Research ArticleImmunology

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells

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Science Signaling  02 Jun 2020:
Vol. 13, Issue 634, eaaz1053
DOI: 10.1126/scisignal.aaz1053

Blocking the loop in lupus

Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects all major organs. Using newly developed inhibitors, mouse models, and blood and serum samples from patients and donors, Corzo et al. examined the roles of the B cell–associated kinases BTK and IRAK4 in SLE development. Whereas both kinases stimulated B cells in response to SLE autoantibodies, IRAK4 also mediated an autoinflammatory loop involving cytokine-secreting plasmacytoid dendritic cells. In mouse models of lupus, inhibiting IRAK4 activity reduced autoantibody production and suppressed the development of disease symptoms, including kidney damage that is often lethal in patients. The findings suggest that, unlike current therapies that only manage the symptoms of SLE, IRAK4 inhibitors may effectively treat the disease itself.


The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)–associated kinase 4 (IRAK4) and Bruton’s tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex– and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)–responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.


Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, known for its heterogeneity in its etiology and clinical manifestations, which may include complications of the renal, gastrointestinal, neurological, cutaneous, and musculoskeletal organs (1). General immunosuppressive agents have been approved and are widely available for the treatment of SLE. More recently, the biologic Benlysta (2), a monoclonal antibody that targets B cell–activating factor (BAFF), became the only biologic approved for clinical use in patients with SLE. BAFF is an essential cytokine for B cell maturation and survival, and its circulatory levels have been associated with disease activity in patients with SLE (3). However, these existing therapeutics are not effective for all patients with SLE, and some are associated with undesirable side effects, including increased risk of infections and allergy-like responses (4). Thus, safer and more effective therapeutics are needed for patients with SLE.

Multiple pathogenic pathways in both adaptive and innate immune cells have been implicated in SLE (4, 5). B cells are believed to play dual roles in SLE pathogenesis, both as professional antigen-presenting cells that can activate autoreactive T cells and through the secretion of autoantibodies that recognize nuclear antigens (6, 7). These antinuclear antibodies (ANAs) can form immune complexes (ICs) that contribute to SLE pathogenesis through activation of both Fc receptors and endosomal Toll-like receptors (TLRs), namely, TLR7, TLR8, or TLR9 (811). Kidney deposition of such ICs activates innate immune cells, resulting in chronic inflammation and glomerulonephritis, a serious complication associated with high morbidity and mortality in more than 50% of patients with SLE. ICs are also captured by plasmacytoid dendritic cells (pDCs), wherein ICs activate endosomal TLRs to drive the overproduction of type I interferons (IFNs) and an increase in the IFN signature metric (ISM), a phenomenon observed in half of patients with SLE (12, 13). In turn, pDCs promote B cell differentiation into antibody-secreting plasma cells (14). Thus, an autoinflammatory loop, driven by multiple complex inflammatory pathways in both pDCs and B cells, arises in patients with SLE and leads to the precipitation of pathogenesis.

Several studies suggest that signaling through TLR7 is one of the major pathogenic pathways in the development and progression of SLE (15, 16). TLR7 activation initiates the recruitment of myeloid differentiation primary response 88 protein (MyD88) and members of the interleukin-1 receptor (IL-1R)–associated kinase (IRAK) family to form the multiunit myddosome signaling complex (17). Myddosome formation promotes the activation and rapid phosphorylation of IRAK4, an essential member of the complex and the function of which is indispensable for the generation of TLR-dependent responses (17, 18). IRAK4 activates IRAK1 and its subsequent association with the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6). Biochemical and structural analyses suggest that IRAK1/TRAF6 dissociates from the receptor to activate the transforming growth factor–β–activated kinase 1 (TAK1 or MAP3K7) binding proteins 2 and 3 and the serine/threonine kinase TAK1, leading to the activation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and the expression of proinflammatory cytokines. Moreover, after TLR7-dependent activation in pDCs, MyD88 forms a complex with IRAK1, TRAF6, TRAF3, and inhibitor of NF-κB kinase α that results in IFN regulatory factor 7 (IRF7) phosphorylation and its translocation to the nucleus to induce the expression of type I IFN (18). Because of its critical role in regulating TLR7-dependent signaling and type 1 IFN expression, blocking the kinase activity of IRAK4 is considered as a potential treatment for SLE (19). Data from genetically predisposed mouse models of lupus demonstrate that IRAK4 inhibition seems to be an effective and promising strategy for ameliorating disease symptoms (20, 21). Multiple IRAK4 inhibitors—including BAY-1834845, CA-4948, and R835—are in the early stages of clinical development, and the IRAK4 inhibitor PF-06650833 is already in phase 2 studies for the treatment of rheumatoid arthritis (RA) with promising outcomes (22, 23).

In addition to IRAK4, modulation of Bruton’s tyrosine kinase (BTK) is considered a highly attractive therapeutic strategy for SLE, and multiple covalent and reversible BTK inhibitors are being evaluated in inflammatory and autoimmune diseases. BTK inhibition has produced encouraging results in various preclinical lupus models (2427), reducing autoantibodies and inflammatory cytokines to an extent superior to that of BAFF blockade or spleen tyrosine kinase (SYK) inhibition (26). BTK is a key mediator of various signaling processes in a number of immune cells—BTK indispensably integrates B cell receptor (BCR) signaling in B lymphocytes, Fc gamma receptor (FcγR) signaling in macrophages and DCs, and FcγR signaling in mast cells and basophils (28, 29)—therefore, modulating its activity has the potential to dampen multiple pathogenic inflammatory responses. Furthermore, various reports propose that BTK activity may be necessary for TLR-dependent responses (3033). Despite its reported role in FcγR and TLR signaling, the exact function of BTK in IC-driven activation of pDCs and type1 I IFN production is currently unclear.

In this study, we used highly selective IRAK4 (20) and BTK (27) inhibitors to compare the function of these kinases in IC-activated TLR7/8 pathways in B cells and pDCs. The results altogether suggest that IRAK4 inhibitors might be superior to BTK inhibitors to ameliorate SLE pathogenesis and inflammatory outcomes.


IRAK4 kinase activity positively regulates IC and TLR sensing in pDCs

ICs carrying nucleic acids are potent activators and inducers of type I IFNs (such as IFN-α) that trigger the activation of endosomal TLRs and the phosphorylation and activation of IRAK4 (34). To compare the impact of IRAK4 versus BTK kinase inhibition on IC-driven activation of primary human pDCs, we used ICs derived from SLE patient serum (35). SLE-ICs induced robust production of IFN-α (and, to a lesser extent, TNF-α and IL-6) from healthy-donor pDCs, whereas the blockade of the dominant FcγR expressed on human pDCs, FcγRIIA, substantially dampened this induction (Fig. 1A), indicating that uptake through this receptor is likely essential for the biological effect of SLE-ICs as previously reported (10, 36). In addition, blockade with a dual TLR7/8 antagonist (37) abolished IFN-α secretion (Fig. 1B), demonstrating that TLR7/8 are also critical mediators of SLE-IC–induced biology.

Fig. 1 IRAK4 regulates SLE-IC–mediated activation of human pDCs.

(A) Human pDCs from blood of healthy volunteers were purified, cultured with hIFN-β (50 U/ml) for 60 min, incubated with either FcγRIIA-blocking antibody or isotype control antibody (5 μg/ml) for 30 min, and then stimulated with ICs generated with SLE patient serum (SLE-ICs). The abundance of the indicated cytokines was measured in the culture medium after 24 hours by multiplex Luminex. Data are means (± SEM) cytokine output from cells from four donors. Paired Student’s t test: *P ≤ 0.05; **P ≤ 0.01. Ab, antibody; Unstim., unstimulated. (B) PBMCs were pretreated with a TLR7/8 antagonist (TLR7/8i; 250 nM) or vehicle and then stimulated with SLE-ICs, and the abundance of IFN-α2 in the cell culture medium was measured by electrochemiluminescence technology. Data are means ± SEM of three independent experiments. One-way ANOVA: *P ≤ 0.05. (C) Whole blood from healthy donors was pretreated with IRAK4i or BTKi for 60 min and then stimulated with gardiquimod (5 μg/ml), and the abundance of IFN-α2 in cell culture medium was measured by electrochemiluminescence technology. Data are representative of four independent experiments. (D) Human pDCs were pretreated with IRAK4i (5 μM) or BTKi (1 μM) for 60 min and then stimulated with three distinct SLE-ICs separately overnight. IFN-α2 was then measured in the cell culture medium by electrochemiluminescence technology, and the percentage of maximum inhibition of IFN-α2 secretion relative to vehicle-treated control was calculated. Each data point is the average of two technical replicates for each SLE-IC, shown as mean ± SEM. Data are representative of similar results obtained with cells from four donors. Paired Student’s t test: **P ≤ 0.01. (E) Human pDCs were pretreated with vehicle, IRAK4i (5 μM), or BTKi (1 μM) for 60 min and then stimulated with SLE-ICs. Controls were completely unstimulated. Twenty-four hours later, the surface expression of CD40 was analyzed by flow cytometry. Representative histogram and quantification of mean fluorescence intensity (MFI) from an individual experiment are shown; each data point represents a different SLE-IC. Data are representative of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons test: **P ≤ 0.01; ns, no significance.

We performed functional analyses using two potent and selective IRAK4 kinase inhibitors (herein, IRAK4i). The first compound, IRAK4i-1, was synthesized for this study with a formula obtained from public data (20, 38). Biochemical analysis of a large panel of kinases (220 kinases) confirmed that IRAK4i-1 is highly selective with the biophysical properties suitable for in vitro studies (fig. S1, A and B). The second IRAK4 inhibitor referred to as IRAK4i-2 (39) is a novel Genentech molecule, the selectivity and pharmacokinetic (PK) properties of which have been described previously (40) and was used in vivo (fig. S1, A and B). To block BTK activity, we used a potent and selective Genentech BTK inhibitor (herein, BTKi) that has been previously described (41). The selectivity and general properties of these inhibitors used in our studies are summarized in (fig. S1, A and B). Both IRAK4i molecules were equally potent in repressing the TLR7/8 agonist gardiquimod–induced IFN-α secretion in a healthy-donor whole-blood assay (Fig. 1C and fig. S2). From these results, we decided to use IRAK4i-1 at 5 μM, which would theoretically provide a 90% inhibitory concentration (IC90) coverage and achieve maximal pathway repression in vitro. BTKi reduced BTK phosphorylation at Tyr223 and impaired CD63 up-regulation in basophils in response to FcεR activation induced by immunoglobulin E (IgE) cross-linking, a pathway tightly regulated by BTK (fig. S3) (42). We found that IFN-α secretion by pDCs in response to SLE-ICs was largely dependent on the kinase activity of IRAK4 but not that of BTK (Fig. 1D). IRAK4, but not BTK, inhibition decreased the up-regulation of CD40, a surface receptor that upon ligand engagement enhances the capacity of pDCs to induce B cell differentiation to plasma cells (14), in response to stimulation with SLE-ICs (Fig. 1E). Given that IRAK4 inhibition did not yield full repression of IFN-α production, we combined both inhibitors to assess whether blockade of both kinases would have a synergistic effect; however, after combinatorial treatment with both IRAK4i and BTKi we found similar IFN-α levels as with IRAK4i treatment alone (fig. S4). These results suggest that only IRAK4 kinase activity positively regulates SLE-IC signaling in pDCs.

Because both FcγR and TLR7 pathways are essential for the activity of SLE-ICs on pDCs, we interrogated the role of IRAK4 downstream of these two signaling pathways. Because complete repression of IFN-α by the TLR7/8 antagonist strongly suggests that SLE-IC biological activity in pDCs is primarily mediated by the TLR7/8 pathway, we then interrogated the roles of IRAK4 and BTK downstream of these TLRs. First, we assessed the phosphorylation state of key signaling molecules at functional residues downstream of receptor engagement (Fig. 1D). Gardiquimod stimulation induced subtle BTK phosphorylation at Tyr223 and substantial phosphorylation of phospholipase C–γ2 (PLCγ2) (at Tyr759), P65 (at Ser529), P38 (at Thr180/Tyr182), and IRF7 (at Ser477/Ser479) (Fig. 1F). IRAK4i treatment of pDCs markedly reduced the phosphorylation of P65, P38, and IRF7 (Fig. 1F). However, the phosphorylation statuses of PLCγ2 and BTK were not affected by IRAK4i treatment, suggesting that these two factors act upstream or in parallel with IRAK4 in the TLR7 signaling cascade. Although BTKi treatment diminished the phosphorylation of BTK and PLCγ2, treatment of pDCs did not reduce the phosphorylation of P38, P65, or IRF7 (Fig. 2A). The increase in IRF7 phosphorylation and its respective inhibition by IRAK4i treatment was confirmed by Western blot (Fig. 2B). In addition, by Western blotting, we detected phosphorylation of IRAK4, TANK-binding kinase 1 (TBK1), and extracellular signal–regulated kinase 1/2 (ERK1/2) induced after gardiquimod stimulation. With the exception of pERK, reduction in the phosphorylation of these other molecules after BTKi treatment was not detected (Fig. 2B). After 24 hours, gardiquimod stimulation induced robust secretion of IFN-α, proinflammatory cytokines TNF-α and IL-6 (Fig. 2C), and expression of CD40 (Fig. 2D). As expected, IRAK4i treatment strongly repressed production of IFN-α, TNF-α, and IL-6 and partially reduced surface expression of CD40. Treatment with BTKi marginally reduced the production of IFN-α, TNF-α, and IL-6, with only TNF-α reaching statistical significance, possibly as a result of decreased ERK phosphorylation, and had no impact on CD40 expression (Fig. 2D). Collectively, our results show that IRAK4 prominently mediates TLR-dependent responses through its regulation of NF-κB and MAPK pathways. In contrast, BTK plays only a modest role in TLR-dependent responses. Hence, our data suggest that IRAK4 plays a more prominent role than BTK in integrating nucleic acid–IC signaling in human pDCs.

Fig. 2 IRAK4 regulates TLR7-mediated activation of human pDCs.

(A) Intracellular staining of human pDCs pretreated with IRAK4i (5 μM) or BTKi (1 μM) for 60 min and then stimulated with gardiquimod (10 μg/ml) for 10 min (for assessment of pBTK and pPLCγ2), 30 min (for pP65 and pP38), or 60 min (for pIRF7). Gray histogram, basal phosphorylation in unstimulated cells. Data shown were normalized to mode and are representative of two independent experiments. (B) Western blot analysis of IRAK4 and BTK phosphorylation in the pDCs pretreated with IRAK4i (5 μM) or BTKi (1 μM) and then stimulated with SLE-ICs (1% final volume) for 20 min. Lysates were then blotted for the indicated total and phosphorylated (p) proteins. Blots are representative of two independent experiments. (C) Cytokines secreted by pDCs pretreated as in (A) and (B) and stimulated overnight with gardiquimod (5 μg/ml). Data are means ± SEM from four independent experiments. One-way ANOVA with Tukey’s multiple comparisons test: *P ≤ 0.05; ***P ≤ 0.01; ****P ≤ 0.0001. (D) Surface expression of CD40 in pDCs after stimulation with gardiquimod (5 μg/ml) for 24 hours. Histogram and quantification of mean fluorescence intensity values are representative and means ± SEM, respectively, from four independent experiments. One-way ANOVA with Tukey’s multiple comparisons test: **P ≤ 0.01.

IRAK and BTK positively regulate TLR7 signaling in memory B cells

Because of their function as antigen-presenting cells and their capacity to produce pathogenic self-reactive antibodies, B cells are implicated in SLE pathogenesis (43, 44). It has been demonstrated that endosomal TLR agonists can direct memory B cells to differentiate into plasmablasts (45, 46); however, the relative contribution of IRAK4 or BTK kinase activity to this process has not been formally compared or understood. To evaluate the roles of IRAK4 and BTK in plasmablast differentiation, we purified human memory B cells based on positive expression of CD27; more than 95% of CD27+ memory B cells expressed CD20 but lacked expression of CD38 markers. Memory B cells were cultured in the presence of a cocktail of cytokines (IL-2, IL-6, IL-10, IL-15, and IFN-α) reported to support TLR-driven differentiation into plasmablasts (46). Cells were stimulated with gardiquimod to induce plasmablast differentiation, and after 4 days, more than 50% of cells differentiated into plasmablasts, characterized by up-regulation of CD38 and down-regulation of CD20. As reported, addition of BTKi during the differentiation process drastically reduced the number of CD38+ plasmablasts recovered after 4 days (Fig. 3A) (27). Treating cultures with IRAK4i also blocked the differentiation of memory B cells into CD38+CD20 plasmablasts, although the phenotype was less robust when compared to BTKi treatment (Fig. 3A).

Fig. 3 Regulation of TLR7-mediated responses in human memory B cells by IRAK4 and BTK converges in NF-κB and MAPK pathways.

(A) Human memory B cells (CD27+CD20+CD38) were purified from buffy coats and cultured in the presence of IL-2, IL-6, IL-10, IL-15, and IFN-α for 4 days. During those 4 days, cells were either unstimulated or pretreated with vehicle, IRAK4i (5 μM), or BTKi (1 μM) and stimulated with gardiquimod (10 μg/ml). Data are the number of plasmablasts (CD38+CD20) recovered from initial culture of 2 × 104 memory B cells (CD20+ and CD38), shown as means ± SD of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons test: *P ≤ 0.05. (B) Mobilization of [Ca2+] after BCR cross-linking of memory B cells with hIgM/IgG antibody (50 μg/ml). Cells were pretreated with vehicle (black line), IRAK4i (5 μM; blue line), or BTKi (1 μM; red line) for 60 min before BCR cross-linking. Control cells (unstim., gray line) were pretreated with vehicle but were not BCR–cross-linked. Data are from a representative one of three independent experiments. (C) Intracellular staining for pBTK, pP38, and pP65 in purified memory B cells stimulated with gardiquimod (10 μg/ml). pBTK was assessed 5 min after stimulation; pP38 and pP65 were assessed 30 min after stimulation. Gray histogram, basal phosphorylation in unstimulated cells. Bar graphs show the average percentage increase in mean fluorescence intensity for phosphorylated BTK, P65, and P38 over those in unstimulated cells from two independent experiments. Data are representative (top) and means ± SEM (bottom) of two independent experiments.

To understand how BTK plays a more dominant regulatory role in TLR7-dependent responses in B cells than pDCs (presented above) and whether IRAK4 regulates similar signaling events, we evaluated the signaling pathways activated in human memory B cells during gardiquimod stimulation. Because Ca2+ is a critical signaling messenger in B cells (47), we first investigated whether Ca2+ mobilization is induced as a result of TLR7 activation. Stimulation with gardiquimod did not lead to an increase in Ca2+ mobilization in memory B cells, suggesting that Ca2+ flux is not an event downstream of TLR7 (fig. S5). Furthermore, although BTKi potently repressed Ca2+ flux immediately after anti–IgG/IgM F(ab)2–mediated BCR cross-linking, IRAK4 inhibition did not (Fig. 3B).

We then evaluated the phosphorylation state of BTK, P65, and P38, which were each activated in pDCs after stimulation with gardiquimod. In memory B cells, basal BTK phosphorylation was not unimodal, possibly reflecting the complex heterogeneity of the memory population (48). After stimulation with gardiquimod, we observed a robust increase in BTK phosphorylation at Tyr221, more robust than we observed in pDCs (Fig. 3C), which may suggest that BTK is a more integral component of the TLR7 pathway in memory B cells. Phosphorylation of both P65 at Ser536 and P38 at Thr180/Tyr182 was increased by gardiquimod as well. As expected, the presence of BTKi during stimulation reduced BTK phosphorylation considerably; however, distinct from pDCs, BTK inhibition in memory B cells blocked the activation of both P65 and P38 (Fig. 3C). Unlike BTKi, IRAK4i did not block the elevation in BTK-pTyr221 but similarly decreased the activation of both P65 and P38 (Fig. 3C). These data suggest that the regulation of NF-κB and MAPK pathways during TLR7 activation may be a common feature shared by IRAK4 and BTK in B cells and that modulation of IRAK4 activity may be a therapeutic option for the development of autoantibody-secreting cells in SLE.

Inactivation of IRAK4 kinase activity reduces the expression of the IFN gene signature

The IFN gene signature in circulating immune cells is a hallmark of SLE and serves as a biomarker to identify patients that may be responsive to therapies targeting type I IFN signaling (49). Components of both endosomal TLRs and ICs are implicated in up-regulating the IFN signature in patients with SLE (12). We therefore explored how IRAK4i and BTKi affect the IFN signature in whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors. Gardiquimod stimulation caused a rapid increase in the expression of IFN-responsive gene (IRG) transcripts in whole blood (fig. S6) and PBMCs (Fig. 4A and fig. S7), with changes observed as early as 2 hours after stimulation. IRG transcripts were dampened by pretreating whole blood or PBMCs with IRAK4i but not BTKi (Fig. 4A). Although less robust than gardiquimod, stimulation of PBMCs with the more disease-relevant SLE-ICs up-regulated the same IRG transcripts, and treatment with IRAK4i produced a noticeable reduction of these transcripts, whereas BTKi did not show any effect (Fig. 4B and fig. S7).

Fig. 4 Expression of IFN response genes is lessened after IRAK4 inhibition.

(A and B) PBMCs were pretreated with vehicle or the indicated inhibitor for 60 min and either (A) stimulated with gardiquimod (5 μg/ml) or (B) stimulated with SLE-ICs for 4 hours. Expression of IRG transcripts was determined by Fluidigm after RNA isolation. Plots show the average of two individual experiments, results of each shown as top and bottom whiskers. One-way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (C) Eight-week-old C57Bl/6 mice were each challenged with a single dose of pristane (0.5 ml) and received IRAK4i (100 mg/kg), BTKi (100 mg/kg), or the respective vehicle intraperitoneally twice daily for 14 consecutive days along with IFNR antibody or isotype control antibody (10 mg/kg each) three times per week. After 14 days, whole blood was collected for RNA extraction, and expression of IRG transcripts was determined by Fluidigm. IRAK4i, n = 6 mice; BTKi vehicle, n = 7 mice; all others, n = 8 mice. Student’s t test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (D) Eight-week-old C57BL/6 WT or IRAK4-KD mice were challenged with one dose of pristane (0.5 ml). After 14 days, whole blood was collected for RNA extraction, and expression of IRG transcripts was determined by Fluidigm. Pristane-challenged WT and IRAK4-KD, n = 10 mice each; naïve WT and IRAK4-KD, n = 5 mice each. One-way ANOVA with Tukey’s multiple comparisons test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

To elucidate the role of IRAK4 in the induction of the IFN gene signature in vivo, we used the pristane-induced lupus mouse model. Pristane is a hydrocarbon mineral oil that, when injected into the peritoneal cavity, induces an inflammatory response in mice and consequently causes the development of antibodies against multiple nuclear antigens and glomerulonephritis. The pristane model is characterized by the manifestation of a detectable IFN signature and is highly dependent on type I IFN signaling and the TLR7 pathway for disease progression because the effects of pristane are abolished in TLR7-deficient animals (50, 51). To test how pharmacologic blockade of IRAK4 and BTK affects the IRG signature, we dosed pristane-challenged mice with IRAK4i-2 and BTKi orally twice a day for 14 days. Two weeks after pristane challenge, increases in IRG transcripts composed in part by the canonical genes Ifit, Oas1a, and Oasl2 were detected in whole blood (Fig. 4C). Dosing IRAK4i and BTKi orally at 100 mg/kg maintained the mean steady-state plasma trough concentration between the IC50 and IC90 for IRAK4i and above the IC90 for BTKi (fig. S8) (27).

IFN receptor (IFNR) was required for IRG expression; treating mice with an IFNR-blocking antibody completely quenched the gene signature (Fig. 4C). Although treating mice with IRAK4i did not repress the up-regulation of IRGs completely, we observed significantly reduced transcript levels of Ifit, Oas1a, and Oasl2 (Fig. 4C). In contrast, pristane-challenged mice treated with BTKi showed similar expression levels of IRGs as the respective controls. Given the central role of IRAK4 in IC and endosomal TLR regulation, we generated IRAK4 kinase–deficient (KD) knockin mice, in both C57BL6 or Balb/c genetic backgrounds, to study IRAK4 kinase function in vivo. Splenocytes isolated from IRAK4-KD (C57BL/6 background) mice appeared to express lower amounts of IRAK4 protein than those isolated from wild-type (WT) mice, and we observed attenuated phosphorylation of IRAK4 and IRAK1 in response to the TLR7/8 agonist R848 (fig. S9). As previously reported, IRAK4-KD splenocytes produced lower levels of cytokines in response to gardiquimod (fig. S10) (52, 53). After challenging IRAK4-KD (in C57BL/6 background) mice with pristane, we observed significantly lower IRG transcripts than pristane-challenged WT mice (Fig. 4D). IRAK4, therefore, plays an important role in IRG induction both in human cells and in the murine pristane lupus model. Thus, therapeutic inactivation of IRAK4 kinase activity may be a promising strategy to diminish the IFN gene signature in patients with SLE.

Absence of IRAK4 kinase activity ameliorates pristane-induced lupus immune pathology

After receiving pristane, WT and IRAK4-KD C57BL/6 mice were monitored for up to 47 weeks and evaluated for kidney pathology and presence of ANAs as two major immune-pathologies in this genetic background. Mild proliferative glomerulonephritis but not nonglomerular kidney inflammation was induced in kidneys of WT mice after pristane challenge but was less pronounced in pristane-challenged IRAK4-KD mice and was not quantifiably different from naïve IRAK4-KD mice (Fig. 5, A and B). Direct immunofluorescence microscopy revealed a marked reduction in pristane-induced glomerular IgG+ and IgM+ ICs in IRAK4-KD mice by comparison to that of WT mice (Fig. 5, C and D). Peculiarly, kidney sections from naïve IRAK4-KD mice also displayed lower IgG+ and IgM+ signal fluorescence than naïve WT mice (Fig. 5D), which was likely a consequence of lower levels of total circulating antibodies in IRAK4-KD animals (Fig. 5E and figs. S11 and S12). Pristane treatment increased the levels of circulatory ANAs in WT mice (Fig. 5E), including those specific for ribonucleoprotein (RNP) and Sm antigens (Fig. 5, F and G). On the other hand, the levels of ANAs after pristane challenge were not increased in IRAK4-KD mice. In this disease model, pristane challenge significantly increased IgG2b, IgG2c, and IgM ANAs in WT mice but not in IRAK4-KD mice (Fig. 5H). ANAs of all evaluated isotypes were lower in IRAK4-KD mice than in WT mice, regardless of animal exposure to pristane (Fig. 5F). To clarify whether the reduced ANA titers in IRAK4-KD resulted from decreased global levels of Igs, we measured the levels of Ig subclasses in the serum of WT and IRAK4-KD mice. Pristane-challenged and naïve IRAK4-KD mice showed reduced amounts of total IgG1, IgG2b, IgG2c, and IgM antibodies than those found in WT counterparts (fig. S11). These observations suggest that IRAK4 kinase activity is important for the development of serum antibodies.

Fig. 5 Absence of IRAK4 activity ameliorates disease activity in pristane-challenged mice.

(A) IRAK4-KD mice or WT littermates were administered pristane (0.5 ml, intraperitoneally) or were untreated (naïve). After 47 weeks, animals were euthanized and evaluated for renal pathology. Representative PAS staining of kidney sections are shown. (B) Quantification of glomerular cellularity as a measure of proliferative glomerulonephritis severity in the mice described in (A). Pristane-challenged and naïve WT mice, n = 9 and 5, respectively; pristane-challenged and naïve IRAK4-KD mice, n = 11 and 3, respectively. One-way ANOVA with Tukey’s multiple comparisons test: *P ≤ 0.05; **P ≤ 0.01. (C) Representative immunofluorescence images (×100 magnification) of kidney sections showing IgG+ and IgM+ IC deposits in pristane-challenged WT and IRAK4-KD mice [as described in (A)]. (D) Quantified intensity of IgG+ and IgM+ immunofluorescence signals from kidney cortical sections from mice represented in (C) and their naïve counterparts. Pristane-challenged and naïve WT mice, n = 7 and 5, respectively; pristane-challenged and naïve IRAK4-KD mice, n = 10 and 2 (IgG) or 3 (IgM), respectively. One-way ANOVA with Tukey’s multiple comparisons test: **P ≤ 0.01; ****P ≤ 0.0001. (E to I) The serum levels of ANAs (E), antibodies to RNP (F), and antibodies to Sm (G), as well as the ANA-specific activity of individual IgG1, IgG2a, IgG2b, and IgM subclasses (H) and the splenic abundance of IgJ transcripts (I), were measured in pristane-challenged WT and IRAK4-KD mice and age-matched naïve controls 47 weeks after pristane challenge by ELISA. Means ± SEM are shown. Pristane-challenged and naïve WT mice, n = 10 and 15, respectively; pristane-challenged and naïve IRAK4-KD mice, n = 12 and 3, respectively. One-way ANOVA with Tukey’s multiple comparisons test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Because of the essential role of IRAK4 kinase function in the development of human memory B cells to plasmablasts, we hypothesized that the reduced levels of circulatory Igs in IRAK4-KD mice was related to inadequate plasma cell development. To investigate this, we measured the expression of genes associated with plasma cell development in RNA extracted from spleens of mice after the end of the study. Transcript levels of IgJ, a marker of plasmablast abundance, were reduced in both naïve and pristane-challenged IRAK4-KD mice relative to WT counterparts (Fig. 5I). In addition, we evaluated splenic plasma cells (CD138+B220) in naïve BALB/c mice expressing inactive IRAK4 kinase, age-matched with the C57BL/6 animals that participated in the pristane-induced model (fig. S12). In the BALB/c background, we found significantly fewer number of splenic IgG2a+ plasma cells relative to WT naïve mice and a trend of reduced IgG2b+, IgG1+, and IgM+ plasma cell numbers in IRAK4-KD mice, although the differences between genotypes did not reach statistical significance (fig. S12). Altogether, these results suggest that the absence of IRAK4 kinase activity impairs plasma cell development and curtails the production of pathogenic autoantibodies in autoimmune disorders.

Absence of IRAK4 kinase activity ameliorates imiquimod-accelerated NZB/W lupus model

The pristane model reproduces important clinical features of SLE; however, certain pathological features, such as mortality, proteinuria, and glomerulonephritis, are weakly manifested in this model. To study the utility of IRAK4 kinase inactivation in the context of more pronounced kidney pathology, we used the NZB/W_F1 nephritis model. We generated a kinase-inactive IRAK4 on both New Zealand black (NZB) and New Zealand white (NZW) backgrounds and crossed the strains together to generate IRAK4-KD NZB/W_F1 progenies. To expedite precipitation of disease in this model, imiquimod was topically applied to 8-week-old NZB/W_F1 mice expressing functional or inactive IRAK4 kinase (54). Unlike NZB/W_F1 mice expressing WT IRAK4, NZB/W_F1 IRAK4-KD mice did not develop proteinuria (Fig. 6A) and showed improvement in overall survival (Fig. 6B). Expression of kinase-inactive IRAK4 also diminished severity of proliferative glomerulonephritis and nonglomerular aspects of kidney disease, such as periarteritis, by comparison to that of the WT IRAK4–expressing control mice (Fig. 6, C to E). In addition, as we had observed in the pristane-induced lupus model, the absence of functional IRAK4 in NZB/W_F1 mice had a profound effect in glomerular Ig deposition, with staining for IgG+ and IgM+ deposits markedly decreased relative to WT IRAK4–expressing NZB/W_F1 controls (Fig. 6, F and G).

Fig. 6 Absence of IRAK4 activity ameliorates disease activity in NZB/W spontaneous lupus model.

(A) Eight-week-old NZB/W.IRAK4-WT and IRAK4-KD mice were topically treated with imiquimod three times per week for 8 weeks to precipitate disease, and proteinuria was scored for 8 weeks after the last imiquimod application. Data are means ± SD from n = 6 mice per group. Paired Student’s t test: **P ≤ 0.01. (B) Percentage of overall survival in mice treated as described in (A). P value was determined by log-rank Mantel-Cox test. (C) Top: PAS stain of kidney tissue assessing proliferative glomerulonephritis. Bottom: H&E stain showing an example of nonglomerular kidney inflammation (periarteritis shown) in kidney sections of imiquimod-treated IRAK4-WT and IRAK4-KD NZB/W mice after 8 weeks from the last imiquimod application. One representative image from each group is shown. Scale bars, 100 (top) and 200 μm (bottom). (D and E) Quantification of glomerular cellularity (D) and semiquantitative scoring of nonglomerular kidney inflammation severity (E) in mice treated as described in (A). Student’s t test and Mann-Whitney test, respectively: *P ≤ 0.05. (F) Immunofluorescence staining of kidney sections showing glomerular IgG+ and IgM+ deposits in mice treated as described in (A). Scale bars, 100 μm. (G) Quantified intensity of kidney cortical IgG+ and IgM+ immunofluorescence (n = 4 to 6 per group). Unpaired Student’s t test: ****P ≤ 0.0001.

IRAK4 target genes are up-regulated in SLE

SLE blood transcriptome is enriched in IFN gene signatures and plasmablast signatures, highlighting the importance of pDC and B cells as potential pathogenic cell types (5). We examined the expression of IRAK4 target genes (55) in a published cohort of patients with lupus (56). As expected, patients with SLE with high levels of IFN signaling, defined by the ISM, showed increased abundance of IRAK4 target genes (Fig. 7, A and B). Likewise, IFN-high patients with SLE also showed higher expression of plasmablast-associated IgJ (Fig. 7C). We found a correlation of higher abundance of IRAK4 target genes and plasmablast abundance (Fig. 7D). Although the expression studies do not establish a causal role or cellular source for the associated gene signature, they do suggest that the IRAK4 pathway is actively engaged in a subset of patients with SLE; thus, targeting its kinase activity may effectively reduce both plasmablast and IFN gene signatures.

Fig. 7 IRAK4 target genes are up-regulated in SLE.

(A) Signature score of IRAK4 target genes in ISM-high and ISM-low patients with SLE and healthy donors (HC). Each point corresponds to a single patient or donor; patients with SLE were grouped into ISM high or ISM low on the basis of the ISM signature score. Signature score values were compared using a two-tailed Student’s t test, ***P < 0.001. (B) Expression of IRAK4 target genes in patients with SLE and healthy donors. Log2-transformed normalized RPKM values for IRAK4 target genes were centered and scaled. Genes are clustered using Euclidean distance and Ward linkage. (C) IgJ, a marker for plasmablast abundance, in patients with SLE as correlated with ISM values. Each point is a patient or donor; patients with SLE were grouped into ISM high or ISM low on the basis of the ISM signature score. The y axis shows the normalized RPKM expression value. Values were compared using a two-tailed Student’s t test, ***P < 0.001. (D) Correlation between ISM and IgJ expression. Each point corresponds to a patient with SLE, the x axis shows the ISM score for each patient, and the y axis shows expression of IgJ as normalized RPKM.


The autoinflammatory loop in lupus is driven by the combinatorial action of endosomal TLRs, type I IFNs, antigen receptors, and FcRs present in different cell types. Therapies that target single pathogenic axes, including anti-IFN receptor, anti-IFNs, and B cell depletion, have not achieved success (4). Hence, targeted therapeutics that simultaneously blunt multiple pathogenic pathways may be more effective at dampening systemic autoimmunity and organ damage (44). IRAK4 and BTK are clinical targets shown to regulate several pathogenic pathways in SLE; our study demonstrates, however, that these kinases hold distinct regulatory roles and have different utilities in treating inflammatory or autoimmune diseases.

A link between TLR7/8 and SLE has been resoundingly implicated by multiple genetic studies (57, 58). In pDCs, nucleic acids carried by ICs are internalized and delivered to the endosomes where they activate TLR7/8 to up-regulate the production of type I IFNs. As the major producers of type I IFNs, pDCs highly contribute to type I IFN levels and associated gene signatures observed in patients with SLE (57). Only IRAK4 blockade diminished the ability of pDCs to secrete IFN-α after activation with SLE-ICs; BTK blockade had no effect, and we observed no synergistic effect with dual blockade of both kinases. TLR7/8 activation in pDCs results in activation of PLCγ2, TBK1/IRF7, MAPK, NF-κB, and IRAK4, while only modestly increasing BTK phosphorylation over basal levels, and, with the exception of PLCγ2 and BTK, the phosphorylation of all signaling proteins was diminished upon treatment with IRAK4i. Treatment with BTKi reduced phosphorylation of BTK, PLCγ2, and also ERK1/2, whose activity has been reported to be partly controlled by PLCγ members (5961) and may explain the reduction of pDC-derived cytokines observed after BTK inhibition. Although BTK and PLCγ2 may quite possibly have a role during TLR activation to boost production of inflammatory cytokines, our data show that IRAK4 is the principal regulator of TLR7/8-driven responses and positively regulates multiple downstream signaling pathways.

Memory B cells express high levels of TLR7/8 that, upon encountering ligand, induce proliferation, activation, and differentiation into plasma cells. In B lymphocytes, nucleic acid uptake can occur via the mechanisms of diffusion or via BCR-dependent internalization (58). BTK is an essential component of BCR-dependent signaling; BCR activation exposes the immunoreceptor tyrosine-based activation motif to kinases Lyn and Syk that participate in the transphosphorylation of BTK at Tyr551, an event that culminates in BTK Tyr223 autophosphorylation and full kinase activation (62). Activated BTK promotes Tyr phosphorylation of PLCγ2, potentiating calcium signaling and the activation of NF-κB and MAPK pathways to positively regulate proliferation, survival, and cytokine expression (59, 60). Our data show that, unlike BTK, IRAK4 kinase activity is not integral for BCR signaling because IRAK4 inhibition failed to block Ca2+ mobilization. Whereas IRAK4 is required for TLR7-driven effector functions in pDCs, in memory B cells, both BTKi and IRAK4i reduced gardiquimod-induced P38 and P65 phosphorylation and blocked plasmablast differentiation. Our results obtained with BTKi treatment are consistent with previously reported findings in murine B cells, in which BTK deficiency was shown to decrease proliferation in response to TLR4 and TLR9 agonists (27, 61, 63). BTK, clearly, has an essential role integrating TLR-dependent signals in B cells. It is unclear, then, why BTKi treatment is unable to repress TLR7-induced IRF7, P38, and NF-κB activation in pDCs. Experiments with cells derived from patients with X-linked agammaglobulinemia (XLA), who have mutations that result in BTK inactivation, blocking B cell development and causing immunodeficiency (61), seem to corroborate our findings; although the loss of BTK function severely affects B cell populations in patients with XLA, pDCs isolated from these patients produce comparable levels of IFN-α when challenged with TLR7 or TLR9 agonists as pDCs from healthy controls (64, 65). We conclude therefore that BTK distinctly regulates TLR responses in B cells than pDCs. Several biochemical studies point to a physical interaction between BTK and different members of the myddosome, including MyD88, IRAK1, Toll–interleukin-1 receptor (TIR) domain–containing adaptor protein (TIRAP), and TIR domain–containing adaptor-inducing IFN-β (TRIF) (29, 66, 67). If this interaction between BTK and myddosome components is weaker and less profuse in pDCs than B cells, or even other leukocytes, it may explain why BTK activity is dispensable during TLR-driven responses in pDCs. Additional experiments will be required to elucidate how BTK is integrated into the TLR pathway in different cell types.

Prominent up-regulation of IFN-regulated genes is found in a large portion of patients with SLE, and we found that IRAK4 activity was critical for their up-regulation in PBMCs from healthy donors after stimulation with either gardiquimod or disease-relevant SLE-ICs. We observed a similar effect after IRAK4 inhibition in vivo in the pristane-induced mouse model of lupus. Genetic ablation of IRAK4 kinase function statistically significantly dampened IRG transcript levels in peripheral blood of mice after challenge with pristane. Daily therapeutic intervention with IRAK4i resulted in reduced IRG expression in peripheral blood at the same time point. The incapability of BTKi to repress type I IFNs and up-regulation of IFN-dependent genes substantiates our conclusion that BTK plays little role on the IFN pathway. The ineffectiveness of BTK inhibition in curtailing TLR7-dependent responses in pDCs or to modulate gene expression of IFN-regulated genes in vivo is also corroborated by previous studies (2326). The efficacy of BTK inhibitors in lupus models likely stems from the blockade of B cell functions, such as autoantibody production, but not from direct modulation of IFN production or IFNR signaling.

In both the pristane-induced and the imiquimod-accelerated NZB/W_F1 mouse models of lupus, genetic inactivation of IRAK4 kinase activity resulted in marked amelioration of accompanying immune pathologies and improved survival. In both models, the kinase activity of IRAK4 played a role in glomerular deposition of IgG and IgM and splenic abundance of IgJ, a surrogate marker of plasma cells. Naïve IRAK4-KD mice also had a subtle decrease of all splenic plasma cells independent of Ig subclass. Our results resemble findings from analysis of IRAK4-deficient individuals, who reportedly have statistically significantly reduced percentages of CD27+IgM+IgD+ and a trend of reduction for IgDCD27+ memory cells (68), and underscore a role for IRAK4 in B cell differentiation.

In conclusion, our findings provide further mechanistic data on the role of IRAK4 kinase in the integration of IC- or TLR7/8-driven responses in myeloid cells to regulate IRG expression and immunomediated pathology. Our studies in both the pristane-induced and the accelerated NZB/W_F1 mouse models of lupus show that IRAK4 kinase inhibition has the potential to reduce type 1 IFN as well as other inflammatory cytokine pathology prevalent in patients with SLE. Both IRAK4 and BTK inhibitors are effective in reducing inflammation in the context of RA or SLE in preclinical rodent models (20, 2527, 29, 69), and several inhibitors of IRAK4 and BTK function are currently under clinical assessment for the treatment of SLE and RA. Upcoming clinical data on inhibitors against BTK or IRAK4 will provide better understanding of the cell-specific functions of each kinase and provide rationale for the pursuit of IRAK4 inhibition in the clinic.



C57BL/6J, BALB/cJ, NZB/BlNJ, and NZW/LacJ mice were purchased from the Jackson laboratory. CRISPR-Cas9 technology (70, 71) was used to generate IRAK4-KD mice (K213A/K214A double substitution) in C57BL/6J and BALB/c. Single-guide RNA (sgRNA) design, microinjection, and off-target analysis were done essentially as described by Anderson et al. (72). The sgRNA target was: 5′TCGTGGCGGTGAAGAAGCT3′ protospacer-adjacent motif: CGG. An oligonucleotide donor (5-′CGGTGGCAACCGGATGGGAGAGGGGGGATTTGGAGTGGTGTACAAGGGCTGTGTGAACAACACCATCGTGGCGGTGgccgcaCTCGGAGCGGTAAGCCATCTTCCTTCCTCCTCTCAGAAGAAGCAGCCAGCTACCCTCACCGGATTCATTATCCCAGTGATT-3′) was used to introduce the mutations in Irak4 exon 5 (mutant bases in lowercase). Founders were generated using either C57BL/6J or BALB/c zygotes and mated with WT C57BL/6J and BALB/c mice, respectively, to transmit the edited chromosome. Subsequent analysis of genomic DNA from G1 pups was used to confirm germline transmission of the targeted gene and absence of off-target hits elsewhere in the genome. Using in-licensed b6.129-Irak4KK213AA congenic mice (73), the Irak4 mutation was backcrossed to NZB/BlNJ and NZW/LacJ separately using speed congenics, and NZBWF1-Irak4KK213AA mice were generated by intercrossing NZW.b6.129-Irak4KK213AA and NZB.b6.129-Irak4KK213AA. All animal experiments were approved by the Genentech Institutional Animal Care and Use Committee.

Cell isolation and culture

Human pDCs were purified from buffy coats by depletion of CD3+ cells (STEMCELL Technologies) followed by positive selection of BDCA-4/Neuropilin-1 (CD304 MicroBead kit; Miltenyi Biotec). Human memory B cells were purified by initial enrichment of B cells (RosetteSep Human B Cell Enrichment Cocktail, STEMCELL Technologies) followed by positive selection of CD27+ cells (CD27+ MicroBeads kit, Miltenyi Biotec). Purity after isolation was consistently more than 90% for memory B cells and for pDCs. After purification, cells were resuspended in RPMI 1640 containing 10% fetal bovine serum (FBS), 1 mM glutamine, and penicillin streptomycin and were pretreated with vehicle (0.5% dimethyl sulfoxide) or the appropriate inhibitors for at least 60 min before stimulations.

Stimulatory assays with human pDCs

For overnight stimulations, purified human pDCs were cultured with freshly prepared SLE-ICs or gardiquimod (5 μg/ml; Invivogen). Cells were primed with human IFN-β (hIFN-β) (50 U/ml; 60 min) before stimulations with SLE-ICs to enhance biological response and cytokine production. Human IFN-α2 was quantified using a combination of electrochemiluminescence and multiarray technology (64). TNF-α and IL-6 levels in supernatants were assessed by multiplex Luminex (Millipore). For intracellular phospho-flow cytometry staining and analysis of signaling pathways, cells were stimulated with gardiquimod (10 μg/ml) or SLE-ICs and fixed immediately after. To block hFcγRIIA (hCD32), pDCs were preincubated with mouse anti-hFcγRIIA blocking antibody (clone IV.3, STEMCELL Technologies) or mIgG2b isotype control (BD Biosciences) for 30 min on ice before addition of SLE-ICs.

Generation of ICs

To generate SLE-ICs, we followed a previously described protocol (35). Briefly, sera from several patients with SLE were individually mixed with supernatants from apoptotic U937 cell cultures. Apoptosis was induced in U937 cells by exposing cells to ultraviolet irradiation and incubating at 37°C for 24 hours. Cell supernatant was collected and mixed with serum from individual patients with SLE at a 10:1 ratio. After 20 min of incubation at room temperature, the mixture was used to stimulate cells in a final volume of 200 μl. The final concentration of SLE serum was 1% of final volume.

Plasmablast differentiation of human memory B cells

To induce differentiation into plasmablasts, purified CD27+ memory B cells were seeded on 96-well plates (2 × 104 cells per well). Differentiation was induced with gardiquimod (5 mg/ml) in medium containing hIL-2 (20 U/ml), hIL-6 (50 ng/ml), hIL-10 (50 ng/ml), hIL-15 (10 ng/ml), and hIFN-α (10 ng/ml) for 4 days in the presence of IRAK4 or BTK inhibitors. Plasmablast differentiation was determined by expression of CD38 and down-regulation of CD20 markers.

Flow cytometry

To stain for surface receptors, cells were first stained with LIVE/DEAD viability marker (Thermo Fisher Scientific) to exclude dead cells from analysis. Cells were then resuspended in phospho-flow cytometry buffer [phosphate-buffered saline (PBS) containing 2% FBS, 2 mM EDTA, and 0.1% NaN3] containing human or mouse Fc block for 30 min on ice and various antibody combinations. For phospho-flow cytometry analysis of intracellular signaling pathways, cells were fixed and permeabilized with an eBioscience Foxp3 fixation/permeabilization kit (Thermo Fisher Scientific). During the permeabilization step, cells were incubated with human or mouse Fc block (Miltenyi Biotec) and various antibody combinations. Analysis was conducted using an LSR2 flow cytometer (BD Biosciences).

Antibodies against hCD80 (2D10.4), hCD83 (HB15e), hCD86 (IT2.2), hCD40 (5C3), HLA-DR (LN3), hCD123 (6H6), hCD27 (O323), hCD19 (HIB19), murine CD80 (mCD80) (16-10A1), mCD86 (GL1), mCD40 (1C10), IA/IE (M5.114.15.2), mCD11c (N418), and mB220 (RA3-6B2) were purchased from Thermo Fisher Scientific. Antibodies against BTK pY221 (N35-86), BTK pY551 (24a/BTK), PLCγ2 pY759 (K86-689.37), IRF7 pS477/pS479 (K47-671), P65 pS529 (K10-8895.12.50), P38 pT180/pY182 (36/p38), hCD303 (V24-785), mCD138 (281-2), mIgG2a (R19-15), mIgG2b (R12-3), mIgG1 (A85-1), mIgM (11/41), hCD20 (2H7), and hCD38 (HIT2) were purchased from BD Biosciences.

BCR cross-linking and Ca2+ flux assay with human memory B cells

Purified cells were resuspended in plain RPMI and loaded with Indo-1 acetoxymethyl ester (Thermo Fisher Scientific) at 37°C following the manufacturer’s protocol. After a 45-min incubation, cells were washed with PBS and incubated with inhibitors for 60 min. After additional wash, cells were maintained at 37°C. For BCR cross-linking, cells were stimulated with F(ab)2 goat anti-hIgM/IgG antibody (50 μg/ml; Thermo Fisher Scientific) immediately before acquisition. To study the effects of TLR7 agonism, cells were stimulated with gardiquimod (50 μg/ml) immediately before flow cytometry analysis.

IRAK4 human whole-blood assay

Human whole blood diluted in RPMI 1640 (50% final blood dilution) was incubated with inhibitors for 60 min. Compounds were used at a starting concentration of 20 μM with a 10-point serial dilution. Blood was then stimulated with gardiquimod (5 μg/ml) for 4 hours. Plates were spun down and supernatants assayed for IFN-α2a levels.

In vivo models of lupus

For the pristane-induced lupus model and dosing, a single intraperitoneal injection of 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Sigma-Aldrich) was given to 8-week-old WT or IRAK4-KD female mice. Control mice received the same volume of saline. Body weight and proteinuria were monitored monthly, starting from 4 weeks after pristane injection. Mice were euthanized after 47 weeks. Sera from individual mice were collected throughout the study for evaluation of autoantibodies. At the end of the study, kidneys were collected and examined for lesions consistent with SLE by histology; spleens were collected for gene expression analysis.

In studies involving therapeutic dosing, pristane-challenged C57BL/6 mice were divided into groups and received either IRAK4i vehicle [MCT; by mouth twice a day (PO BID)], IRAK4i (100 mg/kg; PO BID), BTK vehicle (hydroxypropyl methylcellulose; PO BID), BTKi (100 mg/kg; PO BID), IFNR antibody (10 mg/kg, sc; three times per week), or isotype control antibody [10 mg/kg, subcutaneously (sc); three times per week]. Whole blood was collected from animals 2 weeks after receiving pristane and used to evaluate expression of IRGs.

For the imiquimod-accelerated NZB/W_F1 lupus model, 5% imiquimod (generic pharmaceutical grade) was applied topically three times per week for 8 weeks to the right and left ears of IRAK4 WT or IRAK4-KD NZB/W_F1 mice. Animals were monitored and euthanized 8 weeks after imiquimod application.

For proteinuria and survival scoring, proteinuria was determined weekly by colorimetric measurement using dipstick Multistix 10 SG on a Clinitek Status Analyzer (Siemens). Urine protein levels were scored as trace = 0, 30 mg/dl = 1, 100 mg/dl = 2, 300 mg/dl = 3, >300 mg/dl = 4, and death = 5. Progression-free survival was defined as the duration of remission, measured from the first time point when proteinuria was ≤300 mg/dl, or as survival time if there was no proteinuria progression.

Histopathology and immunofluorescence

To evaluate treatment effects on renal pathology, kidneys were formalin-fixed and paraffin-embedded using routine methods. Sections were stained with hematoxylin and eosin (H&E) or periodic acid–Schiff (PAS) and glomerulonephritis, periarteritis, tubulointerstitial nephritis, and pyelitis assessed using a blinded subjective severity scoring system (0 to 3), and groups were compared to the pristane-treated WT reference group using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons. In addition, glomerular cellularity (proliferation index) was quantified on PAS-stained slides by whole-slide digital imaging at (20×) using a NanoZoomer XR scanner (Hamamatsu Photonics) followed by image import into MATLAB (MathWorks, Natick, MA) for analysis. Briefly, 20 randomly selected glomeruli per animal were manually traced and well-defined glomerular nuclei automatically enumerated using color and size criteria. Groups were compared to the pristane-treated WT reference group using analysis of variance (ANOVA) with Holm-Sidak’s multiple comparisons test. Renal IC deposits were detected on formalin-fixed, paraffin-embedded sections by fluorescence staining on an autostainer (Dako Universal Autostainer) using Target antigen retrieval as per the manufacturer’s instructions followed by incubation with fluorchrome-conjugated anti-mouse IgG (Molecular Probes, A21202), IgM (Invitrogen, A21042), or C3 (MP Biomedicals, 55510). Slides were digitally scanned and manual selections of intact renal cortex analyzed in MATLAB for average fluorescent intensity per square micrometer of analyzed cortical area; group means were compared to the pristane-treated WT reference group using ANOVA with Holm-Sidak’s multiple comparisons test.

RNA isolation and quantitative real-time polymerase chain reaction analysis using Fluidigm

Total RNA was extracted from human or murine whole blood using the MagMAX-96 Blood RNA Isolation Kit (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using an iScript cDNA Synthesis kit (Bio-Rad) from 50 ng of RNA per sample. Following gene-specific preamplification steps (Applied Biosystems), gene expression changes were assessed using Fluidigm 96.96 Dynamic Array, according to the manufacturer’s protocol (Fluidigm, South San Francisco, CA). The sample-loaded chips were then run on the BioMark Real Time PCR System using a cycling program of 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. Data were analyzed using BioMark Gene Expression Data software to obtain Ct values. For mouse studies, all gene expression results are expressed as arbitrary units relative to the geometric mean of mouse Gapdh, Hprt1, and Actb as normalizing genes. For human studies, data were normalized to the geometric mean Ct values of HPRT1, GAPDH, and GUSB housekeeping genes and presented as ∆CTs. The TaqMan probes used are listed in table S1.

In vitro whole-blood FcεR/CD63 assay

Whole-blood CD63 assays were performed using the Basotest (Celonic AG) according to the manufacturer’s instructions. Cocktail of anti-CD63/CD123/HLA-DR antibody (H5C6) was purchased from BD Biosciences. Cross-linking goat anti-human IgE secondary antibody (catalog no. H15700) was purchased from Thermo Fisher Scientific.

Serum antibody enzyme-linked immunosorbent assays

The activities of total Ig autoantibodies against nuclear antigens (ANA IgG, IgA, and IgM), nuclear RNP, and Sm antigens were quantified with enzyme-linked immunosorbent assay (ELISA) kits from Alpha Diagnostic International (catalog nos. 5210, 5410, and 5405, respectively) according to the manufacturer’s instructions. To detect activity of specific Ig isotypes for nuclear antigens, secondary goat horseradish peroxidase–conjugated antibodies against IgG1, IgG2b, IgG2c, IgG3, and IgM (Bethyl Laboratories Inc.) were used in combination with the ANA kit by replacing the original secondary antibody provided with the kit. Secondary antibodies were used at a 1:1000 dilution. Serum total Ig isotypes (IgM, IgG1, IgG2a, IgG2b, and IgG3) were detected by multiplex Luminex using Mouse Immunoglobulin Isotyping Magnetic Panel (EMD Millipore). Serum IgG2c isotypes were detected by ELISA with mouse IgG2c ELISA kit (catalog no. E99-136, Bethyl Laboratories Inc).

RNA sequencing analysis

The RNA sequencing (RNA-seq) data from blood isolated from patients with SLE and matched healthy donors were previously published (56) under Gene Expression Omnibus accession number GSE72509. The raw RNA-seq data were processed as previously described (65). Briefly, reads were aligned to the reference human genome (build 38, GRCh38) using the GSNAP algorithm. We used GENCODE basic gene models to define exon boundaries; values within exons were counted to give a per-gene expression value. Normalized reads per kilobase million were generated using the method provided by the HTSeqGenie R package. For visualizing gene expression in a heat map, we added a pseudocount of 0.0005 to the number of reads per kilobase of transcript per million mapped reads (RPKMs) and log2-transformed the data. The signature score for IRAK4-dependent genes (55) was calculated using a method previously reported (66), as implemented by the GSDecon R package ( We used the ISM to group patients into low or high IFN signaling levels (49).

PK analyses and simulations

PK analyses and simulations were performed using Phoenix WinNonlin version 6.4 (Certara USA Inc.). Mean blood concentration–time data (n = 3 per time point) after a single oral dose (100 mg/kg) of either IRAK4i to female C57BL/6 mice or BTKi to male CD-1 mice were used to generate the PK parameters, such as volume of distribution over fraction of dose absorbed (V/F), absorption rate constant (K01), and elimination rate constant (K10), using a one-compartment PK model. The parameters were then used to simulate steady-state blood concentration time profiles on day 14 after multiple doses (PO BID × 14 days) of the inhibitors. Experimental plasma concentration data were collected 12 hours after dose administration on day 14 after twice-a-day oral doses (100 mg/kg) of the inhibitors for 14 days to female C57BL/6 mice.

Western blotting analysis

Equal numbers of cells (10 × 106/ml) were lysed in cell lysis buffer composed of 20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, supplemented with Halt protease and phosphatase inhibitors cocktails (Thermo Fisher Scientific). Cells were lysed on ice for 30 min and centrifuged at 21 000 rcf for 10 min at 4°C. Proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. For Western blot analysis, antibodies to BTK, phospho-BTK (Tyr223), PLCγ2, phospho-PLCγ2 (Tyr1217), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), TBK1, phospho-TBK1 (Ser172), IRF7, phospho-IRF7 (Ser477/Ser479), β-actin, and HSP90 were purchased from Cell Signaling Technology. Antibodies for total and phospho-specific IRAK4 (Thr345/Ser346) were generated in-house.

Kinase selectivity profile

The kinome selectivity of IRAK4 and BTK inhibitors was tested in duplicate at 1 μM against a panel of 220 to 286 active recombinant human kinases using the SelectScreen Profiling Service (Thermo Fisher Scientific). The average percentage inhibition values were reported as previously described (27, 40, 67). The adenosine triphosphate concentrations used in the activity assays were typically within twofold of the experimentally determined apparent Michaelis constant (Km) value for each kinase, whereas the competitive binding tracer concentrations used in the binding assays were generally within threefold of the experimentally determined dissociation constant (Kd) values. The average percentage inhibition values were reported.


Fig. S1. Selectivity of IRAK4 and BTK inhibitors.

Fig. S2. Activity of IRAK4i-2 in whole blood using the gardiquimod stimulation assay.

Fig. S3. FcεR signaling in basophils.

Fig. S4. Inhibition of IFN-α2 production by inhibitors of IRAK4 or BTK.

Fig. S5. Ca2+ mobilization after stimulation of memory B cells with gardiquimod.

Fig. S6. Expression of IRG transcripts in whole blood.

Fig. S7. Expression of IRG transcripts in PBMCs.

Fig. S8. Plasma concentration of inhibitors in pristane-challenged mice.

Fig. S9. IRAK4 protein abundance in IRAK4-KD mice.

Fig. S10. Functional validation of IRAK4-KD mice.

Fig. S11. Ig subclasses in IRAK4-KD pristane-challenged mice.

Fig. S12. Evaluation of splenic plasma cells in IRAK4-KD mice.

Table S1. TaqMan probes used for Fluidigm expression analysis.


Acknowledgments: We are grateful to C. Jones III, S. Saturnio, and D. Dunlap for assistance with histopathology and immunofluorescence analysis. Funding: All studies were funded by Genentech. Author contributions: C.A.C., E.V., A.F.S., R.F., S.K., K.S., S.S.-B., A.P.-M., V.W.C.L., A.H., A.K., Y.S., V.R.-C., K.B., D.X., R.P., D.V., N.G., A.L., J.R.K., M.J.T., and A.A.Z. contributed to the execution of experiments, generation of data, and/or designing of research in the manuscript. C.A.C. spearheaded the execution and design of the mechanistic in vitro studies including pDC, B cell, and macrophage analysis. E.S., Z.H., C.L.E., W.P.L., H.D.B., B.S.M., C.A.C., and A.A.Z. contributed to the execution and/or design of the in vivo studies. J.D., S.D., M.C.B., and J.R.K. contributed to the design and generation of IRAK4i-2 and synthesized IRAK4i-1. J.J.C. and W.B.Y. designed and generated the BTK inhibitor. J.E.-A., C.D.A., and A.A.Z. contributed to the generation and/or interpretation of histology data. J.A.H. and A.A.Z. contributed to the analysis and presentation of bioinformatic data. J.P. contributed to the analysis, generation, and presentation of the pharmacokinetic data. L.T., E.H., S.W., A.W., M.R.-G., and A.A.Z. contributed to the design and generation of IRAK4-KD mice. M.D., C.D.C., and V.A. contributed to the maintenance and care of animal colonies. All authors read and approved the manuscript. C.A.C. and A.A.Z. prepared and revised the manuscript with input from all authors. A.A.Z. conceived and coordinated the research. Competing interests: The authors were all employees of Genentech at the time of this study. Z.H. is an employee of Amgen. C.L.E. is an employee of AstraZeneca. N.G. is an employee of DiCE Molecules. M.C.B. is an employee of the Janssen Pharmaceutical Companies of Johnson & Johnson. A.A.Z. is currently an employee of TRex Bio. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

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