FXYD5 Is an Essential Mediator of the Inflammatory Response during Lung Injury

Abstract

The alveolar epithelium secretes cytokines and chemokines that recruit immune cells to the lungs, which is essential for fighting infections but in excess can promote lung injury. Overexpression of FXYD5, a tissue-specific regulator of the Na,K-ATPase, in mice, impairs the alveolo-epithelial barrier, and FXYD5 overexpression in renal cells increases C-C chemokine ligand-2 (CCL2) secretion in response to lipopolysaccharide (LPS). The aim of this study was to determine whether FXYD5 contributes to the lung inflammation and injury. Exposure of alveolar epithelial cells (AEC) to LPS increased FXYD5 levels at the plasma membrane, and FXYD5 silencing prevented both the activation of NF-κB and the secretion of cytokines in response to LPS. Intratracheal instillation of LPS into mice increased FXYD5 levels in the lung. FXYD5 overexpression increased the recruitment of interstitial macrophages and classical monocytes to the lung in response to LPS. FXYD5 silencing decreased CCL2 levels, number of cells, and protein concentration in bronchoalveolar lavage fluid (BALF) after LPS treatment, indicating that FXYD5 is required for the NF-κB-stimulated epithelial production of CCL2, the influx of immune cells, and the increase in alveolo-epithelial permeability in response to LPS. Silencing of FXYD5 also prevented the activation of NF-κB and cytokine secretion in response to interferon α and TNF-α, suggesting that pro-inflammatory effects of FXYD5 are not limited to the LPS-induced pathway. Furthermore, in the absence of other stimuli, FXYD5 overexpression in AEC activated NF-κB and increased cytokine production, while FXYD5 overexpression in mice increased cytokine levels in BALF, indicating that FXYD5 is sufficient to induce the NF-κB-stimulated cytokine secretion by the alveolar epithelium. The FXYD5 overexpression also increased cell counts in BALF, which was prevented by silencing the CCL2 receptor (CCR2), or by treating mice with a CCR2-blocking antibody, confirming that FXYD5-induced CCL2 production leads to the recruitment of monocytes to the lung. Taken together, the data demonstrate that FXYD5 is a key contributor to inflammatory lung injury.

Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection

“…HRP-tagged secondary antibodies (Bio-Rad, 1721011 and 1721019) were used in combination with Super-Signal ECL kit (Thermo Fisher Scientific) to develop blots using a LI-COR Odyssey Imager and companion software Image Studio version 5.2. Blots were quantified using densitometry (ImageJ 1.46r, NIH) (19). IAV infection.…”

“…Cells were stained with antibodies listed in Supplemental Tables 2 and 3 and gated as described elsewhere (19,36) and outlined in Supplemental Figure 3, A and B. For lymphoid analysis, cells were first permeabilized with the FOXP3 staining kit (eBioscience, 005523) according to the manufacturer's instructions.…”

“…Data from animal models of severe IAV infection suggest that cytokine storm is the major driver of morbidity and mortality (13)(14)(15)(16)(17). Several studies suggest that reductions in circulating cytokines improve lung injury and survival, highlighting the importance of modulating the host inflammatory response during IAV infection (14,18,19).…”

Multiplexed single-cell transcriptomic analysis of normal and impaired lung development in the mouse

“…Several genes known to be induced by cellular stress or inflammation [29][30][31] were up-regulated in the Hyperoxia EC cluster (Supplementary Figure 5h). We observed increased expression of Ctgf and Fxyd5, known to contribute to inflammatory lung injury 32,33 and of Tgfb2, shown to be associated with profibrotic responses in the lung 34 . In addition, an anti-angiogenic gene expression profile was observed, characterized by a decrease in expression of Vegfa, and an increase in expression of Cdkn1a, Timp3, Serpine1, and Igfbp7 ( Supplementary Figure 5h), consistent with the crucial role of angiogenic growth factors in normal lung development 35 .…”

Mapping gene regulatory networks of primary CD4+T cells using single-cell genomics and genome engineering

“…We also identified three activated populations (Th stim ) defined by the expression of HMGB2 and STMN1 (48,49) and distinguished from each other by the expression of histone markers (Th stim,histone , 19,920 cells, 12%), cell cycling markers PTTG1 (50,51) and KIAA0101 (52-54) (Th stim,cycling , 16,905 cells, 10%), and naive markers CCNB1 (55) (T stim,naive , 12,345 cells, 7.4%). We also found a proliferating population (T prolif ) that expressed genes associated with tumor progression, including FXYD5 (56)(57)(58), LIMD2 (59,60), and PFDN5 (61) (903 cells, 0.05%). Finally, we identified two small clusters likely to be transitional, as they are intermediates in lineage trajectory (62) space either between naive and cytotoxic cells (T naive→cyto 752 cells, 0.04%) or between naive and stimulated cells (T naive→stim : 172 cells, 0.01%; Fig.…”