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Outside-in signals from β2 integrins require immunoreceptor tyrosine-based activation motif adapters in myeloid cells that are known to dampen TLR responses. However, the relationship between β2 integrins and TLR regulation is unclear. Here we show that deficiency in β2 integrins (Itgb2−/−) causes hyperresponsiveness to TLR stimulation, demonstrating that β2 integrins inhibit signals downstream of TLR ligation. Itgb2−/− macrophages and dendritic cells produced more IL-12 and IL-6 than WT cells when stimulated with TLR agonists and Itgb2−/− mice produced more inflammatory cytokines than WT mice when injected with LPS. TLR hypersensitivity was not the result of insufficient ABIN-3, A20, Hes-1, or IRAK-M expression, nor to changes in IL-10 production or sensitivity, though Itgb2−/− macrophages had reduced p38 MAPK phosphorylation after LPS treatment. Furthermore, a Cbl-b-MyD88 regulatory axis is not required for TLR inhibition in macrophages. Instead, Itgb2−/- macrophages presented with enhanced IκBα degradation, leading to changes in NF-κB recruitment to target promoters and elevated cytokine, chemokine, and anti-apoptotic gene transcription. Thus, β2 integrins limit TLR signaling by inhibiting NF-κB pathway activation and promoting p38 MAPK activation, thereby fine-tuning TLR-induced inflammatory responses.
Innate immune cell activation depends on the activity of Toll-like receptors (TLRs) that bind conserved molecular features expressed on invading pathogens . Upon encountering microbes, macrophages and dendritic cells (DCs) respond to TLR stimulation by inducing antimicrobial and antiviral programs that result in the rapid synthesis and secretion of inflammatory cytokines and type I interferons. In turn, this potent response must be restrained to spare host tissues from the deleterious effects of exaggerated inflammation. This is accomplished by a variety of inhibitory mechanisms, including cytoplasmic effectors that block TLR signaling directly as well as secreted negative regulators, which work together to limit the severity of the immune response .
Although originally considered as an archetypal cell activation pathway, signals through immunoreceptor tyrosine-based activation motifs (ITAMs) display functional heterogeneity and have been recently appreciated to cross-inhibit TLR responses [3, 4]. ITAM signaling in myeloid cells is mediated by the ITAM-containing molecules DAP12 and FcRγ, which act as signaling adapters for an extensive collection of cell surface receptors [5-7]. Following ligand binding by paired receptors, ITAM signaling via DAP12 and FcRγ in myeloid cells proximally activates Src-family kinases and Syk kinase to enable downstream signals that are predominantly associated with cellular activation, including inducing NF-κB and MAPK pathways, and prompting the release of intracellular Ca2+ stores . However, depending on the identity of the associated receptor and other undefined parameters, ITAM-based signaling can also induce inhibitory responses. For example, triggering of the DAP12-coupled TREM-2 receptor can dampen TLR activation in macrophages . In addition, TREM-2 and/or DAP12-deficient macrophages and DCs produce more inflammatory cytokines in response to TLR stimulation [9-12], demonstrating that these adapter molecules can transduce signals attenuating TLR activation.
During an inflammatory response, leukocytes in the blood adhere to the activated vascular endothelium through the use of integrins. In particular, members of the β2 integrin family facilitate leukocyte firm adhesion, thereby allowing for cell extravasation into the tissues . In doing so, β2 integrins not only mediate cell migration, but also influence cell functions through signals transmitted through β2 integrin activation. This “outside-in” signaling pathway requires ITAM signals from DAP12 and FcRγ, and also involves early effectors such as the Src family kinases and Syk in neutrophils and macrophages [14, 15]. Because β2 integrins signal through ITAM adapters in myeloid cells, we hypothesized that β2 integrin signaling may also inhibit TLR responses. There have been conflicting reports in the literature regarding the influence of β2 integrin signaling on TLRs, with some studies demonstrating that β2 integrins can promote TLR-induced inflammation [16-18], whereas others have reported negative roles for these integrins in TLR responses [19, 20]. Therefore, the nature in which β2 integrins interface with TLR activation and cytokine secretion is complex and unclear.
To better define the contribution of β2 integrins to regulation of TLR signaling, we have examined inflammatory responses in the absence of all β2 integrins. Here we demonstrate that deletion of all β2 integrins rendered myeloid cells hypersensitive to TLR stimulation in vitro and in vivo, showing an inhibitory role for β2 integrins in TLR responses. Furthermore, we examined potential direct and indirect mechanisms by which β2 integrins caused this inhibition, and found that β2 integrins have a direct effect on IκBα degradation that was pronounced in β2 integrin-deficient cells through both early and late phases of TLR stimulation, thus implicating β2 integrin signals in inhibiting NF-κB pathway activation to calibrate inflammatory responses.
β2 integrin-deficient macrophages hyper-respond to TLR stimulation
The four β2 integrins, LFA-1 (lymphocyte function-associated antigen 1, αLβ2), Mac-1 (macrophage-1 antigen, αMβ2), CR4 (αXβ2), and CD11d-CD18 (αDβ2) are heterodimers that consist of distinct CD11 alpha subunits in association with the common beta chain, CD18 (β2), which is encoded by the Itgb2 gene . To examine whether β2 integrin signaling regulates TLR responses, we compared the cytokine secretion profiles of bone marrow-derived (BM-derived) macrophages from wild-type (WT) and Itgb2−/− mice, which are deficient in CD18 and thus are unable to express any of the β2 integrins on the cell surface (Supporting Information Fig. 1A) . Despite the inability of Itgb2−/− BM-derived macrophages to express Mac-1, these cells exhibited surface F4/80 expression and upregulated MHC II in response to IFN-γ treatment (Supporting Information Fig. 1A and B), demonstrating that they were bona fide macrophages. Furthermore, β2 integrin-deficient macrophages exhibited similar or slightly lower levels of cell surface TLR2, TLR4, and Dectin-1 protein and TLR9 mRNA (Supporting Information Fig. 1C and D).
To determine how β2 integrin signals influence TLR activity, we stimulated Itgb2−/− BM-derived macrophages with a panel of TLR agonists, including LPS (TLR4), CpG B DNA (TLR9), and zymosan (TLR2). Zymosan is a complex yeast particle that, in addition to signaling through TLR2, also signals through Dectin-1. After 24 h of activation, Itgb2−/− BM-derived macrophages secreted significantly more IL-12 p40 than did WT control cells (Fig. 1A and Supporting Information Fig. 2A). To address whether this IL-12 p40 was participating in IL-12 p70 or IL-23 production, we assessed the induction of mRNA encoding IL-12 p35 and IL-23 p19. Itgb2−/− macrophages synthesized enhanced levels of IL-12 p35 mRNA in response to LPS when compared to WT controls, but comparable levels of IL-23 p19 mRNA (Supporting Information Fig. 2B), suggesting that β2 integrin deletion enhances IL-12, but not IL-23, production in macrophages. Similarly, we also noted elevated IL-6 secretion in Itgb2−/− macrophages in response to TLR4, TLR9, and TLR2/Dectin-1 stimulation, though this did not reach statistical significance through multiple experiments (Fig. 1A). TNF secretion was similar in Itgb2−/− macrophages to that from WT cells (Fig. 1A and Supporting Information Fig. 2A).
We investigated the kinetics of inflammatory cytokine secretion after LPS treatment and found that the induction kinetics for IL-12 p40 and TNF release were similar between Itgb2−/− and WT macrophages (Fig. 1B and Supporting Information Fig. 2C). Yet, after 12 h of stimulation, the magnitude of IL-12 p40 secretion was greatly enhanced in Itgb2−/− macrophages as compared with levels in WT macrophages, while TNF production remained unchanged between both macrophage populations throughout the course of the experiment (Fig. 1B and Supporting Information Fig. 2C). To ascertain whether the increase in cytokine levels from Itgb2−/− macrophages was due to β2 integrins controlling cytokine secretion, the synthesis of IL-12 p40 and TNF was assessed by intracellular cytokine staining. We observed a larger population of IL-12 p40-producing macrophages in the absence of β2 integrins, such that at 4 h after stimulation the percentage of Itgb2−/− IL-12 p40-positive cells was approximately twice that of WT controls, whereas there was little difference in TNF production (Fig. 1C and D). Therefore, β2 integrin ablation results in increased TLR responses from BM-derived macrophages, most strongly affecting IL-12 p40 and IL-6 production, with modest effects on TNF protein synthesis. In addition to inflammatory cytokine production, β2 integrin signals also moderated type I IFN production downstream of TLR4 activation as Itgb2−/− macrophages expressed significantly more IFNβ mRNA after LPS treatment than did WT cells (Fig. 1E).
TLR responsiveness was also examined in thioglycollate-elicited peritoneal macrophages to determine whether β2 integrins suppress TLRs in an inflammatory macrophage population. Because β2 integrins contribute to cellular infiltration into the peritoneal cavity [23, 24] and as Itgb2−/− mice present with a profound neutrophilia , we were unable to obtain a pure F4/80+Gr-1low macrophage population, even after 4 days postinjection, unlike in WT mice (Supporting Information Fig. 3A). To minimize any effect that elevated neutrophil levels may have on macrophage responses, we purified peritoneal macrophages at day 5 post thioglycollate injection by magnetic bead enrichment (Supporting Information Fig. 3B) and recovered a population of F4/80+ macrophages. Interestingly, Itgb2−/− macrophages showed a broader range of F4/80 expression than WT macrophages (Supporting Information Fig. 3B). We assessed inflammatory cytokine production in these thioglycollate-elicited macrophages by intracellular cytokine staining. F4/80highItgb2−/− peritoneal macrophages exhibited increased TLR4 responses over WT cells (Fig. 2A and B). The percentage of IL-12 p40- and IL-6-producing Itgb2−/− peritoneal macrophages was significantly elevated over WT cells following LPS stimulation, whereas TNF production remained unaffected by β2 integrin deletion, mirroring the phenotype of BM-derived macrophages (Fig. 2B). Thus, these data demonstrate that, in addition to BM-derived macrophages, β2 integrins also negatively regulate TLR-induced IL-12 p40 and IL-6 production in inflammatory macrophage populations.
Itgb2−/− mice have increased LPS-induced cytokines in vivo
To identify the contribution of β2 integrins to inhibiting TLR responses in vivo, we injected WT and Itgb2−/− mice with LPS i.p. and measured inflammatory cytokine levels in serum up to 4 h after injection. The kinetics for TNF, IL-12 p40, and IL-6 induction were similar between WT and Itgb2−/− mice, with the peak serum concentration of each cytokine occurring at the same time in both (Fig. 2C). However, differences in the magnitude of cytokine production were observed. Serum IL-12 p40 levels were dramatically increased in Itgb2−/− mice such that by 4 h post-injection, Itgb2−/− animals displayed approximately three times the concentrations observed in WT controls. Itgb2−/− mice also presented with significantly elevated serum IL-6 and TNF in response to LPS injection (Fig. 2C). While Itgb2−/− mice have changes in leukocyte populations, including increased circulating neutrophils, that make interpreting in vivo findings challenging, these data did support our in vitro findings that β2 integrins inhibited TLR responses in two distinct macrophage populations, BM-derived macrophages and thioglycollate-elicited macrophages.
TLR inhibition by β2 integrins does not involve changes in IL-10, ABIN-3, A20, or Hes-1 expression
TLR stimulation in macrophages results in secretion of the anti-inflammatory cytokine IL-10 that acts in an autocrine or paracrine manner to dampen TLR activation . Interestingly, culture of human macrophages on fibrinogen-coated plates induces IL-10 expression, as well as the expression of proteins such as A20, Hes-1, and ABIN-3, which are known to inhibit TLR signaling . Fibrinogen is a β2 integrin ligand and plating of human macrophages onto fibrinogen-coated plates presumably induces a β2 integrin signal, though other receptors may also be engaged [26-29]. To examine whether the TLR hypersensitivity of Itgb2−/− macrophages was due to deficiencies in these inhibitors, we analyzed their expression and function after TLR stimulation.
Itgb2−/− macrophages secreted similar or slightly elevated amounts of IL-10 following LPS and CpG DNA stimulation (Fig. 3A), demonstrating that Itgb2−/− macrophages were not hampered in their ability to produce IL-10. These results were mirrored in Itgb2−/− mice, which responded to i.p.-injected LPS by producing IL-10 at similar levels to WT (Fig. 3B). Furthermore, Itgb2−/− macrophages did not have defects in their response to IL-10. Treatment of macrophages with IL-10 prior to stimulation with LPS reduced cytokine production in both populations of macrophages to a similar degree (Fig. 3C and D). These data indicate that neither defects in IL-10 production nor the response to IL-10 can explain Itgb2−/− macrophage TLR hypersensitivity. Moreover, the increased TLR response of Itgb2−/− macrophages is not due to deficiencies in ABIN-3, A20, Hes-1, or IRAK-M expression, as would be hypothesized by the data presented by Wang et al. . Itgb2−/− macrophages expressed significantly higher levels of ABIN-3 and Hes-1 mRNA after TLR4 stimulation and exhibited slightly higher or equivalent expression of induced IRAK-M mRNA and A20 mRNA and protein (Fig. 3E and F).
Interestingly, expression of IL-10, A20, and ABIN-3 is associated with a p38 MAPK-driven inhibitory pathway that diminishes inflammation induced by TLRs or UVB irradiation [20, 30, 31]. Despite observing equal or elevated levels of these inhibitory proteins, we noted reduced p38 phosphorylation in LPS-treated Itgb2−/− macrophages (Fig. 3G), perhaps owing to the observation that signaling through β2 integrins themselves involves p38 MAPK pathway activation, the absence of which could lead to a deficiency in phospho-p38 levels . Interestingly, phosphorylation of ERK was not different between WT and Itgb2−/− macrophages (Fig. 3G). Thus, while Itgb2−/− TLR hypersensitivity may be partially due to suppressed p38 phosphorylation, our data do not implicate IL-10, A20, or ABIN-3 in this process and suggest that other MAPK-derived suppressive mechanisms, such as p38 control of inflammatory cytokine mRNA stability , may be controlled by β2 integrin signals.
CD11a, CD11b, and Cbl-b are dispensable for TLR inhibition
Itgb2−/− BM-derived DCs were also hypersensitive to TLR stimulation and secreted more inflammatory cytokines than WT control DCs (Supporting Information Fig. 4). Because these results generally phenocopied our observations in Itgb2 −/− macrophages, we reasoned that a β2 integrin shared between both cell types could inhibit TLR activation, such as LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) . Itgal−/− (CD11a-deficient) and Itgam−/− (CD11b-deficient) macrophages were examined to determine if either LFA-1 or Mac-1 were required to inhibit TLR signals. Neither Itgal−/− nor Itgam−/− BM-derived macrophages demonstrated increased cytokine production over that of WT macrophages following TLR stimulation (Fig. 4A and Supporting Information Fig. 5A). Additionally, we observed a reduction in cytokine-producing Itgam−/− thioglycollate-elicited peritoneal macrophages when compared to stimulated WT cells, though this difference was not statistically significant (Fig. 4B). Itgal−/− and Itgam−/− BM-derived DCs similarly had no increases in TLR−induced inflammatory cytokine production (data not shown), revealing that neither CD11a nor CD11b acts singly to diminish TLR activation.
Signals through the β2 integrin Mac-1 have been suggested to activate Cbl-b, an E3 ubiquitin ligase that can inhibit inflammatory responses in vivo . The proposed model suggests that CD11b signaling causes Cbl-b to ubiquitinate and degrade MyD88, thereby attenuating TLR responses. However, little is known about the ability of Cbl-b to regulate TLR responses specifically in macrophages. Therefore, we evaluated how Cbl-b deficiency influenced inflammatory cytokine production in these cells. Cblb−/− BM-derived macrophages were not hypersensitive to TLR stimulation and produced equal or lower amounts of inflammatory cytokines in response to LPS, CpG DNA, and zymosan treatment (Fig. 4C and Supporting Information Fig. 5B). Furthermore, Cblb−/− thioglycollate-induced peritoneal macrophages synthesized equivalent or lower levels of inflammatory cytokines when compared with WT controls following TLR4 activation (Fig. 4D), indicating that Cbl-b is dispensable for limiting TLR activity in macrophages. The model proposed by Han et al. would also predict that β2 integrin-deficient macrophages would have less MyD88 degradation after TLR signaling . Stimulation with 10 ng/mL LPS led to similar MyD88 degradation in WT and Itgb2−/−macrophages, suggesting that β2 integrins do not inhibit TLR responses by inducing MyD88 turnover (Supporting Information Fig. 5C). We were also unable to detect changes in MyD88 degradation in WT or Itgb2−/− macrophages treated with a lower dose of LPS (1 ng/mL), with which we observed elevated inflammatory cytokine production in β2 integrin-deficient cells (data not shown). Interestingly, Itgam−/− and Cblb−/− macrophages also retained the ability to degrade MyD88 following LPS stimulation (Supporting Information Fig. 5C). These data reveal that a CD11b-Cbl-b inhibitory mechanism is not required for dampening TLR responses in macrophages.
β2 integrin deficiency enhances NF-κB activation in macrophages
After eliminating several potential indirect mechanisms governing β2 integrin-mediated TLR inhibition, we assessed whether Itgb2−/− macrophage hypersensitivity was due to differences in TLR-induced NF-κB pathway activation. To this end, we noted changes in NF-κB activation that are consistent with Itgb2−/− macrophage hypersensitivity. In canonical NF-κB signaling, NF-κB subunits are retained in the cytoplasm by binding to IκBα, which in turn becomes phosphorylated and degraded after TLR stimulation to allow NF-κB proteins to enter the nucleus and enable transcription. Thus, we assessed changes in IκBα expression at early (0–120 min) and late (2–8 h) phases following TLR stimulation to gauge NF-κB pathway activation. LPS-treated WT and Itgb2−/− macrophages led to rapid NF-κB pathway activation such that the lowest amount of total IκBα occurred at 15 min after LPS treatment (Fig. 5A and B). IκBα was quickly resynthesized in WT macrophages such that near baseline levels were reached after 60 min (Fig. 5A and B). In contrast, a consistent trend toward delayed IκBα resynthesis was observed in the absence of β2 integrins (Fig. 5A and B) suggesting an elevation in NF-κB pathway activation in Itgb2−/− macrophages. To assess phosphorylation of IκBα, we stimulated macrophages in the presence of the proteasomal inhibitor MG-132 to compensate for the rapid degradation of IκBα protein. Both WT and Itgb2−/− cells quickly phosphorylated IκBα, without an increase in phosphorylation in the Itgb2−/− cells over WT cells (Supporting Information Fig. 6A and B).
These results were coupled with similar observations at the late phase of TLR stimulation. Itgb2−/− macrophages displayed consistently lower levels of IκBα up to 4 h post-LPS treatment in comparison with WT cells, though the magnitude of this effect was modest (Fig. 5C and D). Itgb2−/− macrophages displayed similar phosphorylation of IκBα at 2 h post LPS treatment to WT macrophages, but this IκBα phosphorylation was slightly increased in Itgb2−/− macrophages over WT macrophages at 4 h post LPS treatment (Supporting Information Fig. 6C and D). Notably, increases in IκBα degradation in Itgb2−/− macrophages were not due to a defect in IκBα resynthesis in these cells. Itgb2−/− macrophages were able to transcribe IκBα mRNA at or beyond the levels observed for WT macrophages (Fig. 5E and F). Therefore, our data show that β2 integrins can affect the magnitude of the signal leading to NF-κB activation in the cytoplasm.
We thus compared the induction of NF-κB-dependent genes induced during TLR responses in WT and Itgb2−/− macrophages. TLR hyperactivation also generated changes to the NF-κB-dependent gene transcriptional profile of Itgb2−/− macrophages. As expected, β2 integrin-deficient macrophages produced more inflammatory cytokine transcripts than did WT control cells following TLR stimulation, with the greatest differences observed for IL-12 p40 and IL-6 mRNA (Fig. 6A). Consistent with these observations, Itgb2−/− macrophages also presented with higher levels of mRNA for many NF-κB-dependent genes  as compared to WT, including increases in Bfl-1, CXCL1, CXCL2, CXCL10, and GADD45β (Fig. 6B), indicating a global increase in NF-κB activity without β2 integrin-mediated inhibition. The magnitude of the effect of β2 integrin deficiency varied and a curious exception to this increased gene expression profile was that of iNOS, which directs the antimicrobial nitric oxide responses, the synthesis of which was identical between Itgb2−/− and WT macrophages (Fig. 6B). Furthermore, the hyperactive NF-κB pathway activity found in Itgb2−/− macrophages was limited to TLR stimulation and was not observed after treatment of these cells with recombinant TNF, a potent NF-κB inducer (Supporting Information Fig. 6E) .
Activation of the NF-κB subunit p65/RelA controls the intensity of IL-12 p40 transcription . Because of this, we analyzed p65/RelA activation directly by assessing its binding to the promoter of Il12b, which encodes IL-12 p40, by chromatin immunoprecipitation (ChIP) assay. Interestingly, p65/RelA occupancy of the Il12b promoter was elevated in Itgb2−/− macrophages after 8 h of TLR4 stimulation (Fig. 6C), demonstrating a direct effect of β2 integrins on NF-κB subunit binding to the Il12b locus. Taken together with our gene expression data and signaling analyses, these observations clearly show that one way by which β2 integrins suppress macrophage activation and inflammatory cytokine production is by fine-tuning NF-κB pathway activation. While β2 integrin signals direct modest, but consistent, changes in IκBα expression after TLR stimulation, these changes are sufficient to dramatically reduce inflammatory cytokine production in myeloid cells and demonstrate a critical role for β2 integrins in dampening TLR responses.
A variety of cell surface receptors use ITAM-containing adapters to relay external signals and enable appropriate cellular changes, including the β2 integrins, which signal via DAP12 and FcRγ [4, 14]. Yet while signals through DAP12 and FcR-γ have been clearly shown to block inflammation [10, 11, 36], defining the connection between the β2 integrins themselves and inflammatory processes has proven difficult due to conflicting data showing both positive and negative regulatory roles for this family of adhesion molecules [16-20, 37]. We have clarified how β2 integrin activation influences TLR responses by using macrophages and DCs derived from the Itgb2−/− mouse, which lack all β2 integrin surface expression. Itgb2−/− macrophages and DCs produced more IL-12 p40 and IL-6 in response to stimulation with a variety of TLR agonists and Itgb2−/− mice generated more inflammatory cytokines after LPS injection than did WT control animals, demonstrating that β2 integrins are essential for inhibiting TLR activity in vitro and in vivo.
While these phenotypic findings are consistent with other studies reporting a suppressive role for β2 integrins, our use of Itgb2−/− myeloid cells provided a useful system with which to test various aspects of TLR regulation and to define the molecular requirements for β2 integrin-mediated TLR inhibition. To this end, we have identified a novel role for β2 integrins in calibrating NF-κB pathway activation downstream of TLR ligation. Without β2 integrin inhibitory signals, macrophage total IκBα levels remained consistently lower throughout the course of TLR stimulation. Curiously, we did not find consistently enhanced phosphorylated IκBα levels in Itgb2−/− cells after TLR stimulation, though this may be due to complications arising from using the proteasome inhibitor MG-132 in these experiments to inhibit the rapid degradation of IκBα. However, its presence would have also inhibited normal turnover of IκBα, perhaps masking potential differences between WT and β2 integrin-deficient cells. Nevertheless, this fine-tuning of NF-κB activation by β2 integrins contributed to dramatic differences in the ability of macrophages to respond to TLRs and induce NF-κB-dependent gene expression. Importantly, we noted that the affected genes encompassed both “primary response” (Tnf, Cxcl1, Cxcl2) and “secondary response” (Il12B, Il6) genes that encode for inflammatory cytokines, chemokines, and anti-apoptotic functions . We also observed a direct effect of β2 integrin deletion on enhancing p65/RelA binding to the Il12b (IL-12 p40) promoter downstream of LPS stimulation. However, it should be noted that fine-tuning of the NF-κB pathway by β2 integrins did not control expression of all “NF-κB-dependent” genes tested. Peculiar omissions from this list include A20 and iNOS, which were both expressed similarly between WT and Itgb2−/− macrophages, suggesting that other pathways may be influenced by β2 integrin signals to control transcription of these genes.
One such pathway is p38 MAPK signaling. Itgb2−/− macrophages demonstrated a reduced ability to phosphorylate, and therefore activate, p38 following LPS treatment, consistent with the fact that β2 integrin outside-in signals are known to directly activate the MAPK pathway . In addition to its well-regarded proinflammatory activities , activation of p38 and its subordinate protein kinases MSK1 and MSK2 has been implicated in dampening inflammation through several mechanisms. For example, p38 activity limits Th1 responses to Leishmania by destabilizing IL-12 p40, though not TNF, mRNA stability . p38 and MSK1/2-derived signals have also been shown to negatively regulate TLR responses by inhibiting inflammatory cytokine transcription directly or by promoting IL-10 synthesis through activation of CREB and Atf-1 transcription factors [30-32]. In addition to IL-10, p38-directed A20 and ABIN-3 production has previously been linked to TLR suppression by β2 integrins . However, Itgb2−/− macrophage TLR hypersensitivity could not be attributed to deficiencies in A20, ABIN-3, Hes-1 or to changes in IL-10 production or signaling, arguing against a role for these proteins in β2 integrin-medited TLR suppression. Interestingly, Itgb2−/− macrophages presented with higher TLR-induced levels of some of these inhibitors than WT cells, likely owing to enhanced NF-κB activation. The differences between our results and those of Wang et al.  may be due to our use of plastic petri dishes to induce β2 integrin signals instead of plate-bound fibrinogen, which itself is known to bind to additional receptors [26-29]. Indeed, fibrinogen's ability to dampen TLR activity in macrophages may be at least partially β2 integrin-independent as we found that inflammatory cytokine secretion was suppressed in Itgb2−/− macrophages similar to WT cells after plating onto fibrinogen-coated plates (data not shown). Alternatively, our results may differ due to our overnight resting of macrophages after plating or other technical differences. Despite this, β2 integrin signaling may contribute to inhibition of TLR responses through other p38-directed processes, such as by regulating inflammatory cytokine mRNA stability  or by influencing NF-κB crosstalk [34, 40], possibilities that remain to be tested experimentally.
Our findings are consistent with observations made in the Itgb2hypo mouse on the PL/J background, which suffers from a chronic inflammatory skin disease similar to human psoriasis . Macrophages are required for maintenance of this disease and selective disruption of NF-κB activation in macrophages improves the psoriaform lesions in Itgb2hypo mice [41, 42]. While these results suggest a connection between β2 integrins and NF-κB regulation, they are complicated by the ongoing disease of the animals and the presence of residual β2 integrin signaling in all cell types. However, by using myeloid cells isolated from healthy Itgb2−/− mice on a C57BL/6 genetic background, we have avoided these issues and have clearly revealed a role for β2 integrins in fine-tuning the NF-κB pathway, demonstrating that β2 integrin signaling can inhibit TLR activation.
In attempting to identify the specific β2 integrins required for TLR inhibition, we found that deletion of Mac-1 alone is insufficient to render myeloid cells hyperresponsive to TLR stimulation. This was a surprising finding given that Mac-1 activation has been proposed to regulate TLR signaling by inducing Cbl-b activity, leading to degradation of MyD88 and TRIF . Cbl-b is a potent negative regulator of inflammation [43, 44] and it is known to modulate TLR4 activity in neutrophils by facilitating TLR4-MyD88 binding . However, we found that Cbl-b is not required to dampen TLR activation in macrophages. Cblb−/− macrophages were not hypersensitive to TLR stimulation and Cbl-b deficiency did not change the kinetics of MyD88 degradation, as would be predicted based on the model proposed by Han et al.  through experiments in HEK293 cells. Thus, our data suggest that inhibiting TLR4 does not require a CD11b-Cbl-b-MyD88 regulatory axis in primary macrophages. Deleting LFA-1 was also not sufficient to cause hypersecretion of inflammatory cytokines in macrophages. We theorize that one or more integrins shared between both cell types are responsible for TLR inhibition and that compensatory integrin signaling is able to block TLR responses in Itgal−/− or Itgam−/− myeloid cells.
Our data suggest an important role for cell adhesion events in fine-tuning inflammation. β2 integrins first encounter their ligands within the luminal side of blood vessels. By finding that β2 integrins are required for negatively regulating TLR responses, we have highlighted the exciting prospect that cell adhesion events may limit inflammatory cytokine production in the bloodstream and thereby compartmentalize inflammatory cytokine production to the site of inflammation. Beyond this initial β2 integrin binding, myeloid cells also encounter β2 integrin ligands within the extracellular matrix while en route to their intended targets. Here these ligands would be modified by local inflammatory mediators , suggesting that distinct β2 integrin ligands may differentially regulate TLR responses in a manner that targets inflammatory cytokine production to the infected tissue and therefore minimizes damage to the host.
Materials and methods
C57BL/6 mice were purchased from Charles River Laboratories. CD18-deficient (Itgb2−/−) mice  were backcrossed six generations against C57BL/6 mice and were provided by Dr. Clifford Lowell (University of California, San Francisco). CD11a-deficient (Itgal−/−) and CD11b-deficient (Itgam−/−) animals were purchased from Jackson Laboratories [23, 47]. Cbl-b-deficient (Cblb−/−) mice were backcrossed 12 generations against C57BL/6 and were provided by Dr. Phil Greenberg (University of Washington) . All animals were housed in specific-pathogen-free facilities and all experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Benaroya Research Institute.
BM cells were flushed from femurs and tibias, followed by erythrocyte lysis in ACK buffer (Lonza). For macrophages, BM cells were plated onto a 10 cm petri dish (Fisher Scientific) using 10 mL of BM macrophage growth medium, which consisted of DMEM supplemented with 10% FBS (Sigma), 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Lonza), penicillin/streptomycin (Gibco) and 10% CMG14–12 cell conditioned media as a source of CSF-1 . BM-derived DCs were grown in DC medium, which consisted of RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, penicillin/streptomycin and 10 ng/mL GM-CSF (Peprotech). For both macrophages and DCs, an additional 10 mL of growth medium was added after 3 days of culture. Day 6 DCs were isolated from culture by magnetic bead enrichment for MHCII+ cells. Cells were treated with anti-FcγRII/III (2.4G2) followed by staining with anti-MHC II-biotin (M5/114.15.2/eBioscience), antibiotin microbeads (Miltenyi biotech) and sorting with MACS columns according to the manufacturer's instructions. The purity of CD11c+ cells was >90% in WT cultures. BM-derived macrophages and DCs were used at day 6 of culture.
Peritoneal macrophage isolation
Mice were injected i.p. with 3% thioglycollate broth and peritoneal cells were isolated by lavage with Cell Dissociation Buffer (Invitrogen) 5 days after injection. Macrophages were purified by magnetic bead enrichment using anti-F4/80-biotin (BM8/eBioscience) followed by incubation with antibiotin microbeads and then sorted by MACS according to the manufacturer's instructions. F4/80+ macrophages were cultured in DMEM supplemented with 10% FBS (Sigma).
TLR stimulation and ELISA
Macrophages or DCs were plated onto 96-well tissue culture-treated plates at 5 × 104 cells/well and allowed to rest for 16 h prior to stimulation. TLR agonists were added to triplicate wells at the indicated concentrations for up to 24 h. Supernatants were collected and the amount of IL-12 p40, IL-12 p70, IL-6, TNF, or IL-10 were assessed by ELISA (eBioscience). TLR agonists used were as follows: S. Minnesota R595 Ultra Pure LPS (List Biological Laboratories), CPG DNA (ODN1826, Invivogen), and zymosan particles (Sigma). CpG DNA and zymosan particles were incubated in 10 μg/mL polymixin B for 1 h prior to use.
Flow cytometry and intracellular cytokine staining
Macrophages were stimulated with LPS for up to 8 h in the presence of 10 μg/mL Brefeldin A for the final 2 h of stimulation. Macrophage FcRs were blocked with 2.4G2 for 10 min followed by surface staining with anti-F4/80 (BM8/eBioscience). Peritoneal cells were also surface stained with anti-Siglec F (E50–2440/BD Biosciences) and anti-Gr-1 (RB6–8C5/eBioscience). Macrophages were then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences). Intracellular cytokine staining was performed using anti-IL-12 p40 (C15.6/Biolegend), anti-TNF (MP6-XT22/eBioscience), and anti-IL-6 (MP5–20F3/eBioscience). Flow cytometry analysis was conducted using an LSR2 (BD Bioscience) running FACSDiva software (BD Bioscience). All flow cytometry analyses were conducted using FlowJo software (Treestar).
Mice were injected with 1 μg/g LPS i.p. in PBS and blood was collected at indicated time points. Serum concentrations of IL-12 p40, IL-6, TNF, and IL-10 were determined by ELISA.
Gene expression analysis
Total RNA from LPS or TNF (Peprotech) stimulated macrophages was isolated using the RNeasy Plus kit (Qiagen) and reverse transcribed with Superscript III reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed in triplicate wells using the Power SYBR Green PCR master mix (Applied Biosystems) and reactions were run on a 7900HT Real-Time PCR System (Applied Biosystems). All data were normalized to GAPDH endogenous control. Oligonucleotide primers for GAPDH and A20 were previously described . Primer sequences were as follows: Hes1: 5′- tgccagctgatataatggaga-3′ and 5′-ccatgataggctttgatgact-3′; TNIP3 (ABIN-3): 5′- tccttgtcttcccaggacat-3′ and 5′-ttcttcttggtggagcacact-3’; Irak3 (IRAK-M): 5′- tcgac agatta cagtgc acaa-3′ and 5′-ggctatt cctatcaatacgct-3’; Bcl2a1a (Bfl-1): 5′- tttccagttttgtggca gaat-3′ and 5′-tcaaacttctttatgaagccatctt-3′; Gadd45b: 5′- ctgcctcctggtcacgaa-3′ and 5′- ttgcctctgctctcttcaca-3’; Nos2 (iNOS): 5′-gggctgtcacgg agatca-3′ and 5′-ccatgatggtca cattctgc-3′; Cxcl1: 5′- agactccagccacactccaa-3′ and 5′-tgacagcgcagctcattg-3′; Cxcl2: 5′-aaaatcatccaaaagatactgaacaa-3′ and 5′- ctttggttcttccgttgagg-3′ and 5′- ctttggttcttccgttgagg-3′; Cxcl10: 5′- gctgccgtcattttctgc-3′ and 5′- tctcactggcccgtcatc-3′; Il12a (IL-12 p35) 5′- tcagaatcacaaccatcagca-3′ and 5′ cgccattatgattcagagactg-3′; Il12b (IL-12 p40): 5′- gattcagactccaggggaca-3′ and 5′-tggttagcttctgaggacaca-3′; Il23a (IL-23 p19) 5′-tccctactaggactcagccaac-3′ and 5′-agaactcaggctgggcatc-3′; Tnf: 5′-gtcaggttgcctctgtctca-3′ and 5′- tcagggaagagtctggaaag-3′; Il6: 5′- aggcataacgcactaggttt-3′ and 5′-agctggagtcacagaaggag-3′; Ifnb: 5′- gcactgggtggaatgagactattg-3′ and 5′- ttctgaggcatcaactgacaggtc-3′.
ChIP was conducted as described in  with minor variations. Briefly, macrophages were stimulated with 1 ng/mL LPS for 8 h, washed and fixed with a 1% final concentration of formaldehyde (37% HCHO in 10–15% methanol; Fisher). Crosslinking was stopped after 10 min by addition of glycine to a final concentration of 125 mM and incubated for 10 min. Macrophages were then washed three times with ice-cold PBS and spun down, and pellets were flash frozen in a dry ice/ethanol bath and kept at –80°C until further analysis. To isolate nuclei, macrophages were first resuspended in Cell Lysis Buffer (10 mM HEPES pH 7.9, 0.5% IGEPAL-30, 1.5 mM MgCl2, 10 mM KCl) and kept on ice for 25 min, vortexing every 5 min. Nuclei were then centrifuged at 4°C and resuspended in Nuclear Lysis Buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS), followed by sonication in a 4°C water bath to create fragments between 200–800 bp in length. Sonicated samples were then precleared with Protein A Dynabeads (Invitrogen) for 30 min at 4°C and supernatants were collected by magnetic separation. The supernatants were then diluted 1:10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl) and incubated with 2 μg of anti-p65/RelA (Santa Cruz) overnight at 4°C. Immunocomplexes were then collected with Protein A Dynabeads and washed with Low Salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1), High Salt buffer (same as low salt but with 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl) and two times with TE buffer. Complexes were extracted with Elution buffer (1% SDS, 0.1 M NaHCO3) and protein: DNA crosslinks were reversed by treating with RNAse A and Proteinase K at 65°C. DNA was then purified (MoBio UltraClean PCR kit) and analyzed by qPCR. Normalization was accomplished by subtracting Ct values from precleared “input” chromatin. The primer sequences for the Il12b promoter are: 5′-ctttctgatggaaacccaaag-3′ and 5′-ggggagggaggaacttctta-3′.
Macrophages were stimulated with indicated concentrations of LPS for various times and lysed in lysis buffer containing 1% Triton X-100, protease inhibitors (mammalian protease inhibitor cocktail, Sigma) and 1 mM sodium orthovanadate (Sigma). For phospho-IκBα blots, macrophages were pretreated with 10 μM MG-132 (Sigma) for 30 min prior to LPS treatment. Lysates were separated by Tris-bis SDS-PAGE gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Rabbit antibodies specific for IκBα, phospho-IκBα, phospho-p42/44 ERK, phospho-p38, A20, and β actin were from Cell Signaling. Rabbit anti-MyD88 was from Biovision. An HRP-conjugated donkey antirabbit IgG was used as a secondary (GE Healthcare). Antibody binding was detected by the Immobilon chemiluminescence system (Millipore). Densitometry analysis was conducted using ImageJ software (NIH).
Student's unpaired t-test was used to measure statistical significance between two groups and one-way ANOVA with Dunnet's multiple comparison test was used to determine statistical significance between multiple groups against WT control. All statistical analyses were performed by Prism 5 (Graphpad Software).
We thank Dr. Clifford Lowell for providing Itgb2−/− mice and Dr. Hua Gu and Dr. Phil Greenberg for providing Cblb−/− mice. We would also like to acknowledge Dr. Amy Weinmann for advice on chromatin immunoprecipitation and thank members of our laboratory for helpful discussions and review of the manuscript.
This work was supported by NIH grants R01AI073441 and R01AI081948, an Investigator Award from the Cancer Research Institute, a pilot award from the Alliance for Lupus Research and DOD grant W81XWH-10-1-0149 (to J.A.H). N.Y. was supported in part by NIH training grant 5T32CA09537.
Conflict of interest
The authors declare no financial or commercial conflict of interest.