TRPV4 Mechanosensitive Ion Channel Regulates Lipopolysaccharide-Stimulated Macrophage Phagocytosis

Abstract

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

Figure 1. Functional TRPV4 is expressed in murine BMDMs

(A) WT or TRPV4 KO BMDMs (differentiated bone marrow derived macrophages) were incubated ± LPS (100 ng/mL, 24 h). Immunoblot reveals that TRPV4 protein is expressed and unchanged ± LPS in BMDMs. TRPV4 protein is deleted in BMDMs from TRPV4 KO cells and decreased by 80-85% in BMDMs treated with TRPV4-specific siRNA compared to control (CNTL) siRNA after 3 or 4 days (3d, 4d) (panels cut from the same blot and exposure). (B) Calcium influx measured in the presence of TRPV4 agonist (GSK) using fluorescent dye-treated BMDMs from WT mice with TRPV4 downregulation (siRNA) or TRPV4 KO mice. BMDMs have a decreased or absent TRPV4 agonist (GSK)-induced calcium signal in siRNA transfected cells, or in TRPV4 KO BMDMs on glass substrates (* denotes p < 0.05 TRPV4 siRNA vs CNTL, ⁺ denotes p < 0.001 TRPV4 KO vs WT). RFU - Fluorescence intensity reflects intracellular calcium concentration, WT - Wild type cells, CNTL siRNA - Non-targeting siRNA, TRPV4 siRNA - TRPV4-specific siRNA, TRPV4 KO - genetic deletion of TRPV4. (C) Small molecule inhibition of TRPV4 (HC) induces a dose-dependent decrease in the TRPV4 agonist (GSK)-induced calcium signal (IC₅₀ = 7 µM). n ≥ 3 times in quadruplicate.

Figure 2. TRPV4 mediates LPS-stimulated macrophage phagocytosis of E. coli particles

BMDMs or freshly-isolated alveolar macrophages were incubated ± LPS (100 ng/mL, 24 h), in DMEM ± Ca²⁺ [1.802 mM (200 mg/mL)] followed by an incubation with fluorescently-labeled E. coli particles (2 h in HBSS ± Ca²⁺ [1.261 mM (140 mg/mL)]). Phagocytosis was measured as fluorescence intensity per cell. (A) Optimal macrophage phagocytosis requires extracellular calcium during both the LPS incubation (LPS, stimulation - 24 h) and subsequent period of E. coli particle incubation (E. coli, phagocytic phase - 2 h). +Ca denotes the presence of Ca²⁺ during both the LPS (stimulation phase) and E. coli incubations (phagocytic phase); No Ca LPS denotes absence of Ca²⁺ during the LPS incubation; No Ca E. coli denotes absence of Ca²⁺ during the E. coli incubation; No Ca denotes absence of Ca²⁺ during both LPS and E. coli incubation periods. Phagocytosis is quantified as % of induction by LPS (*^,+p < 0.05). (B) LPS stimulates macrophage phagocytosis in WT BMDMs (⁺p < 0.001) that is abrogated upon TRPV4 deletion (KO) BMDMs (*p = 0.002) and TRPV4 downregulation (siRNA) BMDMs (*p < 0.001). UT - untreated cells, LPS - lipopolysaccharide treated cells. (C) TRPV4 inhibitor (HC) blocks LPS-stimulated phagocytosis in BMDMs in a concentration-dependent manner (IC₅₀ = 8 µM, p < 0.001). [HC] ≥ 30 µM completely inhibited LPS-induced phagocytosis (Figure 2C) to a level comparable to that seen in the TRPV4 KO BMDMs (as in Figure 2B). Quantified as % of induction by LPS. (D) TRPV4-dependent phagocytic defect seen in TRPV4 KO primary murine alveolar macrophages (*^,+p < 0.05). AM - alveolar macrophages. + denotes the increase by LPS vs UT, * denotes difference in LPS response from WT mice (B, D) or ± Ca²⁺ (A) under the indicated conditions. n ≥ 3 times in duplicate.

Figure 3. TRPV4 mediates LPS-stimulated macrophage phagocytosis of IgG-coated latex beads in vitro

BMDMs or freshly-isolated alveolar macrophages were incubated ± LPS (100 ng/mL, 6-24 h) ± other indicated molecules and then incubated with IgG-coated latex beads (1.5 h). Phagocytosis was measured as signal intensity/cell. (A) Representative photomicrographs of BMDMs given IgG-coated beads ± LPS ± 30 µM HC. Green- beads, Blue- nuclei, Red- CD45 to show plasma membrane (B) Quantification of signal intensity from panel A (*^,+p < 0.05). (C) Representative photomicrographs of BMDMs given IgG-coated beads ± LPS in TRPV4 siRNA-treated and TRPV4 KO cells. (D) Quantification of signal intensity from panel C (*^,+p < 0.05). (E) Representative photomicrographs of alveolar macrophages (AM) given IgG-coated beads ± LPS ± HC. (F) Quantification of signal intensity from panel E (*^,+p < 0.05). WT denotes WT cells in the absence of HC, WT+HC denotes WT cells in the presence of the TRPV4 inhibitor (HC). + denotes the increase by LPS vs UT, * denotes difference in LPS response as compared to WT (D) ± TRPV4 inhibitor (B,F; HC). n ≥ 3 times in at least duplicate. All photomicrograph panels, 40X Orig. Mag., scale bar 30 µm.

Figure 4. TRPV4 mediates the stiffness induction effect on LPS-stimulated calcium influx and macrophage phagocytosis

BMDMs were treated ± LPS while attached to fibronectin-coated glass (50 × 10⁶ GPa) (A) or polyacrylamide hydrogels of indicated stiffnesses (B-D). (A) Calcium influx was measured as in Figure 1B in WT BMDMs ± LPS ± HC versus KO BMDMs on glass substrate. LPS stimulated an increase in calcium influx that was abrogated with inhibition of TRPV4 (HC) or deletion of TRPV4 (KO) (*^,+p < 0.05). (B) Calcium influx and (C) LPS-stimulated phagocytosis of E. coli particles, measured as % induction by LPS, were dependent on pathophysiologic range stiffness (>8-25 kPa) (⁺p < 0.05 ± LPS). (D) LPS-enhanced phagocytosis in WT BMDMs was decreased 4-fold upon deletion of TRPV4 (KO) or inhibition of TRPV4 (HC) on pathophysiologic range stiffness (25 kPa) (^#p < 0.05). * denotes difference as compared to ± LPS, + denotes the increase in LPS vs UT (A) or difference consistent with 1 kPa (B-C), # denotes difference as compared to LPS treated WT ± HC (WT-No HC). n ≥ 3 times in quadruplicate.

Figure 5. TRPV4 modulates the cytokine response to LPS in a manner that depends on matrix stiffness

ELISAs (IL-1β and IL-10) were performed on macrophage conditioned media from BMDMs cultured on both glass substrate and/or pathophysiologic-range matrix stiffness ± LPS (100 ng/mL, 24 h). (A) LPS-stimulated release of IL-1β was enhanced in TRPV4 KO BMDMs (*p < 0.05) and (B) LPS-stimulated release of IL-1β was suppressed in WT BMDMs as stiffness increased over the pathophysiologic-range (⁺p < 0.05). (C) LPS-stimulated release of IL-10 was suppressed in TRPV4 KO BMDMs (*p < 0.05) and (D) LPS-stimulated release of IL-10 was enhanced in WT BMDMs as stiffness increased over the pathophysiologic-range (⁺p < 0.05). * denotes difference in LPS response compared with WT, + denotes the increase in LPS response compared with 1 kPa. n ≥ 3 times in quadruplicate.

Figure 6. TRPV4 mediates LPS-stimulated macrophage phagocytosis of IgG-coated latex beads in vivo

WT and TRPV4 KO C57BL/6 were treated with IT LPS (3 µg/g) for 16 h followed by intratracheal (IT) IgG-coated latex beads for 6 h. Cell phagocytic analysis was performed on the BAL by microscopic analysis of cytospin preparations. (A) Representative confocal images of WT and TRPV4 KO mice given IT saline (n = 2) or LPS (n = 5) followed by IgG-coated latex beads (white arrow heads) - Green-beads, Blue-nuclei (PMN: multilobular nucleus, macrophage: single concentric nucleus), Red-CD45 to show membrane (All panels, 40X Orig. Mag.). (B) LPS treated WT mice had increased macrophage phagocytosis of IgG-coated latex beads compared to the LPS treated TRPV4 KO. The number of beads per macrophage were quantified from panel A (*^,+p < 0.05). + denotes the increase in LPS vs UT, * denotes difference in LPS response between KO and WT.

Figure 7. Working model illustrating that LPS and TRPV4 signal cooperate to alter macrophage phenotypic change leading to enhanced clearance of infection and resolution of lung injury

Our data suggest that TRPV4 is sensitized by extracellular matrix stiffness in the range of inflamed/fibrotic lung. Interaction between the LPS signal and the matrix stiff signal through TRPV4 promote increased TRPV4 channel activity and macrophage phenotypic change leading to increased clearance of bacteria and resolution of infection-associated lung injury.