1. Myeloid lineage-specific responses to TLR signaling

Fig S1： To understand the basis of myeloid lineage-specific responses, we examined transcriptional footprints that define the differentiation trajectory from monocytes into either MDMs or MDDCs.
作者從人PBMC中分離了外周血單核細胞，分別使用M-CSF或GM-CSF/IL-4將其分化成MDMs或MDDCs（干預了7天）。在分化過程中，作者在9個不同的時間點 (0 h, 4 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 和 7 days) 對細胞進行了RNAseq（n=2）。PCA結(jié)果顯示在誘導分化的前3天，細胞出現(xiàn)了比較顯著的轉(zhuǎn)錄變化，而在4-7天后其轉(zhuǎn)錄改變趨于穩(wěn)定。

作者推測這些轉(zhuǎn)錄改變可能提示三種髓系細胞在PRR刺激下存在不同的反應。因此作者分析了單核細胞、MDMs和MDDCs在不同TLR agonist刺激下的轉(zhuǎn)錄改變。
Fig 1a： 作者首先分離了5例HC的外周血單核細胞，誘導7天，得到MDMs和MDDCs。隨后作者使用分別使用 TLR2 (Pam3CSK4), TLR4 (LPS), TLR7 (R837), 和 TLR7/8 (R848) agonists對三種細胞刺激了18個小時，并進行了RNAseq檢測。
Fig S2a： 作者首先分析了三種細胞在不同干預下不同TLR的表達，發(fā)現(xiàn)三種細胞從TLR1到TLR10都有表達， 但組內(nèi)表達略有差異。有趣的是，這些髓系細胞群的基礎TLR表達同樣存在差異。例如TLR3在分化的MDMs和MDDCs中有較高基礎表達，在單核中則表達很低；TLR7主要在單核和MDMs中表達，不在MDDC中表達；TLR10則幾乎只表達在MDDCs。
Fig S2b： PCA結(jié)果顯示三種細胞在未刺激時就存在顯著的轉(zhuǎn)錄差異，在刺激后，這些細胞也具有各自獨立的表達模式。

Fig 1b-c： 作者篩選了每一種細胞特異性的高變基因，發(fā)現(xiàn)了不被TLR調(diào)控的3個cluster的基因（clusters I, II, III）。這三個cluster的基因在TLR刺激后也在穩(wěn)定表達。此外，TLR誘導后，三群細胞又具有其各自的表達模式。
These data establish the purity of these populations and further indicate that TLR signaling does not promote direct differentiation of monocytes into either macrophages or DCs.
結(jié)合Figure S1的結(jié)果，M-CSF or GM-CSF receptor signaling primarily modulates and imprints the differentiation program that remains unaltered by PRR signaling.

Fig 1d： qpcr對前面的結(jié)果進行了驗證

Overall, these data demonstrate that a common node of the inflammatory response is set in motion following TLR signaling and that this response is independent of the identity of the proximal TLR but dictated by the cell lineage (clusters IV, V, and VI). These genes are different from the lineage-specifying genes (clusters I, II, and II), which remain unchanged. The cellular identity dictates the nature of the inflammatory response, thereby suggesting that the TLR response induced in specific cell types may be imprinted during differentiation.（不同細胞的初始轉(zhuǎn)錄模式就不同，在刺激后的轉(zhuǎn)錄模式也不同，但不同刺激后的轉(zhuǎn)錄模式是類似的）（感覺不太有說服力，作者難道不是直接找的差異基因嗎？shared基因更多吧）

Fig 1

2. TLR8 activation leads to the most potent induction of cytokine and chemokine genes in monocytes compared to MDMs and MDDCs

Fig 2a-b： qpcr對前面的結(jié)果進行了驗證隨后作者鑒定了一個單核細胞unique gene cluster (cluster VII)，這個cluster的gene被TLR8顯著誘導，而且包含多種細胞因子和趨化因子包括IL1B, IL6 和 CCL3。盡管這些基因在單核細胞中的表達 也可以被TLR2, TLR4 和 TLR7 不同程度的活化，TLR7/8 agonist R848 的活化作用最強。而且當單核細胞分化為MDMs和MDDCs時，TLR8誘導的促炎基因活化則消失。
Fig 2c： TLR8 (TL8-506) 和 TLR7/8 (R848) agonists 可以誘導單核細胞IL1-B和IL-6的表達，TLR7 (R837) agonists 則不行。

3. Stimulation of TLR4 in MDMs, but not monocytes, induces persistent expression of ISGs

Fig 3a-b： 隨后作者關(guān)注了gene cluster VIII，這個gene cluster可以在MDMs中被TLR4 agonist LPS所誘導。TLR4 agonist LPS誘導下，這群基因在MDDC中比較弱，而在單核中不表達。但單核細胞可以在TLR7 or TLR7/8 agonists的誘導下表達這群基因。The cluster mainly consists of ISGs such as MX1, OAS1, and IFIT3.
因此，MDMs中的TLR4 signaling，和單核中的 TLR7 或 TLR7/8 signaling，都可以 initiating ISG response。反過來，TLR4刺激單核細胞、TLR7/8刺激MDMs，則不能誘導ISGs。
Fig 3c： 單核和MDMs中TLR4的表達在5個sample中都沒有顯著變化。

很怪哦，表達TLR4差不多，在相同的agonist誘導下的反應卻不一樣，為什么呢

Fig 3d： qpcr的結(jié)果驗證了TLR4 signaling可以引起MDMs中，而不是單核細胞中ISG的表達。

The THP-1 acute monocytic leukemia cell line has been widely used as a model to study human monocyte and macrophage functions and signaling pathways. Following differentiation using phorbol 12-myristate 13- acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics primary human MDMs in terms of cell morphology, cytokine production, and surface markers.

因此作者隨后探究了PMA-induced, MDM-like THP-1 cells是否對TLR4的刺激具有同樣的反應性。
Fig 3e： 作者對未分化的和PMA誘導過的MDM-like THP-1使用LPS干預了18h，隨后進行了RNAseq。結(jié)果顯示cluster VIII中的113/227基因uniquely expressed in MDM-like, compared to monocyte-like, THP-1 cells，其中多種ISGs被顯著誘導，與前面的結(jié)果一致。
Fig 3f： GSE結(jié)果也顯示LPS-stimulated MDM-like THP-1 cells存在著IFN responsive genes 和 IFN-b targets 通路的顯著富集。
Fig 3g： 與前面的發(fā)現(xiàn)相一致，TLR4 stimulation在THP1中引起了更高水平的 MX1 和 OAS1的高表達，supporting the idea that myeloid differentiation imparts changes that elicit distinct responses to TLR activation.

4. Chromatin accessibility coincides with the induction of IFNB1 and ISGs by TLR agonists in monocytes and MDMs

Our results point to the possibility that distinct downstream events engaged by TLR4 in monocytes and MDMs, rather than differences in TLR expression, were responsible for the observed differences in ISG induction.

TLR4刺激引起IRF3的活化和核轉(zhuǎn)位，在細胞核中，IRF3與它的binding site結(jié)合，誘導IFNB1和primary response ISGs的表達。隨后 IFN-b 可以 acts in cis and trans by signaling through IFN-a/bR-ISG factor 3 (ISGF3) complex, thereby amplifying secondary ISG expression

Fig 4a： 作者檢測了LPS刺激后不同時間點的p-IRF3和t-IRF3的表達，發(fā)現(xiàn)MDM比mono的反應性強，但其kinetics是類似的，suggesting that the stark differences in ISGs induction cannot be explained by differential activation of IRF3。
Fig 4b d： 但是LPS在MDMs中而不是單核細胞中非常顯著的上調(diào)了IFNB1, RSAD2 和 IFITM3 的轉(zhuǎn)錄。These data suggest that, despite activation, phospho-IRF3 in monocytes is unable to induce the transcription of IFNB1 and ISGs.

由于組蛋白修飾和染色質(zhì)結(jié)構(gòu)可以影響TFs和調(diào)控轉(zhuǎn)錄的regulatory elements的結(jié)合，作者假設單核和MDMs間的 signal- induced chromatin accessibility 差異可能影響了the ability of IRF3 to access the relevant regions of chromatin。
Fig 4b-e： 因此作者對TLR4刺激后1，6和18h的單核和MDMs進行了ATAC-seq和RNAseq。結(jié)果顯示Chromatin accessibility of the ISG loci, such as the 5', 3' region and proximal gene body of IFNB1, and the promoters of RSAD2 and IFITM3, increased in MDMs 1 h post-LPS stimulation, which correlated with changes in their mRNA expression（紅色是changes in ATAC-seq peaks）
Fig 4f： 作者發(fā)現(xiàn)，LPS刺激 broadly opens the chromatin of ISG promoter loci as early as the 1-h time point in MDMs (單核中沒有)
Fig 4g： thereby enabling transcription from these regions as reflected by ISG induction at 6 and 18 h (單核中沒有)
Fig 4h： The percentage of ISG loci with open chromatin (ATAC-seq peaks) are also significantly higher in MDMs than in monocytes at all time points, indicating a clear correlation between chromatin opening and ISG transcription in MDMs.

Overall, these data suggest that differences in chromatin accessibility, rather than the robustness of early signal transduction, account for the differences in the expression of ISGs upon TLR4 stimulation in myeloid cells.

5. Enrichment of IRF binding motifs in open chromatin and increase in nuclear translocation of IRF1 in MDMs upon TLR4 stimulation

Fig 5a： 作者假設MDMs的 TLR4 signaling 通過一個 modifier 引起了染色質(zhì)重塑，因此作者performed a global analysis of the enrichment of TF binding site motifs in accessible chromatin regions. 作者使用了HOMER 去計算 the enrichment of each TF motif in open chromatin (ATAC-seq peaks) specific to MDMs, compared to monocytes, at various times post-stimulation. These analyses revealed that the open loci in MDMs had enrichment for the binding sites of IRFs, which play essential roles in type I IFN and ISG induction. In contrast, signal transducer and activator of transcription (STAT) binding motifs are relatively lowly enriched in ISG promoters and enhancers, and showed no significant bias be- tween MDM and monocyte
Fig 5b： Furthermore, IRF motifs that are predicted by HOMER to be bound by IRF1 were significantly enriched in MDMs compared to monocytes in response to TLR4 stimulation.
Fig 5c：隨后作者檢測了IRF1蛋白的表達，發(fā)現(xiàn)其表達在TLR4刺激后的MDMs中顯著增加，而在刺激后的單核中則保持穩(wěn)定或低水平。
Fig 5d： 在刺激6和18h后，作者在MDMs和單核中還觀察到了顯著的IRF1表達差異。MDMs, 而不是單核細胞中的IRF1，在TLR4刺激后快速進入了細胞核 (Figure 5C)，提示TLR4 signaling in MDMs enables IRF1 transcription and facilitates its translocation into the nucleus. These results suggest that activated IRF1 is a critical factor for ISG chromatin accessibility in TLR4-activated MDMs.

6. IRF1 deficiency leads to impaired chromatin accessibility and expression of ISGs

為了進一步探究IRF1在人巨噬細胞中對染色質(zhì)可及性的調(diào)節(jié)，作者通過CRISPR/Cas9 editing構(gòu)建了IRF1-deficient (knockout [KO]) 和 IRF-sufficient control (Ctrl) THP-1 細胞。
Fig 6a： 對Ctrl 和 IRF1 KO THP-1細胞進行PMA誘導，隨后給予LPS干預，并在1h和6h后檢測了ISGs的表達。IRF1缺陷completely abrogated the ability of differentiated THP-1 cells to induce ISGs, such as MX1 and OAS1, following LPS stimulation。
Fig 6b： 與在MDMs中觀測到的一致，we did not find any significant differences in TLR4-induced IRF3 phosphorylation between PMA-differentiated Ctrl and IRF1 KO THP-1 clones。
Fig 6c： 隨后作者探究了IRF1 deficiency對ISG loci的染色質(zhì)可及性的影響。作者使用了兩種細胞系在LPS刺激后進行了RNA-seq 和 ATAC-seq。We found a decreased global expression of all ISGs in IRF1 KO clones when compared to Ctrl clones (左). Further- more, the impaired ISG expression directly correlated with a lack of chromatin accessibility in KO cells (右)。While chromatin of ISG loci in Ctrl THP-1 cells became accessible at 1 and 6 h, these regions remained inaccessible in IRF1 KO THP1 cells at all time points. Surprisingly, the deletion of IRF1 also reduced the basal accessibility of ISG loci in resting unstimulated cells.
Fig 6d： To determine whether IRF1 is required for the accessibility of the conserved IFN-sensitive response element (ISRE), we used HOMER to assess the status of ISRE motifs that are occupied by IRF or by the ISGF3 complex. ISRE enrichment in open chromatin regions in IRF1 KO was significantly less compared to the Ctrl clone as early as 1 h.

Overall, these results suggest that IRF1 is an essential factor contributing to chromatin remodeling that facilitates ISG induction in TLR4-stimulated human macrophages.