Genetic Deletion of Toll-Like Receptor 4 on Platelets Attenuates Experimental Pulmonary HypertensionNovelty and Significance
Rationale: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown.
Objectives: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH.
Methods and Results: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4−/− mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4−/− mice were generated (PF4-TLR4−/− mice). TLR4−/− platelets from either global TLR4−/− or PF4-TLR4−/− mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4−/− mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner.
Conclusions: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.
Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Historically, PH research has relied on concepts derived from hypoxic pulmonary vasoconstriction for the development of new therapies. Increasingly, however, thrombotic and inflammatory mechanisms are recognized as complicating and possibly initiating events in PH.1
Editorial, see p 1551
In This Issue, see p 1547
Toll-like receptors (TLRs) are one family of receptors that the innate immune system uses to sense pathogens or tissue damage by recognizing molecular patterns in microbial products or endogenous molecules released by damaged tissues.2 TLR4, perhaps more than any other TLR, interfaces microbial and sterile inflammation by responding to bacterial lipopolysaccharides and endogenous ligands, including hyaluronic acid,3 heparin sulfate,4 high-mobility group box 1,5 and heat shock proteins.6 Recent studies demonstrate that the activation of TLR4 contributes to the pathogenesis of PH.7,8
The lung is equipped to sense and respond to endogenous TLR4 ligands. The lung consists of parenchymal cells, including fibroblasts, endothelial cells (ECs), and smooth muscle cells, and nonparenchymal cells (NPCs), including alveolar macrophages, dendritic cells, granulocytes, and transiting leukocytes and platelets. Both parenchymal cells and NPCs express TLR4 and have intact TLR4 signaling pathways. The aim of this study was to determine the role of TLR4 on NPCs in the pathogenesis of PH.
Animal procedures were performed in accordance with University of Pittsburgh Institutional Animal Care and Use Committee guidelines. TLR4loxP/loxP mice were generated as described9 and interbred with stud males (TLR4loxP/−;Ella-cre, TLR4loxP/−;LysM-cre, or TLR4loxP/−;PF4-cre) to generate the desired genotype. Wild-type (WT) mice were TLR4loxP/loxP mice without the introduction of Cre recombinase. Bone marrow (BM) was isolated from WT or TLR4−/− mice and transplanted into irradiated WT or TLR4−/− mice as described.10 PH was induced by exposure to chronic hypoxia (CH) for 21 days or the combination of SU5416 (a vascular endothelial growth factor receptor inhibitor) plus CH (SUH) as described.7,11 Right ventricular systolic pressure (RVSP) and RV hypertrophy (RVH) were measured by established methods.7 Pulmonary vascular muscularization and percent wall thickness were assessed as described.7 Bleeding time was measured as described.12 Platelet-rich plasma was prepared from whole blood anticoagulated with acid citrate dextrose.12 To isolate platelets, platelet-rich plasma was centrifuged at 800g, and platelets were resuspended in tyrodes buffer. Serotonin and interleukin-6 were measured by ELISA. Platelet activation was determined using flow cytometry.12 The effect of platelets on human pulmonary artery smooth muscle cell (HPASMC) proliferation was assessed by coculturing lipopolysaccharide-activated platelets with HPASMC. Cell proliferation was assessed by [3H]-thymidine incorporation. Statistical analyses were performed using GraphPad Prism software. Data were analyzed by 1-way ANOVA and Tukey post hoc test. Detailed methods are provided in the Online Data Supplement.
Equal Contribution of NPCs and Parenchymal Cell TLR4 to Hypoxia-Induced PH
To deduce a role for TLR4 in NPCs versus parenchymal cells, we performed BM transplantation into irradiated mice to create WT and TLR4−/− BM chimeric mice. After transplantation and recovery, mice were exposed to CH to induce PH. WT mice that received WT BM developed PH as determined by measuring RVSP and RVH. PH was attenuated in TLR4−/− mice that received TLR4−/− BM (Figure 1A and 1B). Interestingly, WT mice that received TLR4−/− BM, or TLR4−/− that received WT BM, showed the same attenuation of PH as whole-body TLR4 −/− chimeric mice.
TLR4 on Myeloid-Derived Cells Does Not Contribute to Hypoxia-Induced PH
TLR4 is highly expressed on cells of myeloid lineage, and these cells are often associated with response to TLR4 agonists. To investigate a role for TLR4 on myeloid-derived cells, we exposed WT or LysM-TLR4−/− mice to 3-week CH. The loss of TLR4 on myeloid-derived cells failed to attenuate the development of PH as assessed by RVSP (Figure 1C) and RVH (Figure 1D).
Functional Characterization of Platelet TLR4 Knockout Mice
To investigate a role for platelet TLR4 in PH, we used platelet-specific TLR4−/− (PF4-TLR4−/−) mice. Platelets were isolated from WT, TLR4−/−, and PF4-TLR4−/− mice and stimulated with lipopolysaccharide. WT platelets upregulated surface P-selectin after lipopolysaccharide stimulation, whereas platelets from TLR4−/− or PF4-TLR4−/− mice did not (Figure 2A–2C). Collagen induced P-selectin expression on platelets isolated from all 3 genotypes, demonstrating that TLR4−/− and PF4-TLR4−/− platelets were functional. Additionally, serotonin was increased in the supernatant of WT platelets, but not TLR4−/− or PF4-TLR4−/− platelets, after stimulation by lipopolysaccharide (Figure 2E).
To demonstrate the specificity of platelet knockout, we tested the effect of lipopolysaccharide on ECs isolated from the lungs of WT, TLR4−/−, or PF4-TLR4−/− mice. The ECs expressed CD31 but not smooth muscle α actin, demonstrating purity (Figure 2E). Lipopolysaccharide induced interleukin-6 release from ECs derived from WT or PF4-TLR4−/− mice, but not from ECs derived from TLR4−/− mice (Figure 2F).
Genetic Deletion of Platelet TLR4 Attenuates Hypoxia-Induced PH
We next exposed WT, TLR4−/−, and PF4-TLR4−/−mice to 3-week CH to induce PH. The deletion of TLR4 globally or on platelets significantly attenuated PH as assessed by measuring RVSP and RVH (Figure 3A and 3B).
Genetic Deletion of Platelet TLR4 Attenuates Platelet Activation in Hypoxia-Induced PH
Exposing mice to CH leads to decreased bleeding time and increased platelet surface P-selectin expression, indicative of platelet activation.12 In both the CH and SUH models, bleeding time decreased in WT mice but not in TLR4−/− or PF4-TLR4−/− mice (Figure 3C). Similarly, platelet surface P-selectin expression was increased in CH and SUH WT but not TLR4−/− or PF4-TLR4−/− mice (Figure 3D).
Genetic Deletion of Platelet TLR4 Attenuates Vascular Remodeling in Hypoxia-Induced PH
TLR4−/− or PF4-TLR4−/− CH or SUH mice exhibited less pulmonary vascular remodeling compared with WT mice (Figure 4A). Morphometric analysis of peripheral arterioles revealed less thickening of the vessel wall in TLR4−/− or PF4-TLR4−/− mice versus WT mice (Figure 4B). Likewise, there was less muscularization of peripheral arterioles in TLR4−/− or PF4-TLR4−/− mice versus WT mice (Figure 4C and 4D). Plasma serotonin was increased in CH WT mice but not TLR4−/− or PF4-TLR4−/− mice. Coculturing HPASMC with lipopolysaccharide-stimulated WT platelets, but not TLR4−/− platelets, promoted HPASMC proliferation (Figure 4E). This effect was blocked by GR127935, a 5HT1B receptor antagonist (Figure 4F). Figure 4G shows the proposed conceptual framework for how platelet TLR4 fits into TLR4 and serotonin signaling in PH.
In addition to their role in hemostasis and thrombosis, platelets are mediators of inflammation and immune responses.13 They express several TLRs (TLR1, 2, 4, 6, 8, 9),14,15 pattern recognition receptors involved in the innate immune response by recognizing microbial structures and endogenous molecules released from damaged, stressed, or activated cells.16 Lipopolysaccharide, a TLR4 agonist, induces platelet P-selectin expression and interleukin-1β and ATP secretion and primes platelets to aggregate in response to low-dose thrombin.17 Thus, platelet TLRs bridge innate immunity and coagulation. Previous studies from our laboratory demonstrated a role for TLR4 in the pathogenesis of PH.7,8 The major finding of this study is that genetic deletion of TLR4 on platelets, but not myeloid cells, attenuates the pathogenesis of PH. Furthermore, platelets are activated to express surface P-selectin and release serotonin during hypoxia by TLR4-dependent mechanisms. Thus, the deletion of TLR4 globally or on platelets prevented CH- or SUH-induced PH, in vivo platelet activation, and pulmonary vascular remodeling. Importantly, this study is the first to show that genetic deletion of a platelet surface receptor can attenuate PH.
There is an emerging role of platelet-derived mediators, such as serotonin, thromboxane-A2, and growth factors in patients with severe PH. These vasoactive mediators promote vasoconstriction (thromboxane-A2, serotonin), thrombosis (thromboxane-A2), and proliferation of vascular smooth muscle cells, ECs, and fibroblasts (serotonin, platelet-derived growth factor). Furthermore, platelet aggregation is enhanced by the altered balance of proaggregatory molecules (thromboxane-A2) and antiaggregatory molecules (nitric oxide, prostacyclin).
The serotonin hypothesis of PH was postulated in the 1960s when it was discovered that women taking an indirect serotonergic agonist developed PH. More recently, it was recognized that patients with PH have markedly elevated plasma serotonin.18 Data demonstrate that serotonin released from ECs binds to serotonin receptors on PASMC or is taken up by PASMC via the serotonin transporter stimulating PASMC proliferation, migration, and contraction, thus contributing to vascular remodeling in PH.19 Furthermore, mice deficient in bone morphgenetic protein receptor 2 are more sensitive to serotonin-induced PH, which was associated with the inhibition of Smad1/5 phosphorylation.20 These data suggest that, in humans, increased serotonin could provide a second hit necessary for the development of PH caused by bone morphgenetic protein receptor 2 haploinsufficiency. In this study, we found that genetic deletion of TLR4 on platelets abrogated platelet activation and prevented the increase in plasma serotonin in 2 experimental models of PH. Furthermore, coculturing HPASMC with lipopolysaccharide-stimulated WT platelets, but not TLR4−/− platelets, promoted HPASMC proliferation via a mechanism that was dependent on the 5HT1B receptor. Together, our data suggest that TLR4 plays a role in platelet activation and serotonin release in PH and that platelets are an important source of serotonin in PH.
It was surprising to find that the loss of TLR4 on myeloid cells did not influence the disease course because myeloid cells are important responders to TLR4 ligands. It is possible that TLR4 on myeloid cells works toward counter purposes in PH, thus masking the role of TLR4 on individual cell types. It may also be a limitation of the CH mouse model that the role of these cells in sensing endogenous TLR4 ligands is diminished or absent because of the mild inflammatory phenotype. Future studies will be necessary to sort out the role of TLR4 on myeloid cells in the pathogenesis of PH.
In summary, this study demonstrates the importance of platelet TLR4 in PH because its deletion from platelets improved disease outcome. These data suggest that platelet TLR4 is a proximate promoter of platelet activation and serotonin release in PH. We proffer that drugs interrupting TLR4 interaction with its endogenous ligands may limit platelet activation and inflammation and lead to better therapies for PH.
Sources of Funding
This work was supported by the National Institutes of Health (HL085134 to P.M. Bauer), the American Heart Association (13GRNT17160004 to P.M. Bauer), and the University of Pittsburgh School of Medicine and Clinical and Translational Science Institute (Pilot Project Program in Hemostasis and Vascular Biology Grant, to P.M. Bauer). The project described was supported by the National Institutes of Health through grant numbers UL1 RR024153 and UL1TR000005.
In February 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.8 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.114.303662/-/DC1.
- Nonstandard Abbreviations and Acronyms
- bone marrow
- chronic hypoxia
- endothelial cell
- human pulmonary artery smooth muscle cell
- nonparenchymal cell
- pulmonary hypertension
- right ventricular
- right ventricular hypertrophy
- right ventricular systolic pressure
- SU5416 plus CH
- toll-like receptor
- Received February 8, 2014.
- Revision received March 12, 2014.
- Accepted March 17, 2014.
- © 2014 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Toll-like receptor 4 (TLR4) contributes to the pathogenesis of pulmonary hypertension (PH) via largely unknown mechanisms.
Platelets participate actively in thrombus formation and produce (eg, thromboxane-A2), store (eg, serotonin), and release mediators that may contribute to the initiation or aggravation of PH.
Platelets express functional TLR4, and TLR4 agonists stimulate release of soluble mediators from platelets.
What New Information Does This Article Contribute?
TLR4 on both parenchymal and nonparenchymal cells (NPCs) contribute to the pathogenesis of PH.
Platelet TLR4 is a proximate promoter of platelet activation and serotonin release in PH.
Serotonin released from platelets may contribute to increased pulmonary vascular smooth muscle cell proliferation and pulmonary vascular remodeling.
Despite evidence for thrombotic and inflammatory mechanisms in PH, no drugs have been developed to target these mechanisms. Recent data from our laboratory demonstrate a role for the innate immune receptor TLR4 in PH. We sought to determine the role of TLR4 on bone marrow (BM)−derived cells in PH. BM chimera experiments revealed an equal contribution of TLR4 on NPCs and parenchymal cells. Although myeloid lineage cells are most often associated with TLR4 responses, the deletion of TLR4 from these cells had no effect on PH. Platelets, in addition to their role in thrombosis, are mediators of inflammation and express functional TLR4. We, therefore, hypothesized that platelet TLR4 contributes to PH. Indeed, the deletion of TLR4 on platelets attenuated platelet activation, serotonin release, pulmonary vascular remodeling, and PH in 2 experimental models of the disease. These data demonstrate that platelet TLR4 is a proximate promoter of platelet activation and serotonin release in PH and suggests that drugs interrupting TLR4 interaction with its endogenous ligands may limit platelet activation and inflammation and lead to better therapies for PH.