Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate  Several studies have demonstrated that tumor-stroma cross talk can play a major role in drug resistance and cancer relapse by retaining minimal residual disease3. The molecular mechanism and molecular players involved in contributing towards minimal residual disease and drug resistance are highly dynamic. This can be achieved by stroma-derived secretory factors such as cytokines and chemokines, which help in homing, survival, and growth of the cancer cell in bone-marrow, as well as provide protection against drug toxicity4. Apart from several secretory factors, microenvironment mediated changes in micro-RNA (miRNA) expression in tumor cells for cancer progression, metastasis, and drug resistance has also been highlighted8. Microenvironment can influence miRNA mediated regulation of gene expression in cancer cells either by regulating their expression in tumor cells via modulating signaling networks or by direct miRNA transfer9. Identifying the molecular mechanisms and dynamics of microenvironment mediated response to therapy and cancer progression is therefore crucial to improve therapy and achieve prolonged disease free survival in cancer patients.Bone-marrow microenvironment is known to be actively involved in an onco-protective role for metastatic cancer cells, as well as in leukemia11. This suggests that there could be other mechanisms by which the leukemic cells in APL evade drug toxicity rather than by clonal evolution or drug binding site mutations. Microenvironment-mediated drug resistance or adaptation of leukemic cells is one of the mechanisms where the relapse of the disease can occur through persistence of minimal residual disease. Previous reports, including data from our laboratory suggests that bone-marrow stromal cell mediated drug resistance, predominantly mediated by NF-kB signaling, is significant in APL and AML cell lines and primary cells13.Acute myeloid leukemia involves abnormal proliferation and accumulation of immature myeloid cells. Currently, World Health Organization (WHO) had classified this disease based on its molecular pathology. Amongst the different AML subtypes, APL (AML-M3) is known to have best response to therapy when arsenic trioxide (ATO) based regimens are used. However, even in APL the relapse rate varies from 10–15% when ATO is used as a single agent. Unlike most cancers it has been noticed that primary resistance to ATO is almost never seen in patients both at initial diagnosis and at relapsemiR-23a-5p. Downregulation of miR-23a-5p in co-cultured leukemic cells results in upregulation of protective autophagy via TLR2, which protects the leukemic cells from chemotherapy induced apoptosis. Using GFP-based miRNA reporter constructs and miR-23a-5p mimic, we demonstrate that this miRNA plays a significant role in protection of leukemic cells against chemotherapy toxicity. We also demonstrate that this molecular mechanism of drug resistance identified in APL, is also relevant in some AML cell-lines and patient samples but not in acute lymphoid leukemia.In this study, we have uncovered the molecular mechanism of stroma mediated drug resistance via NF-kB signaling in APL. We find that stromal cells activate NF-kB signaling in leukemic cells which is directly responsible for downregulation of 14. This stroma-mediated protective effect against ATO is both contact dependent and independent  where the NF-kB pathway was repressed by overexpressing a mutant IkB super-repressor  and miR-23a-5p expression  were identified as top five targets that could be potentially regulated by miR-23a-5p  at 3′end of GFP   Fig. , even inn1) Fig.  suggestills Fig. . Furthersed Fig. . AltogetmiR-23a-5p while myeloid leukemic cells such as U937, HEL, and THP1 cell-lines had lower expression of miR-23a-5p  but not in THP-1 (AML cell-line) or the Sup-B15 and Jurkat-E6.1 cell-lines  and primary AML cells against DNR or Cytarabine (Ara-C) Fig. . Activatnts Fig. .Fig. 5mimiR-23a-5p mimics were re-introduced in AML cells, there was a reversal of resistance in U937 cell-line but not in THP1 cell-line against DNR  in AML patients. Our preliminary results showed that, as observed in APL patient samples  in favorable risk AML samples when compared to intermediate or adverse risk patient’s samples  is sufficient to overcome stroma-mediated protective effect. For this, we treated co-cultured cells with known autophagy inhibitors-Bafilomycin A1 and Hydroxychloroquine (HCQ) along with ATO. The ability of the autophagy inhibitor (at the concentrations used) to inhibit autophagy was verified  so that a synergy between ATO and HCQ, if any, could be observed. We find that when HCQ was combined with ATO, there was significant reduction of tumor burden in the APL mice  involved in activation of NF-kB signaling, we performed cytokine array using conditioned medium from stromal cells line. Our results suggests that several cytokines, up to 43, were upregulated upon stromal co-cultures (data not shown), several of which could potentially activate NF-kB signaling. Apart from cytokines, exosomes derived from stromal cells are also known for their protective effect on the cancer cells via juxtacrine signaling or due to delivery of exosomal miRNAs or other regulatory factors ferried in the lumen of the exosomes28. Recently, even Wnt5 has also been identified as a ligand involved in activate NF-kB signaling29. Wnt proteins are also known to be ferried on exosomes30. Thus NF-kB signaling can be activated by several possible stroma-derived paracrine factors. Secreted cytokines and exosomes can act in additive or redundant manner to activate NF-kB signaling. A more detailed study needs to be initiated to understand if NF-kB activation or ATO resistance can be imparted by stromal cells in exosome dependent or independent manner and to nail the dominant player (if any) in activation of NF-kB signaling in leukemic cells upon stromal co-culture.Accumulating evidence suggests that bone marrow stromal micro-environment act as sanctuary site for leukemic cells and affords protection from conventional chemotherapy agents. We had previously reported in APL that there was significant microenvironment-mediated ATO-resistance by upregulation of the NF-kB pathway32. Here, using NGS based approach and stringent analysis parameters, we identify that miR-23a-5p is significantly downregulated among other miRNAs in co-cultured leukemic cells. Our data using NF-kB inhibitors, NF-kB signaling incompetent leukemic cells, as well as relapsed APL patient samples, strongly suggest that downregulation of miR-23a-5p is a result of upregulated NF-kB signaling upon stromal co-culture. This inverse relationship between miR-23a-5p and NF-kB identified in leukemic cells by our study is in agreement with a study done in Jurkat cells, where NF-kB has been shown to inversely regulate miR-23a-5p expression33; unlike HEK, K562 and HEL cells were NF-kB signaling upregulated expression of miR23a-27a-24 cluster34. The observed difference in regulation of miR-23a-5p levels suggests role of other tissue specific regulators.It has been reported that apart from upregulating cell proliferation and anti-apoptotic genes, NF-kB signaling is also known to exhibit their pro-tumorigenic activity by regulating expression of specific miRNAsmiR-23a-5p overexpression experiments suggests that downregulation of this miRNA plays a significant role in protection of leukemic cells against chemotherapy toxicity. To the best of our knowledge this is the first time that miR-23a-5p has been implicated in leukemia drug-resistance in a NF-kB dependent manner. In order to understand the molecular mechanism of miR-23a-5p downregulation mediated drug-resistance in AML cells, we predicted the targets of miR-23a-5p. As reported for Mycobacterium infected macrophages35, we were also able to validate TLR2 as its direct target using GFP-reporter assays and miR-23a-5p mimics. Further knock-down of TLR2 resulted in reversal of drug-resistance in leukemic cells even in the presence of stroma. Consistent with our findings, it has also been reported that increased expression of TLR2 in AML is associated with poor clinical outcomes36.Further, miR-23a-5p in leukemic cells results in downregulation of cellular autophagic flux, estimated by accumulation of p62 [a well-established autophagy target37], as well as reduced the expression of autophagy proteins. Here, we conclude that this miR-23a-5p-mediated effect on autophagy pathway is via TLR2 since overexpression of miR-23a-5p mimics resulted in downregulation of TLR2 transcripts but did not influence autophagy transcripts. Therefore, TLR2 may activate cellular autophagy probably via post translational modifications of autophagy genes38. Upregulation of autophagy and its association with drug-resistance identified here, is consistent with reported clinical studies in AML that have demonstrated an increased risk of relapse and decreased overall survival associated with an increased expression of autophagy related genes40.Overexpressing 12. Whereas this study suggests that the stromal co-culture induces cytoprotective-autophagy in leukemic cells. It is well noted that autophagy is a ‘double edged sword’ and the threshold where the protective-autophagy can be converted into cytotoxic-autophagy is not known. However, nuclear accumulation of B-catenin is a known marker of cytotoxic-autophagy41; we found that the autophagy induced during drug treatment [based on our previous study12], indeed leads to nuclear accumulation of B-catenin unlike stromal co-cultures  for better treatment outcome. It must be noted that we had demonstrated previously that bortezomib when used in combination with ATO can improve therapy outcome by inducing cytotoxic-autophagymiR-23a-5p-mediated protective influence is valid for myeloid  and lymphoid cells (Jurkat E6.1 and SUP B15). We were able to demonstrate a significant downregulation of miR-23a-5p in myeloid malignant cells (except THP1) but not in lymphoid cells upon co-culture. Data from primary cells suggested that at baseline APL cells had the highest expression of miR-23a-5p followed by AML (with variable expression levels) and ALL samples, respectively. Comparing the conventionally defined risk groups in AML, the favorable risk group patients had highest expression of miR-23a-5p compared to intermediate and adverse risk groups analyzed. The high expression of miR-23a-5p could be one of the reason why APL cells are most sensitive to ATO and anthracycline and being most curable leukemia, while other acute leukemia are not. Further, correlating relapse with miR-23a-5p expression, we identified that those who relapsed had a lower expression of miR-23a-5p in their malignant cells at diagnosis compared to those who did not relapse. We suspect that there will be a significant heterogeneity in the cellular levels of miR-23a-5p in AML patient samples and active regulation of miR-23a-5p-TLR2-Autophagy circuit which might be restricted to rather small subset of cells which are close to the bone-marrow stromal cells which would in turn contribute to disease relapse. While upregulation of autophagy in AML cells upon co-culture has been identified before43, our work resolves the underlying molecular events, as well as their dynamic nature. It must be noted that, the illustrated stroma-mediated mechanism of protection against chemotherapeutic agents was predominantly seen in myeloid leukemia it cannot be generalized to all myeloid leukemia’s, as noted with exception seen with THP1 cell-line.Further, in order to evaluate impact of our findings in other leukemia’s, we attempted to validate if the This study thus demonstrates that myeloid leukemia cells can adapt themselves to the drug-induced stress by interacting with stromal cells in bone-marrow niches. Identifying and targeting such molecular cross talks could potentially prove to be an effective strategy in treating high risk or relapsed patients.The human leukemic cell-line such as U937, Kasumi-1, THP-1, Jurkat E6.1, SUP B15 and stromal cell line HS-5 were obtained from ATCC, USA, NB4 was a kind gift from Dr. Harry Iland, RPAH, Sydney, Australia (with permission from Dr. Michel Lanotte), NB4/GFP and NB4/GFP-MAD cells  were used in this study. Mycoplasma detection was done once in every 6 months and all the cell-lines used were free from Mycoplasma. The study was approved by the institute review board (IRB Min. No. 7826 dated 18.04.2012). AML, ALL, APL patient samples at diagnosis and relapse were collected, prior to treatment, after getting written and informed consent.ATO, a kind gift from Intas Pharmaceuticals Ltd, Ahmedabad, India, was used in the study. Daunorubicin, Cytarabine, Bafilomycin A1, Hydroxychloroquine, Bay11-7086 was procured from Sigma, St. Louis, USA. Antibodies used included those against Actin, ATG12, Beclin1, p62, p65 , LC3 , TLR2  anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase  and with alexaflour 488 and 594  were used for western blotting and immunofluorescence.5 cells/well) on a layer of primary stromal cells or HS-5 stromal cell-line in 24 well plates or seeded in transwells plates. The co-cultured cells were incubated overnight and then exposed to various chemotherapeutic agents along with appropriate controls. After 48 h incubation at 37 °C CO2 incubator, the leukemic cells were carefully pipetted out and their viability was measured using Annexin V/7AAD apoptosis assay kit  as per manufacturer’s protocol. CD105 staining was used to exclude stromal cells if present during acquisition and analysis. The flow data were analyzed using Cell Quest pro software .Leukemic cell-lines were added  and the NB4 co-cultured (treated) were compared to generate statistically significant differentially expressed small RNAs. Small RNA libraries were prepared using the Illumina TruSeq small RNA kit as described by the manufacturer (Illumina). 1 microgram of total RNA were used from each sample for the library preparation. 5' and 3' Small RNA adaptors were ligated to the RNA and the ligated products were reverse transcribed using superscript II reverse transcriptase . The RT products were then amplified by PCR and resolved on an 8% polyacrylamide gel. Bands corresponding to 140–160 nucleotides nt were gel eluted. The size and integrity of each library was verified using the Bioanalyser. The libraries were sequenced on an Illumina Hiseq 1000 (Centre for cellular and Molecular Platforms (CCAMP), Bengaluru, India).44 program. We then mapped these reads to rRNA database and unaligned reads were taken for further analysis. We then segregated reads that are 18–24 nucleotides and mapped to GRCh38 Genome and miRNA databases45 using bowtie v1.0.046 Customized perl script was used to obtain count data for all the miRNAs. The count data was normalized using DESeq47 and the normalized data was used for further analysis. miRNAs which have adjusted p value < 0.05 were considered for further analysis. We plotted volcano plot using R ggplot2 package. For figure esthetic purpose, miRnas with adj.pvalue 1 are replaced with their p-values . Targets for differentially expressed miRNAs were predicted using TargetScan v7.148 miRNA targets were overlapped with microarray data to identify targets that are upregulated/not changing. We did pathway and gene Ontology GO analysis of these genes using Gene Set Enrichment Analysis (GSEA)49.From the sequencing reads, we trimmed TruSeq small RNA adapters using customized perl script and cutadaptmiR-23a-5p was analyzed for expression profiles at different conditions as mentioned in the respective experiments and levels where normalized to cellular U6snRNA levels. The primer sequences used were given below.Quantitative PCR was performed using miRNA-specific primers (MSPs). The primer for reverse transcription was designed with double-stranded stem–loop structure with universal reverse primer binding site at 5′ end and last eight nucleotides at the 3′-end complementary to the 3′-end of miRNA. cDNA generated using this Stem-loop-RT primer was used to carried out real time PCR using miRNA-specific forward primers and universal primer with complementarity to the ‘stem’ sequence of the MSP. All primers used are listed in below. Reverse transcription was performed using SuperScript III RT , and the quantitative PCR was carried out using SYBR Green  as the fluorescent detector on an Applied Biosystems 7900HT machine. mir-23a-5p or TLR-2 3′UTR region were commercially synthesized , phosphorylated using T4 PNK, annealed, and cloned in to EcoRI and XhoI site in pMXCRGb vector50. Positive clones were identified using colony PCR followed by sanger sequencing. Oligo sequences used are:Oligos containing three tandem repeat of the complementary sequence to hsa-miR-23a-5p(forward)-AATTAAATCCCATCCCCAGGAACCCCAAATCCCATCCCCAGGAACCCCAAATCCCATCCCCAGGAACCCC;hsa-miR-23a-5p(reverse)-5′TCGAGGGGTTCCTGGGGATGGGATTTGGGGTTCCTGGGGATGGGATTTGGGGTTCCTGGGGATGGGATTT3′TLR2–3′UTR(forward)-5′AATTGACTTCATGGATGCAGAACCCATGACTTCATGGATGCAGAACCCATGACTTCATGGATGCAGAACCCAT3′,TLR2–3′UTR(reverse)-5′TCGAATGGGTTCTGCATCCATGAAGTCATGGGTTCTGCATCCATGAAGTCATGGGTTCTGCATCCATGAAGTC3′.TLR2–3′UTR mutant 1 (forward): AATTGACTTCATGGATGCAGATGCCATGACTTCATGGATGCAGATGCCATGACTTCATGGATGCAGATGCCATTLR2–3′UTR mutant 1 (reverse): TCGAATGGCATCTGCATCCATGAAGTCATGGCATCTGCATCCATGAAGTCATGGCATCTGCATCCATGAAGTCTLR2–3′UTR mutant 2 (forward): AATTGACTTCTTGGATGCACATGCCATGACTTCTTGGATGCACATGCCATGACTTCTTGGATGCACATGCCATTLR2–3′UTR mutant 2 (reverse): TCGAATGGCATGTGCATCCAAGAAGTCATGGCATGTGCATCCAAGAAGTCATGGCATGTGCATCCAAGAAGTCACTB and the fold differences were calculated using 2−∆∆Ct method. The NF-κB array  was performed according to manufacturer’s instructions.Total RNA was extracted using Trizol reagent . Five hundred nanogram of the extracted RNA was converted into cDNA using superscript II cDNA kit . The expression of genes was studied using SYBR green method . The Ct values were normalized with NB4 homogenates were obtained by cell lysis in RIPA buffer , with complete protease inhibitors . Nuclear extracts were taken from cells using NE-PER kit  according to the manufacturer’s protocol. The lysates and elutes were analyzed in SDS–PAGE. After protein transfer to nitrocellulose membrane, membranes were blocked with non-fat dry milk  followed by incubation with primary antibodies overnight. The protein bands were detected by standard chemiluminescence method .miR-23a-5p mimics  into NB4 cells were performed using Amaxa electroporator unit (program NB4 X-01) and recovered in complete media for 24 h. Same kit was used to electroporate pMXCRGb and pMXCRGb-TLR2 vectors in NB4 cells. For analysis in vector electroporated experimental set up the cells were gated for RFP positive cells and looked for the expression of GFP upon re-introduction of miR-23a-5p mimics.The electroporation of The leukemic cells were co-cultured with stromal cells, the cells were carefully pipetted out and cytospin slides were made after 6 h of co-culture. The cells were fixed in 4% paraformaldehyde followed by blocking using 5% goat serum. It was further incubated with primary antibodies such as p65  overnight at 4 °C. The slides were rinsed with PBS thrice and incubated with secondary antibodies (anti-mouse) conjugated with alexaflour 594,  for 1 h. The slides were again washed, air dried and counterstained with DAPI containing mountant . The images were acquired in fluorescence microscope  at ×100 with oil immersion and images were analyzed using ISIS metasystem, .For the expression of TLR2 (CD282) protein on the leukemic cells, phycoerythrin tagged antibody against TLR2 was purchased (PE Mouse Anti-Human CD282 Clone 11G7) from BD Pharmingen . Briefly the cells were washed and incubated with the antibody in dark for 30 min at 4 °C. The cells were washed to remove unbound antibodies and the re-suspended in PBS-albumin (1%) solution. The cells were acquired in Beckman Coulter Gallios  and the data were analyzed in FlowJo software V10.07 .51. Briefly, APL cells from the spleen of MRP8-PML-RAR transgenic mice (FVB/N) were harvested and cryopreserved (a kind gift from Dr. Christine Chomienne with permission from Dr. Scott Kogan). Mouse APL cells (5 × 104 cells/mouse) were injected intravenously via the tail vein into genetically compatible FVB/N recipients, without conditioning with either radiation or chemotherapy. ATO was given as intra-peritoneal at the concentration of 5 mg/kg of mice starting on day 7 post injection of malignant cells and continued for 28 days, while hydroxychloroquine was also given as intra-peritoneal at the concentration of 60 mg/kg of mice starting on day 7 post injection of malignant cells and continued for 28 days. Blood was collected on day 20 by retro-orbital method and the mice were monitored for their survival.FVB/N mice were obtained from Jackson Laboratory . Mice at 6 to 8 weeks of age were used in all the experiments. The animal study design and euthanasia protocols were approved by the Institutional animal ethics committee . A well-established transplantable APL mouse model was used in this studySupplementary dataSupplementary table1Supplementary table 2


This study proposes the use of PdCl2 as a Pd precursor and activated carbon as the support to fabricate Pd/C catalytic materials (Pd/C-EP-Ar) by using ethanol—which is renewable, easily stored, and safe—combined with AP cold plasma (AP ethanol cold plasma) followed by calcination in Ar gas at 550 °C for 2 h. Both Pd/C-EP and Pd/C-HP fabricated using AP ethanol and hydrogen cold plasma  respectively, exhibit low CO oxidation reactivity. The activity of Pd/C-EP is lower than Pd/C-HP, which is mainly ascribed to the carbon layer formed by ethanol decomposition during plasma treatment. However, the 100% CO conversion temperature (T100) of Pd/C-EP-Ar is 140 °C, which is similar to that of Pd/C-HP-Ar fabricated using AP hydrogen cold plasma . The characterization results of X-ray diffraction, X-ray photoelectron spectroscopy, and transmission electron microscopy indicated that the carbon layer formed by ethanol decomposition enhanced the interaction of metal nanoparticles to the support, and a high Pd/C atomic ratio was obtained. This was beneficial to the high CO oxidation performance. This work provides a safe method for synthesizing high-performance Pd/C catalytic materials avoiding the use of H2, which is explosive and difficult to transport.Treatment with atmospheric-pressure (AP) hydrogen cold plasma is an effective method for preparing highly active supported metal catalytic materials. However, this technique typically uses H Supported Pd catalytic materials are widely used in organic catalytic hydrogenation, CO oxidation, automobile exhaust treatment, and the Suzuki reaction, among others ,3,4,5,6.2 or NH3, CH4 has been added into the working gas in AP cold plasma due to the frequency of collision between its electrons and heavy particles, leading to inadequate electron temperature for direct metal ion reduction [2*, etc.) in AP cold plasma. However, the aforementioned gases are explosive and are difficult to store and transport. Thus, the development of a relatively safe AP cold plasma for the preparation of supported metal catalytic materials bears research significance and exhibits application potential.As a non-equilibrium plasma, Heduction ,16,17,182) as well are produced during microwave discharge [2O3 catalyst by dielectric barrier discharge, which increased the conversion rate of ethanol to hydrogen by nearly 30%, meanwhile, the carbon balance was 97% [Ethanol, a hydrogen-rich renewable resource, can be mixed with water in any proportion and shows reducibility. Research on reforming hydrogen production from ethanol has recently gained interest ,20,21. Pischarge . Wu et a was 97% .2, and Pd/P25 catalytic materials with high activity and small Pd nanoparticles were prepared by AP ethanol cold plasma treatment. The performance was better than that of the catalytic materials prepared using AP hydrogen cold plasma [2·g−1) and rich oxygen-containing functional groups located on the surface. In addition, activated carbon is cheap and conducive to the recovery of precious metals. In this study, activated carbon was selected as the support, and AP ethanol cold plasma was used in the fabrication of Pd/C catalytic materials.In a previous study, metal  ions loaded on Deggusa P25  were successfully reduced by combining AP cold plasma with ethanol instead of Hd plasma ,25. The In this study, Pd/C catalytic materials were successfully prepared using AP ethanol cold plasma. The results of transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and CO oxidation reaction show that the CO oxidation activity of Pd/C-EP-Ar prepared using AP ethanol cold plasma is similar to that of Pd/C-HP-Ar prepared using AP hydrogen cold plasma. A carbon protective layer can be formed during AP ethanol cold plasma preparation, which can effectively protect the Pd nanoparticles.2  was used as the Pd source. Prior to the fabrication of Pd/C catalytic materials, the activated carbon and 30 wt% HNO3 were mixed in a 250 mL conical flask and then stored in a constant-temperature water bath at 85 °C for 5 h. The mixture was washed with deionized water to neutral pH and then dried at 120 °C for 2 h for subsequent use [2 solution for 12 h and then dried at 120 °C for 2 h. The Pd/C precursor was ultimately treated with AP ethanol cold plasma to prepare Pd/C catalytic materials.Activated carbon  was used as the support, and the aqueous solution of PdCluent use ,27. The 2 at 50 SCCM each, the mixture was used as the working gas. After calcination in 100 SCCM of Ar gas at 550 °C for 2 h, Pd/C-EP and Pd/C-HP are denoted as Pd/C-EP-Ar and Pd/C-HP-Ar, respectively.The schematic of the Pd/C catalytic materials prepared using the AP ethanol cold plasma experimental device is presented in The samples were characterized by XRD  with graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å) under the following conditions: Voltage, 40 kV; current, 30 mA; scan range, 5° to 90°. The chemical compositions and binding energies of the samples were investigated by XPS  with a monochromatized Al Kα (1486.6 eV) X-ray source. All binding energies were corrected with C1s orbital binding energies at 284.6 eV. The morphology of the sample was investigated by TEM , and the average size of the Pd nanoparticles was determined by selecting at least 200 nanoparticles. The specific surface area, pore volume, and pore size of the materials were measured using a NOVA 2200e gas sorption analyzer  via nitrogen adsorption and desorption. CO oxidation was selected as a probe reaction to evaluate the activity of the prepared Pd/C catalytic materials in a quartz tube (inner diameter: 4 mm) placed in a temperature-programmed furnace. Detailed test conditions can be found in previous work .T100) of Pd/C-EP-Ar, Pd/C-HP-Ar, and Pd/C-HP were 140 °C, 130 °C, and 215 °C, respectively. Notably, no CO oxidation activity over Pd/C-EP was observed even at 200 °C, and the conversion rate of CO at 230 °C was only 14.5%. The significantly lower CO oxidation reactivity over Pd/C-EP than that over Pd/C-HP is mainly attributed to the carbon layer formed by ethanol decomposition during plasma treatment, which will be discussed later. The results show that the T100 of Pd/C-EP-Ar is similar to the T100 of Pd/C-HP-Ar, indicating that AP ethanol cold plasma can achieve the same effect and ability as that of AP hydrogen cold plasma in the preparation of Pd/C catalytic materials, and its advantage is that the use of explosive H2 gas is prevented. In this work, the theoretical load of Pd is 5 wt%. The T100 of Pd/C-EP-Ar (140 °C) is close to the previously reported 6 wt% Pd/graphene (127 °C) fabricated by the thermal reduction method [The XRD patterns of the Pd/C-EP, Pd/C-EP-Ar, Pd/C-HP, Pd/C-HP-Ar, and Pd/C precursor are illustrated in 2 adsorption and desorption and pore size distribution of all samples were observed , both of which can reduce metal ions. Meanwhile, CO and the excited state of CO* produced by ethanol decomposition can also reduce Pd ions, and the carbon layer generated by ethanol decomposition can inhibit the agglomeration of Pd nanoparticles. Thus, Pd/C-EP with the smallest Pd nanoparticles size was fabricated [2*) in the preparation of AP hydrogen cold plasma. They are produced by high-energy electrons e* colliding with Ar and H2 gases. Pd/C-HP-Ar with larger Pd nanoparticles can be formed when Pd/C-HP is calcined in Ar gas to remove chloride ions.The proposed preparation mechanisms of Pd/C-EP-Ar and Pd/C-HP-Ar were determined from the results of the XPS, TEM, and XRD analyses, among others . The intbricated ,25,35. H0. The low activity of Pd/C-HP is attributed to the influence of Cl− ions, while it is ascribed to the influence of Cl− ions and the protection of the carbon layer formed by ethanol decomposition in plasma for Pd/C-EP. In addition, the Pd/C atomic ratio for Pd/C-EP (0.013) is lower than that for Pd/C-HP (0.026) due to the carbon layer protection. Therefore, Pd/C-EP exhibits lower CO oxidation reactivity than Pd/C-HP. In contrast, both Pd/C-HP-Ar and Pd/C-EP-Ar exhibit high CO oxidation activity notwithstanding the large size of Pd nanoparticles due to the high-temperature calcination. Compared to the Pd/C-HP and Pd/C-EP catalytic materials, the enhanced performance of the Pd/C-HP-Ar and Pd/C-EP-Ar samples is mainly attributed to the removal of Cl− ions. In addition, the average size of Pd nanoparticles in Pd/C-HP-Ar is smaller than that in Pd/C-EP-Ar, while the Pd/C atomic ratio in Pd/C-EP-Ar is higher than that in Pd/C-HP-Ar due to the protection of the carbon layer. Therefore, both of them exhibit high CO oxidation activity.In summary, both AP ethanol cold plasma and AP hydrogen cold plasma can reduce the Pd ions into metallic Pd nanoparticles. The Pd/C-HP and Pd/C-EP catalytic materials exhibited significantly lower CO oxidation activity in spite of their large amounts of metallic PdT100) of Pd/C-EP-Ar is 140 °C, which was similar to that of Pd/C-HP-Ar prepared using AP hydrogen cold plasma. AP ethanol cold plasma and AP hydrogen plasma can be effectively used to fabricate Pd/C catalytic materials. The use of H2, which is explosive and difficult to transport, was avoided in the treatment with AP ethanol cold plasma. This study carries considerable research significance and application potential for the fabrication of supported metal catalytic materials.Pd/C catalytic materials were prepared by treatment with AP ethanol cold plasma and AP hydrogen cold plasma. The structure and properties of the materials were characterized, and the preparation mechanism was investigated. The results of TEM, XPS, and XRD analyses show that both methods can reduce the Pd ions loaded on the activated carbon to the metal state. Not only the active hydrogen species but also reductive species, such as active CO* species, can be formed during treatment with AP ethanol cold plasma. All of these species, produced by ethanol decomposition, can reduce the Pd ions. The Pd nanoparticles in Pd/C-EP were smaller than those in Pd/C-HP because of the carbon layer formed via ethanol decomposition, which effectively prevented the agglomeration of Pd nanoparticles. The results of CO oxidation reaction indicated that the 100% CO conversion temperature (