Wildtype (WT) C57BL/6 were purchased from The Jackson Laboratory (JAX strain #000664) and bred in-house. Tpl2^-/- mice backcrossed at least nine generations onto the C57BL/6 WT strain were kindly provided by Dr. Philip Tsichlis (5). Ifnar1^-/- mice (B6.129S2-Ifnar1^tm1Agt/Mmjax; #032045-JAX) were kindly provided by Dr. Biao He. Tpl2^-/- mice were intercrossed with Ifnar1^-/- mice to produce Tpl2^-/-Ifnar1^-/- mice (44). TPL2 kinase-dead (TPL2-KD) mice with a D270A mutation were generated by Dr. Ali Zarrin (6) and generously provided by Dr. Mark Wilson and Genentech, Inc. Animals were housed in microisolator cages at the University of Georgia Coverdell Rodent Vivarium.

Bone marrow-derived macrophages were isolated from the tibias and femurs of age-matched 6–10-week-old male and female wildtype (WT), Tpl2^-/- , TPL2-KD, Ifnar1^-/- , and Tpl2^-/-Ifnar1^-/- mice. Bone marrow cells were cultured in RPMI-160 medium with glutamine (Mediatech, Inc.), 10% heat-inactivated fetal bovine serum (FBS, Neuromics), penicillin-streptomycin (Mediatech, Inc.), HEPES (VWR Chemicals, LLC), 2-ME (Sigma-Aldrich, Co.), and mouse recombinant M-CSF (10 ng/mL, PeproTech, Inc.). Fresh media and M-CSF were added 4 days after isolation. At day 7 post-isolation, cells were harvested using Cellstripper (Mediatech, Inc.) and seeded at 1 x 10⁶ cells/mL in various formats.

BMDMs were pre-treated with or without TPL2 inhibitor TC-S 7006 (10 μM, Tocris) and left unstimulated or stimulated with 100 ng/mL of lipopolysaccharide (LPS) (E. coli 0111:B4, InvivoGen) at the time intervals indicated. For some experiments, ATP (5 mM, MP Biomedicals and InvivoGen) was added 4 hours after LPS stimulation for the stated duration.

Cell supernatants were removed. Cells were collected in TRK lysis buffer, RNA was extracted from the BMDMs using E.Z.N.A. Total RNA Kit I (Omega Bio-Tek, Inc.) and converted into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The relative gene expression was measured using probes purchased from Applied Biosystems with Sensifast Probe Hi-ROX kit (Meridian Biosciences). Real time quantitative PCR was performed on the QuantStudio3 instrument (Applied Biosystems). Samples were normalized to the actin internal control and the respective wildtype sample for the experiment using the ΔΔCT method. The probes used were: Il1b (Mm00434228_m1), Il18 (Mm00434226_m1), Nlrp3 (Mm00840904_m1), Casp1 (Mm00438023), Gsdmd (Mm00509958_m1), and Ifnb (Mm00439552_s1).

Cell supernatants were collected for cytokine analysis by ELISA. IL-1β cytokine secretion was detected using Mouse IL-1 beta Uncoated ELISA Kit (Invitrogen). IL-18 cytokine was measured by Mouse IL-18 Uncoated ELISA kit with Plates (Invitrogen). IFN-β cytokine secretion was detected using Rapid bioluminescent murine IFN-β ELISA kit (InvivoGen).

Standard statistical analyses were performed with GraphPad PRISM software version 10.4.1 (627). Individual data points are the average of three biological replicates from a single experiment and the data shown represent the mean ± standard error of mean. Differences between groups were analyzed using two- and one-way ANOVA with Tukey’s post hoc test for multiple comparisons and were considered statistically significant if p ≤ 0.05. Further statistical analysis details are provided in figure legends.

Previous research demonstrated that the absence of TPL2 attenuated IL-1β production during lipopolysaccharide (LPS) stimulation by severely impairing Il1b transcription (5, 14, 35, 36). These studies did not distinguish whether TPL2 promotes IL-1β production by solely regulating Il1b mRNA synthesis during inflammasome priming or if TPL2 also mediates IL-1β cleavage and secretion during inflammasome activation. To evaluate TPL2’s function during inflammasome priming, bone marrow-derived macrophages (BMDMs) were stimulated with LPS for 4 hours ( Figure 1A ). LPS stimulation caused a trending increase of Il1b mRNA expression in wildtype BMDMs ( Figure 1B ). Tpl2^-/- BMDMs stimulated with LPS had reduced induction of Il1b mRNA synthesis compared to their unstimulated counterparts ( Figure 1B ).

TPL2 deficiency increases type I IFN cytokine expression, and type I IFNs inhibit IL-1β production (41–43). IFN-β and IFN-α initiate their signaling cascade through the type I IFN receptor, IFNAR1 and IFNAR2, to induce the expression of interferon-stimulated genes (ISGs) (45, 46). Therefore, to test if early type I IFN signaling decreased Il1b mRNA synthesis in Tpl2^-/- BMDMs, Ifnar1^-/- and Tpl2^-/-Ifnar1^-/- BMDMs were simultaneously stimulated with LPS for 4 hours ( Figure 1A ). Tpl2^-/-Ifnar1^-/- BMDMs lack both TPL2 protein and a functional type I IFN receptor. These BMDMs do not respond to or initiate the type I IFN signaling pathway, but they do produce and secrete type I IFN cytokines (44, 47, 48). Stimulating Ifnar1^-/- BMDMs with LPS induced Il1b mRNA levels, similar to LPS-stimulated wildtype BMDMs ( Figure 1B ). If increased type I IFNs were contributing to reduced Il1b mRNA synthesis in Tpl2^-/- BMDMs, then Tpl2^-/-Ifnar1^-/- BMDMs would rescue Il1b expression. There was no difference in Il1b mRNA expression between LPS-stimulated Tpl2^-/- and Tpl2^-/-Ifnar1^-/- BMDMs ( Figure 1B ).

In addition to Il1b, Il18 is a pro-inflammatory cytokine precursor produced during inflammasome priming. We examined if TPL2 ablation reduced Il18 mRNA expression during inflammasome priming by stimulating BMDMs with LPS for 4 hours. There was no difference in Il18 mRNA expression between wildtype and Tpl2^-/- BMDMs ( Figure 1C ), indicating TPL2 is not required for Il18 synthesis. Both Ifnar1^-/- and Tpl2^-/-Ifnar1^-/- BMDMs synthesized lower levels of Il18 mRNA relative to wildtype BMDMs ( Figure 1C ), consistent with previous publications that observed Il18 mRNA synthesis is dependent on type I IFNs signaling (49, 50).

Next, we assessed whether TPL2 and type I IFNs altered inflammasome component mRNA synthesis during priming, which could potentially result in modified inflammasome activity. Tpl2^-/- BMDMs have trending decreases in Nlrp3 mRNA expression ( Figure 1D ). There was no significant difference in Nlrp3 mRNA levels between wildtype and Ifnar1^-/- or Tpl2^-/- and Tpl2^-/-Ifnar1^-/- BMDMs ( Figure 1D ). The absence of TPL2 did not alter Casp1 mRNA expression relative to LPS-stimulated wildtype BMDMs ( Figure 1E ); however, the blockade of type I IFN signaling did significantly lower Casp1 mRNA synthesis ( Figure 1E ). LPS-stimulated Ifnar1^-/- BMDMs had significantly less Gsdmd mRNA expression than wildtype LPS-stimulated BMDMs ( Figure 1F ). Overall, these data indicate that TPL2 deficiency attenuates Il1b mRNA expression, while type I IFN signaling blockade decreases Casp1 and Gsdmd mRNA synthesis.

TPL2 has dual roles as both a scaffolding protein and a kinase. In its scaffolding function, TPL2 regulates the maintenance of ABIN2 and NF-κB1 p105 proteins; the interaction with these two proteins inhibits TPL2 kinase activity (2–4). To determine if the changes in Tpl2^-/- BMDM Il1b mRNA transcription during inflammasome priming were attributed to TPL2’s kinase activity, BMDMs were treated with TPL2 inhibitor 15 minutes prior to LPS stimulation ( Figure 2A ). TPL2 inhibitor treatment significantly decreased Il1b mRNA synthesis in wildtype and Ifnar1^-/- BMDMs relative to their LPS-stimulated counterparts, confirming that TPL2 kinase activity promotes Il1b mRNA transcription ( Figure 2B ). Il1b levels were unchanged in Tpl2^-/- and Tpl2^-/-Ifnar1^-/- BMDMs treated with TPL2 inhibitor ( Figure 2B ). Wildtype BMDMs treated with TPL2 inhibitor showed a modest reduction in Il18 mRNA transcription compared to those stimulated with LPS alone ( Figure 2C ). The addition of TPL2 inhibitor did not alter Nlrp3, Casp1, or Gsdmd mRNA synthesis ( Figures 2D-F ).

Because pharmacological inhibition can potentially cause off-target effects, we further verified the importance of TPL2 kinase activity in Il1b mRNA production during inflammasome priming. We utilized BMDMs from TPL2 kinase dead (TPL2-KD, Tpl2^D270A ) mice, in which the TPL2 protein remains intact but has no kinase activity, to evaluate mRNA synthesis ( Figure 2G ) (6, 51). Unstimulated TPL2-KD BMDMs exhibited no difference in mRNA expression of inflammasome pro-inflammatory precursors or components relative to all genotypes ( Supplementary Figures 1A–E ). LPS-stimulated Tpl2^-/- and TPL2-KD BMDMs expressed significantly decreased Il1b mRNA compared to LPS-stimulated wildtype BMDMs ( Figure 2H ). There was no change in Nlrp3, Il18, Casp1, or Gsdmd mRNA synthesis between LPS-stimulated wildtype, Tpl2^-/- , and TPL2-KD BMDMs ( Figures 2I–L ).

Having established the specific role of TPL2 kinase activity during LPS inflammasome priming, we next examined whether increased type I IFN signaling in Tpl2^-/- BMDMs was a contributing factor to alterations in inflammasome mRNA synthesis. Ifnb mRNA synthesis peaks 4 hours after LPS stimulation ( Supplementary Figure 2A ), and Tpl2^-/- BMDMs secrete elevated IFN-β relative to LPS-stimulated wildtype BMDMs ( Supplementary Figure 2G ). Additionally, mRNA expression of inflammasome-processed cytokines (Il1b and Il18) and components (Nlrp3, Casp1, and Gsdmd) over a 24-hour period indicated that transcription occurred by 4 hours of LPS stimulation and often had the greatest synthesis levels at 8 hours ( Supplementary Figures 2B–F ). Therefore, to evaluate the effects of type I IFNs on the transcription of inflammasome components, BMDMs were LPS-stimulated for 8 hours with ATP addition after 4 hours ( Figure 3A ). Il1b mRNA levels 8 hours after LPS treatment followed a similar expression trend across the different BMDM genotypes found in Figure 2B ( Figure 3C ), and IL-1β cytokine secretion from the various BMDM genotypes matched their Il1b mRNA expression ( Supplementary Figure 3A ). Nlrp3 mRNA expression was not altered by type I IFN signaling ( Figure 3D ). Wildtype and Tpl2^-/- BMDMs expressed significantly higher Casp1 mRNA relative to BMDMs that lack type I IFN receptors ( Figure 3E ). Both Ifnar1^-/- and Tpl2^-/-Ifnar1^-/- BMDMs have trending decreases in Il18 and Gsdmd mRNA expression compared to wildtype, Tpl2^-/- , and TPL2-KD BMDMs ( Figures 3C, F ). There was no difference in IL-18 secretion in BMDMs stimulated under these conditions ( Supplementary Figures 4B ). These data indicate that type I IFNs do not contribute to decreased Il1b mRNA expression during inflammasome function; however, type I IFNs do promote the expression of inflammasome components, such as Casp1.

We have demonstrated that TPL2 kinase activity regulates Il1b mRNA expression during inflammasome priming; however, it is unclear if TPL2 kinase activity also affects inflammasome activation and IL-1β release. First, to evaluate the role of TPL2 in inflammasome priming, BMDMs were treated with TPL2 inhibitor 15 minutes prior to LPS stimulation ( Figure 4A ). Four hours later, inflammasome activation was initiated by ATP stimulation for 30 minutes ( Figure 4A ). Wildtype and Ifnar1^-/- BMDMs treated with TPL2 inhibitor before inflammasome priming secreted significantly less IL-1β than their LPS-stimulated counterparts ( Figure 4B ). To assess the effect of TPL2 inhibition on inflammasome activation directly, BMDMs were stimulated with LPS for 4 hours, then treated with TPL2 inhibitor 15 minutes prior to ATP stimulation ( Figure 4C ). LPS-stimulated wildtype and Ifnar1^-/- BMDMs treated with TPL2 inhibitor just prior to inflammasome activation exhibited no reduction in IL-1β secretion ( Figure 4D ), indicating that pro-IL-1β processing by the inflammasome and secretion are independent of TPL2 kinase activity. Overall, these data suggest that TPL2 kinase activity is crucial for Il1b mRNA transcription during inflammasome priming but is dispensable for inflammasome activation and IL-1β secretion.