ALK1 signaling is required for the homeostasis of Kupffer cells and prevention of bacterial infection

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. The BMP receptor activin receptor–like kinase 1 (ALK1) is well known for its role in blood vessel formation; however, its role within the immune system has never been revealed to our knowledge. Here, we found that BMP9/BMP10/ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen, and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages showed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

ALK1, rather than ALK2, ALK3, and TGFβR2, is responsible for regulating the expression of Id1 and Id3 in KCs and plays an important role in maintaining the KC surface phenotype.
We next performed single-cell RNA-Seq (scRNA-Seq) analysis of sorted CD64 + F4/80 + Tim4 + KCs from Alk1 fl/fl Clec4f Cre mice compared with their controls to understand how ALK1 affected KCs. After sequencing, aggregation of the samples, and removal of poor-quality and contaminating cells, 13,690 cells remained (6295 cells from Alk1 fl/fl mice and 7395 cells from Alk1 fl/fl Clec4f Cre mice). We identified 5 clusters by generating a uniform manifold approximation and projection (UMAP) from the transcriptome data using the Seurat pipeline ( Figure 2A). Cluster 0 was predominantly composed of cells originating from Alk1 fl/fl mice, whereas cells in clusters 1 and 2 mainly originated from Alk1 fl/fl Clec4f Cre mice ( Figure  2A). Because we were unable to determine which cells expressed full-length or floxed mRNA using the 3′ Assay from 10X Genomics, we next analyzed the differentially expressed (DE) genes between these clusters to find markers that could distinguish the different cell populations by flow cytometry. scRNA-Seq analysis revealed 243 DE genes in cluster 0, 347 DE genes in cluster 1, 182 DE genes in cluster 2, 189 DE genes in cluster 3, and 383 DE genes in cluster 4 (Supplemental Table 1) and showed that Clec4F was expressed in clusters 0, 2, 3, and 4, but not in cluster 1 (Figure 2A and Supplemental Figure 5A). Interestingly, we found that expression of Id1 and Id3 was also substantially reduced in cluster 1 (Figure 2A). Given that Id1 and Id3 are target genes of ALK1 signaling in KCs, these results suggested that Clec4F -Tim4 + KCs (cluster 1) may be deficient in Alk1. We performed quantitative PCR (qPCR) and genomic PCR on sorted Clec4F -Tim4 + and Clec4F + Tim4 + KCs from Alk1 fl/fl Clec4f Cre mice to verify this result and found that Clec4F -Tim4 + KCs efficiently deleted Alk1, whereas Clec4F + Tim4 + KCs were heterozygous for the Alk1 deletion (Supplemental Figure 5, B and C). KCs from Alk1 fl/+ Clec4f Cre mice did not display a phenotype similar to that of Alk1 fl/fl Clec4f Cre mice (Supplemental Figure 5D), suggesting no obvious effect of Alk1 haploinsufficiency on KCs.
In Alk1 fl/fl Clec4f Cre mice, Cre recombinase is expressed under the control of the Clec4F promoter, implying that Clec4F was once expressed in Clec4F -Tim4 + KCs. We prepared Alk1 fl/fl Clec4f Cre R26 yfp reporter mice to examine this possibility and observed high expression of the YFP reporter gene in Clec4F -Tim4 + KCs and diphtheria toxin receptor (DTR) was inserted into the 3′-UTR of the Clec4f gene. We crossed Clec4f Cre mice with a conditional reporter strain (R26-tdTomato) to determine the efficiency and specificity of Cre-mediated recombination using flow cytometry. In the liver, the Clec4f Cre strain efficiently recombined in CD64 + F4/80 + KCs (>90%), and almost all tdTomato + cells were KCs (Supplemental Figure 1, A and B, and Supplemental Figure 2A; supplemental material available online with this article; https://doi.org/10.1172/ JCI150489DS1). Furthermore, we did not detect tdTomato expression in other tissues (including CD45 + and CD45cells) (Supplemental Figure 1, C and D). Immunostaining experiments also confirmed that the reporter gene tdTomato was exclusively expressed in KCs and not in hepatocytes (Supplemental Figure 1E). In addition, 24 hours after diphtheria toxin (DT) administration, KCs were efficiently deleted in Clec4f Cre mice, as determined by immunostaining and flow cytometry (Supplemental Figure 1, F and G). Thus, the Clec4f Cre strain is a useful tool to specifically target KCs.
Recently, the expression of both the Id1 and Id3 genes was reported to be restricted to KCs compared with other tissue-resident macrophages, and Id3 deficiency impairs the differentiation of KCs (17). Loss of Id3 results in reduced numbers of KCs (17). Interestingly, Id1 is upregulated in Id3-deficient KCs, suggesting that Id1 may compensate for the function of Id3. The transcription factors Id1 and Id3 are target genes of BMP signaling (18), and thus an intriguing speculation is that BMPs present in the liver environment might be one of the tissue-derived signals that regulates KCs.
We first analyzed the expression of genes encoding BMP receptors and their coreceptors using data from the ImmGen Consortium to investigate which BMP signaling pathway regulates KCs and found that genes encoding BMPR2 and endoglin were expressed at high levels in KCs compared with expression levels in other tissue-resident macrophages (Supplemental Figure 3). Endoglin is required for BMP9/ALK1 signaling (19), and BMP9 is specifically expressed in the liver; therefore, a reasonable hypothesis is that ALK1 signaling might be important for KCs.
We generated Alk1 fl/fl Clec4f Cre mice to test this hypothesis. We also prepared Alk2 fl/fl Alk3 fl/fl Clec4f Cre mice. ALK1, ALK2, and ALK3 belong to the superfamily of TGFβ receptors, and TGFβ signaling has been proposed to be important for KCs (7). Thus, we also generated Tgfbr2 fl/fl Clec4f Cre mice. Alk2, Alk3, and Tgfbr2 were efficiently deleted in KCs from Alk2 fl/fl Alk3 fl/fl Clec4f Cre and Tgfbr2 fl/fl Clec4f Cre mice, respectively, but the deficiency of these genes did not affect the expression of Id1 and Id3 (Supplemental Figure 4, A and B). In contrast, ablation of ALK1 resulted in a dramatic reduction in the expression of Id1 and Id3 in CD64 + F4/80 + hepatic macrophages from Alk1 fl/fl Clec4f Cre mice, although the expression of Alk1 was reduced by approximately 60% in these macrophages ( Figure 1A). We further analyzed the total hepatic macrophage population in Alk1 fl/fl Clec4f Cre mice. We observed no difference in the number of KCs between Alk1 fl/fl Clec4f Cre and Alk1 fl/fl mice ( Figure 1B), but the KC surface phenotype in Alk1 fl/fl Clec4f Cre mice was altered, with a reduced population of Clec4F + Tim4 + KCs and increased populations of Clec4F -Tim4 + KCs, Clec4F + Tim4 -KCs, and Clec4F -Tim4 -KCs ( Figure 1C). In addition, no difference was observed in the cell number and surface phenotype of KCs between Alk2 fl/fl Alk3 fl/fl Clec4f Cre , Tgfbr2 fl/fl Clec4f Cre , and their littermate controls (Supplemental Figure 4, C-F). Taken together, these results suggested that J Clin Invest. 2022;132(3):e150489 https://doi.org/10.1172/JCI150489 respectively, were downregulated ( Figure 2E). Based on these results, the ALK1 signaling pathway is required for KC identity and may negatively regulate Spic expression in KCs.
The maintenance of KCs requires ALK1 signaling. Decreased expression of Tim4 in KCs from Alk1 fl/fl Clec4f Cre mice suggested that circulating monocytes might replenish liver macrophages. We generated shielded chimeras in which the livers of Alk1 fl/fl Clec4f Cre mice and Alk1 fl/fl littermate controls were shielded during irradiation, and these mice were reconstituted with congenic CD45.1 WT BM to examine this possibility ( Figure 3A). As expected, partial shielding resulted in mixed chimerism in blood Ly6C hi monocytes in all groups ( Figure 3B). KCs in Alk1 fl/fl mice were not chimeric, as KCs were self-maintained under steady-state conditions independent of circulating monocytes ( Figure 3B). However, hepatic macrophages from Alk1 fl/fl Clec4f Cre mice displayed chimerism ( Figure 3B), suggesting that the ALK1 deficiency may have led to a loss of KCs and that circulating monocytes repopulated the empty niche to maintain the macrophage pool in the liver. Consistent with this result, the number of Clec4F -Tim4 + KCs decreased with age, and Tim4 -Mo-KCs expanded significantly over time (Figure 3C). 5-ethynyl-20-deoxyuridine (EdU) incorporation assays revealed that Clec4F -Tim4 + KCs had a reduced capacity to proliferate compared with their counterparts ( Figure 3D), indicating that a decrease in the proliferation of Alk1-deficient KCs leads to a severe disadvantage of these cells under competitive conditions. Maf and Mafb function as negative regulators of KC proliferation (24). Consistent with the impaired proliferation, we found that Clec4F + Tim4 + KCs, but very low YFP expression in Tim4 -KCs ( Figure 2B), suggesting that Clec4F -Tim4 + KCs once expressed Clec4F. Moreover, upon ALK1 deletion, KCs no longer expressed Clec4F. We further prepared Alk1 fl/fl Clec4f Cre/Cre mice (homozygous for Cre) to increase the recombination frequency and to support this hypothesis and found that KCs from these mice did not express Clec4F ( Figure 2C). Based on the results described above, we identified cluster 1 as Alk1 -/-KCs from Alk1 fl/fl Clec4f Cre mice and cluster 0 as Alk1 +/+ KCs from Alk1 fl/fl mice. Cells in cluster 2 from Alk1 fl/fl Clec4f Cre mice were identified as Alk1 +/-KCs. Clusters 3 and 4 were proliferating cells expressing DNA replication-associated genes such as Mcm2-7 and Mki67 (Supplemental Figure 5A). Then, we compared the transcriptional profiles between clusters and found that among the 25 top core genes of KCs described previously (20), the expression of 16 core genes was significantly reduced upon the loss of Alk1 in KCs ( Figure 2D), suggesting that ALK1 plays a critical role in maintaining the identity of KCs.
The transcription factors Zeb2 and Nr1h3 are required for the identity of KCs (20). We found that the expression of Nr1h3, but not Zeb2, was significantly decreased in Alk1 -/-KCs ( Figure 2D and Supplemental Table1). The transcription factor SPI-C is required for the development of splenic red pulp macrophages (RPMs), and its expression is induced by heme (22,23). SPI-C expression was significantly upregulated in the absence of ALK1 ( Figure 2E), but the CD163 and CD91 (encoded by Lrp1) receptors that uptake circulating hemoglobin-haptoglobin and heme-hemopexin complexes, in Tim4 + and Tim4 -KCs after tamoxifen administration ( Figure  3E). Because a good antibody is unavailable to stain ALK1 for flow cytometry and we cannot exclude the possibility that Clec4F -Tim4 -KCs were derived from newly arrived WT MoKCs, we used Clec4F -Tim4 + KCs to represent Alk1-deficient cells and determined Clec4F expression in Tim4 + KCs originating from CD45.2 + donor cells 5, 10, and 25 days after the last treatment with tamoxifen. Chimeras not treated with tamoxifen were used as controls. Approximately 30% of donor-derived Tim4 + KCs were Clec4Fon days 5 and 10 after the last treatment, whereas Clec4F -Tim4 + KCs were no longer detected in the liver 25 days after the last treatment ( Figure 3E), indicating that Alk1-deficient KCs were lost over time.
ALK1 is dispensable for the maintenance of macrophages located in the lung, kidney, brain, and spleen. To assess whether ALK1 Maf was expressed at higher levels in Alk1-deficient KCs than in their counterparts ( Figure 2E). Thus, these results suggested that ALK1 may be required for the maintenance of KCs.
To determine whether ALK1 deficiency results in KC disappearance, we established BM chimeras in which CD45.1 + mice were lethally irradiated and injected with congenic CD45.2 + Alk1 fl/fl UBC CreERT2 BM, in which tamoxifen administration leads to deletion of Alk1 in a wide range of cells (25). The chimeric mice were treated with tamoxifen 8 weeks after reconstitution. Based on a previous report (5), Mo-KCs are able to differentiate into mature Clec4F + KCs, but only some of these cells acquire Tim4 expression. Our results also confirmed this finding ( Figure 3E). Consistent with the aforementioned observation that ALK1 is important for Clec4F expression, we observed reduced expression of Clec4F  Figure 4A). After signaling is required for the maintenance of other tissue-resident macrophages, we generated mixed BM chimeras. We used Alk1 fl/fl Vav1 Cre mice, in which Cre recombinase is expressed at high levels in hematopoietic stem cells and maintains robust activity during   Figure 4B), suggesting that liver macrophages deficient in Alk1 are outcompeted by their WT counterparts. In contrast, blood monocytes and other tissue macrophages, including lung macrophages, kidney macrophages, brain macrophages, and splenic macrophages, were reconstituted at an equal ratio in WT (CD45.1/ CD45.2) and Alk1 fl/fl Vav1 Cre (CD45.2) chimeras (Figure 4, C-G). Taken together, these results suggested that ALK1 is dispensable for the survival of macrophages located in the lung, brain, kidney, and spleen.
BMP9 and BMP10 instruct KC signature gene expression. Both BMP9 and BMP10 are ligands of ALK1. In the results described above, we showed that ALK1, rather than ALK2 and ALK3, was required for the expression of Id1, Id3, and Clec4f. Consistent with these results, BMP9 and BMP10 treatment maintained higher expression of Id1, Id3, and Clec4f in cultured KCs than did BMP2 (ALK3 ligand) or BMP6 (ALK2 ligand) ( Figure 5A). However, Clec4F expression in KCs from Bmp9-KO mice was unaltered compared with that in KCs from WT littermate controls ( Figure 5B). In fact, it has been reported that BMP10 is able to compensate for the loss of BMP9 (27,28). To verify this, we administered an anti-BMP10 neutralizing antibody to Bmp9-KO mice and found that it resulted in decreased expression of Clec4F, whereas an injection of this antibody into WT mice did not affect its expression ( Figure 5B). qPCR analysis revealed that KC-specific genes, such as Id1, Id3, Clec4f, Fabp7, Cd5l, and Cdh5, were most significantly ALK1 signaling functions in KCs through the canonical Smad pathway Smad4 functions as a common Smad required for transcriptional regulation in response to BMPs. We generated Smad4 fl/fl Clec4f Cre mice to assess whether ALK1 signaling functions in KCs via the Smad pathway. Similar to the effect of Alk1 deletion, KCs from Smad4 fl/fl Clec4f Cre mice displayed altered expression of Clec4F and Tim4 ( Figure 6A). In protected chimeras, Smad4 deficiency led to the replenishment of KCs from monocytes ( Figure 6B). Similar to our findings in Alk1 fl/fl UBC CreERT2 mice, Clec4F -Tim4 + cells were also detected in KCs from tamoxifen-treated Smad4 fl/fl UBC CreERT2 mice ( Figure 6C). We sorted Clec4F -Tim4 + and Clec4F + Tim4 + KCs and examined the expression of Smad4, Id1, and Id3. qPCR analysis revealed that Clec4F -Tim4 + KCs had efficiently deleted Smad4, whereas Clec4F + Tim4 + KCs maintained the expression of Smad4 at levels comparable to those in KCs isolated from untreated Smad-4 fl/fl UBC CreERT2 mice ( Figure 6D). Accordingly, its target genes, Id1 and Id3, were significantly reduced in Clec4F -Tim4 + KCs but not in Clec4F + Tim4 + KCs. In summary, we found that ALK1 signaling regulated KCs through the canonical Smad pathway.

The functional phenotype of KCs is maintained by ALK1 signaling.
To investigate the functional consequences of ALK1 deletion in KCs, we generated ALK1 conditional-KO mice, in which Alk1 was completely targeted in these cells. Alk1 was not efficiently deleted in KCs from Alk1 fl/fl Clec4f Cre mice, so we analyzed Alk1 fl/fl Vav1 Cre mice. qPCR analysis revealed that Alk1 was successfully deleted in the total population of CD64 + F4/80 + hepatic macrophages (Supplemental Figure 6A). Moreover, Id1 and Id3 expression was significantly reduced in KCs from Alk1 fl/fl Vav1 Cre mice compared with those from Alk1 fl/fl mice (Supplemental Figure 6A), and Clec4F expression was not detected in either Tim4 + or Tim4 -KCs from Alk1 fl/fl Vav1 Cre mice (Supplemental Figure 6B). We also analyzed the number of hepatic myeloid cells, including KCs, neutrophils, monocytes, plasmacytoid DCs (pDCs), and conventional DCs (cDCs), and found that these cell counts were normal in Alk1 fl/fl Vav1 Cre mice (Supplemental Figure 6, C-F). Similarly, Smad4 was also efficiently targeted in KCs from Smad4 fl/fl Vav1 Cre mice (Supplemental Figure 6, G and H).
In addition, we examined E-cadherin and glutamine synthetase expression and interactions between KCs, liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) in Alk1 fl/fl Vav1 Cre and Smad4 fl/fl Vav1 Cre mice to determine whether the liver architecture was affected by ALK1 and Smad4 deficiency. We found  In addition to the cell surface phenotype, KC function is also governed by the cell's 3D morphology within the vasculature, as its elongated and branched shape increases the intravascular surface area available for interacting with circulating pathogens (9). KCs are strategically enriched near periportal regions. This positional asymmetry (immune zonation) is important for KCs to protect against the systemic dissemination of pathogens from local infection sites such as the digestive tract (29). Interestingly, Alk1 fl/fl Vav1 Cre and Smad4 fl/fl Vav1 Cre mice lacked the periportal polarization of KCs observed in their controls (Figure 8, A and B). In addition, KCs from Alk1 fl/fl Vav1 Cre and Smad4 fl/fl Vav1 Cre mice were generally smaller, with a decreased cell surface area and volume compared with KCs from Alk1 fl/fl control mice (Figure 9, A and B). Taken that E-cadherin and glutamine synthetase were expressed in the periportal and central vein regions of the liver lobules, respectively. The regional localization of E-cadherin and glutamine synthetase in the liver lobules of Alk1 fl/fl Vav1 Cre and Smad4 fl/fl Vav1 Cre mice was not affected (Supplemental Figure 7, A and B), and KCs from these mice still closely interacted with LSECs and HSCs (Supplemental Figure 7, C and D), suggesting that the liver architecture was intact.
VSIG4 (also known as CRIg), a new family of complement receptors, was reported to be expressed by KCs at high levels (13) and plays an important role in KC-mediated capture of grampositive bacteria (11,13,14). qPCR analysis revealed that Vsig4 in KCs was obviously upregulated after BMP9 and BMP10 treatment ( Figure 5A). The transcriptomic analysis revealed that Vsig4 expression was significantly decreased following the deletion of Alk1 in KCs ( Figure 2D). Confocal microscopy and flow cytometry also KCs are essential for host survival in L. monocytogenes infection, but most L. monocytogenes are not killed by the KCs (30). Elimination of the pathogen in L. monocytogenes infec tion requires the recruitment of neutrophils to the liver and the specific binding of these neutrophils to KCs (30). We found that neutrophil recruitment to the liver occurred 2 hours after infection, and we observed no difference in their recruitment and interaction with KCs between Alk1 fl/fl Vav1 Cre and Alk1 fl/fl mice (Supplemental Figure 9). IFN-γ is essential for the innate defense against L. monocytogenes infection (31). We observed increased expression of intracellular IFN-γ in NK cells, CD4 + T cells, and CD8 + T cells from Alk1 fl/fl Vav1 Cre mice 24 hours after infection (Supplemental Figure 10A), which coincided with higher bacterial counts in the livers and spleens of Alk1 fl/fl Vav1 Cre mice than in Alk1 fl/fl mice (Supplemental Figure 10B). Recently, it has been shown that IL-17A plays a critical role in innate defense against L. monocytogenes infection in the liver (32). Consistent with the previous report (32), we found that IL-17A together, ALK1 signaling plays an important role in maintaining the functional phenotype of KCs.
The ALK1 signaling pathway in KCs protects the host from infection with L. monocytogenes. Next, we infected Alk1 fl/fl Vav1 Cre and Alk1 fl/fl mice with L. monocytogenes and found that approximately 80% of Alk1 fl/fl Vav1 Cre mice died within 7 days of infection, whereas no Alk1 fl/fl mice succumbed to the infection ( Figure 10A). Similarly, Smad4 fl/fl Vav1 Cre mice exhibited higher mortality following L. monocytogenes infection than did Smad4 fl/fl control mice (Supplemental Figure  8A). Vav1-Cre not only deletes floxed genes in KCs, but also in other cell types, such as hematopoietic stem cells and their progenies. To examine whether the influence of ALK1 on controlling L. monocytogenes infection is KC intrinsic, we infected Alk1 fl/fl Clec4f Cre/Cre and Alk1 fl/fl mice with L. monocytogenes and found that Alk1 fl/fl Clec4f Cre/Cre mice were more susceptible to L. monocytogenes infection than were their controls ( Figure 10B), suggesting that ALK1 has a KCintrinsic role in protecting the host from L. monocytogenes infection. Moreover, we used intravital microscopy (IVM) to visualize and quantify the bacterial capture within the liver and to understand the mechanism underlying the increased susceptibility to infection and found that Alk1/Smad4-deficient mice showed a significant reduction in the capture of circulating L. monocytogenes by KCs (Figure 10  their tissue of residence, they undergo extensive differentiation according to molecular cues provided by their tissue-specific niche (33). This process enables these progenitors to develop into specialized tissue-resident macrophages with a unique transcription profile. However, the precise signals governing this process remain largely unknown. Here, we identified a critical role for BMP9/BMP10/ALK1 signaling in imprinting KC identity. Loss of ALK1 impaired the ability of KCs to proliferate. Notably, we found that ALK1 was dispensable for the survival of macrophages in many organs, which demonstrated a specific role in the maintenance of KCs.
Clec4F is selectively expressed in mature KCs. In contrast to F4/80, which is a constitutively expressed surface marker for resident macrophages (34,35), Clec4F is inducible in the liver microenvironment (36). Embryonic progenitors and monocytes progressively acquire Clec4F expression upon entry into the liver (5,36). Thus, studies aiming to understand what types of signals or molecules are necessary to regulate Clec4F expression in the liver microenvironment would be interesting (36). We and other researchers (6) showed that BMP9 stimulation induces the expression of Clec4f.
In the present study, in vivo blockade of ALK1 signaling by injecting anti-BMP10-blocking antibody into Bmp9-KO mice resulted in decreased expression of Clec4Fat mRNA and protein levels. Moreover, KCs no longer expressed Clec4F when Alk1 was completely deleted, as observed in KCs from Alk1 fl/fl Vav1 Cre and Alk1 fl/fl Clec4f Cre/Cre mice. Together, these results suggested that ALK1 signaling is essential for Clec4F expression in KCs. was mainly expressed by TCR γδ + T rather than CD4 + T cells 5 days after L. monocytogenes infection (Supplemental Figure 10C). The proportion of IL-17A-producing cells in TCR γδ + T cells in the liver of Alk1 fl/fl Vav1 Cre mice was comparable to that in the liver of Alk1 fl/fl mice (Supplemental Figure 10C), indicating that the ability of TCR γδ + T cells to produce IL-17A was not impaired in Alk1 fl/fl Vav1 Cre mice.
To investigate whether KC phagosome maturation and activation are altered in the absence of ALK1, we quantified phagocytosis and ROS production by KCs that engulfed L. monocytogenes in vivo using pH-and ROS-sensitive probe-labeled bacteria. We observed no differences between Alk1 fl/fl Vav1 Cre and Alk1 fl/fl mice in the acidification or ROS production of L. monocytogenescontaining phagosomes in KCs (Supplemental Figure 11, A and B). Furthermore, we also determined cytokine expression by KCs from Alk1 fl/fl Vav1 Cre and Alk1 fl/fl mice in response to L. monocytogenes infection and found that expression of the TNF-α and MCP-1 mRNAs in KCs was slightly reduced in the absence of ALK1 (Supplemental Figure 11C), indicating decreased KC activation in Alk-1 fl/fl Vav1 Cre mice during L. monocytogenes infection. Overall, these results suggested that the ALK1 signaling pathway is critical for host defenses against L. monocytogenes infection and that this effect appears to be KC specific.

Discussion
Most tissue-resident macrophages were derived from yolk sac macrophages or fetal liver monocytes. Once progenitors arrive at visceral arteriovenous malformations (AVMs) (40). It has been reported that patients with HHT are more susceptible to bacterial infection, especially gram-positive Staphylococcus aureus (41,42). We also found that Alk1 fl/fl Vav1 Cre mice had a significant reduction in the capture of circulating S. aureus by KCs (Supplemental Figure  12). Thus, it is possible that the high incidence of infectious diseases observed in patients with HHT may be due to the impaired innate immune function of KCs seen in mice lacking ALK1. In addition, during liver injury or infection in mice, KC homeostasis was also disrupted, with loss of resident KCs and replenishment of monocyte-derived macrophages (43)(44)(45). More important, the alteration of KC homeostasis also occurs in human liver diseases, as the number of liver macrophages is significantly reduced in patients with liver fibrosis (46). It has been reported that patients with acute liver failure and advanced cirrhosis have a high risk of bacterial infections, and dysfunction of liver macrophages may play a role (46,47). This suggests that therapeutic interventions aimed to prevent the loss of KCs and/or promote the maturation of newly arrived monocyte-derived macrophages might help reduce susceptibility to infection in these patients.
Recently, Bonnardel et al. and Sakai et al. proposed a 2-step model in which Notch signaling drives the initial commitment of BM monocytes to the KC lineage, and TGF-β family ligands, including TGF-β and BMP9, establish KC identity (6,7). In addition, Sakai et al. documented an important role for Smad4 in maintaining KC identity (7). The 2 reports provided the background for understanding how engrafted circulating monocytes differentiated into KCs. In the present study, we precluded the roles of TGF-β, BMP2, and BMP6 and showed that, in addition to BMP9, BMP10 also has a critical role in maintaining KC identity. In addition, we also demonstrated that this signaling pathway is important for KCs to accomplish their strategic role in protecting the host from L. monocytogenes infection. Altogether, this study revealed how the liver microenvironment regulates BMP9 is preferentially expressed in the liver. In the present study, in addition to BMP9, BMP10 was critical for controlling the identity of KCs. BMP10 is mainly expressed in the heart and present in blood (27). However, this circulating BMP10 is unable to activate the ALK1 signaling pathway (27). In fact, it is also weakly expressed in the liver (37). The source of BMP9 and BMP10 was reported to be HSCs (38,39), indicative of a paracrine loop that regulates KC identity and self-maintenance.
KCs are enriched near periportal regions. This asymmetric localization also has a critical role in protecting against systemic bacterial dissemination (29). Interestingly, we found that hepatic macrophages lost their tissue-specific localization in Alk1 fl/fl Vav1 Cre mice and Smad4 fl/fl Vav1 Cre mice. CXCR3 expressed on KCs has been reported to play a role in shaping the positioning of resident immune cells in the liver (29). However, CXCR3 expression in hepatic macrophages from Smad4 fl/fl Vav1 Cre mice was not affected (our unpublished observations), suggesting that the alteration of anatomical localization was not due to the lack of CXCR3 expression. In fact, monocytes constantly replenished the macrophage pool in the livers of Alk1 fl/fl Vav1 Cre mice and Smad4 fl/fl Vav1 Cre mice. These less-mature Mo-KCs may have contributed to a uniform distribution of KCs. Nevertheless, our study is the first to our knowledge to provide insights into how the tissue-specific functions of KCs are affected by factors that imprint their identity.
Deletion of Alk1 and Smad4 resulted in disruption of KC homeostasis, exhibited by loss of KCs over time and replacement by monocyte-derived macrophages. These immature monocyte-derived macrophages displayed reduced expression of VSIG4 and altered function of KCs. Finally, Alk1/Smad4-deficient mice showed an increased susceptibility to infection with gram-positive L. monocytogenes. In fact, mutations in Alk1 result in hereditary hemorrhagic telangiectasia (HHT), which is a rare genetic disease characterized by recurrent epistaxis, cutaneous telangiectasia, and

Methods
Mice. Clec4f Cre/DTR mice were generated at the Nanjing BioMedical Research Institute of Nanjing University (NBRI) using CRISPR/ Quantification of captured L. monocytogenes is shown in the graphs on the right. (E) CFU were assayed in liver, lung, and blood of Alk1 fl/fl Vav1 Cre mice and their littermate controls 10 minutes after injection of L. monocytogenes (4 × 10 7 CFU, n = 4 per group). The experiment was repeated twice. Results represent the mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mantel-Cox test (A and B), 2-way ANOVA (C and D), and 2-tailed Student's t test (E). incorporation was measured by flow cytometry using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
qPCR. Total RNA was isolated with RNeasy Plus Mini Kit (QIAGEN), and cDNA was synthesized with Prime Script RT Reagent Kit (Takara). qPCR was performed with a SYBR Green PCR kit in a CFX Connect Real-time PCR detection system (Bio-Rad). The specific qPCR primers used are listed in Supplemental Table 3.
Data availability. The raw RNA-Seq data generated from this study are available in the NCBI's Sequence Read Archive (SRA) under accession code PRJNA705814.
IVM of liver and L. monocytogenes capture. A multichannel confocal microscope was used to image mouse liver as previously described (50). Briefly, mice were anesthetized (2.5% avertin, 20 mL/kg, i.p., Mil-liporeSigma). The tail vein was cannulated to administer fluorescent dyes and labeled bacteria. The abdominal cavity was exposed by removing the skin and muscles. The mouse was placed on a heated stage (37°C), and the largest lobe of the liver was positioned onto a coverslip; a small piece of sterile laboratory wipes was moisturized with saline and placed over the liver to keep it moist and stable. For the visualization of liver macrophages, platelets, and neutrophils, 2 μg anti-F4/80 (BM8) or 2.5 μg anti-Ly6G (1A8) (BioLegend) was administered i.v. Images were acquired using an inverted Olympus FV3000 confocal microscope with a 20×/0.75 UPLANSAPO objective lens. Laser excitation wavelengths of 488 nm, 561 nm, and 647 nm and a high-sensitivity spectral detector with a GaAsP Xu (Fudan University, Shanghai, China). Alk2 fl/fl mice were provided by Vesa Kaartinen (University of Michigan, Ann Arbor, Michigan, USA). Alk3 fl/fl mice were provided by Yuji Mishina (University of Michigan). Vav1 Cre mice were provided by Bing Liu (Fifth Medical Center of Chinese PLA General Hospital, Beijing, China). CD45.1/Ly5.1 mice were provided by Mingzhao Zhu (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). Mice and their littermates were used between 6 and 16 weeks of age unless otherwise specifically indicated. All mice were maintained at the specific pathogen-free (SPF) facilities of the Beijing Institute of Lifeomics.
Cell suspension preparations, flow cytometry, and antibodies. Cell suspensions were prepared as previously described (48). Briefly, CNS, spleen, lung, and kidney were cut into small pieces, incubated in collagenase type IV (MilliporeSigma) at 37°C for 30 minutes, and vigorously pipetted. The cell suspensions were filtered through a 70μm cell strainer to obtain a homogeneous cell suspension. CNS cell suspensions were further enriched by a Percoll gradient. The liver was perfused via the portal vein with approximately 20 mL HBSS, followed by perfusion with digestion buffer containing 0.05% collagenase type IV for 5 minutes. The digested livers were then excised and disrupted, and the cell suspension was passed through a 70μm cell strainer. Parenchymal cells were separated from nonparenchymal cells by centrifugation at 50g for 5 minutes. Liver cell suspensions were further enriched by iodixanol gradient (OptiPrep) as previously described (49).
For surface marker analysis, cell pellets were stained with the appropriate antibodies at 4°C for 20-30 minutes. For intracellular cytokine analysis, cells were stained with the Cytofix/Cytoperm kit according to the manufacturer's instructions (eBioscience). To obtain hepatic mononuclear cells, mice were perfused via the portal vein with HBSS, and the livers were minced through a 70 μm cell strainer followed by lysis with RBC lysis buffer. The hepatic mononuclear cells were stimulated with a cell stimulation cocktail (eBioscience) for 6 hours, followed by intracellular IL-17A staining.
Flow cytometry was performed using an LSR II Fortessa (BD Biosciences). The acquired data were analyzed with FlowJo software (Tree Star). For cell sorting, a FACS Aria III (BD Biosciences) was used. The antibodies used are listed in Supplemental Table 2.
In vitro culture of KCs. Sorted KCs by FACS were seeded in 12-well plated in DMEM (HyClone) containing 10% FBS (Gibco, Thermo Fisher Scientific). After overnight, the culture medium was replaced by serum-free X-VIVO15 media (Lonza), supplemented with 20 ng/mL M-CSF in the presence or absence of 50 ng/mL rmBMP2 (Peprotech), rhBMP6 (Peprotech), rhBMP9 (Peprotech), or 50 ng/mL rmBMP10 (R&D Systems). The half media were changed every other day. On day 7, KCs were acquired, and RNA was extracted using an RNeasy Plus Mini Kit (QIAGEN).
EdU staining. Mice were administrated 0.5 mg EdU (Thermo Fisher Scientific) via i.p. injection. After 20 hours, KCs were obtained and EdU