Single-cell transcriptome profiling of sepsis identifies HLA-DR^(low)S100A^(high)monocytes with immunosuppressive function

Background Sustained yet intractable immunosuppression is commonly observed in septic patients,resulting in aggravated clinical outcomes.However,due to the substantial heterogeneity within septic patients,precise indicators in deciphering clinical trajectories and immunological alterations for septic patients remain largely lacking.Methods We adopted cross-species,single-cell RNA sequencing(scRNA-seq)analysis based on two published datasets containing circulating immune cell profile of septic patients as well as immune cell atlas of murine model of sepsis.Flow cytometry,laser scanning confocal microscopy(LSCM)imaging and Western blotting were applied to identify the presence of S100A9^(+)monocytes at protein level.To interrogate the immunosuppressive function of this subset,splenic monocytes isolated from septic wild-type or S100a9^(–/–)mice were co-cultured with naive CD4^(+)T cells,followed by proliferative assay.Pharmacological inhibition of S100A9 was implemented using Paquinimod via oral gavage.Results scRNA-seq analysis of human sepsis revealed substantial heterogeneity in monocyte compartments following the onset of sepsis,for which distinct monocyte subsets were enriched in disparate subclusters of septic patients.We identified a unique monocyte subset characterized by high expression of S100A family genes and low expression of human leukocyte antigen DR(HLA-DR),which were prominently enriched in septic patients and might exert immunosuppressive function.By combining single-cell transcriptomics of murine model of sepsis with in vivo experiments,we uncovered a similar subtype of monocyte significantly associated with late sepsis and immunocompromised status of septic mice,corresponding to HLA-DR^(low)S100A^(high)monocytes in human sepsis.Moreover,we found that S100A9^(+)monocytes exhibited profound immunosuppressive function on CD4^(+)T cell immune response and blockade of S100A9 using Paquinimod could partially reverse sepsis-induced immunosuppression.Conclusions This study identifies HLA-DR^(low)S100A^(high)monocytes correlated with immunosuppressive state upon septic challenge,inhibition of which can markedly mitigate sepsis-induced immune depression,thereby providing a novel therapeutic strategy for the management of sepsis.

Association between HLA-DR Expression and Multidrug-resistant Infection in Patients with Severe Acute Pancreatitis

Multidrug-resistant （MDR） bacterial infection is a common complication of severe acute pancreatitis （SAP）. This study aimed to explore the association between human leukocyte antigen-antigen D-related （HLA-DR） expression and multidrug-resistant infection in patients with SAP. A total of 24 SAP patients who were admitted to Nanjing Drum Tower Hospital between May 2015 and December 2016 were enrolled in the study. The percentages of CD4^＋, CD8^＋, natural killer （NK）, and HLA-DR （CD14＋） cells and the CD4^＋/CD8^＋ cell ratio on days 1, 7, 14, and 28 after admission were determined by flow cytometry. Eighteen patients presented with the symptoms of infection. Among them, 55.6% patients （10/18） developed MDR infection. The most common causative MDR organisms were Enterobacter cloacae and Acinetobacter baumannii. The CD4＋/CD8＋ cell ratio and the percentage of NK cells were similar between patients with non-MDR and patients with MDR infections. In patients without infection, the HLA-DR percentage was maintained at a high level throughout the 28 days. Compared to the patients without any infection, the HLA-DR percentage in patients with non-MDR infection was reduced on day 1 but increased and reached similar levels on day 28. In patients with MDR infection, the HLA-DR percentage remained below normal levels at all-time points. It was concluded that persistent down-regulation of HLA-DR expression is associated with MDR bacterial infection in patients with SAP.

Expression of HLA-DR genes in gliomas: correlation with clinicopathological features and prognosis

Background: Human leukocyte antigen (HLA)-DR is a classical major histocompatibility complex (MHC) class II molecule encoded by five genes: HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5. The current study aimed to investigate the role of these genes in gliomas by analyzing microarray data. Methods: We enrolled 305 patients with histologically confirmed gliomas, and performed microarray data analysis along with studying their clinical characteristics. A new variable, termed HLA-DR score, was defined to explain the expression information of all five HLA-DR genes by factor analysis. HLA-DR scores in each grade of glioma and normal brain tissue were compared using one-way ANOVA. Lastly, correlations of HLA-DR scores with progression-free survival (PFS) and overall survival (OS) were analyzed with Kaplan-Meier and Cox analysis. Results: Our study indicated that an increased HLA-DR score, i.e. overexpression of HLA-DR genes, was correlated with a more aggressive glioma tumor grade (p < 0.001, One-way ANOVA). Moreover, the HLA-DR score was significantly higher in astrocytic tumors than oligodendroglial tumors (?0.718 ± 3.177 versus ?2.975 ± 2.662, t-test) in low-grade gliomas (LGGs). Kaplan-Meier analysis of both PFS (p = 0.046, log-rank test; p = 0.021, Breslow test) and OS (p = 0.029, Breslow test) showed significant differences in the clinical outcomes between LGG patients with high versus low HLA-DR scores. Finally, the HLA-DR score was further identified to be an independent prognostic factor of clinical outcomes by multivariate analysis (p = 0.042 and p = 0.025, for PFS and OS, respectively) in LGG patients. Conclusion: Expression of HLA-DR genes can be used to predict the tumor grade in gliomas, and the histological subtype in LGG. Furthermore, they are also an independent predictor for LGG patient survival.

不同乙型肝炎病毒感染者中人类白细胞抗原DR基因的表达水平

目的研究不同乙型肝炎病毒（hepatitis B virus，HBV）感染转归者外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）中HLA-DR mRNA表达水平的差异。方法收集哈尔滨医科大学附属第二医院和哈尔滨市传染病院2012年5月至2015年11月感染科就诊的急性、慢性和隐性自限性HBV感染者101例和体检中心健康体检者（包括HBV疫苗免疫者和乙肝五项全阴者）53例；采用荧光定量PCR法检测了其外周血血浆中HBV-DNA含量和PBMCs中HLA-DR mRNA表达水平。结果在急性、慢性、隐性自限、疫苗免疫和五项全阴五组外周血PBMCs中HLA-DR mRNA的表达水平在隐性自限组最高，五项全阴组最低，总体比较差异显著（F＝3.901，P＝0.005）。其中，隐性自限组明显高于慢性组[（2.57±1.65）比（1.86±1.58），t＝2.016，P＝0.047]、疫苗免疫组[（2.57±1.65）比（1.69±1.35），t＝2.383，P＝0.020]和五项全阴组[（2.57±1.65）比（0.94±0.61），t＝4.881，P＝0.000]，而临界高于急性组[（2.57±1.65）比（1.39±1.23），t＝1.780，P＝0.084]；慢性组和疫苗免疫组也都有意义地高于五项全阴组[（1.86±1.58）比（0.94±0.61），t＝3.740，P＝0.000和（1.69±1.35）比（0.94±0.61），t＝2.215，P＝0.008]。在慢性HBV感染组，不同HBV-DNA水平组HLA-DR表达量整体无差异（F＝1.204，P＝0.319），但在（1.000-9.999）e＋005组HLA-DR表达量最高，临界意义地高于（5.000-9.999）e＋006组和≥ 1.000e＋007组[（1.000-9.999）e＋005比（5.000-9.999）e＋006，t＝1.769，P＝0.099；（5.000-9.999）e＋005比≥1.000e＋007，t＝2.114，P＝0.053]；在携带、轻度、中度和重度慢性HBV感染分组中，以及HBeAg^＋和HBeAg^－分组中HLA-DR表达量总体和两组间均无统计学差异。结论不同HBV感染和疫苗免疫均诱导了HLA-DR表达量增高，尤其在清除HBV感染状态下；在慢性HBV感染中HLA-DR表达可能与HBV-DNA水平相关。

Arachnoid cell involvement in the mechanism of coagulation-initiated inflammation in the subarachnoid space after subarachnoid hemorrhage

Objective： To assess if arachnoid cells have the capability to present antigen and activate T-lymphocytes after stimulation by bloody cerebrospinal fluid （CSF）, and to illuminate the mechanism of coagulation-initiated inflammation in the subarachnoid space after subarachnoid hemorrhage （SAH）. Methods： Arachnoid cells were cultured, characterized, and examined by immunofluorescence for the basal expression of human leukocyte antigen-DR （HLA-DR）, Expression of HLA-DR, after co-culturing arachnoid cells in vitro with bloody CSF, was investigated by immunofluorescence and flow cytometry （FCM）. The variation of arachnoid cells＇ ultrastructure was observed by transmission electron microscope （TEM）. Arachnoid cells were co-cultured with peripheral blood mononuclear cells （PBMCs）. The content of soluble interleukin-2 receptor （slL-2r） in culture medium was detected by enzyme-linked immunosorbent assay （ELISA）. Results： （1） Arachnoid cells were successfully cultured for many passages. The immunofluorescent staining was positive for HLA-DR in over 95% of the human arachnoid cells. The punctate HI_A-DR was distributed in cytoplasm and not in the karyon. （2） After co-culturing arachnoid cells in vitro with bloody CSF, numerous particles with strong fluorescence appeared in the cytoplasm on Day 6. On Day 8, the quantity of particles and fluorescent intensity were maximal. FCM showed that the percentage of HLA-DR expressing cells was （2.5＋_0.4）% at the first 5 d, increasing to （60.8＋_3.6）% on Day 7. （3） After co-culturing arachnoid cells in vitro with bloody CSF, many lysosome and secondary lysosome particles were present in the cytoplasm. Hyperplasia of rough endoplasmic re- ticulum and enlarged cysts were observed, with numerous phagocytizing vesicles also observed at the edge of the arachnoid cells. （4） Arachnoid cells stimulated by bloody CSF were co-cultured in vitro with PBMCs. The content of slL-2r in the culture medium, having been maintained at around 1.30 ng/ml during the first 3 d, had increased by Day 4. The content of slL-2r peaked 7.53 ng/ml on Day 7 and then reduced gradually. Conclusions： （1） Basic HLA-DR expression is present in arachnoid cells. （2） After stimulation by bloody CSF, arachnoid cells have the potential to serve as antigen presenting cells （APCs） and the ability to activate T-lymphocytes, indicating that arachnoid cells are involved in the mechanism of coagulation-initiated inflammation in the subarachnoid space after SAH.