Fig 2: STK25-mediated phosphorylation of IRF5 at Thr265 induces transcriptional activation in HEK293T cells.(A) HEK293T cells were co-transfected either alone or in combination with IRF5-FLAG, IRF5-T265A-FLAG, or STK25 in addition to an interferon-stimulated response element (ISRE) firefly luciferase (ISRE-Luc) reporter and a cytomegalovirus (CMV) Renilla luciferase (CMV-RL) internal control. The firefly luciferase signal was normalized to the Renilla luciferase signal to determine the relative light units (RLU). For each experiment, the relative ratio for each sample was normalized to a sample with IRF5-FLAG, ISRE-Luc, and CMV-RL (N = 12–13 transfections from three independent experiments). (B) Immunoblot analysis of HEK293T cells co-transfected with IRF5-FLAG or IRF5-T265A-FLAG. Blots were probed with antibodies against IRF5 and β-actin. (C) Immunoblot analysis of STK25-deficient HEK293T cells generated via CRISPR-Cas9-mediated gene editing. Blots were probed with antibodies against STK25 and β-actin. (A, D) WT and STK25-deficient (KO) HEK293T cells were co-transfected as in (A) to evaluate normalized ISRE-Luc reporter activity (N = 5 biological replicates per genotype). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. Data represent mean ± SEM.

Fig 3: Basal autophosphorylation of STK25 at Thr174 is increased in PBMCs from patients with systemic lupus erythematosus.(A) Immunoblot analysis of STK25 autophosphorylation at Thr174 in PBMCs from healthy donors and patients with systemic lupus erythematosus. Blots were probed with antibodies against p-STK25 (T174), STK25, and β-actin. (B) Densitometric analysis of total STK25 protein levels after normalization to the expression of β-actin (N = 8–9 samples per condition). (C) Densitometric analysis of p-STK25 (T174) protein levels after normalization to total STK25 protein levels and the expression of β-actin (N = 8–9 samples per condition). Data represent mean ± SEM. (D) Proposed model for STK25 as an IRF5 kinase downstream of TLR7/8/9 signaling (pathway highlighted by the red dashed arrows). The activation of TLR7/8 by ssRNA or TLR9 by CpG-B DNA induces the autophosphorylation of STK25 at Thr174 and stimulates the formation of the Myddosome, a signaling complex comprised of MyD88, IRAK1/4, TRAF6, and IRF5. The activation of IRAK1 by IRAK4 leads to the recruitment of TRAF6. The ubiquitination (colored circles) of TRAF6 provides a binding site for TAB2/3, thereby facilitating the activation of TAK1. NEMO, a component of the IKK complex, interacts with ubiquitinated TRAF6 to promote the activation of IKKβ by TAK1. IKKβ-mediated phosphorylation of IκBα induces its ubiquitination and degradation by the 26S proteasome. The inhibition of IκBα permits the activation and nuclear translocation of the RelA and p50 subunits of NF-κB. In the nucleus, NF-κB regulates the expression of pro-inflammatory cytokines. In the alternate arm of the pathway, IRF5 undergoes TRAF6-catalyzed ubiquitination in addition to IKKβ- and/or STK25-dependent phosphorylation at Ser446 and Thr265, respectively. The activation of IRF5 also involves an interaction with the adaptor protein, TASL, which binds to SLC15A4. Whereas IKKβ is required for TASL-mediated activation of IRF5, it is unknown if STK25 modulates the TASL-dependent pathway. Altogether, these modifications induce IRF5 dimerization and nuclear translocation. In the nucleus, transcriptionally active IRF5 modulates the expression of pro-inflammatory cytokines and type I interferons. Created with BioRender.com.

Fig 4: Identification of kinases involved in the regulation of R848-induced inflammation.(A) A comprehensive siRNA screen was conducted in THP-1 cells to identify kinases that modulate R848-mediated pro-inflammatory cytokine release. The 20 proteins that exhibited the most significant reduction in R848-induced TNF-α production following siRNA knockdown are displayed in ranked order from left to right. Previously characterized regulators of TLR signaling are underlined in red. Targets underlined in blue have never been associated with TLR7/8-induced pro-inflammatory cytokine production. The black dotted line represents the mean of the R848-stimulated control group (N = 63 biological replicates). Each letter represents an independent siRNA construct (N = 3 technical replicates). (A, B, C, D) The color scheme used in (A) is conserved in (B, C, D). (B) The strictly standardized mean difference (SSMD) scores for each siRNA construct used in the identification of key regulators of R848-induced TNF-α production in the THP-1 screen are shown in descending order of significance. An SSMD score of −1 for a particular siRNA molecule was considered a positive hit and targets with two or more hits were selected for validation in follow-up studies with human primary cells. The black dotted line represents an SSMD score of 0, and the red dotted line represents an SSMD score of −1. (C, D) Human primary monocytes (C) and monocyte-derived dendritic cells (D) were stimulated with R848 for 24 h following siRNA knockdown of targets identified in the primary screen. SSMD scores for each siRNA construct were determined using the degree of siRNA-mediated inhibition of R848-induced TNF-α and IL-6 production from cells isolated from two independent donors. (E, F, G, H, I) Relative mRNA expression of STK16 (E), MAP3K19 (F), SPHK2 (G), STK25 (H), and IRF5 (I) in sorted peripheral blood leukocytes from healthy control donors (N = 11–26). Normalized transcripts per million values were obtained from GSE149050. T, T cells; B, B cells; PMN, neutrophils; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells; cMo, classical monocytes. (J) Immunoblot analysis of STK25 protein expression in untreated human immortalized cell lines. Blots were probed with antibodies against STK25 and GAPDH. (K) Densitometric analysis of STK25 protein levels after normalization to the expression of GAPDH (N = 3–4 biological replicates). *P < 0.05, U, unstimulated, S, R848-stimulated, ns, not significant. Data represent mean ± SEM.

Fig 5: STK25 responds to TLR7/8 activation in THP-1 cells and regulates TLR-induced pro-inflammatory cytokine production in murine primary immune cells.(A) Immunoblot analysis of STK25 autophosphorylation at Thr174 in THP-1 cells stimulated with R848 for 0.5, 1, 2, 4, 6, or 24 h. Blots were probed with antibodies against p-STK25 (T174), STK25, and GAPDH. (B) Densitometric analysis of p-STK25 (T174) protein levels after normalization to total STK25 protein levels and the expression of GAPDH (N = 2 independent experiments). (C) Immunoblot analysis of STK25 expression in THP-1 cells stimulated with R848 for 24 h. (D) Densitometric analysis of STK25 protein levels after normalization to the expression of β-actin (N = 4 biological replicates). UT, untreated. (E) Immunoblot analysis of STK25 protein expression in Stk25+/+ (WT) and Stk25−/− (KO) splenocytes. Blots were probed with antibodies against STK25 and GAPDH. (F, G) Imaging flow cytometry analysis of IRF5 nuclear translocation in peripheral blood CD11b+ monocytes (F) and B220+ B cells (G) from WT and KO mice following stimulation with R848 or CpG-B for 2 h (N = 3 biological replicates per genotype). (H, I) Flow cytometry analysis of the frequencies of IL-6+CD11b+ (H) and TNF-α+CD11b+ (I) splenocytes from WT and KO mice following stimulation with R848 for 18 h (N = 5–8 biological replicates per genotype). (J, K, L) Quantification of IL-6 production in culture supernatants of WT and KO splenocytes stimulated with R848 (J), CpG-B (K), or LPS (L) for 24 h (N = 4–5 biological replicates per genotype). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. Data represent mean ± SEM.