Specific Deletion of LDL Receptor-Related Protein on Macrophages Has Skewed In Vivo Effects on Cytokine Production by Invariant Natural Killer T Cells

Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ). We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.


Introduction
The activation of invariant natural killer T (iNKT) cells has been shown to impact disease progression in mouse models of human disease such as multiple sclerosis [1,2], atherosclerosis [3,4] systemic lupus erythemathosus [5], cancer [6] and pathogenic infection [7]. In humans and mice, iNKT cells rearrange their T cell receptor (TCR) to express Va24-Ja18 and Va14-Ja18, respectively [8]. This allows iNKT cells from both species to recognize similar glycolipid antigens and elicit potent immune responses. Unlike conventional adaptive immunity mediated by CD4 + and CD8 + T cells that requires 4-6 days to initiate, the activation of iNKT cells occurs quickly after in vivo glycolipid challenge leading to a rapid secretion of effector cytokines including IL-4, IL-12 and IFN-c which can be detected as early as 90 minutes following activation [9]. Additionally, iNKT cell responses are known to enhance the initial phase of immunity by increasing NK, B and T cell activation. This effect, known as immune transactivation, is thought to link innate immunity to adaptive immune responses [10] capable of fending off infection [11] and priming effector CD4 + and CD8 + T cells [12]. Therefore, modulation of iNKT cell responses is a potential therapeutic approach for cancer, autoimmunity and chronic inflammatory disorders.
Although research has vastly increased our knowledge of iNKT cell function, less is known about the cellular components in antigen presenting cells (APCs) that present glycolipid ligands to activate iNKT cells. Studies focused on understanding iNKT cell immunity have shown that proteins classically involved in lipoprotein metabolism, such as the microsomal triglyceride transfer protein (MTP) [13], low density lipoprotein receptor (LDLr) [14], scavenger receptors (SRs) [15], and cholesterol membrane transporters [16] can modulate iNKT cell homeostasis and activation. The mechanisms by which MTP, LDLr and SRs on APCs modulate iNKT cell activation are unknown, but data suggest a critical role in glycolipid uptake and presentation. MTP has been shown to be essential in the loading of endogenous lipids into the hydrophobic pocket of CD1d [17] serving to stabilize this molecule for appropriate expression. MTP-deficient APCs lack CD1d expression on the cell surface and iNKT cells fail to develop in fetal thymus organ culture when treated with MTP inhibitors [18]. On the other hand the LDLr binds to apoE present on the surface of many lipoproteins which can function as vessels for glycolipid transport. Targeting glycolipid uptake via the LDLr in APCs enhances CD1d-mediated antigen presentation and leads to increased iNKT cell activation [14,19]. This shows that molecular pathways related to lipoprotein homeostasis and metabolism are closely linked to the modulation of iNKT cell function.
A similar protein to LDLr initially described in lipoprotein metabolism, but later found to bind approximately 30 different ligands, is the LDL receptor-related protein (LRP, also referred to as CD91) [20]. In mice, the LRP was initially discovered in hepatocytes [21], but is currently thought to be expressed in most cells of the body, including many immune cells [22][23][24][25][26]. The expression of LRP on APCs has been shown to enhance the adaptive immune response by facilitating antigen uptake [27][28][29]. . LRP can modulate innate immune responses by binding pseudomonas exotoxin A [30], rhinovirus particles [31] and collectins [32]. For iNKT cell activation, the LRP may be important by binding apoE-containing lipoproteins [33], facilitating processing of the lipid transport proteins called saposins (necessary for CD1d loading) [34] and/or by interacting with the endoplasmic chaperone protein calreticulin on the cell's membrane leading to phagocytosis of opsonized pathogens and apoptotic bodies [35]. Given these characteristics of LRP and the fact that it can actively recycle through endocytic comparments [36], we hypothesized that this surface receptor plays an active role in glycolipid antigen presentation and subsequent activation of iNKT cells. In this study, we demonstrate that LRP is highly expressed in specialized macrophages (MWs) capable of iNKT cell activation. We also show that MW LRP can modulate iNKT activation in vivo and is necessary for IL-4, but not IFN-c secretion.

Animals
Lysozyme M (LyzM)-Cre +/-,LRP flox/flox (hereafter referred to as LRP-cKO) mice were a kind gift from Dr. Sergio Fazio (Department of Medicine, Vanderbilt University Medical Center) and have been described previously [37]. Briefly, mice harboring loxP sites flanking the LRP gene [38] were crossed with mice expressing cre-recombinase under the control of macrophage LyzM promoter [39]. Littermate LyzM-Cre -/-, LRP fl/fl were used as wild type (hereafter referred to as WT) controls. For some experiments, C57Bl/6 mice were used as indicated. B6.PL-Thy1, a./CyJ mice were obtained from the Jackson Laboratory. All mice were on the C57BL/6 background. Mice were maintained in the Vanderbilt University animal care facility and had access to food and water ad libitum. All procedures were approved by Vanderbilt University Medical Center's Institutional Animal Care and Use Committee.

Cell isolation
Isolation of leukocytes from liver and spleen has been described previously [40]. In short, spleens were digested with 1 mg/mL collagenase type II (Sigma) in Hank's balanced salt solution (HBSS, Mediatech). Digested spleens were passed through a 70 mm cell strainer and red blood cells lysed by osmotic shock. Livers were perfused with cold PBS, digested with collagenase type II and pressed through a 70 mm cell strainer. Hepatic leukocytes were then isolated from the interface of a 40/60% Percoll gradient (GE Healthcare, Piscatway, NJ). Peritoneal macrophages were collected in PBS 3 days after peritoneal injection of 3% thioglycollate (Fluka).

Enzyme-linked Immunosorbent Assay
Mouse IL-2, IL-4, IFN-c and IgE were measured by standard sandwich ELISA according to the manufacturer's protocol (BD Pharmingen).

Measurement of In Vitro and In Vivo Responses to aGC
Splenocytes were plated at 2.5610 5 cells/well in RPMI (Hyclone) media containing 10% FBS (Sigma), penicillin-streptomycin with 50 mmol/L L-glutamine (Gibco) and 50 mmol/L b-2mercaptoethanol (Sigma) with the indicated concentrations of aGC. Supernatants were collected after 72 hours of culture and cytokine levels determined by ELISA. For peritoneal MWs, 1.0610 5 cells/well were incubated with 1.0610 5 of murine iNKT hybridoma DN32.D3 (from Dr. Albert Bendelac, University of Chicago, described in [41]). Supernatants were collected 48 hours after culture and IL-2 measured by sandwich ELISA. For in vivo measurements, 4 mg, 1 mg or 0.5 mg aGC reconstituted in vehicle buffer (0.5% polysorbate) was i.p. injected in a total volume of 200 ml. An equal volume of vehicle was injected in to control mice. At the specified times following injection, plasma was collected and splenocytes were isolated, stained and analyzed by flow cytometry.

Pulse of Peritoneal MWs with BODIPY-aGC
Peritoneal MWs (pMWs) were obtained as described previously. A total of 5610 5 cells/tube were incubated with 1 mg/mL BODIPY-aGC (gift from Dr. Paul Savage, Brigham Young University) and harvested at the time points noted in the text. For the pulse-chase experiment, pMWs were preincubated with aGC for 4 hours. After this time, cells were washed an incubated with BODIPY-aGC.

Immunization with aGC and induction of Immunoglobulin E (IgE)
Mice were injected with 4 mg/mouse of aGC or vehicle buffer (0.5% polysorbate). Serum was collected prior to injection and also 6 days after immunization to measure IgE levels.

Statistical Analyses
Statistical analyses were conducted using PRISM V.5.0 software (GraphPad, La Jolla California, USA). For direct comparison between two groups, an unpaired Student's t-test was used and for comparisons made between three or more groups a one-way analysis of variance (ANOVA) was performed. Values are expressed as mean 6 standard error of the mean unless otherwise noted. A p value ,0.05 was considered statistically significant.

LRP is differentially expressed on immune cells
We measured LRP expression on immune cells isolated from tissues in which iNKT cells are known to reside and encounter antigen. We collected spleen, liver and lymph nodes from C57Bl/6 mice and stained with fluorescently labeled antibody (5A6) specific for the 85 KDa subunit of LRP [42].

CD169 + MWs express high levels of LRP
Recent reports have shown that MWs expressing sialic acid binding immunoglobulin-like lectin 1 (Siglec-1, also known as CD169) can mediate potent activation of iNKT cells in secondary lymphoid tissues [43]. CD169 + MWs can cross-present tumor antigens [44] and take up liposomal particles conjugated to CD169 ligands [45]. One such study demonstrated that aGC conjugated to CD169 ligands elicited an iNKT cell response two orders of magnitude higher that administration of free aGC [46]. Interestingly, we found significantly higher LRP expression levels on F4/80 + CD169 + MWs compared to F4/80 + CD169 -MWs in spleen ( Figure 2A) and liver of C57Bl/6 mice ( Figure 2B). These data demonstrate that LRP is highly expressed on MWs known to elicit iNKT cell responses.

Conditional knockout leads to deletion of LRP on MWs
In order to test MW LRP function in iNKT cell activation, we used mice harboring a MW knockout of LRP [37]. In this mouse model, LRP flox/flox mice were crossed with LyzM-Cre mice, which have cre recombinase under the control of the lysozyme M promoter. Flow cytometry staining of splenic and liver MWs ( Figure 3A and 3B) shows that, compared to WT, LRP-cKO mice have decreased LRP expression on splenic and hepatic MWs (71% and 68%, respectively). As expected, other immune cells in LRP-cKO mice such as T cells, B cells and DCs had no reduction of LRP expression, verifying MW specificity. Further analysis of LRP-cKO MWs subpopulations showed that CD169 + MWs have a 53% reduction in LRP expression (Figure 2A-B). Taken together, these data show that LRP-cKO mice have a deletion of LRP on MW, including specialized CD169 + that are capable of presenting antigens to iNKT cells.

LRP deficiency on MWdoes not alter CD1d expression or iNKT cell homeostasis
We asked whether LRP deletion in MWs leads to changes in CD1d expression or iNKT cell homeostasis. LRP deletion had no effect on CD1d expression either on spleen MWs, liver MWs, or double positive (CD4 + CD8 + ) thymocytes ( Figure S1A). Cocultures of WT or LRP-cKO thymocytes with iNKT hybridoma (DN32.D3) led to similar levels of IL-2 secretion ( Figure S1B), showing that LRP-cKO thymocytes have no defects in presentation of endogenous glycolipid ligands. Analysis of thymic cells demonstrates normal expression of development markers in LRP-cKO iNKT cells when compared to WT ( Figure S2). Additionally, iNKT cells, isolated from spleen and liver were present at normal frequencies ( Figure S3A-C) and expressed normal levels of the prototypic markers of homeostasis and activation ( Figure S3D and S3E). Finally, in vitro uptake of BODIPY-labeled aGC was not affected by LRP deficiency on MWs as measured by flow cytometry (Figure S4A and S4B). However, a pulse-chase experiment where unlabeled aGC was used to pulse perotineal macrophages (pMWs) prior to chase with BODIPY-aGC showed a faster turnover of LRP-cKO pMWs as BODIPY-aGC was higher than WT pMWs ( Figure S4C). This difference is observed as early as 4 hours but reaches statistical significance only at 8 and 16 hours. These data suggests that LRP deletion in MWs does not affect uptake of glycolipids but can alter cellular glycolipid turnover.

LRP deficiency on MWs does not affect iNKT activation in vitro
Although we did not see changes in uptake of aGC by LRPdeficient MWs, LRP is also important for cell signaling. Therefore, to determine whether decreased LRP expression modulates iNKT cell activation, we first harvested thioglycollate-elicited pMWs from WT and LRP-cKO mice and determined their LRP expression. Similar to previous reports [37], we found a 63% decrease in LRP expression on LRP-cKO pMWs when compared to WT ( Figure 4A and B). To measure iNKT cell activation, pMWs were pulsed with aGC and incubated with iNKT cell hybridoma, DN32.D3, and secreted IL-2 was measured in 48 hour culture supernatants by ELISA. We observed a slight but reproducible decrease in IL-2 when iNKT cells were incubated with LRP-cKO pMWs ( Figure 4C). Unfortunately, this did not reach statistical significance. In vitro incubation of freshly isolated splenocytes from WT or LRP-cKO mice with aGC for 24, 48, 72 hours likewise showed no statistically significant difference in IFN-c or IL-4 levels in culture supernatants ( Figure S5), but a slight reduction in IL-4 at 48 hours of culture. These data suggest that in vitro activation of iNKT cells by MWs is not dependent of LRP expression.
Although we did not see an effect of MW-specific deletion of LRP on in vitro iNKT cell responses to aGC, we were interested in understanding whether the magnitude and/or kinetics of the in vivo iNKT cell response to aGC might be altered in LRP-cKO mice where the spatial integrity of the secondary lymphoid tissues would remain intact. To do this, we challenged WT and LRP-cKO mice with aGC (4 mg/mouse i.p.) and measured serum IFNc and IL-4 at 2, 12 and 24 hours. ELISA results from this experiment show the near absence of IL-4 production by iNKT cells in LRP-cKO mice ( Figure 5A) However, the kinetics and levels of IFN-c were not affected by LRP deficiency on MWs).Lower doses of aGC (1 mg and 0.5 mg per mouse) did not recapitulate a similar difference between WT and LRP-cKO mice ( Figure S6). In addition to this we measured a statistically significant reduction of IL-4 + iNKT cells ( Figure 5B) and normal IFN-c + iNKT cells (Data not shown) in LRP-cKO cells by intracellular cytokine stain. The presence of normal IFN-c responses is supported by similar levels of transactivation of T cells and NK cells as measured by CD69 upregulation (data not shown). These results suggest that, in vivo, MW LRP is not required for IFN-c secretion, but can impact production of IL-4 by iNKT cells.

Decreased IL-4 expression by iNKT cells in LRP-cKO mice is cell extrinsic
Although iNKT cells in LRP-cKO mice should not be directly affected we wanted to confirm that decreased IL-4 production in response to aGC was not due to an iNKT cell intrinsic deficiency. To do this we generated bone marrow chimeras using a 1:1 ratio of bone marrow from Thy1.1 and LRP-cKO mice (Thy1.2) transplanted into recipient WT mice at a 1:1 ratio. Four weeks after transplant, mice were injected with aGC and splenocytes analyzed for intracellular IL-4 cytokine stain. The results from this experiment ( Figure 5C) show the production of IL-4 by Thy1.2 + iNKT cells (LRP-cKO) is rescued to Thy1.1 + iNKT cell levels in the 1:1 mixed bone marrow chimeras. This result suggests that the decrease in IL-4 measured in LRP-cKO mice is cell extrinsic and that iNKT cells from LRP-cKO mice can respond normally in the presence of LRP-sufficient macrophages.

LRP-cKO mice have a normal IgE response when challenged with aGC
To determine whether lack of IL-4 response in LRP-cKO mice to aGC could lead to a deficiency in the adaptive immune response, we measured production of IgE. It has been established that a single dose of aGC can lead to a robust increase in the presence of serum IgE 6 days after aGC injection [47]. To test this, we injected WT and LRP-cKO mice and obtained serum at day 6 and measured IgE levels by ELISA ( Figure 6). Titration curves of IgE in this experiment show that, despite a lack of early burst of Effect of Macrophage LRP on NKT Cell Activation PLOS ONE | www.plosone.org IL-4, LRP-cKO mice are still able to produce a normal, although reduced, IgE response. This result suggests that, while LRP deficiency in macrophages decreases iNKT cell IL-4 production, the main cytokine involved in IgE production, it is not necessary for increased IgE.

Discussion
Several studies have reported that activation of the immune system can reciprocally regulate the expression of lipoprotein receptors in APCs. For example, exposure of macrophages to IFNc can exert dramatic changes of LRP [48,49], very low density lipoprotein (VLDL) receptor [50], and scavenger receptors (SRs) [51]. Far less however, is known about how these changes in lipid-binding receptors might influence immune responses. LRP's ubiquitous nature, ligand diversity and involvement in intracellular signaling cascades allow it to be a receptor capable of affecting a multitude of cellular processes. Unfortunately, the examination of LRP's expression and function has been largely confined to human cell lines [52,53], mouse cell lines [54,55] and primary mouse cells [26,37]. The LRP flox/flox mouse model [38] has allowed a wide number of conditional knockouts which verify its multicellular distribution and function [29,56,57]. By crossing it with LyzM-Cre mice, we observed close to a 70% reduction of LRP in primary cells (Figure 3), which is reminiscent to other Cre +/models such as the adipocyte specific aP2-Cre and neuron specific synapsin-Cre [58]. In all these cases a full deletion was not observed, but rather a near complete reduction of LRP. In this study, we show that in the steady state, T cells express low levels of LRP followed by B cells. DCs express a small amount of LRP, but MWs express higher levels of LRP (Figure 1). This demonstrates that on immune cells, LRP expression is largely restricted to phagocytes and professional APCs supporting its role in antigen presentation and modulation of immunity.
Using a conditional mouse model of MW LRP knockout, we show that reduction of LRP expression in MWs does not influence iNKT activation in vitro ( Figure 4C and Figure S5), but abolishes IL-4 production in vivo ( Figure 5). This lack of IL-4 response was not the result of altered development and/or homeostasis of iNKT cells ( Figure S3). Because we did not observe changes in iNKT cell activation using single cell suspensions in vitro, but did see a near absence of early iNKT cell IL-4 production following in vivo challenge, we hypothesize that spatial orientation of the LRP expressing macrophages may be important to early iNKT cell responses. We focused our studies on spleen because 1) marginal zone macrophages express receptors that allow them to capture blood borne antigens ( [59], 2) spleen contains an elaborate distribution network which disseminates molecules present in the blood to white pulp where APCs are located [60], and 3) iNKT cells that reside in the spleen are exposed to blood borne antigen within minutes after they enter the bloodstream [43]. Our attempts to measure iNKT cell activation in liver and LN shortly after aGC injection were not successful. Therefore, whether iNKT cells in liver and lymph nodes undergo a similar exposure to blood borne antigens is an issue that needs to be addressed in future studies.
Indeed, previous studies have demonstrated that MWs in the splenic marginal zone (MZ) are the first APCs to activate iNKT cells in response to blood-borne glycolipids [43]. Using time-lapse multiphoton microscopy the authors showed that, following antigen challenge, splenic iNKT cells quickly localize to the MZ and come into contact with MZ CD169 + MWs. Interestingly, when the MZ integrity was compromised following injection of clodronate liposomes, a method of MW-specific depletion, there was a dramatic decrease in iNKT cell cytokine production in response to in vivo challenge with particulate aGC [41]. In vitro comparison of antigen presenting efficiency of DCs and MZ MWs to iNKT cells showed that, although MZ MWs were able to activate iNKT cells in vitro, DCs were clearly superior. Therefore, the data suggest that the spatial interaction of iNKT cells with the MZ MWs soon after injection of aGC is the most plausible explanation for the observed in vivo decreases in iNKT cell activation.
More specifically, it is possible that at 2 hours the localization of LRP-expressing MWs in the MZ induces iNKT cells to secrete IL-4. However, at later times other APCs (such as DCs) are primarily responsible for iNKT cell activation and the resulting IFN-c response. Because in vitro disruption of the spleen to make single cell suspensions would destroy this spatial integrity allowing for iNKT cells to be activated by DCs, this decrease in IL-4 production in response to aGC would not be observed. In support of this hypothesis, our study demonstrates that splenic CD169 + MZ MWs express higher levels of LRP than the CD169 -MWs ( Figure 2). Therefore LRP expression in these CD169 + MWs may influence the early activation of iNKT cells and consequently lead to decreased IL-4 at 2 hours.
The expression of surface receptors that can direct glycolipid uptake and subsequent iNKT cell activation suggests multiple pathways are responsible for delivery to APCs. To date, no specific receptor has been described that carries out this function. In fact, the hydrophobic nature of glycolipids prevents their individual presence in the serum and would require their association to serum-borne particles. Van Den Elzen et al found that apoEcontaining lipoproteins, specifically VLDL could associate with iNKT cell activating glycolipids such as aGC's close family member galactosyl(a1-2) galactosyl ceramide [14]. The investigators went on to demonstrate that apoE-associated glycolipid was efficiently taken up by the LDLr and enhanced iNKT cell activation. More recent studies demonstrate that, like the LDLr, other lipid receptors such as scavenger receptor (SRA) can bind and facilitate uptake of glycolipid antigens by DCs to activate iNKT cells [15]. Preference for receptor/glycolipid interaction was determined to be more dependent on chemical structure of the lipid. Interestingly, both SRA-and LDLr-deficient DCs showed decreased ability to activate the iNKT cell hybridoma DN32.D3 in response to aGC in vitro. However, in vivo challenge of SRA and LDLr-deficient mice with soluble aGC led to the same blunted IL-4 response we observed in LRP-cKO mice ( Figure 5). How these receptors contribute to such different cytokine responses remains to be clarified. One possibility is that all three receptors (LRP, SRA and LDLr) are required for an optimal IL-4 response in vivo. Another may be that loss of any one of these receptors leads to changes in intracellular lipid compositions and that the iNKT cell IL-4 response is sensitive to these changes. We did not observe changes in uptake of BODIPY-aGC in LRP-cKO MWs in our studies ( Figure S4A-B), However, it is possible that this is the result of the limitation of our in vitro system. Surprisingly, in a pulse chase experiment, we did see increased appearance of BODIPY-aGC in LRP-cKO MW indicating that lipid exchange may be accelerated in the absence of LRP ( Figure S4C and S4D). Therefore, one might hypothesize that more rapid replacement of aGC with endogenous lipids in LRP-cKO MWs results in a blunted, early IL-4 response in vivo. Further investigation is warranted to determine whether there are in vivo changes in antigen-uptake or whether loss of these receptors affects lipid rafts, or if other lipid pools important for cell activation are required.
Collectively, this study demonstrates that LRP expression in MWs does not alter total activation of iNKT cells in vivo but, like the LDLr and SRA, LRP plays a role in IL-4 secretion by iNKT cells. This result is remarkable as it suggests a scenario where LRP can be manipulated to modulate iNKT cell responses. Activation of iNKT cells has been associated with altered progression of cancer, autoimmune disorders and atherosclerosis. Understanding how lipoprotein receptors affect their function can lead to targeted therapies to harness the immunoregulatory potential of iNKT cells.  Figure S4 Peritoneal MWs uptake of fluorescently labeled aGC (BODIPY-aGC). pMWs from WT (n = 3) and LRP-cKO (n = 4) were incubated with 1 mg/ml of BODIPY-aGC and harvested at the indicated times. This was followed by incubation with fluorochrome conjugated antibodies for CD11b, CD11c and B220. Shown are (A) representative histograms and (B) MFI quantification in CD11b + CD11c -B220 -pMWs pulsed with unlabeled aGC for four hours and chased with labeled BODIPY-aGC at the indicated hours. BODIPY-aGC (MFI) was measured by flow cytometry MWs. (C) Representative histograms of WT (n = 3) and LRP-cKO (n = 3) and (D) MFI quantification in CD11b + CD11c -B220 -pMWs. Results shown are representative of an experiment from three independent experiments. Bars represent mean and standard error and * denotes p,0.05. (TIF) Figure 6. Induction of IgE in LRP-cKO mice is normal when compared to WT. Both WT (n = 8) and LRP-cKO (n = 6) mice were challenged with aGC (4 mg/mouse) and vehicle (WT, n = 3, LRP-cKO n = 2). Blood was collected at 0 and 6 days and total serum IgE levels measured by ELISA. Bars represent mean and standard error. These results are from one experiment. doi:10.1371/journal.pone.0102236.g006 Figure S5 WT and LRP-cKO splenocytes challenged with aGC. Splenocytes from WT and LRP-cKO mice were stimulated with indicated concentration of aGC for 24, 48 and 72 hours. Supernatants were assayed for IFN-c and IL-4 by ELISA. Results are from one representative experiment of three independent experiments. Data points show standard error and mean of 3 mice in each group. N.S. stands for data sets that are statistically not significant. (TIF) Figure S6 WT and LRP-cKO mice challenged with aGC. Mice were challenged with 1 mg and 0.5 mg aGC/mouse and blood collected at the indicated time points. Serum was assayed for IFN-c and IL-4 by ELISA. Data points show standard error and mean. WT (n = 3 for 2, 12 and 24 hours, respectively) and LRP-cKO (n = 3 for 12 and 24 hours). Results shown are representative of 3 independent experiments. (TIF)