BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50–60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3–5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPLV501A-W515R and JAK2V617F mutations and a man with PMF displaying an uncommon double MPLV501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF.Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main  BCR-ABL1-negative myeloproliferative neoplasms (MPNs). Over 70% of ET and PMF patients display a specific genetic alteration: 56% present Janus Kinase 2 (JAK2) mutations, 20–25% exhibit calreticulin (CALR) alterations, and 3–5% show nucleotide substitutions in the myeloproliferative leukemia (MPL) virus oncogene [Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main JAK2 displays the common V617F mutation, the ensuing protein acquires constitutive catalytic activity even in the absence of cytokine stimulation [MPL gene encodes for TPO-R. Excessive TPO-R activation promotes megakaryocytic hyperplasia and bone marrow fibrosis [MPL mutation involves the W515 residue on exon 10 [JAK2 is a non-receptor tyrosine kinase that contributes to myeloid cell proliferation and differentiation ,5. When fibrosis ,9. The mMPL mutations have been previously described but represent an extremely infrequent event [MPLW515L mutation associated with the S505N substitution [MPL mutations in three subjects: two ET patients, one presenting the S505C-W515L double mutation and the other with the V501A-W515R sequence alteration, and an individual with PMF displaying the V501A-W515L mutations [Double nt event ,14,15,16titution ,13. Pietutations . Howeverutations ,18. Furtutations ,19.MPL mutations identified by Next Generation Sequencing (NGS) that were investigated for the presence of additional genetic alterations in order to discern the possible clonal evolution of their disease.In this report, we describe two patients diagnosed with MPNs expressing two concurrent 9/L)  .JAK2 and CALR mutations by Sanger sequencing (SS). In particular, the genomic DNA (gDNA) was extracted from 1.7 mL of peripheral blood (PB) using the Qiasymphony DSP DNA Midi kit (Qiagen), according to the manufacturer’s protocol. For both analyses, we performed a Polymerase Chain Reaction (PCR) using 300 ng of gDNA. To amplify exon 14 of JAK2, we employed the forward (FW) primer 5′-GGGTTTCCTCAGAACGTTGA-3′ and the reverse (RV) primer 5′-TCATTGCTTTCCTTTTTCACAA-3′. To amplify exon 9 of CALR, we employed the FW 5′-CCTGCAGGCAGCAGAGAAAC-3′ and the RV 5′-ACAGAGACATTATTTGGCGCG-3′ primers. The PCR conditions consist of an initial denaturation step at 95 °C for 5 min, then 35 cycles of denaturation (95 °C for 45 s), annealing , extension (72 °C for 30 s), and a final extension at 72 °C for 7 min. The final PCR products (460 bp for JAK2 and 288 bp for CALR) were loaded on an agarose gel in TAE 1.2%, purified, and sequenced by SS using the same FW primers employed for the PCR. Both sequencing results were negative for the presence of mutations in the two genes.Following the World Health Organization (WHO) guidelines , the patMPL mutations on exon 10. In particular, 700 ng of gDNA were employed to perform a PCR using the FW 5′-AGTAGGGGCTGGCTGGATGA-3′ and the RV 5′-TGCCTGTTTACAGGCCTTCG-3′ primers. The Hot Start PCR conditions consist of an initial denaturation step at 94 °C for 5 min, then 80 °C for 1 min, followed by 35 cycles of denaturation (94 °C for 30 s), annealing (60.2 °C for 50 s), extension (72 °C for 30 s), and a final extension at 72 °C for 10 min. The final PCR product of 235 bp was loaded on an agarose gel in TAE 1.2%, purified, and sequenced by SS using the same FW primer employed for the PCR. Sequencing of the MPL gene revealed a V501A (c.1502T>C) substitution . A second BM biopsy confirmed her ET diagnosis. Spleen size was normal.The patient underwent a bone marrow (BM) aspiration that revealed megakaryocytic hyperplasia and grade 1 fibrosis. Eight months later (February 2018), she still presented a high platelet count (931 × 10JAK2V617F (VAF 39.77%), PTENQ17H (VAF 27.88%), TP53H178Tfs*69 (VAF 11.5%), PIK3CAN1044D (VAF 6.02%), and KITN566T (VAF 1.62%) (JAK2 mutation (39.77%) might explain why this alteration was not detected in her PB, as it may have been expressed in a limited number of neoplastic clones in the PB.To better analyze the molecular profile of this patient, we performed an NGS analysis. The sensitivity of NGS is much greater than that of SS, as it is able to detect mutant alleles with a frequency lower than 1%, while SS has a limit of detection around 20%. We analyzed the DNA isolated from the patient’s PB and BM, employing the Ion Ampliseq Cancer Hotspot Panel v2  as previously reported . This NGF 1.62%) . The rel9/L) and platelet (785 × 109/L) counts  counts .V617F substitution, but the test was negative as it was for CALR mutations. Subsequent analyses for additional genetic alterations detected the presence of two mutations on the MPL gene: V501A (c.1502T>C) and W515L (c.1544G>T)  a. The BMcis or in trans, we carried out the clonal selection of Burst-Forming Units-Erythroid (BFU-E) and Colony-Forming Units-Granulocyte and Monocyte (CFU-GM), followed by SS on the DNA extracted from these colonies. In detail, 1 × 105 primary mononuclear cells (MNCs) were isolated from BM biopsies by density gradient centrifugation. For the colony-forming assay, MNCs were plated at a density of 10.000 cells/mL (in triplicate) in Methocult H4435  and incubated at 37 °C in 5% CO2 for 12 days. Forty individual colonies (20 BFU-E and 20 CFU-GM) were plucked and transferred into PCR tubes for DNA extraction by thermic lysing (94° for 10 min). Subsequently, DNA was subjected to the same PCR described above for the amplification of MPL exon 10 and sequenced by SS [cis V501A-W515L mutations, while the remaining 8 exhibited the wild-type gene (cis) for the double mutation, while the remaining 16 were wild-type (13 colonies) or only expressed the W515L substitution (3 colonies) (cis) [To establish if the detected double mutations were in ed by SS . Twelve ype gene b. Of theolonies) b. These s) (cis) .M541L mutation (VAF 59.7%), frequently reported in mastocytosis and chronic eosinophilic leukemia [To further analyze the genetic profile of the disease and understand its possible clinical evolution, we performed an NGS analysis on the DNA obtained from the PB of the patient, employing the same procedures described above. Sequencing data confirmed both the V501A and the W515L mutations with a VAF of 38.5% and 34.4%, respectively. The analysis also detected the KITleukemia ,25  and high accuracy, discerning nucleotide variants, small insertions and deletions, copy number variations, and fusion transcripts .V617FJAK2 alteration. The lower frequency of the JAK2 substitution (39.77%) in the BM compared to MPL (100%) might explain the absence of the former alteration in the patient’s PB and suggest progressive disease evolution from a pre-existing dominant MPL-mutated clone [V501A-W515LMPL double mutation, likely generated in cis by a hematopoietic precursor mostly differentiating along the erythroid lineage. While the W515LMPL mutation is known to promote EPO-independent activation of the receptor [We report an ET patient simultaneously carrying the V501A and the W515R mutations. Next-generation sequencing analysis of her BM biopsy also detected a ed clone . The secreceptor , the patreceptor , which cTo date, the presence of numerous (ET patient) or limited (PMF patient) additional genetic alterations has not yet translated into a more aggressive disease, as both patients are currently in excellent clinical conditions while only receiving 100 mg daily acetylsalicylic acid for thromboembolic event prophylaxis, with a follow-up of more than 5 years since diagnosis. However, the employment of a new commercially available NGS panel specific for MPNs or myelodysplastic syndromes could be of great interest to explore other novel genetic and/or epigenetic mechanisms involved in the pathogenesis of the disease. Indeed, we recently extended NGS analysis in MPNs using a DNA/RNA panel including 40 genes and 29 fusion transcripts involved in myeloid malignancies. This new NGS panel is of great interest as it is able to confirm the diagnosis and discriminate genetic profiles involved in clonal evolution and disease progression of MPN/MDS.In conclusion, NGS analyses coupled with colony-forming assays allow the characterization of the molecular landscape and clonal evolution of MPN patients with uncommon genetic mutations. A longer follow-up will be required to address the prognostic impact of this information.


RP gene, we employed a gene overexpression strategy. We found that Arabidopsis lines overexpressing RPL16D (L16D-OEs) display short and curled rosette leaves. Microscopic observations reveal that both the cell size and cell arrangement are affected in L16D-OEs. The severity of the defect is positively correlated with RPL16D dosage. By combining transcriptomic and proteomic profiling, we found that overexpressing RPL16D decreases the expression of genes involved in plant growth, but increases the expression of genes involved in immune response. Overall, our results suggest that RPL16D is involved in the balance between plant growth and immune response.In plant cells, multiple paralogs from ribosomal protein (RP) families are always synchronously expressed, which is likely contributing to ribosome heterogeneity or functional specialization. However, previous studies have shown that most RP mutants share common phenotypes. Consequently, it is difficult to distinguish whether the phenotypes of the mutants have resulted from the loss of specific genes or a global ribosome deficiency. Here, to investigate the role of a specific  RP families contains between two and seven paralogs that share 65–100% protein sequence identity [RP family is expressed in mammals, and one or two genes from an RP family are expressed in yeast [RP family are essential for maintaining the threshold dosage of an RP and always have non-redundant functions [Ribosomes are the ribonucleoprotein complexes that are responsible for protein translation in cells. Like DNA replication and transcription, protein translation is also a basic component of the central dogma of molecular biology. In eukaryotes, the cytosolic ribosome comprises 60S large and 40S small subunits. The 60S large subunit is composed of 25/28S, 5.8S, and 5S rRNA and about 47 RPs (RPLs), while the 40S small subunit is composed of 18S rRNA and approximately 33 RPs (RPSs) [unctions ,10,11. Iunctions ,12.RPs in each family are also specialized by their variation in expression patterns and/or protein dosages. For example, the RPS5 family is encoded by RPS5A and RPS5B. Promoter-driven GUS experiments showed that RPS5A is enriched in meristem regions, while RPS5B is restricted to differentiating tissues [RPL16 family, RPL16A is expressed in the root stele and anthers, while RPL16B is enriched in proliferating tissues [RP mutants normally display similar developmental phenotypes or lack obvious morphological phenotypes [RP mutants [RP mutants hinders the research of specific RPs and ribosome heterogeneity.In Arabidopsis,  tissues . In the  tissues . These senotypes ,4,15. Fo mutants ,20,21,22RPs under the control of the CaMV 35S promoter. We found that increasing the RPL16D protein inhibited plant growth while protecting the plant from pathogen attack. This study provides a new insight into the function of specific RPs and the importance of ribosome heterogeneity in plants.In this study, we investigate the functions of specific RPs and their ribosomes by overexpressing specific RP genes in Arabidopsis, we created different RPs-GFP fusion sequences which were driven by the 35S promoter (35S:RPs-GFP) and introduced them into WT plants. The GFP tag was used to compare the expression levels of RP-GFP among different lines. In this study, Arabidopsis lines overexpressing RPL16D-GFP (L16D-OE) were further investigated, and two representative lines (OE5 and OE9) were used to identify the phenotype. No obvious phenotypic difference was observed between L16D-OEs and WT before 20 DAG (days after germination) . The 35S:RPL16D lines displayed similarly short and curled rosette leaves, ruling out the possibility that the chimeric protein of RPL16D-GFP played a dominant-negative role by competing with the native RPL16D (To study the function of specific ination) A. However 20 DAG A. This pOE9 line A. The pehenotype A. The fisurfaces B. The lese of WT C. These OE5 line C. The leOE5 line C. We alse RPL16D .L16D-OE5, and L16D-OE9 plants were analyzed by scanning electron micrography (SEM) on the adaxial (Ad) and the abaxial (Ab) surfaces  and protein sequences   . The RPL website . RPL16A  tissues . The exp tissues .RPL16D in different tissues and organs. In this experiment, GUS was driven by a 2001 bp promoter of RPL16D in the transgenic plants. GUS signals were clearly detected in active growth regions, such as cotyledon tips, shoot apices, the primary root tip, newly emerged lateral roots, expanded rosette leaves (13 DAG), and flower buds, which was largely consistent with the online data  and 3271 (2293 significantly upregulated genes and 1978 significantly downregulated genes) differentially expressed genes (DEGs) (|log2(fold-change)| ≥ 1 and q-value < 0.05) were identified in L16D-OE5 and L16D-OE9, respectively (L16D-OE5 and L16D-OE9 (2(fold-change)| ≥ 0.25 and p-value < 0.05) were identified in L16D-OE9 -related DEGs and DEPs was significantly increased, including ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), PATHOGENESIS-RELATED GENEs (PR1 and 5), EP1, and ARABIDOPSIS EDS1-DEPENDENT 1 (AED1) C. Furthel growth D.RPL16D overexpression inhibits the accumulation of plant growth-related proteins but induces the expression of many pathogen resistance-related genes, especially PR1 being upregulated by approximately 78-fold in the mRNA level and by 3-fold in the protein level in L16D-OE9  is significantly upregulated upon auxin treatment [RPL16D in proliferating tissues. The independent mutation of paralogous RP genes in yeast has been reported to cause distinguishable phenotypes, suggesting that paralogous genes have distinct functions despite the high similarity of protein sequences [RPL16 genes encode highly similar proteins, we cannot rule out the possibility that RPL16D has a specific function.RPL16 plays an essential role in peptidyl transferase activity in bacteria and yeast by associating with the small subunit, binding aminoacyl-tRNA (aa-tRNA), and organizing the architecture of a functional site in ribosomes ,28. In Areatment . Both ouequences ,29. TherRPL16D overexpression lines 16D-OE5 and 16D-OE9 shows that the transgenic plants display an inhibited-growth phenotype at the late vegetative growth stage (after 21 DAG) in an RPL16D protein dosage-dependent manner. It has been reported that the proper composition of membrane sterol is not only required for membrane integrity, permeability, and fluidity, but it also affects the signaling cascades involved in normal plant growth [L16D-OE5 and L16D-OE9 is likely responsible for the abnormal leaf development.The phenotypic characterization of the t growth ,31. Our t growth ,33. In at growth ,25,26. OEDS1 together with PAD4 was reported to cause an autoimmunity phenotype (short rosette leaves), resembling the phenotypes of L16D-OEs, whereas the overexpression of EDS1 or PAD4 alone did not cause this phenotype [L16D-OEs. Here, upregulated PR1 and PR5 in L16D-OEs might contribute to the enhanced pathogen resistance. Taken together, we found that the overexpression of L16D leads to inhibited growth and enhanced pathogen resistance.Unlike animals, sessile plants have evolved a complex strategy to adapt to different environmental conditions and pathogen attacks. To date, little research has focused on the role of RP and ribosome heterogeneity in plant pathogen resistance. Here, we found that many pathogen resistance genes and proteins were upregulated in our transcriptomic and proteomic results. The constitutive overexpression of henotype . EDS1-PAhenotype ,35,36. HL16D-OE5) display weak growth inhibition but still have enhanced pathogen resistance. Furthermore, a previous study reports that plants overexpressing PR1, PR2, and PR5 show drought-tolerance phenotypes [Transgenic plants with a moderately increased RPL16D dosage (such as enotypes . Therefo2O2 (10%) and plated on solid 1/2 Murashige and Skoog (MS) medium containing 1% sucrose (pH 5.7). After a 3 d stratification, plates were transferred to the growth room .Arabidopsis accession Columbia (Col-0) was used as the wild-type (WT) background in this study. Seeds were surface-sterilized using HRPL16D overexpression construct (with or without a GFP tag), the full-length coding sequences of RPL16D (At5g45775) were amplified with or without a stop codon using specific primers. The latter was fused with a C-terminal GFP-tag. RPL16D and RPL16D-GFP fusion sequences were cloned into pCAMBIA2300 driven by the cauliflower mosaic virus (CaMV) 35S promoter. The 2001 bp promoter of RPL16D was amplified and cloned into pBI121 to generate a pRPL16D:GUS construct. The above constructs were transformed into WT Arabidopsis using the Agrobacterium tumefaciens (GV3101)-mediated floral dip method to transform Arabidopsis plants. Transgenic plants were screened using kanamycin. All primers used in this study were listed in To generate an L16D-OEs. The protoplast isolation was previously described [GFP signals in five DAG cotyledons and leaf protoplasts were detected using confocal laser scanning microscope . Protoplasts were isolated from escribed . At leas4 buffer (pH 7.0), 5mM K3Fe(CN)6, 5mM K4 Fe(CN)6 and 2 mM X-glucuronide) overnight at 37 °C [v/v glycerol for photography.Seedlings and rosette leaves were stained for GUS activity by incubation in the GUS staining buffer (0.05 M NaPOat 37 °C . The mathttp://bowtie-bio.sourceforge.net/Bowtie2/index.shtml). The gene expression level was calculated with RSEM (http://deweylab.biostat.wisc.edu/RSEM 13 March 2023) and normalized using the fragments per kilobase of transcript per million mapped reads (FPKM) method. Differentially expressed genes (DEGs) were identified using DEGseq software version 1.0  and used the Volcano Plot to show the summary of DEGs.Total RNA was extracted from 20 DAG rosette leaves from three independent biological replicates using RNAiso (Takara). DNase I (Takara) was used to remove genomic DNA according to the manufacturer’s protocol as described previously . The RNAL16D-OE9 (20 DAG) from three independent biological replicates were finely ground in liquid nitrogen for protein extraction. Raw MS/MS data were analyzed using ProteoWizard Tool msConvert (version 3.0.1), and then peptides were identified using TAIR10_1015 . At least one unique peptide was necessary for an identified protein. Differential accumulation ratios of proteins were analyzed by the automated software IQuant (version 2.2.1). To calculate the differential accumulation ratios, all identified spectra from a protein were used to obtain an average protein ratio relative to the control label . We used p < 0.05 and the fold change >1.2-fold or <0.83-fold as the thresholds to judge the significantly differentially expressed proteins (DEPs).Rosette leaves of WT and p-value ≤ 0.001. The pictures were made as previously described [GO annotation was classified into biological process ontology using Blast2GO and the significantly enriched GO terms were identified using a hypergeometric test under the standard of the escribed ,41.RPL16A (AT2G42740), RPL16B (AT4G18730), RPL16C (AT3G58700), RPL16D (AT5G45775), EDS1 (AT3G48090), PAD4 (AT3G52430), and PR1 (AT2G14610).The accession numbers of the genes used in this article are as follows:


TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations “hypomorphs.” One of these hypomorphs is a SNP that exists in 6%–10% of Africans and 1%–2% of African Americans, which changes proline at amino acid 47 to serine . We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory  macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression.The tumor suppressor TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.Findings presented here show that the P47S variant of  TP53 gene is subject to mutational inactivation in the majority of tumors, rendering many of these cancers with poor prognosis and poor response to conventional cancer therapies  was reported close to 30 years ago that alters proline at amino acid 47 to serine; data from our group showed that this SNP impairs phosphorylation on serine 46, which is critical for p53-mediated apoptosis , were obtained from the ATCC and grown in McCoy's 5A medium (Gibco) supplemented with 10% FBS  and 1% penicillin/streptomycin (Corning Cellgro). Cells were grown in a 5% COTP53, between 6 and 10 weeks of age were used for all experiments. In most cases, the mice analyzed are littermate mice from P47/S47 heterozygous crosses; in all cases, P47 and S47 mice were no more than two generations removed from a backcross to each other. Mice were housed in plastic cages with ad libitum diet and maintained with a 12-hour dark/12-hour light cycle at 22°C. For tumor engraftment experiments, 1 × 106 MC38 cells were injected subcutaneously into the right flanks of 6–10 weeks old male mice. For CD8+ T-cell depletion experiments, mice were treated with 200 μg anti-CD8b antibody  via intraperitoneal injection on the following schedule: 1 week prior to MC38 injections, 2 days prior to MC38 injections, two times per week for the duration of the study. For PD-L1 blockade therapy experiments, mice were administered three doses of anti-CD274 antibody  or three doses of rat IgG2b isotype control antibody (BioLegend: 400672) by intraperitoneal injection on the following schedule: 7 days post-MC38 injection, 10 days post-MC38 injection, 13 days post-MC38 injection. Tumor volumes were measured using digital calipers and tumor volume was calculated using the formula: volume = (length × width2) × 0.52. All mice were monitored daily for signs of pain or distress. Tumor volumes were measured every other day until the study endpoint, at which point all mice were euthanized.All animal studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH. All protocols were approved by The Wistar Institute Institutional Animal Care and Use Committee. Hupki mice in a pure C57Bl/6 strain background [described previously ], homozy7 cells/15 cm dish. After 5 days of differentiation, BMDMs were harvested using ice-cold detachment buffer containing: 1X PBS, 2% FBS, and 2 mmol/L etheylenediaminetetraacetic acid (EDTA)  and used in polarization experiments. BMDMs were polarized to M1 phenotype by lipopolysaccharide (LPS) alone (100 ng/mL) or LPS + IFNγ  for 8 hours. BMDMs were polarized to M2 phenotype by IL4  for 8 hours.Bone marrow–derived macrophages (BMDM) were generated by culturing bone marrow cells from P47 and S47 mice with MCSF , as described previously . Bone maSpleens and tumors were harvested from P47 and S47 mice. Spleens were crushed through 70 μm cell strainers  using the plunger/piston end of the syringe, washed in 10 mL 1X PBS, resuspended in 10 mL of 1X red blood cell (RBC) Lysis Buffer , and incubated for 10 minutes to remove RBCs. RBC-depleted splenocytes were then washed with R10 medium . Excised tumors were washed in 10 mL 1X PBS and dissociated using the Tumor Dissociation Kit  according to the manufacturer's instructions. For staining surface markers, cell pellets were incubated with fluorochrome conjugated antibody cocktail for staining at 4°C for 30 minutes and were fixed using 1% paraformaldehyde (Sigma) for 20 minutes. All antibodies were used at 1:400 dilution. Samples were then washed and resuspended in 1x PBS and acquired on BD FACS Symphony flow cytometer. Data were analyzed using FlowJo v10 . Fluorochrome conjugated antibodies used for flow cytometry are listed in 7 cells/mL in R10 medium and 1 × 106 total splenocytes were plated per well in a 96-well flat-bottom plate (Corning). Splenocytes were then either left unstimulated or stimulated with 1X (phorbol 12-myristate 13-acetate) PMA/Ionomycin , 1X Concanavalin A , or anti-CD3/CD28 Dynabeads  at a 5:1 bead to cell ratio. To measure degranulation, anti-CD107a antibody  was added to each well at the time of stimulation at a 1:200 concentration. After 1 hour of stimulation, 1X Brefeldin A  and 1X Monensin  were added to each well and cells were incubated for an additional 4 hours. After 5 hours of total stimulation, cells were washed, stained for surface antigens, and then stained for intracellular antigens using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacturer's instructions .Splenocytes derived from naïve, non–tumor-bearing S47 or P47 mice were adjusted to a concentration of 1 × 102+ or Ca2+, 2% FBS, 2 mmol/L EDTA) and stained in V-bottom 96-well plates. Cells were incubated simultaneously with TruStain FcX PLUS  and dextramers for the Rpl18 (KILTFDRL) neoantigen conjugated to APC (Immudex). Cells were incubated with Fc block and dextramers for 20 minutes at room temperature, then washed twice with FACS buffer before surface and intranuclear staining.Tumor-derived cells and splenocytes from tumor-bearing mice (TBM) were first washed and resuspended in FACS Buffer  and transferred to a V-bottom 96-well plate. After dextramer or other surface staining was performed, cells were washed in FACS buffer and fixed using the True-Nuclear Transcription Factor Buffer Set  according to the manufacturer's instructions specific to staining in 96-well plates.Tumor-derived cells and splenocytes from TBM were first washed and resuspended in FACS Buffer . RNA purified from lysates was used to synthesize cDNA using iScript Reverse Transcription Supermix kit . RT-PCR reactions were carried out on Applied Biosystems Quant Studio 7 Flex Real-Time PCR system using 1X SYBR Green PCR Master Mix (Applied Biosystems). The relative mRNA expression was normalized to the expression of β-actin. Primer sequences are listed in Supernatants from BMDM culture polarized with M1 stimuli were assessed for cytokine production by ELISAs. Commercially available kits from Invitrogen were used to measure IL6 , IL12p40 , and IL10  according to the manufacturer's instructions.t test when sample sizes were larger . All mouse experiments had a minimum of 5 animals/experimental group. Linear mixed models were used to analyze longitudinal tumor growth measures. Visualization and statistical comparisons of inhibitory receptor (IR) coexpression on tumor-derived T cells was performed using SPICE software .All statistical analyses between two groups were performed using the Wilcoxon rank-sum test when group sizes were small (<6 datapoints) or the Student er e.g., , except software . WilcoxoThe data generated in this study are available upon request from the corresponding authors.+ T cells via administration of an anti-CD8b antibody. In the control groups, tumors again grew significantly larger in S47 hosts (+ T cells were depleted (+ T-cell depletion in the tissues was confirmed by flow cytometry (We previously showed that the P47S variant of p53 (hereafter denoted S47) shows an intrinsic defect in tumor suppressor function and is associated with increased cancer risk . In the 47 hosts ; howeverdepleted . Successytometry . These d+ T-cell depletion abolished the differences in tumor growth, and because we previously reported that S47 mice show increased populations of M2-like (anti-inflammatory) macrophages (+) CD4+ T cells in S47 mice (+ and CD62L+) CD4+ T cells in S47 mice  CD4+ and CD8+ T cells in S47 mice , we found a significantly reduced frequency of degranulating . To address this, we analyzed tumors and splenocytes from S47 and P47 TBM for immune cell distribution and activation phenotype by flow cytometry. We found no evidence for differences in the levels of populations of immune cells in the tumors of P47 and S47 mice, including myeloid-derived suppressor cells MDSC), tumor-associated macrophages, dendritic cells (DC), or CD4 T cells . However neoantigen of MC38 tumor cells, located in the ribosomal protein L18 (+) CD8+ T cells in the TME between S47 and P47 mice, but that, unexpectedly, S47 mice harbored significantly elevated frequencies of KL8-dex+ CD8+ T cells in the spleen  found in S47 TBM demonstrated alterations in transcription factor profiles or IRs, which would be indicative of attenuated functional capacity, when compared with P47 mice. There was no difference in the percentage of proliferating TILs as evidenced by Ki67 expression, indicating that S47-derived TILs are not anergic and respond to antigen . However S47 TBM . Tbet ph S47 TBM , and we  S47 TBM . Consist P47 TBM . We alsopartment . Collecthe tumor . In KL8-P47 mice , suggest+ and CD8+ T cells for expression of the IRs lymphocyte activating protein 3 (LAG3), programmed cell death protein 1 (PD1), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and T-cell immunoglobulin and mucin domain 3 (Tim3). We observed a significant increase in the frequency of LAG3+, PD1+, and TIGIT+ CD4+ TILs derived from S47 TBM, and a similar profile in CD8+ TILs with the exception of TIGIT which was globally lower on CD8+ TILs compared with CD4+ TILs , and fewer subpopulations that expressed a single IR (red colors) when compared with P47 mice (+/PD1+ population which was found in significantly higher frequencies in both CD4+ and CD8+ TILs from S47 mice (+ TILs from S47 mice were triple positive for LAG3, PD1, and TIGIT or LAG3, PD1, and Tim3 in CD4+ and CD8+ TILs, respectively, and these populations were significantly enriched compared with P47 TBM (The expression of IRs in TILs is another major determinant of T-cell exhaustion . TherefoD4+ TILs  and B. WP47 mice  and D. T3, mice were randomly separated into two groups: one group received an IgG2b isotype control antibody and the other group received anti-PD-L1 antibody. Consistent with our previous results, tumors grew larger over time in S47 hosts compared with P47 (With the alterations in the immune microenvironment of S47 mice in mind, we next evaluated the efficacy of immune checkpoint blockade between genotypes. Toward this goal, we again analyzed MC38 tumor growth, as this tumor is known to respond to anti-PD-L1 therapy . We injewith P47 . Both P4with P47 . Notablywith P47 . In contTP53 shows impaired “tumor-intrinsic” tumor suppressor function. This impaired tumor suppressor function has been attributed to reduced serine 46 phosphorylation (+ and CD8+ T cells, and an increase in polarized CD4+ Th cells in S47 mice. Furthermore, upon stimulation, both CD4+ and CD8+ T cells from S47 mice show lower levels of degranulation, cytotoxicity, and effector cytokine production. Our analysis of BMDMs also demonstrates an anti-inflammatory/immunosuppressive phenotype in S47 mice. In TBM, our analysis of immune cell populations in the spleen shows an increase in MDSCs, along with decreased activation of macrophages, DCs, and B cells. Future studies are needed to more comprehensively analyze the differences in immune populations using single-cell RNA sequencing or other corroborative approaches.Previous work from our lab and others has shown that the S47 variant of rylation , which irylation , increasrylation , decreasrylation , and decrylation . Herein,+ T cells either in the spleens or tumors of S47 mice, suggesting that there are no overt deficits in priming or trafficking of S47 T cells specific for MC38-derived neoantigens. We note that the mismatch between the murine p53 expressed by the transferred MC38 tumor cells and the humanized p53 gene expressed by S47 and P47 mice in the Hupki background could lead to a significant murine p53-specific T-cell response, which was not measured in this study. However, given that a role for CD8+ T cells in suppressing MC38 tumor growth has been reported in C57Bl6 mice with fully murine p53 where a p53 mismatch is absent (+ and CD8+ T-cell exhaustion in S47 mice. All or many of these differences are likely to contribute to the increased tumor growth in S47 hosts, but the contribution of each of these parameters to the altered rate of tumor growth and differing efficacy of immunotherapy in S47 mice is likely to be complex. There have been other studies that have analyzed the impact of p53 loss or mutation on immune cell responses. However, most of the data published to date focuses on the immunologic outcomes associated with p53 knockout or “gain of function” mutants of p53 as opposed to hypomorphs that marginally affect p53 function. When p53 is absent in T cells only, there is a significant increase in T-cell effector functions (With respect to tumor-specific T-cell responses, we did not observe reductions in the frequency of neoantigen-specific CD8s absent , and thaunctions , whereasunctions . This woTwo issues are unresolved in this study. The first is whether the defects in immune cell function evident in our S47 mice also occur in humans. Interestingly, and consistent with our findings, increased markers of T-cell exhaustion have been reported in African-descent individuals . However+ and CD8+ TILs from S47 mice (+ memory T cells (A second issue unresolved in this study is the mechanism whereby the S47 SNP contributes to an altered immune microenvironment in mice. WT p53 has also been shown to activate immune-regulating genes such as TAP1 and CD80 , and PD-S47 mice . mTOR haS47 mice , and it  T cells . Moreove T cells . OverallFigure S1Figure S1 shows BMDMs from S47 mice are more anti-inflammatory and polarize towards a M2 phenotype following IL-4 stimulation.Click here for additional data file.Figure S2Figure S2 shows that the tumor microenvironment in both P47 and S47 mice is comprised of similar proportions of subpopulations of immune cells with similar phenotypes.Click here for additional data file.Table S1 and S2Table S1 and Table S2Click here for additional data file.


STN1 knockdown (KD) sensitized HeLa cells to high doses of H2O2. While H2O2 induced DNA strand breaks throughout the cell cycle, STN1 KD cells were as resistant as control cells to H2O2 treatment when challenged in the G1 phase of the cell cycle, but they were sensitive when exposed to H2O2 in S/G2/M phase. STN1 KD cells showed a failure of DNA synthesis and RAD51 foci formation upon H2O2 treatment. Chemical inhibition of RAD51 in shSTN1 cells did not exacerbate the sensitivity to H2O2, implying that the CST complex and RAD51 act in the same pathway. Collectively, our results suggest that the CST complex is required for maintaining genomic stability in response to oxidative DNA damage, possibly through RAD51-dependent DNA repair/protection mechanisms.Genomic DNA is constantly exposed to a variety of genotoxic stresses, and it is crucial for organisms to be equipped with mechanisms for repairing the damaged genome. Previously, it was demonstrated that the mammalian CST (CTC1-STN1-TEN1) complex, which was originally identified as a single-stranded DNA-binding trimeric protein complex essential for telomere maintenance, is required for survival in response to hydroxyurea (HU), which induces DNA replication fork stalling. It is still unclear, however, how the CST complex is involved in the repair of diverse types of DNA damage induced by oxidizing agents such as H In each condition, the signal intensities of 800 randomly selected nuclei are shown. p-values for unpaired t-tests are shown above the respective bars.(A) Representative images of an indirect immunofluorescence assay for γ-H2AX in untreated, HU-, or H(PDF)Click here for additional data file.S4 Fig2O2-treated HeLa cells. The horizontal axis shows Alexa488 fluorescence (γ-H2AX level), and the vertical axis indicates cell counts for each intensity value. Blue-colored regions indicate γ-H2AX positive fractions. (B) Another data set for the experiment described in 2O2 treatment required for γ-H2AX signals to appear was 10 minutes. HeLa parental cells were treated with 500 μM H2O2 for indicated periods before fixation. Broken lines show nuclei. Scale bars, 20 μm.(A) Histograms of Alexa488 intensity in undamaged, HU- or H(PDF)Click here for additional data file.S5 Fign) are shown above each bar. (A) Classification of the neutral comet assay data. Class 1, tail length shorter than 80 μm; Class 2, tail length longer than 80 μm. Chi-square test (with one degree of freedom) rejected the null hypothesis that the class distributions are the same between KD control and shSTN1 cells (p = 2.4E-9). (B) Classification of the alkaline comet assay data. Class 1, tail length shorter than 155 μm; Class 2, tail length longer than 155 μm. Chi-square test (with one degree of freedom) rejected the null hypothesis that the class distributions are the same between KD control and shSTN1 cells (p < 2.2E-16).  Histograms of comet tail lengths of H2O2-treated HeLa cells. The horizontal axis shows comet tail length (μm) and the vertical axis indicates counts for each binned value. Blue-colored regions indicate “Class 2” fractions. The same data set shown in (PDF)Click here for additional data file.S6 Figt = 0 hr, the arrested cells were synchronously released into S phase by replacing the medium with drug-free fresh medium. To monitor fractions of S-phase cells in the released cell population, pulse-labeling by EdU (incubation with 10 μM EdU for 10 min before fixation) was carried out at t = 0, 1, 2 and 4 hr, followed by conjugation with Alexa Flour 488 by Click chemistry (Click-iT EdU Cell Proliferation Kit for Imaging). EdU incorporation into nuclei was assessed by fluorescence microscopy. (B) Jitter plots showing the signal intensity of EdU . For each condition, the signal intensities of 500 randomly selected nuclei are shown. A nucleus with fluorescence intensity > 3,500 a.u. was defined to be EdU-positive. The EdU-positive rate per condition is shown above each lane. EdU (-), asynchronous cells without EdU labeling; EdU (+), asynchronous cells with EdU labeling. At t = 0 hr, all the analyzed nuclei were EdU-negative, demonstrating highly efficient cell cycle arrest at G1/S. As time elapsed, the fraction of EdU-positive nuclei gradually increased, indicating cell cycle progression into S phase with a modest synchronization rate. (C) Flow cytometric analysis of DNA content (PI fluorescence) in asynchronous (white) and mimosine-treated (colored) samples. After the release, cells were collected at t = 0 (red), 1 (green), 2 (blue), and 4 (purple) hours, respectively. The horizontal axis shows PI intensity (DNA content), and the vertical axis indicates cell counts for each intensity value.(A) Experimental timeline. HeLa cells were treated with 500 μM mimosine for 23 hours to arrest them at the G1/S boundary . At t = (PDF)Click here for additional data file.S7 FigJitter plots showing the signal intensity of EdU . In each condition, the signal intensities of 700 randomly selected nuclei are shown. A nucleus with fluorescence intensity > 500 a.u. was defined to be EdU-positive. The EdU-positive rate per condition is shown above each lane. EdU (-), asynchronous cells without EdU labeling.(PDF)Click here for additional data file.S8 FigThe compiled data shown in (PDF)Click here for additional data file.S9 FigSTN1 knockdown in U2OS cells was evaluated by qRT-PCR. STN1 RNA levels were normalized to GAPDH RNA levels. X-axis, cell lines; Y-axis, relative level of STN1 RNA normalized to the KD control. The mean ± SEM is shown (n = 3). (B) Representative images of RAD51 foci in EdU-positive nuclei of U2OS cells. The enlarged area shown in the rightmost column is indicated by white squares. Scale bar: 10 μm. (C) Box-and-whisker plots showing the number of nuclear RAD51 foci per nucleus (Y-axis). At least 40 nuclei were randomly selected in each experimental condition. Numbers of analyzed nuclei (n) are shown at the top of each lane. p-values for Mann–Whitney U tests are shown above the indicated lanes.The same set of experiments as in (PDF)Click here for additional data file.S10 Figp-values for Mann–Whitney U tests are shown above the indicated lanes. (D) Distributions of the number of RAD51 foci per nucleus in U2OS cells. p-values for Mann–Whitney U tests are shown above the indicated lanes.(A) The experimental timeline. HeLa cells were treated with RAD51 inhibitor B02 starting one hour before the beginning of Etposide treatment . (B) Representative images of RAD51 foci and γ-H2AX signals in HeLa cell nuclei. The enlarged area shown in the rightmost column is indicated by white squares in the RAD51 column. RAD51 foci formation induced by etoposide treatment was suppressed by B02 treatment. Scale bars, 10 μm. (C) Distributions of the number of RAD51 foci per nucleus in HeLa cells. (PDF)Click here for additional data file.S11 FigOxidative stress by hydrogen peroxide can cause a variety of DNA damages including single-strand breaks, double-strand breaks, and base modifications. In S phase, such DNA lesions can cause replication fork stalling and/or generate one-ended DSBs through fork collisions with SSB sites. RAD51 is crucial in both restarting stalled forks and initiating HR-dependent repair of the collapsed forks. The CST complex could contribute to efficient RAD51 loading onto the damaged sites by protecting and stabilizing the exposed ssDNA (which is a substrate for RAD51 filament formation) and/or by directly recruiting RAD51. See (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Over the last decades, epidemiology and functional studies have started to reveal a pivotal role of vitamin D in both type 1 and type 2 diabetes pathogenesis. Acting through the vitamin D receptor (VDR), vitamin D regulates insulin secretion in pancreatic islets and insulin sensitivity in multiple peripheral metabolic organs. In vitro studies and both T1D and T2D animal models showed that vitamin D can improve glucose homeostasis by enhancing insulin secretion, reducing inflammation, reducing autoimmunity, preserving beta cell mass, and sensitizing insulin action. Conversely, vitamin D deficiency has been shown relevant in increasing T1D and T2D incidence. While clinical trials testing the hypothesis that vitamin D improves glycemia in T2D have shown conflicting results, subgroup and meta-analyses support the idea that raising serum vitamin D levels may reduce the progression from prediabetes to T2D. In this review, we summarize current knowledge on the molecular mechanisms of vitamin D in insulin secretion, insulin sensitivity, and immunity, as well as the observational and interventional human studies investigating the use of vitamin D as a treatment for diabetes. A small quantity of vitamin D, including vitamin D2 and vitamin D3, can be acquired from dietary sources. The majority of circulating vitamin D, in the form of vitamin D3, is formed in the skin from 7-dehydrocholesterol (7-DHC) in the presence of sunlight [2D) in the kidney (2D) is the metabolically active form of vitamin D [Vitamin D is a group of fat-soluble secosteroids. This term generally refers to vitamin Dsunlight . Throughe kidney  2]. Whi. Whi2 , a ligand-dependent nuclear receptor that functions as a transcription factor by generating a heterodimer with the retinoid X receptor (RXR) upon ligand binding [2D may bind to membrane-anchored receptors to regulate the activity of signaling molecules or the production of second messengers [Vitamin D exerts its effects via both genomic and nongenomic actions. For the genomic pathway, 1,25(OH) binding . Additiossengers .In addition to the canonical functions in regulating calcium absorption, bone growth and remodeling , vitaminAlready a prevalent endocrine disease, the incidence of T2D is expected to escalate rapidly in the coming decades. T2D is characterized by diminished insulin secretion from pancreatic islets and insulin resistance in peripheral organs. Insulin secretion defects and insulin resistance, triggered by chronic and excess nutritional intake, cause glucose intolerance and hyperglycemia. Both β cell mass and glucose-stimulated insulin secretion (GSIS) are reduced even in the early prediabetic stage . The detVdr) and 1α-hydroxylase (Cyp27b1), which catalyzes the activation of 25(OH)D into 1,25(OH)2D, consistent with the cell-intrinsic role for VDR [INS locus is still lacking. Several in vivo and ex vivo studies in rats have indicated that vitamin D deficiency in vivo resulted in reduced serum insulin levels and impaired islet insulin secretion in isolated islets [Ins2 expression are significantly decreased in VDR-mutant mice [β cells express both the vitamin D receptor transcript ecretion , and celecretion . On the ecretion  by promoecretion . 1,25  toxicity. Vitamin D has long been identified as an anti-inflammatory hormone in the immune response. Vitamin D or over-expression of VDR has also been shown to repress cytokine-induced proinflammatory responses and apoptosis in β cell lines and islets ,40,41. TAlthough the pleiotropic protective role of vitamin D in islets is clear, vitamin D supplementation showed mixed results in glucose metabolism ,44,45,46It is noteworthy that the contributions of vitamin D in regulating islet function may also come from non-β endocrine and non-endocrine cells in islets. Islet macrophages express VDR, which suggests that vitamin D may function in islet-resident immune cells . Interes2D activates VDR to increase insulin receptor expression [2D activated target in several cancer cell lines [2D [2D ameliorated insulin resistance in C2C12 myotube cells triggered by free fatty acid [2D enhanced glucose uptake via the SIRT1/IRS1/GLUT4 axis by activating SIRT1, phosphorylating IRS1, and ultimately translocating GLUT4 in myotubes [2+ concentrations in muscle, enhances the translocation of GLUT4, and increases glucose uptake [Insulin resistance, defined as an impaired ability of insulin to induce glucose uptake in peripheral tissues resulting in hyperglycemia, is a hallmark of prediabetes and T2D. Vitamin D has been suggested to regulate insulin sensitivity in cell lines and peripheral metabolic organs . Severalpression ,50,51, wll lines , while sines [2D ,54. Moretty acid . Manna emyotubes . Moreovee uptake . Togethee uptake . A diffee uptake . In conte uptake . Some eve uptake . In obese uptake ,62 and ce uptake  releasede uptake , to conse uptake . The ante uptake ,67, whice uptake  and IKBae uptake ,69. Moree uptake .A number of studies have shown that vitamin D is involved in lipid metabolism by regulating adipogenesis, lipolysis, and lipogenesis. The exact function of vitamin D in these processes is likely to be context-dependent. For in vitro studies using mesenchymal cells (MSCs) from adipose tissue or bone marrow, vitamin D promotes differentiation of the adipocyte progenitors, likely through upregulating lineage factors, such as PPARγ and AP2 ,72,73,74Several animal models have been used to interrogate vitamin D’s role in adipose tissue function and lipid homeostasis. Mice fed with vitamin D3-containing diet for 3 weeks showed an increase in subcutaneous and visceral fat , while m2D supplement that can activate the Ca2+/CaMKKβ/AMPK pathway to ameliorate insulin resistance and ER stress [In addition to the mechanisms discussed above, the pleiotropic role of vitamin D/VDR in insulin resistance may involve (1) vitamin D-induced increases in parathyroid hormone (PTH), which reduces insulin resistance by increasing the quantity of GLUT1 and GLUT4 in vitamin D-deficient adipose tissue, liver, and muscle ,86; (2) R stress ; and (4)R stress .Several clinical studies also support a protective role of vitamin D in insulin resistance. Chiu et al. performed univariate regression analyses on 126 glucose-tolerant subjects and concluded that patients with hypovitaminosis D have a higher risk of developing insulin resistance . Low plaVitamin D deficiency is defined as a 25(OH)D level of less than 20 ng/mL, according to established consensus . VitaminThe overall link between vitamin D serum levels and metabolic health has been observed in multiple studies. A cross-sectional study including 10,229 subjects showed a negative association between serum 25(OH)D and BMI during winter months . In a co3 did not appreciably decrease the risk of diabetes among people with a high risk of T2D [Based on epidemiological results, it has been postulated that supplementing vitamin D may ameliorate insulin resistance and enhance glycemic control. A single-center, double-blind, randomized placebo-controlled trial performed on 96 non-diabetic participants suggested a significant beneficial effect of vitamin D3 supplement on peripheral insulin sensitivity compared with placebo after six months . A similk of T2D . Moreovek of T2D . Howeverk of T2D . Hence, + and CD8+ T cells induce β cell damage that consequently potentiates the immune response by releasing more self-antigens [Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic β cells, leading to insulin deficiency. The development of T1D is a gradual process of breaking tolerance to autoantigens. β cell-specific autoantigens  are presented by antigen-presenting cells (APCs), triggering cytotoxic T cell responses, which cause β cell damage ,118. Sevantigens ,123,124.antigens ,126,127.2D3 protected the diabetic mice from developing hyperglycemia [2D3 on NOD mice reduced the incidence of insulitis and hyperglycemia [2D3 treatment in subjects with new-onset T1D [Animal models and epidemiological studies strongly support the ability of vitamin D to prevent T1D pathogenesis. In CD-1 mice with diabetes induced by daily intraperitoneal injections of low doses of streptozotocin (STZ), intraperitoneal administration of 1α,25(OH) glycemia . Long-teglycemia ,130,131.glycemia . Similarglycemia , which iglycemia . Zipitisglycemia . A similglycemia . Althougglycemia  suggestenset T1D ,139. The2D3 reduces MHC II and co-stimulatory molecules  expression and prevents T cell activation [2D3 inhibited dendritic cell maturation and stimulatory functions. 1,25(OH)2D3 treatment inhibits the expression of CD1a+ (dendritic cell marker), MHC II, and co-stimulatory genes while maintaining the expression of monocytic markers [2D3 has also been demonstrated to induce dendritic cell apoptosis [2D3 in dendritic cells is to induce the expression of the mannose receptor, the endocytic capacity-related molecule involved in antigen-capturing [2D3. Th1 and Th17 cells are essential in T1D initiation [2D3 inhibits the expression of multiple cytokines, such as IL-12 and IL-23, and consequently drives a T cell subpopulation shift from Th1/Th17 to Th2 [2D3 is able to suppress T cell infiltration by reducing gene expression and/or secretion of multiple cytokines  and chemokines  that manipulate T cell migration [2D3 suppresses autoreactive T cells and maintains tolerance [2D3 also inhibits B cell proliferation, differentiation in memory B cells, and production of immunoglobulins [The beneficial effects of vitamin D in T1D could be rooted in its versatile functions in various immune populations. VDR is expressed in nearly all immune cells, including activated T and B cells, dendritic cells, macrophages, and neutrophils ,142,143.tivation ,150. In  markers ,153,154.poptosis ,155, or poptosis ,157. Tolpoptosis . Anotherapturing . Lymphocitiation ,160. 1,27 to Th2 ,163,164.igration ,165,166.olerance  by promoolerance  and suppolerance . In addilobulins . WhetherIn addition to its canonical role in skeletal function, vitamin D modulates insulin secretion and action in diabetes. Vitamin D/VDR directly regulates functional genes, including critical genes in the secretion pathway and insulin action. As an anti-inflammatory hormone, vitamin D also acts on tissue resident immune cells to reduce local and systemic inflammation, thus preventing islet, liver, and muscle dysfunction. Though vitamin D is known to work on multiple organs and cell types, the relative contribution of individual cell types to the anti-diabetic effects remain to be determined. Mechanistically, how does vitamin D activate essential functional genes while repressing inflammatory targets? The cell type-specific regulatory circuitry of vitamin D-VDR remains to be elucidated. Vitamin D deficiency is prevalent in the general population and is linked to a higher type 2 diabetes incidence. Normalizing the vitamin D levels in deficient patients has slowed T2D progression. However, large-scale clinical trials have not demonstrated the clinical benefit of vitamin D supplements in ameliorating type 2 diabetes . These r


Megakaryocytes (MKs) develop from hematopoietic stem cells after stimulation by the cytokine thrombopoietin. During megakaryopoiesis, MKs enlarge, undergo the process of endomitosis, and develop intracellular membranes . During DMS formation, there is active transport of proteins, lipids, and membranes from the Golgi apparatus to the DMS. The most important phosphoinositide that controls anterograde transport from the Golgi apparatus to the plasma membrane (PM) is phosphatidylinositol-4-monophosphate (PI4P), whose levels are controlled by suppressor of actin mutations 1–like protein (Sac1) phosphatase at the Golgi and endoplasmic reticulum.Here we investigated the role of Sac1 and PI4P in megakaryopoiesis.We analyzed Sac1 and PI4P localization in primary MKs derived from fetal liver or bone marrow and in the DAMI cell line by immunofluorescence. The intracellular and PM pools of PI4P in primary MKs were modulated by expression of Sac1 constructs from retroviral vector and inhibition of PI4 kinase IIIα, respectively.We showed that in primary mouse MKs, PI4P is mostly found in the Golgi apparatus and the PM in immature MKs, while in mature MKs, it is found in the cell periphery and at the PM. The exogenous expression of wild-type but not C389S mutant  Sac1 results in the perinuclear retention of the Golgi apparatus resembling immature MKs, with decreased ability to form proplatelets. The pharmacologic inhibition of PI4P production specifically at the PM also resulted in a significant decrease in MKs that form proplatelets.These results indicate that both intracellular and PM pools of PI4P mediate MK maturation and proplatelet formation. •Phosphatidylinositol-4-monophosphate (PI4P) controls anterograde transport from the Golgi.•We studied PI4P in relation to Golgi apparatus in primary mouse megakaryocytes (MKs).•In immature MKs, PI4P is enriched at the Golgi, but in mature MKs, it is enriched at the plasma membrane.•Disrupting PI4P localization or its levels leads to smaller MKs that produce fewer proplatelets. During Suppressor of actin mutations 1–like protein (Sac1) is a SAC1 domain-containing phosphatase  that maiHere we show that in the early stages of primary mouse MK maturation, PI4P is enriched at the Golgi apparatus, while in the later stages, it is found to be enriched at the PM. The exogenous expression of wild-type Sac1 results in smaller MKs that produce fewer proplatelets. Since the inhibition of PI4P production at the PM by PI4KIIIα also results in a decrease in proplatelet formation, we suggest that both the intracellular and PM pool of PI4P mediate MK maturation and proplatelet formation.22.1Primary antibodies were obtained from the following resources: polyclonal rabbit anti-Sac1 (ABIN949071) was obtained from antibodies-online, polyclonal rabbit anti-Sac1 (HPA069869) was obtained from Atlas Antibodies, monoclonal mouse anti-GM130 (610823) was obtained from BD Transduction Laboratories, monoclonal mouse anti-PI4P IgM (Z-P004) was obtained from Echelon Biosciences, monoclonal mouse anti-KDEL (sc-58774) was obtained from Santa Cruz Biotechnology, polyclonal rabbit anti-TGN46 (ab16059) was obtained from Abcam, monoclonal rat anti-GPIbβ (M050-1) was obtained from Emfret Analytics, rabbit anti-β1 tubulin was a kind gift by Dr Joe Italiano , phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was obtained from Invitrogen, polyclonal rabbit anti-PI4KIIIα (4902S) and polyclonal rabbit anti-GFP (2555S) were obtained from Cell Signaling, monoclonal mouse anti-IgM  was a kind gift from Dr Jelena Ban , and monoclonal mouse anti-GAPDH (MAB374), polyclonal rabbit anti-IgG (12-370), and polyclonal mouse anti-IgG (12-371) were obtained from EMD Millipore. Secondary antibodies were obtained from the following sources: goat anti-mouse conjugated with Alexa Fluor (AF)-555 (A21429), goat anti-rabbit conjugated with Alexa Fluor (AF)-555 (A21429), and goat anti-mouse conjugated antibodies with Alexa Fluor (AF)-488 (A11029) from Life Technologies; goat anti-mouse conjugated with Alexa Fluor (AF)-568 (A21043), goat anti-rabbit conjugated with Alexa Fluor (AF)-488 (A11070), and goat anti-rat conjugated antibodies with Alexa Fluor (AF)-650 (SA5-10021) from Invitrogen; and goat anti-rabbit IgG HRP-linked (7074S) and goat anti-mouse IgG HRP-linked (7076S) antibodies from Cell Signaling.2.2DAMI cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Lonza). On day 0, DAMI cells were differentiated with 100 nM phorbol 12-myristate 13-acetate  and cultivated for 4, 8, 24, and 48 hours in full medium or for 4, 8, and 48 hours in medium lacking 10% FBS. For transfection, DAMI cells were seeded and differentiated with 100 nM PMA and after 24 hours transfected with pEGFP-Sac1, pEGFP-C389S-Sac1 , or pmCherry-PI4P-SidM  with Lipofectamine 3000 transfection reagent (Thermo Fischer). Twenty-four hours after transfection, the cells were fixed and stained for proteins of interest. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (Pan Biotech) supplemented with 10% FBS (Pan Biotech) and 1% penicillin/streptomycin (Lonza). Cells were seeded in full medium for 24 hours before transfection with the EGFP-Murine Stem Cell Virus (MSCV) constructs using Lipofectamine 3000 transfection reagent or before lysis for western blotting.2.3C57BL/6J mice were used for all experiments. Mice were handled following the European Communities Council Directive of November 24, 1986 (86/609/EEC), and with institutional and national guidelines. All experimental procedures were approved by the Ethics Committees of the Department of Biotechnology and Medical School of the University of Rijeka  as well as the Ministry of Agriculture of the Republic of Croatia (UP/I-322-01/15-01/123).2.4Mouse BM from 8- to 12-week-old mice and fetal liver (FL) from 13.5 days postcoitum old embryos were used. Cell aggregates from the BM were dissociated by mechanical aspiration 10 times through a 21G × 1” needle, 10 times through a 23G × 1” needle, and 2 times through a 25G × 5/8” needle. Dissociated cells from the BM and FL were filtered through a 70-μm cell strainer  and centrifuged for 5 minutes at 1000 rpm. The cells were then resuspended and cultured in Dulbecco’s modified Eagle’s medium (Pan Biotech) supplemented with 10% FBS (Pan Biotech) and 1% penicillin/streptomycin (Lonza). For MK production, thrombopoietin (2% of the culture-producing cytokine) was added. Cells were cultivated for different periods, as indicated in the figures and figure legends. MKs were enriched through bovine serum albumin  gradient on days 2 and 4 (FL–derived MKs) or days 3 and 5 (BM-derived MKs) for further analysis.2.5On day 4 after isolation of FL–derived MKs, a BSA gradient was performed, and 18 hours later, the number of MKs forming proplatelets was counted and shown as the percentage of MKs that form proplatelets over all MKs. The counting of MKs and proplatelets was performed using an epifluorescent microscope (Olympus) or a bright-field microscope (Zeiss) at 20× magnification. For counting the number of MKs that form proplatelets after the inhibition of PI4KIIIα, 100 nM GSK-A1  was used.2.6g. Platelet rich plasma was diluted 1:5 in washing buffer  and centrifuged for 6 minutes at 100 g. Platelet rich plasma supernatant was taken and centrifuged for 5 minutes at 1200 g. The pellet was resuspended in 1 mL of washing buffer and again centrifuged for 5 minutes at 1200 g. The pellet was resuspended in 200 μL of resuspension buffer . PLTs were then left to rest for 30 minutes at 37 °C before lysing for western blot assay.Mouse PLTs were isolated from mouse peripheral blood. Peripheral blood was drawn in 1/10 of the Aster-Jandl anticoagulant . Blood was centrifuged for 8 minutes at 100 2.7g for 30 minutes and incubated at 37 °C for 3 hours. After replacement with fresh media, MKs were cultured for an additional 2 days. On day 3, FL–derived MKs were separated via BSA gradient and analyzed for the ability to form proplatelets on day 4. The percentage of MKs that produce proplatelets was calculated from the number of EGFP+ MKs producing proplatelets over the number of all EGFP+ MKs.Human Sac1 (shares a 95.06% homology with the mouse protein) from pEGFP-Sac1 (wild-type [WT]) and C389S-Sac1 (mutated) from pEGFP-C389S-Sac1 were amplified by polymerase chain reaction with primers Fwd 5′ TAGGCGCCGGAATTAGATCACCGGTATGGTGAGCAAGGGCGAG 3′ and Rev 5′ ATCGGCCGCCAGTGTGCTGGAATTCAGTCTATCTTTTCTTTCTGGACC 3′. Both constructs were subcloned into a retroviral MSCV vector expressing EGFP into AgeI and EcoRI sites. For cloning of both constructs, NEBuilder HiFi DNA Assembly DNA Master Mix  was used. To produce retroviruses, HEK293T cells were seeded for 24 hours and then cotransfected with MSCV constructs  and pCL-Eco packaging plasmid using the transfection reagent Lipofectamine 3000. After 24 hours, the medium was changed, and after 72 hours, the viral supernatants were collected. Viral supernatants were filtered through a 0.45-μm filter and stored at −80 °C. On day 2 of the FL culture, the cells were transduced by adding retroviral supernatants in the presence of 8 mg/mL of polybrene . The samples were centrifuged at 800 2.8DAMI cells were grown directly on coverslips, while MKs were spun down on glass coverslips using the Hettich cytospin centrifuge Rotofix 32A. For Sac1 with Golgi or ER markers, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 minutes at RT, rinsed 3 times with PBS, and permeabilized and blocked with 1% goat serum/0.2% BSA/0.05% saponin  for 45 minutes. The cells were incubated with primary antibodies (diluted 1:100) overnight at 4 °C or for 1 hour at RT depending on the manufacturer’s instructions. The cells were rinsed 3 times with PBS and incubated with secondary antibodies (diluted 1:500) for 1 hour at RT. The cells were rinsed again 3 times with PBS, stained with 4’,6-diamidino-2-phenylindole  in PBS for 1 minute, and rinsed and mounted.4Cl, and permeabilized with 0.01% digitonin in buffer A  for 5 minutes. Cells were then rinsed with buffer A, blocked (5% goat serum in PBS with 50 mM NH4Cl) for 45 minutes, and labeled with primary antibodies (diluted 1:100). The cells were then washed with buffer A, stained with secondary antibodies (diluted 1:500), postfixed for 5 minutes in 2% PFA, and mounted.Immunostaining of intracellular pools of PI4P, as well as Golgi markers through MK development, was performed as previously described . Briefly4Cl. All subsequent steps were performed on ice with all prechilled solutions. Cells were blocked and permeabilized with 0.5% saponin (in buffer A containing 5% goat serum and 50 mM of NH4Cl) for 5 minutes and blocked with buffer A containing 5% goat serum and 50 mM of NH4Cl for 45 minutes. Then, the cells were labeled with primary antibodies diluted 1:100 (anti-PI4P) or 1:200 (anti-GPIbβ), washed with buffer A, stained with secondary antibodies (1:500), postfixed for 10 minutes in 2% PFA on ice and an additional 5 minutes at room temperature, and then mounted.The staining of the PM pool of PI4P was performed with the modified protocol as described previously . BrieflyFor the staining of proplatelets, cells were fixed with 4% PFA in PBS for 15 minutes, rinsed with PBS, and permeabilized with 0.01% Triton X-100 in PBS for 10 minutes. Cells were then blocked (5% goat serum in PBS) for 45 minutes, labeled with primary antibodies, and diluted 1:100 or with phalloidin 488 and anti-GPIbβ (1:200). The cells were then washed with PBS, stained with secondary antibodies (1:500), and mounted.2.9Epifluorescent images were obtained using an IX83 Olympus microscope equipped with a Hamamatsu Orca R2 CCD camera. The objective used was a PLAPON 60×/1.42 Oil (Olympus). The Pearson correlation coefficient and the area of the cell were measured in FIJI ImageJ  softwareConfocal images were obtained using an LSM880 confocal microscope (Carl Zeiss) equipped with an Argon-laser multiline (458/488/514 nm) and HeNe laser (543 nm and 633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss). For ultrastructure visualization, the airy scan mode was used. Total fluorescence intensities (TFI) and the area of cells were measured using ZEN black software (Carl Zeiss), quantified on maximum z-projections, and expressed over the area (size) of cells. For fluorescence intensity measurements, imaging conditions between different samples were kept constant. Fluorescence intensity was always expressed over the area (size) of cells. Colocalization studies were performed using ZEN black software (Carl Zeiss).2.10Cells were lysed with 3× SDS STOP/β-mercaptoethanol . Lysates were denatured for 5 minutes at 95 °C and proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide gel at 100 V for 1 hour in the Bio-Rad system for vertical electrophoresis. For protein size tracking, PageRuler Plus Prestained Protein Ladder was used . Proteins from the gel were electrotransferred onto the nitrocellulose membrane (Santa Cruz Biotechnology) in the Bio-Rad system at 0.45 A for 90 minutes. Membranes were blocked for 45 minutes in BSA/Tris-buffered saline with Tween  and incubated with primary antibodies overnight at 4°C. Membranes were then washed 3 times for 10 minutes with Tris-buffered saline with Tween, and secondary antibodies were added. Protein bands were visualized with ECL Prime Western Blotting detection reagents , and the pictures were taken with a Bio-Rad ChemiDoc MP Imaging System.2.11t-test or anova using Prism software (GraphPad). Differences were considered significant when P values were < .05 .All experiments were performed at least in triplicate. Data are represented as mean ± SEM. Data were analyzed by 33.1First, we investigated the expression and localization of Sac1 in primary MKs. We cultivated MKs from mice BM for 3 or 5 days to produce immature or mature MKs, respectively (described in detail in reference ). Briefl3.2In other cells, PI4P is mostly localized on the Golgi apparatus but it can also be found at the PM . In immaThe change in PI4P localization could be due to the changes in the Golgi morphology itself. As shown in 3.3To determine the role of the intracellular pool of PI4P that is controlled by Sac1, we expressed WT and catalytically inactive C389S mutant Sac1 from retroviral vector in FL–derived MKs and assessed MK’s size, PI4P levels, and the ability of MKs to form proplatelets. The successful cloning of the Sac1 constructs in the retroviral vector was proved by transfecting HEK293T cells with the EGFP-MSCV, EGFP-Sac1-MSCV, and EGFP-Sac1-C389S-MSCV. The cells were then lysed and blotted with the anti-EGFP antibody . The EGFTo test the function of the cloned constructs before transducing primary MKs, we transfected DAMI cells. PI4P is mostly localized at the Golgi in untransfected DAMI cells , but itsPrimary MKs were readily infected with the control EGFP as well as WT or mutant Sac1 constructs, with no significant difference in expression shown by western blot where infected cells were blotted for Sac1, and both endogenous and exogenous Sac1 can be detected . The traInterestingly, there was no significant difference in PI4P fluorescence intensity when WT or mutant Sac1 was expressed in MKs A, B. How3.4We performed a proplatelet formation assay to determine the ability of Sac1-expressing MKs to produce proplatelets. MKs expressing WT Sac1 produced significantly fewer proplatelets than MKs expressing only EGFP or mutant Sac1 with no apparent change in proplatelet morphology A, B. TheSince PI4P localizes more on the PM with MK maturation, we wanted to see if perturbation of the PM pool of PI4P would change the production of proplatelets. PI4KIIIα was shown to be the major source of PI4P at the PM in other cells  and it i42], but it can also contribute to the polyanionic lipid pool that defines the inner leaflet of the PM [Here we show that PI4P changes location during MK maturation. In young MKs, PI4P is mostly confined to the Golgi apparatus, while in mature MKs, it is mostly present at the PM. Both the intracellular and the PM pool of PI4P mediate MK maturation and proplatelet formation since the expression of WT-Sac1 and the inhibition of PI4KIIIα significantly reduce the number of MK-forming proplatelets. Although primarily found on the Golgi apparatus, PI4P also resides on the PM and ER in other cells , as wellf the PM . A nonvef the PM .In mature MKs in our experiments, it was not expected for PI4P to maintain its perinuclear localization since the Golgi apparatus itself undergoes morphology changes during megakaryopoiesis. In line with our observations, it has been previously shown that immature MKs have a condensed, perinuclear Golgi, while in mature MKs, the Golgi apparatus is widespread throughout the cell . The GolThe changes in Golgi morphology have been investigated in mice ,18, ratsAlthough we could not detect differences in overall PI4P levels in primary MKs or DAMI cells, the expression of WT Sac1 in DAMI cells dispersed PI4P, while in primary MKs, it caused accumulation of PI4P, indicating that diverse types of cells could differently regulate PI4P levels. For measuring PI4P levels, we used an established immunocytochemistry method  that wasThe lack of difference in PI4P fluorescence intensity levels accompanied by a change in localization of PI4P in WT-Sac1 expressing DAMI or primary MKs might be due to ectopic PI4P formation due to low intracellular PI4P levels. It was shown in HeLa cells that the inhibition of PI4KIIIα, a kinase that produces PI4P at the PM, leads to a decrease in PI4P levels at the PM with a simultaneous increase in intracellular PI4P . PI4KIIISeveral PI4P effector proteins have been shown to directly bind to PI4P in mammalian cells . For somIn conclusion, we demonstrate that both the intracellular pool of PI4P that is controlled by the Sac1 phosphatase and the PM pool of PI4P that is controlled by the PI4KIIIα kinase are necessary for MK maturation and proplatelet formation. These results suggest that tight control of the levels and localization of PI4P is important for megakaryopoiesis.


Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages  with massive viral replication, pro-inflammatory cytokines , and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection. The low-pathogenicity avian influenza virus (LPAIV), H9N2, and highly pathogenic avian influenza virus (HPAIV), H5N1 are the main epidemic subtypes, resulting in great economic losses on poultry and potentially threat to human. Thus, in-depth exploration of AIV pathogenesis in chickens is necessary for developing efficient control methods. In our study, using a single-cell RNA sequencing analysis of 16 cell types in the chicken lung, we revealed complicated and distinct immune response landscapes after H5N1 and H9N2 AIV infection, and identified key factors contributing to H5N1 AIV driven inflammatory lung injury. Therefore, our study potentially provides new targets and direction for AIV control. The low-pathogenic avian influenza virus (LPAIV), H9N2, and highly pathogenic avian influenza virus (HPAIV), H5N1 are the main epidemic subtypes in the ongoing virus circulation among Chinese poultry causing major economic losses in spite of the long-term vaccination programs . NotablyAIV infections of the chicken mainly occur via the respiratory route, and the lung is the important target organ. In lung tissue, AIV infection induces both antiviral and inflammatory factors which play crucial roles in host protection and immunopathogenesis ,9. InfecSingle-cell RNA sequencing (scRNA-seq) has been widely applied to identify the involvement of different cell types and to investigate the immune responses under conditions of virus infection –15. It iIn order to explore more comprehensive and refined immune cell responses to H5N1 and H9N2 AIV infection, we sorted the major immune cell types, including MHC Class II antigen presenting cells and CD3 positive T cells from chicken lung mononuclear cells for performing scRNA-seq analysis. In this study, we analyzed 16,642 immune cells in the lung tissue isolated from chickens after AIV infection with different pathogenic strains at various time points. Our results revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection. Our study has developed a valuable catalog of marker genes for identifying 16 cell types in the lung tissue of chicken via scRNA-seq. We also provided (for the first time) the key immune cell types and pro-inflammatory factors that contribute to H5N1 AIV-driven inflammatory injury in chicken lung tissue.All animal experiments were carried out in ABSL-3 facilities in compliance with approved protocol (CNAS BL0011) by the biosafety committee of South China Agriculture University . All animal procedures were performed according to the regulations and guidelines established by this committee and international standards for animal welfare.7 EID50 in 0.2 mL). The H5N1-infected group was intranasally inoculated with DK383 strain (106 EID50 in 0.2 mL). The control group was inoculated with 0.2 mL of phosphate buffered saline (PBS). Three chickens were dissected at 1-day post-inoculation (DPI) in the H5N1-infected group, 3 DPI in the H9N2-infected group, and 0 DPI in the control group, respectively. The virus titer in selected organs and virus shedding were detected as previously described [Low pathogenic avian influenza H9N2 subtype HN strain (A/Chicken/Hunan/HN/2015), and high pathogenic avian influenza H5N1 subtype DK383 strain (A/Duck/Guangdong/383/2008) were isolated and identified by our research team ,22. NineInfected or mock-infected chickens were humanely sacrificed. Their lungs were then perfused with RPMI-1640 containing 2% fetal bovine serum (FBS)  via the right ventricle. Half of the entire pool of the lung was fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Tissue slides were deparaffinized with xylene, hydrated in a graded series of alcohol solutions to distilled water, and blocked for endogenous peroxidase in 3% hydrogen peroxide. Sections were then stained with mouse anti-Influenza A virus NP primary antibody , followed by staining with anti-mouse-HRP secondary antibody  and visualizing via optical microscopy . Then, 0.1g from the other half of the lung tissue was kept for virus titer detection, and the remaining half of the lung pool was dissected and dissociated into single-cell suspensions, using the Lung Dissociation Kit  in combination with a gentleMACS dissociator (Miltenyi Biotec) and enzymatic dissociation . Followi7 lung cell pooled suspension was prepared from three chickens, with 1×107 lung cell suspension for each treatment group. MHC Class II and CD3 positive cells were sorted out from each pulmonary cell pool after incubation with the FITC-conjugated MHC Class II and APC-conjugated CD3 antibodies  using Fluorescence-activated cell sorting machine . The gating strategy for each population is shown in 6 cells/mL. High quality single cell suspension was subjected to encapsulation using a 10x Genomics v.3 kit . The library preparation and RNA-sequencing were completed by Gene Denovo  as described previously [3×10eviously . An averThe percentage of macrophages from lung single cell suspension was detected via flow cytometry  with the FITC-conjugated mouse anti-chicken MHC Class II and PE-conjugated mouse anti-chicken KUL01 monoclonal antibodies  . Data weFluorescence-activated cell sorting machine  was used to sort a single cell into each well of a 96-well PCR plate containing 2.5μL of 10× Lysis Buffer (Vazyme# N711). For the isolation of macrophage, each pulmonary cell pool was stained with FITC-conjugated mouse anti-chicken MHC Class II and PE-conjugated mouse anti-chicken KUL01 monoclonal antibodies . Macrophages were sorted following the manufacturer’s procedures. Herein, each sorted cell population was analyzed in four replicates, and 100 single cells were sorted in each replicate for subsequent SMART-Seq2 analysis. Four empty wells served as controls in each 96-well plate. Immediately after sorting, each plate was spun down to ensure immersion of cells into the lysis solution, snap-frozen on dry ice, and stored at −80°C until processing. Library construction and sequencing were completed by Gene Denovo  as described previously . PearsonCt-ΔΔ method [Total RNA was extracted from lung cell suspensions or sorted macrophages for qRT-PCR. qRT-PCR was performed on an ABI7500 Real-Time PCR system  using Universal SYBR R Green Supermix Kit reagents  according to the manufacturer’s specifications. Primers used for qRT-PCR are listed in t method .As described previously , the rawCD3D, IL7R and CD4), B cells , CD8 T cells , Cytotoxic T cells , and Dendritic Cells  [http://iocc.hrbmu.edu.cn/CellMarker/) [CD3D, RORA, CCR6 and CCL20), Macrophages , and Smooth muscle cells (MYH11 and TAGLN). Next, identification of most of the remaining cell clusters was based on the specific expression of the marker genes reported in the literature, e.g: Epithelial cells (BMX and EHF) [SFTPA1, SFTPA2 and SFTPC) [COL1A1, COL1A2 and COL6A1) [ADGRL4, VWF, PODXL and SELECTIN) [BLB2, RNASE6 and VSIG4) [CD3D, CD25, IL7R and TNFRSF1B) [BLB2, VCAN, IL1B, HSPH1 and DNAJA4) [CD3D, MAF, CCR4, DRD4, and KK34) [Four strategies were used for cell-type annotation. Firstly, we identified cell types based on the top 5 expressed genes in each cluster and classical marker genes of chicken immune cells, e.g: CD4 T cells (nd XCR1) ,34. SecoMarker/) , e.g: Thand EHF) , type IId SFTPC) –39, Fibr COL6A1) , VasculaELECTIN) –42, M2 md VSIG4) ,44, and NFRSF1B) ,46. Last DNAJA4) , and Th2nd KK34) . The disTo determine the state of H9N2 AIV or H5N1 AIV infection in each cell type, the expression of viral genes and viral load were analyzed in the different cell types in the H9N2-infected group, H5N1-infected group and control group. Viral gene expression was analyzed using Cell Ranger, based on the viral sequence of H9N2 subtype HN strain (A/Chicken/Hunan/HN/2015) or H5N1 subtype DK383 strain (A/Duck/Guangdong/383/2008) used in this study. The ‘viral genome load’ of a cell in scRNA-seq analysis was based on the number of UMIs that mapped to the AIV eight segmented mRNAs and expressed as a percentage of total UMI content of a given cell. To reduce the false positive rate of infected cells, only the cells with highly expressed AIV genes (at least one transcript per gene per cell) were defined as highly infected cells. Therefore, the cells in each cell type susceptible to AIV infection could be divided into highly infected cells , potential or lowly infected cells , and undetected cells  .To explore the response of each cell type to AIV, we further analyzed the DEGs between the H5N1 or H9N2-infected group and the control group using Seurat software. A hurdle model in MAST   was usedWe used cellphone DB  to inferP < 0.05, ** P < 0.01, *** P < 0.001.Data were analyzed using GraphPad Prism 8.0 software . The results were presented as mean ± SEM. The paired t-test and one-way ANOVA were used for statistical comparison. Data were considered significant at * + T cell (Cluster 3 and Cluster 10), Cytotoxic T cell (Cluster 9), CD4+T cell (Cluster 2), Th2 cell (Cluster 5), Th17 cell (Cluster 12), Treg cell (Cluster 13), B cell (Cluster 16), DC (Cluster 11), Macrophage , Macrophage like cell (Cluster 8), and M2 Macrophage (Cluster 6)  and CD3 (T cells)-positive cells derived from the lungs of low pathogenic H9N2 AIV-treated (3 DPI), highly pathogenic H5N1-treated (1 DPI), and PBS-treated (Control) chickens, for scRNA-seq transcriptional profiles using the 10× Genomics platforms . Detailsuster 6) .To explore the effect of AIV infection on the composition of cell subsets in the lungs, we analyzed the proportion of each cell type in the lung from H9N2 AIV infected, H5N1 AIV infected and control chickens. We found that the proportion of macrophages showed a huge increase in the lungs from H9N2 AIV infected or H5N1 AIV infected chickens when compared to the control . MoreoveTo characterize the host responses to H9N2 or H5N1 AIV infection, we analyzed the differential expressed genes between cell populations of the H9N2 or H5N1 AIV-treated and control samples using Seurat package (v4.0.4). Massive changes of the transcriptional landscape were found between the normal lung tissue and tissue from chickens challenged with H9N2 AIV or H5N1 AIV. Compared to the control sample, it was found that most up-regulated host genes were in macrophages and M2 macrophages of H5N1 group, and macrophages of H9N2 group . And theSince the viral mRNA is poly-adenylated, scRNA-seq can capture both viral and host mRNAs within each individual cell. Therefore, host cells infected with AIV were quantitatively identified by tracking the intracellular AIV-segmented mRNAs at single-cell resolution. In AIV infection initiates in the respiratory tract and spreads in the lung, which triggers widespread pulmonary immune responses. Our foregoing results indicated that highly pathogenic H5N1 AIV, instead of low pathogenic H9N2 AIV infection, induced pulmonary inflammatory damage and a strong antiviral immune response with major viral replication. Therefore, we further analyzed the expression of genes involved in the inflammatory response after AIV infection. There are 301 genes related to the GO term of “inflammatory response” (GO: 0006954). The inflammatory response was firstly analyzed within various cells. And we found that macrophage, M2 macrophage and macrophage-like populations generated abundant inflammatory factors . Then, u+CLASS II+) and scRNA-seq showed that Clusters 0, 1, and 14 (annotated as a macrophage population) had high correlation with macrophages (KUL01+CLASS II+)  , which fASS II+)  and FACSASS II+) . TherefoASS II+) .Lastly, in order to depict how cell populations interacted in the lung through pro-inflammatory cytokines and chemokines after AIV infection, we plotted the intercommunication predictions for cell types expressing the receptor or ligand of the pro-inflammatory factors. Intriguingly, we identified the interaction categories based on CCL4, CCL19, and CXCL13 and their receptors after H5N1 AIV challenge, but not H9N2 AIV challenge. Specifically, the possible communications of various cell populations in the lung through CCL4, CCL19, and CXCL13 and their receptors are sketched, which were induced after H5N1 AIV infection . Thus, HMultiple key questions related to the response of the immune cells to AIV infection in chicken lung have yet to be answered. In particular, the identity of the key immune cell types, antiviral and inflammatory factors contributing to the host protection and immunopathogenesis have not been systematically elucidated. Advances in scRNA-seq technology have allowed us to explore the atlas of the immune cells response to AIV infection in chicken lungs.In this study, we performed 10x scRNA-seq on the sorted immune cells including MHC Class II (antigen presenting cells) and CD3 (T cells) positive cells for characterizing the responses of major immune cell populations in the lung tissue isolated from chickens after H5N1 HPAIV(1DPI), H9N2 LPAIV (3DPI) and PBS treatment. Here, 19 distinct clusters in chicken lungs were identified following the analysis of scRNA-seq data. In addition to the 11 specialized immune cell types (14 clusters) that were identified and annotated, we also identified 5 clusters of non-immune cell types which may be accidentally sorted out during the experiment. In total, 16,642 immune cells were profiled and analyzed including various T cell subsets  and antigen presentation cells   Files. MCompared to LPAIV infection, the efficient virus replication of HPAIV can be correlated with tissue damage in the lung ,12. But HE staining results revealed that H5N1 AIV, as against H9N2 AIV infection, induced pulmonary inflammatory damage with inflammatory cell infiltration , which pIn mice studies, it has been reported that the communication networks between various cell types play an important role in influenza A virus (IAV)-induced cytokine storm and pneumonia . Thus, wIn summary, through scRNA-seq analysis, we demonstrated the key factors associated with the pathogenesis of AIV infection in chickens. Importantly, by sequentially analyzing the transcriptome of various cell types, we discovered that infiltrating macrophages with massive viral replication and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, may contribute to the H5N1 AIV driven inflammatory lung injury. Paralleled with the previous findings from the literature, our results and validation studies confirm the fidelity of our analyses and interpretations. Our data also provide extensive resources for future studies to address the function of identified cell types and in response to pathogenic infection in chickens.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.S8 File(XLSX)Click here for additional data file.S9 File(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig−ΔΔCt value and the average log2 (fold change) values of DEGs. Data from qRT-PCR were collected from three biological samples, and each sample was tested in triplicate. Statistical comparisons were performed with paired t-test, and significance was assessed as P-values using GraphPad Prism. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate SEM.Total RNA of lung cell suspensions was extracted from three chickens of the H5N1 or H9N2 AIV-infected and control groups, respectively. The data of relative mRNA expression level was derived from the ratio of the H5N1 AIV (A) or H9N2 AIV-challenge (B) group results to the control group results. qRT-PCR and RNA-seq results are respectively displayed as the 2(TIF)Click here for additional data file.S3 FigFresh lung tissues were firstly stained with hematoxylin and eosin, then immunohistochemically labeled for NP protein antibody, and examined microscopically. Scale bar = 100μm.(TIF)Click here for additional data file.