WNK4–SPAK modulates lipopolysaccharide-induced macrophage activation

Journal Pre-proofs WNK4–SPAK modulates lipopolysaccharide-induced macrophage activation Chin-Mao Hung, Chung-Kan Peng, Sung-Sen Yang, Hao-Ai Shui, Kun-Lun Huang PII: DOI: Reference:

S0006-2952(19)30437-X https://doi.org/10.1016/j.bcp.2019.113738 BCP 113738

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Biochemical Pharmacology

Received Date: Accepted Date:

8 October 2019 26 November 2019

Please cite this article as: C-M. Hung, C-K. Peng, S-S. Yang, H-A. Shui, K-L. Huang, WNK4–SPAK modulates lipopolysaccharide-induced macrophage activation, Biochemical Pharmacology (2019), doi: https://doi.org/ 10.1016/j.bcp.2019.113738

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© 2019 Published by Elsevier Inc.

Category: Inflammation and Immunopharmacology

WNK4–SPAK modulates lipopolysaccharide-induced macrophage activation Running Title: WNK4 modulates macrophage functions

Chin-Mao Hung1,2, Chung-Kan Peng3, Sung-Sen Yang1,4, Hao-Ai Shui1, Kun-Lun Huang1,2,3 *

1Graduate

Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.

2Graduate

Institute of Aerospace and Undersea Medicine, National Defense Medical Center,

Taipei, Taiwan. 3Division

of Pulmonary and Critical Care, Department of Medicine, Tri-Service General

Hospital, National Defense Medical Center, Taipei, Taiwan. 4Division

of Nephrology, Department of Medicine, Tri-Service General Hospital, National

Defense Medical Center, Taipei, Taiwan. *Address all correspondence to: Kun-Lun Huang, MD, PhD. E-mail: [email protected]

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ABSTRACT Dysregulation of alveolar macrophage activation has been recognized as the major mechanism in the pathogenesis of acute lung injury. The aim of the present study was to investigate the role of NKCC1 regulating mechanism in modulating macrophage activation. Knockout (SPAK– /– and

WNK4–/–) and knockin (WNK4D561A/+) mice were used in this study. LPS induced

expression of p-NKCC1 and activation of NFB in the primary culture of alveolar macrophages. WNK4 or SPAK knockout suppressed p-NKCC1 expression and inflammation cascade activation, whereas WNK4 knockin enhanced these responses. Intrapulmonary administration of LPS induced in vivo expression and phosphorylation of NKCC1 in alveolar inflammation cells and caused a shift in the cell population from macrophage to neutrophil predominance. WNK4 or SPAK knockout attenuated the LPS-induced alveolar cell-population shifting, macrophage NKCC1 phosphorylation, and acute lung injury, whereas WNK4 knockin augmented the inflammatory response. In summary, our results demonstrated the presence of NKCC1 in alveolar macrophage, which is inducible by lipopolysaccharide. Our results also showed showed that the WNK4–SPAK–NKCC1 cascade plays an important role in modulating macrophage activation to regulate LPS-induced lung inflammation and lung injury. Keywords: alveolar macrophage, inflammation, Na-K-Cl cotransporter-1, SPAK, WNK4

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1. Introduction Acute lung injury is a consequence of systemic or pulmonary fulminant inflammation. Pathogens elicit the inflammatory response via the activation of leukocytes, including macrophages. Alveolar macrophages play an important role in the front line of the host defense response [1]. Once activated, macrophages produce a variety of inflammatory mediators that facilitate the trafficking of leukocytes to the alveoli, to engulf the invading pathogens [2, 3]. However, excessive recruitment of leukocytes is harmful to the lungs and leads to acute lung injury or acute respiratory distress syndrome (ARDS) [1, 4]. Dysregulation of alveolar macrophage activation has been recognized as the major mechanism in the pathogenesis of acute lung injury. Macrophages are classically activated by the tumor necrosis factor (TNF) in cooperation with interferon- (IFN-) [4] or by a TLR ligand, such as lipopolysaccharides (LPS) [5]. The modulation of macrophage activation involves physical–mechanical alterations of the alveolar microenvironment, interaction between cells that mediate inflammation [6], and paracrine crosstalk between macrophages and the epithelium [7]. Recently, transporter channel activity has been recognized as a modulating factor in macrophage activation. Nguyen et al. [8] used a mouse model of bacterial pneumonia to demonstrate that animals lacking the sodiumpotassium-chloride cotransporter-1 (NKCC1) develop a less severe sepsis-related lung injury. Genetic manipulation [9] or pharmacological modulation [10] of NKCC1 may alter the inflammation cascade in the lungs. Our recent study demonstrated that NKCC1 augments LPSinduced macrophage activation [11], which suggests that NKCC1 plays an important role in the modulation of the immune response. NKCC1 is a unique ion transporter that regulates intracellular volume by coupling the transport of sodium, potassium, and chloride, which creates a driving force for transmembrane

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free water movement [12]. NKCC1 is normally expressed in many tissues and exhibits a variety of important physiological functions, including clearance of the alveolar fluid [10], regulation of the cell volume [11], and modulation of tumor invasion and migration [13]. The activity of NKCC1 is regulated mainly through the lysine-deficient kinase (WNK)/Ste20related proline/alanine-rich kinase (SPAK)/oxidative-stress responsive 1 (OSR1) cascade [14]. In response to hypertonicity or cell shrinkage, binding of WNK4 to SPAK facilitates phosphorylation and activation of SPAK, which in turn phosphorylates NKCC1 [15]. Overexpression of SPAK enhances the production of proinflammatory cytokines [16], whereas mice lacking SPAK are more resistant to inflammatory bowel diseases [17]; these findings suggest that SPAK is involved in the mechanism of regulation of the immune response. Our previous study demonstrated that SPAK knockout attenuates the ischemia–reperfusion-induced lung injury, whereas WNK4 knockin intensifies lung tissue injury, as assessed using an ex vivo model [18]. Lin et al. [10] also reported that the WNK4–SPAK–NKCC1 signaling pathway mediates the pathophysiological mechanism of hyperoxia-induced lung injury. The tissue protection may stem from the attenuation of parenchymal cell injury or the suppression of the overwhelmed inflammation. The aim of the present study was to investigate the role of the WNK4–SPAK–NKCC1 cascade in the regulation of macrophage activation.

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2. Materials and methods 2.1. Animals This study was approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taipei, Taiwan. The knockout (SPAK–/– and WNK4–/– ) and knockin (WNK4D561A/+ ) mice used here were established by Yang et al. [19-21]. These authors reported that the SPAK–/– and WNK4–/– mice exhibited low NKCC1 expression, whereas the WNK4D561A/+ animals showed high NKCC1 expression. Wild-type C57BL/6 mice aged 10–12 weeks were obtained from BioLASCO, Taipei, Taiwan. All animals were kept in a 12h light (day)/12h dark (night) cycle in pathogen-free conditions at the animal center of the National Defense Medical Center, Taipei, Taiwan. Chow diet and water were provided to the animals ad libitum. The genetic status of mice was monitored by PCR of SPAK and WNK4 gene fragments from genomic DNA extracted from mouse tails and using the primers provided by Yang et al. [19-21]. The PCR products were analyzed using the HT DNA High Sensitivity Assay Kit and a Caliper LabChip GX automated electrophoresis system (Caliper Life Science, USA).

2.2. Primary culture of alveolar macrophages (AMs) AMs in the bronchoalveolar lavage fluid (BALF) were used as primary cells [22]. BALF was pelleted by centrifugation at 500g at 4°C for 10 min, resuspended, and cultured overnight at 37℃/humidified air in 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5% Minimal Essential Medium (MEM) essential amino acids, 0.5% MEM nonessential amino acids, 1mM HEPES, 0.1% -mercaptoethanol, 1% penicillin–streptomycin, 100 U/ml of granulocyte–macrophage colony-stimulating factor, and 10% fetal bovine serum. All of the above were purchased from Invitrogen Co. (Carlsbad, CA, USA). The non-adherent cells were

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washed off, while the adherent cells were cultured further. LPS (1μg/ml) (Escherichia coli serotype 026: B6, Sigma, USA) was administered to the cells after pretreatment with bumetanide or PBS for 30 min. The NKCC1 mRNA expression was analyzed 2 h after LPS administration, while the expression of p-NKCC1 and proinflammatory mediators were analyzed at 20 h.

2.3. Modulation of cell size Cell volume was modulated by incubating the cells in media with different osmotic pressures (100, 300, or 500 mOsm) and adding various amounts of NaCl (Sigma-Aldrich Inc.) or distilled water to the HBSS solution (Invitrogen Co., USA), to achieve the desired concentration. The osmolality of the media was verified using an osmometer (Advanced Instruments 3900 Osmometer, EquipNet, Inc., USA).

2.4. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 20 min and incubated with 10% BSA (Sigma-Aldrich, USA) for an additional 20 min. Cells were incubated for 1 h with an antibody against NKCC1 (1:200), followed by incubation with a goat anti-rabbit DyLight 488-conjugated antibody (1:100; as a secondary antibody) and also the AMs were indentified by incubated with anti-F4/80+-APC (1:100) for 1 h. These antibodies were purchased from Invitrogen Co. (Carlsbad, CA, USA). The samples were mounted using VECTASHIELD HardSet Antifade Mounting Medium (Vector Lab, CA, USA) and images were captured using a laser scanning confocal microscope (LSM 880, Carl Zeiss, Germany).

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2.5. Flow cytometry The populations of AMs (F4/80+, CD11c+ ) and neutrophils (Ly6G+ ) in each BALF sample were analyzed using flow cytometry (FACSVerse, Becton Dickinson) and presented using the FACSuite software 4.0 (BD Bioscience). Flow cytometry was also used to detect pNKCC1 expression in macrophages. Antibodies to F4/80, CD11c, and Ly6G were purchased from Invitrogen Co. (Carlsbad, CA, USA). Antibody to phospho (p)-NKCC1 (T206) was customized and provided by Yang [18].

2.6. In-Cell Western Assay An In-Cell Western Assay was performed using an Odyssey Infrared Imaging System (LICOR Biosciences, NE). Primary AMs were cultured at a density of 8104 cells/well in 384well culture plates (Falcon: 353961, Thermo Fisher Scientific co., US). AMs were fixed with 4% paraformaldehyde and stained. Anti-rabbit IRDye® 680RD-labeled (1:5000) and antimouse IRDye® 800-labeled CW (1:5000) antibodies (LI-COR Biosciences, US) were used as secondary antibodies and were detected by the 700 and 800 nm channels, respectively.

2.7. LPS-induced acute lung injury The animals received nebulizing of bumetanide or saline for 30 min prior to intratracheal (IT) spraying of LPS at a dose of 2μg/g of body weight or saline in vivo. BALF cells of WT mice was collected for analysis of NKCC1 mRNA expression at 2, 4, 8, 16, and 24 h after LPS treatment. To determine the expression of phosphorylated NKCC1 in AMs, BALF cells of other WT mice were harvested at 4, 8, 16, 20, and 24 h. Mice from other groups, namely WT, SPAK-/-, WNK4D561/A+, and WNK4-/-, were sacrificed 20 h after LPS administration and BALF was collected from the right lung to analyze cell population, p-NKCC1 expression, level of

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cytokines, and LDH activity. The left lung was resected to evaluate pulmonary edema based on the wet-to-dry lung weight ratio (W/D) and to assess the lung injury by histological analysis. Other mice were pretreated with nebulized bumetanide (100 μM) (Sington Co., Taipei, Taiwan) 30 min before the intratracheal administration of LPS.

2.8. Histological Analysis The histological analysis was performed with Hematoxylin and Eosin staining as described previously [23]. Ten microscopic fields were randomly examined at high magnification (×400 ) to score as histopathologic evidence of lung injury within each field. The level of lung injury severity was based on (1) infiltration or aggregation of neutrophils in the airspace or vessel wall grading from 0 (normal) to 5 (most severe), and (2) thickness of the alveolar wall grading from 0 (normal) to 3 (most thickening). The resulting two scores were added and presented as the lung injury score.

2.9. Enzyme-linked immunosorbent assay (ELISA) TNF-, IL-6, and the macrophage inflammatory protein 2 (MIP-2) were measured in BALF and in culture media via ELISA according to the instructions provided by the manufacturer (R&D Systems Inc., USA).

2.10. Statistical analysis The data are expressed as means ± SD and were evaluated by one-way ANOVA or twoway ANOVA followed by Tukey’s post hoc test using GraphPad Prism 5 (GraphPad Software, US). Significance was set at P<0.05.

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3. Results 3.1. LPS stimulates the expression of NKCC1 in primary culture of AMs LPS stimulates immune responses in the lung, primarily by activating AMs isolated from the WT littermate mice. To determine whether NKCC1 was expressed in these cells, we isolated AMs from the BALF of C57BL/6 mice and found that it contained 93.6% macrophages (Fig. 1A). An immunofluorescence study revealed the expression of NKCC1 in the primary culture of AMs (Fig. 1B). LPS treatment led to a significantly greater upregulation of the NKCC1 mRNA in AMs than those in the control group (Fig. 1C). Furthermore, flow cytometry verified that LPS increased NKCC1 phosphorylation (Fig. 1D), whereas bumetanide pretreatment significantly suppressed the increase in p-NKCC1 (Fig. 1E).

3.2. NKCC1 phosphorylation regulated by WNK4 and SPAK Oxidative stress might activate NKCC1 through the WNK4–SPAK pathway in alveolar epithelium[10]. We validated this regulating pathway in the LPS-induced activation of primary AMs. The abundance of p-NKCC1 was assessed by using an In-Cell Western Assay. LPS induced a significantly lower level of NKCC1 phosphorylation in the AMs of WNK4–/– mice; in contrast, this response was partially suppressed in SPAK–/– mice (Fig. 2A and 2B).

3.3. AM activation after genetic manipulation of WNK4 and SPAK in primary cell cultures The WNK4-SPAK pathway is usually activated by inflammatory mediators [14, 24]. Interestingly, emergent evidences showed that genetic manipulation of WNK4 and SPAK may modulate the activation of the inflammation cascade reciprocally [10]. In this study, we assessed the abundance of IB and p-p65 in primary AMs using an In-Cell Western Assay.

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The degradation of IB (Fig. 3A and 3B) and phosphorylation of NFB/p65 (Fig. 3C and 3D) induced by LPS were significantly suppressed in the AMs of both SPAK–/– and WNK4–/– mice, whereas these responses were significantly enhanced in WNK4D561A/+ mice. Our previous study demonstrated that enlarging the cell volume of RAW264.7 cells amplified the LPS-induced inflammatory cell activation [11]. The present results showed that LPS-induced expression of TNF- (Fig. 4A) and IL-6 (Fig. 4B) was amplified by hypoosmotic culture and suppressed in a hyperosmotic environment in WT and WNK4D561A/+ mice, whereas these responses were blunted in SPAK–/– and WNK4–/– animals. In all groups, hypoosmotic culture augmented the LPS-induced expression of MIP-2, whereas a hyperosmotic medium suppressed this response significantly (Fig. 4C).

3.4. LPS stimulates the expression of NKCC1 in alveolar inflammation cells in vivo The role of WNK4-SPAK in inflammatory lung injury was validated in vivo. Cells in the BALF were collected within 24 h of the intratracheal spraying of LPS (2 µg/g of BW) and analyzed to determine the in vivo activation of NKCC1. At 2 h after its administration, LPS induced a significant increase in the expression of the NKCC1 mRNA, followed by a return to the baseline levels (at 8 h after treatment) (Fig. 5A). LPS caused a time-dependent increase in p-NKCC1 in alveolar cells that mediate inflammation, with peak expression at 16–24h (Fig. 5B and 5C). The LPS-induced phosphorylation of NKCC1 was significantly suppressed in SPAK–/– and WNK4–/– mice, whereas it was enhanced in WNK4D561A/+ mice (Fig. 5D and 5E).

3.5. Modulation of population shifting in the alveolar inflammation cells Intratracheal administration of LPS caused a significant population shift in the alveolar cells that mediate inflammation, from a macrophage to a neutrophil predominance (Fig. 6A).

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This cell population shift was suppressed in SPAK–/– and WNK4–/– mice and was significantly inhibited by bumetanide pretreatment in all groups of mice (Fig. 6B and 6C). In the remaining macrophages, the induction of NKCC1 phosphorylation by LPS was significantly lower in SPAK–/– and WNK4–/– mice compared with WT mice (Fig.6D and 6E).

3.6. LPS-induced lung injury The intratracheal spraying of LPS (2μg/g of body weight) caused significant histological changes associated with inflammatory lung injury (Fig.7A). LPS caused a less-severe lung injury in SPAK–/– and WNK4–/– mice compared with WT mice, as indicated by a lower level of neutrophil infiltration (Fig.7B), lower lung injury score (Fig. 7C), and milder increases in LDH activity in the BALF (Fig. 7D) and in the W/D ratio (Fig. 7E). The LPS-induced expression of TNF- and IL-6 in the BALF was also suppressed in the SPAK–/– and WNK4–/– groups (Fig. 7F and 7G). Bumetanide pretreatment suppressed the LPS-induced lung injury (Fig. 7A–7E) and cytokine production (Fig. 7F and 7G).

4. Discussion Fulminant lung inflammation remains the leading cause of tissue damage in acute lung injury [25]. Although anti-inflammatory drugs have failed in clinical trials for the treatment of ARDS, research efforts in this field remain centered on inflammatory regulation [26]. In the present study, intrapulmonary administration of LPS recruited neutrophils into alveoli and induced a change in cell population from a macrophage to a neutrophil predominance. Normally, neutrophils that are recruited into alveoli die quickly, to prevent excessive inflammation [27]. During acute inflammation, active macrophages may produce a variety of factors, including TNF-, to prolong neutrophil survival [28], and their own lifespan may be

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shortened. Alveolar macrophage cell death and neutrophil infiltration affect each other reciprocally to amplify the inflammatory response, which plays an important role in the exaggeration of inflammatory lung injury [29]. Our results showed that pretreatment with bumetanide or knockout of upstream regulatory genes, such as WNK4 and SPAK, significantly suppressed the population shift of alveolar cells that mediate inflammation. These results suggest the involvement of WNK4–SPAK in the mechanism that regulates alveolar inflammation. The WNK4–SPAK signaling cascade is a regulating mechanism that is important for body fluid homeostasis. Typically, this cascade exists in the renal epithelium and plays a crucial role in regulating salt balance and blood pressure [30, 31]. Overexpression of WNK4 mimics pseudohypoaldosteronism type II, which is characterized by salt-sensitive hypertension and hyperkalemia [32]. The initiation of this cascade by aldosterone [33] promotes the reabsorption of sodium and chloride from the lumen of renal tubules through the sodium– chloride channel (NCC). In addition to the renal homeostasis, a recent review [30] summarized the contributions of the WNK4–SPAK pathway to the functional regulation of a variety of tissues and cells. Kahle et al. [34] demonstrated that this pathway plays an important role in the regulation of cell volume and the excitability of neuronal cells. Clinical evidence also showed that a defect in WNK kinase regulation is associated with peripheral neuropathy [35]. Genetic depletion of SPAK normalized the hypersecretion of cerebrospinal fluid (CSF) in a posthemorrhagic hydrocephalus model [36]. Recently, we demonstrated the presence of the WNK4–SPAK cascade in the lungs of animals [10]. This cascade was activated by an osmotic gradient [37], free oxygen radicals [10], tissue ischemia/reperfusion, and inflammatory cytokines [18]. Via the regulation of NKCC1 phosphorylation and ENaC expression in the pulmonary epithelium, this cascade also creates an osmotic gradient across the blood–gas barrier, causing fluid movement. Therefore, in the respiratory system, the disturbance of the

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alveolar microenvironment may activate the WNK4–SPAK–NKCC1 cascade to maintain the pulmonary homeostasis and an optimal gas exchange. Interestingly, our previous study showed that NKCC1 inhibitors protected animals from lung injury via not only the enhancement of alveolar fluid clearance, but also the suppression of inflammatory responses [10, 11]. Emerging evidence has revealed the existence of an interaction between the cells that mediate inflammation and the epithelial barrier [7, 38]. Nguyen et al. [8] demonstrated that NKCC1-null mice developed a lower level of bacteremia and hypothermic sepsis in a pneumonia model, suggesting that NKCC1 plays an important role in the modulation of the inflammatory response. Those authors proposed that NKCC1 hinders neutrophil migration into alveoli through inter-epithelium gaps. The effects of NKCC1 on inflammation may be a consequence of epithelium swelling, which obstructs neutrophil transmigration. Moreover, it has been reported that mice lacking SPAK, an upstream regulator of NKCC1, are more resistant to inflammatory bowel diseases [17], suggesting that SPAK plays an important role in the modulation of both the function of the epithelial barrier and inflammatory responses [39]. In agreement with our previous study [11], the results reported here showed that the NKCC1 inhibitor suppressed LPS-induced macrophage activation and inflammatory lung injury. Moreover, WNK4 or SPAK knockout reduced LPS-induced neutrophil infiltration, suppressed cytokine production, and attenuated lung tissue edema, whereas WNK4 knockin augmented the inflammatory responses and acute lung injury. Therefore, the WNK4–SPAK–NKCC1 cascade might play an important role in the regulation of the inflammatory response by modulating directly the functions of the cells that mediate inflammation, in parallel to its effects on epithelia. To clarify the critical role of the WNK4–SPAK–NKCC1 cascade in the modulation of the functions of the cells that mediate inflammation, we used a primary culture of AMs. Although we harvested only a few unstimulated AMs from a small animal, the lavage fluid

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collected from three mice was pooled for each experiment. In agreement with our previous study, we demonstrated that culture in a medium with low osmotic pressure caused an increase in cell volume and amplified the LPS-induced phagocytic activity of the RAW 264.7 cells [11]. NKCC1 inhibition may attenuate inflammation-related acute lung injury by reducing cell volume and inhibiting the functions of the cells that mediate inflammation [26]. An increased macrophage cell volume is associated with functional activation in response to chemical [40] or physical [41] stimulation. Compan et al. [40] demonstrated that cell swelling induced by hypotonic culture induces greater cytokine production after LPS stimulation in murine macrophages. Liu et al. [42] also reported that the SPAK/JNK pathway is involved in insulininduced THP-1 cell chemotaxis. Here, we used an In-Cell Western Assay to detect the small amount of inflammation mediators produced by primary AMs. WNK4 or SPAK knockout abolished NKCC1 phosphorylation and suppressed the LPS-induced NFB activation, whereas WNK4 knockin augmented these responses (Fig. 5). In hypoosmotic conditions, LPS induced cytokine and chemokine production to a greater extent in WNK4 knockin AMs compared with WNK4 or SPAK knockout AMs (Fig. 6). These results suggest that the WNK4–SPAK–NKCC1 pathway modulates the cell volume, inflammation-activation capacity, and phagocytic activity of primary AMs and neutrophil chemoattraction during LPS stimulation. In summary, the intrapulmonary administration of LPS induced NKCC1 expression and phosphorylation in the alveolar cells that mediate inflammation and caused inflammatory lung injury. WNK4 or SPAK knockout reduced NKCC1 activation and attenuated LPS-induced neutrophil infiltration and lung injury, whereas WNK4 knockin augmented the inflammatory response. Inhalation of theNKCC1 inhibitor bumetanide produced lung-protective effects, thus, mimicking WNK4 or SPAK knockout. LPS also stimulated the primary culture of alveolar macrophages to express the activated form of NKCC1. WNK4 or SPAK knockout suppressed the activation of NFB and production of cytokines induced by LPS in hypoosmotic

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conditions, whereas WNK4 knockin augmented these responses. Therefore, we concluded that the WNK4–SPAK–NKCC1 cascade plays an important role in the modulation of macrophage activation to regulate LPS-induced lung inflammation and lung injury.

Acknowledgments This study was financially supported by grants from the Ministry of Science and Technology, Taiwan (MOST-105-2314-B-016-032; MOST 106-2314-B-016-036-MY3), Medical Affairs Bureau of the Ministry of National Defense, Taiwan (MAB-106-063), and TriService General Hospital, Taiwan (TSGH-C106-070; TSGH-C107-077; TSGH-C108-103). We would like to acknowledge Instrument Center of National Defense Medical Centre for the technical supports.

Declaration of interest None

Submission declaration and verification The manuscript or portions of it has never been published by another journal or electronic publication and that it has not been submitted simultaneously for publication elsewhere.

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Figure legends Fig. 1. LPS stimulates the expression of p-NKCC1 in alveolar macrophages in vitro. The primary AMs isolated from the BALF of C57BL/6 mice (A) and detected by NKCC1 immunofluorescence staining (Nuclear: blue; NKCC1: green; F4/80: red) (B). AMs expressed a high level of NKCC1 after an in vitro LPS challenge for 2 h (C) (n=4). LPS treatment for 20 h stimulated the expression of p-NKCC1 in AMs (D). Bumetanide pretreatment suppressed the increase in p-NKCC1 expression (E) (n=6). * p< 0.05 versus control (CTRL); # p < 0.05 versus LPS.

Fig. 2. The WNK4-SPAK modulates NKCC1 phosphorylation. LPS induced p-NKCC1 to a lower extent in primary AMs isolated from WNK4–/– and SPAK–/– mice detected by In-Cell Western Assay(A) and the quantitative values from intensity of p-NKCC1 (n=3) (B). * p< 0.05 versus CTRL;

+ p<

0.05 versus WT. WT: wild-type; SK: SPAK–/– ; WI: WNK4D561A/+ ;

WK: WNK4–/–

Fig. 3. The WNK4-SPAK cascade modulates the inflammatory response in vitro. The IB degradation (A and B) and NFB p65 phosphorylation (C and D) induced by LPS were significantly suppressed in both SPAK–/– and WNK4–/– mice, whereas these responses were enhanced in WNK4D561A/+ mice (n=3). * p< 0.05 versus CTRL; + p< 0.05 versus WT.

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Fig. 4. The WNK4–SPAK cascade modulates the effects of osmotic culture on the LPSinduced AM activation. WNK4 knockin augmented the LPS-induced production of TNF-, independent of the osmotic pressure of the culture media (A) (n=6). Hyperosmotic culture suppressed the LPS-induced IL-6 production slightly in WT mice (B), whereas it significantly abolished MIP-2 production in all groups of animals (C) (n=6). * p< 0.05 versus 300 mOsm; + p < 0.05 versus WT.

Fig. 5. The WNK4–SPAK cascade regulates NKCC1 activation in alveolar cells that mediate inflammation. LPS increased the expression of the NKCC1 mRNA (n=3) (A) and the phosphorylation of NKCC1 (B and C) in alveolar cells that mediate inflammation (n=5). The LPS-induced p-NKCC1 expression was suppressed in SPAK–/– and WNK4–/– mice, whereas it was enhanced in WNK4D561A/+ mice (D and E) (n=6). * p< 0.05 versus 0 h. ; + p < 0.05 versus WT

Fig. 6. The WNK4–SPAK cascade modulates the LPS-induced change in the population of alveolar cells that mediate inflammation. Intrapulmonary spraying of LPS caused a population shift from a macrophage to a neutrophil predominance (A). This population shift was suppressed in SPAK–/– and WNK4–/– mice (B and C) (n=5). In WNK4D561A/+ mice, the alveolar neutrophils and macrophages expressed p-NKCC1 at high levels before intrapulmonary LPS spraying (D–G). In SPAK–/– and WNK4–/– mice, LPS induced p-NKCC1 expression to a lower extent in macrophages (D and E) (n=6). * p< 0.05versus control (CTRL); # p < 0.05 versus LPS; + p < 0.05 versus WT.

Fig. 7. The WNK4–SPAK–NKCC1 cascade modulates LPS-induced lung injury. Intrapulmonary spraying of LPS caused a less severe lung injury in SPAK–/– and WNK4–/– mice,

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as indicated by the observation of fewer histological changes (A), lower neutrophil infiltration (B), a lower lung injury score (C), and a less pronounced increase in BALF LDH activity (D) and in the W/D ratio (E) (n=6). The LPS-induced production of BALF, TNF-, and IL-6 was also suppressed in the SPAK–/– and WNK4–/– groups (F and G) (n=6). Bumetanide pretreatment suppressed the LPS-induced lung injury (A–E) and cytokine production (F and G) (n=6). * p< 0.05 versus CTRL; # p < 0.05 versus LPS; + p < 0.05 versus WT. Chin-Mao Hung: Investigation, Software, Methodology, Writing- Original draft preparation Chung-Kan Peng: Project administration, Visualization, Validation Resources Sung-Sen Yang: Supervision, Formal analysis, Resources Hao-Ai Shui: Conceptualization, Methodology Kun-Lun Huang: Conceptualization, Validation, Funding acquisition, Writing - Review & Editing

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