16年8月的一篇JCI Insight, IF: 9.484

为了探究心肌缺血灌注损伤时调控中性粒细胞trafficking的upstream signals，作者使用了小鼠心脏移植模型（An essential advantage of this system is the ability to resolve the roles of resident (donor) and recruited (recipient) immune cell populations.）+双光子血管内成像。

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，作者团队发现心脏存在不同的巨噬细胞亚群。因此作者首先想要去探究组织原位巨噬细胞在心脏移植后是否参与中性粒的招募。

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
氯膦酸盐脂质体不是清除外周趋化来的巨噬的吗？

作者在移植手术24h前对B6 WT donor小鼠给予了氯膦酸盐脂质体清除了组织原位巨噬细胞，并将其心脏移植给了syngeneic LysM-GFP neutrophil reporter hosts。对照组小鼠给予的是PBS脂质体。在reperfusion 2h后使用了双光子血管内成像 (We have previously demonstrated that LysM-GFP nearly exclusively labels neutrophils in the donor heart at this time point)。在移植后，移植了给予PBS liposomes心脏的受体鼠中性粒细胞被趋化到大冠状静脉，在这里中性粒细胞slow down, 黏附到血管, crawl, 并迁移到心脏组织。而移植了给予氯膦酸盐脂质体心脏的受体鼠中性粒细胞向心脏的趋化减少（B图可以明显看到中性细胞在血管壁黏附增加，但不能透过血管）。此外，中性粒的rolling没有受影响，但是crawl显著下降了。

LysM-GFP不是髓系示踪的嘛，移植了清除巨噬的心脏过去之后，外周带GFP的中性粒和巨噬都可以趋化过去被看到啊，为啥只说是中性粒呢？

为了定义相关组织原位巨噬细胞群，作者将B6 CD45.2⁺ WT心脏移植到congenic B6 CD45.1+ WT recipients鼠，在移植2小时后检测了donor心脏巨噬细胞。
Fig 2A-B：鉴定出了donor-derived MHCII^loCCR2^–和 MHCII^hiCCR2^–巨噬细胞、MHCII^hiCCR2⁺巨噬细胞和MHCII^loCCR2⁺单核细胞（图B和D标反了吧...）。

在此前的研究中，作者发现和胚胎来源的 CCR2^–巨噬细胞 相比，CCR2⁺单核细胞来源的巨噬细胞在受损心脏中扩增并表达高水平促炎细胞因子和趋化因子，因此作者推测tissue-resident CCR2+ macrophages 在ischemia reperfusion injury中参与调节neutrophil recruitment。

Fig 2C-F：因此作者对B6 CCR2-DTR donor mice给予了diphtheria toxin (DT)清除了小鼠CCR2⁺单核和巨噬细胞，并将心脏移植给syngeneic LysM-GFP recipients。双光子结果显示清除了CCR2⁺单核和巨噬细胞之后，中性粒细胞的渗出显著减少。同样的，中性粒的rolling没有受影响，但是crawl显著下降了。

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

图标错了，D其实是B，B和C其实是C和D

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

巨噬细胞表达模式识别受体来识别DAMPs。此前的研究显示several endogenous ligands that are released during sterile inflammation signal through MyD88。因此作者就想探究一下donor心脏中MyD88的表达在缺血灌注中中性粒趋化中的作用。

Fig 3A-E：作者将B6 MyD88–deficient hearts移植给了syngeneic LysM-GFP recipients，结果显示MyD88–deficient心脏的中性粒渗出显著下降，中性粒的rolling轻度增加，crawl下降。
Fig 3F-J：由于很多心脏细胞都表达MyD88，作者cross了B6 MyD88–floxed mice 和 B6 LysM-Cre，得到组织原位巨噬细胞特异性MyD88缺陷鼠（尽管髓系细胞都表达LysM，但心脏没有组织原位中性粒）。作者将得到的组织原位巨噬细胞MyD88缺陷鼠的心脏移植给syngeneic LysM-GFP recipients，得到了和前面一样的结果。

随后作者想要去探究巨噬细胞MyD88 signaling如何影响了中性粒细胞趋化因子的表达。因此，作者将B6 CD45.2⁺ WT 或 B6 CD45.2⁺ MyD88缺陷心脏移植给B6 CD45.1⁺ recipients，并分选了donor CCR2^–和 monocyte-derived CCR2⁺巨噬细胞。结果显示和CCR2^–巨噬相比，WT鼠的CCR2⁺巨噬CXCL2和CXCL5显著要高。而在MyD88缺陷鼠中，这种现象消失。

这分子都挑的好主观哦，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.

随后作者想要去探究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. 因此作者检测了TLR2, TLR4, TLR7和TLR9在CCR2^–和 CCR2⁺巨噬细胞的表达，发现TLR2和TLR9在CCR2^–和 CCR2⁺细胞中都高表达，TLR4和7则比较低(附件)。

因此作者将B6 TLR2–deficient、TIRAP缺陷 (an adaptor protein that is critical for TLR2 and TLR4 signaling via MyD88)和B6 TLR9-deficient心脏移植到LysM-GFP hosts，发现只有TLR9 KO鼠的中性粒迁移下降。

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.

最后作者分别block了donor心脏的CXCL2和CXCL5，移植后发现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.

整个文章做的吧，就是“一招鲜，吃遍天”。各种基因鼠是挺多的。