Alike to natural immunoactive macrophages, NMΦ is stably retained in tumors through the specific affinity of TRF to tumor cells. Zn2+ as an “artificial cytokine” is then released from the ZIF-8 layer of NMΦ under tumor microenvironment. Similar as proinflammatory cytokines, Zn2+ can trigger cell anoikis to expose tumor antigens, which are selectively captured by the BaSO4 cavities. Therefore, the hierarchical nanostructure of NMΦs allows them to mediate immunogenic death of tumor cells and subsequent antigen capture for T cell activation to fabricate long-term antitumor immunity. As a proof-of-concept, the NMΦ mimics the biological functions of macrophage, including tumor residence, cytokine release, antigen capture and immune activation, which is hopeful to provide a paradigm for the design and biomedical applications of artificial cells.Artificial cells are constructed from synthetic materials to imitate the biological functions of natural cells. By virtue of nanoengineering techniques, artificial cells with designed biomimetic functions provide alternatives to natural cells, showing vast potential for biomedical applications. Especially in cancer treatment, the deficiency of immunoactive macrophages results in tumor progression and immune resistance. To overcome the limitation, a BaSOThe online version contains supplementary material available at 10.1007/s40820-023-01193-4. Biomimetics utilizes synthetic materials to mimic the biological functions or processes in nature, which has attracted interdisciplinary research interest –3. As a To date, artificial macrophages have been rarely fabricated to mimic natural macrophages, probably due to the following challenges . Firstly2+, Mg2+, Zn2+ and H2S, are highly effective in regulating physiological processes and cellular communication [2+ or Zn2+ overload can effectively impede tumor growth, and activate antitumor specific immune response to arrest tumor metastasis [With the advances in nanotechnology, nanomaterials have been developed to mimic versatile cellular functions , 24. Espnication . The tranication . For exatastasis , 28. Givtastasis . Nanomat4@ZIF-8/TRF nanomacrophage (NMΦ) with hierarchical nanostructure is constructed as an alternative to immunoactive macrophages , sodium sulfide nonahydrate (Na2S·9H2O), zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 2-methylimidazole, and isopropanol were obtained from Aladdin . The transferrin (TRF) was purchased from Yuanye Bio-Technology Co., Ltd . Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, 1% streptomycin/penicillin, phosphate buffered saline (PBS), and lipopolysaccharide (LPS) were obtained from Thermo Fisher . The membrane protein extraction kit was purchased with Invent Biotechnologies . FITC-/Cy3.5-/Cy5.5-fluorescence protein label kits, Cy3.5-PAA and Cy5.5-PAA were obtained from Ruixi Biological Technology Co., Ltd . BCA protein assay kit, 4% paraformaldehyde, phalloidin-iFluor 488, lactic dehydrogenase (LDH) cytotoxicity assay kit, radioimmunoprecipitation (RIPA) buffer lysis, phenylmethanesulfonyl fluoride (PMSF), HRP-labeled goat anti-rabbit IgG, Alexa Fluor 488-labeled goat anti-rabbit IgG, bovine serum albumin (BSA), D-luciferin, electrochemiluminescence (ECL) assay kit, cell counting kit-8 (CCK-8), calcein-AM, propidium iodide, reactive oxygen species assay kit, mitochondrial membrane potential assay kit, and cell cycle analysis kit were purchased from Beyotime . Rabbit anti-E-cadherin, anti-integrin β1, anti-JNK, anti-BIM, anti-BMF, anti-BAK, anti-caspase 3, anti-Bcl-2, anti-Bax, anti-GAPDH antibodies were supplied by ABclonal Technology Co.,Ltd. . Chloroquine (CQ) was obtained from Sigma-Aldrich . CellTracker™ Green CMFDA, CellMaker™ Blue, Zinquin and 4′,6-diamidino-2′-phenylindole (DAPI) were obtained from Maokang Biotechnology Co.,Ltd. . Enzyme linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, IL-10, ATP and IFN-γ were supplied by DAKEWE . Anti-calreticulin (CRT), and anti-HMGB1 antibodies were purchased from Abcam . FITC-conjugated CD86, APC-conjugated CD206, PE-conjugated CD80, APC-conjugated CD11c, PE-conjugated CD4, APC-conjugated CD8a, FITC-conjugated CD3, PE-conjugated F4/80, FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L, and PE-conjugated CD8a antibodies were obtained from Biolegend . Anti-mouse PD-1 was supplied by Bioxcell .Polyacrylic acid (PAA), barium hydroxide octahydrate (Ba(OH)4) nanoparticles were synthesized by chemical coprecipitation method. Briefly, 3.6 mg of PAA was added into 50 mL of Ba(OH)2·8H2O aqueous solution (1 mg mL−1) and vigorously stirred for 30 min. Then, PAA-Ba was gradually precipitated from the solution and collected by centrifugation . Subsequently, PAA-Ba was dispersed in 50 mL of water and mixed with 10 mL of Na2S·9H2O aqueous solution (20 mg mL−1). After reaction for 7 h at room temperature, the reaction solution was mixed with 15 mL of isopropanol and centrifugated  to obtain PAA-BaSO4 nanoparticles. Cy3.5-PAA and Cy5.5-PAA were used to synthesize fluorescense-labeled PAA-BaSO4 nanoparticles.The PAA modified barium sulfate 2·6H2O. After stirring for 30 min, 10 mL of 2-methylimidazole (80 mM) in isopropanol was added and stirred for another 2 h. The BaSO4@ZIF-8 nanoparticles were collected by centrifugation . For modification of BaSO4@ZIF-8 with TRF, 10 mg of TRF was added into 20 mL of BaSO4@ZIF-8 nanoparticles in water (1 mg mL−1) and stirred overnight. The BaSO4@ZIF-8/TRF nanoparticles were collected by centrifugation . Similarly, the Cy3.5 or Cy5.5-labeled BaSO4@ZIF-8/TRF nanoparticles were also synthesized following the above mentioned procedures. To prepare ZIF-8/TRF nanoparticles, 14.8 mg of Zn(NO3)2·6H2O was dissolved in 30 mL of isopropanol, and then mixed with 10 mL of 2-methylimidazole (80 mM) in isopropanol for 30 min. ZIF-8 nanoparticles were collected by centrifugation  and modified with TRF following the above mentioned procedures.Twenty milligrams of PAA-BaSOThe morphology of nanomaterials was captured by transmission electron microscopy  and scanning electron microscopy . Elemental compositions were determined on an X-Ray photoelectron spectroscopy . The hydrodynamic size and zeta potential of nanomaterials were measured on a zeta nanosizer instrument . Crystal structures of nanomaterials were analyzed on a powder X-ray diffractometer (XRD) . Fourier transform infrared (FT-IR) spectra were detected on an FT-IR spectrometer . The absorption spectra were measured on a UV–Vis spectrometer . The pore size was characterized with an automatic BET specific surface area and porosity analyzer .2 atmosphere. Animal experiments were carried out under protocols (No. XMSB-2022–0103) approved by the Department of Laboratory Animals .All the cells were cultured in complete DMEM medium with 10% fetal bovine serum and 1% streptomycin/penicillin at 37 °C in a 5% CO4, ZIF-8/TRF, BaSO4@ZIF-8 and BaSO4@ZIF-8/TRF nanomacrophages (NMΦs) were dispersed in 20 mL of PBS buffer with different pH (7.4 and 6.5) at 37 °C. At desired time points , 1 mL of solution in different groups was taken out and centrifuged at 8000 g for 10 min. The contents of Zn2+ and Ba2+ in the supernatants were detected by an atomic absorption spectrometer .BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) for 18 h. Membrane proteins of 4T1 tumor cells were extracted with membrane protein extraction kit following the manufacturer’s protocol. To study the protein trapping capacity and binding selectivity of different nanomaterials, TRF, BSA and membrane proteins were individually labeled with FITC, Cy3.5 and Cy5.5 using fluorescence label kits. Then, 10 mL of BaSO4, ZIF-8/TRF-FITC, BaSO4@ZIF-8 and BaSO4@ZIF-8/TRF-FITC nanoparticles (1 mg mL−1) were mixed with 5 mL of Cy3.5-BSA (1 mg mL−1) or Cy5.5-membrane proteins (1 mg mL−1) in phosphate buffer (pH = 7.4 or 6.5). After 24 h, the mixture solution was taken out and centrifugated at 10,000 g for 10 min. The protein contents in both the supernatants and precipitations were quantified with BCA protein assay kit. Then, the different protein contents in the supernatants and precipitations were quantified by fluorescence measurement with a microplate reader .The 4T1 cells were treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) for 18 h. Then, cells were fixed in 4% paraformaldehyde and observed with SEM. To further study the cytoskeleton structure, 4T1 cells were seeded into a 6-well plate and treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for 12 h. The stress fibers were stained with phalloidin-iFluor 488 following the manufacturer’s protocol. The fluorescence images were captured with an inverted fluorescence microscope .The 4T1 cells and L02 cells were treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for 24 h. The cell supernatants were collected for cell viability detection using LDH cytotoxicity assay kit. Then, all the cells were collected and lysed with RIPA lysis buffer (containing 1 mM PMSF) on the ice for 30 min. After centrifugation  for 20 min, the obtained supernatants were stored at − 80 °C. Membrane proteins were extracted with membrane protein extraction kit according to the manufacturer’s protocols. The proteins were resolved by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto the surface of polyvinylidene difluoride (PVDF) membranes. Subsequently, PVDF membranes were blocked with 5% BSA and co-incubated with different rabbit anti-mouse primary antibodies  at 4 °C overnight. After washing with TBST three times, the membranes were treated with HRP-labeled goat anti-rabbit IgG at room temperature for 1 h. The protein bands were imaged with a chemiluminescence gel imaging system  using an electrochemiluminescence (ECL) assay kit.Western blot assay was performed to detect the variations of anoikis-related proteins. The 4T1 cells were cultured in a 6-well plate and incubated with BaSO4 , ZIF-8/TRF , BaSO4@ZIF-8 , BaSO4@ZIF-8/TRF , and BaSO4@ZIF-8/TRF  + CQ (3 μg mL−1) for 24 h. Cytotoxicity was detected with CCK-8 assay kit following the manufacturer’s protocol. Cells were stained with calcein-AM (10 μM) and propidium iodide (20 μM) for 20 min. Fluorescent images were captured with the inverted fluorescence microscope.4T1, 3T3 and L02 cells were incubated in 96-well plates and treated with BaSO2+ content, 4T1, 3T3 and L02 cells were treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for different time periods . Then, all the cells were collected and stained with Zinquin (20 μM) for 30 min. The intracellular fluorescence was analyzed on a flow cytometer .To study the intracellular Zn4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for 12 h. To detect mitochondrial membrane potential, cells were stained with JC-1 assay kit following the manufacturer’s protocol. To detect intracellular oxidative stress, cells were stained with DCFH-DA (10 μM) for 20 min. Cellular fluorescence was imaged with an inverted fluorescence microscope.4T1 cells were treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for 24 h. After fixation with 70% ethanol overnight, cell cycles were analyzed by the flow cytometer with a cell cycle analysis kit.4T1 cells were treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), BaSO4@ZIF-8/TRF (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) + CQ (3 μg mL−1) for 12 h. The supernatant in each well was collected for ATP quantification using an ATP assay kit. Moreover, 4T1 tumor cells were fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton-X100. Cells were immunostained with rabbit anti-mouse CRT or HMGB1 antibodies at 4 °C overnight. After washing with PBS three times, cells were stained with Alexa Fluor 488-labeled goat anti-rabbit IgG for another 1 h at room temperature. Finally, the nuclei were stained with DPAI (1 μM) for 30 min, and the fluorescent images were taken with a high-content cell imaging system .4T1 cells were cultured in a 48-well confocal plate and treated with BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), Cy3.5-BaSO4@ZIF-8 (150 μg mL−1), and Cy3.5-BaSO4@ZIF-8/TRF (150 μg mL−1). After 24 h of incubation, the fluorescence co-localization images were captured by a high-content cell imaging system.4T1 tumor cells and J774A.1 macrophages were individually stained with CellTracker™ Green CMFDA and CellMaker™ Blue CMAC following the manufacturer’s protocols. Green fluorescence-labeled 4T1 cells and blue fluorescence-labeled J774A.1 macrophages were seeded into the upper and lower chambers of transwells, respectively. After incubation overnight, cell culture medium in the upper chambers was replaced by medium containing Cy3.5-BaSO−1), BaSO4 (120 μg mL−1), ZIF-8/TRF (30 μg mL−1), BaSO4@ZIF-8 (150 μg mL−1), and BaSO4@ZIF-8/TRF (150 μg mL−1) for 24 h, respectively (n = 5 in each group). The cytokines  in the supernatants of macrophages were detected with corresponding ELISA assay kits. The J774A.1 macrophages in the lower chambers were stained with FITC-conjugated CD86 and APC-conjugated CD206 antibodies. The polarization of M1 (CD86high/CD206low) and M2 (CD86low/CD206high) phenotypes was detected by flow cytometry.For macrophage polarization study, 4T1 cells in the upper chambers were treated with LPS : (1) PBS, (2) BaSO4, (3) ZIF-8/TRF, (4) BaSO4@ZIF-8, (5) BaSO4@ZIF-8/TRF. All the nanoparticles were intratumorally injected into the primary tumors at the dosage of 5 mg kg−1. On day 3 post injection, mouse blood was collected and the serum cytokines  were detected with corresponding ELISA assay kits. On day 5 post injection, Zinquin was injected into the primary tumors at the dosage of 2 mg kg−1 for Zn2+ detection. Moreover, the spleens and primary tumors were isolated to prepare single-cell suspensions. All the cells were fixed with 4% paraformaldehyde and blocked in 5% bovine serum albumin (BSA). To detect dendritic cell maturation, the splenic cell suspensions were stained with FITC-conjugated CD86, PE-conjugated CD80, and APC-conjugated CD11c at 4 °C for 12 h. To detect T cell activation, the splenic cell suspensions were stained with PE-conjugated CD4, APC-conjugated CD8a, and FITC-conjugated CD3 at 4 °C for 12 h. To detect the intratumoral macrophages, cell suspensions of primary tumors were stained with FITC-conjugated CD86, APC-conjugated CD206 and PE-conjugated F4/80 at 4 °C for 12 h. All the cell suspensions were washed with PBS three times and analyzed by flow cytometry. In addition, the distant tumors were sliced and subjected to CD3/CD4/CD8 immunohistological staining and FoxP3/CD3 immunofluorescent staining.Female BALB/c mice (6-week-old) were subcutaneously injected with 4T1 tumor cells to fabricate bilateral tumors model. When the primary tumor volume was over 50 mm3, mice were divided into eight groups (n = 6 in each group): (1) PBS; (2) BaSO4; (3) anti-PD-1; (4) ZIF-8/TRF; (5) BaSO4@ZIF-8; (6) BaSO4@ZIF-8/TRF; (7) ZIF-8/TRF + anti-PD-1; (8) BaSO4@ZIF-8/TRF + anti-PD-1. All the nanoparticles were intratumorally injected into the primary tumors at the dosage of 5 mg kg−1. Anti-PD-1 was intravenously injected into mice at the dosage of 0.75 mg kg−1 on day 1, 4, and 7. To monitor tumor growth, tumor bioluminescence images were captured after intravenous injection of D-luciferin (100 mg kg−1) every 5 days by an IVIS Lumina imaging system . The tumor volumes of mice were recorded by a vernier caliper every other day and calculated according to the following formula: tumor volume = (width2 × length)/2; meanwhile the body weights of mice were measured. On day 15 post injection, blood cells were harvested for blood biochemistry analysis on an auto hematology analyzer  and a blood chemistry analyzer . All tumors were subjected to H&E and TUNEL staining for histopathological analysis. Main organs  were also excised and stained with H&E. To calculate survival rate, mice received the above treatments and were fed for 60 days. When tumor volume was over 2000 mm3, mice were euthanized according to ethical guidelines. The survival rates of mice were calculated on day 60 post-injection.4T1-Luc tumor cells were subcutaneously injected into mice to fabricate bilateral tumor model. When the primary tumor volume was over 50 mmFor tumor anoikis analysis, the harvested primary tumors in different groups were divided into two halves. One half of the tumors were fixed and sliced for immunofluorescence staining of E-cadherin and integrin β1. The other half was prepared into single-cell suspension for western blotting analysis following the aboved-mentioned procedures.6). On day 32, the spleens of one half of the mice were harvested to obtain single-cell suspensions. Following tissue homogenization and erythrocyte lysis, the cells were stained with FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L and PE-conjugated CD8a. On day 42 post-treatment, D-luciferin (100 mg kg−1) was intravenously injected into the mice, and the bio-luminescence images were captured by the IVIS Lumina imaging system. Finally, the lungs of mice were collected and the tumor metastases were histologically examined via H&E staining.4T1 breast tumor-bearing mice received the above-mentioned treatments for 15 days (n = 10 in each group). Residual tumors were removed via surgical excision under anesthesia. On day 28 post-treatment, mice were intravenously injected with 4T1-Luc cells . The experiments were replicated three times, unless otherwise stated. The normality of data was examined by Shapiro–Wilk tests in SPSS Statistics 26. One-way ANOVA of variance was utilized to evaluate significance of difference. * 4@ZIF-8 hierarchical structures, BaSO4 nanoparticles were initially prepared via a chemical precipitation method  at the surface , which can serve as the template for ZIF-8 growth. After the in situ growth of ZIF-8, an obvious shell with thickness of 8 nm can be clearly observed at the surface of BaSO4 nanoparticles . X-ray photoelectron spectroscopy (XPS) data reveal the coexistence of Ba and Zn with atomic ratio of 1.6:1 in the BaSO4@ZIF-8 nanoparticles .To assemble BaSOhod Fig. a. As dis4@ZIF-8 nanoparticles, TRF with specific affinity to tumor cells was decorated at the surface[4@ZIF-8/TRF is − 22.4 ± 0.9 mV, which is higher than that of BaSO4@ZIF-8 nanoparticles (− 29.8 ± 0.6 mV) (Table S1). Besides, the UV–vis absorption spectrum of BaSO4@ZIF-8/TRF displays an obvious absorption band of TRF at 272 nm .Natural macrophages are usually distributed around tumor cells with high tumor-resident ability . To reale surface. After t2+ as an artificial proinflammatory cytokine, we investigated Zn2+ release from the NMΦs in response to tumor pathological conditions, and its regulation on tumor cell death and immune activation. The PXRD data shows that the diffraction peaks of ZIF-8 disappear when BaSO4@ZIF-8 nanoparticles are exposed to tumor acidic condition (pH 6.5)  . The intracellular Zn2+ flux was further investigated in tumor cells (4T1), and normal cells (3T3 and L02) in vitro. The cellular Zn2+ was stained with Zinquin as a fluorescent probe and quantified with flow cytometry  as a typical Zn2+ inhibitor was supplemented to scavenge free Zn2+. Obviously, the intracellular Zinquin fluorescence of tumor cells returned to normal level. Thus, BaSO4@ZIF-8/TRF NMΦ can release Zn2+ ions under tumor acidic conditions, similar to the cytokines secreting profiles of macrophages.The immune response of macrophages highly relies on cytokine secretion to regulate tumor cell death and immune cell activation . To veri2+ as an artificial proinflammatory cytokine, cell viability assay was carried out on 4T1, 3T3 and L02 cells. After the treatment of BaSO4@ZIF-8/TRF NMΦs, the cell viabilities of 3T3 and L02 cells maintained above 80% . Obviously, the majority of tumor cells were dead after the treatment of BaSO4@ZIF-8/TRF. Notably, the toxicity was also observed in the groups of ZIF-8/TRF and BaSO4@ZIF-8 but not in the group of BaSO4 nanoparticles, indicating the Zn2+-mediated tumor cytotoxicity. To confirm this assumption, CQ was used as an inhibitor to scavenge free Zn2+. As expected, tumor cell viability was recovered to 91% in the co-presence of BaSO4@ZIF-8/TRF NMΦs and CQ, verifying the cytotoxic role of Zn2+. Therefore, Zn2+ can mimic the function of proinflammatory cytokines to mediate tumor cell death.Proinflammatory cytokines are able to mediate tumor cell death via multiple apoptotic pathways –36. To d4@ZIF-8/TRF NMΦ, cytoskeleton of tumor cells was examined with fluorescent phalloidin as a specific probe. As shown in Fig. 4@ZIF-8/TRF NMΦs, which are the characteristics of anoikis [4@ZIF-8/TRF NMΦs, respectively ; meanwhile, the activation of JNK and BAK/BAX pathways was suppressed. Therefore, the anoikis of tumor cells in the presence of BaSO4@ZIF-8/TRF NMΦs was mediated by Zn2+ release via activating JNK and BAK/BAX pathways , indicating mitochondrial dysfunction. Given the critical role of mitochondria in cellular redox homeostasis, mitochondrial dysfunction would result in cellular oxidative stress and lactic dehydrogenase (LDH) leakage [4@ZIF-8 and BaSO4@ZIF-8/TRF; whereas little intracellular fluorescence was observed after the addition of BaSO4 or BaSO4@ZIF-8/TRF + CQ, suggesting Zn2+-induced intracellular oxidative stress . Moreover, the LDH efflux increased almost 1.7-fold after the treatment of BaSO4@ZIF-8/TRF NMΦs . Due to the mitochondrial damage, about 33.7% of tumor cells were retarded at the G2/M phase by BaSO4@ZIF-8/TRF NMΦs , including adenosine triphosphate (ATP), calreticulin (CRT) and high-mobility group protein B1 (HMGB1) . Naturalc.a. 20 nm were exposed on the BaSO4 nanoparticles . When exposed to physiological environments (pH 7.4), the binding ability of BaSO4@ZIF-8/TRF NMΦs to TRF is 63.8 mg g−1, but its binding ability to cell protein or serum protein is less than 12.0 mg g−1  was further investigated through molecular dynamics (MD) simulation. Either membrane proteins or serum proteins can interact with BaSO4 by van der Waals interactions and Coulomb force. However, the binding energy of BaSO4 with CRT and E-cadherin was about 1.6- and 2.4-fold lower than that of BaSO4 with serum protein, respectively .In the process of anoikis, the detached cancer cells are likely to migrate and recolonized, resulting in anoikis resistance and tumor metastasis . To prev4@ZIF-8/TRF NMΦs in vitro was examined via transwell invasion assay . Comparatively, after the treatment of BaSO4@ZIF-8/TRF NMΦs, tumor antigens were uptaken by more than 80% of macrophages  and BaSO4@ZIF-8 (30.8%)  approximately quadrupled in the presence of BaSO4@ZIF-8/TRF NMΦs; whereas the secretion of anti-inflammatory IL-10 was decreased by 67%  Figs. c & S21. 67% Fig. d–f. Ther4@ZIF-8/TRF NMΦs to modulate immune cells in vivo. Since tumors are usually surrounded by tumor associated macrophages [4@ZIF-8/TRF NMΦs were intratumorally injected into mice to mimic the spacial distribution of natural macrophages. Owing to the specific affinity of TRF to tumor, the retention of BaSO4@ZIF-8/TRF NMΦs in tumor tissue was 25% higher than that in the BaSO4@ZIF-8 group . The high affinity of TRF to tumors mimics the specific interactions of natural macrophages with tumors, endowing BaSO4@ZIF-8/TRF NMΦs with tumor resident ability [2+ level was examined by fluorescence staining of tumor slices with Zinquin as a Zn2+ indicator. Obviously, the Zn2+ level was remarkably increased about 4 times post injection of BaSO4@ZIF-8/TRF NMΦs . BaSO4@ZIF-8/TRF NMΦs dramatically promoted the M1 macrophage rate from 4.65% to 28.3%, which was more efficient than ZIF-8/TRF nanoparticles . Accompanying with M1 macrophage polarization, the serum levels of IL-6 and TNF-α were increased 11.5- and 2.2-fold by the NMΦs, respectively ; while the serum IL-10 level was decreased by 70%. Furthermore, the maturation rate of dendritic cells (CD11c+CD80+CD86+) was increased from 12.6% ± 2.1% to 42.6% ± 2.5% by the NMΦs . IFN-γ as a prototypical antitumor cytokine was also overexpressed, indicating the immune activation by BaSO4@ZIF-8/TRF NMΦs . FoxP3+ regulatory T cells (FoxP3+ Tregs) are a subset of T cells that can inhibit the immune system from attacking tumors to antagonize CD8+ cytotoxic T lymphocytes and CD4+ T helper cells. BaSO4@ZIF-8/TRF NMΦs decreased the population of FoxP3+ Tregs by nearly 80% . Based on the above analysis, BaSO4@ZIF-8/TRF NMΦs successfully reversed tumor immunosuppression and activated systemic antitumor immunity.Next, we evaluated the ability of BaSOrophages , BaSO4@Z ability . The int4@ZIF-8/TRF NMΦs encourages us to evaluate their therapeutic efficiency in bilateral 4T1 tumor-bearing mice. To suppress immune tolerance of tumors, PD-1 antibody as a typical immune checkpoint blockade agent was administrated during the treatments. Upon injection of BaSO4@ZIF-8/TRF NMΦs, the expressions of adhesion-related proteins  were sharply downregulated . The high antitumor efficacy of BaSO4@ZIF-8/TRF NMΦs could be ascribed to tumor anoikis. When anti-PD-1 was co-administrated with BaSO4@ZIF-8/TRF NMΦs, the tumor suppression rate further reached 95% due to the activation of antitumor immunity. Moreover, cytotoxic T cells and helper T cells were able to migrate into distant tumors for immunological attack, thereby inhibiting the growth of distant tumors by 90% . After the treatment of BaSO4@ZIF-8/TRF NMΦs with anti-PD-1, obvious nucleus condensation and DNA fragmentations of dead cells were found from histological analysis of tumor slices . Because of the immune modulating role of BaSO4@ZIF-8/TRF NMΦs, the mice survival rate was substantially increased to 83% within 60 days  in the group of BaSO4@ZIF-8/TRF + anti-PD-1. Therefore, BaSO4@ZIF-8/TRF NMΦs can successfully elicit the long-term immune memory effect to defend against tumor recurrence . All these aspects suggest the high biosafety of BaSO4@ZIF-8/TRF NMΦs for practical applications.To evaluate the immune memory effect, tumor-bearing mice were rechallenged with intravenous injection of 4T1-Luc tumor cells after various treatments Fig. a. For bl4@ZIF-8/TRF as a paradigm, we present an artificial macrophage to mimic macrophage-tumor interactive pattern for antitumor immune activation. BaSO4@ZIF-8/TRF NMΦs are able to selectively accumulate surrounding tumor cells and gradually release Zn2+ into tumor microenvironment. Zn2+ as a chemical messenger depressed the expression of adhesion proteins on cell membranes to induce tumor immunogenic death via anoikis. As the disintegration of ZIF-8 shell, a large number of mesopores were left on the BaSO4 nanoparticles, providing cavities for capturing tumor antigens. As a result, BaSO4 nanoparticles bearing tumor antigens promote the macrophage polarization and subsequent T cell recruitment for immune activation. Together with immune checkpoint inhibitors, BaSO4@ZIF-8/TRF NMΦ can elicit systemic antitumor immunity against distant metastasis. Moreover, the immune memory effect is successfully achieved to prevent tumor relapse. Overall, the BaSO4@ZIF-8/TRF NMΦ successfully simulates the basic biological functions of natural macrophages, including tumor retention, cytokine release, antigen capture and immune activation. The artificial macrophages with hierarchical nanostructure provide a promising strategy to overcome tumor immunosuppression, and raise the prospect of artificial cells in the biomedical field.In summary, with BaSOSupplementary file1 (PDF 3019 KB)Below is the link to the electronic supplementary material.


Reduced RyR2 shut down basal Ca2+ oscillation in Tregs, which reduced m-calpain activities that are needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency rendered the CD4+ T cell pool immune suppressive and caused it to behave in the same manner as Foxp3+ Tregs in viral infection, asthma, hypersensitivity, colitis, and tumor development. In the absence of Foxp3, Ryr2-deficient CD4+ T cells rescued the systemic autoimmunity associated with scurfy mice. Therefore, Foxp3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.The suppression mechanism of Tregs remains an intensely investigated topic. As our focus has shifted toward a model centered on indirect inhibition of DCs, a universally applicable effector mechanism controlled by the transcription factor forkhead box P3 (Foxp3) expression has not been found. Here, we report that Foxp3 blocked the transcription of ER Ca  Second, the direct DC/Treg binding appears to be commonly observed both in vivo and in vitro  with Ryr2 knockdown (KD) bound to DCs with forces similar to those of Tregs and became suppressive, echoing our early findings that m-calpain blockage imparts Treg-like inhibition to Tconvs. In addition, T cells with CD4+-specific Ryr2 deletion became immune suppressive both in vitro and in vivo in the absence of Foxp3 expression. In the absence of Tregs, Ryr2–/– Tconvs restored immune homeostasis in all aspects of immune deficiency in scurfy mice, reminiscent of Sakaguchi’s reports that cotransfer of untreated CD4+ T cells blocked the ability CD5loCD4+ T cells to induce systemic autoimmunity in athymic mice . To study this in detail, we observed Ca2+ signals in both Tconvs and Tregs in resting state with two different indicators, Fluo-4 AM and ratiometric Cal Red R525/650 AM. While spontaneous Ca2+ oscillations are a common feature shared by many immune cells, including Tconvs, this activity was essentially absent in Tregs  and found that RyR2, an ER-membrane calcium channel protein, showed markedly reduced mRNA and protein levels in Tregs . To. ToRyr2  control . Althoug control , Foxp3 s control , appears control . These dRyr2 in CD4+ T cells with CD4-Cre  cells mice (see below). To further reveal any impact of Ryr2 deletion in T cell biology, we used 50:50 WT/RyR2 CKO bone marrow to restore the immune system in γ-irradiated recipients. Using CD45.1/CD45.2 markers, we repeated the thymic and peripheral comparisons  analyses for Tconvs, CKO Tconvs, and Tregs, both under resting state and following activation. RNA-Seq revealed that with the expected reduction of RyR2 in both Tregs and CKO cells, no other suspected suppression function was significantly elevated in CKO cells  RyR2 CKO OT-II T cells and repeated the experiment. Infusion of OVA peptide increased the binding duration between WT OT-II T cells and DCs, yet it had no effect on contact duration between RyR2 CKO OT-II T cells and DCs, suggesting the adhesion to DCs can be inversely regulated by RyR2 and the antigen specificity may not be essential in our settings  repertoire analysis revealed that RyR2 CKO TCR frequencies were almost identical to WT Tconvs and distant from those of Tregs . Therefosettings . To calcsettings . Ca2+ rentensity . To addrntensity . Ca2+ reose data . TherefoKO cells , D and EKO cells , F and GRyr2–/– Tconvs and Tregs, we tested their behaviors in viral infection, allergic response, autoimmune colitis, and tumor development, with comparison to similarly infused purified Tregs. In a footpad inoculation model  mice, with pervasive inflammation as a result of multiorgan autoimmunity. This pathology leads to death in a defined window of 2–4 weeks of age. If the immune regulatory effects of Ryr2–/– Tconvs represent a missing effector function in scurfy mice, Ryr2–/– Tconvs should correct those autoimmunities in the systemic absence of Foxp3. scurfy mice  were injected with PBS, Tregs, Foxp3– Tconvs, or Ryr2–/– Tconvs 2–3 days after birth. As expected, mice infused with PBS or Foxp3– Tconvs all died within a window of 2–4 weeks after birth , indicating that this mechanism may be of clinical value in the future. In our survey of Ryr2–/– Tconvs, factors that may be associated with immune suppression do not appear to be altered, suggesting that this regulation may be independent of previously proposed models. This certainly does not exclude those factors from participating in Treg suppression in other disease models. Yet, the Ryr2 deletion resulting in an immune suppression similar to that of Tregs suggests that this chain of regulation may be one of the central effector mechanisms of Tregs, fulfilling a missing link in this research field. But caution should also be exercised in how much our findings can be generalized for entire Treg biology. Our model relies on the instantaneous contact between Tregs and DCs, which likely takes away a basal condition for Treg suppression, but many of Treg inhibitory mechanisms are likely late onset and potentially require TCR signaling at some stage of their survival and additional effector functions. Our experimental designs could have unintentionally left out those considerations.In this report, we found that Tregs, in comparison to other T cells, lack basal Caof Tregs . Perhaps+ T cells carrying highly self-reactive TCRs. Tconv TCRs, on the other hand, are shaped more stringently by the thymic deletion and are therefore unlikely to be the same as those on tTregs  and mouse CD25 Regulatory T Cell positive selection Kit (STEMCELL Technologies). Tregs and Tconvs sometimes were sorted by FACS from CD4+ splenocytes of Foxp3GFP or FOXP3-IERS-RFP–transgenic mice. Murine DCs were isolated from spleens with the mouse CD11c selection Kit II (STEMCELL Technologies). OT-II T cells were isolated from OT-II splenocytes by the mouse CD4+ T Cell Isolation Kit and sometimes sorted by FACS with an anti-TCR Vα2 antibody .DC2.4 cells were a gift from Kenneth Rock of UMass Medical School, Worcester, Massachusetts. Vero cells were from Xu Tan of School of Pharmaceutical Sciences, Tsinghua University. Renca cells were from Guangyu An, Chao-Yang Hospital, Beijing, China. HEK293FT cells were a gift from Wei Guo of School of Medicine, Tsinghua University. MC38 and NIH-3T3 cells were purchased from ATCC. MC38, Renca, NIH-3T3, and Vero cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin. All other cells were grown in RPMI-1640 with the same supplements plus 10 mM HEPES (pH 7.0) and 50 μM β-mercaptoethanol. All cell lines were tested for mycoplasma contamination by PCR analysis. Murine CD4Recombinant human IL-2 was from R&D Systems. The Dual-Luciferase Report Assay System was purchased from Promega. Ultra-LEAF purified anti-mouse CD3ε antibody (145-2C11) and Ultra-LEAF purified anti-mouse CD28 antibody (37.51) for T cell activation were from BioLegend. For flow cytometric analysis, CD62L (MEL-14) monoclonal antibody was purchased from BD Pharmingen, and others (named in the next sentence) were purchased from eBioscience. The following clones were used: CD45.1 (A20), CD45.2 (clone 104), CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), FoxP3 (3G3), GITR (DTA-1), CD25 (PC61.5), PD-1 (RMP1-30), CTLA-4 (UC10-4B9), CD39 (24DMS1), TIM3 (8B.2C12), LAG-3 (C9B7W), TCR Vα2 (B20.1), CD44 (IM7), CD5 (53-7.3), Nrp1(3DS304M), Helios (22F6), IFN-γ(XMG1.2), IL-4 (11B11), IL-17A (eBio17B7), Ki-67 (SolA15), and Rat IgG2a kappa Isotype Control (eBR2a).The cells were collected and lysed with RIPA buffer (Beyotime). The cell lysate was centrifuged and the supernatant was collected. Total proteins were quantified with BCA Protein Assay Kit (Beyotime). After being mixed with 3′SDS loading buffer and boiled for 5 minutes, the proteins were loaded onto 7.5% or 5% PAGE Gels (EpiZyme) or 4%–12% Precast Protein Plus Gels (Yeasen). Then, the proteins were transferred onto a Nitrocellulose membrane  and immunoblotted with indicated primary  and secondary antibodies . Finally, the immunostained bands were detected by the Super ECL Detection Reagent (Yeasen).gapdh or 18S RNA was used as reference gene for normalization. The primer sequences were as follows: gapdh, 5′-CATCACTGCCACCCAGAAGACTG-3′ and 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′; 18S RNA, 5′-CGGACAGGATTGACAGATTG-3′ and 5′-CAAATCGCTCCACCAACTAA-3′; Ryr2, 5′-ATGGCTTTAAGGCACAGCG-3′ and 5′-CAGAGCCCGAATCATCCAGC-3′; Ryr1, 5′-GCACACTGGTCAGGAGTCGTATG-3′ and 5′-GGGTGTAGCACAGGATTTAT-3′; Ryr3, 5′-ATCGCTGAACTCCTGGGTTTG-3′ and 5′-TTCATGTCGATGGAACTTAGCC-3′; Foxp3, 5′-CCCATCCCCAGGAGTCTTG-3′ and 5′-ACCATGACTAGGGGCACTGTA-3′; Capn2, 5′-GGTCGCATGAGAGAGCCATC-3′ and 5′-CCCCGAGTTTTGCTGGAGTA-3′; Itpr1, 5′-CGTTTTGAGTTTGAAGGCGTTT-3′ and 5′-CATCTTGCGCCAATTCCCG-3′; Trpm1, 5′-ATCCGAGTCTCCTACGACACC-3′ and 5′-CAGTTTGGACTGCATCTCGAA-3′; Trpm4, 5′-GGACTGCACACAGGCATTG-3′ and 5′-GTACCTTGCGGGGAATGAGC-3′; Trpv2, 5′-TGCTGAGGTGAACAAAGGAAAG-3′and 5′-TCAAACCGATTTGGGTCCTGT-3′; Cacna2d4, 5′-GGCAGCAAGTTATCTCCCAG-3′ and 5′-CCACAGGATGATTGGCGTCTT-3′; Ahnak, 5′-CAGCGCATCTACACCACGAA-3′ and 5′-CACTTCATGCCTTGGTATCTTGA-3′; Stim1, 5′-TGACAGGGACTGTACTGAAGATG-3′ and 5′-TATGCCGAGTCAAGAGAGGAG-3′; Pkd1, 5′-CTAGACCTGTCCCACAACCTA -3′ and 5′- GCAAACACGCCTTCTTCTAATGT-3′; Ncs1, 5′-AGCAAGTTGAAGCCTGAAGTT-3′ and 5′-GCTGGGGCAGTCCTTAATGAA-3′; Slc24a3, 5′-AGCAAGTTGAAGCCTGAAGTT-3′ and 5′-GCTGGGGCAGTCCTTAATGAA-3′; Hspa2, 5′-GCGTGGGGGTATTCCAACAT-3′ and 5′-TGAGACGCTCGGTGTCAGT-3′; P2rx7, 5′-GACAAACAAAGTCACCCGGAT-3′ and 5′-CGCTCACCAAAGCAAAGCTAAT-3′; Itgav, 5′-CCGTGGACTTCTTCGAGCC-3′ and 5′-CTGTTGAATCAAACTCAATGGGC-3′; Ccdc109b, 5′-CCACACCCCAGGTTTTATGTATG-3′ and 5′-ATGGCAGAGTGAGGGTTACCA-3′; Pln, 5′-AAAGTGCAATACCTCACTCGC-3′ and 5′-GGCATTTCAATAGTGGAGGCTC-3′; Gsto1, 5′-ATCCGGCACGAAGTCATCAAC-3′ and 5′-TGACAGATTCGGTGACCAAGT-3′; Itpr2, 5′-CTGTTCTTCTTCATCGTCATCATCATCG-3′ and 5′-GAAACCAGTCCAAATTCTTCTCCGTGA-3′; and Itpr3, 5′-CTTTATCGTCATCATCATCGTGTTG-3′ and 5′-AGGTTCTTGTTCTTGATCATCTGAGCCA-3′.Tconvs and Tregs were cultured overnight in presence of recombinant human IL-2 for subsequent experiments unless noted otherwise. Total RNA was extracted from indicated cells using TRIzol reagent (Invitrogen) and first-strand cDNA was synthesized with Reverse Transcriptase M-MLV (TaKaRa). Real-time PCR was performed using Hieff qPCR SYBR Green Master Mix (No Rox) (Yeasen). 7 cells per assay) cultured overnight were fixed for 15 minutes at room temperature with 1% formaldehyde and then digested with 45 cycles of sonication (Bioruptor). Foxp3 mAb  or control rabbit IgG antibody  were added to bind. Binding of Ryr2 promoter was determined by qPCR. The following primer pairs were used: Gmpr promoter, 5′-CAGCTGGAACAGCCTTGGAA-3′ and 5′-AAATGTCAAGGCCCCTGTGA-3′ and Ryr2 promoter, 5′-TGCAGGGGGACCGACC-3′ and 5′-GTCACTGCTAACCAGGATGTTCTA-3′.ChIP was conducted with the SimpleChIP Plus Sonication Chromatin IP Kit (CST) following the manufacturer’s instructions. Tregs and Tconvs . Data collection was controlled by NIS-Elements 3.0 software (Nikon). The mean fluorescence intensity changes over time for individual cells were analyzed by ImageJ (NIH) and normalized to resting fluorescence F0 (Fluo-4 F/F0) after subtracting background. Calcium concentration was analyzed based on following equation for fluo-4 (T cells cultured overnight were stained with 2 μM fluo-4 AM (Thermo Fisher Scientific) in Hank’s solution (Coolaber) at 37°C for 30 minutes. After washing and incubation, the cells were allowed to adhere to a poly-L-lysine–coated  round glass slips mounted in a sandwiched chamber made in-house at room temperature. Excess nonadherent cells were removed by flushing with Hank’s solution after 15 minutes. Then, the measurement chamber was placed on an Olympus IX-73 microscope equipped with a 20×  or 40×  Olympus objective. Fluorescence Car fluo-4 ,max is the fluorescence intensity at 10 mM saturating [Ca2+], the cytosolic [Ca2+]0 is 50 nM for resting T cells  at room temperature for 30 minutes. The dye-loaded cells were then subjected to wide-field imaging. The Cal Red R525/650 fluorescence dye was excited at 488 nm, and the emitted fluorescence signal was captured at 525 nm (F525) and 650 nm (F650). For Cal Red, the intensity ratios can be converted to intracellular calcium concentration .where R represents the fluorescence intensity ratio F525/F650 during the experiment, in which F525 and F650 are the fluorescence detection wavelengths for the ion-bound and ion-free indicator, respectively. R5 T cells overnight cultured were incubated at 200 μL PBS solution containing 20 μM calpain substrate CMAC  at room temperature in dark. The reaction was terminated by 4% PFA (Biosharp) for up to 2 minutes after a 5-minute or 1-minute (for 4-CmC addition) incubation, and then the cells were immediately placed on the ice at least 5 minutes. Calpain activities as ﬂuorescence signals from the digested substrate were determined by Fortessa cytometers (BD Biosciences) via the Hoechst Blue channel. JTV519 (Sigma-Aldrich) was added 30 minutes before the experiment, and 4-CMC was added at the start of assay.For measurement of calpain activity, 10For surface marker detection, cells were incubated with Fc blocker  for 5 minutes and then incubated with surface antibody for 15 minutes at room temperature while avoiding light. Stained cells were analyzed directly or, occasionally, after being fixed with 1% paraformaldehyde (PFA) using Fortessa cytometers (BD Biosciences) with FACS Diva software. The Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) was used for intracellular staining of mouse Foxp3, Ki67, and Helios. The Intracellular Fixation/Permeabilization Buffer Set (Invitrogen) was utilized for mouse cytokine detection. Data were analyzed with Flowjo V10.6 purified Tregs, Ryr2+/+ Tconvs, and Ryr2–/– Tconvs were stimulated by anti-mouse CD3ε plus anti-mouse CD28 antibodies. After 72 hours, the supernatants were collected. High-binding 96-well ELISA plates (Nunc) were coated with anti-mouse IL-10 (eBioscience) and anti-human/mouse TGF-β1 capture antibody (eBioscience) at 4°C overnight, respectively. After drying, plates were blocked with 2% BSA in PBS for 1 hour at room temperature. After washing, 100 μL of diluted cell supernatant was added in triplicate wells, followed by incubation for 1 hour at room temperature. Plates were then washed with PBST  and were incubated with anti-mouse IL-10 and anti-human/mouse TGF-β detection antibody for 0.5 hours at room temperature. TMB (eBioscience) was added (100 mL/well), and the plates were incubated for 10 minutes at room temperature in the dark, followed by the addition of H2SO4  per well to terminate the reaction. Optical density was immediately read at 450 nm using an ELISA plate reader (Bio-Rad).For IL-10 and TGF-β detection, 10E. coli with the EndoFree Plasmid Midi Kit (CWBIO). Lentivirus production was performed according to the manufacturer’s instruction. In brief, 293FT cells were cultured in a 10 cm dish with 60%–80% conﬂuency. Culture media were replaced 2 hours before DNA transfer. 5 μg pLKO.1 vector (with inserted shRNA) was transfected into 293FT cells with 2.5 μg package vectors pMD2.G and 2.5 μg psPAX2 using Neofect DNA transfection reagent (Neofect). 72 hours later, lentiviruses were harvested and added to infect T cells with Polybrene . 48 hours after the virus infection, cells were sorted with a BD FACSAria cytometer. KD efficiency was verified by real-time PCR. shRNA sequences are as follows: control shRNA, 5′-CCGGcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatcttgttgTTTTTG-3′ and 5′-AATTCAAAAAcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatcttgttg-3′; Ryr1 shRNA, 5′-CCGGcgtcgcatagaacggatctatCTCGAGatagatccgttctatgcgacgTTTTTG-3′and 5′-AATTCAAAAAcgtcgcatagaacggatctatCTCGAGatagatccgttctatgcgacg-3′; Ryr2 shRNA, 5′-CCGGccgctaatgaagccatataaaCTCGAGtttatatggcttcattagcggTTTTTG-3′ and 5′-AATTCAAAAAccgctaatgaagccatataaaCTCGAGtttatatggcttcattagcgg-3′; Ryr3 shRNA, 5′-CCGGccgacatggttcagagagaaaCTCGAGtttctctctgaaccatgtcggTTTTTG-3′ and 5′-AATTCAAAAAccgacatggttcagagagaaaCTCGAGtttctctctgaaccatgtcgg-3′; Ahnak shRNA, 5′-CCGGtgccaccatctactttgacaaCTCGAGttgtcaaagtagatggtggcaTTTTTG -3′ and 5′-AATTCAAAAAtgccaccatctactttgacaaCTCGAGttgtcaaagtagatggtggca-3′; Itpr1 shRNA, 5′-CCGGgcagtaggtaagaagttattaCTCGAGtaataacttcttacctactgcTTTTTG-3′ and 5′-AATTCAAAAAgcagtaggtaagaagttattaCTCGAGtaataacttcttacctactgc-3′; Stim1 shRNA, 5′-CCGGgcagtactacaacatcaagaaCTCGAGttcttgatgttgtagtactgctTTTTTG-3′ and 5′-AATTCAAAAAgcagtactacaacatcaagaaCTCGAGttcttgatgttgtagtactgct-3′; Trpm1 shRNA, 5′-CCGGcggagtgaacatgcagcatttCTCGAGaaatgctgcatgttcactccgTTTTTG-3′ and 5′-AATTCAAAAAcggagtgaacatgcagcatttCTCGAGaaatgctgcatgttcactccg-3′; Trpm4 shRNA, 5′-CCGGgcacatcttcacggtgaacaaCTCGAGttgttcaccgtgaagatgtggTTTTTG-3′ and 5′-AATTCAAAAAgcacatcttcacggtgaacaaCTCGAGttgttcaccgtgaagatgtgc-3′; Trpv2 shRNA, 5′-CCGGccaaggaacttgtttctatttCTCGAGaaatagaaacaagttccttggTTTTTG-3′ and 5′-AATTCAAAAAccaaggaacttgtttctatttCTCGAGaaatagaaacaagttccttgg-3′; and Cacna2d4 shRNA, 5′-CCGGtaggaacgcaatggatattaaCTCGAGttaatatccattgcgttcctaTTTTTG-3′ and 5′-AATTCAAAAAtaggaacgcaatggatattaaCTCGAGttaatatccattgcgttccta-3′. Lower case letter sequences are targeting sequences for the intended genes.Lentivirus-based shRNA was used to KD specific genes. pLKO.1 vectors purchased from shRNA library platform of Center of Biomedical Analysis of Tsinghua University were used for our all gene KD experiments. All predesigned shRNA sequences were synthesized by Ruibiotech. Then, they were inserted into pLKO.1 vector. Plasmids, including shRNA and the package constructs (pMD2.G and psPAX2), were purified from transformed For Foxp3 overexpression, pLVX-IRES-mcherry vectors were a gift from Xiaohua Shen of School of Medicine, Tsinghua University. For vector construction, Foxp3 was amplified by PCR using cDNA from Treg total RNA as template a with the following primers: forward primer 5′-ATCGCTCGAGATGCCCAACCCTAGGCCA-3′ and reverse primer 5′-ATCGGAATTCTCAAGGGCAGGGATTGGA-3′. The amplified fragment was gel purified, digested (XhoI and EcoRI), and cloned into the pLVX-IRES-mcherry plasmid, resulting in pLVX-Foxp3-IRES-mcherry construct. Lentivirus production harboring pLVX-Foxp3-IRES-mcherry– and Foxp3-overexpressed cell line generation was performed according to the above KD protocol. Foxp3 expression was verified by real-time qPCR.Ryr2 were designed with the CRISPR design prediction tool (ref. http://crispor.tefor.net/). Selected gRNAs were cloned into Lenti-sgRNA (MS2)-EF1a-EGFP backbone plasmid by following the SAM target sgRNA cloning protocol. We generated sgRNA insertion by annealing each oligo: sgRNA-2, 5′-CACCGCCTCCGGGCCGCCAAACCCG-3′ and 5′-AAACCGGGTTTGGCGGCCCGGAGGC-3’; sgRNA-4, 5′-CACCGGTGCCCTTCCTGACCTCAAG-3′ and 5′-AAACCTTGAGGTCAGGAAGGGCACC-3′; and sgRNA-5, 5′-CACCGAGGAGCTCAGCTTCCCGCTG-3′ and 5′-AAACCAGCGGGAAGCTGAGCTCCTC-3′.To overexpress RyR2, we attempted to use a CRISPR transcriptional activation (CRISPRa) system. All associated vectors, including pLenti-EF1a-dCas9-VP64-blast, pLenti-sgRNA(MS2)-EF1a-zeo, and pLenti-MS2-P65-HSF1-2A-Hygro, were a gift from Qiaoran Xi, School of Life Sciences, Tsinghua University. BleoR sequence in pLenti-sgRNA(MS2)-EF1a-zeo plasmid was replaced with EGFP to generate Lenti-sgRNA (MS2)-EF1a-EGFP backbone plasmids. For designing and cloning sgRNA, in brief, 20-nucleotide gRNA sequences targeting the promoter of The annealing reaction was performed using T4 polynucleotide kinase (NEB) under the following conditions: 37°C for 30 minutes; 95°C for and 5 minutes; a ramp up to 25°C at 5°C/min. Diluted annealing product (1:10) was mixed with backbone vector at 1:2.5 mass ratio in Golden Gate reaction using BsmBI restriction endonuclease (NEB) and T4 DNA ligase (NEB). The program was performed on a thermal cycler: 37°C for 5 minutes, 20°C for 5 minutes and repeated for 30 cycles; and 60°C for 5 minutes. Through the above steps, a gRNA fragment was inserted between the U6 promoter and sgRNA-MS2 scaffold to result in the pLenti-sgRyR2 (MS2)-EF1a-EGFP construct. Then, pLenti-EF1a-dCas9-VP64-blast, pLenti-sgRyR2(MS2)-EF1a-EGFP, and pLenti-MS2-P65-HSF1-2A-Hygro were cotransfected into MC38 cells, and RyR2 expression was verified by real-time qPCR.2. In all experiments, a minimum of 14 force curves were collected for further analysis. The force curves were processed using the JPK image processing software. Only round and robust cells were selected for AFM gluing. For each SCFS experiment, a T cell–DC pair was used to generate force readings from each up and down cycle over a period of several minutes; these readings are plotted. At least 3 such pairs were used for each condition.The experiments were performed as previously described using a JPK CellHesion unit , 47. In 4 purified DCs from splenocytes were pulsed with 2 μg/mL OVA323–339 peptide, and then suppressor cells (2 × 104 Tregs or Ryr2+/+ Tconvs or Ryr2–/– Tconvs or Ryr2 KD Tconvs) were added onto DCs to occupy the latter for 30 minutes. CD25- OT-II Tconvs were stained by CellTrace CFSE (Thermo Fisher Scientific). 2 × 104 OT-II Tconvs were mixed in DC-suppressor cell culture to compete with OVA-loaded DCs occupied by suppressor cells in a 96-well U-bottom plate. The proliferation of OT-II T cells was assessed by CFSE dilution by Fortessa flow cytometry (BD Biosciences). Inhibition percentage was calculated using 1 – proliferation% and then normalized no-Treg group as 0% inhibition and the Treg group as 100% inhibition.10https://deeptools.readthedocs.io/en/develop/#) and then correlated and clustered by Spearman’s correlation. DEGs were visualized with log10 FPKM.For transcriptome comparison, total RNA of Tconvs and Tregs cultured overnight from WT or CKO mice were extracted and sequenced (Annoroad). Anti-CD3/CD28–activated cells were sequenced as well. For chromatin opening comparison, Tn5-based libraries were built from fixed cells and sequenced . RNA-Seq FPKM files were correlated with the Pearson correlation method, and then hierarchy clustering was calculated and heatmaps were plotted using Euclidean distance. ATAC-Seq data were analyzed with deeptools  with mouse reference genome mm8. Gene tracks were generated as screenshots in specific gene loci.Visible ChIP-seq data were downloaded from GEO data sets . Visualization of ChIP-seq data was facilitated by IGV . 36 hours after transfection, cell lysates were prepared and analyzed using the Dual-Luciferase Report Assay System (Promega).Dual-luciferase report assay was established as described previously . Murine Ryr2+/+ Tconvs (Celltrace Far Red labeled), Ryr2–/– Tconvs (TAMRA labeled), and WT Tregs (Far Red-TAMRA duo labeled) cells were i.v. transferred into CD11c-DTR/eGFP-transgenic mice, at 1:1:1 ratio, 3 × 106 cells each. Approximately 12–18 hours after transfer, inguinal lymph nodes were exposed and imaged at 37°C in the intrafollicular zone. 4D (3D stack + time) videos were imported into Bitplane Imaris software and analyzed. DC channel (eGFP-positive) area was calculated as surfaces and then set as the reference plane for T cell migration. Contacts were designated as T cell–DC distance <1 μm, and contact duration was designated as continuous contact until release . For antigen-specific interactions, 6 hours after Far Red OT-II Tconv transfer, 50 μg OVA323–339 + 1 μg LPS was injected subcutaneously in right abdomen. Both draining inguinal lymph node (right) and control node (left) were imaged at approximately 12–18 hours. For contact disruption of OT-II cells and DCs by suppressor cell occupation , suppressor cells were labeled with 5 μM CellTrace Yellow (Thermo Fisher Scientific), and OT-II responder cells were labeled with 5 μM CellTrace FarRed (Thermo Fisher Scientific). A 1:1 mixture of both cells (5 × 106 each) was i.v. transferred into CD11c-DTR/eGFP-transgenic mice. For calcium fluctuation experiments, OT-II responder cells were first labeled with FarRed and then loaded with 5 μM calcium indicator FuraRed and transferred. Videos were analyzed with Imaris (Bitplane), and contacts between DC and responder T cells with or without suppressor cell occupation were analyzed.For basal contact dynamics between T cells and DCs in vivo, 5 CD45.2 CKO bone marrow cells in conjunction with an equal number of CD45.1 control bone marrow cells into lethally x-ray irradiated  5-week-old CD45.1/2 recipient mice. Mice were sacrificed 8 weeks after bone marrow transplantation. Thymi, spleens, and lymph nodes from mixed bone marrow chimeras were harvested for characterization of lymphocyte development.CD45.1 mice were bred with CD45.2 C57BL/6 mice to generate CD45.1/2 mice  expressing both the allelic variants. Mixed bone marrow chimeras were generated by i.v. injecting 5 × 10Ryr2 deletion affects T cell thymic selection, T cell receptor Vβ chains of splenic CD4+ T cells from CKO and control mice were assessed using a commercial mouse TCR Vβ screening panel (BD Biosciences). Flow cytometry data collection was performed on an LSR II (BD Biosciences). Data on the expression of TCR vβ chains in splenic CD4+ T cells for peripheral TCR usage and the frequencies of CD4+ Tconvs and Tregs expressing commonly utilized TCR Vβ chains were analyzed using FlowJo V10.To assess whether Mice were sacrificed by necropsy. Skin, ears, livers, and other tissues were all fixed in 4% neural buffered formalin at 4°C for over 48 hours before processing. Then samples were embedded in paraffin. Five- to six-micrometer thick slides were cut. All slides were stained with H&E. Inflammation was scored as follows: 0, no inflammatory infiltration; 1, sparse infiltration; 2, obvious infiltration; 3, massive infiltration with normal tissue morphology; 4, massive infiltration with tissue deform; and 5, complete tissue destruction.6 pfu HSV-1 (F strain) in 20 μL PBS into hind footpads at day 0. At 3 days after injection, cells  were adoptively transferred into the footpad with 2 × 105 cells/20 μL PBS. Virus titer was tested 7 days after injection with homogenized footpad tissue on Vero cells. For delayed-type hypersensitivity, the right footpad was rechallenged with UV-inactivated HSV-1 (106 pfu/20 μL PBS) at 6 days after injection, and then footpad swelling was measured at 7 days after injection, with the left footpad as control.Herpes infection was induced by 106 cells  were adoptively transferred i.v. in 100 μL PBS. Bronchoalveolar lavage fluid (BALF) infiltrates and histology were analyzed at day 32.Airway inflammation was induced by OVA. At day 0 and 14, 1 i.p. injections of OVA plus alum adjuvant (100 μg + 4 mg in 200 μL PBS) were given to sensitize mice. Intratracheal OVA rechallenge (50 μg in 50 μL PBS) was repeatedly given at back of the tongue on days 21, 23m and 25. At day 23, 106Ryr2–/– Tconvs, WT Tconvs, or Tregs were transferred into 6-week-old male C57BL/6 mice i.v. The second day, colitis was induced by oral administration of 4% DSS (w/v)  in drinking water for 7 days followed by normal drinking water. Normal control mice were treated with PBS and were given normal drinking water. The mice were sacrificed on day 10, colons were dissected, and colon length was measured. The colons were fixed at 4°C over 48 hours in 4% PFA for subsequent H&E staining. The colon sections were examined by 3DHISTECH Pannoramic SCAN.DSS-induced murine experimental colitis was established as described previously , 50. Bri5 MC38 cells in 200 μL PBS were coinjected with 106 T cells s.c. into the abdomen of 6-week-old male C57BL/6 mice after barbering. Tumor growth was monitored using a slide caliper approximately every 4–5 days from day 7 after tumor injection. The volume was calculated as (length × width × width)/2. Group 1 consisted of MC38 + PBS; group 2 consisted of MC38 + Tconvs; group 3 consisted of MC38 + Tregs; and group 4consisted of MC38 + Ryr2–/– Tconvs. There were 10 mice in each group.The grafted tumor mouse model was established as previously described . Briefly6 purified cells  was performed on day 2 or 3 of life in 50 μL PBS for i.p. injection into newborn syngeneic scurfy mice. Then, in the first 2 weeks, cells transfer was performed every 3 or 4 days and then once every 2 weeks. When transferring, the body weight of scurfy and male WT littermates was recorded and survival was monitored. Genotyping for the sf mutant gene was conducted by PCR and verified by sequencing. Primers for Foxp3 PCR were 5′-CATCCCACTGTGACGAGATG-3′ and 5′-ACTTGGAGCACAGGGGTCT-3′. For the histology, PBS and Foxp3– Tconv-infused mice were analyzed on week 3, and those from Treg- and Ryr2–/– Tconv-infused mice were on week 8–12. Skin, ear, liver, and other tissues were all fixed for H&E staining.The rescue of Foxp3-deficient mice was established as previously described , 53. Scut test was used for comparing endpoint means between 2 groups. One-way ANOVA with nonparametric Kruskal-Wallis test was used for multiple comparisons among 3 or more groups. SuperPlots of data were generated as previously described . Values for all data points in graphs are reported in the Co–first authorship order was decided by weighing productivity and significance of work; to be specific, Xiaobo Wang carried cellular and molecular studies to make concrete progress, SG carried sequencing and animal studies to show the translational value of this work, and JM performed the initial proof-of-concept experiments for this study. Xiaobo Wang and JM performed all the experiments unless noted otherwise. Xiaobo Wang and SG designed and performed all animal experiments with help from JG, ZZ, and XM. JM and NK performed AFM-related experiments. XL performed ChIP-qPCR and in vitro suppression. XX performed calcium imaging. HL and Ying Xu performed shRNA screening of KD cells. BZ analyzed and visualized ChIP-seq data. Yanni Xu and Xin Wang performed RyR2 protein detection. XS and SG performed intravital imaging assays. DZ and Xiaoting Wang performed cytokine detection. Xiaoting Wang and NN performed transcription analysis of calcium channel genes. RS performed histology analysis. RW assisted with RyR2 function analysis. JG provided scurfy and Treg biology expertise. SRWC provided expert insight in RyR2 regulation. XZ provided critical review of Treg biology. TX provided biophysical mechanisms of cell adhesion. HQ provided overall critical insight. XH provided expert insight on gene regulation of RyR2. YS was responsible for the overall design and wrote the manuscript with input from XH, HQ, and RW.


No therapeutic drugs are currently available for nonalcoholic steatohepatitis (NASH) that progresses from nonalcoholic fatty liver via oxidative stress-involved pathways. Three cognate peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) are considered as attractive targets. Although lanifibranor (PPARα/δ/γ pan agonist) and saroglitazar  are currently under investigation in clinical trials for NASH, the development of seladelpar (PPARδ-selective agonist), elafibranor , and many other dual/pan agonists has been discontinued due to serious side effects or little/no efficacies. This study aimed to obtain functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of three current and past anti-NASH investigational drugs: lanifibranor, seladelpar, and elafibranor. Ligand activities were evaluated by three assays to detect different facets of the PPAR activation: transactivation assay, coactivator recruitment assay, and thermal stability assay. Seven high-resolution cocrystal structures –lanifibranor, PPARα/δ/γ-LBD–seladelpar, and PPARα-LBD–elafibranor) were obtained through X-ray diffraction analyses, six of which represent the first deposit in the Protein Data Bank. Lanifibranor and seladelpar were found to bind to different regions of the PPARα/δ/γ-ligand-binding pockets and activated all PPAR subtypes with different potencies and efficacies in the three assays. In contrast, elafibranor induced transactivation and coactivator recruitment  of all PPAR subtypes, but the PPARδ/γ-LBD–elafibranor cocrystals were not obtained. These results illustrate the highly variable PPARα/δ/γ activation profiles and binding modes of these PPAR ligands that define their pharmacological actions. There is serious concern regarding the medical/economic burden of the treatment of the globally expanding number of patients with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). NAFLD is defined by the evidence of hepatic steatosis (either through imaging or histology) and the absence of secondary causes of significant alcohol consumption, long-term use of a steatogenic medication, or monogenic hereditary disorders . The ovePPARs belong to the nuclear receptor superfamily and the ligand-activated transcription factors; they exist in three subtypes in mammals  with considerable amino acid identity (54–71% in humans). The synthetic PPARα agonists known as “fibrates” have been widely used for the treatment of hypertriglyceridemia, while the synthetic PPARγ agonists known as “thiazolidinediones (glitazones)” are anti-diabetic drugs. In the guidelines of the American Association for the Study of Liver Diseases, pioglitazone has been proposed for the treatment of biopsy-proven NASH. The PPAR pan agonist lanifibranor (IVA-337) and the PPARα/γ dual agonist saroglitazar are currently in clinical trials for NASH . However, the development of most glitazars , including muraglitazar, tesaglitazar, and aleglitazar, has been abandoned due to serious safety concerns . TherefoThis study was designed so as to provide functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of lanifibranor, seladelpar, and elafibranor. We have found that all three agents can activate all PPAR subtypes with highly different preferences in the three different PPAR activation assays undertaken herein. Furthermore, we have characterized the high-resolution structures of the PPARα/δ/γ-ligand-binding domain (LBD)–lanifibranor, the PPARα/δ/γ-LBD–seladelpar, and the PPARα-LBD–elafibranor cocrystals through X-ray diffraction analyses.Renilla luciferase control plasmid under the control of a cytomegalovirus promoter were cotransfected into COS-7 cells  that were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, at 37 °C, in a 5% CO2/95% air incubator. The pSG5-GAL–hPPARα/δ/γ plasmids express fusion proteins comprising the yeast transcription factor GAL4 DNA-binding domain and each of the human PPARα/δ/γ-LBDs [Firefly luciferase gene [Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System  as described previously [Firefly luciferase responses induced by potent/specific PPARα/δ/γ agonists: GW7647 (1 µM) for PPARα, GW501516 (0.02 µM) for PPARδ, and GW1929 (1 µM) for PPARγ [Renilla luciferase responses. GW7647, GW501516, and pioglitazone were purchased from the Cayman Chemical Company . Elafibranor, lanifibranor, seladelpar, and saroglitazar were purchased from ChemScene . GW1929 was purchased from Sigma-Aldrich .In order to evaluate the PPARα/δ/γ-mediated transcriptional activation, pSG5-GAL–human PPARα/δ/γ chimera expression plasmids, a MH100(UAS)×4-tk-Luc reporter plasmid, and a pRL-CMV δ/γ-LBDs ,12. The ase gene . The celeviously . The traor PPARγ , after nHuman PPARα-LBD (amino acids 200–468), PPARδ-LBD (amino acids 170–441), and PPARγ-LBD (amino acids 203–477 in isoform 1) were expressed as amino-terminal His-tagged proteins by the pET28a vector  in Rosetta (DE3) pLysS competent cells (Novagen) and were subsequently purified using three-step chromatography, as previously described in detail ,15,16,1750 were performed using the Prism 9 software .The activation status of each PPARα/δ/γ subtype was also determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that detects physical interactions between His-tagged hPPARα/δ/γ-LBD proteins and a biotin-labeled PPARγ coactivator 1α (PGC1α) coactivator peptide (biotin-EAEEPSLLKKLLLAPANTQ (amino acids 137–155) synthesized by GenScript) or a steroid receptor coactivator 1 (SRC1) peptide (biotin-CPSSHSSLTERHKILHRLLQEGSPS (amino acids 676–700) from GenScript) using the LANCE Ultra TR-FRET assay  ,15,17. ATm) that corresponds to the midpoint of the denaturation process. The ligand-induced increases in the Tm values were defined as ∆Tm.PPARα/δ/γ-LBD proteins (10 µM) were incubated with different concentrations of ligands in buffer C consisting of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Tris 2-carboxyethylphosphine (TCEP)-HCl, and 10% glycerol. The circular dichroism (CD) spectra were monitored within a 200–260 nm range at increasing temperatures from 30 °C to 70 °C (2 °C/min) using a J-1500 spectropolarimeter equipped with a PTC-510 thermal controller . The spectra of all PPARs displayed local minima at 208 and 222 nm, which is a typical feature of α-helical proteins . The the50 value of 574 µM in the PGC1α recruitment activity) [We have previously obtained the PPARα-LBD–clofibric acid (a low affinity PPARα ligand with an ECctivity) –SRC1 (LTctivity) , and havctivity) . The PPAn-octyl-β-d-glucoside, and 1 µL reservoir solution (50 mM Bis-Tris propane (pH 7.5), 14% PEG8000, 0.2 M KCl, 6% propanediol, 1 mM EDTA, 1 mM CaCl2 for PPARδ-LBD–lanifibranor; 50 mM Bis-Tris propane (pH 8.0), 14% PEG8000, 0.1 M KSCN, 6% propanediol, 1 mM EDTA, 1 mM CaCl2 for PPARδ-LBD–seladelpar), at 20 °C, for several weeks.The cocrystallization of PPARδ-LBD–lanifibranor/seladelpar was performed in hanging-drop mixtures of 0.5 µL of PPARδ-LBD (10 mg/mL in buffer C), 0.1 µL of 10 mM ligand, 0.3 µL of buffer D (20 mM Tris-HCl (pH 8.0), 500 mM ammonium acetate, 1 mM TCEP-HCl, and 10% glycerol), 0.1 µL of 5% The cocrystallization of PPARγ-LBD–lanifibranor/seladelpar–SRC1 was performed in hanging-drop mixtures of 0.5 µL PPARγ-LBD (20 mg/mL in buffer C), 0.5 µL ligand (2 mM in buffer C), and 1 µL reservoir solution (0.1 M HEPES-NaOH (pH 7.5)/1.0 M trisodium citrate dihydrate for PPARγ-LBD–lanifibranor–SRC1; 0.1 M Tris (pH 8.0)/1.1 M trisodium citrate dihydrate for PPARγ-LBD–seladelpar–SRC1), at 20 °C, for several weeks.All obtained cocrystals were briefly soaked in a cryoprotection buffer . Subsequently, these were flash-cooled in a stream of liquid nitrogen until the X-ray diffraction analysis was conducted.Rmerge < 0.5, Rpim < 0.3, and completeness > 0.9) were set by the highest resolution shell [Datasets were collected by a BL-5A or a BL-17A beamline at the Photon Factory  using synchrotron radiation of 1.0 Å. Diffraction data were collected at a 0.1° oscillation per frame, and a total of 1800 frames (180°) were recorded for a 1.0-Å X-ray crystallography ,15,17. Don shell ,15,17. Aon shell  with Proon shell  and PHENon shell . The strBased on those X-ray cocrystal structures, proximity distances between each amino acid in PPARα/δ/γ-LBD and the three ligands were measured using PyMOL. All PPARα/δ/γ-LBD amino acids that have 4.5 Å or less proximity distances from the ligands were listed in 50 values of 398 nM (for δ) < 572 nM (for γ) < 4.66 µM (for α), and equivalent efficacies  (50: 20.2 nM) and efficacy (99.3%) compared to PPARα (1.64 µM and 41.0%) and PPARγ (3.53 µM and 58.5%) (50) of 388 nM (for α) < 2.12 µM (for γ) < 3.13 µM (for δ) and efficacies of 38.5% (for α/γ) > 14.8% (for δ), thereby acting like a PPARα/γ dual agonist (50: 479 nM) and efficacy (104%) compared to PPARα (4.79 µM and 25.2%) and PPARδ (>20 µM and 0.518%)  B. The PPd 58.5%) C. Unexpe agonist D. In con agonist E. The PP 0.518%) F. These The ligand-bound activated forms of PPARs are stabilized by associations with specific coactivators, thereby enabling the regulation of specific gene expression ,26. The Tm values at 222 nm are considered as the reflection of stable α-helical structures in PPARs, because ligand binding stabilizes the ligand-binding portion (LBP) [Tm values were 49.54 °C ± 0.12 °C (n = 4), 51.76 °C ± 0.17 °C (n = 6), and 48.95 °C ± 0.16 °C (n = 4) for PPARα/δ/γ-LBD, respectively. The PPARα/δ/γ-selective GW compounds  indeed exhibited highly increased Tm values with their highest soluble (in 0.1% DMSO) concentrations  so as to match the 10 µM PPARα/δ/γ-LBD protein in the assay mixture   . The bas mixture A. Lanifi (61.4%) A,B, whic (61.4%) A. SeladePARδ-LBD C, which PARδ-LBD C,D. In complexes D. SaroglPARα-LBD E, whereaRα/δ-LBD F. The thP21 at a 2.98 Å resolution, a monoclinic space group P21 at a 2.46 Å resolution, and an orthorhombic space group P212121 at a 2.29 Å resolution  and electrostatic interactions (blue dotted lines) with the four surrounding consensus amino acids : Ser280/P21 at a 2.01 Å resolution (PDB ID: 8HUN), a monoclinic space group P21 at a 2.67 Å resolution (PDB ID: 8HUO), and an orthorhombic space group P212121 at a 2.36 Å resolution (PDB ID: 8HUP), respectively  between each amino acid in PPARα/δ/γ-LBD and the ligands  were measured using PyMOL, and the MolDock scores  of their interactions were calculated . The fouThis study investigated how lanifibranor, seladelpar, and elafibranor bind to and activate PPARα/δ/γ subtypes. Major antioxidant genes, including catalase, heme oxygenase-1, glutathione peroxidase 3/4, superoxide dismutase 2/3, thioredoxin, CD36, and uncoupling protein 2/3, contain PPAR responsive element (PPRE) in their promoter regions and are transcriptionally regulated by the PPARs ,35,36. I50 values for PPARα/δ/γ were in small ranges  , was fir50 values for PPARδ-LBD were ~2 orders lower than those for PPARα/γ-LBD  . AlthougRα/γ-LBD C,D, and Rα/γ-LBD J, similaRα/γ-LBD .50 values for PPARα/δ/γ-LBD were in small ranges , has been abandoned at the phase 3 clinical trial against NAFLD/NASH due to its non-significant effect on the primary endpoint of the resolution of NASH without a worsening of fibrosis . In a ph3 cavities, the LBPs of PPARα/δ/γ have relatively large (1300–1440 Å3) cavities that are able to accept 1–4 ligand molecules [When compared to the LBPs of other nuclear receptors that have 600–1100 Åolecules . The PPA


Scientific Reports 10.1038/s41598-023-41031-6, published online 28 August 2023Correction to: d’, ‘206Pb/238U’ for the samples “TbP1-10”, “TbP1-8”, “TbP1-5”, “TbP1-3”, “TbP1-9”, “TbP1-13”, “TbP1-11(2)”, “TbP1-7” and “TbP1-6(2)” were omitted. The correct and incorrect columns appear below.The original version of this Article contained errors in Table 2, where values in the column ‘Age [Ma]Incorrect:Correct:Additionally, Table 3 contained errors, where the columns ‘Column’, ‘Lower tephra’, ‘Age Ma’, ‘Upper tephra’, ‘Age Ma’, ‘Thickness between tepras cm’, ‘Rate mm/year ’, ‘Loam thickness (maximum) cm’ and ‘Terrace gravel age Ma’ were omitted. The correct and incorrect tables appear below.Incorrect:Correct:Furthermore, the caption of Table 3 was incomplete.Table 3. Estimation of sedimentation rates, tephra ages, and terrace gravel ages (= terrace emergent ages). See references in main text.”“now reads:Table 3. Estimation of sedimentation rates, tephra ages, and terrace gravel ages (= terrace emergent ages). See references in main text. Significance of bold-italic values: Rate mm/year for loam; significance of underlined values: Rate mm/year for fluvial; significance of bold values: Adopted terrace gravel age Ma.”“The original Article has been corrected.


H-pyrrol-2-yl)prop-2-en-1-one (3HPPP) crystallized as planar molecule together with half a molecule of water in the asymmetric unit in the monoclinic crystal system with space group P2/c. A Hirshfeld surface analysis for the chalcone component showed that H⋯H (40.9%) and H⋯C/C⋯H (32.4%) contacts make the largest contributions to the crystal packing of 3HPPP. In the vicinity of water, the H⋯O/O⋯H and H⋯C/C⋯H contacts are the most significant, at 48.7% and 29.8%, respectively.3-(3-Hydroxyphenyl)-1--1-(1H-pyrrol-2-yl)prop-2-en-1-one (3HPPP), which was recently discovered to be a promising anti-MRSA candidate, and a half-molecule of water. The compound crystallizes in the monoclinic space group P2/c. The crystal structure features intermolecular pyrrole-N—H⋯O (water), carbonyl/keto-C—O⋯H—O-phenol and phenol-C—O⋯H (water) hydrogen bonds, which help to consolidate the crystal packing. A Hirshfeld surface analysis for the components in the asymmetric unit showed that H⋯H (40.9%) and H⋯C/C⋯H (32.4%) contacts make the largest contributions to the intermolecular interactions of 3HPPP. Considering the presence of water, in its vicinity H⋯O/O⋯H and H⋯C/C⋯H are the most significant contacts, contributing 48.7 and 29.8%, respectively.High-quality single crystals of the title compound, 2C In the crystal, molecules are linked into dimers via multiple intermolecular hydrogen bonds  −x + 1, −y + 1, −z + 1) between two molecules of 3HPPP can be observed connecting these non-covalently. Molecules of 3HPPP are linked into inversion dimer–dimer chains through these weak interactions. Moreover, the lattice water molecules act as donors and acceptors in hydrogen bonds with the phenol and pyrrole moieties of 3HPPP [O3—H3O⋯O2ii and N1—H1N⋯O3; symmetry code: (ii) x − 1, −y + 1, z − 3HPPP dimers in different planes comparably strongly and further consolidate the crystal packing.Fig. 2s Table 1. The dim4.et al., 20163HPPP molecule as been reported. However, focusing on the pyrrole ene-one side yielded three [refcodes HIXGAW , the most prominent interactions in the crystal packing are the hydrogen bonds, which are represented by four bright-red spots on the mapped dnorm surface. The bright-red spots around O1 and O2 correspond to the hydrogen bonding between hydroxyl and carbonyl/keto functional groups of two molecules of 3HPPP. The other two bright-red spots are due to hydrogen bonding between the pyrrole-N—H functional group and the water molecule, and between the water molecule and the hydroxyl group of 3HPPP. In addition to these four spots, two faint-red spots appear around O1 and H13, representing the non-classical hydrogen-bond interaction of an aromatic C–H and the carbonyl/keto functional group. The intensities of all these red spots indicate the relative strengths of the interactions, as well as the distances of the contacts. The dnorm Hirshfeld surface for the water molecules present in the crystal lattice was also calculated and mapped . Four bright-red spots are observed, which are due to the pyrrole-to-water and water-to-hydroxyl hydrogen bonds and are thereby mirrors of the interactions involving water described above.PP Fig. 3a, the med Fig. 3b. Four 3HPPP, and those delineated into H⋯H, H⋯O/O⋯H, H⋯C/C⋯H and H⋯N/N⋯H interactions are shown in Fig. 43HPPP show that H⋯H and H⋯ C/C⋯ H are the most significant interatomic interactions in the crystal packing, contributing 40.9 and 32.4%, respectively, to the Hirshfeld surface. The H⋯O/O⋯H (19.4%) and other minor contacts (H⋯N/N⋯H = 2.0%) further contribute to the Hirshfeld surfaces. On the other hand, the most prominent interatomic contacts for the water molecule are H⋯O/O⋯H, as expected, with a 48.7% contribution while H⋯H and H⋯C/C⋯H contacts contribute 16.2 and 29.8%, respectively.The overall two-dimensional fingerprint plots of both molecules, water and 6.3HPPP was synthesized by a Claisen–Schmidt condensation reaction between 2-acetylpyrrole (2 mmol) and 3-hydroxybenzaldehyde (2 mmol) under ethanolic (10 ml) conditions. The resulting mixture was stirred for 5 min followed by the dropwise addition of 3 ml of a 40% aqueous NaOH solution  as solvent system. Multiple spectroscopic analyses confirmed the chemical structure  = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023001925/yz2023sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023001925/yz2023Isup2.hklStructure factors: contains datablock(s) I. DOI: 2233979CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, which includes bone-forming and bone-resorbing cells, i.e., osteoblasts (OBs) and osteoclasts (OCs). OBs originate from mesenchymal progenitors, while OCs are derived from HSCs. Self-renewal, proliferation and differentiation of HSCs are under the control of regulatory signals generated by OBs and OCs within the BM niche. Consequently, OBs and OCs control both bone physiology and hematopoiesis. Since the human developmental and bone marrow failure genetic syndrome fanconi anemia (FA) presents with skeletal abnormalities, osteoporosis and HSC impairment, we wanted to test the hypothesis that the main pathological abnormalities of FA could be related to a defect in OC physiology and/or in bone homeostasis.Fanca−/− mouse is impaired in vitro due to overactivation of the p53–p21 axis and defects in NF-kB signaling. The OC differentiation abnormalities observed in vitro were rescued by treating Fanca−/− cells with the p53 inhibitor pifithrin-α, by treatment with the proinflammatory cytokine TNFα or by coculturing them with Fanca-proficient or Fanca-deficient osteoblastic cells.We revealed here that the intrinsic differentiation of OCs from a Overall, our results highlight an unappreciated role of Fanca in OC differentiation that is potentially circumvented in vivo by the presence of OBs and TNFα in the BM niche.The online version contains supplementary material available at 10.1186/s13578-023-01067-7. Bone marrow failure (BMF), myelodysplasia (MDS) and acute myeloid leukemia (AML) are three pathological conditions with underlying alterations in hematopoietic stem and progenitor cells (HSPCs) due to the cytotoxic consequences of exogenous stress (DNA damage) as well as to acquired or inherited mutations in genes encoding proteins involved in several classes of biological functions, including epigenetics , 2, RNA After birth, HSPCs are localized in a specific microenvironment in the bone marrow (BM), the hematopoietic niche. Niche homeostasis is determined by tightly regulated crosstalk between HSPCs and niche components that regulates the quiescence, proliferation, and differentiation of the former and influences the dynamics of the latter , 18. IndFANCA accounting for more than 60% of all FA cases . Briefly, cells were fixed using a fixative solution composed of 2.5 ml citrate solution, 6.5 ml acetone and 0.8 ml 37% formaldehyde for 30 s at room temperature (RT). Fifty microliters of Fast Garnet GBC Base Solution and fifty microliters of sodium nitrite were mixed for 30 s, left to stand at RT for 2 min and added to 4.5 ml of prewarmed deionized water (37 °C). Then, 50 µl of naphthol AS-BI phosphate solution, 200 µl of acetate solution and 200 µl of tartrate solution were added. The samples were incubated with the complete solution for 20 min at 37 °C. OCs were identified as TRAP-positive cells with 3 or more nuclei and a defined membrane.®594 donkey anti-rabbit secondary antibody for 30 min at 37 °C in the dark. Finally, the cells were incubated with a DAPI solution (1:500) for 3 min at RT and observed under a fluorescence microscope.Mature OCs were fixed in 4% formaldehyde for 15 min at RT and permeabilized in 0.1% Triton X-100 for 5 min at RT. Then, the cells were incubated with 1× phalloidin-iFluor 488 reagent (1:1000)  for 90 min at 37 °C in the dark. For the 53BP1 analysis, the cells were fixed and permeabilized as previously mentioned and then incubated with a primary antibody anti-53BP1  at the same time as phalloidin. After 3 washes with PBS, the cells were incubated with an Alexa Fluor2, 0.1% SDS, and 1% benzonase  with protease and phosphatase inhibitors ). After incubation for 15 min at RT, protein concentrations were determined using the Bradford assay , and the samples were combined with 4× Laemmli buffer containing β-mercaptoethanol and denatured by boiling at 98 °C. The proteins (50 μg) were separated using SDS-PAGE, transferred onto a nitrocellulose membrane , and then exposed to the appropriate antibodies. The proteins were visualized using an enhanced chemiluminescence system  and an ImageQuant LAS 4000 system . The following antibodies were used for Western blot analysis: rabbit anti-M-CSFR , mouse anti-RANK , mouse anti-NFATc1 , mouse anti-Cathepsin K , goat anti-p53 , rabbit anti-FANCA/FAA , rabbit anti-phospho-NF-kB p65 (Ser536) , rabbit anti-RelB , rabbit anti-NF-kB1 p105/p50 , rabbit anti-NF-kB2 p100/p52 , mouse anti-MiTF  and goat anti-β-Actin . Red Ponceau staining was used as a loading control.The collected cells were disrupted in lysis buffer  and reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit . qPCR was performed using iTaq™ Universal SYBR® Green Supermix  and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The following primers (Eurogentec) were used:Total RNA was isolated using the MaxwellRank: forward 5ʹ-GGACAACGGAATCAGATGTGGTC-3ʹ, Tm: 52.0 °C; reverse 5ʹ-CCACAGAGATGAAGAGGAGCAG-3ʹ, Tm: 51.6 °C;M-Csfr: forward 5ʹ-TGGATGCCTGTGAATGGCTCTG-3ʹ, Tm: 51.6 °C; reverse 5ʹ-GTGGGTGTCATTCCAAACCTGC-3ʹ, Tm: 51.6 °C;Nfatc1: forward 5ʹ-GGTGCCTTTTGCGAGCAGTATC-3ʹ, Tm: 52.1 °C; reverse 5ʹ-CGTATGGACCAGAATGTGACGG-3ʹ, Tm: 52.3 °C;Oscar: forward 5ʹ-CGTGCTGACTTCACACCAACAG-3ʹ, Tm: 51.6 °C; reverse 5ʹ-AGTGCAAACCGCCAGGCAGATT-3ʹ, Tm: 51.6 °C;Dc-Stamp: forward 5ʹ-TTTGCCGCTGTGGACTATCTGC-3ʹ, Tm: 51.7 °C; reverse 5ʹ-GCAGAATCATGGACGACTCCTTG-3ʹ, Tm: 51.9 °C;Atp6v0d2: forward 5ʹ-ACGGTGATGTCACAGCAGACGT-3ʹ, Tm: 51.6 °C; reverse 5ʹ-CTCTGGATAGAGCCTGCCGCA-3ʹ, Tm: 53.1 °C;Cathepsin K: forward 5ʹ-AGCAGAACGGAGGCATTGACTC-3ʹ, Tm: 51.7 °C; reverse 5ʹ-CCCTCTGCATTTAGCTGCCTTTG-3ʹ, Tm: 51.7 °C;Trap: forward 5ʹ-GCGACCATTGTTAGCCACATACG-3ʹ, Tm: 51.6 °C; reverse 5ʹ-CGTTGATGTCGCACAGAGGGAT-3ʹ, Tm: 52.0 °C; p21 (Cdkn1a): forward 5ʹ-TCGCTGTCTTGCACTCTGGTGT-3ʹ, Tm: 52.0 °C; reverse 5ʹ-CCAATCTGCGCTTGGAGTGATAG-3ʹ, Tm: 51.9 °C.Fanca−/− progenitors and OCs as described in the “Total RNA was extracted from the WT and We used recombinant murine TNFα , recombinant human TGF-β1 , Nutlin-3 , pifithtrin-α , butyrolactone I  and human/mouse anti-TNF-alpha antibody  at the indicated concentrations. Antisense oligonucleotides directed against p21 were purchased from Eurogentec (5ʹ-ACA-TCA-CCA-GGA-TTG-GAC-ATG-GGG-GGG-GGG-3ʹ).l-glutamine and 1 mM sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO2. For induction of OB differentiation under coculture conditions, the cell line was maintained in the same medium with 50 µg/ml l-ascorbic acid (Sigma #92902) and 4 mM β-glycerophosphate (Merck #G9422).siRNA directed against Fanca was purchased from Dharmacon (#L-058407-01-0005), and cells were transfected using Lipofectamine™ RNAiMAX transfection reagent  following the manufacturer’s instructions.The murine preosteoblastic cell line MC3T3-E1 Subclone 4 was purchased from ATCC (CRL-2593) and was routinely maintained in αMEM with 10% FBS, 100 U/ml penicillin/50 μg/ml streptomycin, 2 mM αMEM was added at the same time as OC precursors 24 h after siRNA transfection.TNFα was evaluated by FACS analysis of MC3T3 cells 3 days after siRNA transfection. The cells were fixed for 15 min in 1% PFA/PBS, washed 1× in PBS, incubated for 30 min in the dark at RT at the appropriate dilution (1:100) with a FITC-conjugated anti-mouse TNFα antibody  and washed 1× in PBS. The cells were then analyzed with a BD Accuri™ C6 Cytometer.Fanca−/−). For the cortical bone analysis of the femur, the following parameters were used: cross-sectional area (TtAr), cortical bone area (CtAr), cortical area fraction (CtAr/TtAr) and cortical thickness (CtTh). For the trabecular bone analysis of the femur, the following parameters were used: bone volume/total volume (BV/TV) ratio, trabecular number (TbN), trabecular separation (TbSp) and trabecular thickness (TbTh). Analyses were performed following published guidelines [Cortical bone and trabecular bone microarchitecture assessment was performed on femurs using a high-resolution X-ray microcomputed tomography (micro-CT) system . Micro-CT datasets were analyzed using the built-in multiplanar reconstruction tool Osirix 5.8 (Pixmeo). The reader was blinded to the status of the mouse . Differences were analyzed with unpaired and/or paired two-tailed t tests. Significance was set at 0.05.Additional file 1: Figure S1. A. Number of OCs for field, number of nuclei for OCand surface of the OCsat days 3, 4 and 5 of differentiation in 1-year-old mice. B. Relative Fanca expression evaluated by qRT-PCR in progenitors and OCs at day 4 of differentiation from WT and Fanca−/− mice. Data are shown as the mean ± SEM. C. Western blot showing Fanca in OCs from WT and Fanca−/− mice at days 4 and 5 of differentiation. b-Actin was used as a loading control. Figure S2. A. From left to right: relative Rank, M-Csfr, Nfatc1, Dc-Stamp and Cathepsin K expression evaluated by qRT-PCR in progenitors and OCs at day 4 of differentiation from 1-year-old WT and Fanca−/− mice. Data are shown as the mean ± SEM. B. Western blot showing Nfatc1 in OCs from 1-year-old WT and Fanca−/− mice at day 4 of differentiation. b-Actin was used as a loading control. Right: Relative protein expression of Nfatc1 in OCs from 1-year-old WT and Fanca−/− mice at day 4 of differentiation. C. Western blotand quantificationshowing Mitf expression in OCs from WT and Fanca−/− mice at day 4 of differentiation. Figure S3. A. Number of OCs for field, number of nuclei for Csand surface of the OCsat day 4 of differentiation in the WT and Fanca−/− cells left untreated or treated with TGFb. Figure S4. A. Representative µCT images of the distal femur and femur sections in WT and Fanca−/− mice. B. Comparison of trabecular status in bone from WT and Fanca−/− mice. Figure S5. A. Number of OCs per field from WT and Fanca−/− cells cultured in the absence or presence of TNFα on day 4 of differentiation. B. Number of OCs per field from the WT and Fanca−/− cells plated on WT or Fanca-depleted MC3T3 cells and treated or not with an inhibitory anti- TNFα antibody at day 4 of differentiation. C. Number of OCs per field from the WT cells plated on WT or Fanca-depleted MC3T3 cells and treated or not with an inhibitory anti- TNFα antibody at day 4 of differentiation. Figure S6. Uncropped gel images used for representative Western blot.


Th. Th+CD8αELs Fig. , revealiELs Fig. . In addie B Fig. , indicat+CD8αα+ DP IEL generation remains questionable because we did not observe a decrease in DP IEL frequencies and numbers in IL-27Rα-deficient mice . Compared to wild-type controls, Bcl-2Tg mice contained a substantially increased numbers of DP T cells in the SI epithelium (Fig. + IELs (Fig. If such were the case, we expected that the forced expression of Bcl-2 would expand the size of the CD4ium Fig. , but witELs Fig. . Thus, t+CD8αα+ DP IEL pool but supports the expression of LAG-3 and induction of IL-10. These findings further put forward a new transcriptional circuitry of DP IEL differentiation and phenotype acquisition, while it remains to be assessed what cellular signals drive and maintain this Runx3d-mediated pathway in CD4+CD8αα+ IELs.Collectively, our results identify Runx3d expression as a new regulatory mechanism that constrains the size of the SI CD4Suppl Figures and Methods


II ions ligated by adamantane-1,3-dicarboxylate and N-(pyridin-3-yl)isonicotinamide ligands forming coordination polymer layers with a  grid topology.The title compound contains octahedrally coordinated Ni 12H14O4)(C11H9N3O)(H2O)]·H2O}n, contains octahedrally coordinated NiII ions ligated by adamantane-1,3-dicarboxylate (adc) and N-(pyridin-3-yl)isonicotinamide (3-pina) ligands forming coordination polymer layers with a  grid topology. These diperiodic layer motifs aggregate in an AAA pattern mediated by supramolecular C—H⋯O interactions to form the full triperiodic crystal structure of the title compound.The title compound, {[Ni(C We have reported nickel adc coordination polymers featuring 4,4′-dipyridylamine (dpa) (Travis 2O)]·H2O}n, contains two NiII atoms on crystallographic inversion centers (Ni1 and Ni2), a complete adc ligand, an N-(pyridin-3-yl)isonicotinamide (3-pina) ligand, one water molecule bound to Ni1, and one water molecule of crystallization (3-pina)2(adc)2]n monoperiodic chain motifs with an Ni1⋯Ni2 through-ligand distance of 9.694 (1) Å 2(adc)2(3-pina)2]n coordination polymer layer motifs with  grid topology 2(adc)2(3-pina)2]n layers into the triperiodic full crystal structure of the title compound. The layers stack in an AAA pattern along both the a and the c crystal directions 2·6H2O , adamantane-1,3-dicarboxylic acid (adcH2) , N-(pyridin-3-yl)isonicotinamide (3-pina) , and 0.75 ml of a 1.0 M NaOH solution were placed in 10 ml of distilled water in a Teflon-lined acid digestion bomb. The bomb was sealed and heated in an oven at 393 K for 48 h, and then cooled slowly to 273 K. Green crystals of the title complex were obtained in 71% yield.Ni(NOUiso(H) values fixed at 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2414314623007587/vm4062sup1.cifCrystal structure: contains datablock(s) I, 1R. DOI: 10.1107/S2414314623007587/vm4062Isup2.hklStructure factors: contains datablock(s) I. DOI: 2291890CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-quaternized ketene N,O-acetal, 1-(1-ethoxyvinyl)-2-(methyl(phenyl)amino)pyridin-1-ium trifluoromethanesulfonate was synthesized and its structure determined, making it a rare example of this class of compounds to be structurally characterized.The  N-quaternized ketene N,O-acetal, C16H19N2O+·CF3O3S−, was synthesized and its structure determined. The title compound is a rare example of a pyridinium ketene hemiaminal for which a crystal structure has been determined, joining the 2-chloro-1-(1-ethyoxyethenyl)pyridin-1-ium trifluoromethanesulfonate salt from which it was synthesized. The cationic species of the title compound can be defined by three individually planar fragments assembling into a non-coplanar cation. The phenyl substituent extending from the amino nitrogen atom and the ethyoxyvinyl substituent extending from the pyridine N atom are oriented on the same side of the molecule and maintain the closest coplanar relationship of the three fragments. Supramolecular interactions are dominated by C—H⋯O interactions from the cation to the SO3 side of the trifluoromethanesulfonate anion, forming a two-dimensional substructure.The novel bench-stable  Mean plane to mean plane angles between the fragments are 71.71 (4)° between A and B, 68.16 (4)° between A and C, and 29.77 (6)° between B and C. The phenyl group attached to the amino-nitrogen atom is folded toward the same side of the aminopyridine fragment as the ethoxyvinyl substituent, likely requiring their mean plane to mean plane angles to be closest to parallel. The orientation of the ethoxyvinyl substituent on the pyridine ring [C1—N1—C6—O1 torsion angle of 116.44 (12)°] is similar to that in the 2-chloro-substituted compound I, CSD refcode JETTOU, which has a mean plane to mean plane angle between the pyridine and ethoxyvinyl fragments of 70.2 (2)° and a C—N—C—O torsion angle of 109.1 (2)° about the exocyclic N—C bond, which was shown to be an energetically favorable arrangement  Å, C—H⋯F = 153.1°] is considered from the C13 atom of the phenyl fragment to the F2 atom of the anion. Only one such contact occurs to the CF3 side of the anions.The triflate anions and substituted pyridinium cations are arranged in individual columns along the ll Fig. 3. All thrll Fig. 3. As a rell Fig. 3. The six4.et al., 2016et al., 2018I, 2-chloro-1-(1-ethyoxyethenyl)pyridin-1-ium trifluoromethanesulfonate, CSD refcode JETTOU   was slowly added. After one minute of stirring at room temperature, the sealed microwave vial was placed in a pre-heated 313 K oil bath and stirred for 24 h. The reaction mixture was cooled to room temperature then concentrated to a residue that was purified by silica gel column chromatography using a 0-70% gradient of isopropanol in chloroform to provide compound IV as a yellow solid . 1H NMR  δ 8.36–8.26 , 7.69 , 7.52 , 7.45–7.38 , 4.69 , 4.36 , 3.74 , 3.63 , 1.27 ; 13C NMR  δ 154.5, 152.6, 145.5, 144.2, 142.6, 129.8, 127.9, 126.4, 121.5 , 119.5, 116.4, 85.9, 65.8, 43.4, 13.2.; LRMS–ES+ m/z (relative intensity) 255.1 ; HRMS–ES+ (C16H19N2O) calculated 255.1497 (M+), found 255.1497. X-ray quality crystals were formed by slow evaporation of a solution of the purified compound IV in acetone over the course of one week.A sealed 0.5-2.0 mL Biotage microwave vial was charged with potassium carbonate , freshly prepared 2-chloropyridinium ketene hemiaminal 6.Uiso(H) = 1.5Ueq(C) for methyl hydrogen atoms and Uiso(H) = 1.2Ueq(C) for other hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023005741/zl5048sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023005741/zl5048Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023005741/zl5048Isup3.cmlSupporting information file. DOI: 2278022CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: