were characterized by greater (p = 0.01) mean NEFA concentrations than C-8h calves at UN2 which was expected because C-0h calves did not have the opportunity to eat prior to the 4 h transport. These results are in agreement with a previous study, where conditioned calves that received no rest (0h) were characterized by greater mean NEFA concentrations at UN2 than calves that received 8 h of rest after 36 h of transport [p > 0.10) between the groups, N-0h calves did have numerically greater NEFA concentrations than N-8h calves. One explanation is that N-8h calves may not have consumed enough feed during the rest period for NEFA concentrations to be different from the N-0h calves. Even though the amount of feed consumed by N calves was lower than C calves, we would have expected to see a difference between N-0h and N-8h calves, as NEFA concentrations have been reported to decrease rapidly when animals are provided feed and water [The C-0h calves were characterized by greater (ransport . We woulnd water .p < 0.01) was observed for mean SAA concentrations. The N-0h calves were characterized by greater mean SAA concentrations than C-0h calves, and N-8h calves were characterized by greater mean SAA concentrations than C-8h calves, at LO1 and on d 1 (p ≤ 0.01) (p = 0.02) mean SAA concentrations than C-0h calves at UN2. A conditioning × rest (nested in time) effect (p < 0.01) was observed for mean HP concentrations. The N-0h calves were characterized by greater (p < 0.01) mean HP concentrations than C-0h calves at UN1, and on d 1 and 2 (p < 0.01) mean HP concentrations than C-8h calves on d 2, 3, and 6.A conditioning × rest (nested in time) effect ( ≤ 0.01) . The N-0 1 and 2 . The N-8The acute phase response (APR) has been reported to peak 2 to 3 d after a stimulus that produces inflammation, tissue damage, infection or trauma . This isp > 0.10) were observed in cortisol concentrations at LO1 or throughout the study. In addition, we did not expect C calves to have SAA and HP concentrations above the normal range at LO1. Conditioned calves may have been stressed when mixed with conditioned calves from other pens and non-conditioned calves prior to LO1 sampling, however, we would not expect to see an increase in concentrations shortly after a stressor as indicated earlier.In the present study, HP and SAA values were greater than the normal range (HP: < 0.1 g/L and SAA 1.3 mg/L) for cattle  prior toDifferences have been reported between SAA and HP responses to the same stimuli , 57. Difp < 0.01) was observed for mean CK concentrations. At LO1, N-0h were characterized by greater mean CK concentrations than C-0h calves, and N-8h calves were characterized by greater mean CK concentrations than C-8h calves (p < 0.01) (A conditioning × rest (nested in time) effect ( < 0.01) . Creatinp < 0.01) mean CK concentrations than C-0h calves. On d 1 and 2, N-8h calves were characterized by greater (p < 0.01) mean CK concentrations than C-8h calves. These results suggest that N calves had greater muscle fatigue than C calves, and that differences were observed for a longer period of time in N-0h compared to N-8h calves, likely due to recovery of the 8h calves during the rest period. This is contrary to the HP findings where differences in 8h calves were observed at a later time point than the 0 h calves. Differences between these parameters may be because CK is associated with muscle fatigue, while HP concentrations can increase due to stimuli that cause inflammation, infection, stress, or trauma.At UN2, d 1, 2, 3 and 6, N-0h calves were characterized by greater (p = 0.01) was observed for mean CK concentrations. At UN2, R-0h calves were characterized by greater (p < 0.01) mean CK concentrations than A-0h calves (A source × rest (nested in time) effect  was observed for osmolality, however no differences were observed between comparisons of interest. Osmolality is a measure of the total number of dissolved solute particles in one kilogram of a solution. Plasma osmolality has been previously used as is an indicator of hydration in cattle [A source × conditioning × rest (nested in time) effect  was observed for L-lactate concentrations. The C-R-8h calves , d14 μ = 1.0 mmol/L  were characterized by greater mean L-lactate concentrations than N-R-8h calves , d 14 μ = 0.6 mmol/L  at UN1 and on d 14 post-transport. L-lactate is an indicator of muscle damage (similar to CK), however, it can also be produced in other tissues in the body [A source × conditioning × rest (nested in time) effect (the body . These fp = 0.05) was observed for WBC count. The N-8h calves were characterized by greater (p ≤ 0.03) WBC count than C-8h calves at LO1, and on d 1 and 2 post-transport (p > 0.10) were observed between N-0h and C-0h calves. WBC counts were higher in all treatments than the reported normal range (4–12 × 103/μL) for beef calves [A conditioning × rest (nested in time) effect  was observed for granulocyte count. The mean granulocyte count for the C-0h calves was greater (p < 0.01) than the N-0h calves, and the mean granulocyte count of the C-8h calves was greater (p < 0.01) than N-8h calves at LO1 (p < 0.01) granulocyte counts than C-8h calves on d 1 and 2 post-transport. Similar to the results for WBC counts, mean granulocyte counts were greater in N calves than C calves except at LO1. This is contrary to what we expected as N calves were recently weaned and transported and we would have expected calves to be more stressed than C calves. A source × rest (nested in time) effect (p < 0.01) was observed for granulocyte counts. The A-0h calves were characterized by greater (p = 0.01) granulocyte counts than R-0h calves on d 3 post-transport (3/ul) reported for beef cattle [A conditioning × rest (nested in time) effect Click here for additional data file.S2 TableLeast squares-means (± upper and lower limits at 95% confidence) of production and behavioural parameters of conditioned (C) and non-conditioned (N), auction market (A) and ranch direct (R) calves rested for 0 (0 h) or 8 (8 h) h.(DOCX)Click here for additional data file.S3 Table1.Least squares-means (± upper and lower limits at 95% confidence) of physiologic parameters of conditioned (C) and non-conditioned (N), auction market (A) and ranch direct (R) calves rested for 0 (0 h) or 8 (8 h) h(DOCX)Click here for additional data file.S1 FileDetailed feeding behavior results and least squares-means of feeding behavior for conditioned (C) or non-conditioned (N), auction market (A) or ranch direct (R) calves rested for 0 (0 h) or 8 (8 h) h. (A) Feeding rate, (B) feeding time, (C) meal frequency, (D) meal size, (E) feeding intake, and (F and G) meal duration.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens.  The proteins consist of four transmembrane domains, which can generate a protein structure containing one small extracellular loop (EC1) and one large extracellular loop (EC2). The function of tetraspanins depends on their ability to be laterally associated with the partner molecules, e.g., immunoglobulin superfamily members, integrins, and signalling molecules, to form functional assemblies as termed tetraspanin enriched microdomains (TEMs)3. Evidence is emerging and suggesting that TEMs are involved in many infectious diseases caused by viruses, bacteria, and protozoa and may act as gateways for infections4. TEMs may participate in various stages of infective processes, from facilitating binding to activating signalling pathways. Disruption of the TEM function may disorganize many potential receptors and interferes with the binding site of pathogens. Novel anti-infections targeting tetraspanins is a new strategy to inhibit the process of these infections.Tetraspanins are a superfamily of four-span transmembrane proteins distributed in multicellular organismsBurkholderia pseudomallei is the causative agent of melioidosis, a fatal disease in tropical regions, endemic in Southeast Asia, and northern Australia5. The predicted burden of disease is about 165,000 cases, which includes 89,000 deaths6. The clinical manifestation of melioidosis ranges from acute to chronic infections with pneumonia and septicaemia being the most common presentations7. The mortality rate is 10 to 50% worldwide, and approximately 35% in Thailand. The death from melioidosis is often caused by delays in treatment or because of complications in clinical recognition and diagnosis9. Due to the high mortality rate, intrinsic antibiotic resistance, low infectious dose, aerosol route of infection, and no vaccine available, melioidosis is a public health concern in tropical countries. B. pseudomallei is an environmental bacterium, but it is classified as a CDC tier 1 select agent, a potential biothreat. It can infect humans and animals by inoculation, inhalation, and ingestion. B. pseudomallei can infect and survive within either phagocytic or non-phagocytic cells. After infection, the bacteria multiply in the cytoplasm and induce cell-to-cell fusion or multinucleated giant cell formation (MNGC). This process is crucial for spreading from infected cells to neighbouring cells. In this way, the bacteria can avoid exposure to host immune response or antibiotics10. Once the infection is established, B. pseudomallei may disseminate to many organs5. Although B. pseudomallei has been reported to use several virulence factors for invasion, the potential host molecules that contribute to bacterial and host interactions are poorly understood.B. thailandensis is a closely-related species of B. pseudomallei. B. thailandensis can infect both phagocytic and non-phagocytic cells and subsequently induce MNGC formation in vitro similar to B. pseudomallei11. Tetraspanins have been widely implicated in the regulation of various cell: cell fusion processes, including viral syncytium formation15. We have also recently demonstrated that reagents targeting tetraspanins CD9, CD63, and CD81 could affect B. thailandensis-induced MNGC in mouse macrophage cell lines. In brief, recombinant proteins corresponding to the EC2s of these tetraspanins inhibited MNGC formation while antibodies to CD9 and CD81 enhanced MNGC formation, and a macrophage cell line generated from a CD9KO mouse showed enhanced MNGC formation16. This led us to hypothesize that tetraspanins might play a role in B. pseudomallei infection and bacterial spreading between host cells during melioidosis.B. pseudomallei infection, we used monoclonal antibodies (MAbs) specific to the large extracellular EC2 domain of tetraspanins and recombinant EC2 proteins of CD9, CD63, and CD81 to pretreat a human epithelial cell line A549 and a mouse macrophage cell line J774A.1 before infection. Mouse macrophage cell lines have been widely used as the in vitro model for B. pseudomallei and B. thailandensis infection and MNGC formation21. The mouse tetraspanin CD9 is about 90% homology to Homo sapiens CD9 and has been used to represent mammalian cells in sperm-egg fusion assay23. We determined the expression of tetraspanins on host cells by flow cytometry and assessed the number of bacterial adhesion and internalization to these cells by colony count. MNGC formation and MNGC size of infected cells was determined by imaging analyses using light microscopy and confocal microscopy. We also performed tetraspanin genes knock out on cells using the CRISPR/Cas9 system and confirmed a role for host tetraspanins for B. pseudomallei infection. Finally, the role of tetraspanins for infections was compared between B. pseudomallei and B. thailandensis.In order to investigate the role of tetraspanins in B. pseudomallei infection and MNGC formation were investigated in two cell lines. A549 represented a non-phagocytic human epithelial cell, and J774A.1 represented a phagocytic mouse macrophage cell. To determine if tetraspanins are expressed on the cell surface of the cells, we used flow cytometry and antibodies that recognise CD9, CD63, and CD81. We observed in both A549 and J774A.1 that the MFI level of cells treated with antibodies against CD9 and CD81 were significantly higher than those treated with isotype controls  , respectively  and 41.1% (P = 0.003), respectively compared with cells treated with isotype-matched controls or GST control  , respectively when compared with cells treated with isotype controls  and 31.5% (P = 0.003) compared with GST control  Figs.  and 3E als Figs.  and 3G. B. pseudomallei-induced MNGC formation by CD9-EC2 and CD81-EC2 may increase at higher EC2 concentrations, pretreatments of J774A.1 were performed with CD9-EC2 and CD81-EC2 at 10, 20, 50, and 100 µg/ml. We did not observe different MNGC formation between cells treated with PBS or GST control and those pretreated with ten µg/ml CD9-EC2, but there was a decrease in MNGC formation when cells were treated with 20, 50, and 100 µg/ml of CD9-EC2 concentrations  CD9 and CD81 localisation in J774A.1 and (2) CD81 localisation in A549 cells during B. pseudomallei-induced MNGC formation by immunofluorescence staining and confocal microscopy. CD9 and CD81 were examined using specific antibodies and LSM, Z-stack analysis. In uninfected cells, immunofluorescence staining showed that CD9 and CD81 distributed in all areas of the cells  effectors facilitate B. pseudomallei entry into host cells33. However, our study did not find that CD9, CD63, and CD81 mediate the adherence of B. pseudomallei to epithelial cells and macrophages. The interplay between host cells and B. pseudomallei is complex and not very well understood but presumably in macrophage uptake is mainly by phagocytosis.Tetraspanins are known to be involved in infections caused by other intracellular pathogens, particularly virusesB. pseudomallei internalisation into A549 cells. The data suggest that anti-CD9 MAb or CD9-EC2 may stabilise or enhance the formation of entry platforms for bacteria. The interaction of CD9 with B. pseudomallei components that explains its involvement with bacterial internalisation is currently unknown. Further study is required to investigate the role of CD9 as well as other tetraspanins for B. pseudomallei internalisation and to identify their partner proteins for the organisation of "entry platforms" for B. pseudomallei infection. Our data cannot suggest that overexpression of CD9 in A549 leads to a decrease in internalisation during B. pseudomallei infection but data from Hassuna NA et al. suggest that overexpression of CD63 in HEK 293 cells is not sufficient to promote S. Typhimurium binding to non-phagocytic cells30.Interestingly, our results demonstrated that anti-CD9 MAb and CD9-EC2 but not CD63 or CD81 reagents enhanced 34 but may be affecting elements of the T3SS-mediated entry. CD81 has been shown to be necessary for internalisation of L. monocytogenes in which the mechanism involving phosphatidylinositol 4-kinase II28. CD81 might also affect actin polymerization, e.g., CD81 interfaces between the plasma membrane and the cytoskeleton by activating spleen tyrosine kinase, colocalizing ezrin-radixin-moesin proteins, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling35. CD9 and CD81 have also been shown to be linked to the actin cytoskeleton via their interaction partner proteins EWI-2 and EWI-F36. Our data indicate that CD81 plays a significant role in MNGC formation in both epithelial cells and macrophages. In contrast, the data indicate that CD9 plays this role in macrophage cells only. The finding from our CRISPR/Cas9 knockout experiment in A549 cells has consistently confirmed the role of CD81 in B. pseudomallei-induced MNGC formation and CD9 in internalization. Although the CD9 and CD81 genes knockout experiment in J774A.1 cells was not successful, our data suggest that CD9 and CD81 are involved in B. pseudomallei-induced MNGC formation in this cell. The evidence for this belief comes from our consistent results that the antibodies and EC2 proteins of CD9 and CD81 can block B. pseudomallei-induced MNGC formation.There was no effect of CD9 or CD81 reagents on J774A.1 cells internalisation which might be expected to occur via phagocytosis but we observed the effect in A549 cells, where the mechanism presumably involves the T3SS. This suggests that the tetraspanins were not affecting phagocytosisB. pseudomallei during cell fusion. Other studies have also demonstrated the relevance of CD9 and CD81 on cell-to-cell fusion in other conditions. For example, anti-mouse CD9 and CD81 MAbs can inhibit sperm-oocyte fusion37. Gene knockout of CD9 or CD81 in mice could impair sperm-egg fusion while double knockout of CD9 and CD81 were completely interfile38. Anti-CD9 MAb can inhibit canine distemper virus-induced cell-to-cell fusion15. CD9-EC2 can inhibit monocyte fusion and reduce MNGC size in monocytes cultured under fusogenic conditions41. To our knowledge, the role of tetraspanins in MNGC formation has never been reported in B. pseudomallei. It is possible that these tetraspanins may not directly interact with bacterial molecules during the infection and MNGC formation processes, but they may be regulated by other host partner TEM proteins that may directly interact with B. pseudomallei. Other host proteins have been shown to be involved with B. pseudomallei infection including E-selectin (CD62E), a fusion regulatory protein (CD98), E-cadherin (CD324) integrin (LFA-1) complex, ICAM-1 (CD54), signal-regulatory protein α (CD172a), and integrin-associated protein (CD47). Blocking these proteins with specific antibodies could inhibit B. pseudomallei-induced MNGC formation in human leukemic monocyte lymphoma cell line42. We also showed the effects of antibodies to CD47, CD98, and dendritic cell-specific transmembrane protein (DC-STAMP) on B. thailandensis-induced fusion in J774.2 cells16. CD9 and CD81 that are involved in B. pseudomallei-induced MNGC formation may be associated with other cell surface molecules that may interact with B. pseudomallei. The antibodies and recombinant EC2 proteins target the EC2 regions of the tetraspanins. However, if the mechanism of action is affecting signalling in the cells, then this could be by the recruitment of intracellular signalling molecules via the C-terminal regions of the tetraspanins. Moreover, tetraspanin peptides are active on the host cell rather than on the bacteria themselves, as they have no toxicity to the bacteria, e.g., they have no anti-S. aureus adherence activity when added to the bacteria before adhesion to host cells31. Blocking of the EC2 domain of tetraspanins by antibodies may interfere with the association between tetraspanins and other partner molecules, leading to alteration of TEM organization and function43. This vital information warrants further investigation.Our immunofluorescence and confocal microscopy analyses showed that CD9 and CD81 were distributed widely on the cell surface and located with B. thailandensis was different from those induced by B. pseudomallei. Specifically, anti-CD9 and anti-CD81 enhanced MNGC formation induced by B. thailandensis in both A549 and J774A.1 cells, whereas anti-CD9 and anti-CD81 inhibited MNGC formation induced by B. pseudomallei, suggesting that the roles of tetraspanins for cell–cell fusion are dependent on the microorganism. Although B. thailandensis is a closely-related species to B. pseudomallei and widely used as a model to study in melioidosis, our study suggests different effects of host tetraspanins transmembrane in the bacterial-induced cell fusion of these two species. The mechanism by which tetraspanins are involved in cell–cell fusion after Burkholderia infection is not well understood. Our data and data from other studies suggest that host factors and probably bacterial factors seem to be involved in the cell-to-cell spread process. The MNGC formation induced by B. pseudomallei or B. thailandensis requires the type VI secretion system (T6SS-5), which is regulated by BsaN, VirAG, and BprC44. This mechanism could lead to cell death by activating autophagy and triggering the cGAS-STING pathway via micronuclear formation20. T6SS-5 plays an essential role in MNGC formation by which the C-terminal of VgrG5 processes fusogenic activity45. It is likely that the different amino acid sequences and conformation of these factors46 might contribute to the interaction with associated tetraspanins. It will be of interest to determine the effect of CD9 or CD81 on MNGC formation during infection with B. pseudomallei T6SS-5 and VgrG5 mutants in future studies.The effect of anti-tetraspanin MAbs on MNGC formation induced by It is not clear why MAb treatments were more efficient in inhibition of MNGC formation and subsequent reduction of MNGC size than recombinant EC2 protein treatments. A possible explanation is that the MAbs may be more specific to the disruption of tetraspanin function compared to the recombinant EC2 proteins.Staphylococccus aureus skin and wound infections to cultured keratinocytes and human skin infection model31. Our finding suggests that targeting these tetraspanins using specific antibodies or recombinant EC2 proteins could inhibit B. pseudomallei-induced MNGC, thus limited bacterial spreading between different cell types and that CD9 and CD81 may be good vaccine targets. Antibodies or recombinant EC2 proteins may be useful for therapeutic purposes. However, this requires further evaluation in other cell types and testing in animal models of melioidosis.Tetraspanins have been reported as a novel treatment for parasitic and bacterial infections. Synthetic peptides of tetraspanin, CD9 prevent B. pseudomallei-induced cell-to-cell fusion of human epithelial and mouse macrophage cells by which tetraspanins are associated with the process by their distribution on the cell surface. Our study in epithelial cells and macrophages revealed that CD9 and CD81 are involved in B. pseudomallei-induced MNGC formation. Antibodies specific to tetraspanins and EC2 proteins could be a potential therapeutic agent to prevent the spread of B. pseudomallei to uninfected cells.Host and bacterial interplay are complicated, but our study provides insights into the understanding of B. pseudomallei strain K96243, a clinical isolate, and B. thailandensis strain E264, an environmental isolate from Thailand, were used in this study. The bacteria were grown on Columbia agar  and incubated at 37 °C overnight. Human lung epithelial cell A549  was cultured in RPMI 1640  supplemented with 10% heat-inactivated fetal bovine serum (FBS) . Mouse macrophage cell line J774A.1 was cultured in DMEM (Gibco BRL). All cell cultures were performed at 37 °C in a humidified 5% CO2 incubator. All experiments involving B. pseudomallei were performed in a biosafety level 3 laboratory.30.Three monoclonal antibodies (MAbs) that recognised human tetraspanins CD9 (clone 602.29), CD63 (clone H5C6), CD81 , and IgG1 isotype control (clone JC1) were used to treat A549 cells. MAbs that recognised mouse tetraspanins, including MAbs specific to CD9 , CD63 , CD81 , and rat IgG2b, rat IgG2a, and hamster IgG1 isotype controls were used to treat J774A.1 cells. The production of recombinant glutathione S-transferase (GST) fusion proteins with CD9, CD63, and CD81 tetraspanin EC2 extracellular domains were previously described30. The cells were cultured in the medium for 2 days. To remove cells from culture flasks, A549 cells were trypsinised with 0.25% of trypsin–EDTA (Gibco BRL), while J774A.1 cells were dislodged by cell scraper. The cells at a concentration of 1.0 × 105 cells were incubated with 5 µg/ml of each primary Mab against tetraspanins, or isotype control at 4 °C for 1 h. The cells were then washed with PBS followed by incubating with 5 µg/ml secondary antibodies  or FITC-conjugated anti-rat IgG; J774A.1: anti-hamster IgG  at 4 °C for 30 min. The cells were centrifuged at 1,000 rpm at 4 °C for 5 min, washed with PBS, and analysed using a FACSCalibur flow cytometer . The data was analysed using a Flowjo software version 10.1 . Two independent experiments were performed for each experiment.Cell surface expression of tetraspanins on A549 and J774A.1 cells was assessed using flow cytometry. The experiment was performed as previously described4 cells in 100 µl per well into a 96-well plate and incubated at 37 °C for overnight. The cells were pretreated with 20 µg/ml anti-tetraspanin MAbs or recombinant EC2 proteins at 37 °C for 1 h, then washed with PBS and infected with B. pseudomallei K96243 at a multiplicity of infections per cell (MOI) at 100 for A549 and 30 for J774A.1. The plates were incubated at 37 °C with 5% CO2 for 1 h. Non-adherent bacteria were detached by gentle washes with PBS for 5 times47, and the cells were lysed with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. The bacterial suspension was serially diluted with PBS, spread on Columbia agar, and incubated at 37 °C for 24 h for colony count. The number of adhered bacteria was enumerated as colony-forming units (CFU). The percentage of adhesion reflected the number of adherent bacteria at 1 h post-infection × 100/ the number of CFU added. The percentage of adhesion was calculated from three independent experiments; each was conducted in triplicate47.A549 and J774A.1 cells were seeded at 1 × 1017. The adhesion assay was then performed as described above.In another experiment, J774A.1 cells were pretreated with 2 µg/ml of cytochalasin D (Sigma-Aldrich) for 2 h to block phagocytosisB. pseudomallei at MOI of 100 for A549 and 30 for J774A.1 for 2 h to allow bacterial internalisation. The cells were washed with PBS before adding a fresh culture medium containing 250 mg/ml of kanamycin (Gibco BRL). The infected cells were further incubated for 2 h to kill residual extracellular bacteria. The infected cells were then washed and lysed with 0.1% Triton X-100. The intracellular bacteria were colony counted. The percentage of internalization reflected the number of intracellular bacteria at 4 h post-infection × 100/ the number of bacteria added. The percentage of internalization and MNGC formation was calculated from three independent experiments; each was conducted in triplicate47.A549 and J774A.1 cells were seeded and pretreated with anti-tetraspanin MAbs or recombinant EC2 proteins, as described above. The cells were then washed with PBS and infected with B. thailandensis E264 and B. pseudomallei K96243, as described by Kespichayawattana W et al.19. A549 and J774A.1 cells were seeded in a 96-well plate and pretreated with anti-tetraspanin MAbs or recombinant EC2 proteins, as described above. The cells were washed and infected with bacteria at MOI of 100 for A549 cells and 30 for J774A.1 cells for 2 h. The extracellular bacteria were then removed, and the cells were further incubated with a fresh culture medium containing 250 mg/ml of kanamycin. MNGC formation was quantified after 12 h of incubation. The cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, washed again with PBS, and stained with Giemsa solution  for 5 min. The cells were rewashed with water and air-dried at RT. The stained cells were imaged with an Operetta High Content analysis system (PerkinElmer) and analyzed using ImageJ software version 1.52n (https://rsb.info.nih.gov/ij/). The cells with 3 nuclei or more were considered to be MNGC. The percentage of MNGC was calculated from many nuclei in MNGC × 100/total number of nuclei counted. MNGC size was calculated from the number of nuclei in MNGC/number of MNGC40. The percentage of MNGC formation was calculated from three independent experiments, each of which was conducted in triplicate.The determination of MNGC formation was performed with 48. A549 or J774A.1cells were seeded at 7 × 105 cells/well on a sterile glass coverslip in a 6-well cell culture plate and incubated overnight at 37 °C with 5% CO2. The monolayer of cells was infected with B. pseudomallei at MOI of 25:1 and 10:1 for two h washed with PBS, and further incubating with a medium containing 250 µg/ml of kanamycin for 6 h. The cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min. The cells were permeabilised with 0.5% triton X-100 for 30 min. The permeabilised cells were then incubated at 37 °C for 1 h with a mixture of (i) an Alexa Fluor 488 conjugated mouse MAb 4B11 specific to B. pseudomallei capsular polysaccharide49 at dilution of 1:200, (ii) 20 µg/ml of an Alexa Fluor 555 conjugated mouse anti-human CD81 or Alexa Fluor 555 conjugated rat anti-mouse CD9 and (iii) Hoechst 33,258 (Invitrogen) at dilution of 1:1000. MAbs 4B11 was conjugated with an Alexa Fluor 488 (Invitrogen) and mouse anti-human CD81 or a rat anti-mouse CD9 antibodies or isotype-matched controls were conjugated with an Alexa Fluor 555 according to the manufacturer's instruction (Invitrogen). After three times washing with PBS, coverslips were mounted on a glass slide with 8 µl of ProLong Gold antifade reagent (Invitrogen). The cells were imaged using a laser scanning confocal microscope  by Z-stack mode with an oil-immersion 100 × objective lens and analysed with a ZEN 2010 version 6.0 software.Immunostaining was performed with CD81 for A549 cells and CD9 for J774A.1 cell, as previously describedCD9 and CD81 genes knock out were performed with A549 and J774A.1 cells using a CRISPR/Cas9 system50. Single guide RNA (sgRNA) sequences targeting both mouse and human CD9 and CD81 were designed with appropriate overhangs: sgRNA1 (Human CD9-Fw 5′-CACCTTAGGGCGCGCGACCCGCTG-3′ and Human CD9-Rw 5′-AAACCAGCGGGTCGCGCGCCCTAA-3′); sgRNA2 (Human CD81-Fw 5′-CACCCCTCCACTCCCATGGCGGCG-3′ and Human CD81-Rw 5′-AAACCGCCGCCATGGGAGTGGAGG-3′); sgRNA3 (Mouse CD9-Fw 5′-CACCCTGAGACACGCTGCGGCTAG-3′ and Mouse CD9-Rw 5′-AAACCTAGCCGCAGCGTGTCTCAG-3′); sgRNA4 (Mouse CD81-Fw 5′- CACCTGGGGACAGGGCGCACCCCA-3′ and Mouse CD81-Rw 5′-AAACTGGGGTGCGCCCTGTCCCCA-3′). sgRNA sequences targeting both mouse and human CD9 and CD81 were cloned into the Bbs1 site of the pSpCas9(BB)-2A-Puro V2.0  by using single-step digestion-ligation and a standard cloning method51. The inserted gRNA sequences were verified by direct sequencing. A549 or J774A.1 cells were transfected for 24 h with individual plasmids expressing Cas9 and the corresponding sgRNA using lipofectamine 3,000 reagent  according to the manufacturer's instruction. After transfection, the cells with plasmids were selected by incubating in culture media containing puromycin (2 μg/mL) for 48 h. Both CD9 and CD81 negative cells were sorted into a 5 ml polystyrene round-bottom tube  using a BD FACSAria III . Single-cell clones were isolated by limiting dilution in a 96 well plate and expanded for gene knockout validation.Genomic DNA was isolated from individual single-cell clones and non-edited control cells using the Gentra Puregene Blood Kit . PCR amplicons spanning the Cas9-sgRNA cleavage site was amplified in a 25 µl total reaction volume consisting of 1 µl of 50 ng DNA template, 12.5 µl of KAPA2G Fast Hotstart PCR kit , 0.5 µl of each 10 µm forward and reverse primers with the following forward and reverse primers: CD9: CD9F 5′-GCGAGGCCTCCACGTAAGTC-3′ and CD9R 5′-AACTTAGCCTGGGCCGAACG-3′; or CD81: CD81F 5′- ATAAGTACTGCGGAGCGAGG-3′ and CD81R 5′-CTCCTGGAGCCCACAGGTGG-3′. The PCR conditions were as follows: initial denaturation at 95 °C for 3 min; 34 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 10 s, and extension at 68 °C for 20 s; followed by a final extension at 68 °C for 5 min. The PCR amplicons were run on a 2% agarose gel. Each PCR fragments were purified and subjected for direct sequencing using the forward and reverse PCR.g at 4 °C for 15 min. The protein concentration of the supernatant was quantified using a BCA kit . Cell lysates were diluted with sample buffer and heated at 100 °C for 5 min. Thirty µg of protein was run on 15% SDS gels, transferred to a nitrocellulose membrane and probed with monoclonal anti-CD9 antibody (clone 602.29) or anti-CD81 antibody  at dilution of 1:1000. Rabbit anti-mouse IgG/HRP  at dilution of 1:2000 was used as secondary antibody. BM Chemiluminescence Blotting Substrate  was added on the membrane before exposure to an X-ray film.Knock out cells were tested for the presence of CD9 and CD81 at the protein level using 15% SDS-PAGE and Western blotting. A549 cells were lysed with 100 µl lysis buffer  and 1 µl protease inhibitor   at 4 °C for 30 min. The lysed cells were centrifuged at 15,000 × t tests for comparison between two groups. A statistically significant difference was considered at P value < 0.05.Statistical analysis was performed using GraphPad Prism software version 6.0 . The data are presented as individual points and means ± standard deviation. The data were tested for the difference using one-way ANOVA for comparison among three or more groups and Supplementary Figures


Ulcerative colitis (UC) causes chronic inflammation and damage to the colonic mucosal layer. Recent studies have reported significant changes in phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in UC patients and oral administration of PC has considerable therapeutic effects against UC, suggesting the metabolism of phosphatidylcholine may be involved in the UC development. Our previous work has demonstrated that berberine effectively suppresses inflammation and protects colonic mucosa injury in DSS-induced colitic mice. However, whether the therapeutic effects of berberine are attributed to its action on the PC metabolism remains unknown. In the present study, we have shown that berberine significantly reduces the lysophosphatidylcholine (LPC) levels in the sera of DSS-induced experimental colitis mice and LPS-stimulated macrophage RAW 264.7 cells. The cytosolic phospholipase A2a (PLA2G4A), an enzyme for hydrolyzing PC to LPC, was found to be up-regulated in the colon tissue of experimental colitis mice and inflamed macrophage RAW 264.7 cells. We then demonstrated berberine inhibits the phosphorylation of cytosolic phospholipase A2a (PLA2G4A) in the colon tissue of experimental colitis mice and inflamed macrophage RAW 264.7 cells. Subsequently, we revealed berberine suppressed the expression of pro-inflammatory factors including TNF-alpha and IL-6 through regulating PLA2G4A dysfunction in macrophage RAW 264.7 cells. Mechanistically, we found that berberine directly binds to PLA2G4A and inhibits MAPK/JNK signaling pathway to inhibit PLA2G4A activity in inflammatory status. Therefore, we concluded that berberine inhibits colonic PLA2G4A activity to ameliorate colonic inflammation in experimental colitic mice, suggesting modulation of the PC metabolism via PLA2G4A might be beneficial for establishing new therapies strategy for UC. Ulcerative colitis (UC) and Crohn’s disease (CD) are classified as severe and chronic inflammatory bowel disease (IBD) that causes inflammation and sores (ulcers) in the gastrointestinal tract, particularly in the large intestine . Lipid mCoptis chinensis Franch. (Chinese goldthread), Berberis vulgaris L. (European barberry), and Berberis aquifolium Pursh. (Oregon grape). Recent studies along with our findings have suggested berberine alleviates experimental colitis by suppressing inflammatory responses in macrophages and T cells and maintaining intestinal barrier function . PLA2 activator, JNK inhibitor and activator, Mouse Pla2g4, and control siRNA were purchased from Santacruz . The recombinant human PLA2G4A protein was obtained from R&D Systems .Berberine chloride was purchased from Shenzhen ChemStrong Scientific Co., Ltd.  at the highest available purity (≥95%). Lipopolysaccharide (LPS), TNF-α, and endotoxin removal solution were purchased from SIGMA . Interferon-γ was obtained from Merck . cPLAα, p-cPLAα, JNK, and p-JNK antibodies were purchased from Cell Signaling . Insulin was purchased from Life Technologies . Lipid standards, including SPLASH2. Berberine hydrochloride was dissolved in DMSO to prepare 50 mM stock solution. Other reagents were dissolved in 1× phosphate-buffered saline (PBS) as stock solution.RAW 264.7 macrophage cell line was obtained from ATCC . RAW 264.7 cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco). RAW 264.7 cells were maintained at 37°C in a humidified atmosphere containing 5% COad libitum. All experimental protocols and procedures were supervised by the Animal Care Ethics Committee of HKBU and the Department of Health, Hong Kong Special Administrative Region. Induction of colitis on mice was through 2.0% (w/v) dextran sulfate sodium (DSS) in drinking water. After 3 days of development, colitis mice were confirmed by fecal occult blood testing and randomly divided into two groups (model group and berberine treatment group). Mice in the berberine treatment group were administered berberine hydrochloride solution  through oral gavage, while mice in the model group were administered water as parallel control. On day 7, the mice were anesthetized by 3% chloral hydrate through intraperitoneal injection. About 1 ml serum was collected through hemostasis from the heart to sacrifice the mice.C57BL/6 background male mice were purchased from the Chinese University of Hong Kong. They were maintained in the animal laboratory of the School of Chinese Medicine, Hong Kong Baptist University (HKBU). The animals were kept in a standard living condition  with diet and water For lipidomics study on serum, 30 μL serum and 30 μL water were added with 240 μL Folch solvent (chloroform:methanol = 2:1) and vortexed vigorously. Two phases were formed after centrifugation at 8,000 rpm for 15 min at 4°C. For lipidomics study on colon tissue, about 10 mg frozen colon tissue was weighed and homogenized in 1× PBS with a 1:10 m/v ratio. Then, 60 μL homogenate was added with 240 μL Folch solvent  for lipid extraction. The samples were vortexed and centrifuged at 8,000 rpm for 15 min at 4°C to induce phase separation. About 150 μL down layer solution was collected and dried under a vacuum concentrator within 30 min at room temperature. The samples were stored in −80°C until analysis.3, 350 μL H2O, and 350 μL CHCl3 were added step by step and vortexed at each step to induce phase separation. After centrifugation at 4,000 rpm for 30 min at 4°C, the down layer (200 μL) was transferred to 1.5 ml tubes. The samples were dried under vacuum at room temperature within 30 min and stored at −80°C until analysis.RAW 264.7 cells were extracted according to a well-agreed protocol from LIPID MAPS using Bligh–Dyer (BD) extraction. In brief, cells from a 100-mm culture plate were washed twice with 5 ml ice-cold 1× PBS after aspirating the culture medium. Then, 10 ml 1× PBS was added to the plate, and the cells were scraped and transferred to 15-ml conical tubes. Then, 200 μL suspension was taken out for protein quantification by a protein BCA kit. Cell pellets were collected after centrifugation at 3,500 rpm for 10 min at 4°C. Then, the BD system: 1 ml of 80°C pre-cold 70% MeOH, 350 μL CHCl2O  and centrifuged at 14,000 rpm for 10 min at 4°C. For LC-MS analysis, the liquid chromatogram was performed on an Agilent 1290 UPLC system and mass spectrometry was employed on an Agilent 6540 quadrupole time-of-flight mass spectrometry system. The Waters ACQUITY UPLC HSS C18 column  was used for separation at 40°C. Mobile phases A and B were prepared as 60:40 water/acetonitrile and 90:10 isopropanol/acetonitrile with 10 mM ammonium formate and 0.1% formic acid. The linear gradient was set as 0–32% B (0–0.8 min), 32–45% B (0.8–2 min), 45–52% B (2–2.5 min), 52–58% B (2.5–4 min), 58–66% B (5–5.5 min), 66–70% (5.5–7 min), 70–75% B (7–8.8 min), and 75–97% B (8.8–10.5 min); then maintained at 97% B from 10.5 to 15 min; and decreased to 32% B from 15 to 18 min. Lipids were detected and identified in both negative and positive ionization modes. The injection volume is 3 μL in the negative mode and 1 μL in the positive mode for each analysis. For lipid identification, the frame m/z values were used to search for information on LIPID MAPS and Human Metabolome Database (HMDB). The matches were confirmed by exact tandem mass (MS/MS) of lipids and retention time based on standards and databases.Before LC-MS analysis, the dried samples were dissolved with 200 μL of acetonitrile/isopropanol/H® Transfection System (Life Technologies) was used following the instructions of the manufacturer for transfection study. For transfection on RAW 264.7 cells, 1 × 106 cells were transfected with 20 nM siRNA with one pulse of 1,680 V for 20 ms. After transfection, cells were seeded in a 6-well cell culture plate to recover overnight in antibiotics-free medium. Then, RAW 264.7 cells were incubated with berberine 10 μM for 2 h before challenged with stimuli (LPS) for 24 h. Mouse Pla2g4 siRNA and matched control siRNA were transfected using the Neon Transfection System according to the manufacturer’s instructions. Quantitative RT-PCR was used to detect transfection efficiency.NeonTotal RNA was isolated from RAW 264.7 cells using TRIzol reagent  following the manufacturers’ instruction, and cDNA was synthesized with a reverse transcriptase kit . For RT-PCR analysis, amplification was performed using SYBR Green for 40 cycles . The primers were designed and synthesized from Invitrogen. The primer sequences are provided in An equal amount of protein (10 μg) from each sample was separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20  at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies were prepared with 5% bovine serum albumin (BSA). After being washed with TBST five times, the blots were hybridized with secondary antibodies conjugated with horseradish peroxidase  in 5% non-fat milk dissolved in TBST at room temperature for 1 h and washed five times with TBST. The membranes were then incubated with enhanced chemiluminescence reagents and exposed to X-ray film .NO production was evaluated measuring nitrite, the stable metabolite of NO, from the culture media of RAW 264.7 by a modified Griess reagent (Sigma) according to the manufacturers' instructions. Duplicates of 100 μL of supernatant or culture media were added to 96-well microtiter plates and mixed with 100 μL Griess solution. The plate was then read on a microtiter plate reader using 540-nm wavelength. TNF-α and IL-10 were measured from supernatants of RAW 264.7 cells through enzyme-linked immunosorbent assay (ELISA) analysis by using ELISA kits according to the manufacturer’s protocol . Duplicates of 100 μL of supernatant or culture media were added to 96-well coating plates for ELISA analysis using 570-nm wavelength. The result obtained from the microtiter plate reader was normalized by measured protein.The PLA2G4A activity was measured by its ability to hydrolyze 1-hexadecanoyl-2-(1-pyrene-decanoyl)-sn-glycero-3-phosphocholine. The berberine was dissolved in DMSO (50 mM) as a stock solution to prepare the enzyme–inhibitor complex. The assay procedure was followed by the protocol online from R&D Systems.Homology modeling of “open lid” conformation of PLA2G4A: The “open lid” conformation of PLA2G4A catalytic domain was constructed by grafting the “open lid” in PLA2G4D (PDB ID: 5IXC) onto the “closed lid” structure of PLA2G4A (PDB ID: 1CJY). First, the catalytic domain structures of PLA2G4A (residue 141–749) and PLA2G4D (residue 277–810) were superposed based on main-chain atoms and refined with Gaussian distance weights in Molecular Operating Environment (MOE) version 2012.10 . Second,Docking of berberine into the “open lid” conformation of PLA2G4A: The 3D structure of berberine was extracted from the crystal structure of the QacR–berberine complex (PDB ID: 3BTI). The whole docking study was performed with MOE: dock. The methyl gamma-linolenyl fluorophosphonate was used as a reference ligand to guide the berberine docking into the active site of PLA2G4A. The placement method was Triangle Matcher, and a flexible receptor was used during the refinement stage. The GBVI/WAS dG was selected as the final scoring function. Docking poses were ranked by the GBVI/WAS dG score and analyzed by manual observation as well as ligand interactions modules. All 3D structures were drawn with VMD 1.8.3 software .p values were calculated using GraphPad Prism 6 from either Students’ t-test or one-way ANOVA, and the p value less than 0.05 was regarded as statistically significant.Results were from multiple, at least three times independent experiments. The lipidomics data were processed by the R package with XCMS. MetaboAnalyst was used for multivariate statistical analysis. Cytoscape was used to match the associated enzyme in the KEGG database. ImageJ was used to process Western blot results. Other data are expressed as average and SD value of at least triplicate samples. Significance We first investigated the effect of berberine on lipid metabolism in experimental colitic mice. DSS treatment caused a decrease in body weight, colon shortening, and increase in stool consistency as well as colonic bleeding. In contrast, treatment of berberine significantly ameliorated colon tissue injury and inflammatory responses in DSS-induced colitis mice . To chartnf-alpha, nos2, il6, and cxcl10 in the LPS-stimulated RAW 264.7 macrophages. Based on the results of cytotoxicity and anti-inflammatory effects of berberine on RAW 264.7 cells, berberine treatment at a concentration of 10 μM was chosen for lipidomics study. In RAW 264.7 cells, the score plot displayed clear classification among the control group, model group, and berberine treatment group, indicating that pro-inflammatory stimulations by LPS affect total lipid profiles. Berberine effectively reduced the elevated LPC levels in LPS-treated RAW 264.7 cells  activity has been shown to be increased in the colon biopsies of UC patients . Herein,t manner . Since Pt manner , we wond® Transfection System was used to establish transfection models using Pla2g4a and control siRNA. Together with the transfection models, a phospholipase A2 (PLA2) activator, namely, sc-3034  was used for activating PLA2G4A. Subsequently, we determined the expression levels of pro-inflammatory genes in knockdown and activating models of PLA2G4A RAW 264.7. We found the expression of pro-inflammatory gene levels decreased in the Pla2g4a siRNA model but increased by sc-3034 treatment. Meanwhile, we found that knockdown of Pla2g4a promoted berberine-induced suppression of pro-inflammatory genes in LPS-activated RAW 264.7 cells. In contrast, activation of PLA2G4A by sc-3034 suppressed the anti-inflammatory effects of berberine on LPS-stimulated RAW 264.7 cells   . Next, wwith PMA , whilst with PMA . Combiniwith PMA .in vivo experimental murine colitis and in vitro RAW 264.7 macrophages. In these in vivo and in vitro models, we showed berberine treatment significantly reduces the phosphorylation of PLA2G4A. Second, we showed berberine suppressed the expression of pro-inflammatory genes by inhibiting PLA2G4A in these models. Mechanically, we revealed that berberine directly inhibits PLA2G4A activity and regulates PLA2G4A activation via the MAPK/JNK signaling pathway in inflammatory responses. Collectively, this study suggested berberine suppresses inflammatory responses through inhibition of PLA2G4A activity to ameliorate DSS-induced experimental colitis.In this study, we have shown a novel phospholipid-related mechanism underlying the anti-inflammatory effects of berberine. First, we demonstrated the aberrant activation of PLA2G4A in inflammatory responses in both Numerous studies have revealed the anti-inflammatory properties of berberine in the digestive system . The antMacrophages are responsible for innate immune responses and play an important role in mediating colonic mucosal inflammation in IBD patients . DefinedPla2g4a siRNA to knock down the expression of cytosolic phospholipase A2α in in vitro cell models but not in in vivo animal models. Second, due to the lack of specific JNK activator, we used a general MAPK activator PMA to induce the activation of MAPK/JNK signaling to determine whether the inhibitory effects of berberine treatment on cytosolic phospholipase A2α are mediated by MAPK/JNK. In the future, we will establish colon-specific Pla2g4a knockout mice to further understand the role of colonic Pla2g4a in the inflammatory responses of IBD. Moreover, we will perform a screening assay of PLA2G4A to investigate the inhibitory activity of bioactive components from herbal extracts on PLA2G4A in order to provide potential therapeutic strategies for IBD.There are several limitations to this study. First, to examine the role of PLA2G4A in mediating the inflammatory responses and whether the anti-inflammatory properties of berberine treatment are through inhibition of PLA2G4A, we have used In conclusion, this study demonstrated that berberine suppresses colonic inflammatory responses through inhibition of PLA2G4A activity to ameliorate DSS-induced experimental colitis, which enhances our understanding on the pharmacological mechanism of berberine treatment on ulcerative colitis from a perspective of lipid metabolism and signaling. This study highlights the therapeutic potential of targeting cytosolic phospholipase A2a dysfunction in the management of IBD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Animal Care Ethics Committee, Hong Kong Baptist University.Z-XB, H-TX, and C-YL designed the study and revised the manuscript. LXZ, C-YL, and P-GW conducted the animal study, cell study and wrote the manuscript. TH conducted molecular docking study. X-JH, Z-WN, and LXZ performed the lipidomics analysis. LZ, SF, HK, and HW provided technical support and advice toward this study.This work was kindly funded by National Natural Science Foundation of China (Grants No. 81973538 and 81560676), Key-Area Research and Development Program of Guangdong Province (Grants No. 2020B1111110003), Shenzhen Science and Technology Innovation Committee Grant (Grants No. JCYJ20170413170320959 and JCYJ20190808164201654), and Logan Charitable Foundation.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.