To appreciate the distinct contribution of individual SR proteins to macrophage gene expression, we generated RAW 264.7 macrophage cell lines (RAW MΦ) stably expressing shRNA hairpins directed against Srsf1, 2, 6, 7, and 9, as previously reported in Wagner et al., 2021. Most SR proteins are ubiquitously expressed across cell types, and we confirmed expression of each of these SRs in our RAW MΦ (Figure 1—figure supplement 1A). Because it is well-established that several members of the SR protein family are essential genes across multiple cell types (Feng et al., 2009; Goldberger et al., 2021; Ortiz-Sánchez et al., 2019; Wang et al., 2001; Xu et al., 2005) and because high-quality transcriptomics analyses have been carried out successfully for SR family members using shRNA or siRNA gene silencing (ENCODE Project Consortium, 2004; Van Nostrand et al., 2020), we opted to stably knockdown these factors, instead of relying on CRISPR-mediated gene editing. To identify major transcriptomic changes due to loss of SR proteins, we isolated total RNA from each of these KD cell lines and a scramble (SCR) control that was selected alongside the SR KDs, performed RNA-seq, and measured differential gene expression using the CLC Genomics Workbench, as in Wagner et al., 2021. Using a simple hierarchical clustering algorithm, we visualized the gene expression profiles of each SR KD macrophage cell line and pinpointed SRSF6 as unique amongst the SR proteins queried (correlation between SRSF6’s node and the rest of the tree = 0.138) (Figure 1A). Manual annotation of genes in clusters that were uniquely impacted by loss of SRSF6 revealed several downregulated genes related to mitochondrial biology (Figure 1B). On the other hand, many upregulated genes fell into the category of interferon stimulated genes (ISGs) (Figure 1C), as defined by Kim et al., 2018; Liu et al., 2012; Thomas et al., 2006; Zahoor et al., 2014. ISGs are a group of genes whose transcription is activated through IFNAR receptor-mediated type I interferon signaling. Generally, very few ISG transcripts accumulate in resting macrophages. Direct visualization of RNA-seq reads using the Integrated Genome Viewer shows elevated reads across all coding exons for representative ISGs Rsad2 (Figure 1D) and Mx1 (Figure 1—figure supplement 1B). Ingenuity Pathway Analysis confirmed overrepresentation of differentially expressed genes in functional categories related to interferon and antiviral responses (Figure 1E), indicating an overall increase in type I IFN signaling. Furthermore, RT-qPCR confirmed basal ISG expression in two independently derived Srsf6 KD cell lines (Figure 1H–J), with the degree of ISG accumulation correlating with SRSF6 KD efficiency and protein expression (Figure 1F–G). We also measured ISG accumulation upon transient siRNA KD of SRSF6 in RAW MΦ cells (Figure 1—figure supplement 1C), arguing against off-target effects resulting from stable selection of KD cell lines. Importantly, this phenotype was recapitulated by Srsf6 siRNA KD in primary cell types including bone marrow derived macrophages (BMDMs) (Figure 1K) and mouse embryonic fibroblasts (MEFs) (Figure 1—figure supplement 1D). Differential expression of genes involved in type I IFN responses was not observed in other resting SRSF KD RAW MΦ cell lines, suggesting this phenotype is unique to loss of SRSF6 and is not a general consequence of interfering with splicing (Wagner et al., 2021).

Having concluded that SRSF6 plays a role in regulating basal ISG expression, we sought to investigate the cell-intrinsic vs. cell-extrinsic nature of this phenotype. Phosphorylation of IFN regulatory factor 3 (IRF3) is a critical step in the initial stages of PAMP and DAMP sensing that lead to production of IFN-β. Immunoblot analysis revealed increased levels of phospho-IRF3 (S396) in resting Srsf6 KD macrophages (Figure 1L). Higher levels of IFN-β protein were also measured in the supernatants of Srsf6 KD RAW MΦ cells via ISRE reporter cells (Hoffpauir et al., 2020; Figure 1M). Consistent with higher levels of IFN-β secretion, supernatants from Srsf6 KD macrophages were sufficient to stimulate ISG expression in naïve wild-type RAW MΦ cells (24 hr incubation) (Figure 1N). Elevated basal ISG expression in Srsf6 KD cells was rescued by treatment with an IFN-β neutralizing antibody (Figure 1O). Together, these data suggest that IRF3-mediated expression of IFN-β drives upregulation of basal ISG expression in Srsf6 KD RAW MΦ cells. Finally, to demonstrate the biological relevance of increased SRSF6-dependent basal ISG expression, we infected cells with the single stranded RNA virus VSV, which is hypersensitive to even low levels of ISG expression (Wagner et al., 2021; West et al., 2019). We observed a dramatic restriction of VSV replication in Srsf6 KD RAW MΦ cells compared with SCR controls (Figure 1P and Figure 1—figure supplement 1E), supporting a bona fide role for SRSF6 as a negative regulator of antiviral immunity.

We next sought to identify the trigger of the higher basal type I IFN expression in Srsf6 KD RAW MΦ cells. Mitochondrial DNA (mtDNA) can activate cytosolic DNA sensing pathways when mitochondria are damaged or depolarized such that mtDNA is released from the inner matrix (West et al., 2015). Because mitochondrial genes were downregulated in Srsf6 KD RAW MΦ (Figure 1B), we hypothesized that the mitochondrial network might be impacted by a loss of SRSF6. To begin to establish mtDNA as a source of the increased ISG expression, we first set out to determine if cytosolic mtDNA was increased in Srsf6 KD macrophages. To measure cytosolic mtDNA we used differential centrifugation to separate cytosolic and cellular membrane fractions (the latter of which include mitochondria) from SCR and Srsf6 KD RAW MΦ (Figure 2A). We detected significant enrichment of several mtDNA genes (Cytb, Dloop2, and Dloop1) in the cytosol of Srsf6 KD RAW MΦ cells (Figure 2B). Additionally, we determined the necessity of mtDNA to drive the increased basal ISG expression in Srsf6 KD macrophages, as mtDNA depletion via treatment with the nucleoside analog 2,3’-dideoxycytidine (ddC) (Figure 2C), returned expression of the ISG Rsad2 to that of SCR control cells (Figure 2D). Elevated basal ISG expression was dependent on cytosolic DNA sensing, as Srsf6 KD did not cause Rsad2 transcript accumulation in the absence of the cytosolic DNA sensor, cGAS (Figure 2E–F, Figure 2—figure supplement 1A). Together, these data argue that loss of SRSF6 causes leakage of mtDNA into the cytosol where it engages cGAS, leading to phosphorylation of IRF3 (Figure 1L), expression of IFN-β (Figure 1M), and activation of ISG expression in resting macrophages (Figure 1A–C).

To understand how loss of SRSF6 disrupts mitochondrial homeostasis to allow for mtDNA cytosolic access, we first visualized the mitochondrial network in Srsf6 KD and control MEFs, which are well-suited for imaging due to their extensive mitochondrial network. Immunofluorescence microscopy (anti-TOM20) revealed increased mitochondrial fragmentation in Srsf6 KD MEFs (Figure 2G), although total levels of mitochondrial proteins like TOM20 and VDAC were comparable between SCR and Srsf6 KD cell lines (Figure 2—figure supplement 1B). Importantly, mitochondria in Srsf6 KD RAW MΦ also exhibited loss of membrane potential (Figure 2H), as measured by tetramethylrhodamine ethyl ester (TMRE) signal (decreased TMRE signal = increased membrane depolarization). As another measure of mitochondrial function, we measured metabolic output in Srsf6 KD vs. SCR RAW MΦ cells normalized to protein abundance via the Agilent Seahorse Metabolic Flex Analyzer. Srsf6 KD cells displayed deficiencies in multiple readouts of oxygen consumption rate (OCR), which serves as a proxy for oxidative phosphorylation (OXPHOS). Loss of SRSF6 reduced mitochondrial OXPHOS, indicated by lower basal and maximal respiration. Additionally, Srsf6 KD mitochondria had lower ATP production and lower respiratory capacity compared with SCR controls (Figure 2I top-J). Glycolysis, as measured by extracellular acidification rate (ECAR) was unaffected by SRSF6 knockdown (Figure 2I, bottom), suggesting that SRSF6 specifically impacts cellular metabolism primarily at the level of mitochondrial OXPHOS.

Our data suggest that SRSF6 limits basal type I IFN expression in macrophages through a role in maintaining mitochondrial homeostasis. SRSF6 is best known as a regulator of alternative splicing (Filippov et al., 2008; Juan-Mateu et al., 2018; Tran and Roesser, 2003; Tranell et al., 2010). Therefore, we hypothesized that loss of SRSF6 could alter alternative splicing of one or more pre-mRNAs that encode proteins involved in mitochondrial biology. To identify SRSF6-dependent alternative splicing events, we mined a list of local splicing variations (LSVs) in Srsf6 KD MΦ cells quantified by the computational algorithm MAJIQ (Modeling Alternative Junction Inclusion Quantification) Vaquero-Garcia et al., 2016 as reported in Wagner et al., 2021. As expected for an SR protein, SRSF6 mostly controls exon inclusion in macrophages (1043 exon skipping LSVs), with changes in intron retention (247 LSVs), and alternative 5’ (206 LSVs) and 3’ (233 LSVs) splice site usage also detected (Figure 3A). Manual annotation of these >1600 LSVs identified alternative splicing events in 31 genes that also demonstrated some change in gene expression (p<0.05) with annotated roles in mitochondrial function (Figure 3B). Semi-quantitative RT-PCR was used to validate hits and SRSF6-dependent splicing of intron 2 in the Xaf1 pre-mRNA, which is predicted to introduce a premature stop codon and target Xaf1 for nonsense mediated decay, was confirmed (Figure 3—figure supplement 1A).

One alternative splicing event with unique protein coding capacity that piqued our interest occurred in Bax, the mitochondrial pore-forming protein and executioner of apoptosis. Specifically, MAJIQ analysis measured preferential retention of intron 1 in Srsf6 KD macrophages (Figure 3C). Considerable Bax intron 1 retention occurred in SCR MΦ cells as well, but to a lesser extent (Figure 3C–E). We validated SRSF6-dependent Bax splicing by RT-qPCR, using a forward primer in exon 1 and a reverse primer in intron 1 (Figure 3F), as well as by semi-quantitative RT-PCR, using a forward primer in exon 1 and a reverse primer in exon 6 (Figure 3—figure supplement 1B). Increased Bax alternative splicing did not impact total BAX protein expression (Figure 3—figure supplement 1C). Retention of intron 1 in Bax causes usage of a downstream ATG start site encoded in exon 2, which creates a 20 amino acid N-terminal truncation, but otherwise leaves the Bax coding sequence completely in-frame and intact (Figure 3G and Figure 3—figure supplement 1D). This annotated isoform of BAX, dubbed BAX kappa (BAX-κ; GenBank accession number AY095934), is upregulated in the rat hippocampus following cerebral ischemic injury and promotes apoptosis when overexpressed in murine hippocampal neuronal cells (Jin et al., 2001). Because they both lack an ART (apoptosis-regulating targeting) sequence (Cartron et al., 2005; Goping et al., 1998), Bax-κ is predicted to be functionally analogous to the human BAX-psi isoform, which has been shown to constitutively associate with mitochondria (Cartron et al., 2005). ESE Finder, a web-based platform to identify exonic splicing enhancer motifs (Cartegni et al., 2003), identified three strong SRSF6 consensus sequences in Bax exons 1, 3, and 6 (Figure 3H). UV Crosslinking-immunoprecipitation (CLIP) experiments in RAW MΦ cells showed a clear enrichment of Bax transcripts bound to 3xFLAG-SRSF6 compared with a 3xFLAG-GFP control (Figure 3I–J). Together, these data strongly suggest that SRSF6 controls splicing of Bax intron 1 by directly binding consensus exonic splicing enhancers in Bax exons.

Previous literature has demonstrated that in addition to promoting release of cytochrome c during apoptosis, BAX pores can release mtDNA capable of stimulating cGAS-dependent type I IFN responses (Rongvaux, 2018; Rongvaux et al., 2014). We hypothesized that high basal ISGs in Srsf6 KD macrophages are a consequence of Bax-κ-dependent release of mtDNA. Consistent with this prediction, even modest knockdown of Bax (all isoforms) via siRNA transfection rescued high Rsad2 and Ifit1 expression in Srsf6 KD macrophages (Figure 3K). Because knockdown of Bax in wild-type RAW MΦ had no impact on Rsad2 or Isg15 expression (Figure 3L), we can conclude that links between aberrant basal ISG expression and BAX are limited to Srsf6 KD cells in which Bax splicing is defective.

In addition to their role in cellular metabolism and energy production, mitochondria serve as gatekeepers of multiple cell death pathways. Based on previous studies of BAX-κ and human BAX-psi, we hypothesized that accumulation of BAX-κ would promote cell death in RAW MΦ cells. We first tested this by measuring incorporation of the cell viability stain propidium iodide (PI) in resting SCR and Srsf6 KD MΦ cells. We observed significantly higher levels of PI incorporation in Srsf6 KD cell lines (Figure 4A), suggesting that even in the absence of cell death inducing agents, a population of stable Srsf6 KD MΦ undergoes cell death. Because Srsf6 KD MΦ secretes unusually high levels of IFN-β, we tested whether cell death was dependent on IFN-β as in Apelbaum et al., 2013; Sarhan et al., 2019. Treatment with an anti-IFN-β neutralizing antibody had no effect on PI incorporation in SCR or Srsf6 KD cells, effectively ruling out a role for IFN−β in mediating SRSF6-dependent cell death (Figure 4B). Flow cytometric analysis of Srsf6 KD cells via PI and annexin V staining (a protein that preferentially binds to phosphatidylserine on cells undergoing apoptosis), confirmed higher levels of PI incorporation in the absence of Srsf6 (Figure 4C–D). It also identified a population of pro-apoptotic cells (annexin V-positive but PI-negative) that is more abundant in Srsf6 KDs (6.3% in KD1 and 9.7% in KD2 vs. 4.2% in SCR) (Figure 4E). Consistent with Srsf6 KD RAW 264.7 cells being prone to apoptosis, significantly more Srsf6 cells stained PI +after treatment with low levels of the apoptosis-inducing drugs staurosporine (Figure 4F) or ABT373 (Figure 4G) and at early time-points following treatment with the cell death agonist etoposide (Figure 4—figure supplement 1A).

We next set out to better define the nature of cell death in Srsf6 RAW MΦ cells. Previous reports have implicated BAX in non-canonical proinflammatory forms of cell death and IL-1β release mediated by caspase 8 (Hu et al., 2020; Vince et al., 2018). We did not suspect inflammasome-mediated cell death could explain our findings in Srsf6 KD RAW MΦ cells as RAW cells lack the inflammasome adapter ASC (Pelegrin et al., 2008). However, given that the cell death observed was not silent, with ISG upregulation, we sought to rule out additional proinflammatory pathways present during Srsf6-mediated cell death. We detected no significant difference in IL-1β release between BMDMs transfected with a non-targeting siRNA vs. Srsf6-targeting siRNA in either unprimed or poly dA:dT primed cells (Figure 4—figure supplement 1B-C). Curiously, despite displaying an apoptotic signature (Figure 4C, E), cell death in Srsf6 KD RAW MΦ cells was caspase-independent, as treatment with the pan-caspase inhibitors Q-VD-OPh (Figure 4H) and Z-VAD-FMK (Figure 4—figure supplement 1D) had no impact on PI incorporation in resting Srsf6 KD cells. Consistent with lack of caspase involvement, resting Srsf6 KD RAW MΦ did not release cytochrome c (Figure 4I). These observations, coupled with the disruption to mitochondrial membrane potential reported in (Figure 2H), suggest a form of mitochondrial-dependent caspase-independent cell death (CICD) (as described by Xiang et al., 1996, reviewed in Tait and Green, 2008) that occurs preferentially in Srsf6 KD macrophages. We hypothesize that BAX-κ, which can trigger mitochondrial outer membrane permeabilization (MOMP) and release of mtDNA without releasing cytochrome c, is a major contributor to this noncanonical form of cell death, which is characterized by low-level sustained damage to mitochondria.

We next sought to more directly associate BAX and BAX-κ with the altered mitochondrial homeostasis observed in Srsf6 KD MΦ. Our data, and that from previous studies of Bax-psi (Cartron et al., 2005), suggest that BAX-κ is a constitutively active form of BAX that readily localizes to mitochondrial membranes in resting cells. To determine whether BAX protein accumulates on mitochondria in resting Srsf6 KD MΦ, we performed a ‘mitoFLOW’ experiment as described in Weindel et al., 2022. Briefly, mitoTRACKER green stained mitochondria were isolated by passive lysis and centrifugation, stained with a BAX antibody, and analyzed by flow cytometry. MitoTRACKER was used to identify and gate mitochondria and then BAX association was quantified (Figure 4J top). We observed a significant increase of BAX protein on mitochondria isolated from resting Srsf6 KD cells compared with SCR controls (Figure 4J bottom). Although our antibody cannot distinguish between BAX and BAX-κ, this data further connects loss of SRSF6 with increased BAX mitochondrial association, which we argue is mainly contributed by the BAX-κ isoform (full-length BAX is predominantly cytosolic in resting cells) (Cosentino and García-Sáez, 2017).

We next wanted to see if Bax-κ transcript expression correlated with loss of mitochondrial membrane polarization (Figure 2H). To this end, we sorted TMRE-treated cells into high TMRE signal (normal mitochondrial membrane potential) or low TMRE signal (low mitochondrial membrane potential/depolarized mitochondria). RNA was isolated from these two pools of cells, alongside a total, unsorted pool, and RT-qPCR was performed as in Figure 3F to measure Bax-κ. Bax-κ transcripts were considerably higher in Srsf6 KD cells with low membrane potential compared with mitochondria with high membrane potential or total mitochondria (Figure 4K). Bax-κ transcripts were most abundant in low membrane potential mitochondria isolated from Srsf6 KD cells, although notably, Bax-κ expression correlated with mitochondrial depolarization in SCR cells as well.

The results of our Bax KD experiment suggest that BAX is necessary to drive type I IFN expression in Srsf6 KD cells (Figure 3K). To specifically implicate BAX-κ in this phenotype, we generated C-terminal 2xSTREP-tagged constructs of BAX, BAX-κ, and a BAX point mutant (BAX^G179P) that is unable to translocate to mitochondria (Kuwana et al., 2020) and introduced them via lentiviral transduction into RAW MΦ cells expressing a doxycycline-inducible transactivator (Figure 5—figure supplement 1A). Because high levels of doxycycline can negatively impact mitochondrial function (Dijk et al., 2020), we experimentally determined the lowest doxycycline concentration that ensured robust and uniform expression of an inducible mCherry construct without significant toxicity (1 μg/mL) (Figure 5—figure supplement 1A-C). All three of our BAX constructs were expressed in a doxycycline-inducible fashion, albeit to much lower levels than that of a 2xSTREP-GFP control (Figure 5A). We found that expression of BAX-κ was sufficient to induce robust basal ISG expression in the wild-type RAW doxycycline-inducible cell line (Figure 5B). Importantly, this ISG induction was dose-dependent and seen even at early timepoints post-DOX treatment when BAX-κ ectopic expression is still quite low (Figure 5A). BAX-κ expression was also sufficient to induce apoptosis and cell death, as measured by flow cytometry of PI and annexin V-stained cells at 15 h post-doxycycline treatment (Figure 5C) or by PI + incorporation over a 15 hr time-course of doxycycline induction (Figure 5D). The accumulation of PI + and annexin V + cells was dependent on the dose of BAX-κ. Unlike the cell death we measured in resting Srsf6 KD RAW MΦ cells, BAX-κ induced apoptosis and cell death at 15 hr (Figure 5C) or 24 hr (Figure 5—figure supplement 1D) post-DOX treatment was largely caspase-dependent (Figure 5C, bottom graphs). We suspect this contrast between Srsf6 KD cells and BAX-κ overexpressing cells is due in large part to the timing and dose of BAX-κ: caspase-inhibition had only a slight impact at 5 hr post-DOX, with a reduction of annexin V+/PI- cells from 8.42% to 6.94% (when BAX-κ expression is low), compared with a rescue of annexin V+/PI- cells from 30% to 12.1% at 24 hr (when BAX-κexpression is high) (Figure 5—figure supplement 1D). We predict that high levels of BAX-κ in these overexpression cell lines at later time points induces a level of MOMP that is sufficient to release cytochrome c and activate downstream caspases. When we directly stimulated apoptosis in these cells via low levels of staurosporine (1 μM), BAX and BAX-κ expression induced similar amounts of enhanced cell death relative to GFP or BAX^G179P-expressing cells, confirming that an activation signal is required for full length BAX to stimulate cell death (Figure 5E).

Last, we sought to measure the amount of total BAX associated with mitochondria in each of the overexpression cell lines. Using mitoFLOW (as in Figure 4J), we measured significantly more mitochondria-associated BAX in BAX-κ expressing cells compared with cells expressing BAX^G179P or BAX (Figure 5F). Collectively, these data argue that expression of BAX-κ is sufficient to drive ISG expression and cell death in resting macrophages and support a major role for BAX-κ in inducing the mitochondrial phenotypes we uncovered in Srsf6 KD macrophages.

Our original interest in SR protein function in macrophages was borne out of a global phosphoproteomics analysis that reported SR proteins, including SRSF6, were differentially phosphorylated in BMDMs at several sites over a 24 hr time course of infection with the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb) (Budzik et al., 2020). Having discovered a role for SRSF6 in alternative splicing of BAX and regulating programmed cell death, we set out to determine whether phosphorylation of SRSF6 impacted its ability to carry out these activities in macrophages. Leveraging data from Budzik et al., 2020, we prioritized three serine residues that were differentially phosphorylated in studies of Mtb-infected or LPS-stimulated macrophages Weintz et al., 2010: S295, S297, and S303 (Figure 6A). To test the contribution of phosphorylation of these serines to SRSF6 function, we generated RAW MΦ cell lines expressing doxycycline-inducible constructs of wild-type SRSF6 alongside phosphodead (Ser(S)-to-Ala(A)) or phosphomimetic (Ser(S)-to-Asp(D)) mutations at each site. Expression of each SRSF6 allele was confirmed by immunoblot at 24 hr following addition of doxycycline (Figure 6B). Because phosphorylation of SRSF6 has been implicated in nuclear/cytoplasmic shuttling (Jeong, 2017), we tested where each SRSF6 mutant accumulated in cells using confocal immunofluorescence microscopy, using an antibody directed against the 3xFLAG tag. We observed no major differences in SRSF6 protein localization in any of our phosphomutant/mimetic cell lines compared with wild-type SRSF6 (predominantly nuclear in all cases) (Figure 6C).

To begin to implicate specific phosphorylation events in SRSF6 activity, we measured the ratio of Bax-κ relative to total Bax, as in Figure 3G, in each of the SRSF6-expressing cell lines. We observed that expression of SRSF6^S303D decreased levels of Bax-κ transcripts, compared with those in wild-type SRSF6-expressing macrophages (Figure 6—figure supplement 1A and Figure 6D). Likewise, SRSF6^S303D-expressing cell lines had higher levels of canonical Bax transcripts, relative to wild-type SRSF6-expressing cells, by semi-quantitative RT-PCR (Figure 6E). Consistent with altered Bax-κ to Bax ratios in SRSF6^S303D-expressing cell lines, expression of the SRSF6^S303D phosphomimetic allele rendered cells less prone to apoptosis (annexin V+/PI-) and cell death (Figure 6F–H). Together, these data suggest that phosphorylation of SRSF6 at S303 promotes splicing of Bax, to generate the canonical BAX protein and limit apoptosis.

Our data support a model wherein SRSF6 maintains cellular homeostasis and basal type I IFN expression by controlling the abundance of BAX isoforms. Therefore, we predicted that expression of SRSF6 itself could be subject to regulation downstream of pathogen sensing, as a way for cells to prime apoptotic cell death and/or cytosolic DNA sensing via mtDNA release. To further appreciate how macrophages regulate SRSF6 activity, we measured Srsf6 transcript levels during infection and in response to immune agonists. We consistently detected an approximately 2-fold decrease in Srsf6 transcript abundance at various time points following M. tuberculosis infection of macrophages, as well as in the lung homogenates collected from Mtb-infected mice (day 21 and 77 post-infection) (Figure 7A–B). Macrophages sense Mtb infection via several pattern recognition receptors, including dsDNA via cGAS and autocrine sensing of IFN-β via IFNAR (Watson et al., 2015). Direct stimulation of these pathways via transfection of dsDNA (Figure 7C) or treatment with recombinant IFN-β (rIFN-β) (Figure 7D) also led to downregulation of Srsf6 transcript abundance, as did infection with the gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium (Figure 7E), engagement of TLR-4 via LPS (Figure 7F) and infection with the RNA virus VSV (Figure 7G). These data suggest that downregulation of Srsf6 mRNA levels is part of the general macrophage pathogen sensing response.

Last, we set out to investigate whether SRSF6 plays a more direct role in dictating the outcome of infection with an intracellular pathogen like Mtb. To this end, we infected monolayers of SCR and Srsf6 KD RAW MΦ cells with a luciferase-expressing strain of Erdman Mtb (MOI = 1) as in Bell et al., 2021; Hoffpauir et al., 2020 and monitored relative light units (RLU) as a measurement of Mtb replication. Remarkably, we found that Mtb grew significantly better in Srsf6 KD cell lines compared with SCR controls (Figure 7H). This increase in Mtb replication was concomitant with hyperinduction of Ifnb1 and interferon stimulated genes, consistent with high basal type I IFN potentiating type I IFN responses (Figure 7I; West et al., 2015). Along with harboring higher Mtb bacterial burdens, more Srsf6 KD RAW MΦ cells stained PI + compared with SCR controls over a 40+h Mtb infection (Figure 7J). Collectively, these results demonstrate a critical role for SRSF6 in controlling inflammatory gene expression and cell death during bacterial infection and suggest that regulation of SRSF6, both at the post-translational and transcriptional levels, constitutes an unappreciated layer of complexity in the macrophage innate immune response.

The Erdman strain was used for all M. tuberculosis (Mtb) infections as well as a luciferase expressing strain. Low passage lab stocks were thawed for each experiment to ensure virulence was preserved. Mtb was cultured in roller bottles at 37°C in Middlebrook 7H9 broth (BD Biosciences, GTIN00382902713104) supplemented with 10% OADC (BD Biosciences, 212351), 0.5% glycerol (Fisher, G33-1), and 0.1% Tween-80 (Fisher, BP338-500). All work with Mtb was performed under Biosafety level 3 containment using procedures approved by the Texas A&M University Institutional Biosafety Committee.

Salmonella enterica serovar Typhimurium (SL1344) was obtained from Dr. Denise Monack at Stanford University. S. T. stocks were streaked out on LB agar plates and incubated at 37 °C overnight.

Recombinant Vesicular stomatitis virus (VSV; Indiana serotype) containing a GFP reporter cloned downstream of the VSV G-glycoprotein (VSV-G/GFP) was originally obtained from Dr. John Rose at Yale School of Medicine and shared with us by Dr. A. Phillip West at Texas A&M Health Science Center.

Mice used in this study were C57BL/6 J (Stock #00064) initially purchased from Jackson labs and afterward maintained with filial breeding. All mice used in experiments were compared with age- and sex- matched controls. Randomization and blinding for in vivo infections was performed by identifying mice based by cage number and ear punch prior to genotyping. Genotype was kept coded until after downstream analysis. Littermates were used for experiments. Mice used to generate BMDMs were males between 10 and 16 weeks old. For in vivo infection male and female mice were infected with Mtb at 10–12 weeks. Embryos used to make primary MEFs were 14.5 days post-coitum. All animals were housed, bred, and studied at Texas A&M Health Science Center under approved Institutional Care and Use Committee guidelines. All experiments for this study were reviewed and approved by the Texas A&M University Institutional Animal Care and Use Committee (AUP# 2019–0083). Mice were fed 4% standard chow and were kept on a 12 hr light/dark cycle and provided food and water ad libitum. Mice displaying known C57BL/6 J defects were excluded prior to experimentation. Mice were group housed (maximum 5 per cage) by sex on ventilated racks in temperature-controlled rooms.

Bone marrow derived macrophages (BMDMs) were differentiated from bone marrow (BM) cells isolated by washing mouse femurs with 10 mL DMEM 1 mM sodium pyruvate (Thermo Fisher, 11995065). Cells were then centrifuged for 5 minutes at 400 rcf and resuspended in BMDM media DMEM, 20% FBS (Millipore, F0926), 1 mM sodium pyruvate (Lonza, BE13-115E), 10% MCSF conditioned media (Watson lab). BM cells were counted and plated at 5x106 cells per 15 cm non-tissue culture treated dishes in 30 mL complete BMDM media. Cells were fed with an additional 15 mL of BMDM media on day 3. Cells were harvested on day 7 with 1 X PBS EDTA (Lonza, BE02-017F).

Mouse embryonic fibroblasts (MEFs) were isolated from embryos. Briefly, embryos were dissected from yolk sacs, washed 2 times with cold 1 X PBS, decapitated, and peritoneal contents were removed. Headless embryos were disaggregated in cold 0.05% trypsin-EDTA (Lonza, CC-5012) and incubated on ice for 20 minutes, followed by incubation at 37°C for an additional 20 minutes. Cells were then DNase treated with 4 mL disaggregation media (DMEM, 10% FBS, 100 μg/mL DNASE I [Worthington, LS002173]) for 20 minutes at 37°C. Isolated supernatants were spun down at 1000 rpm for 5 minutes. Cells were resuspended in complete MEF media (DMEM, 10% FBS, 1 mM sodium pyruvate), and plated in 15 cm tissue culture-treated dishes 1 dish per embryo in 25 mL of media. MEFs were allowed to expand for 2–3 days before harvest with 0.05% trypsin-EDTA (Lonza, CC-5012).

RAW 264.7 macrophages (RAW MΦ) (ATCC, TIB-71, originally isolated from male BALB/c mice), were minimally passaged to maintain genomic integrity and new cell lines were generated from low passage stocks. L929 (ATCC, CCL-1) ISRE reporter and cGAS knockout RAW MΦ cell lines have already been produced and described in Hoffpauir et al., 2020; Wagner et al., 2021, where only low passage cells were used. Cell lines were cultured at 37°C with a humidified atmosphere of 5% CO2 in complete media containing high glucose, DMEM (Thermo Fisher, 11965092) with 10% FBS (Millipore, F0926) 0.2% HEPES (Thermo Fisher, 15630080). All cell lines tested negative for presence of mycoplasma via the Universal Mycoplasma Detection Kit (ATTC, 30–1012 K).

For RAW MΦ stably expressing scramble knockdown and Srsf6 knockdown, lentivirus was made by transfection with a pSICO scramble non-targeting shRNA construct and pSICO Srsf6 shRNA constructs targeted at exon 3 and exon 4 of Srsf6 using polyjet (SignaGen Laboratories, SL100688). Virus was collected 24 and 48 hr post transfection. RAW MΦ were transduced using lipofectamine 2000 (Thermo Fischer, 52887). After 48 hr, media was supplemented with hygromycin (Invitrogen, 10687010) to select for cells containing the shRNA plasmid.

Knockdown of mRNA transcripts was performed by plating 3×10⁵ RAW MΦ, 3.5×10⁵ BMDMs on day 4 of differentiation, or 2×10⁵ MEFs, in 12-well plates and rested overnight. The following day complete media was replaced with 500 μL fresh complete media 30 minutes prior to transfection. Cells were transfected using Fugene SI reagent (SKU:SI-100) or Viromer Blue reagent (VB-01LB-0) and 10 μM of Ambion siRNA stock against either Srsf6 (4390771, IDS86053) or Bax (AM16708, ID100458). For a negative control, Silencer Select Negative Control #1 (Ambion, 4390843) was used. Cells were incubated for 48–72 hr in transfection media at 37°C with 5% CO2 prior to downstream experiments.

For generation of doxycycline inducible RAW MΦ, pLenti CMV rtTA3 Blast (Addgene, w756-1) stably expressing clonal RAW MΦ were transduced with pLenti CMV Puro DEST (Addgene, w118-1) constructs containing GFP-Strep, Bax201-Strep, Bax203-Strep, BaxG179P-Strep, GFP-FL, SRSF6-FL, and all SRSF6-FL phosphorylation mutants. After 48 hr, construct containing cells were selected through addition of puromycin (Invivogen, ant-pr-1). 1 mg/mL doxycycline (Calbiochem, 324385) treatment was used to activate construct expression.

For S. enterica Typhimurium infection, overnight cultures of bacteria were grown in LB broth containing 0.3 M NaCl and grown at 37°C until they reached an OD600 of approximately 0.9 RAW MΦ were seeded in 12-well tissue culture-treated plates at a density of 7×10⁵ cells per well 16 hr before infection. On the day of infection cultures were diluted 1:20. Once cultures had reached mid-log phase (OD600 0.6–0.8) at 2–3 hr, 1 mL of bacteria were pelleted at 5000 rpm for 3 minutes and washed 2 X with PBS. Bacteria were diluted in serum-free DMEM (DMEM Thermo Fisher, 11965092) and added to cells at multiplicity of infection (MOI) of 5. Infected cells were spun at 1000 rpm for 5 minutes then incubated for 10 minutes at 37°C prior to adding fresh media. At indicated times post infection, cells were harvested with TRIzol for RNA isolation described below.

RAW MΦ were seeded in 12-well tissue culture-treated plates at a density of 7×105 cells per well16 h before infection. The next day cells were infected with VSV-GFP virus at multiplicity of infection (MOI) of 1 in serum-free DMEM (DMEM Thermo Fisher, 11965092). After 1 hr of incubation with media containing virus, supernatant was removed, and fresh DMEM plus 10% FBS (Millipore, F0926) was added to each well. At indicated times post infection, cells were harvested with TRIzol for RNA isolation described below.

All infections were performed using procedures approved by Texas A&M University Institutional Care and Use Committee (AUP# 2021–0133). The Mtb inoculum was prepared as described above. Age- and sex-matched mice were infected via inhalation exposure using a Madison chamber (Glas-Col) calibrated to introduce 100–200 bacilli per mouse. For each infection, approximately 5 mice were euthanized immediately, and their lungs were homogenized and plated to verify an accurate inoculum. Infected mice were housed under BSL3 containment and monitored daily by lab members and veterinary staff. At the indicated time points, mice were euthanized, and tissue samples were collected. For cytokine transcript analysis, lungs were homogenized in 500 μL TRIzol, and RNA was isolated as described below.

For immune activation RAW MΦ were plated on 12-well tissue-culture treated plates at a density of 7.5×10⁵ and allowed to rest overnight. Cells were then treated with lipopolysaccharide (LPS) from E. coli (Invivogen, tlrl-pb5lps) at 100 ng/mL, or ISD (IDT, annealed in house) at 1 μg/mL, or IFN-β (PBL Assay Science, 12405–1) at 200 I/U per mL for the respective time points. Cells were collected for RNA isolation using TRIzol reagent. For IFNβ neutralization assays RAW MΦ Srsf6 KD and SCR cells were treated for 24 hr with IFN-β neutralizing antibody (BD Biolegend, 581302 [1:250]) prior to harvest with TRIzol. For cell death assays RAW MΦ were plated on corning black 96-well half bottom plates at a density of 2.5×10⁴ cells per well and allowed to rest overnight. Complete media was exchanged for complete media containing 5 μg/mL PI and 1 uM staurosporine (Tocaris Bioscience, 1285) or IFN-β neutralizing antibody (BD Biolegend, 581302 [1:250]). Total cell numbers were determined using NucBlue (Thermo Fisher, R37605, [2 drops per mL]) in PBS with a subset of the plated cells. Nuclear incorporation of PI was measured by fluorescence at 4 X magnification using a LionheartXF plate reader (BioTek) every 40 minutes for 24 hr with 5% CO2 at 37°C. For image analysis Gen 3.5 software (BioTek) was used.

For 2′,3′-dideoxycytidine (ddC) (Abcam, Ab142240) depletion of mitochondrial DNA, RAW MΦ were treated with 10 μM ddC and RNA was harvested after 8 days of culture with TRIzol.

Seahorse XF mito stress test kits and cartridges were prepared per Agilent’s protocols and analyzed on an Agilent Seahorse XF 96-well analyzer. The day before the assay Srsf6 knockdown RAW MΦ were seeded at 5×10⁴ cells per well and rested overnight. Cells were processed per manufacturer’s directions and analyzed using the Agilent Seahorse Mito Stress Test kit (Agilent, 103015–100). Normalization was performed based on absorbance (Abs 562) of total protein concentration measured using a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, 23225). WAVE software was used for post-acquisition analysis.

RNA-seq analysis was performed on RAW MΦ containing shRNA knockdowns of Srsf6, Srsf2, Srsf1, Srsf7, and Srsf9 compared with SCR control with biological triplicates of each cell line as described in Wagner et al., 2021 using Qiagen CLC Genomics software for analysis.

Alternative splicing events were analyzed using Modeling Alternative Junction Inclusion Quantification (MAJIQ) and VOILA (a visualization package) with the default parameters described by Vaquero-Garcia et al., 2016. Uniquely mapped, junction-spanning reads were used by MAJIQ to construct splice graphs for transcripts by using the RefSeq annotation supplemented with de-novo detected junctions (de-novo refers to junctions that were not in the RefSeq transcriptome database but had sufficient evidence in the RNA-Seq data). The resulting gene splice graphs were analyzed for all identified local splice variations (LSVs). For every junction in each LSV, MAJIQ quantified expected percent spliced in (PSI) value in control and knockdown samples and expected change in PSI (ΔPSI) between control and knockdown samples. Results from VOILA were then filtered for high confidence changing LSVs (whereby one or more junctions had at least a 95% probability of expected ΔPSI of at least an absolute value of 10 PSI units between control and knockdown) and candidate changing LSVs (95% probability, 10% ΔPSI). For these high confidence results (ΔPSI 10%), the events were further categorized as single exon cassette, multi-exon cassette, alternative 5′ and/or 3′ splice site, or intron-retention (Wagner et al., 2021).

siRNA knockdown RAW MΦ were plated in 12-well tissue culture-treated plates at 7.5×10⁵ cells per well and rested overnight. The next day, plates were treated with 10 ng/mL LPS for 3 hr (Invivogen, tlrl-pb5lps) followed by 1 µg/mL poly dA:dT (Invivogen, tlrl-patn-1) or only the 10 ng/mL LPS for 4 hr, and supernatants and RNA was collected using TRIzol. Cytokine levels of IL-1β, were determined using DuoSet ELISA Development Systems (R&D Systems, DY401-05) per manufacturer’s protocol using the undiluted cell culture supernatants.

For cell death and apoptosis assays, RAW MΦ were plated in 12-well tissue culture-treated plates at 7.5×10⁵ cells per well and rested overnight. The next day cells were stimulated with 10 µM etoposide (TCI, E0675), or 1 µM ABT737 (Santa Cruz Biotechnology, 207242), or the pan-caspase inhibitor 10 µM Q-VD-OPh (Cayman Chemical, 15260) for the indicated time points prior to being lifted off culture plates with 1 X PBS EDTA (Lonza, BE02-017F). Single cell suspensions were made in 1 X annexin binding buffer. Cells were stained for 5 minutes at RT in 5 µg/mL PI (Invitrogen, P1304MP), and 25 nM annexin-V (APC) (Biolegend, 640912) and were then immediately analyzed on an LSR Fortessa X20 (BD Biosciences). PI fluorescence was measured under PE (585/15). For TMRE assays to assess mitochondrial membrane potential, cells were lifted from culture plates with 1 X PBS EDTA (Lonza, BE02-017F). Single cell suspensions were made in PBS 4% FBS (Millipore, F0926). Cells were stained for 20 minutes at 37°C in 25 nM TMRE (Invitrogen, 11560796), washed 1 X in PBS 4% FBS (Millipore, F0926) and analyzed on an LSR Fortessa X20 (BD Biosciences). Flow-Jo v10software was used for post-acquisition analysis (BD biosciences).

For submicron analysis of mitochondria by flow cytometry, cells were lifted off tissue culture treated plates using 1 X PBS EDTA and added to a 96-well V bottom plate. Cells were pelleted by centrifugation at 400 rcf for 3 minutes. Cells were resuspended in 1 X PBS 2% FBS 200 nM mitoTRACKER green (Invitrogen) and allowed to stain for 15 minutes at 37°C. Cells were then washed once with PBS 2% FBS and were resuspend in ice cold mitoFLOW buffer containing 300 mM sucrose, 10 mM Tris (pH 7.4), 0.5 mM EDTA, and 1 X Halt Protease Inhibitor Cocktail. Lysis was performed by vortexing cells for 3 minutes followed by removal of debris by centrifugation at 400 rcf for 5 minutes at 4°C. For antibody labeling samples were centrifuged at 12,000 rcf for 10 minutes at 4°C and resuspend in 50 µL blocking buffer (5% BSA in mitoFLOW buffer) and incubated on ice for 15 minutes. Blocking was followed by an additional 20 minutes incubation on ice with antibodies of interest (BAX Cell Signaling 2772 S 1:200, Alexa Fluor-647 1:2000 Invitrogen). Mitochondria were washed 2 times in mitoFLOW buffer and were analyzed on an LSR Fortessa X20 (BD Biosciences). Flow-Jo software was used for post-acquisition analysis mitoTRACKER green was used to gate on mitochondria.

7×10⁶ RAW MΦ were plated in 15 cm tissue culture-treated dishes and incubated at 37°C with 5%CO₂. The next day cells were lifted with 1 X PBS EDTA (Lonza, BE02-017F) and resuspended in 5 mL PBS. Total DNA was isolated from 2% of resuspended cells, treated with 25 mM NaOH, boiled for 30 minutes, and then neutralized with 50 mM TRIS pH 8.0. The remainder of the cell suspension was pelleted at 3000 rcf for 5 minutes. Cell pellets were resuspended in 500 μL cytosolic lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 50 μg/mL digitonin, 10 mM EDTA) and incubated on ice for 15 minutes. Cells were spun down at 1000 rcf to pellet intact cells and nuclei that were then used for obtaining the membrane fraction. The supernatant was transferred to a fresh tube and spun down at 15000 rcf to remove additional organelle fragments and transferred to a fresh tube again. Cytosolic protein was obtained by transferring 10% of supernatant to a fresh tube with 6 X sample buffer +DTT and boiled for 5 minutes. Cytosolic DNA was isolated from the remaining supernatant by mixing an equal volume of 25:24:1 phenol: chloroform: isoamyl alcohol, with vigorous shaking followed by centrifugation for 10 minutes at ~21,130 rcf (max speed). The aqueous phase was transferred to a fresh tube and DNA was precipitated by mixing with 300 mM sodium acetate, 10 mM MgCl₂, 1 μL glycogen, and 3 volumes of 100% ethanol, and then incubated overnight at –80°C. The precipitated DNA was pelleted by centrifugation at max speed for 20 minutes at 4°C. The pellet was washed with 1 mL of cold 70% ethanol and centrifuged for 5 minutes at max speed, and then the pellet was air dried for 15 minutes at RT. The DNA was resuspended with 200 μL DNase free water. For the mitochondrial membrane fraction, the pellet of intact cells, previously collected, was resuspended in 500 μL membrane lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% NP-40), vortexed, and then centrifuged for 3 minutes at 7000 rcf. 50 μL of the cleared lysate was transferred to a fresh tube 6 X sample buffer + DTT was added. Samples were boiled 5 minutes. Immunoblotting was used to check for contaminating mitochondrial proteins in the cytosolic fraction compared with the membrane fraction by probing for mitochondrial ATP5A1. RT-qPCR was performed using total DNA diluted 1:100 and cytosolic DNA diluted 1:2. Tert (nuclear DNA), CytB and Dloop (mtDNA) were measured. Total and cytosolic fractions were normalized to Tert to control for variation in cell numbers.

Macrophage-secreted type I IFN-β levels were determined using a L929 cells stably expressing a luciferase reporter gene under the regulation of type I IFN signaling pathway (L929 ISRE cells). 5x10⁴ L929 ISRE cells were seeded in a clear 96-well flat-bottomed plate and incubated at 37 °C with 5% CO₂ the previous day. Macrophage cell culture media was collected, diluted 1:5 in complete media and transferred to the L929 ISRE cells and incubated for 6 hr. Cells were washed with PBS, lysed in 30 μL cell culture lysis buffer, and transferred to a white 96-well flat-bottomed plate (Costar, 3693). 30 μL of Luciferase Assay System substrate solution (Promega, E1501) was added to the plate and luminescence read immediately using a Cytation5 plate reader (Biotek).

9x10⁶ RAW MΦ were seeded in 15 cm tissue culture-treated plates and rested overnight. The next day treated with 1 mg/mL doxycycline for 24 hr. Cells were washed with PBS and UV treated at 2000μJoules x100 with a UVstratalinker1800. Cell pellets were lysed in NP-40 lysis buffer with Pierce EDTA free protease inhibitor (Thermo Scientific, A32965) for 15 minutes and sonicated 3 X for 10 minutes 30 seconds on/off (Biorupter). Lysates were treated with 200 ng/mL RNaseA (Invitrogen, AM2271) and 1 U/mL RQ1 Dnase (Promega, M6101) and then incubated on 3XFLAG beads (Sigma Aldrich, F2426) for 3 hr rotating at 4°C. Bound FLAG was eluted 3 X using 20 μL 5XFLAG peptide (Sigma Aldrich, F4799) by vortexing continuously for 15 minutes. Protein samples were collected and separated as described below and RNA was isolated using ethanol precipitation. Briefly, samples were treated with 0.1%SDS and 0.5 mg/mL proteinase K (Invitrogen, 25530049). Samples were spun down at 10,000 rcf for 5 minutes at 4°C, and the pellet was vortexed with equal parts RNase free water and TRIzol, spun down at 10,000 rcf for 20 minutes at 4°C, and then pellets were stored in 100% ethanol +3 M sodium acetate +1 μL glycogen at –80°C overnight. Samples were spun down at 10,000 rcf for 10 minutes at 4°C and then the pellets were washed with 70% ethanol, spun down again, and then pellets were air dried for 15 minutes and RNA was resuspended in RNase free water. qPCR downstream analysis was performed as described below. No RT control cDNA samples were used to check for DNA contamination.

Cells were plated on glass coverslips in 24-well plates. At the designated time points, cells were washed with PBS and then fixed in 4% paraformaldehyde for 10 minutes at 37°C. Cells were washed with PBS 3 X and then permeabilized with 0.2% Triton-X100 (Thermo Fisher, A16046.AP). Coverslips were incubated in primary antibody diluted in PBS +5% non-fat milk +0.2% Triton X-100 (PBS-MT) for 2 hr at RT. Primary antibodies used in this study were TOM20 clone 2F8.1(Millipore Sigma, MABT166, 1:100); ANTI-FLAG M2 (Sigma Aldrich, F3165; RRID:AB_259529, 1:500); β-ACTIN (Abcam, 6276; RRID:AB_2223210, 1:500) and DAPI (Invitrogen, D1306, 1:10,000). Coverslips were then washed 3 X in PBS and incubated in secondary antibody (Invitrogen, A-11034, A21235) diluted in PBS-MT for 1 hr in the dark. Coverslips were then washed twice in PBS and then twice in deionized water. Following washes coverslips were mounted onto glass slides using ProLong Diamond antifade mountant (Invitrogen, P36961). Images were acquired on an Olympus Fluoview FV3000 Confocal Laser Scanning Microscope.

Cells were washed with PBS and lysed in 1 X RIPA buffer with protease and Pierce EDTA free phosphatase inhibitors (Thermo Scientific, A32957), with the addition of 1 U/mL Benzonase nuclease (Millipore, 101697) to degrade genomic DNA. Proteins were separated by SDS-PAGE on AnykD mini -PROTEAN TGX precast gel (Biorad) and transferred to 0.45 μm nitrocellulose membranes (Cytiva, 10600041). Membranes were blocked for 1 hr at RT in LiCOR Odyssey blocking buffer (927–60001). Blots were incubated overnight at 4°C with the following primary antibodies: β-ACTIN (Abcam, 6276; RRID:AB_2223210, 1:5000), β-TUBULIN (Abcam, 179513, 1:5000) p-IRF3(S396) (Cell Signaling, 49475, 1:1000); IRF3 (Bethyl, A303-384A-M, 1:1000); ANTI-FLAG M2 (Sigma Aldrich, F3165; RRID:AB_259529, 1:5000); NWSHPQFEK (Genscript, A00626-40,1:5000); CGAS (Cell Signaling, 316595, 1:1000); SRP55 (Bethyl, A303-669A-M, 1:1000); VIPERIN (RSAD2) (EMD Millipore, MABF106, 1:1000); ATP5A1 (Bethyl, A304-940A-T, 1:1000); CYTOCHROME-C (Abcam, 133504, 1:1000). Membranes were washed 3 X for 5 minutes in PBS-Tween20 and incubated with appropriate secondary antibodies (LI-COR, 925–32210, 926–68071) for 1 hr at RT prior to imaging on a LiCOR Odyssey Fc Dual-Mode Imaging System.

For transcript analysis, cells and tissue were harvested in TRIzol and RNA was isolated using Direct-zol RNA Miniprep kits (Zymo Research, R2052) with 1 hr DNase treatment. cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad, 1708891). CDNA was diluted to 1:20 for each sample. A pool of cDNA from each treated or infected sample was used to make a 1:10 standard curve with each standard sample diluted 1:5 to produce a linear curve. RT-qPCR was performed using Power-Up SYBR Green Master Mix (Thermo Fisher, A25742) using a Quant Studio Flex6 (Applied Biosystems). Samples were run in triplicate wells in a 384-well plate. Averages of the raw values were normalized to average values for the same sample with the control gene, Actb. To analyze fold induction, the average of the treated sample was divided by the untreated control sample, which was set at 1.

cDNA was synthesized by iScript cDNA Synthesis Kit (Bio-Rad, 1708891) using an extended 3 hr amplification. Q5 high fidelity 2 X Master mix (New England Biolabs, M0492S) was used for PCR amplification using targeted primers. Loading dye was added to PCR products and samples were run on 2% agarose gel containing ethidium bromide (Sigma Aldrich, E1510) at 100 volts for 1 hr. Gels were imaged on LiCOR Odyssey Fc Dual-Mode Imaging System and bands were quantified.

Statistical analysis of data was performed using GraphPad Prism software. Two-tailed unpaired Student’s t tests were used for statistical analyses based on the assumption that samples were independent and drawn from normal distributions. Unless otherwise noted, all results are representative of at least three biological samples (mean +/- SEM [n=3 per group]). Biological samples refer to independent cell populations. Agarose gel images for semi-quantitative RT-PCR and immunoblots are representative of n>3. For in vivo data, we have standardized our sample sizes (number of animals) based on the estimate that detecting a significant effect requires two samples to differ in CFUs by 0.7e^10. Using a standard deviation of 0.35e^10 for each population, we calculated that a minimum size of 5 age- and sex-matched mice per group per time point is necessary to detect a statistically significant difference by a t-test with alpha (2-sided) set at 0.05 and a power of 80%. Therefore, we used a minimum of 5 mice per genotype per time point to assess infection-related readouts. For statistical comparison, each experimental group was tested for normal distribution. Data were tested using a Mann-Whitney test. Graphs were generated using Graphpad Prism software.

The datasets referenced in this study can be found online at National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171418.

Please contact the corresponding author at kpatrick03@tamu.edu for resource sharing and availability.