DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease.
Upon infection, germline-encoded pattern recognition receptors (PRRs) located on the surface of cells, in endosomal compartments and throughout the cytosol initiate an array of signaling cascades that culminate in the production of type I interferons (IFN-I), pro-inflammatory cytokines and chemokines. These cytokines establish an inflammatory response and an antimicrobial state restraining the spread of the infectious agent. The discovery of Rig-I-like receptors (RLR) as sensors of viral RNA sparked considerable interest in the role of other DExD/H RNA helicases as innate modulators of antimicrobial immune responses [1,2]. DExD/H helicases not belonging with typical RLR also contribute to innate immunity in experimental animals, as recently demonstrated for DDX41 which acts in dendritic cells to limit retroviral growth [3]. We and others have identified the RNA helicase DDX3X as a regulator of IFN-I transcription in cells infected with viruses or with the intracellular bacterial pathogen Listeria monocytogenes [4,5]. DDX3X belongs to the DEAD-box RNA helicase superfamily 2 [6] that has widespread functions in RNA metabolism, including transcription, RNA processing, splicing, decay and translation [7,8]. Moreover, DDX3X is implicated in cellular processes such as apoptosis, cell cycle regulation and tumorigenesis [9]. Deletion of DDX3X in all embryonic tissues causes the death of male embryos at an early postimplantation stage. By contrast, male embryos with epiblast-restricted DDX3X deletion die around E11. 5 with widespread occurrence of apoptotic cells and expression of DNA damage markers [10]. This is most likely a direct consequence of a disturbed cell cycle in embryonic tissue lacking DDX3X. This view is further supported by a study investigating the role of DDX3X in early mouse development using siRNA-mediated knockdown [11]. A homologue of DDX3X called DDX3Y is encoded by the non-recombining region of the Y-chromosome. DDX3X and DDX3Y share around 90% homology. While DDX3X is ubiquitously expressed, DDX3Y protein expression was originally thought to be confined to the male germline [12]. More recent proteomic databases list DDX3Y in cells of the immune system, including T-cells, B-cells and NK-cells. Their high degree of similarity supports the idea that DDX3X and DDX3Y are functionally redundant [13]. The heterosomal origin of the DDX3 isoforms suggests they may contribute to sex-related differences in immunity to microbes, the ability to resolve inflammation and the propensity to develop autoinflammatory syndromes [14–16]. Several studies point to an ambiguous role of DDX3X in viral infections. On the one hand, it may promote replication of viruses like HIV or HCV [17–22]. On the other hand, DDX3X stimulates the production of antiviral IFN-I [23,24]. Antimicrobial pathways leading to IFN-I synthesis converge at two related S/T kinases, TBK1 and IKKε. DDX3X interacts with TBK1 and serves as its substrate [4]. It also interacts with IKKε (Schröder et al, 2008; Gu et al, 2013), the other non-canonical IKK kinase responsible for phosphorylation and activation of the interferon regulatory factors (IRF) 3 and 7 that control Ifnb gene transcription. In addition to its function downstream of TBK1/IKKε, DDX3X reportedly associates with the adaptor protein MAVS which localizes upstream of TBK1 and IKKε and supports signal transduction by the two DExH helicases RIG-I and MDA-5. In this context DDX3X was shown to sense viral RNA and to supplement the function of RIG-I and MDA-5 in the early phase of infection [25]. Strengthening its importance for antiviral immunity, a recent report lists DDX3X among genes conferring intrinsic antiviral immunity to stem cells [26]. Like viruses, the Gram-positive bacterium Listeria monocytogenes (Lm) is a potent inducer of IFN-I [27]. After phagocytosis by macrophages, Lm escapes to the cytosol via the secretion of listeriolysin O (LLO) that disrupts the phagosomal membrane [28,29]. The innate response to Lm is characterized by intracellular effector mechanisms as well as the secretion of many pro-inflammatory chemokines and cytokines, among them the IFN-I. Cytoplasmic nucleic acids derived from Lm were shown to trigger strong induction of the IFN-I genes. Lm DNA promotes IFN-I expression through the DNA sensors cGAS and IFI16, the adaptor molecule STING, as well as TBK1 kinase and its downstream targets IRF3 and IRF7 [30,31]. In addition to DNA, Lm RNA was implicated in the induction of type I IFNs via the cytosolic receptor RIG-I [32]. Knock-down of DDX3X phenocopies the silencing of TBK1 on Ifnb gene induction after Lm infection [4], emphasizing the general importance of DDX3X for the TBK-IRF3 axis. Despite the fact that IFN-I are crucial for protective antiviral responses, their impact on Lm infection appears to be detrimental for the host as evidenced by the observation that mice lacking the receptor for type I IFNs (IFNAR) are more resistant to parenteral infection with Lm [33–35]. Unlike IFN-I, the type II interferon (IFNγ) is strongly associated with protective innate immunity against Lm [36,37]. Rapid production of IFNγ is essential and has been attributed to innate lymphocyte responses that include natural killer cells [38]. The resistance against Listeria provided by IFNγ is strongly associated with its role as a macrophage-activating cytokine [39]. Here we report a crucial role of DDX3X in the innate immune responses of cells and mice. We show that besides its role in the regulation of the TBK1-IRF3 axis, DDX3X controls the NFκB signaling pathway and has a profound impact on inflammatory cytokine production. DDX3Y, either alone or together with additional Y-chromosomal genes, partially compensates for the loss of the Ddx3x gene, as homozygous female cells and mice show more severe loss-of-function phenotypes. Mice lacking DDX3X in the hematopoietic system show alterations of bone marrow and splenic cell populations and are highly susceptible to Lm infection. Our data thus demonstrate a vital role of the sex-specific DDX3 isoforms in innate immunity to Listeria.
Based on the role of DDX3X in IFN-I synthesis we investigated whether loss of the helicase causes a defect in innate immunity to virus. In male fibroblasts, derived from Ddx3xfl/y-CreERT2 mice and rendered DDX3X-deficient by treatment with tamoxifen, we observed reduced synthesis of IFNα as well as IFNβ mRNA following infection with vesicular stomatitis virus (VSV; Fig 2A and 2B). In accordance with this, VSV replicated to higher numbers in DDX3X-ablated cells compared to controls that had not been treated with tamoxifen (4OHT; Fig 2C). Surprisingly however, Ddx3xfl/y Vav-iCre mice infected i. v. with VSV showed only a marginal reduction of viral clearance, in spite of the fact that hematopoietic cells contribute to IFN synthesis when i. v. injection is chosen as infection route (Fig 2D; [41,42]). This result suggests that any defect in IFN-I synthesis caused by deletion of DDX3X in hematopoietic cells can be compensated and that VSV infection does not reveal a major defect in the DDX3X-deficient innate immune system. To further characterize the role of DDX3X in immune responses, we subjected Ddx3xfl/y Vav-iCre mice to intraperitoneal (i. p.) infection with the intracellular bacterial pathogen Listeria monocytogenes (Lm). Compared to Ddx3xfl/y control animals, Ddx3xfl/y Vav-iCre mice were highly susceptible to Lm. All knockout animals succumbed to infection within 6 days, whereas most control animals survived the observation period (Fig 2E). In line with this, the bacterial burdens in spleens and livers of Ddx3xfl/y Vav-iCre animals were strongly increased three days after i. p. infection (Fig 2F). IFN-I deficiency increases the innate resistance of mice against Lm [33–35]. Therefore, pro-survival effects of reduced IFN-I synthesis in Ddx3xfl/y Vav-iCre animals are clearly overwhelmed by immune defects that weaken host resistance. To address the consequences of DDX3X deficiency and to seek an explanation for reduced resistance to Lm, we determined the composition of mature hematopoiesis-derived cell populations in bone marrow and spleen. In the bone marrow total cell numbers were slightly reduced due to a rather selective loss of bone marrow B lymphocytes (Fig 3A). We found splenic composition to be more dramatically changed. Besides B220+ B cells the numbers of CD3+CD4+ and CD3+CD8+ T cells, CD3+CD1d-tetramer+ NKT cells and CD3-NK1. 1+ NK cells were significantly reduced in Ddx3xfl/y Vav-iCre mice. Interestingly, cells of myeloid origin were not generally affected. Numbers of total CD11c+ dendritic cells, CD11b+Ly6G+Ly6Clo neutrophils and CD11b+F4/80+ macrophages unaltered and only CD11b+Ly6Chi monocytes showed a slight reduction (Fig 3B). To assess whether reduced lymphoid cell numbers were due to alterations in hematopoiesis we counted the numbers of lineage (Gr1, Mac1, Ter119, B220, CD3) —Sca1+ cKit+ (LSK), lineage- Sca1- cKit+ (LK), as well as lineage- Sca1/cKitdouble-dim CD127+ common lymphoid progenitor (CLP) cells and found all three populations to be significantly reduced (Fig 3C), indicating a role for DDX3X in early hematopoietic development. In line with this observation, further analysis of bone marrow B cell development showed a slight reduction in the numbers of pre-pro- and early pro B cells, as well as a particularly strong reduction of progenitors from the small pre- B cell stage onwards, i. e. following the proliferation of large pre B cells (Fig 4A). Surprisingly, the development of NK cell precursors in the bone marrow seemed unaffected by loss of DDX3X, with lineage (Gr1, Ter119, B220, CD3) - CD122+ NK1. 1- Dx5- NK progenitors (NKp), lineage- CD122+ NK1. 1+ immature NK cells (iNK) and lineage- CD122+ NK1. 1+ Dx5+ mature NK cells (mNK) all present at normal numbers, indicating that the reduction in CLP cells can be compensated for during NK cell development (Fig 4B). Since the numbers of splenic T and NKT cells were also dramatically affected in Ddx3xfl/y Vav-iCre mice we more closely examined development of these lineages in the thymus. Interestingly, the number of thymic CD4/CD8 double-positive (DP) and CD8 single-positive (SP) thymocytes was increased in absence of DDX3X, while CD4 SP thymocytes seemed unaffected, suggesting that a developmental block is not responsible for the reduced numbers of mature T cells observed in the periphery (Fig 4C). In contrast, NKT cell development in the thymus was more dramatically affected with CD1d-tetramer+ CD24- stage 1, CD1d-tetramer+ CD24- CD44+ stage 2 and CD1d-tetramer+ CD24- CD44+ NK1. 1+ stage 3 NKT cells all strongly reduced in numbers (Fig 4D). In summary, our analysis of hematopoiesis in Ddx3xfl/y Vav-iCre mice indicated a differential impact on distinct lineages. While the reduced numbers of peripheral B and NKT cells can be at least partially explained by developmental alterations, the lack of splenic T and NK cells does not seem to result from an obvious developmental block. Given that DDX3X-deficient embryos show increased apoptosis we investigated whether cell death might contribute to the reduction of DDX3X-deficient splenic leukocytes. As shown in Fig 5, the absence of DDX3X caused elevated rates of cell death in B220+ B cells, CD3+ T cells and CD3- NK1. 1+ NK cells, and to a lesser extent in CD11b+ myeloid cells. These results suggest that DDX3X, besides its variable roles in the development of different immune cell lineages, also plays a role in the maintenance of immune cells in the periphery, with the most profound effects observed in lymphocytes and splenic NK cells. To determine whether Lm infection exacerbates defects in hematopoiesis, splenic cell populations were analysed in infected mice. 24 hours of infection increased the number of total splenocytes in both control and Ddx3xfl/y Vav-iCre mice (first panels of Figs 3A and 6A, Table 1), mainly due to recruitment of Ly6Chi inflammatory monocytes (approx. 13-fold versus 12-fold mean increase, respectively), suggesting no obvious recruitment defects caused by the absence of DDX3X. By and large, the cell populations suffering from DDX3X deficiency were the same as in uninfected animals with B220+ B cells, CD3+ CD8+ T cells, CD3+ CD1d-tetramer+ NKT cells and CD3- NK1. 1+ NK cells being particularly compromised. By comparison, DDX3X-deficient myeloid cells showed a rather mild decrease that did not reach statistical significance (Fig 6A). Table 1 summarizes the infection-induced relative changes in splenic leukocyte composition. While some differences in the recruitment and/or proliferation of different leukocyte populations was observed, this display of the data emphasizes that most changes in leukocyte numbers precede infection rather than being result of the innate response to Lm. In our animal experiments, the primary site of Lm infection is the peritoneal cavity. We therefore analysed cell recruitment to this anatomical location. The data summarized in Fig 6B show that unlike the spleen hematopoietic DDX3X deficiency per se has little effect on immune cells residing in the peritoneum, with only the number of Ly6Chi monocytes slightly reduced in PBS treated control animals. Upon infection with Lm we observed a rise in peritoneal exudate cell (PEC) numbers owing primarily to recruitment of Ly6CloLy6G+ neutrophils, Ly6Chi monocytes and CD11b+F4/80+ macrophages. While neutrophil recruitment seemed unaffected, the number of inflammatory monocytes was significantly decreased in Ddx3xfl/y Vav-iCre animals and we observed a tendency for lower macrophage numbers. These results indicate a requirement for DDX3X in the generation, maintenance or recruitment of inflammatory monocytes particularly in the peritoneal cavity. To address potential protective immune mechanisms under DDX3X control we determined the levels of several serum cytokines and chemokines in mice infected with Lm. Most of the factors we assessed were not significantly different between Ddx3xfl/y Vav-iCre mice and control animals. At 24 hours after infection, serum levels of Il-6, IL-17, IL-10, TNFα and IL1β were unchanged and while the levels of Il-6, IL-10, TNFα and IL1β increased in Ddx3xfl/y Vav-iCre relative to control animals at 72h, the difference only reached significance in the case of IL-10. In addition, we observed a small but significant reduction in IL-4 levels at both time points as well as reduced CCL5/RANTES concentrations at 24 hours (S1 Fig). The most striking difference was the reduction of serum IFNγ and IL-12 in Ddx3xfl/y Vav-iCre mice at 24 hours after infection. At 72hrs IFNγ levels of Ddx3xfl/y Vav-iCreand control mice were similar, either due to a recovery of the former, or to more rapid subsiding of IFNγ production in the latter animals (Fig 7A). The results suggest an inhibited IL12-IFNγ axis in the early stage of Lm infection of Ddx3xfl/y Vav-iCre mice. Upon L. monocytogenes infection, early production of IFNγ by NK-cells, NKT cells and CD8+ T-cells is crucial for the activation of macrophage effector functions and subsequent bacterial clearance [43–45]. Early IFNγ production requires IL-12, as IL-12 depletion leads to abrogated IFNγ levels and reduced resistance to infection [36,38,46,47]. To address the contribution of these cell populations to the differences seen in IFNγ levels 24 hours post-infection, flow-cytometric analysis of IFNγ production was performed. We found that NK cells were an important source of IFNγ in wt animals at this early time point, because the fraction of producer cells was the largest among the investigated cell types (Fig 7B). Numbers of NK cells are much lower in Ddx3xfl/y Vav-iCre mice and of the few remaining cells a smaller fraction produced IFNγ, possibly as a consequence of reduced IL-12 amounts. The fraction of IFNγ-producing splenic iNKT cells was low and not significantly affected by genotype, while IFNγ– producing CD8+ T cells were below the limit of detection (Fig 7B). These data suggest that NK cells make an important contribution to serum IFNγ in the early phase of Lm infection, and their absence in Ddx3xfl/y Vav-iCre mice is largely responsible for reduced serum levels of IFNγ at the 24 hour time point. To verify our assumption that NK cells are the primary producers of IFNγ at 24 hours after Lm infection, we depleted NK cells from Ddx3xfl/y and Ddx3xfl/y Vav-iCre mice using an anti-NK1. 1 antibody. 72 hours after intraperitoneal administration about 98% of NK cells were depleted, while splenic CD1d-tet+ iNKT cells remained unaffected under these conditions. The NK-depleted mice were then infected with Lm and 24 hours later serum was collected. Depletion of NK-cells strongly reduced serum IFNγ from about 104pg/ml to less than 103pg/ml after NK depletion (leftmost panel in Fig 7A and 7C). NK depletion also abolished the differences between wild type controls and Ddx3xfl/y Vav-iCre mice (Fig 7C). In line with earlier findings [48] [49], depletion of NK cells did not reduce the bacterial burden in infected organs of wt mice (Fig 7D). Therefore, although our data emphasize the contribution of NK cells to serum IFNγ production during the early, innate immune response against Lm, these results suggest that residual serum IFNγ in NK-depleted mice, or local production by other cells, suffices for macrophage activation in the early phase of Lm infection. Our data also support our notion that NK cells are the primary cell type responsible for the lack of sufficient early serum IFNγ production in mice lacking hematopoietic DDX3X. Macrophages are essential effector cells against Lm. Reduced proinflammatory activity and/or cell-autonomous antimicrobial states in absence of DDX3X might thus explain the reduced innate immunity of Ddx3xfl/y Vav-iCre mice. To study antimicrobial responses of both male and female sexes we generated bone marrow derived macrophages (BMDMs) from Ddx3xfl/fl CreERT2 and Ddx3xfl/y CreERT2 mice. In vitro deletion of DDX3X by tamoxifen treatment allowed us to address the effect of Ddx3x deletion in the presence or absence of the Ddx3y gene. Tamoxifen treatment after 5 days of culturing bone marrow cells in CSF-1-containing medium did not impair the terminal differentiation of macrophages from Ddx3xfl/fl CreERT2 female and Ddx3xfl/y; CreERT2 male mice. However, the fully differentiated, female Ddx3x-/- cells showed a reduced life span and were used no later than 3 days after the termination of tamoxifen treatment. To determine the impact of DDX3X on cell-autonomous clearance of Lm, BMDMs of male (Fig 8A, left panel) and female mice (Fig 8A, right panel) were infected with Lm at an MOI of 10 and colony forming unit (CFU) assays were performed to monitor bacterial growth. DDX3X-deficient cells from both sexes contained higher bacterial loads compared to the wild-type cells 8 hours after infection. Activation of DDX3X-deficient female cells with IFNγ resulted in increased killing of intracellular Lm. The relative drop in bacterial loads were similar to wt (2. 3 versus 2. 7-fold) but in absolute numbers about twice as many bacteria persisted in DDX3X-deficient macrophages (Fig 8A, left panel). The data suggest that IFNγ responsiveness per se is unaffected, but that the remaining difference in bacterial numbers may result from a higher initial burden compared to wt cells. The consequences of DDX3X deficiency on the expression of genes contributing to the innate antimicrobial response of macrophages were examined by RNA-Seq analysis of untreated or Lm-infected cells. Genes differentially expressed upon Lm-infection in either male or female cells (S1 Table) are enriched for GO Immune System terms, and the extent to which DDX3X deficiency altered the transcriptome depended on the sex chromosomes of the cells (Fig 8B, heatmap and upper right panel). This suggests that one or more Y-chromosomal genes partially compensate for the loss of DDX3X at the level of transcript synthesis. The data demonstrate control of the macrophage transcriptome by DDX3 isoforms and, in line with the mouse phenotypes, an incomplete redundancy between DDX3X and DDX3Y and/or additional genes present on the Y chromosome. wt mice of female sex show higher susceptibility to Lm infection and immune-related genes may show sex-dependent differences in expression [50]. In line with this, we observed a considerable variation in the differentially expressed genes in macrophages of infected wt male and wt female mice (heatmap of Fig 8B). Comparison of gene sets differentially expressed upon Lm infection at the same significance threshold in the four experimental conditions revealed that a set of 311 genes responded with altered expression in wt males, but not in wt females, whereas only 7 genes responded to infection in females, but not in males (Fig 8B, Venn diagram). 230 of the 311 male-specific genes and all of the 7 female-specific genes were unresponsive to infection in macrophages of the same sex lacking DDX3X. The data suggest a contribution of DDX3X to sex-specific gene expression in response to infection with Lm. Validation of critical antimicrobial genes affected by the lack of the Ddx3x gene in RNA-Seq experiments by qPCR is shown in Fig 8C. Cytokines with a strong impact on innate resistance to Lm, such as IL-1, IL-6, IL-12, TNFα as well as chemokines were controlled by DDX3X. Further consistent with the RNA-Seq results, the reduction in expression for most of the examined genes was more pronounced in female (lower panel) than in male macrophages (upper panel). The broad effect of DDX3X deficiency on cytokine production is consistent with the impact of the protein on both IRF3/7 and NFκB pathways (Fig 1C, 1E and 1F). To determine whether the DDX3X effect on these genes is at the level of transcription rather than transcript processing, primary transcript synthesis of a number of mRNAs was examined at 2hrs or 4hrs after infection in female macrophages. Consistent with our earlier observation that DDX3X increases Ifnb gene expression at the level of transcription [4], most primary transcripts were reduced, the Tnfa transcript being a noteworthy exception (Fig 8D). DDX3X-deficiency also blunted responses to the defined pathogen-associated molecular patterns (PAMPs) poly (I: C), poly (dA: dT) and LPS (Fig 9A). With exception of the Il6 gene, the impact of DDX3X deficiency on signalling by the respective pattern recognition receptors was more pronounced in female cells (upper versus lower panels). These results show that DDX3X affects both primary transcription as well as transcript processing of genes targeted by microbial sensor molecules. Finally, to assess the production of Interferon stimulated genes (ISGs) in absence of DDX3X we treated DDX3X-ablated and control BMDMs with IFNs. As shown in Fig 9B, the induction of Mx1, Oas2, Ifit3 and Isg15 mRNAs was not significantly altered between genotypes upon stimulation with IFNβ (left panels). Similarly, induction of Irf1 mRNA after addition of IFNγ was comparable between the genotypes (rightmost panel). These data indicate that DDX3X-deficient macrophages respond normally to exogenous sources of IFN.
In spite of being described as a substrate of the TBK1 and IKKε kinases we observed that DDX3X enhances the induction of the Ifnb gene also in the absence of TBK1/IKKε when a constitutively active phosphomimetic mutant of IRF7 is provided. This demonstrates that DDX3X retains the ability to enhance IRF-mediated transcription in absence of TBK1/IKKε. Therefore, activating phosphorylation in this situation is either dispensable or performed by other kinases, for example, the conventional IKK. The latter interpretation is in line with the ability of DDX3X to enhance NFκB-dependent transcription, but requires experimental confirmation. Ddx3x is essential for development, as its deletion leads to prenatal lethality in mice, where DDX3X is necessary for both embryonic and extraembryonic tissue development [10]. Both DDX3X and DDX3Y are among essential genes in some, but not all investigated human leukemic cell lines [52]. Hematopoietic loss of DDX3X via Vav-Cre mediated deletion resulted in viable male but not female mice, emphasizing the pivotal role of DDX3X in hematopoietic development. The Y-chromosomal homologue DDX3Y shares 90% (mouse) or 92% (human) identity at the protein level. The high degree of homology manifested in its redundant functions in enhancing Ifnb gene expression. Therefore, it is tempting to speculate that DDX3Y alone is responsible for the hematopoietic rescue provided by the Y-chromosome. However, this has not been directly tested and a participation of additional Y-chromosomal genes cannot be ruled out. Sofar we have not examined the hematopoietic defect underlying the death of female Ddx3xfl/y Vav-iCre mice. A failure to produce erythrocytes would seem a likely explanation. Analysis of hematopoiesis-derived cells in bone marrow and spleen revealed three degrees of susceptibility to the loss of DDX3X. Particularly B cells were impeded in their development, leading to reduced numbers in both organs. NK cells on the other hand developed normally in the bone marrow, but showed reduced presence in the spleen. This defect may result either from reduced recruitment to the spleen, or a difference in survival conditions between splenic and bone marrow NK cells. Finally, mature myeloid cells such as macrophages, neutrophils, or CD11c+ DC showed little dependence on DDX3X in either organ. One likely explanation for reduced hematopoiesis is an increase in apoptosis in DDX3X-deficient cells, particularly B cells and NK cells. Based on studies in mouse embryos this may result from a p53-mediated response to DNA damage and reflect an activity of DDX3X in cell cycle regulation and/or DNA damage repair (Bol et al, 2015; Sun et al, 2013; Li et al, 2014; Chen et al, 2016). In keeping with this notion a preliminary proteomic screen in macrophages identified multiple DDX3X interactors with potential impact on DNA damage and repair pathways. Among these factors are the DBHS family members SFPQ, NONO and PSPC1 [53]. The idea that the increase in apoptosis found in DDX3X-deficient cells is linked to proliferation is supported by cell types found to be particularly sensitive, such as the small preB stage in B cell development, or inflammatory monocytes that, unlike resident splenic macrophages, showed increased apoptosis when lacking DDX3X. Given the role of DDX3X in the IFN-I synthesis of infected cells we were surprised to find innate immunity to VSV unimpaired. Hematopoietic cells, particularly pDC, are thought to be essential IFN producers during systemic VSV infection [41,42]. One possibility to explain our results is that pDC may not require DDX3X for IFN-I production. Alternatively, serum levels of IFN-I may not accurately reflect the local need to clear infection. This hypothesis is consistent with our findings in Lm-infected mice: NK cells were responsible for high serum IFNγ amounts, but their depletion still left enough IFNγ production for unimpaired innate immunity. An alternative explanation is therefore that our findings with VSV reflect a greater than hitherto suspected need for IFN-I from nonhematopoietic cells. We show that induction of antiviral genes by IFN-I is unimpaired in DDX3X-deficient cells. This further suggests that any IFN-I production defect in Ddx3xfl/y Vav-iCre mice does not reduce the levels of the cytokines below the threshold needed for the antiviral state. The susceptibility of Ddx3xfl/y Vav-iCre mice to Lm shows that DDX3Y alone is not generally sufficient to compensate for the lack of DDX3X in the immune system. Due to the lethality of DDX3X deficiency in female mice, we cannot assess the relative contribution of DDX3Y to the innate immune response against Lm in vivo. Specifically, we cannot address a potential contribution of the DDX3 isoforms to the increased susceptibility of female C57BL/6 mice noted by others [50,54]. Bone-marrow derived macrophages from Ddx3xfl/fl CreERT2 mice, however, allowed us to compare expression levels of innate immune genes induced upon Lm infection of cells representing both sexes. This approach again revealed an incomplete rescue by DDX3Y. It correlated well with the strongly altered gene expression of infected macrophages in vitro. Since macrophages are ubiquitous primary targets of Lm in infected tissues they are major players in shaping the local cytokine milieu and, concomitantly, the activation status of surrounding cells. Consistent with this notion macrophages lacking one or both DDX3 isoforms showed a significant impairment in limiting the growth of intracellular Lm. Activation by IFNγ increased their antibacterial activity, but not to the same level observed in control cells. Thus, reduced cell-autonomous immunity as a consequence of less activating stimuli in the extracellular environment is likely to cause an impairment of mice to reduce the bacterial burden by innate mechanisms. The data in S1 Fig show that the broad effect observed upon DDX3X deficiency particularly in female macrophages did not translate into a similarly widespread impact on serum cytokine levels in infected mice. On the one hand this may result from the fact that male mice had to be used for infection experiments, hence from the weaker impact of DDX3X deficiency in cells expressing DDX3Y. Alternatively, macrophages may not be responsible for building up high levels of serum cytokines. In summary, loss of DDX3X impacts on the innate immune system both through its role in hematopoiesis and its regulation of the innate response to Listeria infections. It alters lymphoid organ cellularity and the cytokine cocktail needed for cell recruitment and functional activation of innate immune cells.
Animal experiments were carried out at the University of Veterinary Medicine Vienna and have been approved by the institutional ethics and animal welfare committee and the national authority (Austrian Federal Ministry of education, Science and Research) according to §§26ff of Animal Experiments Act (Tierversuchsgesetz TVG 2012, BGBl. I Nr 114/2012) under the permission license numbers BMWF 68. 205/0032-WF/II/3b/2014 and BMWFW-68. 205/0212-WF/V/3b/2016. Animal husbandry and experimentation was performed under the Austrian national law and the ethics committees of the University of Veterinary Medicine Vienna and according to the guidelines of FELASA which match those of ARRIVE. Mouse embryonic fibroblast (MEFs) deficient in either TBK1/IKKε (kindly provided by S. Akira, Osaka University, Osaka, Japan, or in DDX3X (derived from Ddx3xfl/y CreERT2 or Ddx3xfl/fl CreERT2 embryos and immortalized by the 3T3 method), as well as HEK293 cells (American Type Culture Collection CRL-3216) were cultured in DMEM (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) supplemented with 10% FBS (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) and with penicillin and streptomycin (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria). Deletion of DDX3X was carried out by Tamoxifen treatment (4-OHT; Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) for 48 hours at a final concentration of 500nM. The plasmids pIE-NHA-hDDX3X, pIE-NHA-hMAVS, pCS2-N-Myc-mTBK1 and pEF-HA-mIRF3 were described previously [4]. The expression vector pIE-NHA-DDX3Y was generated based on the RefSeq ID: NM_012008. 2 using the EcoRI and BspeI restriction sites. FLAG-IRF7-M15 [40] was obtained from Isabelle Marié (New York University, NY, USA). The NFκB-reporter plasmid was kindly provided by Ann J. Richmond (Vanderbilt University School of Medicine, Nashville, Tennessee, USA) [55]. Anti-GAPDH (Clone ABS16; Millipore GesmbH, Vienna, Austria) was used in 1: 3000 dilution; anti-DDX3X (Clone A300-474A; Bethyl Laboratories Inc. , distributed by Sanova Pharma, Vienna, Austria) was used in 1: 1000 dilution; anti-HA. 11 Epitope Tag (Clone MMS-101P-200; Cambridge Bioscience, Cambridge, UK) was used in 1: 1000 dilution; anti-Myc (Clone 9B11; CST, distributed by New England Biolabs, Frankfurt am Main, Germany) was used in 1: 1000 dilution; anti-Flag (anti-ESC; Clone A190-101A; Bethyl Laboratories Inc. , distributed by Sanova Pharma, Vienna, Austria) was used in 1: 1000 dilution. Viability stains were carried out according to manufacturer' s protocol (Fixable Viability Dyes; formerly ebioscience, now Thermo Fisher Scientific, distributed by Fisher Scientific GmbH, Vienna, Austria). Cells were pre-incubated with TruStain fcX (formerly ebioscience, now Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria) and stained with appropriate surface antibodies in PBS 2% FCS. For intracellular cytokine stainings, cells were fixed in 2% PFA and permeabilized using a saponin-containing buffer (BD Perm/Wash; Becton Dickinson Austria GmbH, Schwechat, Austria). Anti-CD11b (Clone M1/70), anti-CD3e (Clone 145-2C11), anti-CD8a (Clone 53–6. 7), anti-B220 (Clone RA3-6B2), anti-Ly6G (Clone 18A), anti-CD11c (Clone HL3), anti-CD49b (Clone Dx5) anti-CD44 (Clone IM7), anti-Gr1 (Clone RB6-8C5), anti-Ter119 (Clone TER119) and anti-CD122 (Clone 5H4) antibodies were purchased from BD Bioscience (Becton Dickinson Austria GmbH, Schwechat, Austria) and used according to manufacturer' s instructions. Anti-CD4 (Clone GK1. 5), anti-NK1. 1 (Clone PK136), anti-NKp46 (Clone 29A1. 4), anti-IFNγ (Clone XMG1. 2), anti-F4/80 (Clone BM8), anti-Ly6C (Clone HK1. 4), anti-BP1 (Clone 6C3), anti-CD43 (Clone ebioR2/60), anti-IgM (Clone eB121-15F9), anti-CD24 (Clone M1/69), anti-cKit (Clone 2B8), anti-Sca1 (Clone D7) and anti-CD127 (Clone A7R34) were purchased from Thermo Fisher Scientific (formerly ebioscience, now distributed by Fisher Scientific Austria GmbH, Vienna, Austria) and used according to manufacturer' s instructions. PBS-57 loaded and PE-conjugated CD1d tetramers were obtained from NIH Tetramer Core facility (NIH Tetramer Core Facility at Emory University, Atlanta, US) and used according to manufacturer' s instructions. Fluorochrome labelled Annexin V and 7-AAD were purchased from Thermo Fisher Scientific (formerly ebioscience, now distributed by Fisher Scientific Austria GmbH, Vienna, Austria) and used according to manufacturer' s instructions. Data was acquired on BD FACSAria or BD LSRFortessa and analyzed using FlowJo Software. MEFs were transiently transfected in 6-well plates (3,5x 105 cells/well) using Turbofect (Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria) according to manufacturer’s instructions. DNA amount across the samples was equalized by transfecting an empty vector. Bone-marrow derived macrophages were obtained from Ddx3xfl/fl CreERT2 and Ddx3xfl/y CreERT2 mice via flushing the tibia and femur. Macrophages were differentiated in DMEM (10% FBS, pen/strep) containing recombinant M-CSF (a kind gift from L. Ziegler-Heitbrock, Helmholtz Center, Munich, Ger). On day 5, medium was changed to starvation medium (DMEM, 2% FBS, pen/strep, M-CSF) and half of the cells was treated with 4-OHT (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) for 48 hours at a final concentration of 500nM to induce deletion of Ddx3x. After 48 hours medium was replaced (DMEM, 10% FBS, pen/strep, M-CSF). On day 8–9 cells were seeded for experiments. BMDMs were stimulated with 30 μg/ml poly (I: C) (formerly Amersham Biosciences, now GE Healthcare Services Europe, distributed by Fischer Scientific Austria GmbH, Vienna, Austria) or 10 μg/ml poly (dA: dT) (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) using Hyperfect (Qiagen, Vienna, Austria) and Polyfect (Qiagen, Vienna, Austria) respectively according to the manufacturer’s instructions. LPS (Escherichia coli 055: B5; Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) was added dropwise to a final concentration of 100ng/ml. Where indicated, cells were treated with IFNβ (PBL, distributed by Enzo Life Sciences, Lörrach, Germany) at a final concentration of 250U/ml or IFNγ (formerly ebioscience, now Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria) at a final concentration of 5ng/ml. L. monocytogenes strain LO28 was grown for 16 hours in brain heart infusion medium (Becton Dickinson Austria GmbH, Schwechat, Austria). BMDMs (seeded the day before in antibiotic free DMEM supplemented with 10% FBS) were infected at a multiplicity of infection (MOI) of 10 for 1 h at 37°C. After this, cells were washed with PBS and complete DMEM containing 50 μg/ml gentamicin (MP Biomedicals, Santa Ana, US) was added to kill extracellular bacteria. After another 1 h, BMDMs were washed with PBS again, and medium was replaced with DMEM containing 10 μg/ml gentamicin and left for another 2 hours. After stimulation, BMDMs were washed two times with PBS and used for RNA isolation. MEFs were transfected with either 50pmol DDY3Y siRNA or with 50pmol non-targeting siRNA (Dharmacon, distributed by THP Medical Products GmbH, Vienna) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria) under antibiotic-free conditions. After 48 hours, medium was exchanged and the cells transfected for 4 hours with 10μg/ml poly (dA: dT) (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) using Polyfect (Qiagen, Vienna, Austria) according to the manufacturer’s instructions. Subsequently, cells were washed once with PBS and used for RNA isolation. Floxed Ddx3x (Ddx3xfl/fl or DDX3Xfl/y) mice were generated via homologous recombination in ES cells. Specifically, loxP sites flanking exon 2 were introduced by gene targeting. Ddx3xfl/fl CreERT2, Ddx3xfl/y CreERT2 and Ddx3xfl/y Vav-iCre mice on a C57BL/6 genetic background were housed under specific pathogen-free conditions according to FELASA guidelines. NK cell depletion of Ddx3xfl/y, Ddx3xfl/y Vav-iCre and C57BL/6 mice was carried out by injecting the anti-Nk1. 1 antibody (150 μg/mouse) 3 days prior to infection. Depletion efficiency was confirmed by flow cytometry of blood and splenic leukocytes. For infection experiments an overnight culture of Listeria monocytogenes was recultured in BHI medium to late logarithmic phase, pelleted and diluted in PBS. The concentration of L. monocytogenes was quantified by optical density measurements at 600 nm. The infectious dose was controlled by plating serial dilutions on BHI agar plates and counting the colonies. For infection, bacteria were diluted in PBS and 200 μl were injected into the peritoneum of 8- to 12-week-old mice. For the infection with VSV, mice were infected i. v. with 100 μl of 1x106 plaque forming unit (pfu) of VSV. The progress of the disease was monitored every 2–4 h during the “day phase” (7 a. m. to 7 p. m.) or both during the “day” and the “night phase” depending on the condition of the animals. In survival or terminal stage experiments, humane endpoint by cervical dislocation was conducted if death of the animals was expected within next few hours. MEFs were infected with VSV at an MOI of 0,1 under FCS-free conditions. After 1 hour of infection, the medium was exchanged to remove unbound virus. After the indicated periods of time, the supernatants were collected. On the next day, L-929 cells (American Type Culture Collection CCL-1) cells were infected by a dilution series prepared from the collected supernatants in DMEM. After 1 hour of infection, the medium was replaced with DMEM containing 0,5% low melting point (LMP) agarose (Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria). After 24 hours, crystal violet solution (Sigma, distributed by Sigma-Aldrich Handels GmbH, Vienna, Austria) was added onto the agarose to stain the cells. Subsequently, plaques were counted and the viral titer was determined. Bone-marrow-derived macrophages were infected with L. monocytogenes strain LO28 as described above. Additionally, BMDMs were stimulated with the combination of heat-killed Listeria (MOI: 50) and IFNβ (250U/mL) for 4hours. RNA was extracted from bone-marrow-derived macrophages using the NucleoSpin RNA II kit (Macherey-Nagel, distributed by VWR International GmbH, Vienna, Austria). RNA samples were poly (A) selected, libraries were prepared using TruSeq RNA Sample Preparation Kit (Illumina Inc. , San Diego, US) as per manufacturers instruction, and sequenced (50 bp single-end read) on an Illumina HiSeq 2000. Sequencing data have been deposited to NCBI Gene Expression Omnibus and are accessible through GEO series accession number GSE86591. 50-bp single-end Illumina mRNA sequencing reads were aligned to the mm10 reference genome using STAR (v 2. 5. 0a), and gene-level read counts were obtained using htseq-count (HTSeq v 0. 6. 1p1) [56]. Heat map of log 2-fold gene expression changes between Listeria-treated and untreated samples were generated for genes differentially expressed upon treatment in WT samples of either sex (padj <0. 01, abs (logFC) >1; DeSeq2 v1. 18. 1). Functional enrichment analysis of the differentially expressed genes using DAVID v6. 8- top 10 enriched GO terms ranked by p-value for the category GOTERM_BP_3 are shown [57]. The assay was performed a recently described [39]. Briefly, 5x104 BMDMs were seeded into 96-well plates in DMEM (10% FBS, no antibiotics) and stimulated with IFNγ as indicated. On the next day, an overnight culture of L. monocytogenes strain LO28 was used to infect BMDMs at an MOI of 10. After uptake of the bacteria medium containing gentamicin was added to kill extracellular bacteria. BMDMs were washed two times with PBS followed by lysis in dH2O at the indicated time points. Serial dilutions of the lysates were plated onto brain heart infusion plates followed by incubation for one day at 37°C. Intracellular bacteria were quantified by counting the number of colonies. Data shown are representatives of at least 3 independent experiments. Proteins were isolated during RNA isolation with NucleoSpin RNA kit (Macherey-Nagel, distributed by VWR International GmbH, Vienna, Austria). In brief, after adjusting RNA binding conditions with 70% ethanol, lysate were centrifuged. At this step nucleic acids are bound to the silica membrane of the column, whereas proteins flow through. After collecting the flow-through containing the proteins, equal amounts of Protein Precipitator (Macherey-Nagel, distributed by VWR International GmbH, Vienna, Austria) was added and samples were left at room temperature for 10 minutes. After this, samples were centrifuged at 11. 000g for 15 minutes and supernatants were discarded. Precipitated proteins were washed with 50% ethanol and centrifuged again. Supernatants were discarded, pellets were dryed and 30 μl of 1x Laemmli sample buffer (62,6 mM Tris pH: 6,8, 10% Glycerol, 2% SDS, 7,1% β-mercaptoethanol, bromophenol blue) was added to them. The samples were then subjected to western blot analysis as described above. After primary antibody binding the blots were probed with fluorescence-labelled secondary antibodies (formerly Invitrogen, now Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria) at a dilution of 1: 15000 and detected by the Odyssey infrared imaging system (LI-COR, Lincoln, NE). For cytokine analysis blood was taken by cardiac puncture and serum was isolated via centrifugation at 10. 000g for 1 minute. Cytokine concentrations were determined using the FlowCytomix system (formerly ebioscience, now Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria). In some experiments, IFNγ concentrations were measured by ELISA (formerly ebioscience, now Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria). Total RNA was extracted from mouse embryonic fibroblasts and bone-marrow-derived macrophages using the NucleoSpin RNA II kit (Macherey-Nagel, distributed by VWR International GmbH, Vienna, Austria). The cDNAs was prepared using Oligo (dT18) Primer and the RevertAid Reverse Transcriptase (Thermo Fisher Scientific, distributed by Fisher Scientific Austria GmbH, Vienna, Austria). Real-time qPCR experiments were run on an Eppendorf Mastercycler to amplify the Gapdh (housekeeping gene), using SybrGreen (Promega, Mannheim, Germany). Real-time qPCR assays targeting mRNA of Gapdh, Ifnb, Il-1b, Il-6, Il-12p40, Nos2, Tnfα, Irf1, Mx1, Isg15, Ifit3 and Oas2 and pre-mRNA of Il-1b, Il-6, Nos2, Tnfα, Ccl5 and Cxcl10 were performed with the following forward (f) and reverse (r) primers: mRNA: Gapdh-f: 5’-CATGGCCTTCCGTGTTCCTA-3’; Gapdh-r: 5’-GCGGCACGTCAGATCCA-3’; Ifnb-f: 5’-TCAGAATGAGTGGTGGTTGC-3’; Ifnb-r: 5’-GACCTTTCAAATGCAGTAGATTCA-3’; Il-1b-f: 5’-AGATGAAGGGCTGCTTCCAAA-3’; Il-1b-r: 5’-AATGGGAACGTCACACACCA-3’; , Il-6-f: 5’-CTGCAAGAGACTTCCATCCAG-3’, Il-6-r: 5’-AGTGGTATAGACAGGTCTGTTGG-3’; Il-12p40-f: 5’-TGGTTTGCCATCGTTTTGCTG-3’; Il-12p40-r: 5’-ACAGGTGAGGTTCACTGTTTCT-3’; Nos2-f: 5’- GAGCAACTACTGCTGGTGGT-3’; Nos2-r: 5’- CGATGTCATGAGCAAAGGCG-3’, Tnfα-f: 5’- CAAAATTCGAGTGACAAGCCTG-3’; Tnfα-r: 5’- GAGATCCATGCCGTTGCC-3’; Irf1-f: 5' -CCG AAG ACC TTA TGA AGC TCT TTG-3' Irf1-r: 5' -GCA AGT ATC CCT TGC CAT CG-3' ; Mx1-f: 5' -GAC TAC CAC TGA GAT GAC CCA GC-3' , Mx1-r: 5' -ATT TCC TCC CCA AAT GTT TTC A-3' ; Isg15-f: 5' -ATG GCC TGG GAC CTA AAG-3' ; Isg15-r: 5' -TTA GGC ACA CTG GTC CCC-3' ; Ifit3-f: 5' -CCT CGC AGC CCT GGA GTG TT-3' ; Ifit3-r: 5' -TGC GTT GCC TCC CAA ACC CC-3' ; Oas2-f: 5' -AAACCTCACACCCAACGAAAA-3' ; Oas2-r: 5' - CCACCCTTAGCCACTTCCT-3' . pre-mRNA: Il-1b-f: 5’-AAGATGAAGGTGAGACTCTGAG-3’; Il-1b-r: 5’-CTTGGTGTGTGGCTGTGGTA-3’; Il-6-f: 5’-AATGGAGTTGTTAGGCATGGG-3’; Il-6-r: 5’-TGTAAATCTTTTACCTAAAGGAGGA-3’; Nos2-f: 5’-TCCTTTAAAGAGTAAGTCTGGCTT-3’; Nos2-r: 5’-CAGGACTCAGCAGTGACCT-3’; Tnfα-f: 5’-AGGGATGAGGTGAGTGTCTG-3’; Tnfα-r: 5’-ACGTGTGAACACACTTGTTCGT-3’; Ccl5-f: 5’-GCCTCACCATGTAAGTCGAG-3’; Ccl5-r: 5’-CACAGAAAAGTTCCTCAGAGGA-3’; Cxcl10-f: 5’-TGGGACTCAAGGTAAGGGAC-3’; Cxcl10-r: 5’-CTTTCTTCCCTTCTTCGTTCCT-3’. Luciferase activity was measured with the Dual Luciferase Reporter Assay System (Promega, Mannheim, Germany). In short, the cell lysates were mixed with the substrate for the Firefly luciferase, then luciferase activity was determined with the luminometer. After this, the stop solution ending the first enzymatic reaction and containing the substrate for the Renilla luciferase was added and the samples were measured again. Quantification was performed normalizing the Firefly luciferase activity to the Renilla luciferase activity. Relative induction levels were derived comparing the samples to an empty control vector. Bacterial loads in spleens and livers were compared using unpaired t-test. The lines represent mean with the standard error of the mean (SEM). Bacterial loads in in vitro CFU assays were compared with the unpaired t-test and bars on the graph represent the mean values with standard deviation (SD). Significances of survival curves after either Lm or VSV infections were calculated using the Mantel-cox-test. Serum cytokine levels were compared using the unpaired t-test where the lines represent means with the standard error of the mean. The mRNA expression data are represented with the mean values with standard deviation (SD). The differences in mRNA expression data were compared using the paired t-test. All statistical analyses were performed using the GraphPad Prism (GraphPad) software. Asterisks denote statistical significance as follows: ns, P > 0. 05; *P ≤ 0. 05; **P ≤ 0. 01; ***P ≤ 0. 001.
The establishment of innate immunity to pathogens requires cells to sense microbial molecules and to initiate a de novo transcription-based antimicrobial response. With the identification of Rig I and Mda5, two RNA helicases were shown to serve as pivotal receptors of viral RNA. Subsequently, a considerable number of RNA helicases were proposed to function as sensors or signal transducers for both microbial RNA and DNA. X-chromosome-encoded RNA helicase DDX3X was discovered as an interactor of the S/T kinase TBK1 which regulates the production of type I Interferons (IFN-I). However, the importance of DDX3X for innate immunity in an organismic context remained elusive. Here we describe and analyze mice lacking DDX3X in hematopoietic cells. We show contributions of DDX3X to hematopoiesis and a striking loss in resistance against Listeria monocytogenes. Our data reveal that DDX3X is critically involved in enhancing the expression of numerous antimicrobial genes. Consistently, production of important cytokines such as IL12 or IFNγ is reduced. Furthermore, DDX3X-deficient macrophages show reduced ability to restrict L. monocytogenes growth. Owing to partial redundancy with its close Y-chromosomal homologue, DDX3Y, the observed effects differ between mouse sexes. Thus, DDX3X may contribute to sex differences in immunity to pathogens and inflammatory disease.
blood cells
innate immune system
medicine and health sciences
immune cells
immune physiology
pathology and laboratory medicine
cytokines
pathogens
immunology
microbiology
cloning
bone marrow cells
developmental biology
molecular development
molecular biology techniques
bacterial pathogens
research and analysis methods
white blood cells
animal cells
medical microbiology
gene expression
microbial pathogens
molecular biology
immune system
listeria monocytogenes
antibody-producing cells
cell biology
b cells
nk cells
physiology
genetics
biology and life sciences
cellular types
macrophages
The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity