21年12月份发表在Gastroenterology (IF: 33.883) 上的文章

1. Immune Profiling of Pancreas Identified Highly Altered Monocyte Clusters and Dynamic Changes in CD206⁺ Macrophages During AP and Recovery

Fig 1a：作者使用腹腔注射雨蛙素 (50 mg/kg) 的方法构建了急性胰腺炎(AP)的模型，在0h, 12h, 24h, 48h, 168h这五个时间点取了小鼠的外周血和胰腺，对其中的白细胞进行了cyTOF检测（panel见文章最后）。

急性胰腺炎 Acute pancreatitis (AP)模型：

雨蛙素（Caerulein）是一种功能和组成上类似于胆囊收缩素（cholecystokinin，CCK）的胃调节分子，能够刺激胃、胆管和胰腺分泌。雨蛙素可用于研究由NF-κB上调蛋白如细胞间黏附分子（ICAM-1），炎症相关因子如NADPH氧化酶以及Janus激酶介导的信号转导途径。已经成功运用于大鼠、小鼠、狗和叙利亚仓鼠等动物急性胰腺炎（AP）模型的建立，建模机制在于：

• 通过刺激胞内NF-KB来上调胰腺腺泡细胞内的细胞间黏附分子（ICAM-1）表达。表面ICAM-1反过来促进中性粒细胞粘附到腺泡细胞上从而增强胰腺炎症效应；
• 通过诱使消化酶分泌失调和细胞质空泡化导致腺泡细胞死亡和胰腺水肿，从而诱导胰腺炎；
• 活化炎症促进因子。

Fig 1b：得到的所有细胞类型
Fig 1c-g：Monocytes的比例在损伤期 (12h, 24h) 出现了显著上升，在恢复期 (48h, 168h) 又下降；CD206⁺巨噬细胞则相反。

Fig 1

2. Seven Novel Monocyte Subsets Were Identified With Altered Expression of CD140a, MHCII, CD54, and Podoplanin in the Pancreas During AP and Recovery

Supplementary Figure 1：不同时间点的差异分析鉴定出一些AP和恢复期的差异基因，包括CD140a, MHCII, PDPN, CD54。

⚠️外周血单核细胞MHC分子的表达下调也出现在：
COVID-19： 重症COVID-19患者的外周免疫反应单细胞图谱；
急性川崎病：急性川崎病外周血PBMC单细胞测序；
BNT162B2诱导的心肌炎：单细胞测序解析BNT162B2诱导的心肌炎中经典单核细胞的转录改变；
trauma：Previous reports from trauma patients indicate a similar reduction in HLA-DR expression on monocytes during initial injury, with potential link to clinical outcome^1,2.

Fig 1h-i：使用MHCII, Ly6Gc, 和 CD45RB可以将单核细胞分为7个亚群。(Some monocyte subsets expressed
Ly6Gc, a neutrophil marker previously reported on murine monocytes)
Fig 1j：随后作者对这7个亚群进行了差异分析，鉴定出表达PDPN, CD140a, 和 CD54 的 MHCII^loLy6Gc^loCD45RB^lo 单核细胞 signatures。这个signature在12和24h降低，在48和168h恢复。（但是这群细胞占比不是先上升后下降的嘛？）

These novel pancreatic monocyte signatures may serve as a primer for future studies to develop immune markers of AP pathogenesis and rapid recovery.

Fig 1

3. Immune Profiling of Pancreas Identified Monocytes as Top Altered Immune Cells During SAP

Fig 2a ： 作者使用CDE diet构建了SAP模型，在0h, 24h, 48h, 72h这几个时间点取了胰腺和血的白细胞进行了cyTOF检测。

重症胰腺炎 severe AP (SAP)模型：

无胆碱乙硫氨酸饲料（choline-deficient diet with DL- ethionine, CDE diet）诱导的动物模型可出现腹水、缺氧、低血容量、酸中毒等症状，致病机制可能是乙硫氨酸干扰细胞内的甲硫氨酸的代谢，从而干扰了细胞膜磷脂的合成，通过饮食中胆碱缺乏的协同功能使这一作用加强，导致胰腺细胞外放作用受阻，雌性激素在其中起着重要作用，因此该模型一般选择雌鼠作为模型动物。
CDE方法建立的模型优势在于：成本低，造模非常简单，不需要外科手术，重复性高；另外通过限制喂养时间可控制获得不同水平的致死率，0%-100%之间，因此非常适合诊断试验和细胞生物学研究。另外其临床变化、生化过程及胰腺大体组织学特征与人体AP相似，非常适用于AP病因、病理生理研究，以及通过测定存活率、 生化特性、组织学、血细胞比容变化、pH值、血气来判断实验性治疗方法的潜力。

Fig 2b：得到的所有细胞类型
Fig 2c-f：Monocytes的比例随着时间而显著增加；CD206+巨噬细胞在72h也增加，但是没有统计学意义。

These findings indicate that the monocytes and their tissue influx likely play an important role in the pathogenesis of both mild AP and SAP.

Fig 2

4. SAP is Associated With Novel Monocyte Subsets With Altered Expression of Several Phenotypic and Functional Markers

Supplementary Figure 2：差异分析显示SAP中CD54, MHCII, PDPN, CD140a, CD196 和转录因子 T-bet, GATA-3, RoRgt 以及 TNF-a, LAP–TGFb 和 IL-10 出现时间依赖性下降。

Fig 2g-h：根据MHCII, Ly6Gc 和 CD45RB的表达可以将SAP的单核细胞分为6类。
Fig 2i：与AP和recovery相对，SAP单核的差异分析提示72h (和0h相比)，MHCII^loLy6Gc^hiCD45RB^lo单核的胞内和胞外marker (CD54, CD140a, CD196, PDPN, TNF-a, LAP-TGF-b, T-bet 和 RoRgt) 表达显著降低。

CD196 (CCR6) is a trafficking receptor implicated in promoting inflammation in mice, Thus, CD196 expression herein might suggest its involvement in monocytes recruitment in SAP.

Fig 2j：而72h时，MHCII^hiLy6Gc^hi的CD196和TNFa显著降低。
Fig 2k：MHCII^loLy6Gc^loCD45RB^lo单核的CD54在48h增加，在72h又出现下降。

Fig 2

Our data also suggests that pancreatic monocytes in SAP are more het- erogeneous in nature with distinct transcriptional regula- tion profiles and functional marker expression and are associated with sustained local inflammatory responses, thereby contributing to disease progression from mild to SAP.

5. Seven Novel Subsets of CD206þ Macrophage With Distinct Marker Expression Profile Were Identified in the Pancreas During AP, Recovery, and SAP

前面主要是关注了单核细胞，在这一部分，作者主要关注了CD206⁺巨噬细胞（Fig 1 E, F中存在着随时间点的比例变化）。

Supplementary Fig 3：差异分析结果显示在AP的急性期和恢复期一些分子如MHCII, CD54, CD44, PDPN, LAP-TGF-b存在着显著的表达变化。(MHCII is a known activation marker for macrophages; whereas CD44 is an adhesion molecule expressed on alveolar macrophages.)

Fig 3a-b：Unbiased profiling 鉴定出CD206⁺巨噬细胞可以根据MHCII, Ly6Gc 和 CD44 的表达分为7个亚群。
差异分析的结果提示CD206⁺巨噬细胞的CD54, PDPN 和 IL22的表达存在有趣的趋势。
Fig 3c：MHCII^hiLy6Gc^loCD44^hi CD206⁺巨噬的CD54和IL22在12h显著下降，24-48h又恢复。PDPN的水平则在24和48h都显著升高。
Fig 3d-e：MHCII^hiLy6Gc^loCD44^lo 和 MHCII^loLy6Gc^loCD44^lo亚群则在12h时CD54表达增加，168h又下降。
Fig 3f：MHCII^loLy6Gc^loCD44^hi亚群和MHCII^hiLy6Gc^loCD44^hi亚群一样，存在着PDPN的表达增加，但是其LAP-TGF-b表达在24h时也显著增加，在168h时恢复正常。

The differential phenotypic and functional marker expression patterns on distinct CD206⁺ macrophage clusters during disease course likely reflect functional diversification of these macrophages in response to microenvironmental cues and crosstalk between the cell populations to fulfill a tissue level need for specialized functions during disease evolution and recovery.

作者对SAP的CD206⁺ 巨噬也进行了分析，结果放在Supplementary Figure 4。

6. Novel Monocyte Subsets are Also Present in Circulation During AP, Recovery, and SAP

前面都是小鼠胰腺的数据，作者对AP小鼠外周血的数据也进行了分析。
Fig 4a-b：AP小鼠外周单核细胞在12h时显著增加，随后又下降。
Fig 4c：和胰腺单核类似，外周血的单核细胞分为了7个亚群。
Fig 4d：外周血单核占比在12h达到peak （在胰腺则是48h）
Fig 4e-h：分别描述了不同亚群的时间差异性表达的分子

However, DE analysis of cir- culatory monocyte subsets did not identify any signifi- cantly (FDR adjusted) altered markers in the blood of the SAP mice. Overall, these novel monocyte subsets with distinct signatures during AP progression and recovery highlight monocytes as key dynamic immune subsets in pancreas and circulation. Thus, the disease can be studied and monitored with less invasiveness via repeated blood sampling.

7. Flow Cytometry Validates Novel Monocyte Subsets Identified Using CyTOF Analysis in the Pancreas and Blood

为了验证CyTOF的结果，作者对AP模型的不同时间点进行了流式检测，结果和CyTOF较为一致。

8. CyTOF Analysis Identified 6 Novel Subsets Among the Abundant Inflammatory Monocytes (CD14⁺CD16^-) in Patients With Pancreatitis

Fig 6a：最后作者对AP(n=12)和 recurrent AP (RAP; n=11) 的外周血进行了CyTOF检测。
Fig 6b-c：AP和RAP患者外周血中经典单核的占比都是最大的。
Fig 6d-e：根据cyTOF的结果，作者根据IL1b, IL27 和 CD11c的表达将外周血单核分为了6个亚群。

The findings in human AP are similar to observations in our mouse models in that inflammatory monocytes were the most significantly altered cells during pancreatitis. It high- lights monocytes as important and heterogenous subsets in mouse and human pancreatitis that can be monitored in the blood. Future studies focused on circulating monocytes with frequent blood sampling from patients with AP to evaluate temporal immune compartment changes during disease progression and recovery will be transformative in our understanding of AP pathogenesis.

讨论

This study has several strengths:
(1) extensive differently timed sampling reflecting various disease states (AP, recovery, and SAP) using 2 independent AP models, (2) simultaneous analysis of blood and tissue immune responses during acute injury and recovery offers unparalleled insights into local and peripheral dynamics that can be translated to human disease where tissue access is prohibitive, and (3) evaluation of circulating monocytes in blood of patients with AP and RAP demonstrates evidence of novel monocyte populations and heterogeneity similar to experimental models.

质谱流式panel

参考文献：

1. Hershman MJ, Cheadle WG, Wellhausen SR, et al. Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient. Br J Surg 1990; 77:204–207.
2. Haupt W, Riese J, Mehler C, et al. Monocyte function before and after surgical trauma. Dig Surg 1998; 15:102–104.