研究巨噬功能能時(shí)，我們經(jīng)常用荷蘭liposoma的巨細(xì)胞清除劑Clodronate Liposomes氨騰酸二鈉脂質(zhì)體(貨號(hào)CP-005-005)清除體內(nèi)巨噬細(xì)胞、然后回輸修飾或者感興趣的目的巨噬細(xì)胞。既要確保原位的巨噬細(xì)胞被很好的清除，又要讓回輸?shù)木藜?xì)胞在早起階段及時(shí)發(fā)揮作用且不能被巨細(xì)胞清除劑清除了。這種既要，又要，就要求注射清除劑后，合適的時(shí)間點(diǎn)回輸目的細(xì)胞。鑒于回輸實(shí)驗(yàn)比較難做，回輸細(xì)胞的數(shù)量，活率，純度以及功能性活性都決定了Transfer實(shí)驗(yàn)的成功與否。

回輸?shù)募?xì)胞類型：

WT和CD1d-KO小鼠。GM-CSF cultured bone-marrow derived cells (BMDCs) 骨髓來(lái)源GM-CSF培養(yǎng)刺激后的細(xì)胞

回輸細(xì)胞的準(zhǔn)備：

Generation of BMDCs and isolation of pMacs

Bone marrow cells were flushed from the femurs and tibias with cold PBS, filtered through a 45?μm strainer, pelleted by centrifugation and resuspended in complete RPMI media (Gibco; 10% FCS, 100 U/ml penicillin, 100?μg/ml streptomycin, 14.3?μM β-Mercaptoethanol) supplemented with 20?ng/ml of GM-CSF (Biolegend). Medium was changed at days 3 and 4 of culture and cells were harvested on day 6. For stimulation experiments, CD11c+ cells were enriched by positive selection with CD11c magnetic beads (CD11c MicroBeads UltraPure, Miltenyi).

To isolate pMacs, mice were sacrificed using CO2 and 4?ml of ice-cold PBS injected into the peritoneal cavity. The peritoneal cavity was briefly massaged and cells recovered using a 10?ml syringe. For stimulation experiments pMacs were enriched by positive selection with F4/80 magnetic beads (Anti-F4/80 MicroBeads UltraPure, Miltenyi).

Clodronate Liposomes清除巨噬細(xì)胞后回輸細(xì)胞解決方案：

清除劑注射方式：腹腔注射（不一定優(yōu)選）

清除劑注射劑量：200ul

回輸細(xì)胞的數(shù)量：3x10^6

回輸細(xì)胞的時(shí)間點(diǎn)：清除劑給藥后72h

實(shí)驗(yàn)數(shù)據(jù)一：

WT小鼠腹腔注射荷蘭Liposoma巨噬細(xì)胞清除劑，72h后，Transfer回輸WT，CD1d-KO小鼠的骨髓來(lái)源細(xì)胞。造模LPS誘導(dǎo)的炎性模型。根據(jù)實(shí)驗(yàn)數(shù)據(jù)，支持在LPS造模后，用CD1d-KO細(xì)胞重建的小鼠表現(xiàn)出對(duì)LPS誘導(dǎo)的炎癥更高的易感性，與用WT細(xì)胞重建的小鼠相比，體溫降低以及血液中細(xì)胞因子的濃度提高。數(shù)據(jù)支持CD1d在髓系細(xì)胞中對(duì)TLR反應(yīng)的負(fù)向調(diào)節(jié)作用。

實(shí)驗(yàn)數(shù)據(jù)二：

WT小鼠腹腔注射荷蘭Liposoma巨噬細(xì)胞清除劑，72h后，Transfer回輸WT，CD1d-KO小鼠的骨髓來(lái)源細(xì)胞。調(diào)查CD36是否會(huì)導(dǎo)致CD1d-KO細(xì)胞中脂質(zhì)攝取的增加。引入實(shí)驗(yàn)變量CD36，sulfosuccinimidyl oleate (SSO)：是一種長(zhǎng)鏈脂肪酸,抑制脂肪酸向細(xì)胞轉(zhuǎn)運(yùn)。可以封閉阻斷CD36。

CD36 是一種多功能糖蛋白，可作為多種配體的受體，包括血小板反應(yīng)蛋白、纖連蛋白、膠原蛋白或β淀粉樣蛋白等蛋白質(zhì)實(shí)體，以及氧化低密度脂蛋白 (oxLDL)、陰離子磷脂等脂質(zhì)成分。長(zhǎng)鏈脂肪酸和細(xì)菌二酰化脂肽。這些配體具有多價(jià)性，可以同時(shí)與多個(gè)受體結(jié)合，促進(jìn) CD36 簇的形成，從而啟動(dòng)信號(hào)轉(zhuǎn)導(dǎo)和受體-配體復(fù)合物的內(nèi)化。對(duì)輔助受體信號(hào)傳導(dǎo)的依賴性明顯是配體特異性的。細(xì)胞對(duì)這些配體的反應(yīng)在腸道內(nèi)的血管生成、炎癥反應(yīng)、脂肪酸代謝、味覺和膳食脂肪加工中發(fā)揮著關(guān)鍵作用。此外，CD36 結(jié)合長(zhǎng)鏈脂肪酸，促進(jìn)其轉(zhuǎn)運(yùn)到細(xì)胞中并參與肌肉脂質(zhì)利用、脂肪能量?jī)?chǔ)存和腸道脂肪吸收。從機(jī)制上講，脂肪酸結(jié)合會(huì)激活下游激酶 LYN，磷酸化棕櫚酰轉(zhuǎn)移酶 ZDHHC5，并導(dǎo)致 CD36 去棕櫚酰化和小凹胞吞作用。在小腸中，CD36 可能通過激活 MAPK1/3 (ERK1/2) 信號(hào)通路在膳食脂肪酸和膽固醇的近端吸收中發(fā)揮作用。它還涉及口腔脂肪的感知和偏好，介導(dǎo)味覺受體細(xì)胞中細(xì)胞內(nèi)鈣水平的增加。此外，CD36 是長(zhǎng)鏈脂肪酸腹內(nèi)側(cè)下丘腦神經(jīng)元感知以及能量和葡萄糖穩(wěn)態(tài)調(diào)節(jié)的重要因素。作為血小板反應(yīng)蛋白的受體，CD36 介導(dǎo)其抗血管生成作用，并與 THBS1 合作，響應(yīng)游離脂肪酸升高而誘導(dǎo)足細(xì)胞凋亡。作為 TLR4:TLR6 異二聚體的輔助受體，CD36 在配體結(jié)合后促進(jìn)單核細(xì)胞/巨噬細(xì)胞的炎癥，通過 NLRP3 炎癥小體的啟動(dòng)和激活，導(dǎo)致 NF-κ-B 依賴性細(xì)胞因子的產(chǎn)生和 IL1B 的分泌。此外，CD36 作為微生物二酰化脂肽的選擇性和非冗余傳感器，觸發(fā) NF-κ-B 依賴性 TNF 產(chǎn)生，并隨后通過 TLR2:TLR6 異二聚體信號(hào)傳導(dǎo)靶向脂筏依賴性途徑中的高爾基體。在微生物感染的情況下，CD36 直接介導(dǎo)惡性瘧原蟲寄生的紅細(xì)胞的細(xì)胞粘附和顆粒的內(nèi)化，與 TLR 信號(hào)傳導(dǎo)無(wú)關(guān)。

中文摘要：

細(xì)胞代謝的變化支撐著巨噬細(xì)胞的激活，但目前對(duì)關(guān)鍵免疫分子如何調(diào)節(jié)巨噬細(xì)胞的代謝程序知之甚少。在這里，我們揭示了抗原呈遞分子CD1d在脂質(zhì)代謝控制中的作用。我們展示了缺乏CD1d的巨噬細(xì)胞表現(xiàn)出代謝重編程，脂質(zhì)代謝途徑下調(diào)，外源性脂質(zhì)進(jìn)口增加。這種代謝重組為巨噬細(xì)胞增強(qiáng)對(duì)先天信號(hào)的反應(yīng)做好了準(zhǔn)備，因?yàn)镃D1d缺失的細(xì)胞在刺激類 Toll 樣受體后表現(xiàn)出更高的信號(hào)傳導(dǎo)和細(xì)胞因子分泌。在機(jī)制上，CD1d通過控制脂質(zhì)轉(zhuǎn)運(yùn)蛋白CD36的內(nèi)吞作用來(lái)調(diào)節(jié)脂質(zhì)進(jìn)口，而阻斷通過CD36的脂質(zhì)攝取可以恢復(fù)巨噬細(xì)胞的代謝和免疫反應(yīng)。因此，我們的數(shù)據(jù)揭示了CD1d作為巨噬細(xì)胞中一個(gè)炎癥-代謝回路的關(guān)鍵調(diào)節(jié)因子，這一功能獨(dú)立于其對(duì)T細(xì)胞反應(yīng)的調(diào)控。

英文摘要：

Alterations in cellular metabolism underpin macrophage activation, yet little is known regarding how key immunological molecules regulate metabolic programs in macrophages. Here we uncover a function for the antigen presenting molecule CD1d in the control of lipid metabolism. We show that CD1d-deficient macrophages exhibit a metabolic reprogramming, with a downregulation of lipid metabolic pathways and an increase in exogenous lipid import. This metabolic rewiring primes macrophages for enhanced responses to innate signals, as CD1d-KO cells show higher signalling and cytokine secretion upon Toll-like receptor stimulation. Mechanistically, CD1d modulates lipid import by controlling the internalization of the lipid transporter CD36, while blocking lipid uptake through CD36 restores metabolic and immune responses in macrophages. Thus, our data reveal CD1d as a key regulator of an inflammatory-metabolic circuit in macrophages, independent of its function in the control of T cell responses.

論文信息：

論文題目：CD1d-dependent rewiring of lipid metabolism in macrophages regulates innate immune responses

期刊名稱：Nature Communications

時(shí)間期卷：13, Article number: 6723 (2022)

在線時(shí)間：2022年11月7日

DOI：doi.org/10.1038/s41467-022-34532-x

產(chǎn)品信息：

貨號(hào)：CP-005-005

規(guī)格：5ml+5ml

品牌：Liposoma

產(chǎn)地：荷蘭

名稱：Clodronate Liposomes and Control Liposomes

辦事處：Target Technology（靶點(diǎn)科技）