End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines.A transcriptomic analysis of the immune response to the Covid-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells (PBMCs) was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and seven days after the second dose (V2D7) using anti-Spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified six months after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response.Transcriptomic analyses demonstrated differing time courses of immune responses, with predominant T cell activity in controls one week after the first vaccination dose, compared to predominant myeloid cell activity in HD at this time point. HD demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p < 0.05). Anti-spike IgG remained elevated above baseline at six months in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development.Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance hemodialysis subjects (HD) comparable to healthy controls (HC) and identify transcriptomic and clinical predictors of anti-Spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of end stage renal disease (ESRD).F30HD102093, F30HL151182, T32HL144909, R01HL138628This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003 The most used form of renal replacement therapy for ESRD patients in the U.S. is hemodialysis (HD). Despite significant improvements in hemodialysis technology, the mortality rate in ESRD patients is still as high as 20% annually 2, with infections being the most common cause of hospitalization and mortality after cardiovascular disease 3. The immunocompromised state of ESRD is characterized by simultaneous immunodepression due to the impact of uremic milieu on immunocompetent cells and immunoactivation due to the accumulation of proinflammatory cytokines 3. There are alterations to both innate and adaptive immunity, including elevated levels of mannose-binding lectin 4, impaired maturation of monocytes and dendritic cells 5, increased B cell apoptosis 6, and decreased T-cell proliferation with elevated Th1/Th2 ratio7.End-stage renal disease (ESRD) is the most advanced stage of chronic kidney disease (CKD), with prevalence in the U.S. reaching 809,000 in 2019 10. Two other studies have demonstrated impaired expression of genes involved in oxidative phosphorylation and mitochondrial function 12. A study identifying a group of inflammatory genes playing a causative role in oxidative stress in dialysis patients showed unique gene expression alterations in maintenance HD patients compared to un-dialyzed CKD patients and compared to patients undergoing peritoneal dialysis 13. These studies indicate a range of immune pathways that may impair vaccination response, and further suggest that dialysis leads to unique immune profile alterations. These alterations may lead to higher susceptibility to infection and lower response to vaccination 8. For example, while more than 90% of patients without CKD develop protective antibodies against HBV after vaccination, only 50–60% of patients with ESRD seroconvert. There have also been higher vaccination failure rates demonstrated against influenza virus, Clostridium tetani, and Corynebacterium diphtheriae in ESRD 9.Studies of genome-wide expression (i.e. transcriptome) profiles of peripheral blood mononuclear cells in ESRD demonstrate a complex picture of immune alterations. One study found upregulation of genes involved in the complement and oxidative metabolism pathways, and downregulation of genes associated with the clathrin-coated vesicle endosomal pathway and T-cell receptor signaling 15. However, certain immunosuppressed populations remain at risk of infection. Given the widespread transmission of COVID-19, detailed assessments of degree, duration, and determinants of immune protection conferred by these vaccines are vitally needed in immunocompromised patient populations including those with ESRD.Understanding the immune response to vaccines in ESRD is particularly important for the new mRNA vaccines developed in response to the COVID-19 pandemic. The COVID-19 mRNA-based vaccines, BNT162b2 and mRNA-1273, have proven to be efficacious in non-immunocompromised individuals, with initial reports showing 95% and 94.1% reduction of COVID-19 disease in recipients 19, they also demonstrate quantitatively reduced SARS-CoV-2 IgG antibodies. We posit that characterization of the transcriptomic underpinnings of antibody titer development on a continuous scale may identify biomarkers for weaker or less durable immune protection in this population. Furthermore, transcriptomic analyses may identify strategies for the development of new, effective vaccines against other infectious diseases for this population. Thus, we characterized the immune response of the HD population to the COVID-19 mRNA-based BNT162b2 vaccine using mRNA sequencing, anti-spike antibody ELISA and neutralization titers across multiple time points. We then integrated these data to identify transcriptomic and clinical determinants of the humoral immune response in HD patients.While recent studies of the SARS-CoV-2 BTN162b2 vaccine in HD demonstrate high levels of seroconversion ranging from 84–96% The study was approved by the University of Illinois at Chicago IRB (#2018-1038) Ethics Review Committee. Maintenance HD patients undergoing vaccination with the BNT162b2 mRNA COVID-19 vaccine in February 2021 were recruited from the outpatient HD unit at the University of Illinois Hospital (UIH) in Chicago, IL. Control subjects consisted of UIH employees undergoing BNT162b2 mRNA COVID-19 vaccination at UIH from December 2020 to January 2021 with no self-reported history of kidney disease or immune disorders. A subset of control subjects matched for age, gender, and COVID-19 history was also analyzed for this study. Blood was collected at 0 – 48 hours prior to and at multiple time points after both the first (V1) and second vaccination doses (V2), which were administered three weeks apart. Control samples were collected prior to each vaccination dose (D0) and at one day (D1) and seven days (D7) after each dose, corresponding to six time points: V1D0, V1D1, V1D7, V2D0, V2D1, V2D7. Blood was collected from HD subjects prior to each vaccination dose and at two days (D2) and seven days after each dose, corresponding to six time points: V1D0, V1D2, V1D7, V2D0, V2D2, V2D7. A final blood sample was drawn six months after initial vaccination (M6) for measurement of antibody titers, prior to additional vaccination doses. Serum and peripheral blood mononuclear cells (PBMCs) were extracted within two hours of blood collection, then stored at −80°C. PBMCs were extracted using density gradient centrifugation at 400g with Ficoll-Paque PLUS. The extracted buffy coat was stored in RNAlater (Invitrogen).20. Baseline clinical lab values were calculated as the median of three lab values across the three months prior to vaccination. Demographic and clinical data was collected from a medical questionnaire at time of consent for control subjects, and included medical history, medications, and self-reported prior SARS-CoV-2 positive test results.Demographic and clinical data was collected from the electronic health record (EHR) for HD subjects, including medical diagnoses, medications, and laboratory values. Laboratory values included monthly SARS-CoV-2 test results, as well as urea reduction ratio , hemoglobin (Hgb), ferritin, transferrin saturation, albumin levels, white blood cell (WBC) count and WBC differential counts obtained during standard of care monthly blood draws for the three months preceding vaccination. Within our analyses, ferritin was coded as either low risk (200ng/ml – 1200 ng/ml) or high risk (<200 ng/ml or >1200 ng/ml), since ferritin levels 200ng/ml – 1200 ng/ml have been shown to be associated with lowest all-cause mortality in HD patients > 5 time points. PBMCs stored in RNAlater were thawed and diluted 1:1 with 1X phosphate buffered saline. The mixture was then pelleted and RNA was extracted using the PureLink RNA Mini kit (Invitrogen). DNase treatment to remove genomic DNA contamination was performed using either the PureLink DNase kit or DNA-free DNA Removal Kit (Invitrogen). Purified RNA in sterile water was stored at −80°C. Each RNA sample was quantified using the Qubit RNA High Sensitivity kit (Invitrogen) and Bioanalyzer RNA Pico kit (Agilent) with RIN>=8.RNA sequencing was performed on PBMCs at all V1 and V2 time points for all subjects for whom RNA libraries were successfully built at For library construction, 50ng of RNA from each sample was aliquoted in 96 well plates. Libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with the optional NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). Each individual sample library was barcoded during PCR amplification using unique dual indexed i5 and i7 primers from the NEBNext Multiplex Oligos for Illumina kit. Each sample library was quantified using the Qubit DNA High Sensitivity kit and Bioanalyzer DNA High Sensitivity kit. Samples were then pooled and sequenced using the MiSeq Nano V2 kit (Illumina) to check read proportions between samples. Samples with lower-than-expected percentage of reads detected were supplemented with an additional spike-in of sample library to the main pool. The supplemented pooled library was sequenced again using the MiSeq Nano V2 kit to verify adequate adjustment. The finalized library was sequenced using a NovaSeq S2 flow cell configured for 75bp paired end output.21. Trimmed reads were then quantified using the Salmon pipeline with an hg38 reference transcriptome index 22. Quantified data was imported into R using the tximeta package 23 to convert Salmon quantification and index data to a count matrix. Transcript names were extracted and matched using Entrez IDs with the AnnotationHub package 23. This finalized count matrix was then imported into a DESeqDataset object and normalized using the variance stabilizing transformation in DESeq2.Raw demultiplexed reads were filtered using fastp to remove adapters and short reads DESeq2 R package was used to identify genes that were differentially expressed at each time point after vaccination for each subject group. Specifically, we implemented a design incorporating group-specific condition effects with individual subjects nested within groups. We performed the classical Deseq2 workflow of estimation of size factors, estimation of dispersion, and negative binomial GLM fitting for βi and Wald statistics, increasing the maximum number of iterations for estimation of the negative binomial distribution to 500. We then generated contrasts to obtain differentially expressed genes for controls at V1D1 and V1D7 (compared to V1D0), and at V2D1 and V2D7 (compared to V2D0). Differentially expressed genes for HD were similarly obtained at V1D2 and V1D7 (compared to V1D0), and at V2D2 and V2D7 (compared to V2D0). We also directly compared gene expression between controls and HD at V1D7 and at V2D7. The significance threshold to determine differential expression was FDR-adjusted (p < 0.05).The Spark® multimode microplate reader. Samples that produced signals greater than the upper limit of the standard curve were diluted 1:2000 and assayed again. IgG concentration was calculated by fitting four-parameter logistic curves to the standard controls and taking the average concentrations of duplicates.The Human SARS-CoV-2 Spike (Trimer) IgG ELISA Kit from Invitrogen was used to quantitate IgG to the SARS-CoV-2 spike protein in serum samples at V1D0, V2D7, and M6 time points. All samples were initially diluted 1:100 (in addition to the 1:10 assay buffer dilution on the 96-well plate) and assayed in duplicate, with two-fold serial dilution of the 150,000 units/mL standard control in duplicate for relative quantification. Absorbance at 450 nm was quantified using a Neutralization assays were performed on serum samples from V1D0 and V2D7 using SARS-CoV-2 pseudotyped virus (pseudovirus). To produce pseudoviruses, an expression plasmid bearing codon-optimized SARS-CoV-2 full length S plasmid was co-transfected into HEK293T cells using the SARS-CoV-2 Spike-pseudotyped lentiviral particle Kit (BEI # R-52948). The cell supernatants were collected 72h after transfection, divided into aliquots and cryopreserved at −80 °C.3 293T-ACE2 cells were seeded per well in a 96-well plate in DMEM containing 10% FBS and 1% penicillin streptomycin. Twenty-four hours later, the pseudovirus was diluted 1:10, followed by five-fold serial dilutions for a total of nine dilutions, with each dilution performed in six replicate wells. After incubation at 37 °C and 5% (vol/vol) CO2 for 72h, the luciferase substrate was added to the 96-well plate for chemiluminescence detection. The 50% tissue culture infectious dose (TCID50) of the pseudovirus was calculated according to the Reed-Muench method in the titration macro template 24.To titrate the pseudovirus, 5×1025. 5×103 293T-ACE2 cells were seeded per well in a 96-well plate. Twenty-four hours later, serial dilutions of the serum samples were performed, incubated for one hour at 37 °C with ~1000 TCID50/ml of pseudovirus, then added to monolayers of ACE2-overexpressing 293T cells in quadruplicate. The cell control with cells alone and the virus control (VC) with pseudovirus were set up in each plate. The target cells were incubated for 65h-72h at 37 °C and 5% (vol/vol) CO2. Fifty μL of Bright-Glo, reconstituted following manufacturer’s instructions, was added to each well of the 96-well plate and incubated for five minutes at room temperature. The 96-well plate was read by a 96-well luminescence plate reader (Tecan Genius Pro plate reader) 26. Percent neutralization was calculated as 100*([Virus-only control] – [Virus plus serum])/[Virus-only control], and neutralizing titer levels are reported as the serum dilution required to achieve 50% neutralization (50% inhibitory dilution [ID50]) 27. The input dilution of serum was 1:20, thus 20 is the lower limit of quantification.Neutralization activity against SARS-2-CoV was measured in a single-round-of-infection assay with pseudoviruses as previously described 28. BTMs with FDR-adjusted p < 0.05 were considered significantly enriched. Enriched BTMs were further characterized using the distribution of Wald statistics of membership genes from DESeq2. To summarize BTM analyses, BTMs were categorized into different families: B cells, cell cycle, dendritic cell/antigen presentation, type I interferon (IFN type I), myeloid activity/inflammation/ T/NK cells, and “others” 29. The percentage of BTMs in each BTM family with significant enrichment at each time point was then quantified over time.Gene set enrichment analysis was performed for each contrast generated in the DESeq2 analysis above using blood transcription module (BTMs) gene sets To determine the effect of vaccination on anti-spike IgG titers at V2D7 and M6, Kruskal-Wallis tests were performed separately for HD subjects and controls. For each group, anti-spike IgG titer levels were compared to assess for the significant effect of time , and Wilcoxon rank sum tests were performed with FDR correction to assess significant differences between each pair of time points . To determine the effect of vaccination on antibody neutralization activity (ID50) at V2D7, Wilcoxon rank sum tests were performed for each group to compare V2D7 vs. V1D0.Linear models were constructed to establish the effect of prior SARS-CoV-2 infection and subject group on anti-spike IgG titer development at V2D7 and M6 and neutralization activity at V2D7. Specifically, log-transformed V2D7 anti-spike IgG titers or V2D7 neutralization activity (ID50) were modeled as the dependent variable, with subject group (HD or controls), log-transformed V1D0 anti-spike IgG titers or V1D0 neutralization activity (ID50), gender, age, race, and ethnicity as independent predictors. To determine predictors of anti-spike IgG at six months, a linear model was constructed with the log-transformed M6 anti-spike IgG titers as the dependent variable, and V2D7 anti-spike IgG titers, SARS-CoV-2 history, gender, age, race, and ethnicity as independent predictors.BTM predictors of antibody response in HD were identified by first calculating a representative expression level of each BTM per sample, which we will refer to as the eigengene. Specifically, the first principal component of each BTM was calculated using DESeq2-derived variance-stabilized gene counts from each module’s member genes across the HD V1 time points, and then across the HD V2 time points. Signs (positive or negative) were assigned to the eigengenes such that samples with higher expression of member genes in a BTM would be given a positive sign, while those with lower overall gene expression would be given a negative sign. This was accomplished for each BTM by (1) computing the median gene expression level across membership genes in a given BTM for each sample, (2) computing the Pearson correlation between the eigengene of the BTM and the median gene expression level across all samples, and (3) multiplying the eigengene of the BTM by −1 if the correlation was negative.Subsequently, we constructed linear models with log-transformed anti-spike IgG at V2D7 as the dependent variable and change in BTM eigengene expression after vaccination as the independent variable, controlling for SARS-CoV-2 history. Separate models were constructed for each BTM that was enriched at each time point after vaccination in HD . Change in BTM expression was calculated as the BTM eigengene after vaccination minus the BTM eigengene before vaccination. P-values were FDR-adjusted across number of enriched BTMs per time point.Additionally, baseline clinical laboratory values predictive of antibody response in the HD subjects were identified. Linear models were separately constructed using URR, ferritin (high risk vs low risk), transferrin saturation, hemoglobin, and WBC count to predict log-transformed anti-spike IgG at V2D7 and M6.Finally, clinical laboratory values responding to vaccination that predicted antibody titer response in the HD subjects were identified. Linear models were separately constructed using log-fold change (LFC) from baseline measurements of ferritin (continuous instead of binarized low- and high-risk), transferrin saturation, and WBC count to predict log-transformed anti-spike IgG titers at V2D7 and M6.Demographic and clinical data of the 20 maintenance hemodialysis (HD) and controls (HC) are summarized in There were eight HD subjects who previously tested positive for SARS-CoV-2, with positive test dates ranging from 7 months to four weeks preceding vaccination. Five control subjects self-reported a prior positive SARS-CoV-2 test, with positive test dates ranging from 8 months to four weeks preceding vaccination. Detailed clinical characterization of HD subjects is summarized in All subjects received two BTN162b2 vaccination doses with the second dose (V2) administered three weeks after the first (V1). Anti-spike IgG binding and neutralizing assay data were obtained for all subjects prior to V1 (V1D0) and seven days after V2 (V2D7). RNA sequencing data was obtained for all control subjects prior to each vaccination dose (D0), and at one day (D1) and seven days (D7) after each dose, corresponding to six time points: V1D0, V1D1, V1D7, V2D0, V2D1, V2D7. One control subject is missing V2D0 data, and one is missing V2D1 data. RNA sequencing data was obtained for 12 HD subjects prior to each vaccination dose, and at two days (D2) and seven days after each dose, corresponding to six time points: V1D0, V1D2, V1D7, V2D0, V2D2, V2D7. Two HD subjects are missing V2D2 data. Sequencing data was not obtained for subjects with fewer than five time points of successfully constructed RNA libraries, due to time points without sample collection or failure to extract high quality mRNA from PBMCs. Six-month follow-up (M6) anti-Spike IgG binding titers were obtained for 15 HC subjects and 19 HD subjects. One HD subject tested positive for SARS-CoV-2 14 days after the second vaccination dose, demonstrating mild symptoms. None of the other subjects reported SARS-CoV-2 infection up to 6 months follow up after the second vaccination.nd vaccine dose, followed by V2D7, V1D1, and V1D7. HD follows a similar pattern, with the largest number of DEGs found at V2D2, followed by V2D7, V1D2, and V1D7. Notably, HD subjects with no SARS-CoV-2 history (n = 6) have substantially lower numbers of DEGs than HD subjects with positive SARS-CoV-2 history (n = 6) at each time point, and particularly at V2 time points.To characterize the molecular basis of immune responses to vaccination in HC and HD, we performed differential gene expression analyses of the PBMC RNA sequencing data. There are substantially more differentially expressed genes (DEGs) in response to V2 compared to V1, and at D1 and D2 post-vaccination compared to D7 . For HC,30 and natural killer cells, and BH3, a pro-apoptotic Bcl-2 family member and mediator of lymphocyte apoptosis 31.Direct comparison of gene expression between HC and HD with no prior reported SARS-CoV-2 infection at V1D7 yielded five DEGs in HD versus HC including increased expression of chemokine CCL19 in HD . Comparison of these same groups at V2D7 yielded 18 DEGs including increased expression in HD of TIA1, which encodes a granule-associated protein expressed in cytolytic lymphocytes TableS1). The immune response transitions to V1D7 enrichment of four BTMs including significant T cell activation and downregulation of monocytes (TableS2). Following V2, HC demonstrate early (V2D1) enrichment of 82 BTMs, with substantial upregulation of innate antiviral activity, similarly to V1D1 (TableS3). The immune response transitions to V2D7 enrichment of ten BTMs, with significant upregulation of plasma cells and immunoglobulins (TableS4).BTM enrichment analysis of subjects without SARS-CoV-2 history reveals the vaccine-induced progression of various immune processes at each time point after vaccination . FollowiTableS5). The immune response transitions to V1D7 enrichment of 17 BTMs, with substantial upregulation of myeloid modules (TableS6). The V1D7 positive enrichment of monocyte/myeloid modules in HD contrast the negative enrichment of these modules in HC (TableS7). The immune response progresses to V2D7 enrichment of one BTM: PLK signaling events  enrichment of 12 BTMs after the first vaccination dose, most significantly involving upregulation of innate antiviral responses , TableS5 modules , TableS6es in HC . Followies in HC , TableS7g events .While there were no significant BTM enrichments in HC with positive SARS-CoV-2 history, most likely due to the insufficient number of subjects, BTM enrichments for HD with positive SARS-CoV-2 demonstrated notable upregulation of plasma cell activity at V1D7. This contrasts with V1D7 for HD with negative SARS-CoV-2, which show primary enrichment of myeloid BTMs . The remSummary enrichments using BTM families show many positive early V1 enrichments of Type 1 IFN activity that dissipate by V1D7 in both HC and HD . HoweverWe next aimed to assess immune protection conferred by the vaccine through quantification of anti-spike IgG antibodies and functional assessment of neutralizing antibodies. All subjects demonstrated an increase in anti-spike IgG at V2D7, with titers for all subjects except one still elevated above baseline at six months. The exception was one HD subject with prior SARS-CoV-2 infection who demonstrated the highest baseline titers of all subjects prior to vaccination. Both HC and HD subjects demonstrated a statistically significant increase in anti-spike IgG and neutralization activity (ID50) from V1D0 to V2D7 (p < 0.001), followed by an expected decrease at M6 from V2D7 levels (p < 0.001) . DespiteHigher anti-spike IgG at V2D7 was significantly predicted by higher pre-vaccination anti-spike IgG, control group assignment, and younger age , while gender, race, and ethnicity were not. Higher anti-spike IgG at M6 was significantly predicted by higher V2D7 anti-spike IgG (p < 0.001), with no additional predictive value conferred by SARS-CoV-2 history, subject group, age, gender, race, or ethnicity. Higher neutralization activity (ID50) at V2D7 was significantly predicted by higher pre-vaccination ID50, with no additional predictive value conferred by subject group, age, gender, race, and ethnicity.Linear models to predict anti-Spike IgG at V2D7 and at M6 in HD using enriched BTMs, controlling for SARS-CoV-2 history, identified BTM predictors at all time points except for V1D2. Of the 18 enriched BTMs at V1D7, increased expression (from V1D0) of “LI.M156.1 plasma cells, immunoglobulins” was predictive of higher anti-spike IgG at V2D7 , controlling for SARS-CoV-2 history. Of the 30 enriched BTMs at V2D2, increased expression of 18 BTMs was predictive of higher anti-Spike IgG at V2D7 . These include innate immune, antigen presentation, and T cell pathways . Increas20Linear models to predict anti-Spike IgG at V2D7 and at M6 In HD using clinical predictors yielded significant baseline and post-vaccination predictors. Baseline ferritin levels in the intermediate range (200 – 2000 ng/ml) were associated with higher anti-spike IgG at V2D7 and M6 , controlling for history of SARS-CoV-2. URR, WBC counts, transferrin saturation, and hemoglobin were not significant predictors of antibody development. The LFC of WBCs from baseline after the first vaccination dose was significantly predictive of antibody titer levels at both V2D7 (p <0.01) and M6 (p < 0.05), controlling for SARS-CoV-2 history and number of days after vaccination that labs were collected . The preOur results demonstrate differing expression of BTMs and differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance hemodialysis subjects (HD) compared to controls. Controls demonstrated expected transitions from early Type I interferon and myeloid activity to T cell activity after the first vaccination dose . The prein vitro apoptosis of lymphocytes in uremia, with a particularly pronounced effect on B cells, mediated by dysregulation of Bcl-2. These results suggest a state of heightened cellular stress in HD after vaccination leading to increased apoptotic signaling.Following the second vaccination dose, both groups demonstrated early enrichment of innate immune modules, with HC alone transitioning to a plasma cell response by V2D7 . Direct 34. Notably, the HD population studied here is younger and more racially and ethnically diverse. The average age of HD cohorts in prior studies was predominantly in the 60s, compared to an average age of 54 in our study. Jahn et al. found in a subset analysis that HD patients under 60 years of age responded equally to healthy controls, suggesting an interaction between increasing age and less effective antibody response in HD patients 17.Despite differing transcriptomic time courses in the two group, our results demonstrate significant elevation of anti-spike IgG titers after two doses of BNT162b2 mRNA COVID-19 vaccination in both HD and controls. HD demonstrated only a slight decrease of IgG levels at V2D7 when controlling for SARS-CoV-2 history (p < 0.05) and no statistically significant difference at six months. Prior studies comparing short-term antibody response to BNT162b2 mRNA COVID-19 vaccination in HD versus controls find antibody response rates of 84–96% in HD after two vaccination doses, but with lower mean IgG levels compared to controls HD subjects with documented SARS-CoV-2 infection prior to vaccination had wider variance of antibody titers at all time points in this study, with two subjects demonstrating V1D0 antibody titer levels similar to that of uninfected subjects. These two subjects consistently had the lowest titer levels at V2D7 and M6 within the group of previously infected subjects and amongst the lowest titers across all subjects. One subject is the oldest enrolled patient, and both are diagnosed with hyperlipidemia.20. Iron deficiency has been linked to impaired immune response and vaccine efficacy in other infections, while inflammation induces macrophage release of the heavy chain component of ferritin, FTH, which has been reported to inhibit lymphocyte proliferation and function 35. Additionally, increased LFC in WBC count 1–3 weeks after vaccination was predictive of higher antibody titers.Given previously and presently demonstrated the wider variance of protective immune responses in HD and altered interactions with risk factors including age, it is valuable to identify predictors of the strength of immune response to vaccination in this population. We identified both transcriptomic and clinical predictors of anti-spike IgG development at both V2D7 and six months after the second vaccination dose (M6). Increased gene expression of blood transcription modules (BTMs) including monocyte activity, dendritic cell and antigen presentation activity, IFN type I activity, and T cell activation two days after the second vaccination dose (V2D2) in HD were predictive of V2D7 anti-spike IgG. Additionally, increased expression of PLK1 signaling events, indicating increased cell cycle activity, at V2D7 was predictive of V2D7 and M6 anti-spike IgG. Clinically, serum ferritin values in the intermediate range at baseline predicted stronger anti-spike IgG development. A prior study of 58,058 maintenance HD subjects found serum ferritin levels between 200 and 1200 ng/ml to be associated with lower all-cause mortality, due to ferritin <200 ng/ml representing low iron status, and >1200 ng/ml representing a hyper-inflammatory state due to ferritin’s status as an acute phase reactant 9. One explanation is that, in mRNA vaccines, the mRNA both encodes the viral antigen and acts as an adjuvant due to its innate immunostimulatory properties; the mRNA is recognized by endosomal and cytosolic innate sensors upon cell entry, resulting in cellular activation and production of type I interferons and other inflammatory mediators 36. This elevated innate immune stimulus could overcome immune desensitization in ESRD, evidenced by diminished TLR4 expression on monocytes 37 and downregulation of activating receptors on natural killer cells in this population 38. If so, the mRNA vaccine delivery vehicle could prove particularly valuable in vaccine development for ESRD and HD going forward.Our study is limited by different early time points between controls and HD (Day 1 vs Day 2 after each vaccination dose) and by sample size, particularly when subdividing SARS-CoV-2 history. The smaller sample size additionally limits our ability to characterize differential immune pathways in clinical subsets of the dialysis population, such as those with low, medium, and high baseline ferritin levels. Future studies are needed for more comprehensive characterization of the immune pathway recruitment in response to the Covid-19 vaccinations in this population. The Covid-19 mRNA vaccines are proving more efficacious than other vaccines in the ESRD population; for example, while more than 90% of patients without chronic kidney disease develop protective antibodies against hepatitis B after vaccination, only 50–60% of patients with ESRD seroconvert Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance hemodialysis subjects (HD) and identify transcriptomic and clinical predictors of anti-Spike IgG titers in HD. Given the efficacy of this vaccination in our HD cohort, the differential transcriptomic responses likely represent immune alterations with sub-clinical effects on humoral immune response to the BTN162b2 mRNA COVID-19 vaccine. Our results warrant further characterization of the immune dysregulation of ESRD and of immune biomarkers that underlie efficacious immune responses to vaccination in this population.Supplement 1


Incident diabetes risk is inversely proportional to 25-hydroxyvitamin D [25(OH)D] levels among non-Hispanic white but is unclear among African American (AA) populations. Serum 25(OH)D2 may be an important component of total 25(OH)D among AA populations due to higher levels of melanin.To assess the association of serum 25(OH)D with incident diabetes among AAs and stratify by detectable 25(OH)D2.Serum 25(OH)D2 and 25(OH)D3 were collected from 2000 to 2004 among AA participants in the Jackson Heart Study. A cosinor model was used to adjust for the seasonality of 25(OH)D3; 25(OH)D3 and 25(OH)D2 were combined to ascertain total 25(OH)D. Incident diabetes  was assessed over 12 years among adults without diabetes at baseline. Participants with missing baseline covariates or diabetes follow-up were excluded. Hazard ratios (HR) were estimated using Cox modeling, adjusting for age, sex, education, occupation, smoking, physical activity, alcohol use, aldosterone, and body-mass index.n = 1671), 25(OH)D ≥ 20 ng/ml compared to <12 ng/ml was associated with a 35%  lower risk of diabetes.Among 3311 adults  584 participants developed diabetes over a median of 7.7 years. After adjustment, 25(OH)D ≥20 compared to <12 ng/ml was associated with a HR 0.78 . Among participants with detectable 25(OH)D2 and 25(OH)D3 (Higher levels of 25(OH)D may be protective against the development of diabetes among AA individuals, particularly among those with detectable 25(OH)D2 and 25(OH)D3. Vitamin D is a fat-soluble paracrine and endocrine signaler important for bone, glycemic and general health . VitaminPre-clinical studies reveal a link between vitamin D and insulin secretion . PancreaEvidence for an inverse association of vitamin D with incident diabetes comes from observational cohort studies, interventional trials, and meta-analyzes with the majority non-Hispanic white (NHW) participants –12. TherGiven the conflicting data on the role of 25(OH)D in the development of diabetes in the AA population compared to NHWs, we examined the association of 25(OH)D with incident diabetes in the Jackson Heart Study (JHS). We hypothesized: (1) serum levels of 25(OH)D at baseline will be negatively associated with the risk of incident diabetes during follow-up, (2) the association of categorically higher levels of baseline 25(OH)D with incident diabetes based on Institute of Medicine (IOM) and Endocrine Society (ES) guidelines would yield similar findings, and (3) detectable 25(OH)D2 may influence the association of 25(OH)D with incident diabetes.n = 1152), or were missing data on baseline or follow-up diabetes status (n = 769) or total 25(OH)D (n = 69). After these exclusions, 3311 participants were included in the analysis  , 19. Ser1*cos(2*π*t/365) + β2*sin(2*π*t/365) +  [A seasonal pattern was evident for 25(OH)D3, but not for 25(OH)D2 levels, thus we used a cosinor model to adjust for the seasonality of 25(OH)D3 over the calendar year. In this model, 25(OH)D concentration is the dependent variable and the sine and cosine of calendar date  are the independent variable: 25(OH)D = βo + βesidual) . The peresidual)  and (1) esidual) . Genotypesidual) .Fasting glucose and insulin concentrations were measured on a Vitros 950 or 250, Ortho-Clinical Diagnostics analyzer  using standard procedures that met the College of American Pathologists accreditation requirement . A high-2 (meters). Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg or the use of antihypertensive therapy. Serum aldosterone was measured by radioimmunoassay (Siemens) and the intra-assay coefficients of variation are reported elsewhere [Baseline information was obtained during clinic visits or at home using standardized questionnaires including demographics, occupation , level of education (≥ Bachelor’s degree versus < Bachelor’s degree), tobacco use (current smoking versus not), alcohol use (in the past 12 months versus not) medical conditions and current prescription medication usage. Calibrated devices were used by certified technicians and nurses to measure participants’ weight, waist circumference (average of two measurements around the umbilicus) and height. BMI was calculated as weight (kilograms)/heightlsewhere .Physical activity in minutes (min) per week was defined according to the American Heart Association (AHA) categorization  as poor Total 25(OH)D values were log-transformed prior to continuous analyzes due to a non-normal distribution. To explore potential non-linear relationships and evaluate for dose-response relationships, a non-log-transformed total 25(OH)D was categorized as described above. Descriptive statistics were used to compare the baseline characteristics of participants by categories of total 25(OH)D using one-way analysis of variance for normally distributed continuous variables, Mann–Whitney and Kruskal–Wallis tests for non-normally distributed continuous variables, and the Chi-square test for categorical variables.2). We performed sensitivity analyses, including analyzing 25(OH)D2 and 25(OH)D3 individually and limiting the analytic sample to participants with detectable 25(OH)D2 to confirm the robustness of our results. To test for possible effect modification of the relationship between 25(OH)D and incident diabetes by DBP genotype, we added interaction terms to the models. The likelihood ratio test was used to determine evidence of a statistically significant interaction. Statistical significance was defined as two-sided alpha <0.05 in the main analysis and <0.10 for interactions [We defined the time of incident of diabetes as the midpoint between last exam without diabetes and the exam at which diabetes was detected . For parractions . AnalyzeA previous version of this manuscript was published as a preprint.P < 0.01; Fig. The baseline characteristics of the 3311 participants across 25(OH)D categories are presented in Table P < 0.0001) . There was a graded decrease in incidence rates across higher IOM 25(OH)D categories : 26.2, 24.4, and 20.7 per 1000-person years from lowest to highest category (The unadjusted and adjusted HRs for the association of 25(OH)D with incident diabetes categorically and continuously are presented in Table The unadjusted and adjusted HRs for incident diabetes associated with 25(OH)D in categories and log-25(OH)D SD among participants with detectable 25(OH)D2 at baseline are presented in Table A 1-SD increase in log-25(OH)D2 was associated with a 12% lower risk of incident diabetes prior to adjustment for BMI (Model 2) and 11% lower risk  after adjustment for BMI among 1671 participants with detectable 25(OH)D2 Table . A 1-SD P value for interaction by DBP genotype in Table The unadjusted and adjusted HRs for the continuous association of total plasma 25(OH)D with incident diabetes are presented alongside the In this prospective study of AA adults, we found that, among individuals without diabetes at baseline, higher log-25(OH)D, 25(OH)D ≥20 compared to <12 ng/ml and 25(OH)D ≥30 compared to <20 ng/ml were associated with a lower diabetes risk prior to adjustment for BMI. These findings were attenuated by adjustment for BMI, except when using IOM categorization. Among participants with detectable 25(OH)D2, higher log-25(OH)D and 25(OH)D ≥20 compared to <12 ng/ml were associated with a lower risk of diabetes, irrespective of BMI. These results suggest that higher levels of 25(OH)D may be protective against the development of diabetes among AA individuals with detectable 25(OH)D2, regardless of obesity status.Consistent with observational studies in majority NHW cohorts –12, we fn = 2102) compared to 3311 in JHS; (2) in ARIC, there were only 97 AA participants in the 25(OH)D referent group (quartile 5), compared to 2,044 NHWs; and (3) ARIC used a more limited definition of diabetes than was used in the JHS, possibly excluding some incident cases.Our results are divergent from two prior multi-ethnic analyzes. First, in the ARIC study, lower 25(OH)D was associated with a higher risk of incident diabetes in quintiles among NHWs, but not among AA participants . Many ofn = 1736 AAs) [Second, in the Third National Health and Nutrition Examination Survey, odds ratios for diabetes were lower in higher quartiles of 25(OH)D among NHWs and Hispanic Americans but not AA participants (736 AAs) . This ap736 AAs) .This study suggests higher levels of 25(OH)D may lower the risk of diabetes in AA populations. Serum 25(OH)D levels are increased readily through vitamin D3 supplementation and may present an effective, broad-scale, low-cost opportunity for diabetes prevention in the AA community. However, results of two recent randomized controlled trials of vitamin-D supplementation show no statistically significant difference in progression to diabetes between supplement and placebo groups of individuals with prediabetes at study inception recruited irrespective of vitamin-D deficiency , 36. TheTesting for effect modification of the relationship between 25(OH)D and incident diabetes by DBP genotype did not yield significant differences. Previous investigations have found associations between single-nucleotide polymorphisms (rs7041 and rs4588) and increased risk and odds of incident and prevalent diabetes, respectively , 38, andThe participants in our study with detectable 25(OH)D2 had a 2.7 ng/ml higher 25(OH)D with slightly better dietary intake D concentration. The IOM and ES guidelines used similar mineral metabolism outcomes, yet reached different conclusions for levels of 25(OH)D deficiency, insufficiency, and sufficiency . The tarThe strengths of our study include a large, socioeconomically diverse, AA cohort. JHS used liquid-based tandem mass spectroscopy—the gold standard—to measure 25(OH)D2, 25(OH)D3 and total 25(OH)D at baseline. JHS used validated questionnaires and a comprehensive documentation of diabetes over time, including fasting glucose, HbA1c, and medication use with improved detection of incident diabetes cases using American Diabetes Association criteria. Despite these strengths, there are some limitations. First, participants in JHS are from one geographic area in the southeastern U.S. and may not be representative of all AA individuals. Second, JHS did not measure vitamin-D binding protein levels, thus was unable to assess bioavailable vitamin D. Third, JHS measured serum 25(OH)D concentrations at only a single time and thus was unable to determine whether changes in 25(OH)D occurred during the follow-up period. Fourth, participants with detectable 25(OH)D2 had a slightly better ideal dietary intake based on American Heart Association’s Life’s Simple 7 classification with 0.4% and 1% in the ideal category for those without vs. with detectable 25(OH)D2 D may be associated with a lower risk of diabetes in AA populations, particularly those with detectable 25(OH)D2. Given the increased prevalence of diabetes in the AA population, it is important to develop biomarkers of diabetes risk and targets for diabetes prevention therapies. Thus the further investigation of the role of vitamin D in diabetes is warranted to advance diabetes equity.SupplementSupplemental Methods


MYB71, a R2R3 MYB transcription factor in regulating ABA responses in Arabidopsis. RT-PCR results show that the expression level of MYB71 was increased in response to ABA treatment. Arabidopsis protoplasts transfection results show that MYB71 was specifically localized in nucleus and it activated the Gal4:GUS reporter gene when recruited to the Gal4 promoter by a fused DNA binding domain GD. Roles of MYB71 in regulating plant response to ABA were analyzed by generating Arabidopsis transgenic plants overexpression MYB71 and gene edited mutants of MYB71. The results show that ABA sensitivity was increased in the transgenic plants overexpression MYB71, but decreased in the MYB71 mutants. By using a DEX inducible system, we further identified genes are likely regulated by MYB71, and found that they are enriched in biological process to environmental stimuli including abiotic stresses, suggesting that MYB71 may regulate plant response to abiotic stresses. Taken together, our results suggest that MYB71 is an ABA responsive gene, and MYB71 functions as a transcription activator and it positively regulates ABA response in Arabidopsis.Abscisic acid (ABA) regulates plant responses to abiotic stresses via regulating the expression of downstream genes, yet the functions of many ABA responsive genes remain unknown. We report here the characterization of  Arabidopsis encoding MYB transcription factors [Arabidopsis [MYB transcription factors are one of the largest transcription factor families in plants, and there are ~200 genes in  factors . Accordibidopsis ,2.Arabidopsis have several different functions. Some of the R2R3 MYB transcription factors are able to regulate plant growth and development, as examples, MYB7 and MYB70 regulate seed germination [It has been shown that members of the R2R3 MYB transcription factors in mination , MYB76 rmination , ASYMMETmination ,9, AtMYBmination , whereasmination ,12. Somemination , AtMYB15mination ,15,16,17mination , PRODUCTmination ,20, and mination . Some ofmination , MYB20, mination ,24,25,26mination , and MYBmination . Yet theSnRK2s genes, and the ABF/AREB/ABI5 transcription factor genes led to reduced abiotic stress tolerance in plants [As the key stress hormone, ABA (abscisic acid) regulates the expression of ABA responsive genes via signal transduction and thereby plant responses to abiotic stresses including drought, salinity, cold and heat ,34,35,36n plants ,41,42,43MYB44 was increased in response to ABA treatment, and Arabidopsis transgenic plants overexpressing MYB44 showed an enhanced tolerance to drought and salt stresses [HSFA6b is an ABA responsive gene, and it plays a role in regulating drought and salt tolerance [AFBA1 (ABA-responsive FBA (F-box-associated) domain-containing protein 1) enhanced plant drought [bHLH112 is induced by ABA, and it positively regulate drought and salt tolerance in Arabidopsis [As mentioned above, ABA regulates the expression of ABA responsive genes and thereby plant responses to abiotic stresses, suggest that ABA response genes are involved in the regulation of plant responses to abiotic stresses. Indeed, some ABA response genes have been shown to be involved in the regulation of plant abiotic stresses responses. For example, the expression level of the R2R3 MYB transcription gene olerance , overexp drought , and thebidopsis . Yet theArabidopsis. For example, we found that the expression of two closely WD40 genes, ABA induced WD40-repeat 1 (AIW1) and AIW2, the non-DNA binding bHLH transcription factor gene Paclobutrazol Resistance 6 (PRE6), and a novel transcription factor family genes ABA-induced transcription repressors (AITRs) were all induced by ABA treatment [aiw1 aiw2 mutants and the transgenic Arabidopsis plants overexpressing PRE6 showed increased sensitivity to ABA [AITR genes in Arabidopsis led to reduced sensitivity to ABA [Arabidopsis, indicating that they may be involved in the regulation of plant responses to abiotic stresses. Indeed, salt tolerance was reduced in Arabidopsis transgenic plants overexpressing AIW1 [Arabidopsis transgenic plants overexpressing PRE6 showed enhanced salt tolerance [AITR genes led to enhanced tolerance to abiotic stresses including drought as well as salt [By analyzing uncharacterized ABA responsive genes, and examining their roles in regulating ABA responses first, we have successful identified several abiotic stress response regulator in reatment ,48,49, ay to ABA ,36, sugg as salt ,36. ThesMYB71, a R2R3 MYB transcription factor in regulating ABA response in Arabidopsis. We found that the expression level of MYB71 is increased in response to ABA, MYB71 is able to activate reporter gene expression, and overexpression or mutation of MYB71 affected plant response to ABA.We report here the characterization of Arabidopsis R2R3 MYB transcription factor gene MYB71 was induced by ABA treatment .Since there is only one T-DNA insertion mutant, we decided to generate gene edited mutants for  mutants ,51,52,53selected B, and gemyb71-c1 mutant, a single nucleotide insertion was occurred in the first target sequence, whereas in the myb71-c2 mutant, an 11 bp deletion was occurred in the second target sequence . Under normal condition, the fusion proteins cannot get into the nucleus, therefore are not functional. However, when the transgenic plants were treated with dexamethasone (DEX), the fusion proteins can move into nucleus, and MYB71 is able to regulate the expression of its downstream genes.Since 35S:MYB71-GR transgenic seedlings with DEX, isolated RNA from the treated and control seedlings, and performed transcriptome analysis. Since MYB71 functions as a transcription activator  analysis show that the 26 upregulated genes are involved in multiple biological processes including plant growth and development, signal transduction, and secondary metabolic . However35S:MYB71-GR transgenic seedlings, we found several genes have been functional characterized, for example, LURP1 and CYP82C2 are involved in pathogen defense [At1g14550 is a tandem repeat gene of PER4, and encodes a protein closely related to PER4 [Among the 26 upregulated genes in DEX treated  defense ,55,56, P defense ,58, and  to PER4 . We therArabidopsis with multiple functions [MYB71 is an ABA responsive gene, and MYB71 positively regulates ABA response in Arabidopsis.ABA is an important hormone that can trigger plants responses to various abiotic stresses, including salinity, drought, high temperature, chilling, and oxidative stress ,60. Sincunctions ,2. Severunctions ,44,61. IMYB71 increased dramatically in response to ABA treatment, in addition, its closely related R2R3 MYB genes MYB79 and MYB121 are also ABA responsive genes  of MYB71 in the mutants only encodes about 20 amino acids before the R2R3 MYB domain in the MYB71 protein, whereas the T-DNA insertion mutant myb71-1 may not be a loss-of-function as the CDS (coding sequence) before the T-DNA insertion encodes the entire R2R3 MYB domain and part of the C-terminal domain , ABI4 and ABI5 [MYB71, a total of 26 genes were found to be unregulated no less than 2 folds in the DEX treated 35S:MYB71-GR transgenic plants  was used for protoplasts isolation, and for plant transformed to generate transgenic plants and gene edited mutants. Seeds of the SALK_013451 T-DNA insertion line in Col background were obtained from ABRC , and used to isolate the myb71-1 mutant.The wild-type Columbia-0 (Col) ecotype −2 s−1 and 8h for darkness, the temperature in the growth room was set at 22 °C.To growth plants for transformation and seeds production, seeds were soaked with sterile water and kept at 4 °C in darkness for 2–3 days, then sown into the soil pots directly and kept in a growth room. The pots were covered with plastic covers for 3–4 days to facilitate generation. The growth room was set with a cycle of 16 h for light with light density at approximately 125 µmol mMYB71 (At3g24310), MYB79 (At4g13480) and MYB121 (At3g30210) to ABA, 10-day-old Col wild type seedlings grown in 1/2 MS plates were collected and treated with 50 μM ABA solution by shaking in darkness for 4 h, and the seedlings treated with solvent methanol were used as a control. The seedlings were then frozen in liquid nitrogen, and use for RNA isolation as described previously [To examine the response of eviously ,35,62,63MYB71 in the transgenic plants, 10-day-old seedlings of the T3 homozygous transgenic plants grown in 1/2 MS plates were collected, frozen in liquid nitrogen and used for RNA isolation.To examine the expression levels of 18, Enzyme Mix and Rnase-free water, incubated at 42 °C for 30 min and then 85 °C for 5 s.Total RNA was isolated by using an EasyPure PlantRNA Kit  according to the manufacturer’s instruction, DNA removal was conducted by treat the RNA isolated with DNase I for 15 min, and cDNA was synthesized by using an EasyScript First-strand DNA Synthesis Super Mix kit . If brief, 2 μg RNA was mixed with Reaction Mix, oligo(dT)MYB71, MYB79 and MYB121, and 24 for ACT2 control with an annealing temperature of 56 °C. The primers used for RT-PCR analysis of MYB71 are 5′-CAACATATGATGAGTTTGTGGGGAGGGATG-3′ and 5′-CAAGAGCTCTTAACAGAAGGGAATGACCATGTTC-3′, and for MYB79 are 5′-CAACATATGATGGTGGAAGAAGTTTGGA-3′ and 5′-CAAGAGCTCTTAACAAAATGGAATCACC-3′, and for MYB121 are 5′-CAACATATGATGCTTGATTGGGGAGTTCAAGGTC-3′ and 5′-CAAGAGCTCTCAAAAAATATAGCCTCCCATGTAAACTCCAG-3′. The primers for ACT2 has been descried previously [The cDNA was used as templates for RT-PCR analysis, 28 cycles were used for amplifying eviously .MYB71, MYB79 and MYB121 were amplified, respectively by RT-PCR, and used to the generation of GD-MYB71, GD-MYB79 and GD-MYB121 by cloning into the N-terminal GD tag-containing pUC19 vector under the control of the CaMV double 35S promoter [GD and the Gal4:GUS reporter construct used for protoplast transfection were as described previously [To generate constructs for transcription activity assays, the full length ORF sequences of promoter ,65. The eviously ,67.GD-MYB71, GD-MYB79 and GD-MYB121 constructs were replaced with a GFP tag to generated GFP-MYB71, GFP-MYB79 and GFP-MYB121 constructs respectively. The NLS-RFP nuclear indicator was as described previously [To generated constructs for the subcellular localization assay, the GD tag in the eviously .35S:MYB71 construct for plant transformation, the GD tag in the GD-MYB71 construct was replaced with a HA tag. To generate the 35S:MYB71-GR construct for plant transformation, the full-length ORF of MYB71 without stop condon was PCR amplified and cloned into the N-terminal HA tag- and C-terminal GR tag-containing pUC19 vector. The 35S:MYB71 and 35S:MYB71-GR constructs generated were then digested with corresponding restriction enzymes and cloned into the binary vector pPZP211 [To generate  pPZP211 .MYB71, the exon sequences of MYB71 were used to identify target sequences on CRISPRscan , and then the identified target sequences were evaluated on Cas-OFFinder . Two specific overlapped target sequences, i.e., 5′-GGGGAGGGATGGGAGGAGGA(TGG)-3′ and 5′-GGATGGGGAATGGTAGAAGA(AGG)-3′ were selected and cloned into the pHEE401E vector following the procedure as described previously [MYB71 gene editing CRISPR/Cas9 construct were DT1-BsF, 5′-ATATATGGTCTCGATTGGGGAGGGATGGGAGGAGGAGTT-3′ and DT1-F0, 5′-TGGGGAGGGATGGGAGGAGGAGTTTTAGAGCTAGAAATAGC-3′, DT2-R0, 5′-AACTCTTCTACCATTCCCCATCCAATCTCTTAGTCGACTCTAC-3′ and DT2-BsR, 5′-ATTATTGGTCTCGAAACTCTTCTACCATTCCCCATCC-3′. The U626-IDF and U629-IDR primers used for colony PCR and sequencing the CRISPR/Cas9 construct generated have been described previously [To generate CRISPR/Cas9 genome editing construct for eviously . The prieviously .GV3101 agrobacterium cells with the 35S:MYB71, 35S:MYB71-GR and the CRISPR/Cas9 constructs to obtain overexpression transgenic plants and gene edited mutants for MYB71, respectively. T1 seeds were collected from transgenic plants selected.The Col wild type plants about 5-week-old with a few flowers in the main inflorescence were transformed by the floral dip method . The plaTo select transgenic plants, the T1 seeds collected were sown on the plates with proper antibiotic. For overexpression transgenic plants selection, T1 seeds were sown on 1/2 MS plates containing 100 μg/mL Carbenicillin and 50 μg/mL Kanamycin. For gene editing transgenic plants selection, T1 seeds were sown on 1/2 MS plates containing 30 μg/mL Hygromycin and 100 μg/mL Carbenicillin.35S:MYB71 and 35S:MYB71-GR homozugous transgenic plants, T2 seeds from T1 transgenic plants were sown on 1/2 MS plates with 25 μg/mL Kanamycin to selected transgenic plants with a single T-DNA insertion (3:1 segregation of transgenic and wild type seedlings), and T3 seeds from T2 plants with a single T-DNA insertion were selected on 1/2 MS plates with 25 μg/mL Kanamycin to identify homozygous transgenic plants. The expression level of MYB71 in the homozygous transgenic plants was examined by RT-PCR to identify overexpression transgenic plants.To identify MYB71, gene editing status of MYB71 in T1 plants was examined by amplifying and sequencing the genomic sequence of MYB71. T2 seeds from gene edited T1 plants were sown in soil pots, and transgene-free T2 plants were identified by amplifying Cas9 fragment as described previously [MYB71 in the transgene-free T2 plants was then examined to identify homozygous mutants.To identify transgene-free gene edited mutants for eviously ,72. Gene35S:MYB71 and 35S:MYB71-GR transgenic plants obtained, DNA was isolated from leaves of T1 plants, a few days after the seedling were transferred into soil pots, by using DNA isolation buffer as described previously [HA primer 5′-CCATGGGATACCCTTACGATG-3′ and the reverse primer used to amplify MYB71 CDS were used to confirm the 35S:MYB71 and 35S:MYB71-GR T1 transgenic plants.To confirm the T1 eviously , and useMYB71 was PCR amplified, for 32 cycles with an annealing temperature of 56 °C by using primers 5′-AGGTTTCACAAGACAGAGAGAC-3′ and 5′-CAAGAGCTCTTAACAGAAGGGAATGACCATGTTC-3′ and sequenced.To examine gene editing status of the T1 transgenic plants of the gene editing transgenic plants, and the T2 transgene-free plants, DNA was isolated from leaves of T1 plants and T2 plants a few days after the seedling were transferred into soil pots, and genome sequence of GFP and NLS-RFP subcellular localization constructs were isolated by using a GoldHi Endo Free Plasmid Maxi Kit  from transformed E. coli cells, and DNA concentration was measured by using a NanoDrop  machine.The plasmids DNA including the reporter and effector constructs, the GD, GD-MYB71, GD-MYB79 and GD-MYB121 effects were co-trasfected, respectively, with the Gal4:GUS reporter construct plasmids DNA into the protoplasts isolated, and the transfected protoplasts were incubated at room temperature for 20–22 h under darkness. GUS activities were then assayed by using a Synergy TM HT fluorescence microplate reader .Rosette leaves from ~3 to 4 weeks old Col wild type plants were used to isolation protoplasts, and protoplasts were transfected by using a procedure described previously ,73,74. FMYB71-GFP, MYB79-GFP and MYB121-GFP were co-transfected respectively with NLS-RFP, and the transfected protoplasts were incubated at room temperature for 18–20 h under darkness. GFP and RFP fluorescence were then observed under a confocal microscope .For the subcellular localization assay, plasmids of MYB71, by using a procedure described previously [35:MYB71 overexpression transgenic plants and MYB71 mutants were sterilized and sowed on the 1/2 MS plates containing 1 µM ABA, or the solvent methanol as control. The plates were kept at 4 °C for 2 days in darkness and then transferred into the growth room. The numbers of seeds germinated were counted every 12 h, and photographs were taken 12 days after the transfer.ABA inhibited seed germination and cotyledon greening were used to examine ABA response of the overexpression transgenic plants and gene edited mutants of eviously ,75,76,7735S:MYB71-GR seeds were sown on 1/2 MS plates and kept for 2 days at 4 °C in darkness. Then the plates were transferred into the growth room and grown for 10 days. The 35S:MYB71-GR seedlings were then treated with 10 µM DEX or solvent only as control by shaking for 4 h. The seedlings were then frozen in liquid nitrogen, and send to the company  for transcriptome sequencing and analysis. A potion of RNA isolated was returned by the company for result verification purpose. Genes upregulated at least 2 folds by DEX treatment were subject to functional categorization on TAIR Gene Ontology (GO) Annotation  under the term of biological process.MYB71 is an ABA responsive gene, MYB71 functions as a transcription activator, and it regulates plant ABA response in Arabidopsis.Our results in this research show that the


In vitro experimental measurements were implemented to validate the function of COL11A1 and LINC00665 in LUAD cells. Our study demonstrated that LINC00665-2 and COL11A1 were significantly upregulated in LUAD tissues compared with nontumor tissues. COL11A1 was positively correlated with multiple immune cell enrichment, suggesting that COL11A1 may be a prospective therapeutic target to enhance the efficacy of immunotherapy in LUAD. A regulatory mechanism LINC00665-2/microRNAs (miRNAs)/COL11A1 axis was identified to facilitate the tumorigenesis of LUAD. si-LINC00665 transfection induced the inhibition of growth and migration, and apoptosis was reversed by the overexpression of COL11A1 in LUAD cells. In conclusion, LINC00665 as a competing endogenous RNA sponging multiple miRNAs to modulate COL11A1 expression in LUAD, suggesting that LINC00665/miRNAs/COL11A1 axis may contribute to the pathogenesis of LUAD.Collagen type XI alpha 1 (COL11A1) as an oncogene has been reported in several malignant tumors. Herein, we aimed to explore the function of COL11A1 and its upstream regulators in lung adenocarcinoma (LUAD). COL11A1 expression prognostic significance, gene ontology, Kyoto Encyclopedia of Genes and Genomes, and immune infiltration were explored in LUAD.  In addiNumerous long noncoding RNAs (lncRNAs) have been authenticated to play important biological functions in physiological and pathological processes ,10. Clashttp://gepia.cancer-pku.cn/index.html). We also found that nine miRNAs  were downregulated in LUAD tissues and might be potential regulators of COL11A1. Previous studies have revealed that several miRNAs, including miR-20a-3p [http://starbase.sysu.edu.cn/), LINC00665 was predicted as a coregulator of nine miRNAs. Furthermore, the biological functions of LINC00665/miRNAs/COL11A1 were validated in LUAD cells and nude-mouse transplanted tumor model.In our study, COL11A1 expression was significantly upregulated in LUAD tissues using the GEPIA database  as described previously . Nomogra2.2http://gepia.cancer-pku.cn/detail.php) [The expression of COL11A1 in LUAD tissues and corresponding nontumor tissues was excavated using the GEPIA platform (ail.php) . The exp2.3https://david.ncifcrf.gov/).Analysis of single gene difference of COL11A1 in LUAD was prepared for GSEA using TCGA database with DESeq2 package (version 1.26.0) as described previously . Top 50 2.4The correlation of COL11A1 with immune cells was analyzed by GSVA package (version 1.34.0) with ssGSEA algorithm ,38.2.5http://www.targetscan.org/vert_72/) and ENCORI database [http://gepia.cancer-pku.cn/detail.php) [miRNAs and lncRNA were predicted by TargetScan (database  with theail.php) . The exp2.616HBE, H1975 and A549 cells were cultured as described previously . LncRNA 2.7Reverse transcription–polymerase chain reaction and western blot were performed as described previously ,41. Anti2.8Cell proliferation and migration were implemented as our previous study . Cell ap2.9U test and Wilcoxon signed-rank test, log-rank test, univariate Cox regression analysis and spearman analysis were implemented to evaluate the significant difference with P < 0.05.The Shapiro-Wilk normality test, unpaired and paired tests with Mann-Whitney Ethics approval and consent to participate: Not applicable.33.1COL11A1 expression in 24 cancer types was evaluated using the TCGA database. As shown in 3.2The association between COL11A1 and OS in 10 cancer types was determined by the GEPIA database. COL11A1 high expression in LUAD exhibited an unfavorable prognosis. However, no significant prognostic value of COL11A1 was observed in other cancer types .3.3P < 0.001), N , M  and pathologic stage  and high COL11A1  expression were unfavorable prognostic factors of OS for LUAD patients. In addition, we established a prediction model of OS by fitting with clinical parameters  and COL11A1 expression based on the TCGA database. As shown in As mentioned earlier, COL11A1 high expression was correlated with a poor OS of patients with LUAD. We also performed univariate COX regression analysis to evaluate OS-related risk factors in LUAD patients. As shown in 3.4P = 0.030), Fc epsilon receptor (FCERI)-mediated nuclear factor-kappa B (NF-κB) activation , FCERI-mediated mitogen-activated protein kinase (MAPK) activation , collagen formation , immunoregulatory interactions between a lymphoid and a non-lymphoid cell  and cell cycle checkpoints . These findings suggested that COL11A1-related genes were enriched in tumor-associated signaling pathways NF-κB and MAPK activation, cell growth, collagen formation and immunoregulatory activity. Next, GO and KEGG pathway enrichment analyses were executed based on the top 50 related genes. One hundred thirty-two terms of BP, 9 terms of CC and 32 terms of MF were significantly enriched by the GO analysis, and the top four terms of BP, CC and MF were listed as shown in To further analyze the biological functions of COL11A1 in LUAD, GSEA was implemented using the TCGA database. Top six terms were listed as follows . As shown in The upstream lncRNAs of miRNAs were also predicted using the ENCORI database (4In our study, a regulatory mechanism LINC00665-2/miRNAs/COL11A1 axis was identified to facilitate the tumorigenesis of LUAD. COL11A1 as a downstream gene target of LINC00665-2 and miRNAs was significantly elevated in LUAD tissues and served as a prognostic and diagnostic indicator. In addition, COL11A1 was positively correlated with multiple immune cells enrichment, suggesting that COL11A1 may be a prospective therapeutic target to enhance the efficacy of immunotherapy in LUAD.Human pan-cancer studies reveal that COL11A1 expression is upregulated in approximately 14 cancer types and mediates inflammation and epithelial-mesenchymal transition phenotype in the tumor microenvironment that deteriorates cancer invasion and metastasis ,44, reflPrevious studies indicate that the growth, invasion and metastasis of lung cancer have interacted with immune cells enrichment in the tumor microenvironment that may facilitate tumor cell escape and an unfavorable factor of antineoplastic immunotherapy ,47. T anIn vitro experiments revealed that knockdown of LINC00665 downregulated COL11A1 protein expression in H1975 and A549 cells. Inhibition of LINC00665 abrogated the malignant phenotypes of H1975 and A549 cells. However, overexpression of COL11A1 neutralized the antineoplastic activities of si-LINC00665. Several studies have corroborate that LINC00665 expedites malignant progression and chemotherapy resistance of NSCLC [The other major finding was that lncRNA LINC00665 might be a regulator of COL11A1. of NSCLC ,52,53.In conclusion, bioinformatics analysis of the TCGA database and functional identification validated the oncogenic role of LINC00665 and COL11A1 in LUAD. Our findings provided a new insight into LINC00665 as a ceRNA sponging multiple miRNAs to modulate COL11A1 expression in LUAD, suggesting that LINC00665/miRNAs/COL11A1 axis may contribute to the pathogenesis of LUAD.


Background: Chitinase-3-like protein 1 (CHI3L1) is overexpressed in various types of tumors, especially in glioma, and contributes to tumor progression. However, the definite role of CHI3L1 and involved pathway in glioma progression are not completely understood.Methods: CHI3L1 expression in human gliomas and its association with patient survival was determined using enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and public databases. Single-cell RNA-seq was used to characterize the landscape of tumor and myeloid cells. Human proteome microarray assay was applied to identify the binding partners of CHI3L1. Protein-protein interactions were analyzed by co-immunoprecipitation and cellular co-localization. The roles of CHI3L1 in glioma proliferation and invasion were investigated in tumor cell lines by gain- and loss- of function, as well as in vivo animal experiments.Results: CHI3L1 was up-regulated in all disease stages of glioma, which was closely related with tumor survival, growth, and invasion. CHI3L1 was primarily expressed in glioma cells, followed by neutrophils. Moreover, glioma cells with high expression of CHI3L1 were significantly enriched in NF-κB pathway. Pseudo-time trajectory analysis revealed a gradual transition from CHI3L1low to CHI3L1high glioma cells, along with the NF-κB pathway gradually reversed from inhibition to activation. Intriguingly, CHI3L1 binds to actinin alpha 4 (ACTN4) and NFKB1, and enhances the NF-κB signaling pathway by promoting the NF-κB subunit nuclear translocation in glioma cells. Further, CHI3L1 were released into the tumor microenvironment (TME) and interacted with CD44 expressed on tumor-associated macrophages to activate AKT pathway, thereby contributing to M2 macrophage polarization. In addition, CHI3L1 positively correlated to the expression of immune checkpoints, such as CD274 (PD-L1) and HAVCR2 (LAG3), which then remodeled the TME to an immunosuppressive phenotype.Conclusion: Our research revealed that CHI3L1 facilitated NF-κB pathway activation within glioma cells and reprogramed the TME, thereby serving as a promising therapeutic target for glioma. Glioma, a heterogeneous group of primary central nervous system (CNS) tumors, accounts for 80% of all brain tumors Nuclear factor (NF)-κB belongs to a family of transcription factors that are involved in viral infection, immune response, cell proliferation and survival, and tumorigenesis In addition to parenchymal cells, immune cells in the tumor microenvironment (TME) undergo substantial reprogramming, thereby acquiring pro-tumor phenotypes to facilitate tumor progression Chitinase-3-like protein 1 (CHI3L1), a member of the glycoside hydrolase family 18 in vivo animal experiments.Here, we analyzed the mRNA-seq data from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and Chinese Glioma Genome Atlas (CGGA) to explore the expression profile of CHI3L1 in glioma at mRNA level. CHI3L1 protein levels were detected via immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and western blot. Spearman correlation analysis was used to explore the association between CHI3L1 expression and immune cells infiltration. Single-cell RNA-seq data were applied to characterize the behavior of tumor cells and delineate the landscape of myeloid cells. The roles of CHI3L1 in glioma survival, proliferation and invasion were further investigated in four glioblastoma (GBM) cell lines and Glioma transcriptomic profiles and matched clinical information were obtained from the GEO (GSE100675), TCGA, and CGGA, while brain tissues from Genotype-Tissue Expression (GTEx) were chose as controls. A total of 479 samples with clinical information were enrolled in further analysis. Single-cell RNA-seq (GSE117891) that included 70 sample points from 13 patients with glioma were obtained from GEO which was publicly available online. For the comparison of gene expression among different human glioma cell lines, we applied the “Expression 21Q3 Public” file downloaded from the Cancer Cell Line Encyclopedia (CCLE) website.The estimation of the total immune cell infiltration within each glioma sample and the immune cell subsets was determined by Cibersort using the LM22 gene set on the Cibersort website We collected serum samples from 69 patients with staged glioma and 25 healthy volunteers, and 41 pairs of clinical diagnosed glioma and peritumoral tissues from the Department of Neurosurgery of The Affiliated Drum Tower Hospital of Nanjing University Medical School  between January 2020 and September 2021 as approved by the Clinical Ethics Committee.Serum levels of CHI3L1 in human were measured by ELISA kits  according to the manufacturer's protocol.Human glioma and paired normal brain tissues were fixed in 10% formalin, embedded in paraffin and cut into 4 μm thick sections. Sectioned tissues were further performed for Hematoxylin and eosin (H&E) staining and IHC staining with anti-CHI3L1 . For immunofluorescence (IF), frozen brain sections (6 μm) were incubated with anti-CHI3L1  and anti-NF-κB p65  antibody. Meanwhile, we incubated frozen sections with anti-human CD163  and anti-human CD206  antibodies to evaluate M2 macrophage infiltration.We utilized a human proteome microarray from Wayen Biotechnologies (Shanghai), Inc. The recombinant human CHI3L1 (rhCHI3L1) was purchased from Sino Biological lnc . The proteome microarray assay was performed as published previously 2. The glioma cells were treated with TNFɑ (50 ng/mL or 200 ng/mL)  to study the effect of CHI3L1 on NF-κB pathway activation after shRNAs transinfection. All cells were negative for mycoplasma.Human GBM cell lines  authenticated by STR profiling were purchased from Procell Life Science & Technology Co., Ltd  and Cell Bank of Chinese Academy of Sciences . Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) , and incubated in a humidified incubator at 37 °C with 5% COBMDMs were isolated and cultured for seven days in the presence of recombinant murine M-CSF (10 ng/mL)  for further studies.Scrambled and human CHI3L1 shRNA plasmids were purchased from GenePharma  and performed according to the manufacturer's instructions. Sequences for control and target specific shRNAs are as following: control shRNA, 5'- TTC TCC GAA CGT GTC ACG T-3'; CHI3L1 shRNA-1, 5'-CAA GGA AAT GAA GGC CGA ATT-3'; CHI3L1 shRNA-2, 5'-TAG CAT CAT GAC CTA CGA TTT-3'. U87MG and A172 cell lines were transfected with shRNAs using Lipo3000 Transfection Reagent  and western bolt was applied to assess the transfection and knockdown efficiencies.6 of HEK293 cells in one 10 cm dish. Briefly, the three plasmids were resuspended in 1.5 mL Opti-MEM, and mixed with 1.5 mL Opti-MEM containing 60 μL polyetherimide (PEI) for 20 min at room temperature. Then, the mixture were added to HEK293 cells dropwise. The culture medium was changed to fresh medium after 8 h. Subsequently, the culture medium containing lentivirus was collected after 24 h and 48 h. Then, U251 cells were infected with lentiviruses and screened by puromycin (2 μg/mL) to obtain stable cell lines. For inducible CHI3L1 expression, doxycycline (Dox) (1 μg/mL) was added to the resultant U251 cells for 24 h.The lentiviral shRNA vector (hU6-MCS-CBh-gcGFP-IRES-puromycin) targeting human Chi3L1 was constructed and the particles were produced by Shanghai Genechem Co.,Ltd. . The shRNA targeting sequence is CAA GGA AAT GAA GGC CGA ATT. The lentiviral expression vector pLV2-TRE3GS-CHI3L1 (human)-TetOne-Puro was constructed by MiaoLing Plasmid Platform . The lentiviruses were packaged using recombinant lentiviral vector plasmid , packaging plasmid  and envelope plasmid  for 4 × 106 cells in 0.1 mL PBS) were subcutaneously injected into nude mice   per site in each mouse. Tumors were measured every other day using calipers and the volume determined using the formula: V = (S2 × L)/2, where V is the volume, S is the shortest diameter, and L is the longest diameter. The mice were euthanized on day 16, and the tumors were measured and photographed.U87MG cells stably infected with lenti-shCHI3L1 or lenti-shControl  (10 ng/mL)  for 24 h, as well as differentiated BMDM, were subsequently incubated with recombinant CHI3L1 (500 ng/mL)  in the presence of recombinant IL-4 (100 ng/mL)  as a positive control. Macrophages polarization was assessed by IF staining using CD163 and CD206 to identify M2 macrophages.Nuclear and cytoplasmic proteins were isolated for further western blot detection using Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer's instructions.U87MG and A172 cells were harvested and lysed using IP buffer  on ice. Cell extracts were incubated with anti-rabbit IgG (0.5 μg) or anti-CHI3L1 antibody  (0.5 μg) overnight at 4 °C on a rotator, followed by addition of protein G agarose beads for 2 h at 4 °C. Next morning, the complexes were resuspended and then subjected to 10% SDS-PAGE for further immunoblotting.Tissues and cells were lysed in lysis buffer and electrophoresed in 10% SDS-PAGE gels, and then probed with specific antibodies listed in HEK293 cells were seeded in 12-well plates and transfected on the following day by PEI 40K Transfection Reagent  according to the manufacturer's instructions. In brief, the cells were transfected with the NF-kB firefly luciferase activity reporter plasmid in combination with either empty vector, or CHI3L1, or ACTN4, or the combination of CHI3L1 and ACTN4 plasmids. To normalize for transfection efficiency, pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Then, the cells were treated with TNFα (20 ng/mL) for 24 h. Subsequently, the cells were harvested and measured luciferase activity using a Dual Luciferase Reporter Assay Kit . Firefly luciferase activities were normalized on the basis of Renilla luciferase activities.HA-hCHI3L1 plasmid was constructed by Jiangsu Genecefe Biotechnology Co., Ltd . The plasmids were transfected into U251MG or U118MG cells by PEI 40K Transfection Reagent. Forty-eight hours after transfection, the cells were harvested, blocked, and stained by PE anti-human CD274  antibody . Then, flow cytometry was performed using BD FACSC II flow cytometer, and the data were analyzed by Flow-Jo software, v10.0.Total RNA was extracted with RNA Isolation Kit , and 1 μg of RNA was immediately applied for reverse transcription . Gene expression was normalized to GAPDH, and the primer sequences were available in Approximately 1000 glioma cells were seeded in a 6-well plate and were grown for 14 days. Cell colonies (≥50 cells) were fixed and then stained with Giemsa solution to facilitate counting.EdU incorporation was performed using BeyoClick™ EdU-488 kit  per manufacturer's instructions after transfected with control or CHI3L1 shRNA to evaluate the proliferative activity of U87MG and A172 cells.U87MG and A172 cells transfected with control or CHI3L1 shRNA were cultured in Two-well culture-inserts  in a 24-well cell culture plate. Remove the culture-inserts and then fill with serum-free medium, and the cell-free gaps were continuously observed for 12 h under a microscope. FLAG-hACTN4 plasmid was purchased from (Cat#: HG10971-CF) .4 cells per well in serum-free medium in a 24-well plate. The absorbance at 450 nm of each well was measured at 0, 24, 48, 72 and 96 hours.The proliferation of glioma cells were evaluated by Cell Counting Kit-8 (CCK8) assay. Cells were seeded at 1 × 10R version 4.0.4 software was used for raw high-throughput RNA sequencing and single cell RNA sequencing data analysis. Fiji was utilized for image analysis, and GraphPad Prism 9.0 was applied for quantification and statistical analysis. Survival analysis was performed by log-rank test in Kaplan-Meier curves. For comparisons, Student's t-test and Wilcoxon test were applied to compare the differences between groups; one-way ANOVA was used for comparisons among three or more groups.CHI3L1 are positively correlated with the WHO grade, IDH status, and TCGA subtypes of gliomas in TCGA and CGGA datasets  mRNA data to compare the expression levels of CHI3L1 in 33 tumor types and paired normal tissues. CHI3L1 was found to be over-expressed in cancer lesions than in the normal adjacent tissues  . CHI3L1 was plotted as an over-expressed gene on the volcano plot and heatmap in TCGA cohorts  891 CST3,high glioma cells were significantly enriched in TNFɑ signaling via the NF-κB pathway, while the CHI3L1low glioma cells exhibited inhibited NF-κB pathway enrichment  and high (200 ng/mL) concentration of TNFα in the temporal gradient, and the phosphorylation of p65 subunit was obviously suppressed from five minutes in -/-Chi3l1 BMDMs, both in the low and high groups  portal, and 18 immune related genes We inferred the absolute levels of 22 types of infiltrated immune cells within samples in TCGA cohort using Cibersort algorithm according to the LM22 signature matrix We then explored the roles of CHI3L1 in regulating the expression of immune checkpoints in glioma based on TCGA and CGGA datasets. In TCGA cohort, CHI3L1 expression was positively associated with the expression of CD274 (PD-L1) and HAVCR2 (TIM3), but had no relationship with PD-1 Figure G-H. In MTo better characterize the specific phenotype and function of myeloid populations in glioma, we analyzed a public single-cell RNA-seq dataset (GSE117891) CHI3L1 and CD44 increased collaboratively in the evolution process of TAMs over pseudo-time , microtubules, non-muscle myosin II (NM II), focal adhesion kinase (FAK), and cytoskeleton tension were used to determine the mechanical components involved CD68  increased markedly in macrophages after IL-4, rhCHI3L1, and culture supernatant of U87-MG and A172 cells, whereas M1 markers showed no difference or slight alterations   and M2-like  8 Figure G-H. We t8 Figure I. Meanwh-/-Cd44 mice. Consequently, -/-Cd44 BMDMs treated with recombinant mouse CHI3L1 (rmCHI3L1) showed no differences of Arg1 and Chil3 expression compared to the control group, while rmCHI3L1 treated +/+Cd44 BMDMs exhibited a significant increase  were not significantly changed  in mouse models Cd44-/- BMDMs displayed decreased phosphorylation of AKT at five minutes compared to +/+the Cd44 group after rmCHI3L1 treatment, which was consistent with a published study showing that the AKT pathway was involved in macrophage polarization Microglia- or monocyte-derived TAMs are the largest population among TME, which predominantly manifest with the M2 phenotype Intriguingly, we demonstrated that F-actin contributed to extracellular secretion of CHI3L1 in GBM cells. Actin has two types of structural forms, F-actin and free G-actin. G-actin polymerizes to form F-actin. Two parallel F actin chains are twisted to form the double helix structure of microfilaments. In non-muscle cells, actin filaments form a track system to transport cargo such as vesicles and organelles. Recently, it was reported that Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis high glioma cells. Increased infiltration of neutrophils in the TME is also positively correlated with increasing glioma malignancy We elucidated in unprecedented detail how the intracellular and released CHI3L1 played pro-tumor roles in the development of glioma. We unexpectedly identified two distinct populations of neutrophils at the single-cell level, whose expression of CHI3L1 was second only to CHI3L1In summary, our study revealed that nuclear CHI3L1 contributed to tumor cell proliferation and survival via sustained activation of the NF-κB signaling pathway. In contrast, CHI3L1 binds to CD44 to activate the AKT pathway to facilitate M2 macrophages polarization after secreted into the TME, which is responsible for the pro-tumorigenic environment reprogramming. In conclusion, our findings provided a basis for the potential role of CHI3L1 as a therapeutic target for glioma.Supplementary figures and tables 1-4.Click here for additional data file.Supplementary table 5.Click here for additional data file.


Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y Even if patients have no obvious drinking history, nonalcoholic fatty liver disease (NAFLD) is a liver illness that resembles alcoholic liver disease. The main contributing factor to the onset of NAFLD is obesity ,2. Nonal12-like receptors  share 45–50% sequence similarity and pair mostly with pertussis toxin-sensitive Gi/o, whereas P2Y1-like receptors  share 28–52% sequence homology anthracene/12-O-tetradecanoylphorbol-13-acetate [6R deletion prevented the neuronal and memory loss caused by tubulin-associated unit causes [P2Ynditions . The synrophages . Endothet aortas . P2Y6R Kammation ,15,22. C-acetate . P2Y6R dt causes .6R is highly expressed in immune cells such as macrophages. P2Y6R regulates phagocytosis and the production of inflammatory cytokines in macrophages or microglia [6R is also expressed in hepatic stellate cells (HSCs) [6R agonist) tripled the mRNA levels of procollagen-1 [6R ameliorates the pathology of the alcoholic steatohepatitis (ASH) model [6R deficiency in adipocytes protects mice from diet-induced obesity due to enhanced energy expenditure and reduced inflammation, with white adipose tissue browning also being reported [6R may contribute to the progression of liver inflammation and fibrosis. However, we demonstrated that the degree of liver fibrosis in P2Y6R KO mice fed with CDAHFD was similar to that in WT mice with CDAHFD, but inflammation and liver injury were rather exacerbated in P2Y6R KO mice. P2Y6R KO mice have been shown to exhibit different phenotypes depending on the environment and experimental site, even with the same inflammation model [6R in muscle has been shown to increase insulin resistance [6R KO mice fed with CDAHFD were reduced in comparison to WT mice fed with CDAHFD. The liver weight increases in proportion to the severity of fatty liver present in the CDAHFD model, but it has been reported that the liver weight decreases when fibrosis also progresses [6R KO mice fed with CDAHFD was increased compared to that in WT mice fed with CDAHFD. These data suggest that P2Y6R knockout slightly promotes liver fibrosis and inflammation. Urine volume in P2Y6R KO mice fed with CDAHFD was reduced compared to that in WT mice fed with CDAHFD. It has been reported that P2Y6R KO mice display more frequent micturition, with smaller bladder capacity compared to WT mice [6R is also expressed in the kidney [6R contributes to the pathogenesis of heart failure [6R signaling in the cardiovascular and renal tissues, causing the difference in urine output between P2Y6R KO and WT. Future investigations using tissue-specific P2Y6R KO mice are necessary to identify which cells are regulated by P2Y6R in the pathogenesis of NASH.Hepatocytes, immune cells such as Kupffer cells and macrophages, and hepatic stellate cells are involved in the progression of NASH . Accordiicroglia ,28,29. Ps (HSCs) . Treatmellagen-1 . The inhH) model . P2Y6R dreported . These ron model ,33. In asistance . Liver wogresses . The mRN WT mice . P2Y6R ie kidney ,27, but  failure ,13 and h failure . CDAHFD 1R, P2Y6R, P2Y11R, P2Y13R, and P2Y14R are increased in patients with NASH compared with patients with NAFL. Among them, P2Y6R showed the best positive correlation with CCL2 and Col1a1. These data suggest that P2Y6R expression level is increased according to the degree of inflammation and fibrosis. Infiltration of inflammatory cells with high P2Y6R expression may contribute to elevated P2Y6R expression in the NASH liver. Since the expression of P2Y6R is also increased by LPS [6R proteins are internalized and degraded by cysteine modification by electrophiles and nitric oxide [6R at the protein level, including not only regulation of transcriptional level but also post-translational modification in patients, to clarify the role of P2Y6R in NASH pathogenesis.We showed that among the P2Y subtypes, P2Yd by LPS , its expic oxide . Oxidati6R by smoking may exacerbate the pathology of NASH. Conversely, there is an epidemiological report that cigarette smoke suppresses the risk of onset, progression, and recurrence of ulcerative colitis (UC) [6R through covalent binding with electrophilic molecules promotes redox-dependent (β-arrestin-independent) P2Y6R internalization, which contributes to the attenuation of UC progression in mice [6R.Smoking is positively correlated with NAFLD . Involvetis (UC) . We rece in mice . As smok2R is known to promote high-fat diet (HFD)-induced hepatic steatosis [12R-dependent CD40L release in NAFLD [14R selectively in adipocytes were protected from obesity and displayed reduced liver weight compared to HFD control mice [14R KO mice [6R is known to couple with G12/13. G12-mediated signaling is known to regulate hepatic lipid metabolism, and G12-knockout mice are reported to display aggravated fatty liver [6R signaling, despite its elevated expression, is not involved in pathogenesis and production of inflammatory markers in fatty liver. P2Y6R may maintain liver homeostasis through G12 signaling.P2YRs are GPCRs that have been further divided into two subfamilies based on G-protein selectivity and sequence similarity. Among the P2YRs, P2Yteatosis . Platelein NAFLD . Anotherrol mice . Reduced KO mice . On the ty liver . We show7R prevents inflammation, fibrosis and hepatocyte apoptosis [7R activation on Kupffer cells enhances TNFα and monocyte chemotactic protein-2 (MCP-2) production in HFD mice treated with CCl4 [7R antagonism might be a beneficial strategy for the treatment of NASH. Adenosine A1AR expression in the liver of streptozotocin (STZ)-induced diabetic rats was elevated [1AR expression remained unchanged while A2AAR and A3AR receptor levels were dramatically elevated in STZ-treated rat liver [2AAR activation reduces inflammation [2AAR exacerbated HFD-induced NAFLD and hepatic inflammation [2AAR deficiency in hepatocytes and macrophages contributed to increased inflammation [2AAR was also observed in a study of the methionine- and choline-deficient (MCD)-induced NASH animal model. The A2AAR KO mice, treated with the MCD-NASH model show a larger body weight, increased liver inflammation, and more severe hepatic steatosis than the control mice [2AR activation in reducing inflammation caused by lipotoxicity can lead to the protection of mouse liver against NASH progression [2BAR has also been shown to have an important function in the regulation of fatty liver disease. A2BAR deficiency protected mice from hepatic steatosis and the formation of fatty liver [Y mice, inhibiting A2BAR with the specific antagonist ATL-801 reduced glucose production during hyperinsulinemic-euglycemic clamp tests [2BAR suppressed lipogenic genes such as sterol regulatory element-binding protein-1 (SREBP-1). The A2BAR KO mice fed with HFD caused hepatic steatosis with increased plasma triglyceride and cholesterol levels [2BAR overexpression and activation lowered lipid production in the liver and enhanced whole-body metabolism [2BAR KO mice lost weight and had enhanced de novo lipogenesis, resulting in raised liver triglyceride levels. Increased glucokinase and fatty acid synthase mRNA levels revealed poor lipid metabolism in the liver of A2BAR KO mice [2BAR KO mice fed with HFD show poor glucose tolerance and insulin sensitivities [2BAR agonist/partial agonist, BAY60-6553, reportedly enhance glucose and insulin tolerance as well as lower fasting blood glucose levels [2AAR and/or A2BAR activations will be a promising therapeutic target for the treatment of liver disorders.Other purinergic receptors, namely P2XRs and adenosine receptors (ARs), have also been reportedly involved in NASH/NALFD progression. P2X7R expression level was elevated in hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells of NASH model mice . In micepoptosis ,44. P2X7elevated . A separat liver . A2AAR aammation ,48, but ammation . Furtherammation . As a reammation . The antrol mice . The biogression ,53. A2BAty liver . In diabmp tests . Accordil levels . Furthertabolism . On a ty KO mice . The A2Btivities . WT micee levels . These r3AR in NAFLD/NASH. A3AR expression was reduced 1.9-fold in NAFLD patients’ livers compared to controls, indicating possible involvement by the receptor in NAFLD pathogenesis [3AR deficiency in mice given an HFD increased the expression of genes implicated in hepatic inflammation and steatosis [AR agonist prodrug (MRS7476) protected the STAM mouse model from the development of NASH [3AR agonist Cl-IB-MECA (namodenoson) was effective in treating NASH in mice [Recent research has underlined the significance of the Aogenesis . A3AR deteatosis . The res of NASH . MRS7476 in mice . The dru in mice .6R knockout slightly promotes NASH pathology. However, we have not investigated whether P2Y6R activation inhibits the progression of NASH. UDPβS and Up3U are likewise P2Y6R agonists [6R [6R agonists suppress the pathological progression of NASH.In this study, we show that P2Yagonists . Severalagonists . Severalists [6R . In the 6R in human NASH pathogenesis might in fact differ from that in mice. In this study, the CDAHFD model was used as the NASH model [A limitation of this study is that almost all experiments were analyzed using NASH/NAFLD model mice, when the role of P2YSH model . One of SH model . One disSH model ,64. CDAH6R expression level is increased in NAFLD patients according to the degree of inflammation and fibrosis. We also revealed using NASH model mice that knockout of the P2Y6R fails to improve (but instead aggravates) liver injury and inflammation. Our findings will prompt reconsideration of P2Y6R-G12 signaling as a therapeutic strategy for NASH.In conclusion, we demonstrated using an online database that P2YGEO RNA-Sequencing Experiments Interactive Navigator  was used6R KO mice were backcrossed onto C57BL/6J mice background, as described previously [6R KO and cohoused littermates as WT . Animals were maintained under a 12 h/12 h light/dark cycle.All animal care and experimental procedures used in this study were approved by the ethics committees at the Animal Care and Use Committee of Kyushu University. Systemic P2Yeviously ,14,15. Wg for 10 min.We fed male mice CDAHFD  or a control diet  for 6 weeks . After 66R was detected using rabbit anti-P2Y6R antibodies . The immunoreactivity of P2Y6R was detected using an Alexa Fluor 488-labeled goat anti-rabbit IgG antibody . Non-specific immunoreactivity was blocked with 10% normal goat serum, 1% BSA, and 0.3% Triton X-100 in PBS. After incubation with the secondary antibody, images were captured using a confocal laser-scanning microscope .For tissue sections, the liver tissue was fixed by 4% paraformaldehyde. Frozen sections (5 μm thick) were cut and prepared for immunofluorescent staining . The expWe measured serum levels of AST and ALT by using Fuji Dry-Chem NX5000  .We fixed the liver with 10% neutral buffered formalin and embedded these in paraffin. We performed hematoxylin and eosin (H&E) staining, as previously described . We quanWe extracted total RNA from the liver, as previously described . The RNAt tests (for two groups) or using the one-way ANOVA method with Tukey’s post hoc test (for three or more groups).Statistical analysis were performed by using GraphPad Prism 9.0  . All res