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Negative regulation of innate immune responses is essential in order to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration and activation, and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G-protein coupled receptors, the function of which in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. Here we show that DC from lpa2−/− mice are hyperactive compared to their wild-type counterparts, and are also less susceptible to inhibition by different LPA species. In transient transfection assays, we found that lpa2-overexpression inhibits NF-κB-driven gene transcription. Using an adoptive transfer approach, we found that allergen-pulsed lpa2−/− DC induced substantially more lung inflammation than wild-type DC after inhaled allergen challenge. Finally, lpa2−/− mice develop greater allergen-driven lung inflammation than their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits dendritic cell activation and allergic airway inflammation.
Originally described as an intermediate in intracellular lipid biosynthesis, LPA (or monoacyl-sn-glycero-3-phosphate) is now recognized as a pluripotent extracellular mediator that regulates cell growth, migration and activation (1). Extracellular LPA is generated primarily by hydrolysis of lysophosphatidylcholine (LPC) by the enzyme autotaxin (or lysophospholipase D) (2, 3). A major advance in the field came from the molecular cloning of specific LPA receptors (4–6). There are at least five established LPA receptors three of which belong to the Edg G-protein coupled receptor (GPCR) superfamily: LPA1 (Edg2), LPA2 (Edg4) and LPA3 (Edg7) (7). Signal transduction via these classical LPA receptors leads to activation of mitogen-activated protein kinases, phosphoinositide-3 kinases (PI3K), and Rho kinases, which affect cell activation, survival and migration (see (8) for recent review). Activation of Gαi and PI3K/Akt is emerging as particularly important for LPA-directed cell migration (9–13).
LPA has physiologic roles in wound repair and development (14–20), and emerging roles in disease states including cancer, atherosclerosis, lung fibrosis, and asthma (21–31). LPA may play a broader role in regulating innate and adaptive immune responses (32). For example, constitutive expression of autotaxin in high endothelial venules contributes to lymphocyte homing to secondary lymphoid organs, presumably by inducing local LPA production and T cell emigration from the vasculature(33). Although LPA has pro-inflammatory effects (28), it can also inhibit inflammation in some contexts. For example, intravenously injected LPA protected mice from lipopolysaccharide (LPS)-induced peritonitis (34), and LPA attenuated cytokine secretion in human monocyte-derived dendritic cells (DC) (9, 35). However, the inhibitory mechanisms and receptor(s) by which LPA attenuates immune responses remain poorly defined.
Here we report that LPA2 (edg4) negatively regulates dendritic cell activation and allergic airway inflammation using different mouse models of asthma. Compared to wild-type controls, lpa2−/− dendritic cells exhibit a hyper-active phenotype both in vitro in DC:T cell co-culture, and also following adoptive transfer into the mouse airway. Whereas LPA inhibits activation of wild-type DC in response to different pattern recognition receptor ligands, lpa2−/− DC are resistant to LPA-dependent inhibition. In transfection studies, we show that expression of LPA2 inhibits LPS-induced NF-κB activation. Finally, we studied allergic airway inflammation using ovalbumin (Ova) as model allergen and protocols known to induced DC activation by either systemic immunization (Ova + alum i.p.) or mucosal immunization (inhaled Ova + low dose LPS), and found that lpa2−/− mice develop greater allergen-driven lung inflammation than their wild-type counterparts. Taken together, these studies uncover a novel anti-inflammatory role for lpa2, and identify a new pathway involved in suppression of innate immune responses.
Wild-type mice on the C57BL/6 background were from Jackson Labs. LPA2-deficient (lpa2−/−) mice were derived from frozen gene-targeted embryos provided by Deltagen Inc. (San Mateo, CA) in collaboration with GlaxoSmithKline, and backcrossed more than six generations onto the C57BL/6 background. Wild-type and gene-targeted mice were maintained at the University of Rochester, and age- and gender matched littermate controls were used in all experiments. C57BL/6.PL OT-II T cell receptor (TCR) transgenic mice recognizing Ova peptide Ova323–339 in the context of Iab were a gift of Dr. David Topham (University of Rochester). Mouse protocols were reviewed by the University of Rochester Committee on Animal Resources, and GSK Institutional Animal Care and Use Committee, and were conducted in accordance with institutional guidelines and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals.
Dendritic cells were derived from bone marrow precursors using modifications of previously published protocols (36). Briefly, wild-type and lpa2−/− mice were euthanized and prepared aseptically to remove femurs and tibias for bone marrow harvest. On day 0, bone marrow cells were seeded at a density of 1×106 cells/ml in RPMI-1640 media supplemented with 10% heat inactivated FBS (Tissue Biologicals, Los Alamitos CA, lot # 103057), 1 M Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 20 mM βME and 50 mg/ml of gentamycin using 6 well plates (media and additives from Gibco, Carlsbad CA), supplemented with 25 ng/ml of GM-CSF (Peprotech, Rocky Hill NJ). The media was changed and supplemented with the same concentration of GM-CSF together with 10 ng/ml of IL-4 (Peprotech) on days 2, 5, and 7. BM-DC grown using this protocol typically express CD11c, high levels of MHCII (Ia/Ie) and CD11b, and low levels of CD8α and PDCA1 (antibody panels available upon request). On day 8, dendritic cells were harvested and co-cultured with allogeneic T cells at dendritic cell to T cell ratios of 1:1, 1:5, 1:25, 1:125, and 1:625. Cell proliferation was then analyzed 72 hours following co-culture using a BrdU Cell Proliferation ELISA (Roche). Cell supernatants were analyzed by ELISA or multiplex cytokine/chemokine bead array using commercially available kits according to manufacturer’s instructions (ELISA [Detection limit]: IL-6 [1.6 pg/ml], TNFα [1.8 pg/ml], VEGF [3 pg/ml], Quantikine kits from R&D Systems; Bead Array: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, TNF-α, eotaxin, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α, MIP-1β, RANTES, and KC, Bio-Plex Pro™ Mouse Cytokine 23-plex Assay, Biorad, Hercules, California).
HEK293T cells stably transfected with TLR4 and MD2 (a kind gift of Dr. Jian Dong Li, URMC) were maintained in Dulbeco’s modified Eagle medium (Gibco Invitrogen Corporation, Carlsbad, CA) containing heat inactivated 10% fetal bovine serum (Tissue Culture Biological, Los Alamitos, CA), 100 µg/ml streptomycin, 100 U/ml penicillin and 0.5 mg/ml geneticin (Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. Confluent HEK293-TLR4/MD2 cells were detached with 0.05% Trypsin-EDTA (Invitrogen, Carlsbad, CA) and passed every 2–3 days. A full-length lpa2 cDNA expression vector was obtained from University of Missouri-Rolla cDNA Resource Center (Rolla, MO). HEK293-TLR4/MD2 cells were grown to ~80% confluency and transfected using jetPRIME transfection reagent (Polyplus Transfection, New York, NY) according to the manufacturer’s protocol. Briefly, cells were seeded in 10% serum DMEM on poly-D-lysine coated 24-well plates for 24 hours. 1 ug of plasmid DNA and 2 ul of jetPRIME transfection reagent were mixed with transfection buffer and incubated for 10 minutes at RT, after which 25 ul of the mixture was added to each well. For NFκB reporter assay, cells were transfected with pNFκB-luc (Stratagene) and pcDNA-3HA-LPA2 or pcDNA empty plasmid at 1:1 ratio using jetPRIME transfection reagent as described above. Twenty-four hours after transfection, cells were washed with PBS and the medium was replaced with 1% serum DMEM containing 0.5 mg/ml geneticin with or without LPS as indicated, and then incubated with reporter lysis buffer and reporter gene activity measured by luminometry using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Inc., San Diego, CA) according to the manufacturer’s protocol. Protein concentration was determined by Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL).
HEK293T cells were collected and lysed with RIPA buffer supplemented with protease inhibitors. Protein concentrations were determined using Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Protein samples were prepared by adding 6x Laemmli sample buffer to a final concentration of 1x plus an equal amount of 8 M urea and incubated at RT for 20 min with occasional mixing. The samples were then loaded on 10% SDS gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% milk/TBST for 1 hour at RT. Anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted at 1:200 in 5% milk/TBST and incubated with the membrane overnight at 4°C. After washing with TBST, the membrane was incubated with 1:1000 anti-mouse-HRP conjugate (Cell signaling Technology, Danvers, MA) for 1 hour at RT. The signal was developed using Amersham ECL Plus (GE Healthcare, Buckinghamshire, UK). As a loading control the membrane also was also probed for GAPDH (ab8245, 20 ng/ml final, Abcam Inc., Cambridge, MA).
Cell culture was carried out using medium supplemented with 10% FCS unless otherwise indicated. The following reagents were used in cell culture experiments to stimulate cells. E. coli lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO) was prepared in PBS. Pertussis toxin was from Calbiochem (San Diego, CA). Different species of lysophosphatidic acid including 16:0 (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate), 18:1 (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate), and 20:4 LPA (1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphate, all from Avanti Polar Lipid, Inc., Alabaster, AL) were stored in methanol:H20. Working solutions were prepared fresh by evaporating methanol under N2 gas and dissolving the residue in PBS containing 1% fatty acid free BSA. Ki16425 was kindly provided by Dr. Andrew Tager (Harvard University), and wortmannin was purchased (Calbiochem).
Wild type and lpa2−/− bone marrow derived dendritic cells were generated as described above. On day 8, dendritic cells were plated at 8×106 cells per condition and pulsed with 400 µg/ml of Grade V Ova (Sigma) or saline control. On day 9, 1×106 Ova and control wild type and lpa2−/− dendritic cells were intratracheally instilled by orophayngeal aspiration (50 ul) into wild type recipients. The recipients were then aerosol challenged with Grade V Ova inhalation on days 19, 20, 21 and 22 (1%, 30 minutes) and sacrificed for analysis of airway inflammation.
Female wild-type and lpa2−/− littermates were used at 6–8 weeks of age. We used systemic and mucosal protocols to sensitize and challenge mice using ovalbumin (Ova) as a model allergen. In the systemic protocol, mice were immunized by intraperitoneal injection of Grade II Ova (20 µg and 100 µg) plus alum (1.3 mg and 6.5 mg Thermo Scientific) on days 0 and 14 respectively, followed by aerosol challenge with Grade V Ova inhalation (1%, 30 minutes) on days 24, 26, and 28. Some mice were sacrificed on day 10 for analysis of Ova-specific serum IgE levels using a commercially available ELISA (mdbiosciences). In order to immunize mice via the airway, separate groups of mice were exposed to Grade V Ova (100 µg, Sigma) purified by Endotoxin Removing Columns (Thermo Scientific) either alone or together with low-dose LPS (100 ng, Sigma) by orophayngeal aspiration (50 ul), followed by aerosol challenge with Grade V Ova inhalation on days 14, 15, and 16 (1%, 30 minutes) as indicated in the text. Bronchoalveolar lavage (BAL) was collected for analysis of cell counts and differentials using standard techniques as previously reported (37). BAL supernatants were analyzed for expression of IL-13 and VEGF using commercially available ELISA kits (eBioscience). Some lungs were inflated with 4% PFA, embedded in paraffin, and 8 micron sections stained using H&E staining. Lung sections were scored by blinded observers using a semi-quantiative scoring system that takes into account extent and severity of inflammation on a 0–4 scale.
Female wild-type and lpa2−/− littermates were exposed to 1000 rads delivered via a 137Cs source using whole body irradiation without shielding. The radiation was delivered in a split dose (500 rads) 4 hours apart. Immediately following the second round of irradiation, the mice were infused with 1×107 bone marrow cells from wild-type and lpa2−/− donor mice via the tail vein. To permit complete chimerism, we allowed 6 weeks of reconstitution before sensitizing and challenging wild type and lpa2−/− bone marrow chimeras using the mucosal immunization protocol described above.
Using primers specific for the classical Edg family of LPA receptors, we confirmed that bone-marrow derived dendritic cells (DC) constitutively express lpa1, lpa2 and lpa3, as previously reported for human monocyte-derived DC (Figure 1A) (35). The yield of cells and cell surface expression of MHC II and co-stimulatory molecules including CD40, CD80 and CD86 was similar when comparing DC derived from wild-type and lpa2−/− littermates (data not shown). However, when comparing the ability of DC to stimulate allogeneic naïve CD4+ T cells in co-culture assays, we found that lpa2−/− DC induced significantly more T cell proliferation than their wild-type counterparts, especially at lower DC:T ratios (black bars, Figure 1B). In co-culture supernatants, we detected almost twice as much IL-13 secretion from T cells stimulated with lpa2−/− DC compared to their wild-type counterparts (Figure 1C). In contrast, DC obtained from lpa1 gene-targeted mice behaved similar to wild-type mice in these assays (gray bars, Figure 1). Pre-treating DC with the LPA1/3 antagonist Ki16425 had no effect on the ability of DC to induce T cell proliferation or activation(38), whereas the PI3K inhibitor wortmannin (0.1–10µM) inhibited the ability of both wild-type and lpa2−/− DC to stimulate T cells equally well (see Supplementary Figure 1, and data not shown). These data indicate that in the absence of lpa2, DC acquire a hyperactive phenotype, and suggest that LPA2 is an inhibitory receptor that attenuates DC activation.
We explored this possibility further using two different approaches. First, we studied the secretion of inflammatory and immunoregulatory cytokines by wild-type and lpa2−/− DC in response to lipopolysaccharide (LPS), which is known to activate DC in a TLR4/Myd88-dependent manner. We first confirmed that the expression of TLR4 was not affected by lpa2-deficiency (data not shown). When compared to their wild-type counterparts, we found that when stimulated in the presence of 10% serum, lpa2−/− DC secreted significantly more VEGF than their wild-type counterparts, whereas secretion of other cytokines including IL-6, IL-12p70 and TNF-α was similar between genotypes (Supplementary Figure 2 and data not shown). One explanation for this finding is that LPA present in serum-containing tissue culture medium is sufficient to suppress LPS-dependent DC activation in an lpa2-dependent manner. To test this possibility, we next stimulated wild-type and lpa2−/− DC under reduced serum conditions with LPS (1 µM) alone or together with 16:0, 18:1, or 20:4 LPA (1–10 µM). If lpa2 normally inhibits DC activation, then wild-type DC should be more susceptible to LPA-dependent inhibition than lpa2−/− DC. Figure 2 shows that exogenous LPA inhibited LPS-driven IL-6 secretion from wildtype (open bars) but not lpa2−/− DC (closed bars), where the data are expressed as relative inhibition compared to cells stimulated with LPS alone and the dotted line indicates no inhibition. Table 1 shows that under reduced serum conditions, LPS-stimulated lpa2−/− DC secreted significantly less IL-10 and more TNF-α than their wild-type counterparts, whereas IL-12p70 secretion was comparable between genotypes. Furthermore, three exogenous LPA species partially suppressed LPS-driven TNF-α secretion in wild-type DC, an effect that was lost using lpa2−/− DC (Table 1). Interestingly, both saturated and unsaturated LPA species inhibited LPS-driven IL-6 and TNF-α secretion, which contrasts with the preferential ability of unsaturated LPA species to induce DC migration in vitro (39) (see Discussion).
Signal transduction via the TLR4 receptor complex is known to induce cytokine secretion in an NF-κB-dependent manner. To test the possibility that lpa2 interferes with NF-κB-dependent gene expression, we used HEK293T cells stably expressing TLR4 and MD2, which do not express LPA2 at baseline (data not shown). We first confirmed that after co-transfection with a full-length expression vector, LPA2 is expressed in these cells and localizes to the cell membrane (Supplementary Figure 3, and data not shown). As expected, LPS induced transcriptional activation of an NF-κB-driven reporter construct in cells co-transfected with an empty expression vector (Figure 3). In contrast, LPS-dependent NF-κB activation was significantly attenuated in LPA2-expressing cells. Levels of secreted IL-6 were at or below detection limits in these experiments (data not shown). Treatment with exogenous16:0 LPA alone or in combination with LPS did not result in additional inhibition of reporter gene activity (data not shown). Interestingly, transient transfection of an LPA1 expression vector also attenuated LPS-dependent NF-κB activation in HEK293T cells expressing TLR4/MD2 (N. Meednu, unpublished observations): the mechanisms and consequences of this effect are being pursued in a separate study. Taken together, these data support the idea that endogenous serum LPA inhibits LPS-induced NF-κB-dependent gene expression at least in part in an lpa2-dependent manner.
Many of the effects of LPA2 are mediated by coupling with Gαi. If lpa2 were inhibiting DC activation in a Gαi-dependent manner, we reasoned that we should be able to augment the activation of wild-type more than lpa2−/− DC using pertussis toxin (PTX). Figure 4 shows that pre-treatment with PTX significantly augmented the ability of wild-type DC to induce T cell proliferation in a dose-dependent manner. Similar to Figure 1, we found that untreated lpa2-deficient DC induced more proliferation in responding T cells than their wild-type counterparts (white bars, p<0.05 vs. wild-type), and in contrast to wild-type cells, pre-incubation with PTX had no significant effect on the T cell stimulating capacity of lpa2-deficient DC. Since no exogenous LPA was supplied in this experiment, these data further support the idea that LPA present in serum transduces a tonic inhibitory signal that dampens DC activation via LPA2.
Using in vitro assays, we found that lpa2-deficient DC induced more T cell proliferation and IL-13 production, and secreted more VEGF than their wild-type counterparts, suggesting that they may promote Th2-driven allergic immune responses in vivo (40, 41). In order to test this possibility, we used an adoptive transfer model in which wild-type mice received allergen-pulsed wild-type or lpa2−/− DC by intra-tracheal administration, followed by aerosol allergen challenge using ovalbumin (Ova) as model allergen (modified from (42–44)). Mice receiving control, saline-pulsed DC followed by Ova aerosol challenge developed little or no lung inflammation as determined by BAL cell counts (>95% macrophages), whereas significant lung eosinophilia developed in mice that received Ova-pulsed wild-type DC followed by Ova aerosol challenge (Figure 6B, and not shown). Interestingly, adoptive transfer of lpa2-deficient, Ova-pulsed DC resulted in substantially more lung inflammation than transfer of wild-type DC following Ova challenge (Figure 5). Taken together with results shown in Figures 1–4, these data indicate that lpa2-deficient DC are hyperactive in both in vitro and in vivo assays.
Finally, we compared wild-type with lpa2-deficient mice in two models of allergic airway inflammation known to involve DC activation. First, we used the well-established model of systemic immunization using intraperitoneal injection of Ova plus alum. Second, we used a mucosal immunization approach and sensitized mice with inhaled endotoxin-free Ova using low-dose LPS (100 ng) as adjuvant (45). Using both systemic and mucosal immunization approaches, we found that lpa2-deficient mice developed greater allergic sensitization, airway inflammation, and airway hyperreactivity, and than their wild-type counterparts (Figures 6–7). In the Ova plus alum model, lpa2-deficient mice developed more airway eosinophilia and higher BAL IL-13 and VEGF levels at 48 and 72 hours following allergen challenge (Figure 6A–C, and data not shown), indicative of greater Th2-driven allergic airway inflammation. In order to determine whether lpa2-deficiency resulted in augmented allergen sensitization, we sacrificed a separate group of mice 10 days after Ova plus alum immunization, and found that serum Ova-specific IgE levels were almost twice as high in lpa2–deficient mice compared to wild-type controls (Figure 6D).
Mucosal immunization protocols result in less severe airway inflammation than systemic immunization following recall allergen challenge, but are a more physiological route of allergen encounter. Using the approach described by Eisenbarth et al. with low-dose LPS as inhaled adjuvant(45), we found that lpa2-deficient mice developed greater influx of inflammatory cells into BAL fluids than their wild-type counterparts 48 hours after allergen challenge (Figure 7A). Interestingly, although the percentages of eosinophils and neutrophils were similar between groups (eosinophils: 22±2% vs. 17±2% and neutrophils: 8±2% vs. 11±3%, wild-type vs. lpa2 knock-out, respectively, mean±SEM of n=9–11), airway hyper-reactivity measured in sedated and paralyzed mice was significantly greater in lpa2-deficient mice compared to wild-type controls (Figure 7B). In order to investigate the requirement for LPA2 in hematopoietic and non-hematopoietic cells in suppressing allergic lung inflammation following mucosal immunization, we used reciprocal bone marrow chimeras. We found that wild-type mice reconstituted with lpa2-deficient bone marrow developed significantly greater airway inflammation than those reconstituted with wild-type bone marrow (Figure 7C). Furthermore, lpa2-deficient recipients of either wild-type or lpa2-deficient bone marrow reacted similarly to wild-type recipients of wild-type bone marrow. These data indicate that lpa2 expression by a radiosensitive bone marrow-derived cell(s) normally restrains allergic lung inflammation.
Using complementary approaches, we uncovered a novel role for lpa2 (Edg4) in suppressing dendritic cell activation and allergic immune responses. Dendritic cells from lpa2-deficienct mice were hyperactive using in vitro assays when compared to their wild-type counterparts, and induced greater allergic airway inflammation after adoptive transfer in vivo. Wild-type (but not LPA2 deficient) DC were susceptible to inhibition by different exogenous LPA species, and pertussis toxin enhanced the ability of wild-type DC to induce T cell proliferation, an effect not seen using LPA2 deficient DC. Collectively, these data support a model in which LPA acting via LPA2 coupled to Gαi acts to tonically inhibit DC activation. Thus in addition to regulating cell recruitment and survival, our data establish a novel role for LPA as a negative regulator of innate immunity.
Negative regulation of innate immune responses is important in order to prevent excess inflammation and tissue injury (46). Negative regulatory mechanisms have been identified that suppress activation of innate immune cells, dysfunction of which may be associated with disease states (47). Since LPA is constitutively present in serum and BAL fluids (48, 49), one possibility is that the extravasation of LPA-containing serum into tissues that occurs during inflammation may be sensed as an anti-inflammatory or “pro-resolution” signal (50). In support of this notion, LPA promotes epithelial barrier function, a key step in the restoration of normal tissue integrity following inflammatory insults (51). A corollary of this hypothesis is that dysfunction of the LPA/lpa2 axis may contribute to persistent inflammation in chronic disease states. Taken together with the observation that mice deficient in G2A, a receptor for lysophosphatidylcholine, develop spontaneous autoimmunity (52, 53), these findings suggest that lysolipids may play a broader role in dampening immune responses than previously suspected.
Our data support a model in which LPA2 coupling to Gαi suppresses NF-κB-dependent dendritic cell activation. Precedence for the idea that pertussis toxin can augment DC activation is provided by the work of Ausiello et al. (54), and our data firmly implicate a role for LPA2 in this regard. The C-terminal tail of LPA2 contains unique sequences that support macromolecular complex formation (55), and it is attractive to speculate that this complex negatively regulates TLR4-dependent activation of NF-κB. Future studies will be needed to explore this and other mechanistic possibilities.
We found that allergic lung inflammation was substantially greater in lpa2−/− mice compared to wild-type littermates using both systemic and mucosal immunization strategies. Our data contrast with the observations of Zhao et al. that heterozygous lpa2+/− mice were partially protected from lung inflammation following challenge with shistosoma egg antigen (56). Reasons for this apparent discrepancy are not immediately obvious, but may relate to the nature of the antigens used, immunization protocols, or genetic backgrounds. Using bone marrow chimeras, we uncovered a key role for lpa2 expression by radiosensitive hematopoietic cells in suppressing allergic airway inflammation. Our results using adoptive transfer experiments firmly implicate DC in this regard, and are supported by the observation that Ova-specific IgE responses are enhanced in the absence of LPA2.
LPA is constitutively present in epithelial lining fluids of the human lung, and significantly enriched during the late-phase response following segmental allergen challenge (49). Based on our findings and previously published research, we can construct a working model in which LPA has both pro- and anti-inflammatory effects in asthma. Pro-inflammatory effects can result from the ability of LPA to promote cell recruitment or activation (9, 27–29), especially in response to submaximal stimuli (57). LPA can also augment airway hyperresponsiveness by direct effects on smooth muscle cells (30, 31). However, since LPA also restores epithelial barrier function (51), enhances IL-13Rα2 expression (58), inhibits epithelial RANTES production (59), and attenuates DC activation (this report), it has the potential to dampen airway inflammation. The observation that LPA is constitutively present in epithelial lining fluids supports its potential role in maintaining lung homeostasis. One intriguing possibility is that LPA contributes to airway remodeling in long-standing asthma. By promoting fibroblast recruitment (25) and smooth muscle mitogenesis (60), local generation of LPA during cycles of airway injury and repair could lead to airway fibrosis and smooth muscle hypertrophy. More research is needed to understand the mechanisms and timing of LPA generation in the airway, and the roles of different LPA receptors and target cells in airway inflammation and remodeling.
Taken together, our results establish a novel role for lpa2 as an inhibitory receptor of a key innate immune cell type, and uncover a new pathway involved in tonic dampening of allergic immune responses. Future studies investigating the function of the inhibitory LPA/lpa2 axis in inflammatory diseases should prove worthwhile. We also speculate that LPA2-specific agonists may have anti-inflammatory properties and therapeutic efficacy in allergic and inflammatory diseases.
We gratefully acknowledge the technical assistance of Siva Sugunan, and thank Drs. Andrew Tager (Harvard University) and Viswanathan Natarajan (University of Illinois) for reagents and/or helpful advice.
Declaration of all sources of funding: NIH R01HL071933 (SG), Pilot Project Funding supported by NIH P30ES01247 (SG), NIH T32 HD057821 (FR), NIH T32 HL066988 (TC), and the University of Rochester Department of Medicine.