SCIMP is a transmembrane adaptor for TLR4 in macrophages

Examination of pTRAP family member expression across immune and non-immune cell populations highlighted the marked expression of SCIMP in primary mouse bone marrow-derived macrophages (BMM), as well as in the RAW264.7 and WR19M macrophage-like cell lines, whereas it was not detectable in the non-myeloid cells that were screened (Fig. 1a). No other pTRAP displays this selective expression pattern (Supplementary Fig. 1a), and we were drawn to further investigate possible SCIMP functions in macrophages. The cellular localization of SCIMP was examined by immunostaining in primary macrophages (BMM; Supplementary Fig. 1b) and in RAW264.7 cells (Fig. 1b). In both cases, SCIMP staining was apparent on intracellular punctate membranes, but was particularly prominent on the macrophage plasma membrane, where it was further concentrated in cell surface projections, notably the filopodia and ruffles of some cells. This localization was replicated by expression of green fluorescent protein (GFP)-SCIMP (Fig. 1c), and at an ultrastructural, cryo-EM level, immunogold labelling of GFP clearly depicts GFP-SCIMP labelling on the plasma membrane at sites of protrusions and ruffles (Fig. 1d). Quantification of the gold labelling shows SCIMP to be enriched ∼4 fold in ruffles compared with other stretches of plasma membrane (Fig. 1d). Both the dorsal ruffles and filopodia of macrophages are cholesterol-rich membranes replete with lipid raft microdomains, and these sites are also enriched with immune receptors, including some TLRs10,22,23. Thus SCIMP at these sites is positioned to participate in pathogen detection and/or receptor-mediated activation of macrophages.

Figure 1: Immune-restricted SCIMP is a TLR4-associated cell surface protein enriched in microdomains. (a) mRNA expression of SCIMP was assessed in the indicated fibroblast, lymphoid and myeloid cell lines. Data represents mean+s.e.m. (n=3). (b) Immunostaining of endogenous SCIMP in LPS-activated RAW264.7 cells (green) on filopodia; cells were co-stained with phalloidin (red) and 4′,6-Diamidino-2′-phenylindole dihydrochloride (DAPI; blue). (c) Fluorescent imaging of LPS-treated (30 min) RAW264.7 cells transiently transfected with SCIMP-GFP (green). The cells were co-stained with DAPI (blue). (d) Immunogold labelling on cryo-EM sections of RAW264.7 cells stably expressing GFP-SCIMP. GFP labelling ruffles at the cell surface. N=nucleus. Gold particles on ruffle or filopodia membranes versus other stretches of plasma membrane were counted (n=5 cells). Significance was assessed using the Student’s t-test (**P<0.01). (e) GST-SCIMP-T1 coupled to GSH-Sepharose was used for pull-downs from LPS-activated RAW264.7 cell extracts. Bound proteins were eluted by a protease cleavage elution method and separated by SDS–PAGE. Excised bands were identified by liquid chromatography and mass spectrometry (LC/MS/MS). A band at ∼100 kDa, absent from the GST control, was identified as TLR4. (f) List of the top hits from the LC/MS/MS analysis of SCIMP-GST pull downs. Data in a–f are representative of at least three independent experiments. Scale bars in b–d represent 10 μm, 20 μm and 10 nm, respectively. Full size image

To determine whether this adaptor is associated with specific receptor pathways, we performed unbiased screens to identify possible SCIMP binding partners in activated macrophages. GST-SCIMP was used for pull-down assays, along with a protease cleavage and elution strategy24, to optimize capture of genuine binding partners from lysates of activated macrophages. LC/MS/MS analysis of SCIMP-bound proteins in LPS-activated macrophages identified the SFK Lyn and the adaptor protein Grb2 (Fig. 1e), which were previously identified as SCIMP partners in B cells18. In addition, one of the top hits identified from this analysis was TLR4 (Fig. 1e,f and Supplementary Fig. 1c), the prototypical TLR and a previously unidentified binding partner of SCIMP. To verify this association in cells, we performed immunoprecipitation (with a V5 antibody) from macrophages expressing V5-tagged SCIMP. Consistent with the LC/MS/MS data (Fig. 1f), immunoblotting confirmed the co-immunoprecipitation of Lyn and Grb2, which were constitutively bound to SCIMP (Fig. 2a). In addition, TLR4 co-immunoprecipitated with SCIMP, and in this case, the interaction was strictly LPS-induced (Fig. 2a), occurring rapidly after ligand activation. A phospho-specific antibody recognizing active SFKs (pY416 Src) reveals that LPS acutely and transiently activates an SFK in this complex (Fig. 2a), which is consistent with, and likely to be, Lyn. Thus, we reveal SCIMP as a component of LPS-activated TLR4 complexes in macrophages.

Figure 2: SCIMP is recruited to TLR4 in macrophages on LPS activation. (a) V5-labelled SCIMP was stably expressed in RAW264.7 macrophages, cells were treated with LPS, and at the indicated times, the V5 antibody was used for immunoprecipitation. Samples were probed for TLR4, pSFK(Y416), Lyn, Grb2 and V5-SCIMP. (b) Confocal imaging of LPS-treated (30 min) RAW264.7 cells transiently co-expressing TLR4-HA (green) and SCIMP-V5 (red), with phalloidin (blue). TLR4 and SCIMP are colocalized (white) on dorsal surface ruffles, as shown for separate channels in the Z projections. Data in a and b are representative of three independent experiments. The scale bar in b represents 10 μm. Full size image

pTRAPs are typically associated with cholesterol-rich membrane microdomains and detergent-resistant fractions. For instance, SCIMP is enriched in tetraspanin-enriched microdomains in B cells18. Here we show that in LPS-treated macrophages, SCIMP and Lyn are also enriched in detergent-resistant membrane fractions, as marked by flotillin (Supplementary Fig. 1d). This is consistent with the nature of pTRAPs and with the localization of SCIMP in cholesterol-rich filopodia and ruffles (Fig. 1b–d). This is also significant for the association of SCIMP with TLR4, which is also in lipid raft domains and detergent-resistant fractions6. However, the apparent interaction between SCIMP and TLR4 in activated macrophages (Fig. 2a) was not merely a consequence of SCIMP being partitioned into LPS-induced microdomains, since there was no non-specific pull-down of the lipid raft marker flotillin in immunoprecipitates (Supplementary Fig. 1e). Finally, we sought verification that SCIMP and TLR4 colocalize in cells by co-expressing HA-tagged TLR4 and V5-SCIMP in activated macrophages. We have previously shown that TLR4 is concentrated in dorsal ruffles, which act as sites for initiation of signalling in LPS-activated macrophages23. Here the joint labelling depicts a concentration of HA-TLR4 in these actin-rich ruffle membranes where it colocalizes with V5-SCIMP (Fig. 2b). Together these results indicate that SCIMP and LPS-activated TLR4 are co-located in plasma membrane domains that are also signalling-competent locales.

SCIMP exerts selective effects on macrophage TLR responses

Given the contributions of other pTRAPs to immune receptor signalling in T and B cells, we next examined a role for SCIMP as a signalling adaptor in its guise as a direct, LPS-induced binding partner of TLR4. The LPS-mediated phosphorylation of ERK, p38 and JNK MAPKs is impaired after small interfering RNA (siRNA) silencing of SCIMP in primary macrophages (BMM). SCIMP-silenced cells also have a modest but significant impairment in LPS-triggered degradation of IκB (Fig. 3a,b). Interestingly, this effect on signalling responses is very transient; for example, SCIMP silencing impairs p38 MAPK activation at 30 min, but not at 60 min, post-LPS, and by 120 min most of the signalling responses are unaffected by SCIMP (Fig. 3a,b and Supplementary Fig. 2a). The transient effect of SCIMP on TLR signalling responses was not explored further, but these findings suggest that SCIMP is likely to have partial or selective effects on downstream biological responses. To test this, we examined LPS-induced cytokine outputs in SCIMP-silenced BMM. Indeed, after depletion of SCIMP with two specific siRNAs, the synthesis and secretion of the proinflammatory cytokines interleukin 6 (IL-6) and IL-12p40 are substantially reduced, while remarkably, tumor necrosis factor (TNF) is unaffected (Fig. 3c,d). These effects are discriminating for TLR signalling, as TNF-inducible IL-6 and IL-12p40 production were unaffected by SCIMP silencing in BMM (Fig. 3e). Further verifying this response, we find that retroviral transduction and overexpression of SCIMP in BMM selectively amplifies LPS-inducible production of IL-12p40, but not of TNF (Supplementary Fig. 2b). Examination of additional cytokines further attests to this selectivity, since levels of the TLR-inducible cytokines IFN-β, IL-10 and IL-12p70 are all unaffected by SCIMP silencing in BMM (Supplementary Fig. 2c). SCIMP additionally does not regulate inducible expression of specific MyD88-dependent (for example, Il1β and Ccl7) or MyD88-independent (for example, Ifnβ and Cxcl10) TLR4 target genes (Supplementary Fig. 2d).

Figure 3: SCIMP is required for a subset of proinflammatory cytokine responses in macrophages. (a) SCIMP was silenced by siRNA in BMM. Representative immunoblots showing levels of SCIMP, IκB, phospho-JNK, phospho-p38 and phospho-ERK1/2 at 0, 30 and 60 min post-LPS stimulation. (b) Relative chemiluminescence of IκB, phospho-JNK, phospho-ERK1/2 and phospho-p38 was assessed at 30 min and 60 min post-LPS stimulation in SCIMP-silenced BMM. Graphs represent pooled data from n=3 experiments (mean+s.e.m.). (c,d) SCIMP silencing in BMM reduces proinflammatory cytokine production at the mRNA (c) and protein (d) level. Levels of individual mRNAs, relative to Hprt, at 4 h post-LPS stimulation were assessed by qPCR (n=4 experiments), and levels of secreted cytokines at 24 h post-LPS stimulation were assessed by enzyme-linked immunosorbent assay (ELISA; n=4 experiments). Graphs depict mean+s.e.m. (e) IL-6 and IL-12p40 protein levels were assessed by ELISA in SCIMP-silenced BMM treated with LPS or TNF for 24 h. Data is representative of two independent experiments. Graphs depict mean+range from technical repeats (n=2). Data in (a–d) are representative of, or combined from, at least 3 independent experiments. For (b–d), significance was assessed using one-way ANOVA (*P<0.05 and **P<0.01; NS, not significant). Full size image

Having examined the effects of SCIMP depletion on TLR4-triggered cytokine outputs, we next extended our analysis to examine possible roles downstream of other TLRs. siRNA silencing of SCIMP in BMM reduced the inducible production of IL-6 and IL-12p40, but not TNF, in response to agonists of TLR3 (Poly(I:C)), TLR7 (imiquimod) and TLR1/2 (Pam3CSK4) (Supplementary Fig. 3a–c). As an alternative and complementary approach, we also assessed the effect of CRISPR/Cas9-mediated SCIMP deletion on TLR responses in RAW264.7 cells. As expected, deletion of SCIMP in multiple clonal cell lines (Supplementary Fig. 4a) did not impair either LPS-inducible TNF production (Supplementary Fig. 4b) or LPS-/Pam3CSK4-induced TNF secretion (Supplementary Fig. 4c,d). However, the secretion of IL-6 at the behest of LPS-activated TLR4 or Pam3CSK4-activated TLR1/2 was significantly reduced (Supplementary Fig. 4c,d). The low levels of LPS-inducible IL-12p40 secretion were also reduced, whilst Pam3CSK4-inducible IL-12p40 secretion from RAW264.7 cells could not be detected. Thus, CRISPR/Cas9-mediated SCIMP deletion corroborated the results obtained with siRNA depletion of SCIMP in BMM. Taken together, these findings show that, in the context of an otherwise broad cytokine programme induced by TLRs, the adaptor SCIMP is responsible for driving a uniquely selective subset of key proinflammatory cytokines, namely IL-6 and IL-12p40. To uncover the mechanism for this selective cytokine regulation, we next dissected the interaction between SCIMP and TLR4 in more detail.

Phosphorylation of SCIMP at Y96 enhances binding to TLR4

The canonical model for adaptor interactions with TLRs is via direct, homotypic interactions between the TIR domains of TLRs and TIR domain-containing adaptor proteins (MAL, MyD88, TRIF, TRAM and SARM)4,5,6. Despite SCIMP lacking a TIR domain, our pull-down experiments suggested at least a close association between SCIMP and TLR4. Like other TRAPs, SCIMP has a short extracellular domain and a long cytoplasmic tail. The intracellular domain contains a proline-rich domain (PRD) and several tyrosine residues that can be phosphorylated by Lyn (refs 11, 18). Two truncated forms of the SCIMP cytoplasmic tail were produced (Fig. 4a), T1 (entire intracellular region: amino acids 29–150) and T2 (C terminal region: amino acids 93–150). The cytoplasmic TIR domain (amino acids 670–835) of TLR4 was also produced, and in in vitro pull-downs using recombinant proteins, we find that both GST-SCIMP-T1 and T2 bind to the His-TLR4-TIR domain (Fig. 4b). Thus, the C-terminal TLR4-TIR domain and the C-terminal region of SCIMP (amino acids 93–150) can directly interact. To confirm this binding in cell lysates, GST-SCIMP T1 and T2 were used for pull-downs from LPS-activated macrophage extracts. SCIMP-T1, and to a lesser extent SCIMP-T2, pulled down endogenous TLR4 (Fig. 4c). Importantly, neither GST-SCIMP T1 nor T2 interacted with endogenous TLR4 in extracts of unstimulated macrophages, whereas GST-SCIMP T1 pull-downs show constitutive binding of Lyn (Supplementary Fig. 5a). These data confirm that, whereas SCIMP interacts constitutively with Lyn in macrophages, its interaction with TLR4 is agonist-induced. Moreover, the pull-down experiments (Fig. 4c) confirm binding of TLR4 to the C-terminus of SCIMP, but also suggest that other regions of SCIMP, in addition to S93-F150, contribute to the stronger binding offered by the longer T1 construct under in-cell conditions. Hence, analysis of the interaction between recombinant SCIMP-T1 and recombinant TLR4-TIR (Fig. 4b) is unlikely to capture all of the factors involved in the interactions between these proteins within macrophages. As expected, SCIMP-T2, which lacks the PRD, does not interact with Lyn (Fig. 4c and Supplementary Fig. 5a). Finally, the in vitro interaction between SCIMP and TLR4 was analysed in fluorescence binding assays using recombinant, fluorescently labelled SCIMP-T1 and TLR4-TIR (Fig. 4d, left panel); these experiments show that the direct in vitro interaction occurs with moderately high affinity (212 nM; Fig. 4d, right panel). Based on these findings, we reveal novel binding, through an unconventional mode, between a non-TIR adaptor and the TIR domain of TLR4.

Figure 4: SCIMP interacts directly with TLR4. (a) A schematic diagram depicting T1, T2 or full-length (FL) murine SCIMP with transmembrane domain (TMD), PRD, tyrosine residues 58, 96 and 120 (green bars) and N- and C-terminal amino acids. (b) Bacterially expressed GST tag alone, or GST-tagged SCIMP T1 or T2 was used to pull-down recombinant His-TLR4-TIR. (c) GST-SCIMP T1 or T2 fusion proteins were used in GST pull downs with extracts from LPS-activated RAW264.7 cells and probed for TLR4 and Lyn. (d) Titration of fluorescently labelled SCIMP-T1 with TLR4-TIR (left panel), with Kd (212 nM) determined by curve fit analysis (right panel). Data in b and c are representative of three independent experiments. Full size image

As the SCIMP-TLR4 interaction is not only direct (Fig. 4b,d), but also ligand-dependent (Fig. 2a), we next examined whether tyrosine phosphorylation of SCIMP regulates binding. Three tyrosines in the cytoplasmic domain are conserved between human and mouse SCIMP (Fig. 4a). Phosphorylation-deficient mutagenesis of each of these residues reveals that only the Y96F mutation attenuates binding of GST-SCIMP-T1 to endogenous TLR4 in extracts from LPS-activated macrophages (Fig. 5a). By contrast, phosphorylation of these tyrosines is not required for binding to Lyn, which binds to the PRD of SCIMP18. Mutation of a residue (W95) adjacent to Y96 in GST-SCIMP-T1 also substantially reduces binding to recombinant His-TLR4-TIR (Fig. 5b), further highlighting the importance of this region of SCIMP for its interaction with TLR4. In contrast, testing the capacity of GST-SCIMP mutants to bind recombinant His-TLR4-TIR (rather than TLR4 from LPS-activated macrophages) shows the (unphosphorylated) Y96F SCIMP mutant still binds TLR4 (Fig. 5b), thus indicating that phosphorylation is not required for in vitro binding. These data therefore suggest that extracts from LPS-activated macrophages trigger phosphorylation of GST-SCIMP at Y96, enhancing its binding to endogenous TLR4. Hence, the affinity of the interaction between phosphorylated SCIMP and TLR4 in LPS-activated cells is likely to be much greater than that observed when using recombinant proteins (Fig. 4d). To directly address this, we performed in vitro binding assays, using recombinant TLR4-TIR domain and short SCIMP-derived peptides (amino acids 90–107) containing either unphosphorylated or phosphorylated Y96. These experiments show that the SCIMP-derived peptide-containing phosphorylated Y96 binds to TLR4 with much greater affinity than the unphosphorylated peptide (Fig. 5c,d). Finally, the requirement for SCIMP Y96 phosphorylation for in vivo binding to TLR4 was tested in cells stably expressing full-length proteins. As previously observed (Fig. 2a), LPS triggers an association between SCIMP and TLR4, however this does not occur with the SCIMP-Y96F mutant (Fig. 5e). Collectively, the above data define SCIMP as a new direct binding partner and non-TIR adaptor for TLR4, and delineate a new recruitment mechanism, in which tyrosine phosphorylation of SCIMP, most likely by Lyn, enables it to directly bind to the TLR4 TIR domain. The consequences of SCIMP binding to TLR4, and the direct involvement of Lyn in these processes, were next examined.

Figure 5: Phosphorylation of SCIMP at Y96 enhances binding to TLR4. (a) Pull-down assays using LPS-activated RAW264.7 cell lysates and GST-tagged tyrosine to phenylalanine (Y to F) mutants of SCIMP-T1. SCIMP interactions with either TLR4 or Lyn were assessed by immunoblotting. (b) GST pull-down assays were performed using GST-SCIMP mutants and recombinant His-tagged TLR4-TIR domain, and binding was evaluated using immunoblotting against His-TLR4-TIR (upper panel). Quantification of immunoblots was performed using densitometry. Data are combined from two independent experiments (mean+range, lower panel). (c) Phosphorylation at Y96 enables SCIMP to bind to TLR4. Titration of fluorescently labelled p-Y96-containing (red) or Y96-containing (green) SCIMP-derived peptides (aa 90–107) with TLR4-TIR. Inset shows fluorescence recovery over a longer time course with unlabelled p-Y96 peptide. (d) The Kd (159 nM and>2.5 μM for p-Y96- and Y96-containing peptides, respectively) was determined by curve fit analysis. (e) SCIMP phosphorylation at Y96 is required for in vivo TLR4 binding. RAW264.7 cells stably expressing wild type (WT) or Y96F SCIMP were used to assess the agonist-induced SCIMP-TLR4 interaction, by co-immunoprecipitation of SCIMP followed by immunoblotting for TLR4 and SCIMP (left). The level of TLR4 pull-down was quantified relative to SCIMP levels (right) to correct for minor differences in levels of WT versus Y96F SCIMP expression (mean+s.e.m.). Data in a are representative of three independent experiments, data in b are representative of (top) or combined from (bottom) n=2 independent experiments, and data in e are representative of three independent experiments (left) or combined from n=3 independent experiments (right). Full size image

SCIMP is required for tyrosine phosphorylation of TLR4

The interaction of SCIMP with TLR4 likely underpins SCIMP-mediated regulation of cytokine production, but several possible mechanisms could account for this effect. For instance, LPS induces the endocytosis of surface-activated TLR4, which is critical for eliciting specific cytokine outputs generated by endosomal signalling events that are distinct from those at the cell surface25,26. To gauge whether SCIMP alters TLR4 endocytosis, the surface expression and ligand-dependent internalization of TLR4 were measured in BMM after siRNA silencing of SCIMP. In these experiments, both basal TLR4 expression and endocytosis of TLR4 were unaffected (Supplementary Fig. 5b,c). We next investigated whether there may be interplay between SCIMP and TIR-containing adaptors. Here we find that, although SCIMP interacts with TLR4 on LPS activation, it does not interact with the proximal TLR4 adaptor MAL, under the same conditions (Supplementary Fig. 5d). Furthermore, SCIMP is still recruited to TLR4 in the absence of MyD88 (Supplementary Fig. 5e). These findings further highlight the distinct nature of the SCIMP-TLR4 interaction, and suggest that other mechanisms likely account for SCIMP-dependent, TLR4-inducible cytokine production.

TLR4 (ref. 27), as well as several other TLRs including TLR2 (refs 28, 29, 30), TLR3 (ref. 31) and TLR8 (ref. 32), are tyrosine phosphorylated in a ligand-dependent manner. Given that SCIMP is required for responses to multiple TLRs (Supplementary Figs 3a–c and 4c,d) and since other TRAPs act as adaptors linking SFKs to receptors for phosphorylation11, we considered the possibility that SCIMP may be recruited to facilitate the ligand-dependent phosphorylation of TLR4. Indeed, we find that LPS-induced tyrosine phosphorylation of TLR4 is dramatically reduced on siRNA silencing of SCIMP in primary macrophages (Fig. 6a). The opposite effect is apparent when SCIMP is overexpressed in RAW264.7 cells, with markedly increased levels of LPS-induced phospho-TLR4 being observed (Fig. 6b). Thus, the direct interaction of SCIMP with TLR4 facilitates tyrosine phosphorylation of TLR4 in response to LPS, offering a possible mechanism for delineating selective cytokine outputs. To determine whether the SCIMP-TLR4 interaction is indeed required for LPS-inducible, SCIMP-dependent cytokine production, we examined the functional capacity of the SCIMP Y96F and W95A mutants, which do not interact with TLR4 in cells or in vitro, respectively (Fig. 5a,b,e). As predicted, these mutants are unable to amplify LPS-inducible production of the SCIMP-dependent cytokine IL-12p40 when overexpressed in primary macrophages (BMM), whereas wild-type SCIMP selectively promotes LPS-inducible IL-12p40 but not TNF production (Fig. 6c). Interestingly, there was a modest but statistically significant decrease in LPS-inducible IL-12p40 production for the Y96F mutant versus empty vector. This could relate to the fact that, although this mutant is unable to be phosphorylated, it can still bind to Lyn (Fig. 5a) and thus may have a dominant-negative effect on this signalling response.

Figure 6: SCIMP drives cytokine selectivity by scaffolding the SFK Lyn for TLR4 phosphorylation. (a) siRNA-mediated SCIMP knockdown in BMM reduces tyrosine phosphorylation of TLR4. After SCIMP silencing (lysates, bottom panels), cells were stimulated for 30 min with LPS, TLR4 was immunoprecipitated and blots were probed for anti-phospho-tyrosine. (b) SCIMP overexpression increases tyrosine phosphorylation of TLR4. RAW264.7 cells stably transfected with empty vector or SCIMP were treated with 30 min with LPS, after which TLR4 was immunoprecipitated and tyrosine phosphorylation was assessed by immunoblotting. (c) IL-12p40 and TNF cytokine production was assessed in primary macrophages (BMM), retrovirally transduced with empty vector (EV), wild-type SCIMP (WT), Y96F SCIMP or W95A SCIMP, at 24 h post-LPS stimulation (mean+s.e.m.). SCIMP protein expression in transduced BMM is shown in the panel on the right. (d) The Src kinase inhibitor SU6656 impairs SCIMP tyrosine phosphorylation and its interaction with TLR4, as assessed by SCIMP immunoprecipitation experiments from lysates of RAW264.7 cells stimulated with LPS for 5 min. (e) Lyn is essential for LPS-induced SCIMP phosphorylation, as well as the interaction between SCIMP and TLR4 as assessed by SCIMP immunoprecipitation from lysates of WT and Lyn−/− BMM stimulated with LPS for 5 min. (f) BMM were pre-treated with SU6656 for 30 min and then stimulated with LPS for 4 h or 8 h. Secreted cytokines (IL-12p40, IL-6 and TNF) were measured by enzyme-linked immunosorbent assay (ELISA; mean+s.e.m.). (g) The effect of SU6656 on cell death in LPS-activated BMM was assessed using the LDH release assay. LDH in culture supernatants was measured as a percentage of total cellular LDH at 8 h post-LPS stimulation from the same samples as in f (mean+s.e.m.). (h) Levels of LPS-induced cytokines (IL-6, IL-12p40 and TNF; ELISA) in culture supernatants from wild type (WT) and Lyn−/− (KO) BMM were assessed at 24 h post-stimulation (mean+s.e.m.). All experiments were repeated at least 3 times, and all graphical data are pooled from at least n=3 independent experiments (mean+s.e.m.). Significance was assessed using one-way ANOVA in c, f and h (*P<0.05 and **P<0.01). Full size image

Lyn phosphorylates SCIMP to enable an interaction with TLR4

In B cells, MHC-II cross-linking triggers SCIMP phosphorylation18. In macrophages treated with either LPS or Pam3CSK4, SCIMP was rapidly phosphorylated (Supplementary Fig. 5f), consistent with the requirement for this pTRAP in cytokine responses downstream of multiple TLRs (Supplementary Figs 3a–c and 4c,d). Although SCIMP is constitutively associated with Lyn in macrophages (Fig. 2a), the role of this SFK in TLR signalling is complex, and somewhat controversial. It has been reported to both inhibit and enhance TLR responses in different cell types and in different in vivo settings33,34,35,36. We therefore more directly assessed the involvement of Lyn in the TLR4-SCIMP pathway. In support of a model in which activated Lyn phosphorylates SCIMP to enable an interaction with TLR4, we found that a SFK inhibitor impairs LPS-induced phosphorylation of endogenous SCIMP, as well as the interaction between SCIMP and TLR4 (Fig. 6d). Moreover, both SCIMP phosphorylation and its inducible association with TLR4 were ablated in BMM from Lyn−/− mice (Fig. 6e). Thus, both pharmacological and genetic approaches posit Lyn as an essential upstream kinase for the agonist-induced association between SCIMP and TLRs. A further prediction of this model is that, if SCIMP is unable to bind to TLR4 for its tyrosine phosphorylation, TLR4 signalling for production of SCIMP-dependent cytokines will be impaired. Accordingly, a SFK inhibitor selectively reduces IL-6 and IL-12p40, but not TNF, production from BMM (Fig. 6f), without affecting cell viability (Fig. 6g). Similarly, Lyn−/− BMM were compromised for LPS-inducible IL-6 and IL-12p40 production (Fig. 6h). In this case, TNF production was also somewhat reduced, which likely reflects a broader role for Lyn beyond its function in the SCIMP pathway. We thus conclude that Lyn-mediated phosphorylation of immune-restricted SCIMP at Y96 enables an unconventional interaction with the TIR domain of LPS-activated TLR4. SCIMP-mediated phosphorylation of TLR4 then serves as a mechanism to initiate a transient proinflammatory signalling code that selectively produces the inflammatory mediators IL-6 and IL-12p40 in the context of acute inflammatory responses. Given that several TLRs are tyrosine phosphorylated27,28,31,32, and that SCIMP directs similar cytokine outputs from multiple TLRs (Supplementary Figs 3a–c and 4c,d), this pTRAP is likely to control other TLR responses through a shared mechanism. Overall, SCIMP is thus revealed as a key, agonist-inducible signalling adaptor and scaffold for phosphorylation of TLRs to enable specific proinflammatory cytokine responses.