16年8月的一篇JCI Insight, IF: 9.484

為了探究心肌缺血灌注損傷時調(diào)控中性粒細(xì)胞trafficking的upstream signals，作者使用了小鼠心臟移植模型（An essential advantage of this system is the ability to resolve the roles of resident (donor) and recruited (recipient) immune cell populations.）+雙光子血管內(nèi)成像。

1. Heart-resident CCR2+ monocytes and monocyte-derived macrophages are critical to promote extravasation of neutrophils into cardiac tissue during ischemia reperfusion injury.

在此前的研究中，作者團(tuán)隊發(fā)現(xiàn)心臟存在不同的巨噬細(xì)胞亞群。因此作者首先想要去探究組織原位巨噬細(xì)胞在心臟移植后是否參與中性粒的招募。

作者此前的研究：We have previously demonstrated that a single dose of clondronate liposomes is sufficient to deplete resident cardiac macrophages
氯膦酸鹽脂質(zhì)體不是清除外周趨化來的巨噬的嗎？

作者在移植手術(shù)24h前對B6 WT donor小鼠給予了氯膦酸鹽脂質(zhì)體清除了組織原位巨噬細(xì)胞，并將其心臟移植給了syngeneic LysM-GFP neutrophil reporter hosts。對照組小鼠給予的是PBS脂質(zhì)體。在reperfusion 2h后使用了雙光子血管內(nèi)成像 (We have previously demonstrated that LysM-GFP nearly exclusively labels neutrophils in the donor heart at this time point)。在移植后，移植了給予PBS liposomes心臟的受體鼠中性粒細(xì)胞被趨化到大冠狀靜脈，在這里中性粒細(xì)胞slow down, 黏附到血管, crawl, 并遷移到心臟組織。而移植了給予氯膦酸鹽脂質(zhì)體心臟的受體鼠中性粒細(xì)胞向心臟的趨化減少（B圖可以明顯看到中性細(xì)胞在血管壁黏附增加，但不能透過血管）。此外，中性粒的rolling沒有受影響，但是crawl顯著下降了。

LysM-GFP不是髓系示蹤的嘛，移植了清除巨噬的心臟過去之后，外周帶GFP的中性粒和巨噬都可以趨化過去被看到啊，為啥只說是中性粒呢？

為了定義相關(guān)組織原位巨噬細(xì)胞群，作者將B6 CD45.2⁺ WT心臟移植到congenic B6 CD45.1+ WT recipients鼠，在移植2小時后檢測了donor心臟巨噬細(xì)胞。
Fig 2A-B：鑒定出了donor-derived MHCII^loCCR2^–和 MHCII^hiCCR2^–巨噬細(xì)胞、MHCII^hiCCR2⁺巨噬細(xì)胞和MHCII^loCCR2⁺單核細(xì)胞（圖B和D標(biāo)反了吧...）。

在此前的研究中，作者發(fā)現(xiàn)和胚胎來源的 CCR2^–巨噬細(xì)胞 相比，CCR2⁺單核細(xì)胞來源的巨噬細(xì)胞在受損心臟中擴(kuò)增并表達(dá)高水平促炎細(xì)胞因子和趨化因子，因此作者推測tissue-resident CCR2+ macrophages 在ischemia reperfusion injury中參與調(diào)節(jié)neutrophil recruitment。

Fig 2C-F：因此作者對B6 CCR2-DTR donor mice給予了diphtheria toxin (DT)清除了小鼠CCR2⁺單核和巨噬細(xì)胞，并將心臟移植給syngeneic LysM-GFP recipients。雙光子結(jié)果顯示清除了CCR2⁺單核和巨噬細(xì)胞之后，中性粒細(xì)胞的滲出顯著減少。同樣的，中性粒的rolling沒有受影響，但是crawl顯著下降了。

Thus, heart-resident CCR2+ monocytes and macrophages are critical to facilitate extravasation of neutrophils into injured myocardial tissue.

圖標(biāo)錯了，D其實(shí)是B，B和C其實(shí)是C和D

2. MyD88 signaling in heart-resident macrophages facilitates extravasation of neutrophils into heart tissue during ischemia reperfusion injury

巨噬細(xì)胞表達(dá)模式識別受體來識別DAMPs。此前的研究顯示several endogenous ligands that are released during sterile inflammation signal through MyD88。因此作者就想探究一下donor心臟中MyD88的表達(dá)在缺血灌注中中性粒趨化中的作用。

Fig 3A-E：作者將B6 MyD88–deficient hearts移植給了syngeneic LysM-GFP recipients，結(jié)果顯示MyD88–deficient心臟的中性粒滲出顯著下降，中性粒的rolling輕度增加，crawl下降。
Fig 3F-J：由于很多心臟細(xì)胞都表達(dá)MyD88，作者cross了B6 MyD88–floxed mice 和 B6 LysM-Cre，得到組織原位巨噬細(xì)胞特異性MyD88缺陷鼠（盡管髓系細(xì)胞都表達(dá)LysM，但心臟沒有組織原位中性粒）。作者將得到的組織原位巨噬細(xì)胞MyD88缺陷鼠的心臟移植給syngeneic LysM-GFP recipients，得到了和前面一樣的結(jié)果。

隨后作者想要去探究巨噬細(xì)胞MyD88 signaling如何影響了中性粒細(xì)胞趨化因子的表達(dá)。因此，作者將B6 CD45.2⁺ WT 或 B6 CD45.2⁺ MyD88缺陷心臟移植給B6 CD45.1⁺ recipients，并分選了donor CCR2^–和 monocyte-derived CCR2⁺巨噬細(xì)胞。結(jié)果顯示和CCR2^–巨噬相比，WT鼠的CCR2⁺巨噬CXCL2和CXCL5顯著要高。而在MyD88缺陷鼠中，這種現(xiàn)象消失。

這分子都挑的好主觀哦，MyD88，CXCL2和CXCL5都是

Thus, MyD88 signaling in CCR2⁺ macrophages regulates the expression of neutrophil chemokines, and MyD88 signaling in heart-resident macrophages is critical to promote the entry of neutrophils into injured myocardial tissue.

3. Donor TLR9 expression regulates expression of neutrophil chemokines and neutrophil transendothelial migration.

隨后作者想要去探究MyD88的上游。Endogenous ligands released during sterile inflammation have been shown to signal via several pattern-recognition receptors that are upstream of MyD88. 因此作者檢測了TLR2, TLR4, TLR7和TLR9在CCR2^–和 CCR2⁺巨噬細(xì)胞的表達(dá)，發(fā)現(xiàn)TLR2和TLR9在CCR2^–和 CCR2⁺細(xì)胞中都高表達(dá)，TLR4和7則比較低(附件)。

因此作者將B6 TLR2–deficient、TIRAP缺陷 (an adaptor protein that is critical for TLR2 and TLR4 signaling via MyD88)和B6 TLR9-deficient心臟移植到LysM-GFP hosts，發(fā)現(xiàn)只有TLR9 KO鼠的中性粒遷移下降。

Together, these data indicate that TLR9 signaling in heart-resident cells promotes the expression of neutrophil chemokines and is a critical regulator of neutrophil extravasation.

4. Donor CXCL5 expression is critical to facilitate the extravasation of neutrophils into heart tissue during ischemia reperfusion injury.

最后作者分別block了donor心臟的CXCL2和CXCL5，移植后發(fā)現(xiàn)block CXCL2 增加了rolling velocity，降低了adhere, crawl 和 extravasation。而block CXCL5 增加了 neutrophil rolling velocity, 降低了 crawling velocity, diminished extravasation。

Thus, CXCL5 expression by donor cells plays a critical and nonredundant role in facilitating the migration of neutrophils into injured heart tissue.

Figure 8. Model of CCR2+ macrophage–mediated neutrophil extravasation.

整個文章做的吧，就是“一招鮮，吃遍天”。各種基因鼠是挺多的。