TRPV4 Mechanosensitive Ion Channel Regulates Lipopolysaccharide-Stimulated Macrophage Phagocytosis

Macrophage phagocytosis (particle engulfment) is a complex, multistep physiologic process that determines the host’s capacity to defend against foreign particulates, pathogens, or apoptotic cells, and it mediates resolution of inflammation and tissue homeostasis (1–5). Phagocytosis requires a coordinated interaction among macrophage surface receptors, particles, and the surrounding matrix, which ultimately drive the cytoskeletal rearrangements required for efficient engulfment (6–10). In fact, the phagocytic function of the macrophage depends on the biophysical properties of the matrix itself (9, 10). For example, studies with prepatterned matrix substrates reveal that matrix stiffness results in cell shape changes that can influence macrophage phenotypic properties (9–13). The mechanism by which macrophages sense extracellular matrix stiffness remains unknown.

Calcium is known to be an essential second messenger in many physiologic cell processes, including phagocytosis (14–16). Many studies show that macrophage phagocytic function depends on a finely tuned orchestration of the intracellular calcium signal and the actin cytoskeleton (17). For example, studies show that particle binding to macrophages induces calcium transients, and calcium appears to be required for both FcR-dependent and -independent phagocytosis (18–20). Intracellular calcium is tightly regulated in a spatiotemporal manner through a system of ion channels and membrane pumps (21). One such channel is the transient receptor potential vanilloid 4 (TRPV4). TRPV4 is a ubiquitously expressed, plasma membrane–based, calcium-permeable cation channel that is sensitized and activated by both chemical (5,6-epoxyeicosatrienoic acid and 4α-phorbol 12,13-didecanoate) and physical stimuli (temperature, stretch, and hypotonicity) (22–25).

In fact, TRPV4 has been implicated in lung diseases associated with lung parenchymal stretch, such as pulmonary edema due to pulmonary venous hypertension, acute lung injury due to pulmonary parenchymal overdistension, and, most recently, pulmonary fibrosis (26–34). As TRPV4 can be sensitized by changes in matrix stiffness, can regulate calcium flux into the cell, and induces its effect, in part, through modulating cytoskeletal remodeling (27, 35), we reasoned that TRPV4 may mediate macrophage phenotypic function. We undertook this study to determine whether the TRPV4 channel modulates the LPS signal for macrophage phagocytosis and cytokine release in a matrix stiffness–dependent manner. This study is potentially applicable to lung host defense, resolution of inflammation, infection, and fibrosis.

Primary Abs to intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), GAPDH (Fitzgerald Industries International, Acton, MA), anti-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO) were purchased. Secondary Ab to rabbit was obtained from Jackson ImmunoResearch Laboratories and rat Alexa Fluor 594 was obtained from Life Technologies (Grand Island, NY). HC067047 (HC) was obtained from EMD Millipore, and GSK1016790A (GSK) was obtained from Sigma-Aldrich. Escherichia coli LPS 0111:B4 for the in vitro experiments and E. coli LPS 055:B5 for the in vivo experiments was obtained from Sigma-Aldrich.

All animal protocols were performed as approved by the Cleveland Clinic Institutional Animal Care and Use Committee. Primary murine bone marrow–derived macrophages (BMDMs) and alveolar macrophages were harvested from 8- to 12-wk-old C57BL/6 wild-type or TRPV4-null mice. BMDMs were differentiated in recombinant M-CSF (50 ng/ml, R&D Systems) as previously published (36). BMDMs and alveolar macrophages were plated on fibronectin-coated (10 μg/ml) glass or polyacrylamide hydrogels with varying stiffness (1, 8, and 25 kPa) (Matrigen, Brea, CA). Cells were treated with LPS (100 ng/ml) alone or with LPS and pretreated for 1 h with TRPV4 inhibitor (HC) for a total of 6–24 h. Primary isolates of alveolar macrophages obtained from bronchoalveolar lung lavage were purified by adherence and cultured in DMEM/10% FBS as previously described (37). TRPV4 expression was downregulated by transfecting BMDMs with TRPV4-specific mouse small interfering RNA (siRNA) duplexes or scrambled siRNA controls (OriGene Technologies) using electroporation, as previously published (36). Immunoblotting was performed for the indicated proteins as previously published (38). ELISAs (IL-1β, TNFα, and IL-10 from R&D systems) were run on conditioned media from WT BMDMs with/without LPS with/without HC and TRPV4 knockout (KO) BMDMs.

BMDMs were stimulated in vitro with/without LPS (100 ng/ml, 24 h) with/without Ca²⁺ (1.802 mM [200 mg/ml]) in DMEM (stimulation phase). To measure phagocytic function, the media were replaced with fluorescently labeled heat-inactivated E. coli with/without Ca²⁺ (1.261 mM [140 mg/ml]) for 2 h, per the manufacturer’s instructions (phagocytic phase) (K-12 strain, Vybrant phagocytosis assay kit, V-6694, Molecular Probes). In selected conditions, TRPV4 inhibitor (HC) was added 1 h before LPS stimulation. Nonopsonized phagocytosis was measured as fluorescence intensity in the FlexStation system (Molecular Devices). Preliminary experiments determined that 50 μl E. coli particles per well was the optimal concentration. A dose response of TRPV4 inhibitor (HC) determined its maximal effects, which were noted at 30 μM. Opsonized phagocytosis was measured by uptake of IgG-coated latex beads (Molecular Probes, F8853, 2-μm beads) and imaged via confocal (Leica DM6000 CFS SP5) microscopy per a previously published protocol (39). Fluorescence intensity was measured as image-integrated pixel intensity per cell using ImageJ software. Alveolar macrophages were maintained in RPMI 1640 containing Ca²⁺ (0.427 mM [100 mg/ml]), and phagocytosis was measured.

LPS-stimulated phagocytosis of IgG-coated latex beads in intact TRPV4-null mice and age-matched 8- to 12-wk-old female congenic WT C57BL/6 mice was performed by intratracheal instillation of LPS (3 μg/g) or PBS per previously published protocols (40, 41). Sixteen hours after LPS or saline injection, 1.5 × 10⁸ IgG-coated latex bead particles in 40 μl saline was intratracheally instilled for 6 h. Lung lavage was performed to determine total WBC counts or cell differentials as described previously (36). Cytospin preparations were performed for each lavage, and images were taken via confocal microscope as per a previously published protocol (39). Neutrophils and macrophages were distinguished by nuclear shape (polymorphonuclear neutrophil, multilobular nucleus; macrophage, single concentric nucleus). Phagocytosis was analyzed by quantifying the number of beads per cell using Image J software. All animal protocols were performed according to guidelines approved by the Cleveland Clinic Institutional Animal Care and Use Committee.

The calcium response to increasing concentrations of a TRPV4 agonist (GSK) was analyzed using fluorescent Calcium 5 dye (Molecular Devices)–treated cells in a microplate reader as previously published (27). Cytosolic calcium increases (Ca²⁺ influx) are presented as relative fluorescence unit (RFU; maximum–minimum) as published previously (27).

All data are presented as means ± SEM, unless otherwise specified. Comparison of data from two groups was performed with the Student t test. Comparing change scores of more than two groups was performed via ANOVA followed by a Dunnett test or Student–Newman–Keuls test. Significance was accepted at the p ≤ 0.05 level.

To determine whether BMDMs express TRPV4 with/without LPS, immunoblots for TRPV4 were performed in WT BMDMs, TRPV4 KO BMDMs, and TRPV4-specific siRNA-treated BMDMs (serving as negative controls). TRPV4 protein expression was unchanged with/without LPS in WT BMDMs and absent in TRPV4 KO whole-cell lysates (Fig. 1A). Downregulation of TRPV4 protein with TRPV4-specific siRNA resulted in an 80–85% reduction of TRPV4 protein (Fig. 1A). To determine whether BMDMs express functionally active TRPV4, varying doses of a TRPV4-specific agonist (GSK) were examined for their ability to induce calcium influx (Fig. 1B, EC₅₀ of 50 nM). Downregulation of TRPV4 by siRNA reduces the maximal calcium influx in response to the TRPV4 agonist by 50%, when compared with scrambled siRNA-transfected controls (Fig. 1B, *p < 0.05). Concordantly, genetic deletion of functional TRPV4 (BMDMs from TRPV4 KO mice) completely abrogates agonist-induced (GSK) calcium influx (Fig. 1B, ⁺p < 0.001). Blockade of TRPV4 with a selective small molecule inhibitor of TRPV4 (HC) reduces calcium influx in a dose-dependent manner (Fig. 1C, IC₅₀ of 7 μM). Taken together, these data clearly demonstrate that TRPV4 is expressed in a functionally active, nonredundant manner in murine BMDMs.

We and others have shown that TRPV4 can play a role in force-dependent cytoskeletal changes in other systems/cell types, and thus it was hypothesized that TRPV4 may play a role in macrophage phagocytosis (27). LPS stimulates phagocytosis of E. coli particles in the presence of calcium by 151 ± 3% in murine BMDMs (Fig. 2A, ⁺p < 0.001). For LPS to induce macrophage phagocytosis of E. coli particles, extracellular calcium is required during both the LPS stimulation and E. coli phagocytic phases (Fig. 2A, *p < 0.05). LPS stimulation of phagocytosis is completely abrogated in BMDMs from TRPV4 KO mice (Fig. 2B, *p < 0.001) and decreased by 67 ± 9% upon downregulation of TRPV4 with siRNA (Fig. 2B, *p = 0.002). Lastly, the small molecule TRPV4 inhibitor (HC) completely abrogates the LPS stimulation of phagocytosis in a dose-dependent manner (Fig. 2C, p < 0.001), with an IC₅₀ (8 μM) that is comparable to the TRPV4 inhibitor’s effect on calcium influx (7 μM, Fig. 1C). In contrast, TRPV4 inhibition has no effect on basal phagocytosis (data not shown). LPS-stimulated phagocytosis was similarly dependent on TRPV4 in both a murine macrophage cell line (RAW 267.4, data not shown) and in freshly isolated murine alveolar macrophages (Fig. 2D, *^,+p < 0.05). Collectively, these results demonstrate that LPS-stimulated phagocytosis of nonopsonized E. coli bacteria requires TRPV4.

To determine whether TRPV4 mediates specific receptor-initiated phagocytosis, we evaluated FcR-dependent phagocytosis by incubating macrophages with IgG-coated latex beads. LPS stimulates uptake of IgG-coated beads by 4-fold, as compared with untreated BMDMs (Fig. 3A, 3B, ⁺p < 0.05). The LPS-stimulated increment in uptake is reduced by 44 ± 16% upon inhibition of TRPV4 (HC) (Fig. 3A, 3B, *p < 0.05). Similarly, LPS-stimulated macrophage phagocytosis is abrogated upon either deletion (TRPV4 KO) or downregulation (TRPV4 siRNA) of TRPV4 (Fig. 3C, 3D, *^,+p < 0.05). LPS-stimulated phagocytosis was similarly dependent on TRPV4 in freshly isolated murine alveolar macrophages (Fig. 3E, 3F, *^,+p < 0.05). The LPS-stimulated cell-spreading response is also completely abrogated upon inhibition, deletion, or downregulation of TRPV4 (data not shown). Overall, these data demonstrate that the LPS-stimulated, FcR-dependent phagocytosis and the cell-spreading response are mediated by TRPV4.

Because lung inflammation causes changes in matrix stiffness and TRPV4 is a mechanosensitive ion channel, we sought to determine whether the LPS response is altered by matrix stiffness sensing through TRPV4. We first noted that LPS itself upregulates TRPV4 activity (calcium influx; Fig. 4A, 62 ± 4%, *^,+p < 0.05) on a supraphysiologically stiff substrate (glass as used in standard culture conditions, 50 × 10⁶ kPa). Importantly, this LPS-inducing effect increases directly with the matrix stiffness over the pathophysiologic range seen in inflamed or fibrotic lungs (1–25 kPa) (Fig. 4B, ⁺p < 0.05). The matrix stiffness dependency was also demonstrable for LPS-stimulated phagocytosis over the same pathophysiologic range (Fig. 4C, ⁺p < 0.05). The LPS-stimulated phagocytic response on fibrotic range matrix stiffness (25 kPa) was lost upon either deletion (TRPV4 KO, 55 ± 5%), or pharmacologic inhibition (HC, 63 ± 3%) of TRPV4 (Fig. 4D, ^#p < 0.05). No stiffness effect was noted in either calcium influx or phagocytic function in the absence of LPS (data not shown). In summary, these data show that TRPV4 cooperates with LPS to effect calcium influx and phagocytosis in a matrix stiffness–dependent manner.

As TRPV4 is sensitized by matrix stiffness and increases its activity in response to LPS, we examined the role of TRPV4 on other LPS-induced macrophage functions related to lung inflammation or infection. As expected, LPS induces macrophage cytokine production of IL-1β and IL-10. Upon deletion of TRPV4 (KO), the LPS induction of IL-1β increased by 50 ± 3% and IL-10 decreased by 47 ± 5% on a glass substrate (Fig. 5A, 5C, *p < 0.05). Both of these effects were dependent on extracellular matrix stiffness in the pathophysiologic range in WT BMDMs (Fig. 5B, 5D, ⁺p < 0.05). These data show that TRPV4 mediates macrophage release of key anti-inflammatory/proresolution cytokines (suppressed IL-1β, enhanced IL-10) in response to LPS in a stiffness-dependent manner.

Lastly, to examine if TRPV4 also mediates macrophage phagocytosis in vivo, we examined macrophage phagocytosis of IgG-coated latex beads intratracheally in intact mice. LPS induces a similar increment in alveolar neutrophil and macrophage numbers in the WT and TRPV4 KO mice (Data not shown). Concordant with the in vitro data, LPS induces macrophage phagocytosis in WT mice in a manner that is completely lost in the TRPV4 KO mice (Fig. 6, *,⁺p < 0.05). The data show that TRPV4 is required for LPS-stimulated macrophage phagocytosis of IgG-coated latex beads in mouse lungs in vivo.

In this study, we describe a novel role for TRPV4 in macrophage function as follows: LPS-stimulated phagocytosis of both nonopsonized E. coli in vitro and FcR-dependent (IgG-coated beads) in vitro and in vivo is mediated by TRPV4. The ability of LPS to induce phagocytosis through TRPV4 is dependent on matrix stiffness in a range comparable to that seen in inflamed or fibrotic lung. Finally, TRPV4 mediates the LPS signal to release anti-inflammatory/proresolution cytokines in macrophages. Taken together, these data implicate the mechanosensing channel, TRPV4, in the pathogenesis of lung infection/bacterial pneumonia by integrating the LPS and the matrix stiffness signals for macrophage phagocytosis and proresolution cytokine release.

Our data demonstrate that LPS-stimulated macrophage phagocytosis on stiff matrices (>8–25 kPa) is TRPV4-dependent. This was shown using two independent methods of inhibiting TRPV4 activity, including small molecule inhibition (HC) and TRPV4 deletion (KO). Furthermore, the TRPV4 dependency of LPS-stimulated phagocytosis was demonstrable in BMDMs, a macrophage-like cell line (RAW cells, data not shown), in primary alveolar macrophages in vitro, and in live intact mice, using both opsonized and nonopsonized particles. This suggests that the TRPV4 mediation of the LPS response is fundamental to phagocytosis and is an inherent property of the macrophage cell lineage.

Studies show that LPS-stimulated macrophage phagocytosis depends on MAPK (p38) activation of small GTPases (Rac, Rho, and Cdc42) and on actin polymerization (17, 42). Although not specifically studied in LPS-stimulated phagocytosis, TRPV channels have been shown to modulate p38 MAPK in response to thermal stimuli in neurons and osmotic stress in chondrocytes, and they have been shown to modulate small GTPase/actin remodeling in keratinocytes (43–45). Future studies in our laboratory will identify the downstream molecular effectors of the TRPV4-mediated effect on LPS-stimulated phagocytosis.

Recent studies have demonstrated that resident alveolar macrophages are relatively quiescent and capable of self-renewal under basal homeostatic conditions (2). Our data are consistent with the hypothesis that the macrophage response to LPS is downmodulated under conditions of uninflamed lung (i.e., 1–3 kPa), thereby supporting maintenance of tissue integrity (46–51). In contrast, during an acute inflammatory or infectious process, studies demonstrate that overall lung compliance is significantly reduced and alveolar level vessel stiffness is increased >10-fold (from 3 to 45 kPa) after intratracheal LPS instillation in mice (52, 53). Thus, bone marrow–derived monocytic lineage cells are recruited to an inflamed/infected lung that possesses enhanced rigidity, in the range noted in our study (52). Taken together with our findings, we hypothesize that TRPV4 mediates a feed-forward upregulation of phagocytosis in lung macrophages when they sense infection/injury-associated lung matrix stiffening. This concept is further supported by the observation of altered alveolar macrophage shape and function in association with increased alveolar surface tension in surfactant protein B–deficient mice (11). TRPV4 also mediates the LPS signal to secrete proresolution cytokines (suppressed IL-1β and enhanced IL-10) in a stiffness-dependent manner.

Although we have convincingly shown, using multiple complementary methods, that TRPV4 is a key mediator of LPS-stimulated macrophage phagocytosis and cytokine production, there are some limitations to our study. Although the intratracheal LPS murine model is a neutrophil-predominant inflammatory stimulus, we consistently observed >20% macrophages in the alveolar space, and the neutrophil uptake of IgG-coated beads was <10% that of the macrophages. Therefore, macrophages are the key phagocytic cell in our in vivo assay system. Additionally, TRPV2 has previously been mechanistically linked to macrophage phagocytosis, however, its effects were on basal phagocytosis and were found to be calcium-independent (54). Thus, it is unlikely that TRPV2-mediated effects are confounding our results. IL-1β and IL-10 are the best studied proinflammatory and proresolution cytokines, respectively (55). However, given the complex pleiotropic, overlapping, and cell type-specific effects of cytokines, one cannot predict the biologic response based on measurements of individual or multiple cytokines with certainty (56–61).

As shown in our proposed schematic model (Fig. 7), our study suggests that macrophage TRPV4 is sensitized by a stiff matrix (inflamed, infected lung) and cooperates with LPS to mediate macrophage phenotypic change. The macrophage phenotypic change results in increased phagocytosis and altered cytokine expression (decreased IL-1β and increased IL-10). The primed macrophage phenotype has increased ability for bacterial clearance and for resolution of infection-associated lung tissue injury. Thus, we envision that TRPV4 would play a significant role in the cytokine-based lung injury response to Gram-negative infection in the setting of underlying lung injury (adult respiratory distress syndrome), underlying lung fibrosis, and/or high volume mechanical ventilation (26, 62, 63). Our observations may also underlie the observed protective effect of LPS on experimental lung fibrosis (64).

In summary, TRPV4 is essential in LPS-stimulated macrophage phagocytosis under conditions of pathological matrix stiffness. This work reveals a novel role of TRPV4 in macrophage phagocytosis that is applicable to lung homeostasis, inflammation, and host defense.

We thank Dr. Serpil Erzurum for critical reading of this manuscript.

The authors have no financial conflicts of interest.