21年12月份發(fā)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) 的方法構(gòu)建了急性胰腺炎(AP)的模型，在0h, 12h, 24h, 48h, 168h這五個時間點(diǎn)取了小鼠的外周血和胰腺栖忠，對其中的白細(xì)胞進(jìn)行了cyTOF檢測（panel見文章最后）崔挖。

急性胰腺炎 Acute pancreatitis (AP)模型：

雨蛙素（Caerulein）是一種功能和組成上類似于膽囊收縮素（cholecystokinin，CCK）的胃調(diào)節(jié)分子庵寞，能夠刺激胃狸相、膽管和胰腺分泌。雨蛙素可用于研究由NF-κB上調(diào)蛋白如細(xì)胞間黏附分子（ICAM-1）捐川，炎癥相關(guān)因子如NADPH氧化酶以及Janus激酶介導(dǎo)的信號轉(zhuǎn)導(dǎo)途徑脓鹃。已經(jīng)成功運(yùn)用于大鼠、小鼠古沥、狗和敘利亞倉鼠等動物急性胰腺炎（AP）模型的建立瘸右，建模機(jī)制在于：

• 通過刺激胞內(nèi)NF-KB來上調(diào)胰腺腺泡細(xì)胞內(nèi)的細(xì)胞間黏附分子（ICAM-1）表達(dá)娇跟。表面ICAM-1反過來促進(jìn)中性粒細(xì)胞粘附到腺泡細(xì)胞上從而增強(qiáng)胰腺炎癥效應(yīng)；
• 通過誘使消化酶分泌失調(diào)和細(xì)胞質(zhì)空泡化導(dǎo)致腺泡細(xì)胞死亡和胰腺水腫太颤，從而誘導(dǎo)胰腺炎苞俘；
• 活化炎癥促進(jìn)因子。

Fig 1b：得到的所有細(xì)胞類型
Fig 1c-g：Monocytes的比例在損傷期 (12h, 24h) 出現(xiàn)了顯著上升龄章，在恢復(fù)期 (48h, 168h) 又下降吃谣；CD206⁺巨噬細(xì)胞則相反。

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：不同時間點(diǎn)的差異分析鑒定出一些AP和恢復(fù)期的差異基因瓦堵，包括CD140a, MHCII, PDPN, CD54基协。

??外周血單核細(xì)胞MHC分子的表達(dá)下調(diào)也出現(xiàn)在：
COVID-19： 重癥COVID-19患者的外周免疫反應(yīng)單細(xì)胞圖譜；
急性川崎病：急性川崎病外周血PBMC單細(xì)胞測序菇用；
BNT162B2誘導(dǎo)的心肌炎：單細(xì)胞測序解析BNT162B2誘導(dǎo)的心肌炎中經(jīng)典單核細(xì)胞的轉(zhuǎn)錄改變澜驮；
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可以將單核細(xì)胞分為7個亞群。(Some monocyte subsets expressed
Ly6Gc, a neutrophil marker previously reported on murine monocytes)
Fig 1j：隨后作者對這7個亞群進(jìn)行了差異分析惋鸥，鑒定出表達(dá)PDPN, CD140a, 和 CD54 的 MHCII^loLy6Gc^loCD45RB^lo 單核細(xì)胞 signatures杂穷。這個signature在12和24h降低，在48和168h恢復(fù)卦绣。（但是這群細(xì)胞占比不是先上升后下降的嘛耐量？）

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構(gòu)建了SAP模型，在0h, 24h, 48h, 72h這幾個時間點(diǎn)取了胰腺和血的白細(xì)胞進(jìn)行了cyTOF檢測滤港。

重癥胰腺炎 severe AP (SAP)模型：

無膽堿乙硫氨酸飼料（choline-deficient diet with DL- ethionine, CDE diet）誘導(dǎo)的動物模型可出現(xiàn)腹水廊蜒、缺氧、低血容量溅漾、酸中毒等癥狀山叮，致病機(jī)制可能是乙硫氨酸干擾細(xì)胞內(nèi)的甲硫氨酸的代謝，從而干擾了細(xì)胞膜磷脂的合成添履，通過飲食中膽堿缺乏的協(xié)同功能使這一作用加強(qiáng)屁倔，導(dǎo)致胰腺細(xì)胞外放作用受阻，雌性激素在其中起著重要作用暮胧，因此該模型一般選擇雌鼠作為模型動物锐借。
CDE方法建立的模型優(yōu)勢在于：成本低，造模非常簡單往衷，不需要外科手術(shù)钞翔，重復(fù)性高；另外通過限制喂養(yǎng)時間可控制獲得不同水平的致死率席舍，0%-100%之間嗅战，因此非常適合診斷試驗(yàn)和細(xì)胞生物學(xué)研究。另外其臨床變化、生化過程及胰腺大體組織學(xué)特征與人體AP相似驮捍，非常適用于AP病因、病理生理研究脚曾，以及通過測定存活率东且、 生化特性、組織學(xué)本讥、血細(xì)胞比容變化珊泳、pH值、血?dú)鈦砼袛鄬?shí)驗(yàn)性治療方法的潛力拷沸。

Fig 2b：得到的所有細(xì)胞類型
Fig 2c-f：Monocytes的比例隨著時間而顯著增加色查；CD206+巨噬細(xì)胞在72h也增加，但是沒有統(tǒng)計(jì)學(xué)意義撞芍。

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：差異分析顯示SAP中CD54, MHCII, PDPN, CD140a, CD196 和轉(zhuǎn)錄因子 T-bet, GATA-3, RoRgt 以及 TNF-a, LAP–TGFb 和 IL-10 出現(xiàn)時間依賴性下降秧了。

Fig 2g-h：根據(jù)MHCII, Ly6Gc 和 CD45RB的表達(dá)可以將SAP的單核細(xì)胞分為6類。
Fig 2i：與AP和recovery相對序无，SAP單核的差異分析提示72h (和0h相比)验毡，MHCII^loLy6Gc^hiCD45RB^lo單核的胞內(nèi)和胞外marker (CD54, CD140a, CD196, PDPN, TNF-a, LAP-TGF-b, T-bet 和 RoRgt) 表達(dá)顯著降低。

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時帝嗡，MHCII^hiLy6Gc^hi的CD196和TNFa顯著降低晶通。
Fig 2k：MHCII^loLy6Gc^loCD45RB^lo單核的CD54在48h增加，在72h又出現(xiàn)下降哟玷。

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 CD206t Macrophage With Distinct Marker Expression Profile Were Identified in the Pancreas During AP, Recovery, and SAP

前面主要是關(guān)注了單核細(xì)胞狮辽，在這一部分，作者主要關(guān)注了CD206⁺巨噬細(xì)胞（Fig 1 E, F中存在著隨時間點(diǎn)的比例變化）巢寡。

Supplementary Fig 3：差異分析結(jié)果顯示在AP的急性期和恢復(fù)期一些分子如MHCII, CD54, CD44, PDPN, LAP-TGF-b存在著顯著的表達(dá)變化喉脖。(MHCII is a known activation marker for macrophages; whereas CD44 is an adhesion molecule expressed on alveolar macrophages.)

Fig 3a-b：Unbiased profiling 鑒定出CD206⁺巨噬細(xì)胞可以根據(jù)MHCII, Ly6Gc 和 CD44 的表達(dá)分為7個亞群。
差異分析的結(jié)果提示CD206⁺巨噬細(xì)胞的CD54, PDPN 和 IL22的表達(dá)存在有趣的趨勢讼渊。
Fig 3c：MHCII^hiLy6Gc^loCD44^hi CD206⁺巨噬的CD54和IL22在12h顯著下降动看，24-48h又恢復(fù)。PDPN的水平則在24和48h都顯著升高爪幻。
Fig 3d-e：MHCII^hiLy6Gc^loCD44^lo 和 MHCII^loLy6Gc^loCD44^lo亞群則在12h時CD54表達(dá)增加菱皆，168h又下降。
Fig 3f：MHCII^loLy6Gc^loCD44^hi亞群和MHCII^hiLy6Gc^loCD44^hi亞群一樣挨稿，存在著PDPN的表達(dá)增加仇轻，但是其LAP-TGF-b表達(dá)在24h時也顯著增加，在168h時恢復(fù)正常奶甘。

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.

作者對SAP的CD206⁺ 巨噬也進(jìn)行了分析篷店，結(jié)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數(shù)據(jù)，作者對AP小鼠外周血的數(shù)據(jù)也進(jìn)行了分析疲陕。
Fig 4a-b：AP小鼠外周單核細(xì)胞在12h時顯著增加方淤，隨后又下降。
Fig 4c：和胰腺單核類似蹄殃，外周血的單核細(xì)胞分為了7個亞群携茂。
Fig 4d：外周血單核占比在12h達(dá)到peak （在胰腺則是48h）
Fig 4e-h：分別描述了不同亞群的時間差異性表達(dá)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

為了驗(yàn)證CyTOF的結(jié)果，作者對AP模型的不同時間點(diǎn)進(jìn)行了流式檢測诅岩，結(jié)果和CyTOF較為一致讳苦。

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

Fig 6a：最后作者對AP(n=12)和 recurrent AP (RAP; n=11) 的外周血進(jìn)行了CyTOF檢測。
Fig 6b-c：AP和RAP患者外周血中經(jīng)典單核的占比都是最大的吩谦。
Fig 6d-e：根據(jù)cyTOF的結(jié)果鸳谜，作者根據(jù)IL1b, IL27 和 CD11c的表達(dá)將外周血單核分為了6個亞群。

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.

討論

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.

質(zhì)譜流式panel

參考文獻(xiàn)：

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.