TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

Abstract

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

Figure 1:. TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model.

Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (*p < 0.05). C. TRPV4 KO mice have decreased bacterial clearance as measured by retained bacterial CFU in the combined BAL/lung homogenate as compared to WT (*p = 0.012). TRPV4 KO mice have greater BAL content of D. IL-6; *p = 0.028, E. CXCL2 (MIP-2); *p = 0.049, and F. CXCL1 (KC); *p = 0.009 than WT control by ELISA. G. TRPV4 KO hematoxylin and eosin (H&E) lung sections show greater parenchymal inflammatory cell infiltration (quantified as % lung consolidation) as compared with WT. n ≥ 5 per sterile bead group and n = 20 per P. aeruginosa group on Day 2–3. The box plots (B-F) indicate the 25^th-75^th percentile for each measure. The error bars denotes maximum and minimum values (5–95^th percentile). The horizontal white line denotes the median value. * denotes WT vs TRPV4 KO.

Figure 2:. TRPV4 mediates clearance of P. aeruginosa by macrophages.

WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP-P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa; anti-CD45, red; dapi, blue). B, D. Quantification of A, C. *^{, #}p < 0.05; % WT vs KO. E. Flow cytometry of macrophage populations (+F4/80, CD64) from collagenase digested lung ± GFP-P. aeruginosa from WT and TRPV4 KO mice. Cell debris was excluded on a FSC-A/SSC-A plot and cell aggregates were excluded on a FSC-A/FSC-H plot. Viable cells were selected on a DAPI/SSC-A plot. Pseudocolor plots for CD45, neutrophil, and macrophage gating thresholds are shown. Gate boundaries for CD45 positive leukocytes and F4/80 positive macrophages were set using fluorescence minus one (FMO) controls. Immunofluorescence with anti-GFP (green) performed on cytospins. F. Quantification of % cell phagocytosis (*p = 0.035). n=20 per group. All images 63X original magnification, 10μm scale bars. *,# denotes WT vs TRPV4 KO. macs: macrophages and PMNs: neutrophils.

Figure 3:. p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa.

BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 (*p < 0.001), C. p-ERK/total ERK, and D. p-JNK/total JNK (*p = 0.027). E. Representative immunoblot for phosphorylated and total MK2 in WT vs TRPV4 KO BMDMs. F. Band density quantified from immunoblot (n = 5, *p < 0.05). G. Representative immunoblot for phosphorylated and total MKK3/MKK6. H. Band density quantified from immunoblot (n = 3). I. Representative immunoblot for phosphorylated and total p38 in homogenized mouse lung after sterile or P. aeruginosa beads (3 days). J. Band density quantified from p38 immunoblot (n = 6) (*p < 0.001). * denotes WT vs TRPV4 KO.

Figure 4:. TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1).

WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) (*p < 0.05). C. Representative immunoblot ± DUSP1 pharmacologic inhibitor, BCI 5μM, for p-p38/total p38 and p-JNK/total JNK in WT BMDMs. Band density quantified for D. p-p38/total p38 or E. p-JNK/total JNK from immunoblot (n = 4) (*p = 0.004). * denotes WT vs TRPV4 KO, + denotes ± pharmacologic inhibitor.

Figure 5:. Macrophage phagocytosis in response to LPS is mediated by p38 MAPK and DUSP1.

BMDMs were incubated ± LPS (100ng/mL, 24h) ± small molecule inhibitors of p38 (SB203580 and BIRB796) and MK2 (PF3644022). Inhibition of p38 by A. SB203580 and B. BIRB796 induces a dose-dependent decrease in LPS-induced phagocytosis (*p < 0.05). Immunoblots show phosphorylation of p38 in the presence of BIRB796 and MK2 in the presence of SB203580 per manufacturer stated mechanism. C. Downregulation of p38α by siRNA (96h) decreases LPS-induced macrophage phagocytosis (*p = 0.019, max 60–70% decrease p38α protein at 96h). Small molecule inhibition of D. MK2 by PF3644022 and E. DUSP1 by BCI blocks LPS-induced phagocytosis. F. In contrast, small molecule inhibition of JNK by SP600125 had no significant effect on LPS-induced phagocytosis (*p < 0.05). n = 3 for all experiments. * denotes difference in LPS response ± inhibition or downregulation of p38/MK2.

Figure 6:. p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness.

Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes (*p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses (*p < 0.05). n = 3–5 for all experiments. * denotes difference in LPS response ± inhibition of p38.

Figure 7:. TRPV4 modulates pro-inflammatory cytokine production through JNK.

BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 (^*,#p < 0.05). n = 3–5, one-way ANOVA followed by Dunnett’s test or Student-Newman-Keuls used for statistical analysis. * denotes WT vs TRPV4 KO, # denotes difference in LPS response ± inhibitor.

Figure 8:. TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK.

Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified (*p < 0.05). One-way ANOVA followed by Dunnett’s test or Student-Newman-Keuls used for statistical analysis, * denotes ± LPS, # denotes difference in LPS response ± inhibitor.

Figure 9:. Matrix mechanical signal transduction through TRPV4 modulates the LPS signal through MAPK switching.

A. In the presence of a sub-threshold mechanical signal, as seen in normal lung, TRPV4 does not influence the LPS/TLR4 signal, which results in limiting the phagocytic response to LPS, thereby maintaining lung homeostasis. B. In the presence of an above threshold mechanical signal, as seen with lung stiffening during injury, TRPV4 influences the LPS/TLR4 signal. We have previously published that TRPV4 regulates the stiffness-dependent responses of increased macrophage phagocytosis, and cytokine secretion in response to LPS (23). We now show a molecular switch from JNK activation to predominantly p38 activation, which results in abrogation of enhanced DUSP1 expression. DUSP1 regulates the MAPK molecular switch by deactivating JNK resulting in enhanced bacterial clearance, inhibiting pro-inflammatory cytokine secretion, and thereby ameliorating lung injury/ARDS. This defines a novel molecular mechanism linking inflammation-induced changes in the mechanical properties of the extracellular matrix with innate immunity.