FGF 13 functions as a regulator of the ERK/aerobic glycolysis axis in the inflammatory state during septic lung injury

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Fibroblast growth factor 13 (FGF13) belongs to the FGF homologous factor subfamily, members of which lack signal peptides. In this study, we demonstrate that FGF13 is significantly downregulated in endothelial cells and macrophages in the lungs of septic patients and septic mice. However, the role of FGF13 in sepsis and the underlying mechanism are largely unknown. By using mice with conditional Fgf13 knockout and FGF13 overexpression, we find that FGF13 accelerates septic lung injury by promoting inflammatory activation of endothelial cells and macrophages. Specifically, FGF13 functions as a scaffold protein in TAK1/MEK/ERK signaling to promote hypoxia-inducible factor (HIF)−1α-regulated aerobic glycolysis in the inflammatory state. Meanwhile, the protective effect of conditional Fgf13 knockout is abolished in HIF-1α-overexpressing mice. In addition, SCH772984 (a selective antagonist of ERK signaling) abolishes the aggravation of inflammation in lungs induced by FGF13 overexpression. Our findings demonstrate that FGF13 promotes inflammatory activation upon septic lung injury through the ERK/aerobic glycolysis axis, thereby accelerating the progression of septic lung injury.

Introduction
As a critical public health emergency, sepsis has had an average annual rate of about 50 million cases globally since 2017, with a mortality rate of approximately 20%, resulting in a financial burden of around $20 billion in the United States alone1. Despite decades of clinical research on sepsis, treatment strategies for this disease remain limited to antibiotics, steroids, supplements, and organ support. These broad-spectrum interventions not only exhibit limited effectiveness but also frequently lead to various side effects, which impact patient prognosis2. Therefore, gaining a deeper understanding of the pathogenesis of sepsis and developing more targeted and safer therapeutic strategies based on this enhanced understanding has significant clinical implications.

During the progression of sepsis, the direct damage caused by pathogens and the excessive inflammatory response induce multi-organ damage, potentially culminating in organ failure and mortality. Among these complications, septic lung injury is the most prevalent and serious manifestation3,4. When pathogen-associated molecular patterns (e.g., lipopolysaccharide (LPS), mitochondrial DNA, and lipoteichoic acid) produced by pathogens destroy the barrier integrity of lung epithelial and endothelial cells or even kill them, they lead to the inflammatory activation of these cells, which results in recruitment of immune cells (e.g., neutrophils and monocytes (macrophages)) into the lung microenvironment to fight pathogens5–7. Although immune cells clear pathogens and cell debris from the lungs, they also lead to “an inflammatory storm” (e.g., interleukin (IL)−1β, IL-6, and tumor necrosis factor (TNF)-α) and production of reactive oxygen species (ROS). These inflammatory cytokines and ROS aggravate the injury of lung epithelial and endothelial cells, further exacerbate the infiltration of immune cells, impair gas exchange, and ultimately lead to respiratory failure8,9. In this process, endothelial cells serve as “initiators” in the development of lung inflammation, while macrophages function as “amplifiers” of inflammatory injury, and both play crucial roles as mediators of septic lung injury. Consequently, inhibition of inflammatory cell infiltration resulting from inflammatory activation of endothelial cells or suppression of the inflammatory storm induced by macrophages can effectively mitigate septic lung injury and reduce the mortality rate of sepsis10.

Fibroblast growth factor 13 (FGF13) belongs to the FGF homologous factor subfamily, which differs from other FGF subfamilies because its members all lack signal peptides and cannot be secreted conventionally11. FGF13 has been extensively studied in the nervous system, and our recent research revealed that it disrupts endothelial cell homeostasis by modulating mitophagy through Parkin, exacerbating diabetic nephropathy12. Additionally, another study of ours demonstrated that FGF12 (homologous to FGF13) regulates macrophage polarization to promote liver fibrosis13. However, it remains unknown whether FGF13 participates in the pathological progression of septic lung injury, which is mainly mediated by endothelial cells and macrophages.

Here, we found that FGF13 was downregulated in endothelial cells and macrophages in the lungs of septic mice and septic patients. FGF13 promoted inflammatory activation of both endothelial cells and macrophages upon LPS stimulation in vitro.

Here, we show, by using two mouse models (injection of LPS (endotoxic shock) and cecal ligation and puncture (CLP)), we find that septic lung injury is alleviated in mice with endothelial cell-specific (Fgf13△Endo) and myeloid cell-specific (Fgf13△mye) Fgf13 knockout, but is aggravated in mice with endothelial cell-specific (CDH5-CreDIO-Fgf13) and myeloid cell-specific (LyzM-CreDIO-Fgf13) FGF13 overexpression. To further investigate the mechanisms of FGF13 in sepsis, we performed co-immunoprecipitation and mass spectrometry. This demonstrates that FGF13 functions as a scaffold protein in the TAK1/MEK/ERK axis to regulate hypoxia-inducible factor (HIF)−1α-induced aerobic glycolysis, which promotes inflammatory activation of endothelial cells and macrophages, ultimately contributing to septic lung injury.

Results
FGF13 is downregulated in endothelial cells and macrophages in septic lungs

FGFs are critical regulators of lung development and microenvironment homeostasis14. Prior studies indicate that specific FGFs (e.g., FGF1, FGF2, FGF9, and FGF10) modulate pulmonary immune responses and repair processes15–18. During sepsis, perturbation of homeostasis in the lung microenvironment substantially alters FGF expression. To systematically profile these changes, we performed RNA sequencing of lung tissues from healthy and LPS-treated mice, and validated the findings via qRT-PCR using human samples (non-septic patients vs. septic patients). FGF2 was consistently upregulated in both species during sepsis, while FGF1, FGF10, FGF13, FGF14, and FGF18 were downregulated (Fig. 1A–C). While the roles of most of these FGFs (except FGF13/FGF14) have been characterized16,19–21, our recent discovery of the regulatory function of FGF13 in endothelial homeostasis prompted us to investigate its specific role in septic lung injury and the underlying mechanisms.

Consistent with its mRNA level, the protein level of FGF13 was decreased in lungs of septic mice and septic patients (Fig. 1D, E). Additionally, the protein level of FGF13 in lungs of moribund mice in LPS injection model was negatively correlated with survival time (Fig. 1F). After confirming through H&E staining that CLP induced significant pulmonary pathological changes (with evident inflammatory cell infiltration) (Fig. 1G), to further clarify the main cell types that express FGF13 in the lungs, we performed immunofluorescence analysis of FGF13 and various cell markers (AQP5, SPC, CD34, and F4/80) in mice. Endothelial cells were the major cell type that expressed FGF13, followed by macrophages. Expression of FGF13 was relatively low in both types of epithelial cells. Upon CLP operation, the number of FGF13-positive endothelial cells and macrophages greatly decreased in lungs of mice (Fig. 1H–L). Consistently, the alterations in the protein level of FGF13 and colocalization of FGF13 with different cell markers were similar in LPS-treated mice (Fig. S1A–G). Consistent with the findings in septic mice, colocalization of FGF13 with endothelial cells and macrophages was decreased in septic patients (Fig. 1M–O). Furthermore, we confirmed these observations in vitro. After LPS treatment, the protein level of FGF13 was decreased in human pulmonary microvascular endothelial cells (HPMECs) and THP-1 cells (Fig. S1H, I). These results suggest that expression of FGF13 in endothelial cells and macrophages is downregulated in the inflammatory state, which might modulate the progression of septic lung injury.

FGF13 promotes inflammatory activation of endothelial cells to aggravate immune cell infiltration upon septic lung injury

To explore the role of FGF13 in septic lung injury, we used Ad-shFgf13/Ad-Fgf13 to knockdown or overexpress FGF13 in HPMECs (Fig. S2A, B). In an endothelial cell inflammatory activation model, the LPS-induced increases of the protein levels of VCAM-1 and ICAM-1, as well as the mRNA levels of Ccl2, Vcam-1, and Icam-1 were attenuated in Fgf13-knockdown HPMECs but enhanced in Fgf13-overexpressing HPMECs (Fig. S2C–F). Fgf13 knockdown reduced monocyte adhesion to LPS-stimulated HPMECs (Fig. S2G–J), whereas LPS induced greater monocyte adhesion to Fgf13-overexpressing HPMECs (Fig. S2K–N). These results indicate that FGF13 promotes LPS-induced inflammatory activation of endothelial cells.

To further validate these findings in vivo, we generated endothelial cell-specific Fgf13 knockout (Fgf13△Endo) mice by crossbreeding transgenic mice, as well as endothelial cell-specific FGF13 overexpression (CDH5-CreDIO-Fgf13) mice by injecting AAV-lung-DIO-Fgf13 (Fig. S3A–C). In both models (CLP and injection of LPS), Fgf13△Endo mice had a higher survival rate than CDH5-Cre controls, while CDH5-CreDIO-Fgf13 mice had a lower survival rate (Fig. 2A, S4A). Assessment of bronchoalveolar lavage fluid (BALF) protein concentrations/cell counts, lung wet-to-dry weight ratios, and lung morphology revealed that sepsis-induced lung edema and hemorrhage were significantly alleviated in Fgf13△Endo mice but exacerbated in CDH5-CreDIO-Fgf13 mice (Figs. 2B–F, S4B–F). Consistent with the in vitro data, lung tissue expression of endothelial cell inflammatory activation markers (iNOS, ICAM-1, and VCAM-1) was lower in Fgf13△Endo and higher in CDH5-CreDIO-Fgf13 mice than in CDH5-Cre controls in both models (Figs. 2G, H, S4G, H). Reflecting this endothelial cell inflammatory activation, hematoxylin-eosin (H&E) staining and myeloperoxidase (MPO)/F4/80 immunofluorescence analysis demonstrated that inflammatory cell infiltration (neutrophils and macrophages) was significantly reduced in lungs of septic Fgf13△Endo mice but increased in those of septic CDH5-CreDIO-Fgf13 mice (Figs. 2I–M, S4I–M). Collectively, these findings demonstrate that FGF13 promotes endothelial cell inflammatory activation during sepsis, thereby exacerbating inflammatory cell infiltration and aggravating septic lung injury.

FGF13 promotes inflammatory activation of macrophages to amplify inflammation upon septic lung injury

Considering that FGF13 also exhibited expression changes in macrophages, which play a pivotal role in septic lung injury, we were intrigued by the possibility that it influences inflammatory activation of these cells. Building upon the methodologies employed in endothelial cells, we used Ad-shFgf13/Ad-Fgf13 to knock down or overexpress FGF13 in THP-1 cells (Fig. S5A, B). Upon LPS stimulation, the increase of the protein level of iNOS and the increase of the mRNA levels of Il-1β, Il-6, and Tnf-α were relieved in Fgf13-knockdown THP-1 cells and aggravated in Fgf13-overexpressing THP-1 cells (Fig. S5C–F). Compared with the culture medium of Ad-vector-transfected THP-1 cells, that of Fgf13-knockdown THP-1 cells induced less apoptosis and barrier loss in HPMECs, while that of Fgf13-overexpressing THP-1 cells induced more (Fig. S5G–N). These results indicate that FGF13 promotes LPS-induced inflammatory activation of macrophages.

To further validate these findings in vivo, we generated macrophage-specific Fgf13 knockout (Fgf13△mye) mice by crossbreeding transgenic mice as well as macrophage-specific FGF13 overexpression (LyzM-CreDIO-Fgf13) mice by injecting AAV-lung-DIO-Fgf13 (Figure S6A–C). Consistent with the results in endothelial cells, Fgf13△mye mice had a higher survival rate than CDH5-Cre mice in both models, while LyzM-CreDIO-Fgf13 mice had a lower survival rate (Fig. 3A, S7A). BALF analysis and lung morphology observation revealed that sepsis-induced lung edema, hemorrhage, and inflammatory cell infiltration were significantly alleviated in Fgf13△mye mice, but aggravated in LyzM-CreDIO-Fgf13 mice (Figs. 3B–G, S7B–G). In line with the in vitro results, inflammatory activation of macrophages was restrained in lungs of Fgf13△mye mice in both models, but aggravated in lungs of LyzM-CreDIO-Fgf13 mice (Figs. 3H, I, S7H, I). Furthermore, compared with LyzM-Cre mice during sepsis, Fgf13△mye mice displayed lower serum levels of IL-1β, IL-6, and TNF-α, while the opposite was true in LyzM-CreDIO-Fgf13 mice (Figs. 3J, S7J). Likewise, sepsis-induced cell apoptosis and barrier loss in lungs were mitigated in Fgf13△mye mice, but aggravated in LyzM-CreDIO-Fgf13 mice (Figs. 3K–N, S7K–N). Collectively, these findings suggest that FGF13 accelerates inflammatory activation of macrophages during sepsis, leading to the production of large amounts of inflammatory cytokines and exacerbation of septic lung injury.

FGF13 promotes aerobic glycolysis in the inflammatory state

Beyond its established functions in the nervous system, FGF13 plays significant roles in colorectal cancer, breast cancer, and several other malignancies22–24. Cancer cells undergo metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in order to fuel their rapid proliferation, migration, and invasion. This metabolic shift is strikingly similar to that observed in endothelial cells and macrophages during inflammatory activation25,26. Notably, elevated lactate levels resulting from enhanced aerobic glycolysis correlate strongly with mortality rates in septic patients27. Recent investigations of glucose homeostasis revealed an inverse relationship between FGF13 expression and the peripheral blood glucose concentration28. Considering the critical role of aerobic glycolysis in mediating pro-inflammatory responses of both endothelial and immune cells, we postulated that FGF13 may regulate inflammation progression by modulating glucose metabolism pathways.

As expected, in both models, the levels of lactate were lower in the peripheral blood of Fgf13△Endo and Fgf13△mye mice than in the peripheral blood of corresponding control mice, whereas the opposite was true in CDH5-CreDIO-Fgf13 and LyzM-CreDIO-Fgf13 mice (Fig. S8A–D). Consistent with the findings of the in vivo studies, the results of the Seahorse XF glycolysis stress test suggested that the aerobic glycolysis capacity of pro-inflammatory HPMECs and THP-1 cells was significantly reduced by knockdown of FGF13, but amplified by overexpression of FGF13 (Fig. 4A–D). Both 2-NBDG fluorescence staining and glucose uptake experiments revealed that glucose uptake and lactate production in pro-inflammatory HPMECs and THP-1 cells were notably disrupted by knockdown of FGF13 (Fig. 4E–L), but enhanced by overexpression of FGF13 (Fig. 4M–T). Subsequent investigations revealed that PFK15 (a glycolysis inhibitor29) mitigated inflammatory activation of Fgf13-overexpressing HPMECs, resulting in decreased monocyte adhesion (Fig. S8E–H). Moreover, PFK15 reduced the elevated levels of iNOS protein and secretion of IL-6 and TNF-α in Fgf13-overexpressing THP-1 cells (Figure S8I, J). Overall, these findings underscore the role of FGF13 in promoting aerobic glycolysis in endothelial cells and macrophages under inflammatory conditions, contributing to the amplification of their inflammatory response.

FGF13 regulates aerobic glycolysis via ERK/HIF-1α signaling

During inflammatory responses, aerobic glycolysis is sophisticatedly regulated by multiple signaling cascades, including the AMPK-mTOR, ERK/HIF-1α, and NF-κB pathways25. To elucidate the precise molecular mechanism by which FGF13 modulates inflammatory aerobic glycolysis, we performed unbiased proteomic analysis via mass spectrometry of lysates of FGF13-overexpressing HEK293 cells (Fig. 5A). This comprehensive screening identified MAPK1 (ERK2) as a high-confidence interaction partner of FGF13 (Fig. 5B). Based on this protein–protein interaction, we hypothesized that FGF13 regulates inflammation-induced aerobic glycolysis primarily through the ERK/HIF-1α signaling axis, which is a well-established pathway in metabolic reprogramming during inflammatory activation.

As expected, knockdown of FGF13 significantly reduced the elevated phosphorylation levels of MEK1/2 and ERK1/2 as well as the protein level of HIF-1α in LPS-treated HPMECs and THP-1 cells, while overexpression of FGF13 further upregulated the phosphorylation levels of MEK1/2 and ERK1/2 as well as the protein level of HIF-1α (Fig. 5C–F). When cells were pretreated with SCH772984 (a specific ERK1/2 inhibitor)30, the enhanced aerobic glycolysis capacity of Fgf13-overexpressing HPMECs was abolished, and the increased glucose uptake and lactate production were also abrogated (Figs. 5G, S9A–E). Consistently, SCH772984 counteracted the increased monocyte adhesion to LPS-treated Fgf13-overexpressing HPMECs (Fig. S9F–I). Similarly, pretreatment with SCH772984 abolished the enhanced aerobic glycolysis capacity of Fgf13-overexpressing THP-1 cells, leading to decreased glucose uptake and lactate production in LPS-treated THP-1 cells (Figs. 5H, S9J–N). Moreover, SCH772984 reduced the elevated levels of iNOS protein and secretion of IL-6 and TNF-α in Fgf13-overexpressing THP-1 cells (Fig. S9O, P). Collectively, these results indicate that the role of FGF13 in promoting aerobic glycolysis to exacerbate inflammatory activation of endothelial cells and macrophages depends on ERK/HIF-1α signaling.

The protective effect of FGF13 knockout on septic lung injury is abolished in HIF-1α-overexpressing mice

To further substantiate our findings in vivo, we engineered mice with endothelial cell-specific and macrophage-specific HIF-1α overexpression by administering AAV-lung-DIO-hif1a. Following confirmation of successful HIF-1α overexpression in endothelial cells, we observed that HIF-1α overexpression reversed the protective effects seen in Fgf13△Endo mice in both models, including reduced peripheral blood lactate levels and improved survival rates (Fig. S10A–E). Comprehensive assessment of lung pathology, including wet-to-dry weight ratios, morphological observation, H&E staining, and immunofluorescence detection of MPO/F4/80 demonstrated that the attenuated lung edema, hemorrhage, and inflammatory cell infiltration (neutrophils and macrophages) in Fgf13△Endo mice in both models were completely negated by AAV-lung-DIO-hif1a administration (Figs. 6A–H, S10F–M). Similarly, after injection of AAV-lung-DIO-hif1a, the peripheral blood lactate levels and survival rate of Fgf13△mye mice did not significantly differ from those of LyzM-Cre mice in both models (Fig. S11A–E). Following LPS challenge or the CLP procedure, HIF-1α overexpression counteracted both the suppression of the inflammatory activation of pulmonary macrophages and the downregulation of serum pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) observed in Fgf13△mye mice (Figs. 6I, J, S11F-I). Likewise, measurement of lung wet-to-dry weight ratios, observation of lung morphology, H&E staining, and fluorescence staining of VE-cadherin in lungs of each group revealed that the reductions of hemorrhage, inflammatory cell infiltration, and barrier loss in Fgf13△mye mice were abolished by AAV-lung-DIO-hif1a in both models (Figs. 6K–P, S11J–O). These findings suggest that FGF13 worsens septic lung injury by promoting HIF-1α-induced aerobic glycolysis in endothelial cells and macrophages.

FGF13 acts as a scaffold protein in TAK1/MEK/ERK signaling

Figure S12A shows the activation process of the ERK signaling pathway upon LPS stimulation. Knockdown of Fgf13 in HPMECs and THP-1 cells did not affect phosphorylation of TAK1 at Thr184/Thr187 (which is critical for ERK signaling activation31) (Fig. S12B, C), indicating that FGF13 does not affect the upstream signaling of TAK1. Liquid chromatography-mass spectrometry (LC-MS) revealed that FGF13 can interact with ERK2; therefore, we speculated that FGF13 is involved in activation of the TAK1-MEK-ERK signaling axis through protein binding. Co-immunoprecipitation experiments showed that FGF13 interacted with TAK1, MEK1/2, and ERK1/2 in HPMECs and THP-1 cells, and these interactions were enhanced upon LPS stimulation (Figs. 7A, B, S13A–D). Similarly, immunofluorescence experiments demonstrated the clear colocalization of FGF13 with TAK1, MEK1/2, and ERK1/2 in these cells (Fig. 7C, D). To further validate our hypothesis, we transfected HEK293 cells with plasmids encoding Flag-FGF13, HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2. As expected, exogenous FGF13 interacted with exogenous TAK1, MEK1, MEK2, ERK1, and ERK2 in HEK293 cells (Fig. 7E–I). Additionally, GST pull-down assays indicated that FGF13 interacted directly with TAK1, MEK1, MEK2, ERK1, and ERK2 (Fig. 7J). Based on these findings, we speculated that FGF13 acts as a scaffold protein in the TAK1-MEK-ERK axis. In LPS-treated HPMECs and THP-1 cells, the interaction between MEK1 and TAK1, as well as ERK1/2, was reduced by knockdown of Fgf13, but enhanced by overexpression of Fgf13 (Fig. 7K–N). Thus, FGF13 promotes activation of the TAK1-MEK-ERK axis by functioning as a scaffold protein to strengthen the interactions between components.

Despite our finding that FGF13 is a critical scaffold protein that facilitates activation of the TAK1-MEK-ERK signaling axis, we observed a paradoxical decrease in FGF13 protein expression in both HPMECs and THP-1 cells under inflammatory conditions. This intriguing contradiction prompted further mechanistic investigation. Previous studies reported p53-mediated transcriptional repression of miR-504 and its host gene Fgf13, while other evidence indicates that septic conditions activate p53 signaling pathways, consequently accelerating apoptosis and DNA damage during disease progression32,33. Integrating these molecular insights, we postulated that inflammatory downregulation of FGF13 might occur through p53-dependent mechanisms. To test this hypothesis, we employed RNA interference strategies targeting p53 in both HPMECs and THP-1 cells. Remarkably, p53 knockdown abolished the LPS-induced downregulation of FGF13 protein, providing compelling evidence that p53 is the critical mediator of inflammatory FGF13 downregulation in both endothelial cells and macrophages (Figure S14A–D). This regulatory mechanism reveals an important homeostatic circuit in which inflammatory stimuli trigger p53-dependent suppression of an otherwise pro-inflammatory signaling scaffold molecule.

SCH772984 mitigates the exacerbation of septic lung injury by FGF13

To further confirm that the role of FGF13 in exacerbating septic lung injury relies on ERK signaling, we administered the ERK signaling antagonist SCH772984 to CDH5-CreDIO-Fgf13 and LyzM-CreDIO-Fgf13 mice in order to inhibit ERK activation following LPS challenge or the CLP procedure (Figs. S15A, B, S16A, B). In CDH5-CreDIO-Fgf13 mice, SCH772984 robustly counteracted the pathological elevation of peripheral blood lactate levels and substantially ameliorated the aggravated pulmonary edema and hemorrhage in both models (Figs. 8A–C, S15C–G). Histopathological analysis through H&E staining and fluorescence staining of MPO/F4/80 revealed that ERK inhibition substantially diminished the enhanced neutrophil and macrophage infiltration in lungs of CDH5-CreDIO-Fgf13 mice following LPS challenge or the CLP procedure (Figs. 8D–H, S15H–L).

In parallel experiments with LyzM-CreDIO-Fgf13 mice, SCH772984 similarly attenuated the pathological elevation of peripheral blood lactate levels under septic conditions (Fig. S16C, D). Notably, ERK inhibition profoundly suppressed both the augmented pulmonary accumulation of pro-inflammatory macrophages and the elevated systemic levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in LyzM-CreDIO-Fgf13 mice in both sepsis models (Figs. 8I, J, S16E- H). Consequently, SCH772984 significantly mitigated the enhanced pulmonary pathologies (edema, hemorrhage, inflammatory cell infiltration, and endothelial barrier disruption) in LyzM-CreDIO-Fgf13 mice following LPS challenge or the CLP procedure (Fig. 8K–P, S16I–N). Collectively, these pharmacological intervention studies provide compelling evidence that FGF13 amplifies septic lung injury primarily by augmenting ERK signaling pathways in both endothelial cells and macrophages.

Methods
Cell culture
HPMECs were obtained from ScienCell Research Laboratories (3000) and cultured according to the instructions. THP1 cells (CL-0233) and HEK293T cells (CL-0005) were purchased from Procell. To knockdown or overexpress FGF13 in HPMECs and THP1 cells, Ad-shFgf13 or Ad-Fgf13 was transfected to HPMECs and THP1 cells. After 48 h, the culture medium was removed, and the cells were used in the following experiments. For experimental purposes, Ad-vector-transfected HPMECs or THP1 cells were used as control group in subsequent experiments. To induce THP-1 cells into macrophages, PMA (200 ng/ml; Selleck, S7791) was used to treat THP-1 cells for 48 hours before adenovirus transfection. Primary endothelial cells in lungs and primary macrophages in lungs were isolated as previously51,52.
LPS (1 μg/ml; Sigma Aldrich, L6386) was used to stimulate HPMECs and THP1 cells. In signaling pathway analysis, each antagonist: PFK15 (30 μM; Selleck, S7289), SCH772984 (10 μM; Selleck, S7101) was pretreated for 12 h before LPS administration.

Detection of 2-NBDG fluorescence
After 12 hours of LPS stimulation, the culture medium from HPMECs or THP-1 cells was washed twice with PBS. Then added complete culture medium containing 2-NBDG (20 μM; GLPBIO, GC10289). After incubating at 37 °C for 30 minutes, the culture medium was again washed twice with PBS, and then the complete culture medium was added. Then, the phenomenon of 2-NBDG in each group was analyzed in 30 minutes (ZEISS LSM 980 with Airyscan2 Confocal microscope (ZEISS, Germany)).

ELISA analysis of biochemical parameters
Before measuring the biochemical parameters, peripheral blood was collected, allowed to settle overnight, and centrifuged (3000 × g, 10 min, 4 °C). The supernatant was used to measure the levels of inflammatory cytokines. Mouse-interleukin-1β (Proteintech, cat. no. KE10003), mouse-tumor necrosis factor-α (Proteintech, cat. no. KE10002), mouse-interleukin-6 (Proteintech, cat. no. KE10007), and Lactate (Nanjing Jiancheng Bioengineering Institute, A019-2-1) were analyzed using a double antibody sandwich method according to the manufacturer’s instructions.

Ethics statement
All human participants' research in this study was conducted in accordance with the principles of “World Medical Association Declaration of Helsinki: ethical principles for medical research involving human participants”. Before collecting samples from non-septic patients (patients requiring surgical removal of lung nodules) and septic patients (patients who developed sepsis due to lung nodule-related infections and requiring surgical intervention), we provided informed consent forms (Supplementary materials: Informed consent form) to both categories of patients, and selected lung tissue distant from the nodule regions from the resected lung specimens for subsequent research. The biochemical indicators and information for each group of patients are included in Table S1: Information of patients. Patient data and pathological evaluations were approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Approval ID: KY2023-197-01). All animals in our study received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals.” Animal protocols were approved by the Ethics Committee of Wenzhou Medical University.

Extracellular metabolic flux analysis
Extracellular acidification rate (ECAR) of HPMECs or THP-1 cells was analyzed with an XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) as described previously53. Briefly, HPMECs or THP-1 cells were plated on Seahorse XF96 polystyrene tissue culture plates (Seahorse Bioscience) and incubated at 37 °C overnight. The next day, after 1 ug/ml LPS treatment for 12 hours, the medium was changed to XF base Medium (Seahorse Bioscience) supplemented with 2 mM glutamine, and then the plate was incubated for 1 h in a non-CO2 incubator at 37 °C. The ECAR assay was performed on the XF96 extracellular flux analyzer (Seahorse Bioscience), and the ECAR values were normalized using protein concentration. Inhibitors and activators used in these tests at the following concentrations: glucose (10 mM), oligomycin (2 μM), 2DG (50 mM).

Glucose Influx and Lactate Efflux Kinetics
HPMECs or THP-1 cells were plated in a 6-well plate. On the day of treatment, culture medium was refreshed, and cells were then stimulated with LPS. One milliliter of medium in each experimental condition was collected immediately after medium change and stimulus addition (time = 0 h), and after 12 h of incubation (time = 12 h). Glucose and lactate content in the medium were measured using glucose and L-lactate assay kits, respectively, according to the manufacturer’s protocols (Cayman, Ann Arbor, MI).

GST-Pull-down assay
HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2 plasmids were transfected into HEK293 cells, respectively, and cells were lysed with IP lysates. HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2 were purified by the kit (Beyotime, P9808). GST and GST-FGF13 were transfected into DH5α cells and were purified by GST beads (Beyotime, P2138) at 4 °C. The purified proteins (GST and GST-FGF13) were incubated with purified HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2 proteins overnight at 4 °C. Then, the beads were washed 3 times with IP lysates. The complexes were boiled with 2× SDS loading buffer at 95 °C for 10 min, followed by western blotting analyses.

Histological analysis of lungs section
Lungs from mice in each group were divided into 5 μm sections. Histological analysis of tissue sections was performed by H&E staining with a Hematoxylin-Eosin Kit (Solarbio, cat. no. G1120). After sealing with neutral resin, images of the stained sections were captured under a microscope (Motic, PA53BIO).

Immunoblotting analysis
After measuring the protein concentration using the BCA method, 30 μg protein for per sample was resolved by SDS-PAGE in Tris-glycine gels and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk (2 h, room temperature), washed three times in 1× TBST (5 min each time), and incubated (overnight at 4 °C or for 3 h at 37 °C) with primary antibodies. This was followed by incubation with HRP-goat-anti-mouse (Abcam, cat. no. ab205719) or HRP-goat-anti-rabbit (Abcam, cat. no. ab205718) secondary antibodies (2 h, room temperature). Prior to visualization with a Chemiluminescent HRP Substrate (Millipore, cat. no. WBKLS0500), the membranes were washed in 1× TBST (three times, 5 min each). After visualization, the proteins in the membranes were analyzed (ImageQuant 800, Amersham, Cytiva) and quantified (ImageQuant 5.2 software, Molecular Dynamics, Sunnyvale, CA). Primary antibodies were specific for: FGF13 (Thermofisher, MA5-27705), ICAM-1 (Affinity Biosciences, AF6088), VCAM-1 (HUABIO, ET1601-18), iNOS (Cell Signaling Technology, 13120), Bcl-2 (Cell Signaling Technology, 3498), Bax (Cell Signaling Technology, 14796), cleaved-caspase 3 (Cell Signaling Technology, 9664), HIF-1α (Affinity Biosciences, BF8002), phospho-MEK1/2 (HUABIO, ET1609-50), MEK1/2 (HUABIO, ET1602-3), phospho-ERK1/2 (Cell Signaling Technology, 4370), ERK1/2 (Cell Signaling Technology, 4695), Flag (HUABIO, HA601080), HA (Cell Signaling Technology, 3724), GST (Cell Signaling Technology, 2622), phospho-TAK1 (Affinity Biosciences, AF4379), p53 (Cell Signaling Technology, 2524), and TAK1 (Affinity Biosciences, AF7616). And the expression of β-actin (Cell Signaling Technology, 3700) was used as a loading control. The detailed information on antibodies is provided in Table S2. Information on Antibody.

Immunoprecipitation
500 μg protein of HPMECs or THP1 cells was incubated with primary antibody (overnight, 4 °C). Add immobilized antibody beads (Millipore, LSKMAGAG10) to cell lysates for 4 h on the next day. Washed beads with TBS for 3 times, and eluted (glycine-HCl (0.1 M, pH 3.5)), then subjected to immunoblotting analysis. Immobilized antibody included: Flag (HUABIO, HA601080), HA (Cell Signaling Technology, 3724), TAK1 (Santa Cruz Biotechnology, sc-7967), MEK1/2 (Santa Cruz Biotechnology, sc-81504), ERK1/2 (Cell Signaling Technology, 4695), and MEK1 (Santa Cruz Biotechnology, sc-6250). The detailed information on antibodies is provided in Table S2. Information on Antibody.

Immunofluorescence staining
Lung sections (5 μm) were heated at 96 °C for 5 min for antigen retrieval, followed by permeabilization with 0.5 % Triton X-100 (30 min, room temperature). The sections were washed three times in 1× PBST (5 min each time), blocked with 5 % BSA (30 min, room temperature), and incubated overnight at 4 °C with primary antibodies. The next day, lung sections were washed three times with 1× PBST (5 min each), followed by incubation with a secondary antibody (2 h, room temperature). Finally, the sections were washed three times with 1× PBST (5 min each) and nuclei stained with DAPI (30 min, room temperature). The primary antibodies were specific for AQP5 (Proteintech, 20334-1-AP), SPC-Ⅱ (Proteintech, 10774-1-AP), CD34 (HUABIO, ET1606-11), MPO (Abcam, ab208670), F4/80 (Cell Signaling Technology, 30325), CD86 (Santa Cruz Biotechnology, sc-28347) and VE-cadherin (Abcam, ab33168). After washing 3 times with PBS, lung sections were incubated with secondary antibody (2 h, room temperature). The secondary antibodies included: Goat Anti-Rabbit IgG H&L (Abcam, ab150077), Goat Anti-Mouse IgG H&L (Abcam, ab150113), Goat Anti-Rabbit IgG H&L (Abcam, ab150083) and Goat Anti-Mouse IgG H&L (Abcam, ab150115). DAPI was used to stain the nucleus.
In immunofluorescence staining of cells, HPMECs or THP1 cells in each group were fixed (4 % formaldehyde, 15 min, room temperature), permeabilized (0.5 % Triton X-100, 30 min, room temperature), blocked (5 % BSA, 30 min, room temperature), and incubated with primary antibodies (overnight, 4 °C). The primary antibodies included: VE-cadherin (Abcam, ab33168), FGF13 (Thermofisher, MA5-27705), TAK1 (HUABIO, ER1802-90), MEK1/2 (HUABIO, ET1602-3) and ERK1/2 (Cell Signaling Technology, 4695). After washing 3 times with PBS, HPMECs or THP1 cells were incubated with secondary antibody (2 h, room temperature). The secondary antibodies used in immunofluorescence staining of HPMECs or THP1 cells were consistent with immunofluorescence staining of lung sections. DAPI was used to stain the nucleus. The phenomenon in lung sections and cell sections of each group was analyzed (ZEISS LSM 980 with Airyscan2 Confocal microscope (ZEISS, Germany)). The detailed information on antibodies is provided in Table S2. Information on Antibody.

Measurement of cell number in BALF
BALF was collected as described previously54. The collected BALF was centrifuged at 2000 g to collect cells for further counting. And the supernatant was used to determine total protein concentration by Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific, 23228).

Mice cohorts and treatment
C57BL/6 male mice in our study were purchased from the Model Animal Research Center of Nanjing University. To generate CDH5-CreDIO-Fgf13 mice and CDH5-CreDIO-hif1α mice, we injected AAV-lung-CMV-DIO-Fgf13-P2A-EGFP-tWPA (5 × 1012 vg/ml, OBIO technology (Shanghai), reference sequence of Fgf13: NM_010200.3) or AAV-lung-CMV-DIO-hif-1α-P2A-EGFP-tWPA (5 × 1012 vg/ml, OBIO technology (Shanghai), reference sequence of hif1α: NM_010431.2) intravenously to four-week-old CDH5-Cre mice. Steadily overexpressed for three weeks, CDH5-CreDIO-Fgf13 mice and CDH5-CreDIO-hif1α mice were prepared for subsequent experiments. And AAV-lung-CMV-DIO-EGFP-tWPA-injected CDH5-Cre mice were used as the control group in subsequent experiments.
The LyzM-Cre mice were obtained from Jackson Laboratories (004781). The construction of LyzM-CreDIO-Fgf13 mice and LyzM-CreDIO-hif1α mice followed the same methodology as described above, with the only difference being that CDH5-Cre mice were replaced with LyzM-Cre mice. And AAV-lung-CMV-DIO-EGFP-tWPA-injected LyzM-Cre mice were used as the control group in subsequent experiments.
And we crossed Fgf13 flox/flox mice with CDH5-Cre mice or LyzM-Cre mice to generate Fgf13△Endo mice or Fgf13△mye mice. For experimental purposes, CDH5-Cre mice or LyzM-Cre mice were used as the control group in subsequent experiments.
All mice in our study were housed in a specific pathogen-free (SPF) environment maintained at constant temperature (22 ± 2 °C) and humidity (40-60%) with a 12/12 h dark-light cycle (light on at 7:00 a.m.), with free access to food and water. Prior to sepsis model induction, male mice (6–8 weeks, 18–23 g) were randomized into experimental groups by laboratory personnel not involved in subsequent experimental procedures. Animals were housed in standard polycarbonate cages (n=5 per cage) and acclimated to the facility environment for 1 week before experimentation. Housing conditions and dietary regimens remained consistent with previously described protocols. Following sepsis model establishment and specimen collection, all phenotypic assessments and biochemical parameter analyses were conducted under single-blind conditions, with evaluators unaware of treatment allocation, to minimize potential observer bias.

Monocyte adhesion assay
HPMECs were plated in a 6-well plate and stimulated with LPS for 12 h. After counting, 1 × 10^6 of THP-1 cells were added to HPMECs in each group at 37 °C for 30 min and then washed twice with warm PBS. Images of each well were photographed using a Nikon Eclipse TE2000-S fluorescence microscope. And the immunofluorescence staining of THP-1 cells in the monocyte adhesion assay was performed according to the process in Immunofluorescence staining. The primary antibodies included: F4/80 (Cell Signaling Technology, 30325). And the fluorescent THP-1 cells were photographed using ZEISS LSM 980 with Airyscan2 Confocal microscope (ZEISS, Germany).

Mass spectrometry analysis
HEK293T cells were transfected with Flag-Fgf13 plasmids, and cells were immunoprecipitated with immunoprecipitation by Flag antibody or IgG (CST, 3900) with protein A/G beads (Meck, LSKMAGAG10). Coimmunoprecipitation was carried out in HEK293T cells and visualized with silver staining by using the Beyotime Fast Silver Stain Kit (Beyotime, P0017S). Then, mass spectrometry was used to detect all the proteins connected with Flag-FGF13. The assay was carried out by Shanghai Bioprofile Technology Company Ltd. In brief, the gradient consisted of 5~35% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nL/min for 10 min, 35~100% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nL/min for 2 min and 100% acetonitrile in 0.1% formic acid at a flow rate of 200 nL/min for 8 min. The eluted peptides were ionized and introduced into a Thermo Fisher LTQ Velos Pro mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using a Proxeon nanoelectrospray ion source. Survey full-scan MS spectra (from m/z 200~1800) were acquired. A full mass spectrum (m/z 200~1800) was followed by fragmentation of the ten most abundant peaks, using 35 % of the normalized collision energy for obtaining MS/MS spectra. All peptide assignments were verified by manual inspection. The information about the proteins connected with Flag-Fgf13 was summarized in the Supplementary Materials: Proteins connected with Flag-FGF13.

Plasmid construction and transfection
The Flag-Fgf13 plasmids were constructed by OBiO Technology (Shanghai, China), and HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2 plasmids were constructed by GENEWIZ (America). Transient transfections for plasmids were performed with Lipo 2000 (Invitrogen, 11668-019) and transfected into HEK293T cells. After transfection for 48 h, HEK293T cells were harvested for co-immunoprecipitation.

Quantification of hemorrhages in the lungs
Mouse lung tissues were washed twice with 0.9% saline and then lysed for 2 h in 1 ml of tissue lysis buffer. Then, 200 µl of lysate was transferred to a 96-well plate at room temperature, and the OD value was measured spectrophotometrically at 400 nm, as described previously55.

Quantification of Wet/Dry weight ratio
After harvest, the lungs of mice from each group were excised and the excess blood was removed. The wet weight of the lungs was measured, and then the lungs were dried in an oven at 37.5 °C for 48 h to determine the dry weight.

Quantitative real-time PCR
Before tested by qRT-PCR, total RNA from samples in each group was extracted using TRI reagent (Sigma, T9424). After measuring the total RNA concentration in each sample, the cDNA was synthesized from 1 μg of RNA according to the manufacturer’s instructions of iScript cDNA Synthesis kit (Bio-Rad, 1708890). Then, qRT-PCR was used to amplify cDNA from samples in each group with specific primers. In addition, the information on specific primers was provided in Table S3. Primer sequences for qRT-PCR.

RNA Interference in HPMECs and THP-1 cells
The Si-Scramble or Si-p53 (Santa Cruz Biotechnology, sc-29435) was mixed with Lipofectamine 2000 in Opti-MEM (Gibco, 51985034) and incubated for 20 min at 37 °C. Then, cells were washed with PBS and incubated in culture medium containing the siRNA mixture. After culturing for 6-8 h, the Opti-MEM mixture was discarded and the cells were washed with PBS. Fresh culture medium was added and the cells were cultured for 48 h prior to use in subsequent experiments.

RNA-seq analysis
RNA-sequencing was performed on the Illumina HiSeqTM 2500/4000 by Gene Denovo Biotechnology Co., Ltd (Guangzhou, China). Total RNA was extracted using Trizol reagent kit (Invitrogen, 12183555) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase-free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments using the fragmentation buffer and reverse transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were end-repaired, A base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1.0×). And the polymerase chain reaction (PCR) amplified. The resulting cDNA library was sequenced using Illumina Novaseq 6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Sepsis model
Based on the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines, CLP was employed to establish murine sepsis models. The specific CLP procedure was conducted according to the methodology described in a previous investigation56, the specific procedure for CLP is as follows: C57BL/6 male mice (8-10 weeks) were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium (50 mg/kg). Sham-operated mice underwent identical procedures, except for the CLP. In the CLP-induced sepsis model, the caecum of mice was ligated 50% and the ligated cecal stump was then perforated by one “through and through” puncture (21-gauge needle). For LPS-induced endotoxemia sepsis, mice were injected intraperitoneally with a single dose of LPS (10 mg/kg) for 12 h. In both models of sepsis, to inhibit the activation of ERK signaling, SCH772984 (10 mg/kg, i.p.) was used to treat mice 1 h after LPS injection or CLP operation. The serum sample and lungs were harvested after 12 h for further experimentation. After collecting the blood sample, the mice underwent perfusion through the left ventricle with 10 ml of 0.9% saline for 2 minutes. After perfusion, the lungs were extracted from the mice. Following this, the lungs were positioned in a 10 cm diameter culture dish filled with 0.9% saline, and images of the lungs’ appearance were taken. And the remaining tissues were fixed or stored at low temperatures as per the experimental needs.

TUNEL staining in HPMECs
Before incubation with the TUNEL reaction mixture, the process of TUNEL staining in HPMECs is consistent with Immunofluorescence staining. The HPMECs were incubated with a TUNEL reaction mixture (1 h, 37 °C) in a humidified atmosphere in the dark. After washing 3 times with PBS, DAPI was used to stain the nucleus. The phenomenon of TUNEL stain in each group was analyzed (Leica TCS SP8 Confocal microscope (Leica, Wentzler, Germany)), and quantified (ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA)).

Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as mean ± standard error of the mean (SEM). For comparisons between two groups, unpaired Student’s t-tests were employed. Multiple group comparisons with a single variable were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to identify specific intergroup differences. For experimental designs with two variable factors, two-way ANOVA was conducted, followed by Tukey’s post hoc test to determine significant differences between specific groups. Survival analyses were performed using the log-rank (Mantel-Cox) test. Statistical significance was established at p < 0.05 for all analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Thank you for allowing me to review this manuscript. This is a functional genomics paper aiming to establish the biological role and mechanisms of action of FGF13 in a model of pulmonary endotoxemia. The data would be more compelling if clear explanation of what was done were included in the manuscript. As is it is impossible to get a sense without have to access the supplemental materials making the reading hard since the reader has to go back and forth from the different documents wo get a clear story.\nVery nice use of SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor -this perhaps should be highlighted in the methods and results\nHuman lung – presumably these are autopsy samples? Please provide study number and comment regarding consent\nHuman cells – collection from septic versus healthy volunteers – is it possible to add “ill” but not septic rather than healthy? It is unclear how specific to sepsis is the decrease in FGF13 – for all we know it may be seen in multiple models of endothelial activation? E.g. is it regulated in CHF? Post-MI? how specific is it to sepsis?\nStatistics – given if you have 2 unknown variables - genotype and treatment - this should be a nonparametric (without normal distribution, ordinal and/or nominal), you can use two-way anova on ranks (kruskal Wallis) when the groups are independent - with a post-hoc multiple correction test. (not a one-way ANOVA).\nNo comments were made regarding experimental design parameters of importance – e.g. housing and breeding of mice, whether microbiomes are comparable, randomization, blinding, group assignment, blinding of assessors - Minimum Quality Threshold in Pre-Clinical Sepsis Studies (Mqtipss). The authors do not comment or complete requirements from The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) are a checklist of recommendations for the full and transparent reporting of research in animals.\nLSP is a model of toxemia (as per Mqtipss not really a model of sepsis). Probably just requires accepting this as a limitation of the study and changing the wording throughout the manuscript.\nUnclear where the RNAseq in figure 1 came from – what was the experiment? The results section begins with ONE sentence: “In the initial phase of our study, we conducted RNA-sequencing of lung tissues from normal and septic mice to detect changes in FGFs upon sepsis” – no information is provided about the experimental design and WHY would they focus their attention of FGFs.\nThe subsequent sentence in the results section is: Furthermore, we conducted qRT-PCR validation using lung tissues from healthy individuals and sepsis patients. “Healthy” is a relatively uninteresting comparison – the better comparison would be an informative control of “sick’ but non septic e.g. CHF. Also – No ethics approval is provided for the use of histological samples from humans. This needs to be provided.\nHUVECs are not pulmonary endothelial cells – this may be cited as a limitation of the study – since pulmonary microvascular endothelial cells may respond differently from HUVECs.\nIt is unclear why they used HEK293 cells for FGF13 overexpression experiments. Why not take BM-macrophages from KO and overexpression mice? Address specifically endogenous levels of FGF13 in HEK293 and confounders for experimental design\nHow selective is SCH772984? What are the off-target effects? What is the clinical utility of SCH772984?\nThe schematic of the mechanism of action diagram provided does not include FGF13. What figure is this? It is unclear to me how a scaffold protein is promoting Hif1a induced aerobic glycolysis? I understand that FGF13 may bind to other scaffolding proteins and promote ERK1/2 phosphorylation not sure why this would happen on the cytoskeleton and how this is linked to cellular activation and/or Hif1A activity. Why is this specific to endothelial cells and macrophages? How are other cells not using this same mechanism to switch to a glycolytic anaerobic metabolism? How come FGF13 is not expressed in other cells? Is this mechanism specific to LPS or is it important in generalized cell stress?\nDiscussion – please discuss limitations of the study.\nReferences – Usually when we cite references we use the authors LAST name (or family name) rather than the first name: e.g. Line 354 – the author’s last name is dos Santos, first name Claudia, middle initial C. so the citation should read: dos Santos CC, ERJ 2021. This needs to be corrected for all references cites in the text.", "reviewer_2": "Fibroblast growth factor 13 (FGF13), also referred to as FGF homologous factor (FHF) 2, is a member of the FGF11 subfamily that is characterized as having sequence similarities to classical FGF receptor (FGFR)-binding FGFs, but functionally do not bind FGFRs. Similar to other FHFs, FGF13 has been considered a non-secreted protein that lacks an amino signal and is prominently expressed in developing and mature neurons of the central and peripheral nervous systems, as well as the heart. The expression of FGF13 is not limited to early embryonic stages and has been shown to persist in adult tissues. As well, FGF13 is known to localize subcellularly, both within the cytoplasm and the nucleus. In this study, the authors found that FGF13 was down-regulated in endothelial cells and macrophages in lungs from septic mice and sepsis patients. They showed that specific knockout of FGF13 in endothelial cells or macrophages reduced acute lung injury and improved survival in mice with LPS-challenged endotoxic shock. On the other hand, targeted overexpression of FGF13 was found to aggravate acute lung injury and worsen survival in mice with LPS-challenged endotoxic shock. Furthermore, they demonstrated that FGF13 appeared to act as a scaffold protein for the TAK1-MEK-ERK signaling axis and thereby facilitated transmission of the signaling pathway upon LPS stimulation, which promoted HIF-1α-induced aerobic glycolysis in endothelial cells and macrophages, leading to their pro-inflammatory activation. While this study includes interesting findings, I have a number of concerns for the authors to consider.\nMajor comments are summarized below.\n1) In the Methods section, the authors describe that “Before collection the sample from healthy volunteers and sepsis patients, the authors provided the human patient consent forms to healthy volunteers and sepsis patients”. It’s really hard to understand that ‘healthy volunteers’ provided their own lung tissues. In that sense, it’s inconceivable that such a study plan was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. In addition, patient information should be more fully and specifically provided.\n2) The authors should mention what was a post-hoc test following ANOVA.\n3) In some animal experiments, an n = 3-4 is not a sufficient experimental number. The insufficient number of samples per test group makes it clear that the study is greatly underpowered. Indeed, in animal experiments, we empirically learn that there are great differences between individuals in mice with lethal endotoxic shock.\n4) While LPS plays a pivotal role in the pathogenesis of sepsis, it is well known that LPS-challenged murine models do not fully reflect all factors associated with human sepsis. Thus, LPS infusion is unsuitable animal model for sepsis. LPS-challenged mice should be recognized as an endotoxic shock model but not as a septic model. In this study, the authors used mice with CLP-induced sepsis, which should have been centrally placed in this study.\n5) It is very hard to understand the reason why the authors used HUVEC for in vitro experiments. This is a very lame approach. In terms of flow in this study, the authors should have used pulmonary vascular endothelial cells. In the first place, HUVEC is not so much sensitive to LPS. I doubt it’s true that HUVEC responded to 1 μg/mL of LPS in this study.\n6) Lung histological images, shown in this paper, are somewhat poor. For routine histology, the authors did not probably use inflation-fixed lungs. Lung histological images are structurally disrupted even in controls. Unfortunately, the readers who are so familiar with the lung histology may not bear to see the histological images presented in this paper.\n7) The authors conclude that “targeting FGF13 is a potential therapeutic strategy to alleviate septic lung injury”. However, the authors found that FGF13 was down-regulated in endothelial cells and macrophages in lungs from mice with endotoxic shock or CLP-induced sepsis and patients suffering from sepsis. Given the down-regulation of FGF13 under endotoxic shock or sepsis conditions, whether targeting FGF13 is of high clinically therapeutic value is extremely doubtful.\n8) Finally, a major language polishing is required. The manuscript has to definitively use more review for grammar and to help with readability."}, "author_response": "Response to Reviewers\n\nReviewer #1\nThank you for allowing me to review this manuscript. This is a functional genomics paper aiming to establish the biological role and mechanisms of action of FGF13 in a model of pulmonary endotoxemia. The data would be more compelling if clear explanation of what was done were included in the manuscript.\nResponse: We would like to express our sincere gratitude for taking the time to review our manuscript and providing your valuable feedback. We deeply appreciate your recognition of our research efforts in elucidating the biological role and mechanisms of action of FGF13 in the septic lung injury.\nYour suggestion that the manuscript would benefit from clearer explanations of the experimental procedures is particularly insightful, and we wholeheartedly agree with this assessment. We will thoroughly revise our paper according to your recommendations, ensuring that the experimental design, methodologies, and data interpretation sections are more comprehensive and transparent, thereby strengthening the persuasiveness of our findings. We look forward to implementing these improvements to elevate the manuscript to meet your expectations and make a more valuable contribution to the field. Once again, thank you for your expert guidance and constructive comments.\n1.As is it is impossible to get a sense without have to access the supplemental materials making the reading hard since the reader has to go back and forth from the different documents wo get a clear story.\nResponse: We sincerely appreciate your insightful suggestions and thoughtful feedback. In response to your recommendations, we have thoroughly revised our manuscript by incorporating the detailed experimental methodology from the supplementary materials directly into the main text. This structural modification provides greater transparency and immediate access to our experimental procedures. We trust that this enhancement addresses your concerns and meets with your approval.\n2. Very nice use of SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor -this perhaps should be highlighted in the methods and results\nResponse: Thank you for acknowledging our experimental methods for constructing transgenic animals. We have made the adjustments to our manuscript as per your requirements and hope these modifications meet with your approval.\n3. Human lung – presumably these are autopsy samples? Please provide study number and comment regarding consent. Human cells – collection from septic versus healthy volunteers – is it possible to add “ill” but not septic rather than healthy?\nResponse: Regarding the ethics statement section of our manuscript, we sincerely apologize for the errors in our writing that led to some misunderstandings. In the revised manuscript, we have corrected this section. The ethics approval documents and participants' informed consent forms have been included with our submission for your review.\n4.It is unclear how specific to sepsis is the decrease in FGF13 – for all we know it may be seen in multiple models of endothelial activation? E.g. is it regulated in CHF? Post-MI? how specific is it to sepsis?\nResponse: Regarding FGF13 expression patterns across various endothelial cell activation models, we appreciate this excellent question that extends the scope of our research. In our previous investigation of diabetic nephropathy, we observed that FGF13 expression was significantly elevated in renal tubular endothelial cells of both diabetic mice and human patients—a finding that contrasts with our observations in sepsis (PMID: 36256844). Unfortunately, due to resource constraints and methodological limitations, we have been unable to obtain human congestive heart failure (CHF) samples or establish a viable CHF model. Nevertheless, our investigations in a murine acute myocardial infarction model revealed that FGF13 expression was markedly decreased in cardiac endothelial cells following infarction, paralleling our findings in sepsis (Fig. R1(A, B)). This observation aligns with existing literature demonstrating reduced FGF13 expression in hypertrophic cardiac tissue (PMID: 28461495). Furthermore, our fluorescence-based detection methods confirmed diminished FGF13 protein levels in cardiac tissue of mice with myocardial infarction. These consistent findings across different cardiovascular pathologies suggest that FGF13 downregulation may represent a common molecular signature in cardiac injury and potentially plays a significant role in the pathophysiological progression of myocardial infarction.\n5. Statistics – given if you have 2 unknown variables - genotype and treatment - this should be a nonparametric (without normal distribution, ordinal and/or nominal), you can use two-way anova on ranks (kruskal Wallis) when the groups are independent - with a post-hoc multiple correction test. (not a one-way ANOVA).\nResponse: Thank you for bringing this to our attention. We sincerely apologize for the statistical errors in our manuscript. In the revised version, we have reanalyzed all data using a new statistical approach. A detailed description of the statistical methods can be found in our updated manuscript. If errors still persist, please do not hesitate to point them out, and we will certainly make careful revisions again.\n6. No comments were made regarding experimental design parameters of importance – e.g. housing and breeding of mice, whether microbiomes are comparable, randomization, blinding, group assignment, blinding of assessors - Minimum Quality Threshold in Pre-Clinical Sepsis Studies (Mqtipss). The authors do not comment or complete requirements from The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) are a checklist of recommendations for the full and transparent reporting of research in animals.\nResponse: Thank you for your thorough review of our manuscript. We have addressed this oversight in our revised submission and incorporated all necessary explanations. If there are still any deficiencies, please point them out to us, and we will certainly make careful revisions again.\n7. LSP is a model of toxemia (as per Mqtipss not really a model of sepsis). Probably just requires accepting this as a limitation of the study and changing the wording throughout the manuscript.\nResponse: We greatly appreciate your constructive suggestion regarding experimental validation. In response, we have comprehensively implemented the cecal ligation and puncture (CLP) model in our revised manuscript to rigorously validate all our conclusions.\n8. Unclear where the RNAseq in figure 1 came from – what was the experiment? The results section begins with ONE sentence: “In the initial phase of our study, we conducted RNA-sequencing of lung tissues from normal and septic mice to detect changes in FGFs upon sepsis” – no information is provided about the experimental design and WHY would they focus their attention of FGFs.\nResponse: Thank you for your suggestion. We have rewritten this section in the revised manuscript to make it more logical and clear. Please review the updated content. If you still have concerns, please don't hesitate to point them out. We are committed to addressing any remaining issues thoroughly in our revision.\n9. The subsequent sentence in the results section is: Furthermore, we conducted qRT-PCR validation using lung tissues from healthy individuals and sepsis patients. “Healthy” is a relatively uninteresting comparison – the better comparison would be an informative control of “sick’ but non septic e.g. CHF. Also – No ethics approval is provided for the use of histological samples from humans. This needs to be provided.\nResponse: Regarding the ethics statement section of our manuscript, we sincerely apologize for the errors in our writing that led to some misunderstandings. In the revised manuscript, we have corrected this section. The ethics approval documents and participants' informed consent forms have been included with our submission for your review.\n10. HUVECs are not pulmonary endothelial cells – this may be cited as a limitation of the study – since pulmonary microvascular endothelial cells may respond differently from HUVECs.\nResponse: Thank you for identifying this limitation in our manuscript. To ensure our experimental results and conclusions are more scientifically sound and reliable, we have replaced HUVECs with HPMECs and systematically updated all related experimental results. All of these changes have been incorporated into the revised manuscript for your review.\n11. It is unclear why they used HEK293 cells for FGF13 overexpression experiments. Why not take BM-macrophages from KO and overexpression mice? Address specifically endogenous levels of FGF13 in HEK293 and confounders for experimental design\nResponse: Thank you for your constructive suggestions. Here are the main reasons why we chose HEK293 cells for our experiments. First, HEK293 cells are widely used as tool cells for plasmid transfection experiments due to their high transfection efficiency, well-developed protein processing systems, and good stability, making them particularly common in protein interaction studies. Second, at the time of our experiments, there was a lack of commercially available FGF13 antibodies proven to meet the standard requirements for immunoprecipitation experiments, which prevented us from conducting protein interaction studies using cells derived from FGF13 knockout transgenic mice or FGF13 overexpressing transgenic mice. Additionally, as primary macrophages, BMDMs actively eliminate exogenous DNA in cells, resulting in very low plasmid transfection efficiency, making it difficult to achieve the level of target gene overexpression possible with viral vector systems or plasmid transfection in HEK293 cells. Therefore, based on practical considerations, we chose HEK293 cells for Flag-FGF13 plasmid overexpression experiments to verify its binding with components of the TAK1-MEK-ERK axis.\nFurthermore, transfecting plasmids in HEK293 cells, combined with co-immunoprecipitation and LC-MS techniques to screen for potential protein interactions, followed by returning to HEK293 cells for plasmid co-transfection and pull-down experiments to verify protein interactions, is currently a highly recognized research approach widely applied in numerous high-quality publications (PMID: 37777507, 36896602, 40202104, 39950300). Additionally, all the plasmids we used contained tag proteins: Flag-FGF13 with a Flag tag, and HA-TAK1, HA-MEK1, HA-MEK2, HA-ERK1, and HA-ERK2 with HA tags. Therefore, in our plasmid overexpression co-immunoprecipitation experiments, we used antibodies against their respective tag proteins (Flag and HA) rather than antibodies against FGF13, TAK1, MEK1, MEK2, ERK1, or ERK2 themselves.This method effectively improved the specificity of immunoprecipitation experiments, significantly reduced background signals and non-specific binding, while also avoiding the potential influence of endogenous FGF13 in HEK293 cells. This method has also been used in the aforementioned high-quality publications to validate protein interactions. We hope you can understand the practical difficulties we encountered. If you still have concerns, please let us know what issues remain, and we will try our best to address them.\n12. How selective is SCH772984? What are the off-target effects? What is the clinical utility of SCH772984?\nResponse: Thank you for your question. Below is a point-by-point response to your inquiries.\nSCH772984 is a highly selective ERK1/2 inhibitor with a unique binding mechanism. Structural studies revealed that SCH772984 induces the formation of a previously unknown binding pocket between the αC helix and P-loop in ERK1/2 (PMID: 25195011). Its selectivity stems from this distinctive binding mode, which involves three key interactions: indazole binding to the hinge region, pyrrolidine linker interactions with the conserved active site, and phenyl-pyrimidine positioning in the induced P-loop pocket. This creates a highly specific binding profile that distinguishes it from other kinase inhibitors. And the slow dissociation kinetics (25-80 minute half-life) also contributes to its sustained activity. These properties confer high selectivity and low nanomolar inhibition (8.3 nM and 2.7 nM for ERK1 (IC50) and ERK2 (IC50), respectively) of SCH772984 for ERK1/2.\nRegarding SCH772984's off-target effects, this study (PMID: 25195011) demonstrates that its inhibitory potency against alternative kinases-including CLK2, DRAK1, and TTK-is dramatically reduced, displaying merely 1/70th to 1/40th of the potency observed against its primary ERK1/2 targets. This pronounced differential in binding affinity highlights the compound's exceptional selectivity profile. Furthermore, structural analyses reveal that SCH772984 adopts canonical yet distinct Type I binding modes when interacting with off-target kinases such as haspin (IC50=398 nM) and JNK1 (IC50=1080 nM), which differs from its unique binding mechanism with ERK1/2.\nSCH772984 demonstrates robust inhibition of the ERK signaling pathway, exhibiting remarkable efficacy in suppressing the proliferation of tumor cells resistant to BRAF and MEK inhibitors in preclinical studies (PMID: 23614898, 28143781). Beyond its promising antineoplastic properties, this compound has also shown therapeutic potential in mitigating hepatic ischemia-reperfusion injury (PMID: 38638379). Despite these encouraging findings, SCH772984 has yet to advance to clinical trials due to significant pharmaceutical limitations-specifically its high clearance rate and poor membrane permeability—resulting in sub-optimal absorption and diminished bioavailability in rodent models (PMID: 30934534). To address these pharmaceutical shortcomings, researchers developed MK-8353, an optimized derivative that maintains the therapeutic efficacy while substantially improving drug delivery characteristics. MK-8353 exhibits enhanced oral bioavailability across various species, achieves potent inhibition with IC50 values in the low nanomolar range, and importantly, avoids cross-inhibition of MEK1/2. The phase I clinical trial (MK-8353-001, NCT01358331) evaluated this compound's safety profile, dosing parameters, and preliminary efficacy in 26 patients with metastatic melanoma, colorectal cancer, and other solid tumors. Results indicated that MK-8353 was well-tolerated at doses up to 400 mg twice daily, with measurable anti-tumor responses observed in three of the fifteen evaluable patients—all of whom harbored BRAFV600-mutant melanomas (PMID: 29467321).\n13. The schematic of the mechanism of action diagram provided does not include FGF13. What figure is this? It is unclear to me how a scaffold protein is promoting Hif1a induced aerobic glycolysis? I understand that FGF13 may bind to other scaffolding proteins and promote ERK1/2 phosphorylation not sure why this would happen on the cytoskeleton and how this is linked to cellular activation and/or Hif1A activity. Why is this specific to endothelial cells and macrophages? How are other cells not using this same mechanism to switch to a glycolytic anaerobic metabolism? How come FGF13 is not expressed in other cells? Is this mechanism specific to LPS or is it important in generalized cell stress?\nResponse: We appreciate your thoughtful questions, which raise several points of significant scientific importance. Please find our detailed responses below.\nThe regulatory role of ERK signaling in HIF-1α is well-established in the literature (PMID: 28928465, 33638620, 37500654, 26579469), thus we did not elaborate extensively on how FGF13 influences this particular mechanism. Nevertheless, to address your specific concerns, we conducted additional experiments to examine this relationship. As is widely recognized in the field, PHD2 (Prolyl Hydroxylase Domain 2) catalyzes the hydroxylation of specific proline residues in HIF-1α, which subsequently targets this transcription factor for degradation via the canonical ubiquitin-proteasome pathway (PMID: 32853548). Previous research has demonstrated that ERK signaling can phosphorylate PHD2 at its Ser125 residue, thereby inhibiting its enzymatic activity. This inhibition consequently reduces HIF-1α hydroxylation, ultimately resulting in HIF-1α protein accumulation (PMID: 31285371). Our investigations yielded results consistent with this established mechanism. Notably, knockdown of Fgf13 produced effects comparable to those observed with attenuated ERK signaling. Specifically, in both HPMECs and THP-1 cell lines, knockdown of Fgf13 significantly diminished the phosphorylation of PHD2 at Ser125, while concurrently enhancing HIF-1α hydroxylation (Fig. R2(A, B)). Furthermore, we observed a marked increase in K48-linked polyubiquitination of HIF-1α following Fgf13 knockdown, providing compelling evidence for accelerated HIF-1α proteasomal degradation under these conditions (Fig. R2(C, D)).\nAt the same time, there might be a small misunderstanding here. In fact, we did not propose the concept that \"FGF13's promotion of ERK signaling occurs on the cytoskeleton.\" We only demonstrated that FGF13 is a scaffold protein for the TAK1-MEK-ERK signaling axis. Scaffold proteins within cells are not exclusively localized to the cytoskeleton; some scaffold proteins (KSR, caveolin, et al.) are predominantly found in the cytoplasm and on the cell membrane (PMID: 11309192, 11741534, 9045678, 22142403). Previous studies have shown that FGF13 binds to microtubules and can stabilize them (PMID: 22726441, 34533854); therefore, we believe the concept that \"FGF13's promotion of ERK signaling occurs on the cytoskeleton\" may also be reasonable.\nTo further validate our hypothesis regarding the expression-dependent function of FGF13, we extended our studies to include A549 cells as an additional experimental model. This choice was informed by two key considerations: first, A549 cells, consistent with many cancer cell lines, exhibit relatively high endogenous FGF13 expression levels that significantly exceed those observed in BEAS-2B cells (Fig. R5A); second, as A549 cells are characterized by constitutively elevated ERK signaling typical of cancer cells, we deemed LPS stimulation unnecessary for ERK activation in this context (PMID: 34533854, 31256425). Our metabolic analyses employing 2-NBDG fluorescence and glucose uptake/lactate secretion assays demonstrated that knockdown of Fgf13 significantly attenuated both glucose consumption and lactate production in A549 cells. Conversely, overexpression of Fgf13 further augmented these glycolytic parameters, an effect that was abolished by co-treatment with SCH772984 (Fig. R5(B-E)).\nCollectively, these findings support our conclusion that the cell-type selectivity of FGF13's effects on aerobic glycolysis primarily reflects its differential expression patterns across cell types. In cells where FGF13 is abundantly expressed (endothelial cells, macrophages, and certain cancer cells), it serves as a critical regulator of glycolytic metabolism. Moreover, our gain-of-function experiments indicate that when expressed at sufficient levels in other cell types, such as epithelial cells, FGF13 is competent to enhance aerobic glycolysis through ERK-dependent mechanisms.\nAddressing the question of whether FGF13's enhancement of aerobic glycolysis is LPS-specific or extends across diverse cellular stimuli, we present here relevant findings from our recent unpublished investigations. The scientific literature has firmly established that both hyperglycemic conditions and OX-LDL exposure can induce aerobic glycolysis in macrophages, thereby promoting their pro-inflammatory phenotype (PMID: 33044562, 36878047). In our experimental paradigm utilizing THP-1 cells, we observed that knockdown of Fgf13 significantly attenuated multiple parameters of glycolytic metabolism under high glucose conditions, including overall glycolytic capacity, glucose uptake, and lactate production. Interestingly, this dependency on FGF13 exhibited stimulus specificity, as knockdown of Fgf13 failed to modulate aerobic glycolysis in THP-1 cells exposed to OX-LDL (Fig. R6(A-C)). These differential responses provide compelling evidence that FGF13's metabolic regulatory function is stimulus-dependent rather than universally applicable across all pro-inflammatory activators. Further mechanistic investigation revealed that the ERK inhibitor SCH772984 effectively suppressed the enhanced glycolytic phenotype induced by Fgf13 overexpression in high glucose-stimulated THP-1 cells-a pattern identical to our observations in LPS-stimulated conditions (Fig. R6(D-F)). This pharmacological evidence suggests a conserved molecular mechanism underlying FGF13's promotion of aerobic glycolysis in both LPS and high glucose contexts, likely operating through the ERK signaling pathway.\nRegarding the differential expression pattern of FGF13 across cell types, it is important to clarify that FGF13 is not absent in non-endothelial or non-macrophage cells, but rather exhibits substantially lower expression levels in these other cell populations. In addition to the pronounced expression observed in endothelial cells and macrophages, FGF13 has been documented to be expressed in neurons and cardiomyocytes, suggesting tissue-specific regulatory mechanisms (PMID: 28162808, 23804213). Despite our extensive experimental investigations, the molecular determinants governing this cell type-specific expression profile of FGF13 remain elusive. To our knowledge, no comprehensive studies have systematically addressed the underlying mechanisms responsible for this selective expression pattern. We postulate that this phenomenon may be attributed to several potential regulatory mechanisms: 1.Transcriptional regulation: The transcription factors responsible for activating FGF13 gene expression may themselves display cell type-specific distribution patterns, thereby restricting robust FGF13 expression to particular cellular lineages. 2.Epigenetic regulation: The Fgf13 locus may be subject to differential epigenetic modifications across cell types, including DNA methylation patterns, histone modifications, and chromatin accessibility states that collectively determine transcriptional permissiveness.\nWe anticipate that employing multidisciplinary approaches will be instrumental in elucidating these regulatory mechanisms. Specifically, techniques such as single-cell lineage tracing, comprehensive transcriptomic profiling, proteomic analysis of chromatin-associated complexes, and genome-wide epigenetic mapping will likely provide valuable insights into the determinants of FGF13's cell type-specific expression. These methodologies, particularly when applied in an integrated fashion, hold considerable promise for resolving this fundamental question in future investigations.\n14. Discussion – please discuss limitations of the study.\nResponse: We appreciate your thoughtful feedback. In response to your suggestions, we have incorporated a comprehensive discussion addressing the limitations of our research in the revised manuscript. We kindly request your review of these additions.\n15. References – Usually when we cite references we use the authors LAST name (or family name) rather than the first name: e.g. Line 354 – the author’s last name is dos Santos, first name Claudia, middle initial C. so the citation should read: dos Santos CC, ERJ 2021. This needs to be corrected for all references cites in the text.\nResponse: We sincerely appreciate your attentiveness in identifying the citation inconsistencies in our manuscript. Please accept our apologies for this oversight. In response to your feedback, we have thoroughly revised all references to align precisely with citation format of Nature Communications.\n\nReviewer #2\nFibroblast growth factor 13 (FGF13), also referred to as FGF homologous factor (FHF) 2, is a member of the FGF11 subfamily that is characterized as having sequence similarities to classical FGF receptor (FGFR)-binding FGFs, but functionally do not bind FGFRs. Similar to other FHFs, FGF13 has been considered a non-secreted protein that lacks an amino signal and is prominently expressed in developing and mature neurons of the central and peripheral nervous systems, as well as the heart. The expression of FGF13 is not limited to early embryonic stages and has been shown to persist in adult tissues. As well, FGF13 is known to localize subcellularly, both within the cytoplasm and the nucleus. In this study, the authors found that FGF13 was down-regulated in endothelial cells and macrophages in lungs from septic mice and sepsis patients. They showed that specific knockout of FGF13 in endothelial cells or macrophages reduced acute lung injury and improved survival in mice with LPS-challenged endotoxic shock. On the other hand, targeted overexpression of FGF13 was found to aggravate acute lung injury and worsen survival in mice with LPS-challenged endotoxic shock. Furthermore, they demonstrated that FGF13 appeared to act as a scaffold protein for the TAK1-MEK-ERK signaling axis and thereby facilitated transmission of the signaling pathway upon LPS stimulation, which promoted HIF-1α-induced aerobic glycolysis in endothelial cells and macrophages, leading to their pro-inflammatory activation. While this study includes interesting findings, I have a number of concerns for the authors to consider.\nMajor comments are summarized below.\nResponse: We sincerely appreciate your comprehensive review of our manuscript and your acknowledgment of our research contributions. We have carefully considered each of your valuable suggestions and concerns, and have provided detailed responses to each point below. Should you find any of our explanations insufficient or identify additional areas requiring refinement after reviewing our responses, we welcome your further guidance. We remain fully committed to incorporating your expert recommendations to strengthen the scientific rigor and clarity of our manuscript.\n1) In the Methods section, the authors describe that “Before collection the sample from healthy volunteers and sepsis patients, the authors provided the human patient consent forms to healthy volunteers and sepsis patients”. It’s really hard to understand that ‘healthy volunteers’ provided their own lung tissues. In that sense, it’s inconceivable that such a study plan was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. In addition, patient information should be more fully and specifically provided.\nResponse: Regarding the ethics statement section of our manuscript, we sincerely apologize for the errors in our writing that led to some misunderstandings. In the revised manuscript, we have corrected this section. The ethics approval documents and participants' informed consent forms have been included with our submission for your review.\n2) The authors should mention what was a post-hoc test following ANOVA.\nResponse: Thank you for bringing this to our attention. We sincerely apologize for the statistical errors in our manuscript. In the revised version, we have reanalyzed all data using a new statistical approach. A detailed description of the statistical methods can be found in our updated manuscript.\n3) In some animal experiments, an n = 3-4 is not a sufficient experimental number. The insufficient number of samples per test group makes it clear that the study is greatly underpowered. Indeed, in animal experiments, we empirically learn that there are great differences between individuals in mice with lethal endotoxic shock.\nResponse: We appreciate your attentive guidance regarding the animal experiment protocols in our study. In response to your valuable feedback, we have thoroughly revised the manuscript to include comprehensive details about the sample sizes used across all animal experiments. This information has been systematically incorporated into the figure legends and statistical analysis descriptions in the newly submitted version. We believe these additions substantially enhance the reproducibility and statistical validity of our research findings.\n4) While LPS plays a pivotal role in the pathogenesis of sepsis, it is well known that LPS-challenged murine models do not fully reflect all factors associated with human sepsis. Thus, LPS infusion is unsuitable animal model for sepsis. LPS-challenged mice should be recognized as an endotoxic shock model but not as a septic model. In this study, the authors used mice with CLP-induced sepsis, which should have been centrally placed in this study.\nResponse: We greatly appreciate your constructive suggestion regarding experimental validation. In response, we have comprehensively implemented the cecal ligation and puncture (CLP) model in our revised manuscript to rigorously validate all our conclusions.\n5) It is very hard to understand the reason why the authors used HUVEC for in vitro experiments. This is a very lame approach. In terms of flow in this study, the authors should have used pulmonary vascular endothelial cells. In the first place, HUVEC is not so much sensitive to LPS. I doubt it’s true that HUVEC responded to 1 μg/mL of LPS in this study.\nResponse: Thank you for identifying this limitation in our manuscript. To ensure our experimental results and conclusions are more scientifically sound and reliable, we have replaced HUVECs with HPMECs and systematically updated all related experimental results. Based on previous studies investigating HPMECs (PMID: 29965781, 26112597), we determined that 1 μg/ml represents the optimal concentration of LPS for effective stimulation and activation of HPMECs. All of these changes have been incorporated into the revised manuscript for your review.\n6) Lung histological images, shown in this paper, are somewhat poor. For routine histology, the authors did not probably use inflation-fixed lungs. Lung histological images are structurally disrupted even in controls. Unfortunately, the readers who are so familiar with the lung histology may not bear to see the histological images presented in this paper.\nResponse: We sincerely appreciate your meticulous review of the histological sections presented in our manuscript. Your expert assessment of the tissue images has been invaluable. We acknowledge the technical limitations in our original submission and thank you for highlighting this important area for improvement. In response to your constructive feedback, we have thoroughly revised the manuscript with completely new tissue section images that feature higher quality and more effectiveness. We welcome any additional suggestions you might have regarding the presentation of our histological data.\n7) The authors conclude that “targeting FGF13 is a potential therapeutic strategy to alleviate septic lung injury”. However, the authors found that FGF13 was down-regulated in endothelial cells and macrophages in lungs from mice with endotoxic shock or CLP-induced sepsis and patients suffering from sepsis. Given the down-regulation of FGF13 under endotoxic shock or sepsis conditions, whether targeting FGF13 is of high clinically therapeutic value is extremely doubtful.\nResponse: We appreciate your concern regarding this apparent paradox. The observation that FGF13 promotes septic lung injury progression despite its downregulated protein expression indeed represents an intriguing phenomenon in our research. Notably, this contradictory pattern extends beyond septic lung injury; similar paradoxical findings have been documented in cardiac hypertrophy and myocardial aging studies, where FGF13 facilitates pathological processes while exhibiting decreased protein expression (PMID: 40184605, 28461495).\nMechanistically, existing literature demonstrates that p53 suppresses transcription of miR-504 and its host gene Fgf13 (PMID: 27994142, 28082725). Importantly, p53 activation has been consistently observed across cardiac hypertrophy, myocardial aging, and sepsis - aligning perfectly with the downregulation pattern we observed (PMID: 30375404, 40184605, 30512967, 17618303). Our experimental evidence further validates this relationship, as we demonstrated that LPS-induced downregulation of FGF13 in endothelial cells and macrophages is abolished following p53 knockdown.\nOur investigations revealed another critical insight: despite overall FGF13 protein reduction under LPS stimulation, its interaction with components of the TAK1-MEK-ERK axis is paradoxically enhanced. As elaborated in our discussion, FGF13 overexpression alone under basal conditions fails to trigger inflammatory responses or activate the TAK1-MEK-ERK pathway, suggesting that the pro-inflammatory function of FGF13 requires exogenous stimulation as a prerequisite. Whether this involves conformational alterations or activity modulation of FGF13 remains an important question requiring further comprehensive investigation in future studies.\n8) Finally, a major language polishing is required. The manuscript has to definitively use more review for grammar and to help with readability.\nResponse: We sincerely appreciate your thorough assessment of our manuscript's linguistic quality and grammatical structure. In response to your valuable feedback, we have undertaken an extensive language enhancement process for the revised submission. We trust that the revised version now meets the publication's language requirements and facilitates a more engaging reading experience for the intended audience."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for their extensive and careful responses to both sets of reviewer comments, and for undertaking substantial revisions and additional experiments. The manuscript is clearly improved in clarity, methodological transparency, and scope. Below I provide my assessment of how the major issues have been addressed.\n1. Methods, Transparency, and Ethics\nThe incorporation of methodological details from the supplemental materials into the main text significantly improves readability. The corrected and clarified ethics statement, together with inclusion of approval and consent documents, addresses concerns from both reviewers.\n2. Models of Sepsis and Experimental Design\nThe addition of the CLP model is a major improvement that strengthens the translational relevance of the work and addresses the limitation of relying solely on LPS. Revisions regarding randomization, blinding, housing, and ARRIVE/MQTiPSS guidelines improve rigor and reporting.\n3. Statistical Analyses\nThe re-analysis of data using appropriate statistical approaches is appreciated. I encourage the authors to ensure that figure legends clearly specify the statistical tests, post-hoc corrections, and sample sizes, as this was a central concern from both reviewers. The issue of relatively small n (3–4 in some experiments) is partly mitigated by transparency but remains a limitation that should be explicitly acknowledged.\n4. Cell Models\nReplacing HUVECs with HPMECs is an important correction. The justification for using HEK293 cells for protein–protein interaction studies is well explained, though it remains a limitation in terms of physiological relevance and should be discussed explicitly.\n5. Histology\nReviewer #2’s concerns about poor lung histology images were significant. The authors state that new, higher-quality images have been provided. If the revised figures indeed meet standard quality, this point is resolved; otherwise, histology remains a weakness.\n6. Specificity of FGF13 Regulation\nThe additional data in myocardial infarction and previous findings in diabetic nephropathy suggest that downregulation of FGF13 is not specific to sepsis, but may reflect a broader injury/stress signature. This is an important contextualization and should be framed in the discussion to avoid overstating specificity.\n7. Mechanism and Scope\nThe additional experiments on ERK/HIF1α signaling, glycolysis, and cell-type specificity strengthen the mechanistic claims. The evidence that effects are stimulus- and expression-dependent is convincing. However, conclusions should be moderated to emphasize context dependence rather than universal application.\n8. Therapeutic Implications\nThe paradox that FGF13 is downregulated in sepsis yet contributes to injury is intriguing. The authors provide thoughtful mechanistic explanations (p53 regulation, enhanced protein interactions despite downregulation). Nevertheless, therapeutic claims should be tempered, given these complexities and the translational uncertainties.\n9. Presentation and Language\nThe manuscript has been substantially revised for clarity and language. The addition of a limitations section and corrections to references are appreciated.\nMinor Comments –\nClarifications - in page 5 line 105 please re-word or clarify LPS is a model sterile injury.\nLanguage – there are still minor problems with editing language: e.g., Line 118 “FGFs critically regulate lung development…” the phrasing does not make much sense: Do you mean FGF is a critical regulator of lung development OR FGF’s critical regulation during lung development? Removing redundancies would also help reading e.g. in both no-sepsis patients and sepsis patients – this could be changed to both non-septic and septic patients. Again – employing a language editor to go through the manuscript would significantly improve understanding by the reader.\nOverall Assessment\nThis manuscript has undergone extensive revision and addresses many of the key methodological and interpretive issues raised in the first round of review. Remaining limitations include the breadth of claims regarding specificity, the paradox of downregulation versus pathogenicity, and the reliance on certain cell models.", "reviewer_2": "I appreciate the effort the authors have made in addressing my original comments. Specifically, the authors have added the data with CLP-induced septic mice in the main figures. These are good points for improvement. However, as indicated in the original review, LPS-challenged mice should be recognized as an endotoxic shock animal model but not as a septic animal model.\nIn this regard, it would be better to replace the LPS mice data with the CLP-induced septic mice data in Figure 1.\nIn addition, the figures, such as Figures 2, 3, 6, and 8, in which the data with LPS-challenged mice and those with CLP-induced septic mice are mixed, should be tidied up. Alternatively, all the data with LPS-challenged mice data should be moved to supplementary figures.\nFigure legends are detail oriented. The authors should write them down to make the readers more understandable.\nFinally, lung histological images are still lousy. Ideally, the authors were supposed to get advice from scientists specializing histology. Furthermore, the magnification should have been increased to ×100 or ×200.\nMinor:\n1) Figure 2G and 3H: The X axis is not “pot-injection hours”.\n2) Insert a figure number."}, "author_response": "Reviewer#1\nI thank the authors for their extensive and careful responses to both sets of reviewer comments, and for undertaking substantial revisions and additional experiments. The manuscript is clearly improved in clarity, methodological transparency, and scope. Below I provide my assessment of howthemajorissueshavebeenaddressed.\nResponse:Wearedeeplygratefulfortheinvaluable feedbackyouprovided throughoutthereview process and for your recognition of our work. We sincerely hope that our current manuscript revision will meet your expectations and standards. Should any aspects still require further refinement, we would greatly appreciate your continued guidance and will be pleased to address themwithcarefulattentiontodetail.\n1.Methods,Transparency,andEthics\nThe incorporation of methodological details from the supplemental materials into the main text significantly improves readability. The corrected and clarified ethics statement, together with inclusionofapprovalandconsentdocuments,addressesconcernsfrombothreviewers.\nResponse: Thank you for acknowledging the modification here. This improvement was made possiblebyyoursuggestions.\n2.ModelsofSepsisandExperimentalDesign\nTheadditionoftheCLPmodelisamajorimprovementthatstrengthensthetranslationalrelevance of the work and addresses the limitation of relying solely on LPS. Revisions regarding randomization,blinding,housing,andARRIVE/MQTiPSSguidelinesimproverigorandreporting.\nResponse: Thank you for acknowledging the modification here. Your suggestions helped us addressthisgap.\n3.StatisticalAnalyses\nThe re-analysis of data using appropriate statistical approaches is appreciated. I encourage the authors to ensure that figure legends clearly specify the statistical tests, post-hoc corrections, and sample sizes, as this was a central concern from both reviewers. The issue of relatively small n (3–4insome experiments)ispartlymitigatedbytransparencybutremainsalimitationthatshould beexplicitlyacknowledged.\nResponse: Thank you for your reminder. We have conducted another thorough review and refinementofthestatisticalmethodsandresultsthroughouttheentiremanuscript.\n4. CellModels\nReplacingHUVECswithHPMECsisanimportantcorrection.ThejustificationforusingHEK293 cells for protein–protein interaction studies is well explained, though it remains a limitation in termsofphysiologicalrelevanceandshouldbediscussedexplicitly.\nResponse: Thank you for recognizing our work. Regarding the limitations of HEK293 cells, we havealreadyaddressedtheminthediscussion(line427-431).\n5.Histology\nReviewer #2’s concerns about poor lung histology images were significant. The authors state that new,higher-qualityimageshavebeenprovided.Iftherevisedfiguresindeedmeetstandardquality, thispointisresolved;otherwise,histologyremainsaweakness.\nResponse:Thank you for your reminder.Wehave already addressedthis deficiencyin the revised manuscript.\n6.SpecificityofFGF13Regulation\nTheadditionaldatainmyocardialinfarctionandpreviousfindingsindiabeticnephropathysuggest that downregulation of FGF13 is not specific to sepsis, but may reflect a broader injury/stress signature. This is an important contextualization and should be framed in the discussion to avoid overstatingspecificity.\nResponse:Thank you for your guidance. We have already raised this point in the discussion (line 354-361).\n7.MechanismandScope\nThe additional experiments on ERK/HIF1α signaling, glycolysis, and cell-type specificity strengthen the mechanistic claims. The evidence that effects are stimulus- and expression-dependent is convincing. However, conclusions should be moderated to emphasize contextdependenceratherthanuniversalapplication.\nResponse: Thank you for acknowledging our supplementary experimental content. We have also revisedthisconclusioninthediscussionsection(line380-402).\n8.TherapeuticImplications\nThe paradox that FGF13 is downregulated in sepsis yet contributes to injury is intriguing. The authors provide thoughtful mechanistic explanations (p53 regulation, enhanced protein interactions despite downregulation). Nevertheless, therapeutic claims should be tempered, given thesecomplexitiesandthetranslationaluncertainties.\nResponse:Thankyouforyourreminder.Wehaverevisedthesesectionsinthenewmanuscript.\n9.PresentationandLanguage\nThe manuscript has been substantially revised for clarity and language. The addition of a limitationssectionandcorrectionstoreferencesareappreciated.\nMinorComments–\nClarifications-inpage5line105pleasere-wordorclarifyLPSisamodelsterileinjury.\nLanguage – there are still minor problems with editing language: e.g., Line 118 “FGFs critically regulate lung development…” the phrasing does not make much sense: Do you mean FGF is a critical regulator of lung development OR FGF’s critical regulation during lung development? Removingredundancieswouldalsohelpreadinge.g.inbothno-sepsispatientsandsepsispatients – this could be changed to both non-septic and septic patients. Again – employing a language editortogothroughthemanuscriptwouldsignificantlyimproveunderstandingbythereader.\nResponse: Our manuscript had already undergone language editing prior to the previous submission.Additionally, we have made our best efforts to further improve the issues you raised thistimeintherevisedmanuscript.\nOverallAssessment\nThis manuscript has undergone extensive revision and addresses many of the key methodological and interpretive issues raised in the first round of review. Remaining limitations include the breadth of claims regarding specificity, the paradox of downregulation versus pathogenicity, and therelianceoncertaincellmodels.\nResponse: Thank you once again for your careful review of our manuscript during the review process and for providing numerous valuable suggestions. The improvement of our manuscript hasgreatlybenefitedfromyourguidance.\nReviewer#2:\n1.I appreciatethe effortthe authorshave made in addressingmy originalcomments. Specifically, theauthorshaveaddedthedatawithCLP-inducedsepticmiceinthemainfigures.Thesearegood points for improvement. However, as indicated in the original review, LPS-challenged mice shouldberecognizedasanendotoxicshockanimalmodelbutnotasasepticanimalmodel.Inthis regard, it would be better to replace the LPS mice data with the CLP-induced septic mice data in Figure 1. In addition, the figures, such as Figures 2, 3, 6, and 8, in which the data with LPS-challenged mice and those with CLP-induced septic mice are mixed, should be tidied up. Alternatively, all the data with LPS-challenged mice data should be moved to supplementary figures.\nResponse: Thank you for your valuable suggestions and for acknowledging our supplementary experimental work.According to your request, we have made our best efforts to transfer the LPS -challenged mice-related data to the supplementary data. Some data that was not transferred was retained due to considerations for figure layout and visual presentation, and we hope for your understanding.\n2. Figure legends are detail oriented. The authors should write them down to make the readers moreunderstandable.\nResponse: Thank you for bringing this to our attention. We have thoroughly revised all figure legendsinthenewlysubmittedmanuscriptandhopethisaddressesyourconcernssatisfactorily.\n3.Finally,lunghistologicalimages arestilllousy.Ideally,theauthorsweresupposedtogetadvice from scientists specializing histology. Furthermore, the magnification should have been increased to×100or×200.\nResponse: Thank you for your valuable suggestions. We sincerely apologize for the confusion regarding the magnification specifications. In the original manuscript, we incorrectly referenced only the objective lens magnification, which does not follow standard notation practices. We greatly appreciate your guidance in correcting this oversight. Given that our eyepiece magnification is 10×, the total magnification of our histological images should have been properly reported as 100× and 200×, respectively. We deeply regret any confusion caused by this error in our methodology. Following consultation with histology experts, we have replaced several histological images and increased the magnification to 400× to provide readers with clearer visualization of inflammatory cell infiltration within the lung tissue. We believe these revisions will enhance the quality and interpretability of our data. We hope these modifications address your concerns adequately. Should you identify any remaining issues with this section of our data, we would be grateful if you could provide specific feedback. We are committed to makingallnecessaryrevisionstoensurethemanuscriptmeetsyourstandardsandexpectations.\n4.Minor:\n1)Figure2Gand3H:TheXaxisisnot“pot-injectionhours”.\n2)Insertafigurenumber.\nResponse: Thank you for your reminder. We have further improved our manuscript according to yoursuggestions."}, {"round": 3, "reviewer_comments": {"reviewer_1": "If the second reviewer is happy with the NEW histological images - I have no further comments", "reviewer_2": "As indicated in the original review, LPS-challenged mice should be recognized as an endotoxic shock/endotoxemia animal model but not as a septic animal model. From this point of view, the authors should not put the data from LPS-challenged mice in those from mice with CLP-induced sepsis. Thus, this study has to still be improved.\n1) Figure 1A: Data should be compared between sham-operated control mice and CLP mice.\n2) Figure 1F: This graph is the same as the graph shown in the original manuscript. Alternatively, this relationship was obtained from LPS-challenged mice.\n3) Figure 1G: In the Results section, there is no explanation for the difference between H&E stained lung sections from sham-operated control and CLP mice.\n4) Figure 6D: The authors mention that “H&E staining demonstrated that the attenuated lung edema, hemorrhage, and inflammatory cell infiltration in Fgf13∆Endo mice were negated by AAV-lung-DIO-hifla administration”. However, the difference between H&E stained lung sections from CDH5-Cre and Fgf13∆Endo mice treated with AAV-lung-vector is apparently less pronounced.\n5) Figure 6N: The authors mention that “H&E staining revealed that the reduction in hemorrhage, inflammatory cell infiltration in Fgf13∆mye mice was abolished by AAV-lung-DIO-hif1a”. However, the difference between H&E stained lung sections from LyzM-Cre and Fgf13∆mye mice treated with AAV-lung-vector is apparently less pronounced.\n6) Overall, lung histological images, shown as H&E sections in this paper, are still poor. This reviewer is just wondering that lung histological images are structurally disrupted even in sham controls.\nMinor:\n1) Figure 3A: The X axis is not “pot-injection hours”."}, "author_response": "Reviewer #1\nIf the second reviewer is happy with the NEW histological images - I have no further comments\nResponse: Regarding the issues with histological image quality, we have consulted with relevant experts and made further improvements to address the identified concerns. We are deeply grateful for your meticulous review of our manuscript and valuable suggestions throughout this year-long review process. Without your dedicated efforts, our manuscript would not have had the opportunity for such substantial enhancement. We once again extend our heartfelt thanks and profound respect to you and both reviewers.\nReviewer #2 :\nAs indicated in the original review, LPS-challenged mice should be recognized as an endotoxic shock/endotoxemia animal model but not as a septic animal model. From this point of view, the authors should not put the data from LPS-challenged mice in those from mice with CLP-induced sepsis. Thus, this study has to still be improved.\nResponse: Regarding this point, we have clarified this matter as thoroughly as possible in our manuscript. By incorporating both models, we believe this approach better demonstrates the role of FGF13 in sepsis. The presentation of data from both models together in certain figures was indeed considered for experimental rigor and figure layout aesthetics. We are deeply grateful for your meticulous review of our manuscript and valuable suggestions throughout this year-long review process. Without your dedicated efforts, our manuscript would not have had the opportunity for such substantial enhancement. We once again extend our heartfelt thanks and profound respect to you and both reviewers.\n1. Figure 1A: Data should be compared between sham-operated control mice and CLP mice.\nResponse: Regarding this point, we respectfully hold a different perspective. While CLP is indeed the most representative mouse sepsis model, it often exhibits considerable inter-individual variability due to surgical procedures and anesthesia levels, which can lead to significant within-group differences in RNA sequencing results. To reliably identify FGF family members that meet our research requirements, we employed a strategic approach: first exploring changes in FGF family members through transcriptomic sequencing in the LPS injection model, followed by further validation in the CLP model using Western blot, immunofluorescence, and other experimental techniques. We believe this approach is more efficient and rational. We hope you can understand and appreciate our experimental strategy, and we are deeply grateful for your consideration.\n2. Figure 1F: This graph is the same as the graph shown in the original manuscript. Alternatively, this relationship was obtained from LPS-challenged mice.\nResponse: Thank you for bringing this issue to our attention. This was an oversight on our part during the revision process, and we sincerely apologize for this error. We have corrected this in the newly submitted manuscript.\n3.Figure 1G: In the Results section, there is no explanation for the difference between H&E stained lung sections from sham-operated control and CLP mice.\nResponse: Thank you for your reminder. We have further improved our manuscript according to your suggestions.\n4.Figure 6D: The authors mention that “H&E staining demonstrated that the attenuated lung edema, hemorrhage, and inflammatory cell infiltration in Fgf13∆Endo mice were negated by AAV-lung-DIO-hifla administration”. However, the difference between H&E stained lung sections from CDH5-Cre and Fgf13∆Endo mice treated with AAV-lung-vector is apparently less pronounced.\nResponse: Thank you for bringing this issue to our attention. We have replaced the images with more representative ones to better illustrate the differences between the two groups.\n5.Figure 6N: The authors mention that “H&E staining revealed that the reduction in hemorrhage, inflammatory cell infiltration in Fgf13∆mye mice was abolished by AAV-lung-DIO-hif1a”. However, the difference between H&E stained lung sections from LyzM-Cre and Fgf13∆mye mice treated with AAV-lung-vector is apparently less pronounced.\nResponse: Thank you for bringing this issue to our attention. We have replaced the images with more representative ones to better illustrate the differences between the two groups.\n6.Overall, lung histological images, shown as H&E sections in this paper, are still poor. This reviewer is just wondering that lung histological images are structurally disrupted even in sham controls.\nResponse: Regarding the issues with histological image quality, we have consulted with relevant experts and made further improvements to address the identified concerns. Regarding this issue, upon reflection, we believe the problem primarily stems from the perfusion procedure. To eliminate blood from the pulmonary vasculature and facilitate observation of interstitial hemorrhage, we performed perfusion with 10 ml of saline within 2 minutes, which may have caused some structural damage to the lungs, resulting in apparent abnormalities in the normal lung architecture of control mice in H&E staining. However, we believe that our description of H&E staining results throughout the manuscript focuses primarily on inflammatory cell infiltration within the lungs. While the perfusion method may cause some structural damage to normal lung architecture, it should not affect the phenomenon of inflammatory cell infiltration in the lungs. Therefore, we consider our H&E staining results reliable for observing pulmonary inflammatory infiltration. We hope our explanation will meet with your approval and express our gratitude once again.\nMinor:\n1) Figure 3A: The X axis is not “pot-injection hours”.\nResponse: Thank you for your reminder. We have further improved our manuscript according to your suggestions."}]

Decision letter