七月份 (22.7.22) 發(fā)表在Science Immunology (IF: 30.630) 的文章
主要關(guān)注了TREM2巨噬細(xì)胞稠炬。之前寫過的梗死心臟中巨噬細(xì)胞的時空動力學(xué)和Trem2hi巨噬細(xì)胞的潛在功能也是關(guān)注的TREM2巨噬壮虫。
1. Cell types recovered in lesional and nonlesional samples of patients with acne
作者對6例活動期痤瘡患者的early lesional (papules) 和非損傷的皮膚進(jìn)行了10x單細(xì)胞測序。在質(zhì)控之后浮还，兩組分別得到了32966和29202個細(xì)胞桑孩。
Fig 1a-c：所有細(xì)胞的注釋拜鹤，得到7種細(xì)胞群。c圖展示了7個細(xì)胞類型中12個樣本來源的細(xì)胞占比變化流椒。（為什么不展示每個樣本各個細(xì)胞類型的變化敏簿？）
Fig 1d-f：對髓系細(xì)胞進(jìn)行了進(jìn)一步細(xì)分，得到6個細(xì)胞亞群。其中TREM2+巨噬細(xì)胞群占比很大且?guī)缀踔辉诓±龢颖局写嬖凇?br>
Fig 1g-i：對角質(zhì)細(xì)胞也進(jìn)行了亞群細(xì)分惯裕，得到5個亞群温数，看了一下比例變化。
2. TREM2 macrophages in acne lesions express lipid metabolism and inflammatory genes
We focused on the role of TREM2 macrophage in acne because they were abundant in acne lesions as opposed to nonlesional skin.
Fig 2a：展示了M1樣巨噬轻猖、M2樣巨噬和TREM2+巨噬的marker基因帆吻。
Fig 2b：TREM2+巨噬特異性表達(dá)多種脂質(zhì)相關(guān)蛋白域那。
Fig 2c：和正常組相比咙边，病例組的TREM2+巨噬高表達(dá)多種促炎性因子和趨化因子。
Fig 2d：6種髓系細(xì)胞的擬時序次员，發(fā)現(xiàn)M2樣巨噬細(xì)胞可以向TREM2+巨噬和CD1C+ DC分化败许，但是和M1樣巨噬不存在明顯分化關(guān)系。
Fig 2e-f：M2樣巨噬細(xì)胞和TREM2+巨噬的分化關(guān)系
Fig 2g：M2樣巨噬細(xì)胞向TREM2+巨噬的分化之后淑蔚，促炎性增強(qiáng)市殷。
These data suggest a trajectory linking M2-like to TREM2 macrophages, characterized by the increased expression of proinflammatory genes in the TREM2 macrophages.
3. KCs in the hair follicles in acne lesions are capable of squalene synthesis
由于毛囊KCs是毛囊皮脂腺的重要組成部分，作者隨后探究了它們的基因表達(dá)刹衫。
Fig 3a：鯊烯合成相關(guān)基因FDFT1和SQLE在一些亞群中高表達(dá)醋寝。
Fig 3b：高表達(dá)鯊烯合成相關(guān)基因的KCs在Acne組表達(dá)顯著比control要高，而且與TREM2巨噬細(xì)胞存在空間共定位带迟。
4. Spatial and Seq-Scope sequencing spatially localize cell populations in acne
為了進(jìn)一步探究細(xì)胞間的空間位置關(guān)系音羞，作者進(jìn)行了空間轉(zhuǎn)錄組測序。
用的是
Seq-Scope(using as low as0.5 umspot-to-spot resolution, compared with the 100 um spot-to-spot resolution of the 10X Visium platform)仓犬。
??10x的spots大小是55um嗅绰，spots-spots之間的距離是100um。Seq-Scope的spots-spots之間的距離是0.5um搀继，精度還是很高的窘面。（類似10X納米球技術(shù)？）
Fig 3c：在55-um大小下（和10x一樣）叽躯，得到394個spots财边，平均每個spots測到1349 genes 和 2950 transcripts（好少），分為六種細(xì)胞点骑。
10X一般每個spot基因中位數(shù)都是3000以上制圈，這個測到的基因數(shù)好少哦
Fig 3d：在切片上可以觀測到縱向的毛囊結(jié)構(gòu)，周圍被炎癥細(xì)胞包繞畔况。通過調(diào)整分辨率鲸鹦，作者發(fā)現(xiàn)KRT5+ KCs存在于真皮的基底層和毛囊的outer root sheath。KRT16+ KCs則存在于毛囊的inner root sheath跷跪。1um分辨率下馋嗜，作者發(fā)現(xiàn)在毛囊的TREM2outer root sheath中，巨噬細(xì)胞和 KRT5+ KCs 相鄰近吵瞻。
Fig 3e：基因表達(dá)也顯示KRT5 和 KRT16的表達(dá)與TREM2巨噬的marker基因APOE相鄰近葛菇。
Fig 3f：免疫熒光的結(jié)果顯示TREM2巨噬和痤瘡丙酸桿菌(C.acnes)的抗體存在共定位甘磨。
These data indicate that TREM2 macrophages containing C. acnes antigens are located near hair follicle epithelium expressing squalene oxidase.
5. Squalene induces TREM2 expression in macrophages in vitro
隨后作者進(jìn)行了體外試驗(yàn)。首先作者想要去建立in vitro model of TREM2 macrophages in acne lesions眯停。為了達(dá)到這個目的济舆，作者使用Ingenuity Pathway Analysis (IPA)分析了TREM2巨噬細(xì)胞群，鑒定了可能驅(qū)動TREM2巨噬細(xì)胞分化的上游調(diào)節(jié)分子莺债，得到了IFNG, IL13, TNF, IL1B, 和 IL4滋觉。其中IL4已被證實(shí)可以與M-CSF一起在體外誘導(dǎo)TREM2的表達(dá)。
Fig 4a：作者首先檢測了這些分子誘導(dǎo)巨噬細(xì)胞的TREM2表達(dá)的能力（with and without M-CSF）齐邦，發(fā)現(xiàn)在M-CSF存在的條件下椎侠，鯊烯誘導(dǎo)TREM2形成的能力顯著上升。
也就是說措拇，M-CSF誘導(dǎo)單核向M2巨噬分化之后我纪，鯊烯可以誘導(dǎo)M2巨噬向TREM2巨噬分化。
Fig 4b：用上述方法誘導(dǎo)的TREM2巨噬細(xì)胞的基因表達(dá)與單細(xì)胞測序檢測到的一些關(guān)鍵基因表達(dá)類似丐吓。
Fig 4c：隨后作者對squalene/M-CSF體外誘導(dǎo)的TREM2巨噬細(xì)胞進(jìn)行了單細(xì)胞測序浅悉，并和此前人的樣本中檢測到的M1, M2和TREM2巨噬細(xì)胞進(jìn)行了比較，發(fā)現(xiàn)和TREM2巨噬細(xì)胞表達(dá)譜類似券犁。
Fig 4d：胞內(nèi)脂滴染色結(jié)果顯示和IFN-r/LPS–treated M1-like macrophages相比术健，squalene/M-CSF–treated macrophages were replete with lipids as measured by BODIPY。
6. Squalene blocks oxidative killing of C. acnes
由于M1樣巨噬細(xì)胞可以對痤瘡丙酸桿菌C. acnes產(chǎn)生抗菌反應(yīng)族操，作者隨后比較了IFN-r/LPS巨噬細(xì)胞（M1）和squalene-derived TREM2巨噬細(xì)胞的抗菌反應(yīng)苛坚。
痤瘡相關(guān)菌株：HL005PA1, HL043PA1, HL096PA1（IA-2型）；
健康相關(guān)菌株：HL042PA3, HL110PA3, HL11-PA4（II型）色难；
Fig 4e：結(jié)果顯示M1巨噬對痤瘡相關(guān)菌株(和健康相關(guān)菌株相比)顯示出更強(qiáng)的抗菌性泼舱。squalene-derived TREM2的抗菌性則很低。
Fig 4f：隨后作者探究了鯊烯存在的情況下M1和M2巨噬細(xì)胞的抗菌性枷莉，發(fā)現(xiàn)都出現(xiàn)顯著下降娇昙。
Squalene consists of a methyl group attached to six double bonds that act as a highly effective reactive oxygen species (ROS) scavenger with a rate constant much larger than other lipids on human skin. 此外，鯊烯在體外可以降低胞內(nèi)ROS和ultraviolet-induced ROS水平笤妙。巨噬細(xì)胞使用phagosomes中的ROS和 reactive nitrogen intermediates (RNIs)來殺傷胞內(nèi)病原體冒掌。
Fig 4g：為了探究是哪種intermediates發(fā)揮了針對C. acnes的抗菌作用，我們使用了NO-donor diethylenetriamine NONOate (DETA NONOate)和兩種oxygen donors, alkyl peroxide tertiary-butyl hydroperoxide (TBHP) and hydrogen peroxide (H2O2)蹲盘，結(jié)果顯示和DETA NONOate相比股毫，C. acnes對ROS的殺傷更敏感。
Fig 4h：Squalene was able to block the killing of C. acnes in the presence of TBHP at similar efficacy to the oxygen scavenger N-acetyl cysteine (NAC), which served as a control.
Together, these data suggest that squalene, which is overproduced in acne lesions, induces TREM2 macrophages with enhanced phagocytic capacity for lipids and C. acnes, but scavenges oxygen radicals, thus blocking the macrophage antimicrobial response. These TREM2 macrophages are not able to reduce the bacterial load but secrete IL-18 and up-regulate chemokine expression, thus contributing to the inflammation that is characteristic at the site of disease
