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.
作者从人PBMC中分离了外周血单核细胞，分别使用M-CSF或GM-CSF/IL-4将其分化成MDMs或MDDCs（干预了7天）。在分化过程中，作者在9个不同的时间点 (0 h, 4 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 和 7 days) 对细胞进行了RNAseq（n=2）。PCA结果显示在诱导分化的前3天，细胞出现了比较显著的转录变化，而在4-7天后其转录改变趋于稳定。
作者推测这些转录改变可能提示三种髓系细胞在PRR刺激下存在不同的反应。因此作者分析了单核细胞、MDMs和MDDCs在不同TLR agonist刺激下的转录改变。
Fig 1a： 作者首先分离了5例HC的外周血单核细胞，诱导7天，得到MDMs和MDDCs。随后作者使用分别使用 TLR2 (Pam3CSK4), TLR4 (LPS), TLR7 (R837), 和 TLR7/8 (R848) agonists对三种细胞刺激了18个小时，并进行了RNAseq检测。
Fig S2a： 作者首先分析了三种细胞在不同干预下不同TLR的表达，发现三种细胞从TLR1到TLR10都有表达， 但组内表达略有差异。有趣的是，这些髓系细胞群的基础TLR表达同样存在差异。例如TLR3在分化的MDMs和MDDCs中有较高基础表达，在单核中则表达很低；TLR7主要在单核和MDMs中表达，不在MDDC中表达；TLR10则几乎只表达在MDDCs。
Fig S2b： PCA结果显示三种细胞在未刺激时就存在显著的转录差异，在刺激后，这些细胞也具有各自独立的表达模式。
Fig 1b-c： 作者筛选了每一种细胞特异性的高变基因，发现了不被TLR调控的3个cluster的基因（clusters I, II, III）。这三个cluster的基因在TLR刺激后也在稳定表达。此外，TLR诱导后，三群细胞又具有其各自的表达模式。
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.
结合Figure S1的结果，M-CSF or GM-CSF receptor signaling primarily modulates and imprints the differentiation program that remains unaltered by PRR signaling.
Fig 1d： qpcr对前面的结果进行了验证
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.（不同细胞的初始转录模式就不同，在刺激后的转录模式也不同，但不同刺激后的转录模式是类似的）（感觉不太有说服力，作者难道不是直接找的差异基因吗？shared基因更多吧）
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对前面的结果进行了验证随后作者鉴定了一个单核细胞unique gene cluster (cluster VII)，这个cluster的gene被TLR8显著诱导，而且包含多种细胞因子和趋化因子包括IL1B, IL6 和 CCL3。尽管这些基因在单核细胞中的表达 也可以被TLR2, TLR4 和 TLR7 不同程度的活化，TLR7/8 agonist R848 的活化作用最强。而且当单核细胞分化为MDMs和MDDCs时，TLR8诱导的促炎基因活化则消失。
Fig 2c： TLR8 (TL8-506) 和 TLR7/8 (R848) agonists 可以诱导单核细胞IL1-B和IL-6的表达，TLR7 (R837) agonists 则不行。
3. Stimulation of TLR4 in MDMs, but not monocytes, induces persistent expression of ISGs
Fig 3a-b： 随后作者关注了gene cluster VIII，这个gene cluster可以在MDMs中被TLR4 agonist LPS所诱导。TLR4 agonist LPS诱导下，这群基因在MDDC中比较弱，而在单核中不表达。但单核细胞可以在TLR7 or TLR7/8 agonists的诱导下表达这群基因。The cluster mainly consists of ISGs such as MX1, OAS1, and IFIT3.
因此，MDMs中的TLR4 signaling，和单核中的 TLR7 或 TLR7/8 signaling，都可以 initiating ISG response。反过来，TLR4刺激单核细胞、TLR7/8刺激MDMs，则不能诱导ISGs。
Fig 3c： 单核和MDMs中TLR4的表达在5个sample中都没有显著变化。
很怪哦，表达TLR4差不多，在相同的agonist诱导下的反应却不一样，为什么呢
Fig 3d： qpcr的结果验证了TLR4 signaling可以引起MDMs中，而不是单核细胞中ISG的表达。
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.
因此作者随后探究了PMA-induced, MDM-like THP-1 cells是否对TLR4的刺激具有同样的反应性。
Fig 3e： 作者对未分化的和PMA诱导过的MDM-like THP-1使用LPS干预了18h，随后进行了RNAseq。结果显示cluster VIII中的113/227基因uniquely expressed in MDM-like, compared to monocyte-like, THP-1 cells，其中多种ISGs被显著诱导，与前面的结果一致。
Fig 3f： GSE结果也显示LPS-stimulated MDM-like THP-1 cells存在着IFN responsive genes 和 IFN-b targets 通路的显著富集。
Fig 3g： 与前面的发现相一致，TLR4 stimulation在THP1中引起了更高水平的 MX1 和 OAS1的高表达，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的活化和核转位，在细胞核中，IRF3与它的binding site结合，诱导IFNB1和primary response ISGs的表达。随后 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： 作者检测了LPS刺激后不同时间点的p-IRF3和t-IRF3的表达，发现MDM比mono的反应性强，但其kinetics是类似的，suggesting that the stark differences in ISGs induction cannot be explained by differential activation of IRF3。
Fig 4b d： 但是LPS在MDMs中而不是单核细胞中非常显著的上调了IFNB1, RSAD2 和 IFITM3 的转录。These data suggest that, despite activation, phospho-IRF3 in monocytes is unable to induce the transcription of IFNB1 and ISGs.
由于组蛋白修饰和染色质结构可以影响TFs和调控转录的regulatory elements的结合，作者假设单核和MDMs间的 signal- induced chromatin accessibility 差异可能影响了the ability of IRF3 to access the relevant regions of chromatin。
Fig 4b-e： 因此作者对TLR4刺激后1，6和18h的单核和MDMs进行了ATAC-seq和RNAseq。结果显示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（红色是changes in ATAC-seq peaks）
Fig 4f： 作者发现，LPS刺激 broadly opens the chromatin of ISG promoter loci as early as the 1-h time point in MDMs (单核中没有)
Fig 4g： thereby enabling transcription from these regions as reflected by ISG induction at 6 and 18 h (单核中没有)
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： 作者假设MDMs的 TLR4 signaling 通过一个 modifier 引起了染色质重塑，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 去计算 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：随后作者检测了IRF1蛋白的表达，发现其表达在TLR4刺激后的MDMs中显著增加，而在刺激后的单核中则保持稳定或低水平。
Fig 5d： 在刺激6和18h后，作者在MDMs和单核中还观察到了显著的IRF1表达差异。MDMs, 而不是单核细胞中的IRF1，在TLR4刺激后快速进入了细胞核 (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
为了进一步探究IRF1在人巨噬细胞中对染色质可及性的调节，作者通过CRISPR/Cas9 editing构建了IRF1-deficient (knockout [KO]) 和 IRF-sufficient control (Ctrl) THP-1 细胞。
Fig 6a： 对Ctrl 和 IRF1 KO THP-1细胞进行PMA诱导，随后给予LPS干预，并在1h和6h后检测了ISGs的表达。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： 与在MDMs中观测到的一致，we did not find any significant differences in TLR4-induced IRF3 phosphorylation between PMA-differentiated Ctrl and IRF1 KO THP-1 clones。
Fig 6c： 随后作者探究了IRF1 deficiency对ISG loci的染色质可及性的影响。作者使用了两种细胞系在LPS刺激后进行了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.
