Toll-like Receptor 2 Ligands Regulate Monocyte Fcγ Receptor Expression and Function*

Background: Toll-like receptors recognize bacterial components, leading to immune activation. Results: TLR2 ligands alter monocyte/macrophage FcγR and phosphatase expression to improve function both in vitro and in vivo. Conclusion: TLR2 ligands enhance monocyte/macrophage function. Significance: FcγR function is critical for antibody therapy. Deciphering the effect of TLR2 ligands provides a potential means to enhance therapy. Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy.

The use of therapeutic antibodies is a common form of treatment for cancer, and efforts are ongoing to optimize its effectiveness (1). Here, antibodies bind antigens on tumor cells and effect their destruction through several possible means, including induction of apoptosis, blockage of aberrant intra-or intercellular signaling, and destruction by host immune effector cells (2,3). By far the most critical mechanism of antibodymediated tumor cell clearance appears to be through the activity of Fc␥R 3 on host immune effector cells (4). The vast majority of immune cells (with the exception of T cells) express one or more Fc␥R (5). Fc␥R bind the Fc portion of antibodies, and their activation leads to effector functions such as phagocytosis, cytokine release, and direct cytotoxicity against the antibodycoated target (5). Among the immune effector cells, monocytes/macrophages play a critical role in that they directly attack opsonized targets and also release a variety of chemokines and cytokines that can attract and activate neighboring cells (6 -8). Indeed, in a murine model of CD20 antibody-mediated depletion of B cells, depletion of macrophages abrogated the effect of the antibody (6). Monocytes express Fc␥R that activate, as well as dampen, downstream events such as phagocytosis and cytokine release. This is mediated through immunoreceptor tyrosine-based activation motifs or immunoreceptor tyrosine-based inhibitory motifs, and Fc␥R serve to activate or inhibit signaling on the basis of the motif they contain (5).
Toll-like receptors (TLR) are evolutionarily conserved pattern recognition receptors for a wide variety of pathogens and potentially harmful molecules (9). However, utilizing TLR activation as a means of strengthening responses to vaccine treatments or to threats such as viruses and tumors is an area of active research. Purified and/or synthetic TLR agonists such as CpG (for TLR9), imiquimod (for TLR7), flagellin (for TLR5), Pam3CSK4 (for TLR2), and others continue to be evaluated, with many showing promise toward treating asthma, cancer, and viral infections and for strengthening vaccine responses (10).
Toll-like receptor 2 (TLR2) responds to a variety of natural stimuli such as Gram-positive bacterial cell wall components  and zymogens as well as synthetic compounds such as tri-and  diacylated lipopeptides. TLR2 recognizes these components  through interacting with other host cell receptors such as  TLR1, TLR6, CD14, CD36, and Dectin-1 (10 -12). As with most TLR, it involves the downstream adapter MyD88, resulting in cellular responses such as activation of MAPKs and NF-B (13). The TLR2 agonist Pam3CSK4 is currently being investigated for its ability to induce vaccine responses (14), for protection against ischemia/reperfusion injury (15) and sepsis (16), and for modulating allergic responses (17), as well as for the study of TLR2 activation within the context of other conditions such as rheumatoid arthritis (18) and acute lung injury (19). Hence, such TLR2 agonists may prove to be effective, clinically relevant immunomodulators.
Here we explored the possibility that TLR2 activation could interact with the Fc␥R pathway, leading to enhancements in Fc␥R function. We found that the TLR2 agonist Pam2CSK4 enhanced Fc␥R-mediated cytokine production as well as phagocytic ability. In addition, Pam2CSK4 plus antibody was significantly more effective than antibody alone in slowing the rate of tumor growth in a murine model of antibody therapy. An examination of the underlying mechanisms revealed an up-regulated expression of Fc␥RIIa, the common ␥ subunit on monocytes, and, to some small degree, the inhibitory Fc␥RIIb. However, Pam2CSK4 also up-regulated miR-155 and down-regulated the inhibitory phosphatase SHIP, which is the major mediator of Fc␥RIIb function. We also found that the related triacylated Pam3CSK4 was equally effective in modulating Fc␥R expression and in enhancing monocyte-mediated phagocytosis in vitro. These results suggest that TLR2 activation is an effective means of strengthening monocyte Fc␥R expression and function and that such agonists could be further examined as potential adjuvants for antitumor antibody therapy.
Western Blotting and ELISAs-Cells were lysed in TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na 4 P 2 O 7 , 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na 3 VO 4 , and 10 g/ml each aprotinin and leupeptin). Postnuclear proteinmatched lysates were boiled in Laemmli sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with the antibody of interest, and then developed by Pierce ECL 2 Western blotting substrate (Thermo Scientific, Rockford, IL). Densitometry was performed using ImageJ software, and graphs of the ratios between the indicated probes and their respective anti-actin reprobes were plotted. Cell supernatants were collected, centrifuged at full speed to clear cellular debris, and then assayed for cytokine via sandwich ELISA (R&D Systems, Minneapolis, MN) according to the protocol of the manufacturer.
Real-time RT-PCR-Cells were lysed in TRIzol reagent (Invitrogen/Invitrogen), and RNA isolation was completed according to the instructions of the manufacturer. Reverse transcription was done with 10 -100 ng of total RNA. The cDNA was run in triplicate for each donor on an Applied Biosystems Step One Plus system with automatically calculated thresholds. Relative expression was calculated as 2ˆϪ ⌬Ct , with ⌬Ct calculated by subtracting the average Ct of two housekeeping controls (CAP-1 and GAPDH for SHIP and RNU44 for miR-155) from the experimental sample Ct (21).
Bone Marrow-derived Macrophage Isolation and Culture-Wild-type Balb/cJ mice were sacrificed according to institution-approved animal care and use protocols. Briefly, bone marrow cells were cultured in RPMI containing 10% fetal bovine serum plus 10 g/ml polymixin B (Calbiochem, San Diego, CA) and supplemented with 20 ng/ml M-CSF (R&D Systems) for 7 days before the differentiated cells were used in experiments.
Phagocytosis-Phagocytosis assays were performed as described previously (20) Briefly, IgG-coated PKH26-labeled sheep red blood cells (SRBC) were added to the PBM. Cells were briefly pelleted by slow centrifugation followed by 30 min of incubation at 37°C. Unphagocytosed RBC were subjected to hypotonic lysis with water and PBS wash prior to fixation with 1% paraformaldehyde. The samples were analyzed by fluorescence microscopy in a blinded fashion. The phagocytic index is defined as the total number of SRBCs ingested by 100 phagocytes.
Murine Solid Tumor Model-CT26-HER2/neu colon carcinoma cells (22) were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin/streptomycin, and L-glutamate; washed to remove non-adherent cells; and then resuspended using enzyme-free cell dissociation buffer (Invitrogen). Cells were centrifuged and resuspended at 10 ϫ 10 6 cells/ml in RPMI 1640. The murine tumor model (22) was used as described previously (23,24). Briefly, 5-week-old female Balb/cJ mice (The Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously with 1 ϫ 10 6 of syngeneic CT26-HER2/neu cells. Mice were left for 7 days to allow tumors to develop. Intraperitoneal injections with treatments (PBS vehicle, 4D5 at 1 mg/kg, Pam2CSK4 at 5 g/kg, or 4D5 plus Pam2CSK4) were then performed three times per week, with tumor measurements recorded on each treatment day. Tumor volumes were calculated as [0.5 ϫ (length measurement) ϫ (height measurement) 2 ] (23), where length was the longest diameter of the tumor. Treatments consisted of 4D5 anti-HER2 antibody at 3 mg/kg, Pam2CSK4 at 100 ng/g, 4D5 plus Pam2CSK4, or PBS control. All in vivo experiments were performed in strict accordance with guidelines set by the Institutional Animal Care and Use Committee.
Statistical Analyses-For all experiments performed in vitro, Student's t tests were used to test for statistically significant differences. Statistical analyses for the murine solid tumor model experiment were done at the Center for Biostatistics, Ohio State University. Briefly, data were transformed by cube root, and then a linear mixed model was applied to identify significant differences between treatment groups using SAS (SAS Institute, Inc., Cary, NC) analysis software.

RESULTS
Pam2CSK4 Enhances Fc␥R Function-We first determined whether TLR2 activation could strengthen monocyte Fc␥R activity with regard to both cytokine production and phagocytosis. We first treated PBM overnight with or without 100 ng/ml of the TLR2 agonist Pam2CSK4 and then incubated them for 30 min with fluoresceinated, IgG-opsonized sheep red blood cells SRBC. The number of ingested SRBC was counted using fluorescence microscopy. Results showed that agonisttreated PBM ingested significantly more SRBC than vehicletreated PBM (Fig. 1A). Next, we treated PBM overnight with or without 100 ng/ml of Pam2CSK4 and then incubated them with or without immobilized IgG. After 24 h, cleared supernatants were collected, and TNF␣ was measured by ELISA. Results (Fig.  1B) showed that Pam2CSK4 elicited synergistic increases in IgG-mediated TNF␣ production with very little background TNF␣ from Pam2CSK4 treatment alone. Collectively, these results show that treatment with the TLR2 agonist Pam2CSK4 can significantly enhance monocyte Fc␥R function.
Pam2CSK4 Modulates Fc␥R Expression-To examine the mechanism by which TLR2 activation leads to increased Fc␥R function, we next tested whether the agonist treatment influenced Fc␥R expression itself. We treated PBM with vehicle or Pam2CSK4 overnight and subsequently measured Fc␥R expression by Western blotting. As shown in Fig. 2A, agonist treatment increased the expression of Fc␥RIIa and the common ␥-chain that associates with Fc␥RI and Fc␥RIII (left and center panels). Expression of the inhibitory Fc␥RIIb was largely unchanged, although it did show increases in some donors tested (right panel, showing an increase). The increase in Fc␥RIIa was verified in subsequent experiments (see Fig. 5). We then examined transcript levels of Fc␥R following overnight treatment with Pam2CSK4. The results (Fig. 2B) showed increases in Fc␥RIIa (left panel), the ␥-chain (center panel), and Fc␥RIIb (right panel). These results suggest that TLR2 activation can up-regulate expression of Fc␥R.
TLR2 Modulates miR-155 and SHIP-Results showed that Pam2CSK4 significantly enhanced Fc␥R function despite also leading to up-regulation of the inhibitory Fc␥RIIb. This suggests that Fc␥RIIb activity is suboptimal, perhaps because of alteration of downstream mediators. Hence, we examined the expression of SHIP, the inositol phosphatase responsible for the majority of Fc␥RIIb-mediated Fc␥R inhibition (25)(26)(27). Following overnight treatment of PBM with Pam2CSK4, the transcript for SHIP was significantly reduced (Fig. 3A). Because recent studies implicated the microRNA miR-155 as a regulator of SHIP expression (28 -30), we also examined the response of miR-155 to Pam2CSK4. As shown in Fig. 3B, Pam2CSK4 significantly up-regulated miR-155. These results suggest that, although this TLR2 agonist may increase expression of Fc␥RIIb, it also down-regulates the major downstream mediator of this inhibitory Fc␥R.
Dose Response to Pam2CSK4-To determine the lowest effective concentration of Pam2CSK4 and to see whether treatment with greater than 100 ng/ml (used above) would lead to still further Fc␥R enhancements, we treated PBM overnight FIGURE 1. Pam2CSK4 enhances Fc␥R function. Human PBM were treated overnight with or without 100 ng/ml of Pam2CSK4. A, PBM were then incubated with fluoresceinated, IgG-coated SRBC for 30 min at 37°C for phagocytosis. Phagocytosis was measured by counting the total number of SRBC ingested by 100 monocytes and is plotted as the phagocytic index. Three independent experiments were performed and scored in a blinded fashion using fluorescence microscopy. B, treated versus untreated PBM were then incubated in 96-well plates with or without immobilized IgG for 24 h at 37°C. Supernatants were harvested and analyzed for TNF␣ by sandwich ELISA. The mean of three separate donors is plotted. Data were analyzed by Student's t test. *, p Ͻ 0.05. UT, untreated. APRIL 26, 2013 • VOLUME 288 • NUMBER 17 with Pam2CSK4 at concentrations ranging from 0 -200 ng/ml. PBM were then tested for phagocytic ability using fluoresceinated, opsonized SRBC. Results showed that as little as 1 ng/ml Pam2CSK4 was sufficient to drive increases in phagocytic ability (Fig. 4A). We also treated with dosages from 0 -200 ng/ml followed by incubation for 24 h with or without immobilized IgG. TNF␣ levels in cleared supernatants were measured and compared between dosages. Results, in accordance with those from the phagocytosis assay, showed that as little as 1 ng/ml could significantly enhance IgG-mediated cytokine production (Fig. 4B). We also measured Fc␥R expression by Western blot-ting, and as shown in Fig. 5A, levels as low as 0.1 ng/ml led to greater expression of activating Fc␥RIIa (top panel) and the ␥-chain (center panel) as well as modestly up-regulating Fc␥RIIb (bottom panel). As in Fig. 2, there was some donor variability with regard to changes in Fc␥RIIb expression. We tested dosages as high as 200 ng/ml and found that changes in Fc␥R were minimal above the 1-5 ng/ml range (Fig. 5B). Hence, we conclude that as little as 0.1 ng/ml of Pam2CSK4 is sufficient to significantly modulate expression of monocyte Fc␥R. Dosages higher than this led to minimal further enhancements and, possibly, to small declines.  Murine Macrophages Respond to Pam2CSK4-We then sought to determine whether the murine system would provide a good model in which to test the effect of Pam2CSK4 on Fc␥R and antibody therapy. To do this, it was first necessary to examine the effects of this agonist on murine Fc␥R function. To do this, we treated mouse BMM overnight with or without 100 ng/ml of Pam2CSK4 and then tested the BMM in a phagocytosis assay. As anticipated, agonist treatment significantly enhanced the phagocytic ability of BMM (Fig. 6A). We also treated overnight with or without Pam2CSK4 and then incubated the BMM for 24 h with or without immobilized IgG. Supernatants were collected and analyzed for TNF␣ levels by ELISA. Results showed that Pam2CSK4 increased IgG-mediated TNF␣ production in these murine cells (Fig. 6B). These experiments suggest that murine cells respond similar to human cells when treated with TLR2 agonist and that the murine system would be a suitable model to test the effect of this agonist on antibody therapy in vivo.

TLR2 Ligands Regulate Fc␥R Expression and Function
Pam2CSK4 Enhances Antibody Therapy in Vivo-We next tested whether Pam2CSK4 could enhance antibody-mediated antitumor responses using a murine model of antibody therapy (22)(23)(24). Here, syngeneic CT26 colon adenocarcinoma cells engineered to express human Her2/neu were injected into the flanks of Balb/cJ mice. Following tumor development, intraperitoneal injections of the 4D5 anti-Her2 antibody with or without Pam2CSK4 were done, and the rate of tumor growth was measured for ϳ18 days. Using this model, we found that cotreatment with Pam2CSK4 and 4D5 antibody was significantly better than antibody alone at slowing the rate of tumor growth (Fig. 7). These results suggest that Pam2CSK4 could potentially serve as an effective adjuvant for antitumor antibody therapy.
Pam3CSK4 Also Enhances Fc␥R Function-We have performed these studies using the diacylated Pam2CSK4, but the related triacylated TLR2 agonist Pam3CSK4 is also being investigated as a putative immunomodulatory agent. Hence, we wished to determine whether this agonist, similar to Pam2CSK4 in structure but leading to TLR2/TLR1 heterodimerization instead of TLR2/TLR6, could lead to similar changes in Fc␥R expression and function. To begin, we tested function by treating PBM overnight with or without 100 ng/ml Pam3CSK4 and measuring phagocytic ability. Results showed that Pam3CSK4 significantly enhanced the number of targets ingested (Fig. 8A). We then treated PBM overnight with 0, 5, 10, or 100 ng/ml Pam3CSK4 and further incubated cells with or without immobilized IgG for an additional 24 h. ELISAs of cleared supernatants showed that the lowest dose of 5 ng/ml could strongly increase IgG-mediated cytokine production (Fig. 8B). We also measured levels of SHIP and miR-155 after overnight treatment of PBM with 5 ng/ml Pam3CSK4. Pam3CSK4 led to a trend toward decreased SHIP, with 60 -65% decreases in two donors and a 10% increase in one donor (p ϭ 0.11, Fig. 8C). However, there was a significant increase in miR-155 in the same three donors (Fig. 8D).
As with Pam2CSK4, we tested the effects of increasing Pam3CSK4 dosages on Fc␥R expression. Results showed that levels as low as 0.1 ng/ml could increase expression of Fc␥RIIa (Fig. 8A, top panel), and dosages between 1 and 5 ng/ml increased ␥-chain expression (Fig. 9A, bottom panel). As with FIGURE 4. Minimal dose required to enhance Fc␥R-mediated function. PBM were treated overnight with vehicle or with the indicated concentrations of Pam2CSK4 at 37°C. A, following treatment, phagocytosis was measured as described in Fig. 1. B, following treatment, PBM were incubated in 96-well plates with or without immobilized IgG for 24 h at 37°C. Supernatants were harvested and analyzed for TNF␣ by sandwich ELISA. The mean of three separate donors is plotted. UT, untreated. Pam2CSK4, dosages above 5 ng/ml did not lead to further increases (Fig. 9B). These results suggest that TLR2 activation itself, whether through heterodimerization of TLR2 with TLR1 (Pam3CSK4) or, as with Pam2CSK4, through TLR6-(31) or non-TLR6-containing complexes (32), can drive positive changes in Fc␥R expression and function.

DISCUSSION
Immunomodulatory agents have been extensively studied for their potential to enhance antitumor responses. Here we have shown that the TLR2 agonist Pam2CSK4 and the related triacylated Pam3CSK4 can modulate both the expression and function of Fc␥R. Both compounds led to up-regulation of all Fc␥R including, to some degree. the inhibitory Fc␥RIIb. Pam2CSK4 increased monocyte phagocytic ability in vitro and significantly enhanced the effect of antitumor antibody treatment in vivo.
Although the TLR2 agonists up-regulated the activating Fc␥R, they also led to increases in the inhibitory Fc␥RIIb. These higher levels of Fc␥RIIb should have resulted in at least partially attenuated Fc␥R activity. However, although expression of Fc␥RIIb was elevated, we simultaneously observed consistent decreases in the inositol phosphatase SHIP (Fig. 3A), which mediates the inhibitory function of Fc␥RIIb. Hence, Fc␥RIIb function was likely compromised despite modestly increased expression. In this sense, the up-regulated Fc␥RIIb would have likely functioned primarily to enhance binding to opsonized target, as its downstream inhibitory functions were compromised.
Up-regulation of miR-155 appears to be a characteristic of TLR activation, as agonists for TLR2, TLR3, TLR4, and TLR9 (33), as well as TLR7/8 (34), induce miR-155 expression. Within the context of Fc␥R, the concomitant down-regulation of SHIP (28) following miR-155 induction would permit stronger signaling events, leading to more effective responses such as cytokine release and phagocytosis. Although clearly not the only effect of TLR2 activation, this miR-155-SHIP response is likely one of the major mechanisms of enhanced Fc␥R response. Indeed, the importance of SHIP as a negative regulator within the monocyte Fc␥R pathway has been well documented (35)(36)(37).
The response of Fc␥RIIb is also interesting in that it is in stark contrast to results seen with the TLR7/8 agonist R-848. (24) R-848 led to rapid decreases in Fc␥RIIb protein, followed by a decrease in transcript. Pam2CSK4, conversely, elicited modest increases in Fc␥RIIb protein as well as marked increases in tran- FIGURE 6. Pam2CSK4 enhances Fc␥R-mediated function and expression in murine BMM. BMM were treated overnight with or without 100 ng/ml of Pam2CSK4. A, treated and untreated BMM were incubated with fluoresceinated SRBC for 30 min at 37°C and scored by fluorescence microscopy for the phagocytic index. B, treated versus untreated BMM were incubated in 96-well plates with or without immobilized IgG for 24 h at 37°C. Supernatants were harvested and analyzed for TNF␣ by sandwich ELISA. The graphs represent data obtained from three independent donors. Data were analyzed by Student's t test. *, p Ͻ 0.05. UT, untreated. script. Results were similar when testing Pam3CSK4, suggesting that the difference is more related to TLR2 itself than to which TLR2-binding partner was employed. This is in agreement with Farhat et al. (13), who found that TLR2/1 and TLR2/6 activation led to similar downstream signaling activities. Regarding TLR2 versus TLR7/8, perhaps a kinomics approach comparing the intracellular signaling events downstream of the different TLR would shed light on why activation of one but not the other TLR would down-regulate Fc␥RIIb.
In summary, we have found that TLR2 agonists are effective agents for enhancing Fc␥R expression and function. These agonists are currently being examined as putative agents to aid with vaccine efficacy (14), ischemia/reperfusion injuries (15), sepsis (16), and allergies (17). Results from this study suggest that they are powerful modulators of Fc␥R and should, therefore, also be tested as candidate adjuvants for antitumor antibody therapy.