Peritoneal Neutrophil Extracellular Traps contribute to septic AKI via peritoneal IL-17A and distant organ CXCL-1/ CXCL-2 pathway in abdominal sepsis.

Peritoneal Neutrophil Extracellular Traps contribute to septic AKI via peritoneal IL-17A and distant organ CXCL-1/ CXCL-2 pathway in abdominal sepsis. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Peritoneal Neutrophil Extracellular Traps contribute to septic AKI via peritoneal IL-17A and distant organ CXCL-1/ CXCL-2 pathway in abdominal sepsis. Yoshitaka Naito, Daiki Goto, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7474386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract There are no specific treatments for Sepsis-associated acute kidney injury (SAKI). We previously reported that Il-17a -knockout mice had dramatically improved survival after cecal ligation and puncture (CLP). Neutrophil extracellular traps (NETs) induce IL-17A, which causes harm in some diseases, but this pathway is poorly understood in sepsis. We found that knockout of Pad4 (Peptidyl Arginine Deiminase 4), an enzyme essential for NET formation, improved survival and AKI, and suppressed neutrophil infiltration into remote organs, involving a peritoneal IL-17A/distant organ CXCL-1/CXCL-2 pathway after CLP. NETs were detected in the peritoneal cavity, and not in plasma or distant organs. Adoptive transfer of peritoneal NETs restored the IL-17A/CXCL-1/CXCL-2 pathway in Pad4 KO mice, leading to neutrophil infiltration and damge to remote organs. These results revealed a pathway from peritoneal NET formation to remote organ injury/inflammation via production of IL-17A at the infectious site and distant organ CXCL-1/CXCL-2. While NETs promoted intraperitoneal IL-17A production, we also showed that conversely, peritoneal IL-17A or CXCL-1/CXCL-2 promoted intraperitoneal NET formation after CLP. This peritoneal vicious cycle that includes NET formation, IL-17A, CXCL-1/CXCL-2 that may amplify organ injury in sepsis. Breaking this vicious cycle by inhibiting NET formation and/or IL-17A might be a promising therapeutic target for sepsis treatment. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Health sciences/Nephrology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction About 60% of septic patients develop sepsis-associated acute kidney injury (SAKI) 1 – 3 , with a mortality rate of 60–70% 1, 4 . There are no specific treatments for SAKI. Neutrophil extracellular traps (NETs), expelled from activated neutrophils, contain cfDNA, histones, proteases, and antimicrobial proteins, and may be the primary source of cfDNA in early sepsis 5 – 7 . Peptidylarginine deiminase 4 (PAD4) is essential for NET formation through histone citrullination 8 . NETs have dual effects in sepsis. They can immobilize bacteria, which is advantageous; but their components (histones, proteases, and cfDNA) can harm host cells[6, 7]. Due to these opposing effects, targeting NETs in sepsis may be difficult. Martinod et al. observed that Pad4 knockout did not enhance survival in a mild CLP model and worsened survival in a severe CLP model 9 . Others have shown that Pad4 knockout or DNase treatment improved organ damage in a mouse model of E. coli sepsis 10 – 12 . Moreover, blocking NETs with a non-specific PAD inhibitor Cl-amidine, antibodies against citrullinated histone H3 (H3Cit), or DNase enhanced survival post-CLP 13 , 14 . These apparently contradictory effects imply that NETs exert both beneficial and detrimental effects in sepsis, contingent upon the particular context. Interestingly, NETs increase IL-17A, a cytokine we previously demonstrated to be downstream of TLR9 15 , a known mediator of sepsis 16 , 17 . NETs promote Th17 cell differentiation through one of their components, histone 18 . Pad4 knockout halted IL-17A accumulation in atherosclerosis within the aorta 19 . However, the contribution of NET formation to IL-17A production in sepsis remains unclear. We hypothesized that NETs increase IL-17A and its downstream factors, CXCL-1 and CXCL-2, causing neutrophil infiltration and organ injury in sepsis. Using two knock out mouse models ( Pad4 KO mice in which NET formation was completely abolished; and Il-17a KO), we evaluated the 1) effect of NET formation on CLP outcomes; 2) main sites of NET formation in CLP-treated mice; 3) effect of NET formation on neutrophil infiltration into remote organs and IL-17A and CXCL-1/CXCL-2 pathway after CLP; 4) relationship between NET formation and IL-17A in the peritoneal cavity; and 5) effect of peritoneal NETs on remote organ injury using an adoptive transfer model of WT neutrophils into Pad4 KO mice after CLP. Results Effect of knockout of Pad 4 on survival, AKI, and neutrophil infiltration into kidney and lung after CLP To investigate the role of NET formation in our moderate severity, clinically relevant model of polymicrobial sepsis, we examined whether knockout of Pad4 alters survival, renal function and morphology, and neutrophil infiltration into kidney and lung after CLP. Pad4KO mice had significantly improved survival at 168 h after CLP, with a survival rate of 80% compared to 47% for WT mice (Fig. 1 A). WT mice developed kidney injury 18 h after CLP, whereas Pad4 KO mice showed decreased serum BUN levels (Fig. 1 B) and improved cortical tubular damage scores (Fig. 1 C and Supplemental Fig. 1). Neutrophil infiltration into kidney and lung were increased at 18 h after CLP, (Fig. 1 D and E,, and Supplemental Fig. 2A and B), but Pad4 KO mice exhibited significantly lower neutrophil infiltration. Knockout of Pad4 did not alter the number of bacterial colonies 18 h after CLP (Supplemental Fig. 3A). Effect of knockout of Pad 4 on CXCL-1 and − 2 production in kidney and lung, and IL-17A production in PLF and plasma after CLP To investigate the role of NET formation in CXCL-1 and − 2 production in kidney and lung after CLP, CXCL-1 and − 2 levels in kidney and lung were evaluated in Pad4 KO and WT mice 18 h after sham or CLP surgery (Fig. 2 A and B). CXCL-1 and − 2 levels were significantly higher in kidney and lung from WT mice vs. Pad4 KO mice at 18 h after CLP. Levels of IL-17A, a known upstream factor of CXCL-1 and − 2 20 , in PLF and plasma were also assessed after CLP (Fig. 2 C and D). IL-17A levels were significantly higher in PLF and plasma of WT mice vs. Pad4 KO mice 18 h after CLP. Effect of knockout of Il-17a on AKI, neutrophil infiltration into kidney and lung, and levels of CXCL-1 and − 2 in kidney and lung after CLP Il-17a KO improves survival in sepsis 15 , but the mechanism is unknown. To investigate the role of IL-17A in SAKI, plasma BUN levels (Fig. 3 A) and renal tubular damage scores (Fig. 3 B and Supplemental Fig. 4) were assessed 18 h after CLP. WT mice developed kidney injury 18 h after CLP, whereas Il-17a KO mice showed decreased BUN levels (Fig. 3 A) and improved tubular damage scores in the cortex (Fig. 3 B and Supplemental Fig. 4) similar to Pad4 KO mice. Knockout of Il-17a significantly decreased neutrophil infiltration into kidney and lung at 18 h after CLP compared with WT mice by naphthol AS-D chloroacetate esterase staining (Fig. 3 C and D, and Supplemental Fig. 5A and B). Neutrophil infiltration into kidney was also assessed by flow cytometry (Supplemental Fig. 6A, B, C, and D). Neutrophil infiltration was upregulated by CLP at 3 and 18 h after CLP, which was attenuated in knockout of Il-17a KO mice. Furthermore, knockout of Il-17a , similar to knockout of Pad4 , significantly decreased CXCL-1 and − 2 levels in kidney and in lung at 18 h after CLP compared to WT mice (Fig. 3 E and F). Knockout of Il-17a did not alter the number of bacterial colonies in PLF collected at 18 h after CLP (Supplemental Fig. 3B). NET formation in peritoneal cavity after CLP We next investigated where NETs can form in CLP-treated mice. Spleen, kidney, and lung were harvested from WT or Pad4 KO mice 18 h after CLP and stained for citrullinated histone H3 (H3Cit) (Fig. 4 A). Few H3Cit-positive cells were observed in a very limited area in the spleen, and not at all in kidney and lung. H3Cit levels significantly increased in CLP treated mice compared to sham only in PLF, but not in plasma, spleen, or kidney (Fig. 4 B). Since evidence of NET formation was detected only in the PLF in our CLP model, we focused on neutrophils in the peritoneal cavity. CLP significantly increased neutrophil accumulation in PLF at 18 h after CLP (Fig. 4 D). Also, among live PLF cells the percentage of neutrophils was as high as 65.36% (95%CI: 51.27–83.30%) at 3 h and 69.78% (95%CI: 63.38–76.86%) 18 h after CLP, even without neutrophil purification (Fig. 4 E). Approximately 40% of PLF cells from WT mice formed NETs after CLP without any ex vivo stimulation (Fig. 4 F), and NETs were almost completely absent in cells from Pad4 KO mice (Fig. 4 G). A similar trend was obtained when the H3Cit-positive area was normalized by cell number, which we interpreted as NET extension in PLF cells (Fig. 4 G). The effect of knockout of Il-17a on CXCL-1 and − 2 production in PLF and plasma and NET formation in peritoneal cavity In PLF and plasma, CXCL-1 and − 2 levels were upregulated 18 h after CLP in WT mice, whereas these levels were significantly decreased in Il-17a KO mice (Fig. 5 A and B). Knockout of Il-17a did not significantly alter the absolute number or percentage of neutrophils infiltrating into the peritoneal cavity 18 h after CLP (Supplemental Fig. 7). However, H3Cit levels in PLF, which were elevated 18 h after CLP in WT mice, were significantly decreased in PLF from Il-17a KO mice (Fig. 5 C). The effect of IL-17A, CXCL-1, and − 2 on NET formation in PLF cells ex vivo. PLF cells from Il-17a KO mice significantly decreased NET extension, measured as SYTOX green positive area normalized to cell number, compared to WT mice (Fig. 6 A and B). NET formation was also assessed by staining with H3Cit antibody using confocal microscopy; PLF cells from Il-17a KO mice vs cells from WT mice had a decreased percentage of NET formation or NET extension (Fig. 6 C, and D). Next, we evaluated the effects of recombinant IL-17A, CXCL-1, or -2 stimulation on NET formation in PLF cells. Ex vivo incubation of PLF cells collected 3 h after CLP with recombinant IL-17A, rCXCL-1, or -2 increased the percentage of NET formation or NET extension (Fig. 6 E and F). Effect of intraperitoneal adoptive transfer of WT neutrophils into Pad4 KO mice on septic AKI, lung inflammation, and IL-17A/CXCL-1/CXCL-2 axis. For further investigation of the relationship between intraperitoneal NET formation and distant organ injuries, we assessed whether adoptive transfer of WT neutrophils into Pad4 KO mice can reverse the CLP-induced AKI or lung inflammation attenuated by Pad4 knockout. First, donor WT or Pad4 KO mice were subjected to CLP. PLF cells were collected at 18 h after CLP and neutrophils were purified from these PLF cells. Then, recipient Pad4 KO mice were subjected to CLP and neutrophils collected from WT or Pad4 KO donor mice were intraperitoneally administered into Pad4 KO mice immediately after CLP of recipients (Supplemental Fig. 8A). In CLP-treated Pad4 KO mice, WT donor neutrophil administration reconstituted AKI, in contrast with Pad4 KO donor neutrophil administration or vehicle injection (Fig. 7 A and B, and Supplemental Fig. 9A). WT neutrophil administration into Pad4 KO mice also increased neutrophil infiltration into kidney and lung compared with injection of Pad4 KO neutrophils or vehicle (Fig. 7 C, and Supplemental Fig. 9B, C, and D). We then assessed CXCL-1 and − 2 levels in kidney (Fig. 7 D) and lung (Fig. 7 E) and IL-17A levels in PLF (Fig. 7 F) and plasma (Fig. 7 G) at 18 h after CLP. Adoptive transfer of WT neutrophils counteracted the attenuation of CXCL-1 and − 2 production in kidney and lung and IL-17A production in PLF and plasma by Pad4 knockout, whereas adoptive transfer of Pad4 KO neutrophils did not alter these levels. These findings indicate that the beneficial effect on SAKI and lung inflammation induced by Pad4 knockout are attenuated by intraperitoneal administration of WT neutrophils. Discussion Neutrophils can have both beneficial and harmful effects in sepsis, potentially explaining why neutrophil depletion does not change overall survival in CLP sepsis models 21 , 22 . For example, NETs formed from activated neutrophils during sepsis can trap bacteria and fungi to contain the infection, while also inducing local and distant inflammation through DAMPs like cfDNA 6 , 23 , 24 . PAD4, a crucial enzyme, regulates NET formation through histone citrullination 8 . Consequently, neutrophils lacking PAD4 fail to produce NETs, even when stimulated by chemokines, LPS, or bacteria 8 , 25 . In this study, we explored the function of ‘local’ peritoneal NETs formed at site of infection, and their impact on distant organ function. The main findings of this paper are: 1) elimination of NETs by knockout of Pad4 improved survival and AKI after CLP; 2) NETs were detected only in the peritoneal cavity, not in plasma or distant organs; 3) knockout of Pad4 suppressed neutrophil infiltration into remote organs via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway; 4) knockout of Il-17a suppressed NET formation and CXCL-1/CXCL-2 production in peritoneal cavity after CLP and recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells; and 5) adoptive transfer of peritoneal NETs restored the peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in Pad4 KO mice, leading to neutrophil infiltration into remote organs and remote organ injury. Our findings are summarized in Fig. 8 . Elimination of NETs by Knockout of Pad4 improves survival and AKI after CLP. We found that Pad4 knockout improved survival and AKI in a CLP model in which the cecum was punctured with 21-gauge needles, and animals were treated with fluids (1 ml of 2/3 normal saline) and antibiotics (s.c.) every 12 h for 7 days. Survival on day 4 after CLP was 53% for WT mice vs. 85% for Pad4 KO and on day 7 was 47% for WT mice vs. 80% for Pad4 KO. NETs defend against bacteria by trapping or killing them 7 . Inhibiting NETs during severe infections could be detrimental for sepsis. Interestingly, Pad4 knockout did not change the peritoneal bacterial count 18 h after CLP. Similar positive results on CLP survival have been seen with Cl-Amidine, a non-specific PAD inhibitor or antibodies against H3Cit 12 , 13 , 26 . Our findings contrast with Martinod et al. 9 , who found no effect of Pad4 knockout in a mild CLP model (using 21-gauge needles, no antibiotics, 75% survival at 4 days, with 7–8 animals per group), and worse outcomes in a severe CLP model (18-gauge needles, with antibiotics, no survival at 10 days). They only partially replaced surgical fluid losses (0.5ml of normal saline once immediately after surgery) 9 . Our model resembles their mild CLP model but includes antibiotics, more fluid, and longer intermittent fluid resuscitation. Variations in CLP severity, fluid resuscitation, antibiotic administration (none, subcutaneous, or intraperitoneal), and our larger sample size (19 to 20 mice per group) could explain the differing results between laboratories. Interestingly, even in their severe CLP model, knockout of Pad4 did not increase bacterial loads in the blood, liver, or lungs at 24 h post-CLP 9 . However, NETs might have aided in reducing bacterial abundance later 9 . Losing NET protection against bacteria could be harmful in severe CLP models, yet dampen excessive inflammation in milder CLP models. NET formation was detected only in the peritoneal cavity, and not in plasma or distant organs. Due to the positive effect of PAD4 inhibition/deletion on reducing distant organ damage and improving survival, we anticipated widespread NET presence, contributing to multiple organ failure. The detrimental effects of NETs in sepsis are attributed to tissue-based NET formation, intravascular coagulation promotion, and the release of enzymes like neutrophil elastase and serine proteases during NETosis 6 . Initially, we hypothesized that intraperitoneal NETs would induce distant NET formation in remote organs via the production of proinflammatory cytokines, chemokines, and damage-associated molecular patterns (DAMPs) like nuclear and/or mitochondrial cfDNA. Surprisingly, we discovered that NETs were confined to the peritoneal cavity and not detected in plasma or other distant organs (lung, liver, kidney, spleen). The location of NETs following sepsis is complex, and it appears to be quite sensitive to the severity of sepsis. NETs have been identified in lungs, kidneys, and plasma after CLP; however, these CLP models were considerably more severe. For example, H3Cit-positive cells were detected in the lungs post-CLP 27 – 31 – but survival was 0% at 24 h 30 or 15% at 48 h 31 , compared to 100% at 24 and 48 h in our model. H3Cit-positive cells have been found in the kidney at 24 or 48 h after CLP 32 ; however, the model was more severe than our model, with 40% survival in WT mice at 24 h post-CLP 32 . Elevated serum H3Cit levels have also been detected post-CLP 12 , 33 ; again, in more severe sepsis models. Yuzi et al. reported survival rates after CLP: around 60% at 24 h and 0% at 60 h post-CLP 12 . In contrast, Bethany et al. found that plasma H3Cit levels were not increased by CLP surgery compared to sham, consistent with our results 13 . Their CLP model showed 80% survival at 24 h and 50% at 60 h, indicating a milder outcome compared to Yuzi’s model, which exhibited elevated blood H3Cit after CLP, and relatively similar to ours 12 , 13 . These findings imply that NETs may form in lungs, kidneys, or blood during severe CLP. However, in a less severe sepsis model, we found that NET formation in PLF induces (see below) lung inflammation or kidney injury even without NET formation in blood or these remote organs. Knockout of Pad4 suppressed neutrophil infiltration into remote organs via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway. We then investigated the mediator(s) from NETs circulating systemically causing organ injury. Pad4 knockout reduced IL-17A in PLF and plasma after CLP. Interestingly, a myeloid-specific Pad4 deletion greatly reduced IL-17A in an atherosclerosis model 19 . Furthermore, Pad4 and Il-17a knockout notably reduced CXCL-1 and CXCL-2 levels in lung and kidney and decreased neutrophil infiltration. This indicates IL-17A, promoted by NET formation, is vital for CXCL-1, CXCL-2 production, and neutrophil infiltration in distant organs post-CLP. IL-17A stimulates epithelial cells to produce chemokines, and promotes myeloid cell mobilization to inflammatory sites 20 . Notably, CXCL-8 in humans and its functional homologs, CXCL-1 and CXCL-2 in mice, are potent inducers of neutrophil migration into inflamed tissues 34 . Il-17a knockout reduces CXCL-1 and − 2 levels, neutrophil infiltration, and kidney impairment after CLP 35 . Thus, IL-17A plays a crucial role in distant organ CXCL-1/CXCL-2 production and neutrophil infiltration. Knockout of Il-17a suppressed NET formation and CXCL-1/CXCL − 2 production in peritoneal cavity after CLP and recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells. After finding NET formation promoted the IL-17A pathway in the CLP model, we assessed if the IL-17A pathway affects NET formation. Il-17a knockout decreased H3Cit, CXCL-1, and CXCL-2 levels in PLF after CLP in vivo and reduced NET formation in PLF cells after CLP ex vivo . Additionally, recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells ex vivo . Previous studies support our results, indicating IL-17A, CXCL-1, or CXCL-2 can promote NET formation 30 , 36 – 38 . Pad4 knockout markedly reduced IL-17A production in PLF or plasma, while Il-17a KO partially decreased NET formation in PLF, though it was statistically significant. In our CLP model, factors beyond IL-17A and the CXCL-1/CXCL-2 pathway in the peritoneal cavity may also influence intraperitoneal NET formation. However, these findings suggest a vicious cycle of IL-17A, CXCL-1/CXCL-2 pathway, and NETs in the peritoneal cavity, potentially exacerbating CLP-induced organ inflammation/injury. Adoptive transfer of peritoneal NETs restored upregulated the peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in Pad4KO mice, leading to neutrophil infiltration into remote organs and remote organ injury. In adoptive transfer experiments, we examined the role of intraperitoneal NET formation in distant organ injury/inflammation in CLP. Transferring WT peritoneal neutrophils into Pad4 KO mice reversed the attenuation seen with Pad4 knockout, unlike Pad4 KO neutrophils. This reversal also included IL-17A levels in PLF and plasma, CXCL-1 and CXCL-2 levels in kidney and lung, neutrophil infiltration into kidney and lung, and AKI. These findings support our hypothesis that intraperitoneal NET formation critically contributes to remote organ inflammation/injury via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in our mouse CLP model. Several limitations exist in this study. First, we utilized mice with systemic Pad4 knockout. PAD4 is expressed not only in neutrophils but also in other immune cells like eosinophils, monocytes, macrophages, and natural killer cells 39 . Furthermore, extracellular traps are known to occur not only on neutrophils but also on mast cells, eosinophils, basophils, and monocytes/macrophages 40, 41 . Although the large majority of immune cells in the peritoneal cavity after CLP were neutrophils, the extracellular traps in our experiments may have included extracellular traps derived from these immune cells other than neutrophils. Given the use of whole-body Pad4 KO mice, we cannot ascertain if the effect stems from neutrophil PAD4. To address this, we conducted adoptive transfer of purified neutrophils from WT to Pad4 KO mice, indicating the significance of intraperitoneal neutrophil PAD4 in the inflammation/injury pathogenesis in remote organs. Secondly, PAD4 has other effects beyond its critical role in NET formation. It mediates apoptosis, inflammation, and pluripotency 39 . PAD4 promoted thrombin activity via antithrombin inactivation in rheumatoid arthritis 42 . The function of PAD4 other than NET formation might contribute to the beneficial effect of knockout of Pad4 on our CLP model. Another limitation is interspecies differences in NET formation. Human neutrophils form NETs more readily than mouse neutrophils 6 , 43 , 44 . This implies that NETs might more likely form in peripheral blood or distant organs in humans than in mice. Further research is necessary to understand the role of local NET formation to distant organ injury via local IL-17A and remote CXCL-1/ CXCL-2 in humans. In summary, this study is the first to elucidate how intraperitoneal NET formation can trigger distant organ injury and inflammation via the IL-17A and CXCL-1/CXCL-2 pathway without remote organ NET formation after CLP. Pad4 knockout improved survival in a clinically relevant abdominal sepsis model with broad-spectrum antibiotics and fluids resuscitation. Pad4 knockout reduced IL-17A production in PLF and plasma. Both Pad4 and Il-17a knockout ameliorated AKI and reduced neutrophil infiltration into the kidney and lung by lowering CXCL-1/CXCL-2 levels, known downstream factors of IL-17A, in these organs. Adoptive transfer of WT neutrophils restored CLP-induced AKI and neutrophil infiltration into kidney and lung, as well as CXCL-1 and CXCL-2 levels in these organs, and IL-17A levels in PLF and plasma attenuated by Pad4 knockout. These findings suggest a pathway from peritoneal NET formation to distant organ injury/inflammation via peritoneal IL-17A production and distant organ CXCL-1/CXCL-2. While NETs promoted IL-17A production in PLF and plasma, we demonstrated reciprocally that IL-17A or CXCL-1 and CXCL-2 promoted NET formation in PLF after CLP. These results highlight a potential vicious cycle among NET formation, IL-17A, and CXCL-1/CXCL-2 amplifying organ injury and inflammation in sepsis. Disrupting this cycle by inhibiting NET formation or IL-17A could be promising therapeutic strategies for sepsis treatment in carefully selected patients. Methods Animals All animal studies were approved by the NIDDK Animal Care and Use Committee (K100-KDB). All experiments were performed in accordance with relevant guidelines and regulations and with ARRIVE guidelines. Il-17a KO (Strain #:016879), Pad4 KO mice (Strain #:030315), and C57BL/6J WT controls (Strain #:000664) were obtained from Jackson Laboratory (Bar Harbor, ME). Mice had an acclimation period of at least 7 days prior to use for any experiments. CLP CLP was performed as previously described 15 . Briefly, 9–12-week-old male mice were anesthetized (isoflurane 5% for induction and 3% to maintain anesthesia). The cecum was ligated at 1 cm from the cecal tip, punctured twice with a 21-gauge needle, and gently squeezed to express a 1-mm column of cecal material. Sham surgeries were identical (without cecal ligation and puncture). Post-surgery, mice received subcutaneous Buprenorphine ER (1.2 mg/kg) and intraperitoneal normal saline (1.0 mL). Mice were euthanized 3 or 18 h post-CLP. Blood, tissues, and peritoneal lavage fluid (PLF) were collected following peritoneal injection of 2 mL PBS with 2mM EDTA. Adoptive transfer of isolated neutrophils into CLP treated mice. Male or female WT or Pad4 KO mice (9–12 weeks old) underwent CLP surgery. PLF was collected at 18 h post-CLP. Neutrophils from PLF were purified using a mouse Neutrophil Isolation Kit (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany), labeled with anti-mouse Ly6G Pacific Blue and anti-mouse CD11b APC/Cy7 antibodies, and analyzed via flow cytometry (~ 95% pure Ly6G + CD11b + cells; Supplementary Fig. 4B). Neutrophils were counted using Countess II (Invitrogen, Carlsbad, CA). Neutrophils (10 6 ) were intraperitoneally injected into Pad4 KO mice immediately after CLP. Mice were euthanized 18 h post-CLP, and tissue specimens, blood, and PLF were harvested. Survival study Mice were monitored every 6–12 h post-CLP, with euthanasia of survivors at 168 h. Antibiotic and fluid resuscitation began 6 h post-CLP via subcutaneous injection of imipenem/cilastatin (14 mg/kg) in 1 mL of 2/3 NS, repeated with 7 mg/kg in 1 mL of 2/3 NS every 12 h. Additional doses of Buprenorphine ER were given at 72 and 144 h post-CLP. Collection of PLF cells PLF cells were harvested after injection of 4 mL PBS with 2mM of EDTA into the peritoneum and centrifuged for 5 min at 500 x g 4 ℃. The cells were resuspended in MACs buffer (Miltenyi Biotech, Auburn, CA) for flow cytometry, or RPMI-1640 medium (containing 10 mM HEPES and 0.5% BSA) without phenol red for immunocytochemistry or NET visualization using SYTOX Green (Invitrogen). In vitro NET generation PLF cells were collected and pooled from 4 WT mice 3 h after CLP. 5 × 10 4 cells were transferred to a poly-L-lysine-coated coverslip (Corning) in 24 well plates, stimulated with 20 ng/ml mouse recombinant IL-17A, CXCl-1, -2 protein (R&D Systems), or 100 nM of phorbol myristate acetate (PMA) for 2–3 h in a humidified incubator (37°C, 5% CO 2 ). Initially, we applied stimuli to PLF cells collected 18 h after CLP, but it was difficult to detect any stimulation, likely because the cells were already highly activated. Therefore, we collected PLF cells 3 h after CLP, because they were less activated. The coverslips were stained with anti-Histone H3 (citrulline R2 + R8 + R17) antibody (Abcam) and Hoechst 33342 (Thermo Fisher Scientific), see also Supplemental Methods. 30 images were obtained using a confocal microscope (Zeiss LSM780, Zeiss) from different areas from 2–3 slides in each group. These images were analyzed for % NETs formed and NET extension. Statistical analyses The results are expressed as means ± standard error of means (SEM). The normality of the data distribution was visually checked using histograms. Differences between two groups were analyzed using unpaired Welch’s t-tests and differences among three or more groups were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test or two- way ANOVA followed by Šídák test. Mouse survival was depicted with Kaplan–Meier curves (log-rank test). All statistical calculations were performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). P values ≤ 0.05 were considered a statistically significant. Further details are provided in Supplemental Methods. Declarations Competing Interests The authors declare no competing interests, as this work was funded entirely by the National Institutes of Health (Z01 DK43403). Funding Declaration This research was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH). The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Author Contribution Y.N., N.H., P.Y., and R.S. conceived and designed research; Y.N., D.G., N.H., and X.H. performed experiments; Y.N., D.G., and N.H. analyzed data; Y.N., P.Y., and R.S. interpreted results of experiments; Y.N., P.Y., and R.S. prepared figures; Y.N., D.G., N.H., P.Y., and R.S. drafted the manuscript; Y.N., D.G., N.H., P.Y., and R.S. edited and revised the manuscript; all authors approved the final version of manuscript. Acknowledgement This research was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH). The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Data Availability Underlying data are published on Mendeley Data (Reserved DOI 10.17632/y2tbj7h96t.1). References Peerapornratana, S. et al. Acute kidney injury from sepsis: current concepts, epidemiology, pathophysiology, prevention and treatment. Kidney Int. 96 , 1083–1099 (2019). Bagshaw, S. M. et al. Acute kidney injury in septic shock: Clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy. Intensive Care Med. 35 , 871–881 (2009). Poston, J. T. & Koyner, J. L. Sepsis associated acute kidney injury. BMJ 364 , k4891–k4891 (2019). Bagshaw, S. M. et al. Septic acute kidney injury in critically ill patients: Clinical characteristics and outcomes. Clin. J. Am. Soc. 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14:11:40","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115917,"visible":true,"origin":"","legend":"","description":"","filename":"7e4f9681a53f4faab11fc5a445e54e341structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/574a7118cd83055ec78878de.xml"},{"id":91998201,"identity":"764d5bba-9557-4db3-84aa-c38f0e8791f2","added_by":"auto","created_at":"2025-09-23 14:03:41","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132973,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/f0c6edb8f828dccdc92bb849.html"},{"id":91999373,"identity":"810a2500-13e8-4ecd-9fe6-017b8f5cfdb4","added_by":"auto","created_at":"2025-09-23 14:11:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1477134,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of \u003cem\u003ePad4\u003c/em\u003e improved survival, AKI, and neutrophil infiltration into kidney and lung after cecal ligation and puncture (CLP). (A) Survival curves of wild-type (WT) mice (n =19) or \u003cem\u003ePad4\u003c/em\u003eknockout (\u003cem\u003ePad4\u003c/em\u003eKO) mice (n = 20) after CLP surgery. The survival data were analyzed by a log-rank test. *P \u0026lt; 0.05 vs. WT mice. (B) Plasma BUN levels in WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after sham (n = 5 per group) or CLP (n = 11 per group) surgery. (C) Tubular damage score in kidney cortex of WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at18 h after sham (n = 5 per group) or CLP surgery (n = 11 per group). (D and E) Number of neutrophils in kidney (D) and lung (E) of WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after sham (n = 5 per group) or CLP surgery (n = 11 per group) using naphthol AS-D chloroacetate esterase staining. The data sets were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Values represent the means ± SEM. **P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/68ae423d7133004b179e5795.jpg"},{"id":91999374,"identity":"5593b377-1021-412a-8963-174022648f53","added_by":"auto","created_at":"2025-09-23 14:11:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":521733,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of \u003cem\u003ePad4\u003c/em\u003e inhibited CXCL-1 and -2 production in kidney and lung and IL-17A production in peritoneal cavity and plasma at 18 h after CLP. (A-B) CXCL-1 and -2 concentration in kidney (A) and lung (B) of WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at18 h after sham (n =5 per group) or CLP (n = 7 per group) surgery. (C-D) IL-17A concentration in PLF (C) and plasma (D) at 18 h after sham (n =5 per group) or CLP surgery (n = 7 per group). The data sets were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Values represent the means ± SEM. **P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/6b694db1f2475ac1fe2d48be.jpg"},{"id":91999919,"identity":"9bd78378-5145-4d98-8b2c-fc2a4d5cdf2d","added_by":"auto","created_at":"2025-09-23 14:19:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1739044,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of \u003cem\u003eIl-17a\u003c/em\u003e attenuated AKI and neutrophil infiltration into kidney and lung, and inhibited CXCL-1 and -2 production in kidney and lung at 18h after CLP. (A) Plasma BUN levels in WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after sham (n = 6 per group) or CLP (n = 10 per group) surgery. (B) Tubular damage score in kidney cortex of WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after sham (n = 5 per group) or CLP surgery (n = 8 per group). (C) Number of neutrophils in kidney of WT or\u003cem\u003e Il-17a\u003c/em\u003eKO mice at 18 h after sham (n = 5 per group) or CLP surgery (n = 8 per group) using naphthol AS-D chloroacetate esterase staining. \u0026nbsp;Neutrophils were counted in ×400 fields and averaged per mouse. (D) Number of neutrophils in lung of WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after sham (n = 5 per group) or CLP surgery (n = 9 per group) using naphthol AS-D chloroacetate esterase staining. \u0026nbsp;Neutrophils were counted in ×400 fields and averaged per mouse. (E-F) CXCL-1 and -2 concentration in kidney (E) and lung (F) of WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at18 h after sham (n =5 per group) or CLP (n = 8-10 per group) surgery. The data sets were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Values represent the means ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/a0546399c9faa652f687f8bc.jpg"},{"id":91998160,"identity":"0990edf2-8da4-4dcc-986e-97073096544b","added_by":"auto","created_at":"2025-09-23 14:03:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5501517,"visible":true,"origin":"","legend":"\u003cp\u003eNET formation was detected only in the peritoneal cavity, and not in plasma, spleen, kidney, or lung at 18 h after CLP. (A) H3Cit staining in spleen, kidney, and lung of WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP. Original magnification, ×400. (B) H3Cit levels in PLF, plasma, spleen, kidney, and lung at at18 h after sham (n =5-6 per group) or CLP (n = 5-12 per group) surgery. \u0026nbsp;(C) Representative images of flow cytometry analysis for Ly6G+ neutrophils in PLF cells at 18 h after sham or CLP surgery. Living (zombie violet negative), CD45+ cells were gated from singlets. Then Ly6G expression was analyzed in this population. \u0026nbsp;(D) Ly6G+ neutrophil population in peritoneal cavity at 18 h after sham or CLP surgery (n=7-8 per group). (E) The course of absolute number (right) or percentage (left) of Ly6G+, CD11b+ neutrophil accumulation in peritoneal cavity after CLP surgery (3 and 18 h). Living cells (7-AAD negative) were gated from singlets. Then Ly6G and CD11b positive cells were gated and counted as neutrophils (n=5 per group). (F) Representative images of NET formation in PLF cells of WT or \u003cem\u003ePad4\u003c/em\u003eKO mice at 18h after CLP. PLF cells were placed on poly-L-lysine coated cover slips without additional stimulation and incubated at 37℃ for 2h. These cells were stained for H3Cit (Green) or Hoechst (Red Pseudo color). Original magnification, ×400. (G) Summary analysis of NET formation in PLF cells of WT (n = 5) or \u003cem\u003ePad4\u003c/em\u003eKO mice (n = 4) at 18h after CLP. Percentage of cells with NET formation was calculated as the number of cells extruding H3Cit-positive structures divided by the number of cells identified by Hoechst and multiplied by 100 (upper). \u0026nbsp;NET extension in PLF cells was also measured as the H3Cit-positive area divided by the number of cells evaluated by Hoechst (lower). The data sets were analyzed by unpaired t-test. Values represent the means ± SEM. ns: not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, **** P \u0026lt; 0.0001. Scale bars = 20 µm.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/6e0260ca8a7546563989c24c.jpg"},{"id":91999376,"identity":"ef42c279-5a34-441b-b7c4-09934221a753","added_by":"auto","created_at":"2025-09-23 14:11:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":461876,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of \u003cem\u003eIl-17a\u003c/em\u003e suppressed CXCL-1 and -2 content in PLF and plasma and H3Cit levels in PLF at 18h after CLP. (A,B) CXCL-1 and -2 concentration in PLF (A) and plasma (B) of WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after sham (n = 5) or CLP (n = 8) surgery. (C) H3Cit levels in PLF of WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after sham (n = 5) or CLP (n = 7-8) surgery. The data sets were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Values represent the means ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/2708b98c492625494ba05033.jpg"},{"id":92001179,"identity":"7f03e119-a327-41c1-b2a7-660a40f371b1","added_by":"auto","created_at":"2025-09-23 14:27:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4299017,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of \u003cem\u003eIl-17a\u003c/em\u003e suppressed NET formation in PLF cells \u003cem\u003eex vivo\u003c/em\u003e at 18h after CLP. \u003cem\u003eEx vivo\u003c/em\u003e stimulation with recombinant IL-17A, CXCL-1, or -2 promoted NET formation in PLF cells. (A) Representative images of PLF cells from WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18h after CLP. PLF cells were applied to a 24 well plate without additional stimulation and incubated at 37℃ for 2 h. These cells were stained with SYTOX green (green) and Hoechst (blue). (B) NET extension in PLF cells from WT (n=10) or\u003cem\u003e Il-17a\u003c/em\u003eKO (n=10) was measured as the SYTOX green-positive area divided by the number of cells detected by Hoechst. (C) Representative images of NET formation in PLF cells from WT or \u003cem\u003eIl-17a\u003c/em\u003eKO mice at 18 h after CLP. PLF cells were placed on poly-L-lysine coated cover slips without additional stimulation and incubated at 37℃ for 2 h. These cells were stained for H3Cit (Green) and Hoechst (Red pseudocolor). Original magnification, ×400. (D) Summary analysis of NET formation in PLF cells of WT (n = 9) or \u003cem\u003eIl-17a\u003c/em\u003eKO mice (n = 8) at 18h after CLP. Percentage of cells with NET formation (upper) and \u0026nbsp;NET extension (lower) in PLF cells were calculated. (E) Representative images of NET formation in PLF cells from WT mice at 3h after CLP. PLF cells were stimulated with recombinant IL-17A, CXCL-1, or -2 at 37℃ for 3h. These cells were stained for H3Cit (Green) and Hoechst (Red pseudocolor). Original magnification, ×400. (F) Summary analysis of NET formation in PLF cells collected 3 h after CLP stimulated with recombinant IL-17A, CXCL-1, -2, or PMA for 3 h (30 fields from 2-3 coverslips per group). Percentage of cells with NET formation (left) and \u0026nbsp;NET extension (right) in PLF cells were calculated. The data sets were analyzed by unpaired t-test (B and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test (F). Values represent the means ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001. Scale bars = 300µm (A) and 20 µm (C and E).\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/6fa521f223187476985ff875.jpg"},{"id":91998167,"identity":"f4c19ff9-c163-46f3-9c74-7987b1f3b001","added_by":"auto","created_at":"2025-09-23 14:03:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2414243,"visible":true,"origin":"","legend":"\u003cp\u003eIntraperitoneal adoptive transfer of WT neutrophils counteracted the attenuation of septic AKI, neutrophil infiltration into kidney, IL-17A production in PLF and plasma, CXCL-1/CXCL-2 production in kidney and lung caused by knockout of \u003cem\u003ePad4\u003c/em\u003e. (A) Plasma BUN levels in \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP surgery injected with WT (n = 12) or \u003cem\u003ePad4\u003c/em\u003eKO (n = 7) neutrophils, or vehicle (n = 5). (B) Tubular damage score in kidney cortex in \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP surgery injected with WT (n = 12) or \u003cem\u003ePad4\u003c/em\u003eKO (n = 7) neutrophils, or vehicle (n = 5). (C) \u0026nbsp;Number of neutrophils in kidney of \u003cem\u003ePad4\u003c/em\u003eKO mice at18 h after CLP surgery injected with WT (n = 12) or \u003cem\u003ePad4\u003c/em\u003eKO (n = 7) neutrophils, or vehicle (n = 5) using naphthol AS-D chloroacetate esterase staining. Neutrophils were counted in ×400 fields and averaged per mouse. (D, E) CXCL-1 and -2 concentration in kidney (D) and lung (E) of \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP surgery injected with WT (n = 9) or \u003cem\u003ePad4\u003c/em\u003eKO (n = 7) neutrophils, or vehicle (n = 5). (F, G) IL-17A concentration in (F) PLF and (G) plasma of \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP surgery injected with WT (n = 12) or \u003cem\u003ePad4\u003c/em\u003eKO (n = 7) neutrophils, or vehicle (n = 5). The data sets were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Values represent the means ± SEM. ns: not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, *** P \u0026lt; 0.001. Scale bars = 20 µm.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/c4b4e3e72e465ec2f75429be.jpg"},{"id":91999923,"identity":"9a428313-c19e-4a8b-b41a-013832bff15d","added_by":"auto","created_at":"2025-09-23 14:19:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1460913,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic presentation of the pathway from NET formation and IL-17A production in the peritoneal cavity to remote organ injuries in the CLP model. CLP upregulated neutrophil accumulation and PAD4 mediated NET formation in the peritoneal cavity (Figure 4). CLP increased IL-17A production in PLF and plasma, which was significantly decreased by \u003cem\u003ePad4\u003c/em\u003e knockout (Figure 2 C and D). Knockout of \u003cem\u003eIl-17a\u003c/em\u003e significantly decreased CXCL-1 and -2 production in PLF after CLP (Figure 5A). Knockout of \u003cem\u003eIl-17a\u003c/em\u003edecreased NET formation in peritoneal cavity after CLP (Figure 5C and 6 A-D). Recombinant IL-17A, CXCL-1 and -2 upregulated NET formation in PLF cells collected from WT mice at 3 h after CLP \u003cem\u003eex vivo\u003c/em\u003e (Figure 6 E and F). \u003cem\u003ePad4\u003c/em\u003eKO as well as\u003cem\u003e Il-17a\u003c/em\u003eKO significantly decreased CXCL-1 and -2 production in kidney and lung, neutrophil infiltration into kidney and lung, and AKI after CLP (Figure 1 and 3). \u003cem\u003ePad4\u003c/em\u003eKO significantly improved survival after CLP (Figure 1A). Adoptive transfer of WT neutrophil into \u003cem\u003ePad4\u003c/em\u003eKO mice restored IL-17A production in PLF and plasma, CXCL-1 and -2 production in kidney and lung, neutrophil infiltration into kidney and lung, and AKI (Figure 7).\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/d0a8b3dd4975ceb52b63e06f.jpg"},{"id":101691871,"identity":"6724cf74-74d9-4515-bd64-78e3722e4e01","added_by":"auto","created_at":"2026-02-02 16:15:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18818094,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/8bf07d34-e240-4188-a406-9fdae8ea6a6f.pdf"},{"id":91998152,"identity":"f6c80daf-9571-4510-ad45-237434997d7b","added_by":"auto","created_at":"2025-09-23 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14:19:40","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":93161,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEGuidelinesAuthorChecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474386/v1/85ad080bc96f0629abfde965.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Peritoneal Neutrophil Extracellular Traps contribute to septic AKI via peritoneal IL-17A and distant organ CXCL-1/ CXCL-2 pathway in abdominal sepsis.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAbout 60% of septic patients develop sepsis-associated acute kidney injury (SAKI)\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, with a mortality rate of 60\u0026ndash;70%\u003csup\u003e1, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. There are no specific treatments for SAKI. Neutrophil extracellular traps (NETs), expelled from activated neutrophils, contain cfDNA, histones, proteases, and antimicrobial proteins, and may be the primary source of cfDNA in early sepsis\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Peptidylarginine deiminase 4 (PAD4) is essential for NET formation through histone citrullination\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. NETs have dual effects in sepsis. They can immobilize bacteria, which is advantageous; but their components (histones, proteases, and cfDNA) can harm host cells[6, 7]. Due to these opposing effects, targeting NETs in sepsis may be difficult. Martinod et al. observed that \u003cem\u003ePad4\u003c/em\u003e knockout did not enhance survival in a mild CLP model and worsened survival in a severe CLP model\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Others have shown that \u003cem\u003ePad4\u003c/em\u003e knockout or DNase treatment improved organ damage in a mouse model of \u003cem\u003eE. coli\u003c/em\u003e sepsis\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, blocking NETs with a non-specific PAD inhibitor Cl-amidine, antibodies against citrullinated histone H3 (H3Cit), or DNase enhanced survival post-CLP\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These apparently contradictory effects imply that NETs exert both beneficial and detrimental effects in sepsis, contingent upon the particular context.\u003c/p\u003e\u003cp\u003eInterestingly, NETs increase IL-17A, a cytokine we previously demonstrated to be downstream of TLR9\u003csup\u003e15\u003c/sup\u003e, a known mediator of sepsis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. NETs promote Th17 cell differentiation through one of their components, histone\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePad4\u003c/em\u003e knockout halted IL-17A accumulation in atherosclerosis within the aorta\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the contribution of NET formation to IL-17A production in sepsis remains unclear.\u003c/p\u003e\u003cp\u003eWe hypothesized that NETs increase IL-17A and its downstream factors, CXCL-1 and CXCL-2, causing neutrophil infiltration and organ injury in sepsis. Using two knock out mouse models (\u003cem\u003ePad4\u003c/em\u003eKO mice in which NET formation was completely abolished; and \u003cem\u003eIl-17a\u003c/em\u003eKO), we evaluated the 1) effect of NET formation on CLP outcomes; 2) main sites of NET formation in CLP-treated mice; 3) effect of NET formation on neutrophil infiltration into remote organs and IL-17A and CXCL-1/CXCL-2 pathway after CLP; 4) relationship between NET formation and IL-17A in the peritoneal cavity; and 5) effect of peritoneal NETs on remote organ injury using an adoptive transfer model of WT neutrophils into \u003cem\u003ePad4\u003c/em\u003eKO mice after CLP.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffect of knockout of\u003c/b\u003e \u003cb\u003ePad\u003c/b\u003e\u003cb\u003e4 on survival, AKI, and neutrophil infiltration into kidney and lung after CLP\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of NET formation in our moderate severity, clinically relevant model of polymicrobial sepsis, we examined whether knockout of \u003cem\u003ePad4\u003c/em\u003e alters survival, renal function and morphology, and neutrophil infiltration into kidney and lung after CLP. \u003cem\u003ePad4KO\u003c/em\u003e mice had significantly improved survival at 168 h after CLP, with a survival rate of 80% compared to 47% for WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). WT mice developed kidney injury 18 h after CLP, whereas \u003cem\u003ePad4\u003c/em\u003eKO mice showed decreased serum BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and improved cortical tubular damage scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Supplemental Fig.\u0026nbsp;1). Neutrophil infiltration into kidney and lung were increased at 18 h after CLP, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E,, and Supplemental Fig.\u0026nbsp;2A and B), but \u003cem\u003ePad4\u003c/em\u003eKO mice exhibited significantly lower neutrophil infiltration. Knockout of \u003cem\u003ePad4\u003c/em\u003e did not alter the number of bacterial colonies 18 h after CLP (Supplemental Fig.\u0026nbsp;3A).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of knockout of\u003c/b\u003e \u003cb\u003ePad\u003c/b\u003e\u003cb\u003e4 on CXCL-1 and \u0026minus;\u0026thinsp;2 production in kidney and lung, and IL-17A production in PLF and plasma after CLP\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of NET formation in CXCL-1 and \u0026minus;\u0026thinsp;2 production in kidney and lung after CLP, CXCL-1 and \u0026minus;\u0026thinsp;2 levels in kidney and lung were evaluated in \u003cem\u003ePad4\u003c/em\u003eKO and WT mice 18 h after sham or CLP surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). CXCL-1 and \u0026minus;\u0026thinsp;2 levels were significantly higher in kidney and lung from WT mice vs. \u003cem\u003ePad4\u003c/em\u003eKO mice at 18 h after CLP. Levels of IL-17A, a known upstream factor of CXCL-1 and \u0026minus;\u0026thinsp;2\u003csup\u003e20\u003c/sup\u003e, in PLF and plasma were also assessed after CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). IL-17A levels were significantly higher in PLF and plasma of WT mice vs. \u003cem\u003ePad4\u003c/em\u003eKO mice 18 h after CLP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of knockout of\u003c/b\u003e \u003cb\u003eIl-17a\u003c/b\u003e \u003cb\u003eon AKI, neutrophil infiltration into kidney and lung, and levels of CXCL-1 and \u0026minus;\u0026thinsp;2 in kidney and lung after CLP\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIl-17a\u003c/em\u003e KO improves survival in sepsis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, but the mechanism is unknown. To investigate the role of IL-17A in SAKI, plasma BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and renal tubular damage scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplemental Fig.\u0026nbsp;4) were assessed 18 h after CLP. WT mice developed kidney injury 18 h after CLP, whereas \u003cem\u003eIl-17a\u003c/em\u003eKO mice showed decreased BUN levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and improved tubular damage scores in the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplemental Fig.\u0026nbsp;4) similar to \u003cem\u003ePad4\u003c/em\u003eKO mice. Knockout of \u003cem\u003eIl-17a\u003c/em\u003e significantly decreased neutrophil infiltration into kidney and lung at 18 h after CLP compared with WT mice by naphthol AS-D chloroacetate esterase staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D, and Supplemental Fig.\u0026nbsp;5A and B). Neutrophil infiltration into kidney was also assessed by flow cytometry (Supplemental Fig.\u0026nbsp;6A, B, C, and D). Neutrophil infiltration was upregulated by CLP at 3 and 18 h after CLP, which was attenuated in knockout of \u003cem\u003eIl-17a\u003c/em\u003e KO mice. Furthermore, knockout of \u003cem\u003eIl-17a\u003c/em\u003e, similar to knockout of \u003cem\u003ePad4\u003c/em\u003e, significantly decreased CXCL-1 and \u0026minus;\u0026thinsp;2 levels in kidney and in lung at 18 h after CLP compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Knockout of \u003cem\u003eIl-17a\u003c/em\u003e did not alter the number of bacterial colonies in PLF collected at 18 h after CLP (Supplemental Fig.\u0026nbsp;3B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eNET formation in peritoneal cavity after CLP\u003c/h3\u003e\n\u003cp\u003eWe next investigated where NETs can form in CLP-treated mice. Spleen, kidney, and lung were harvested from WT or \u003cem\u003ePad4\u003c/em\u003eKO mice 18 h after CLP and stained for citrullinated histone H3 (H3Cit) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Few H3Cit-positive cells were observed in a very limited area in the spleen, and not at all in kidney and lung. H3Cit levels significantly increased in CLP treated mice compared to sham only in PLF, but not in plasma, spleen, or kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Since evidence of NET formation was detected only in the PLF in our CLP model, we focused on neutrophils in the peritoneal cavity. CLP significantly increased neutrophil accumulation in PLF at 18 h after CLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Also, among live PLF cells the percentage of neutrophils was as high as 65.36% (95%CI: 51.27\u0026ndash;83.30%) at 3 h and 69.78% (95%CI: 63.38\u0026ndash;76.86%) 18 h after CLP, even without neutrophil purification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Approximately 40% of PLF cells from WT mice formed NETs after CLP without any \u003cem\u003eex vivo\u003c/em\u003e stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), and NETs were almost completely absent in cells from \u003cem\u003ePad4 KO\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). A similar trend was obtained when the H3Cit-positive area was normalized by cell number, which we interpreted as NET extension in PLF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effect of knockout of\u003c/b\u003e \u003cb\u003eIl-17a\u003c/b\u003e \u003cb\u003eon CXCL-1 and \u0026minus;\u0026thinsp;2 production in PLF and plasma and NET formation in peritoneal cavity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn PLF and plasma, CXCL-1 and \u0026minus;\u0026thinsp;2 levels were upregulated 18 h after CLP in WT mice, whereas these levels were significantly decreased in \u003cem\u003eIl-17a\u003c/em\u003eKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Knockout of \u003cem\u003eIl-17a\u003c/em\u003e did not significantly alter the absolute number or percentage of neutrophils infiltrating into the peritoneal cavity 18 h after CLP (Supplemental Fig.\u0026nbsp;7). However, H3Cit levels in PLF, which were elevated 18 h after CLP in WT mice, were significantly decreased in PLF from \u003cem\u003eIl-17a\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effect of IL-17A, CXCL-1, and \u0026minus;\u0026thinsp;2 on NET formation in PLF cells ex vivo.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePLF cells from \u003cem\u003eIl-17a KO\u003c/em\u003e mice significantly decreased NET extension, measured as SYTOX green positive area normalized to cell number, compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). NET formation was also assessed by staining with H3Cit antibody using confocal microscopy; PLF cells from \u003cem\u003eIl-17a\u003c/em\u003eKO mice vs cells from WT mice had a decreased percentage of NET formation or NET extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, and D). Next, we evaluated the effects of recombinant IL-17A, CXCL-1, or -2 stimulation on NET formation in PLF cells. \u003cem\u003eEx vivo\u003c/em\u003e incubation of PLF cells collected 3 h after CLP with recombinant IL-17A, rCXCL-1, or -2 increased the percentage of NET formation or NET extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of intraperitoneal adoptive transfer of WT neutrophils into\u003c/b\u003e \u003cb\u003ePad4\u003c/b\u003e \u003cb\u003eKO mice on septic AKI, lung inflammation, and IL-17A/CXCL-1/CXCL-2 axis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor further investigation of the relationship between intraperitoneal NET formation and distant organ injuries, we assessed whether adoptive transfer of WT neutrophils into \u003cem\u003ePad4\u003c/em\u003eKO mice can reverse the CLP-induced AKI or lung inflammation attenuated by \u003cem\u003ePad4\u003c/em\u003e knockout. First, donor WT or \u003cem\u003ePad4\u003c/em\u003eKO mice were subjected to CLP. PLF cells were collected at 18 h after CLP and neutrophils were purified from these PLF cells. Then, recipient \u003cem\u003ePad4\u003c/em\u003eKO mice were subjected to CLP and neutrophils collected from WT or \u003cem\u003ePad4\u003c/em\u003eKO donor mice were intraperitoneally administered into \u003cem\u003ePad4\u003c/em\u003eKO mice immediately after CLP of recipients (Supplemental Fig.\u0026nbsp;8A). In CLP-treated \u003cem\u003ePad4\u003c/em\u003eKO mice, WT donor neutrophil administration reconstituted AKI, in contrast with \u003cem\u003ePad4\u003c/em\u003eKO donor neutrophil administration or vehicle injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B, and Supplemental Fig.\u0026nbsp;9A). WT neutrophil administration into \u003cem\u003ePad4\u003c/em\u003eKO mice also increased neutrophil infiltration into kidney and lung compared with injection of \u003cem\u003ePad4\u003c/em\u003eKO neutrophils or vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, and Supplemental Fig.\u0026nbsp;9B, C, and D). We then assessed CXCL-1 and \u0026minus;\u0026thinsp;2 levels in kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) and lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and IL-17A levels in PLF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) and plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG) at 18 h after CLP. Adoptive transfer of WT neutrophils counteracted the attenuation of CXCL-1 and \u0026minus;\u0026thinsp;2 production in kidney and lung and IL-17A production in PLF and plasma by \u003cem\u003ePad4\u003c/em\u003e knockout, whereas adoptive transfer of \u003cem\u003ePad4\u003c/em\u003eKO neutrophils did not alter these levels. These findings indicate that the beneficial effect on SAKI and lung inflammation induced by \u003cem\u003ePad4\u003c/em\u003e knockout are attenuated by intraperitoneal administration of WT neutrophils.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNeutrophils can have both beneficial and harmful effects in sepsis, potentially explaining why neutrophil depletion does not change overall survival in CLP sepsis models\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For example, NETs formed from activated neutrophils during sepsis can trap bacteria and fungi to contain the infection, while also inducing local and distant inflammation through DAMPs like cfDNA\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. PAD4, a crucial enzyme, regulates NET formation through histone citrullination\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consequently, neutrophils lacking PAD4 fail to produce NETs, even when stimulated by chemokines, LPS, or bacteria\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we explored the function of \u0026lsquo;local\u0026rsquo; peritoneal NETs formed at site of infection, and their impact on distant organ function. The main findings of this paper are: 1) elimination of NETs by knockout of \u003cem\u003ePad4\u003c/em\u003e improved survival and AKI after CLP; 2) NETs were detected only in the peritoneal cavity, not in plasma or distant organs; 3) knockout of \u003cem\u003ePad4\u003c/em\u003e suppressed neutrophil infiltration into remote organs via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway; 4) knockout of \u003cem\u003eIl-17a\u003c/em\u003e suppressed NET formation and CXCL-1/CXCL-2 production in peritoneal cavity after CLP and recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells; and 5) adoptive transfer of peritoneal NETs restored the peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in \u003cem\u003ePad4\u003c/em\u003eKO mice, leading to neutrophil infiltration into remote organs and remote organ injury. Our findings are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eElimination of NETs by Knockout of Pad4 improves survival and AKI after CLP.\u003c/em\u003e We found that \u003cem\u003ePad4\u003c/em\u003e knockout improved survival and AKI in a CLP model in which the cecum was punctured with 21-gauge needles, and animals were treated with fluids (1 ml of 2/3 normal saline) and antibiotics (s.c.) every 12 h for 7 days. Survival on day 4 after CLP was 53% for WT mice vs. 85% for \u003cem\u003ePad4\u003c/em\u003eKO and on day 7 was 47% for WT mice vs. 80% for \u003cem\u003ePad4\u003c/em\u003eKO. NETs defend against bacteria by trapping or killing them\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Inhibiting NETs during severe infections could be detrimental for sepsis. Interestingly, \u003cem\u003ePad4\u003c/em\u003e knockout did not change the peritoneal bacterial count 18 h after CLP. Similar positive results on CLP survival have been seen with Cl-Amidine, a non-specific PAD inhibitor or antibodies against H3Cit\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our findings contrast with Martinod et al.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, who found no effect of \u003cem\u003ePad4\u003c/em\u003e knockout in a mild CLP model (using 21-gauge needles, no antibiotics, 75% survival at 4 days, with 7\u0026ndash;8 animals per group), and worse outcomes in a severe CLP model (18-gauge needles, with antibiotics, no survival at 10 days). They only partially replaced surgical fluid losses (0.5ml of normal saline once immediately after surgery)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our model resembles their mild CLP model but includes antibiotics, more fluid, and longer intermittent fluid resuscitation. Variations in CLP severity, fluid resuscitation, antibiotic administration (none, subcutaneous, or intraperitoneal), and our larger sample size (19 to 20 mice per group) could explain the differing results between laboratories. Interestingly, even in their severe CLP model, knockout of \u003cem\u003ePad4\u003c/em\u003e did not increase bacterial loads in the blood, liver, or lungs at 24 h post-CLP\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, NETs might have aided in reducing bacterial abundance later\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Losing NET protection against bacteria could be harmful in severe CLP models, yet dampen excessive inflammation in milder CLP models.\u003c/p\u003e\u003cp\u003e\u003cem\u003eNET formation was detected only in the peritoneal cavity, and not in plasma or distant organs.\u003c/em\u003e Due to the positive effect of PAD4 inhibition/deletion on reducing distant organ damage and improving survival, we anticipated widespread NET presence, contributing to multiple organ failure. The detrimental effects of NETs in sepsis are attributed to tissue-based NET formation, intravascular coagulation promotion, and the release of enzymes like neutrophil elastase and serine proteases during NETosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Initially, we hypothesized that intraperitoneal NETs would induce distant NET formation in remote organs via the production of proinflammatory cytokines, chemokines, and damage-associated molecular patterns (DAMPs) like nuclear and/or mitochondrial cfDNA. Surprisingly, we discovered that NETs were confined to the peritoneal cavity and not detected in plasma or other distant organs (lung, liver, kidney, spleen). The location of NETs following sepsis is complex, and it appears to be quite sensitive to the severity of sepsis. NETs have been identified in lungs, kidneys, and plasma after CLP; however, these CLP models were considerably more severe. For example, H3Cit-positive cells were detected in the lungs post-CLP\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e \u0026ndash; but survival was 0% at 24 h\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e or 15% at 48 h\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, compared to 100% at 24 and 48 h in our model. H3Cit-positive cells have been found in the kidney at 24 or 48 h after CLP\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e; however, the model was more severe than our model, with 40% survival in WT mice at 24 h post-CLP\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Elevated serum H3Cit levels have also been detected post-CLP\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e; again, in more severe sepsis models. Yuzi et al. reported survival rates after CLP: around 60% at 24 h and 0% at 60 h post-CLP\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In contrast, Bethany et al. found that plasma H3Cit levels were not increased by CLP surgery compared to sham, consistent with our results\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Their CLP model showed 80% survival at 24 h and 50% at 60 h, indicating a milder outcome compared to Yuzi\u0026rsquo;s model, which exhibited elevated blood H3Cit after CLP, and relatively similar to ours\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These findings imply that NETs may form in lungs, kidneys, or blood during severe CLP. However, in a less severe sepsis model, we found that NET formation in PLF induces (see below) lung inflammation or kidney injury even without NET formation in blood or these remote organs.\u003c/p\u003e\u003cp\u003e\u003cem\u003eKnockout of Pad4 suppressed neutrophil infiltration into remote organs via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway.\u003c/em\u003e We then investigated the mediator(s) from NETs circulating systemically causing organ injury. \u003cem\u003ePad4\u003c/em\u003e knockout reduced IL-17A in PLF and plasma after CLP. Interestingly, a myeloid-specific \u003cem\u003ePad4\u003c/em\u003e deletion greatly reduced IL-17A in an atherosclerosis model\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003ePad4\u003c/em\u003e and \u003cem\u003eIl-17a\u003c/em\u003e knockout notably reduced CXCL-1 and CXCL-2 levels in lung and kidney and decreased neutrophil infiltration. This indicates IL-17A, promoted by NET formation, is vital for CXCL-1, CXCL-2 production, and neutrophil infiltration in distant organs post-CLP. IL-17A stimulates epithelial cells to produce chemokines, and promotes myeloid cell mobilization to inflammatory sites\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, CXCL-8 in humans and its functional homologs, CXCL-1 and CXCL-2 in mice, are potent inducers of neutrophil migration into inflamed tissues\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIl-17a\u003c/em\u003e knockout reduces CXCL-1 and \u0026minus;\u0026thinsp;2 levels, neutrophil infiltration, and kidney impairment after CLP\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Thus, IL-17A plays a crucial role in distant organ CXCL-1/CXCL-2 production and neutrophil infiltration.\u003c/p\u003e\u003cp\u003e\u003cem\u003eKnockout of Il-17a suppressed NET formation and CXCL-1/CXCL \u0026minus;\u0026thinsp;2 production in peritoneal cavity after CLP and recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells.\u003c/em\u003e After finding NET formation promoted the IL-17A pathway in the CLP model, we assessed if the IL-17A pathway affects NET formation. \u003cem\u003eIl-17a\u003c/em\u003e knockout decreased H3Cit, CXCL-1, and CXCL-2 levels in PLF after CLP \u003cem\u003ein vivo\u003c/em\u003e and reduced NET formation in PLF cells after CLP \u003cem\u003eex vivo\u003c/em\u003e. Additionally, recombinant IL-17A, CXCL-1, or CXCL-2 promoted NET formation in PLF cells \u003cem\u003eex vivo\u003c/em\u003e. Previous studies support our results, indicating IL-17A, CXCL-1, or CXCL-2 can promote NET formation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePad4\u003c/em\u003e knockout markedly reduced IL-17A production in PLF or plasma, while \u003cem\u003eIl-17a\u003c/em\u003eKO partially decreased NET formation in PLF, though it was statistically significant. In our CLP model, factors beyond IL-17A and the CXCL-1/CXCL-2 pathway in the peritoneal cavity may also influence intraperitoneal NET formation. However, these findings suggest a vicious cycle of IL-17A, CXCL-1/CXCL-2 pathway, and NETs in the peritoneal cavity, potentially exacerbating CLP-induced organ inflammation/injury.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAdoptive transfer of peritoneal NETs restored upregulated the peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in Pad4KO mice, leading to neutrophil infiltration into remote organs and remote organ injury.\u003c/em\u003e In adoptive transfer experiments, we examined the role of intraperitoneal NET formation in distant organ injury/inflammation in CLP. Transferring WT peritoneal neutrophils into \u003cem\u003ePad4\u003c/em\u003eKO mice reversed the attenuation seen with \u003cem\u003ePad4\u003c/em\u003e knockout, unlike \u003cem\u003ePad4\u003c/em\u003eKO neutrophils. This reversal also included IL-17A levels in PLF and plasma, CXCL-1 and CXCL-2 levels in kidney and lung, neutrophil infiltration into kidney and lung, and AKI. These findings support our hypothesis that intraperitoneal NET formation critically contributes to remote organ inflammation/injury via a peritoneal IL-17A and distant organ CXCL-1/CXCL-2 pathway in our mouse CLP model.\u003c/p\u003e\u003cp\u003eSeveral limitations exist in this study. First, we utilized mice with systemic \u003cem\u003ePad4\u003c/em\u003e knockout. PAD4 is expressed not only in neutrophils but also in other immune cells like eosinophils, monocytes, macrophages, and natural killer cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Furthermore, extracellular traps are known to occur not only on neutrophils but also on mast cells, eosinophils, basophils, and monocytes/macrophages\u003csup\u003e40, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Although the large majority of immune cells in the peritoneal cavity after CLP were neutrophils, the extracellular traps in our experiments may have included extracellular traps derived from these immune cells other than neutrophils. Given the use of whole-body \u003cem\u003ePad4\u003c/em\u003eKO mice, we cannot ascertain if the effect stems from neutrophil PAD4. To address this, we conducted adoptive transfer of purified neutrophils from WT to \u003cem\u003ePad4\u003c/em\u003eKO mice, indicating the significance of intraperitoneal neutrophil PAD4 in the inflammation/injury pathogenesis in remote organs. Secondly, PAD4 has other effects beyond its critical role in NET formation. It mediates apoptosis, inflammation, and pluripotency\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. PAD4 promoted thrombin activity via antithrombin inactivation in rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The function of PAD4 other than NET formation might contribute to the beneficial effect of knockout of \u003cem\u003ePad4\u003c/em\u003e on our CLP model. Another limitation is interspecies differences in NET formation. Human neutrophils form NETs more readily than mouse neutrophils\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This implies that NETs might more likely form in peripheral blood or distant organs in humans than in mice. Further research is necessary to understand the role of local NET formation to distant organ injury via local IL-17A and remote CXCL-1/ CXCL-2 in humans.\u003c/p\u003e\u003cp\u003eIn summary, this study is the first to elucidate how intraperitoneal NET formation can trigger distant organ injury and inflammation via the IL-17A and CXCL-1/CXCL-2 pathway without remote organ NET formation after CLP. \u003cem\u003ePad4\u003c/em\u003e knockout improved survival in a clinically relevant abdominal sepsis model with broad-spectrum antibiotics and fluids resuscitation. \u003cem\u003ePad4\u003c/em\u003e knockout reduced IL-17A production in PLF and plasma. Both \u003cem\u003ePad4\u003c/em\u003e and \u003cem\u003eIl-17a\u003c/em\u003e knockout ameliorated AKI and reduced neutrophil infiltration into the kidney and lung by lowering CXCL-1/CXCL-2 levels, known downstream factors of IL-17A, in these organs. Adoptive transfer of WT neutrophils restored CLP-induced AKI and neutrophil infiltration into kidney and lung, as well as CXCL-1 and CXCL-2 levels in these organs, and IL-17A levels in PLF and plasma attenuated by \u003cem\u003ePad4\u003c/em\u003e knockout. These findings suggest a pathway from peritoneal NET formation to distant organ injury/inflammation via peritoneal IL-17A production and distant organ CXCL-1/CXCL-2. While NETs promoted IL-17A production in PLF and plasma, we demonstrated reciprocally that IL-17A or CXCL-1 and CXCL-2 promoted NET formation in PLF after CLP. These results highlight a potential vicious cycle among NET formation, IL-17A, and CXCL-1/CXCL-2 amplifying organ injury and inflammation in sepsis. Disrupting this cycle by inhibiting NET formation or IL-17A could be promising therapeutic strategies for sepsis treatment in carefully selected patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e All animal studies were approved by the NIDDK Animal Care and Use Committee (K100-KDB). All experiments were performed in accordance with relevant guidelines and regulations and with ARRIVE guidelines. \u003cem\u003eIl-17a\u003c/em\u003eKO (Strain #:016879), \u003cem\u003ePad4\u003c/em\u003eKO mice (Strain #:030315), and C57BL/6J WT controls (Strain #:000664) were obtained from Jackson Laboratory (Bar Harbor, ME). Mice had an acclimation period of at least 7 days prior to use for any experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCLP\u003c/h2\u003e\u003cp\u003eCLP was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Briefly, 9\u0026ndash;12-week-old male mice were anesthetized (isoflurane 5% for induction and 3% to maintain anesthesia). The cecum was ligated at 1 cm from the cecal tip, punctured twice with a 21-gauge needle, and gently squeezed to express a 1-mm column of cecal material. Sham surgeries were identical (without cecal ligation and puncture). Post-surgery, mice received subcutaneous Buprenorphine ER (1.2 mg/kg) and intraperitoneal normal saline (1.0 mL). Mice were euthanized 3 or 18 h post-CLP. Blood, tissues, and peritoneal lavage fluid (PLF) were collected following peritoneal injection of 2 mL PBS with 2mM EDTA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdoptive transfer of isolated neutrophils into CLP treated mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMale or female WT or \u003cem\u003ePad4\u003c/em\u003eKO mice (9\u0026ndash;12 weeks old) underwent CLP surgery. PLF was collected at 18 h post-CLP. Neutrophils from PLF were purified using a mouse Neutrophil Isolation Kit (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany), labeled with anti-mouse Ly6G Pacific Blue and anti-mouse CD11b APC/Cy7 antibodies, and analyzed via flow cytometry (~\u0026thinsp;95% pure Ly6G\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;cells; Supplementary Fig.\u0026nbsp;4B). Neutrophils were counted using Countess II (Invitrogen, Carlsbad, CA). Neutrophils (10\u003csup\u003e6\u003c/sup\u003e) were intraperitoneally injected into \u003cem\u003ePad4\u003c/em\u003eKO mice immediately after CLP. Mice were euthanized 18 h post-CLP, and tissue specimens, blood, and PLF were harvested.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSurvival study\u003c/h3\u003e\n\u003cp\u003eMice were monitored every 6\u0026ndash;12 h post-CLP, with euthanasia of survivors at 168 h. Antibiotic and fluid resuscitation began 6 h post-CLP via subcutaneous injection of imipenem/cilastatin (14 mg/kg) in 1 mL of 2/3 NS, repeated with 7 mg/kg in 1 mL of 2/3 NS every 12 h. Additional doses of Buprenorphine ER were given at 72 and 144 h post-CLP.\u003c/p\u003e\n\u003ch3\u003eCollection of PLF cells\u003c/h3\u003e\n\u003cp\u003ePLF cells were harvested after injection of 4 mL PBS with 2mM of EDTA into the peritoneum and centrifuged for 5 min at 500 x g 4 ℃. The cells were resuspended in MACs buffer (Miltenyi Biotech, Auburn, CA) for flow cytometry, or RPMI-1640 medium (containing 10 mM HEPES and 0.5% BSA) without phenol red for immunocytochemistry or NET visualization using SYTOX Green (Invitrogen).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eNET generation\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePLF cells were collected and pooled from 4 WT mice 3 h after CLP. 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were transferred to a poly-L-lysine-coated coverslip (Corning) in 24 well plates, stimulated with 20 ng/ml mouse recombinant IL-17A, CXCl-1, -2 protein (R\u0026amp;D Systems), or 100 nM of phorbol myristate acetate (PMA) for 2\u0026ndash;3 h in a humidified incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). Initially, we applied stimuli to PLF cells collected 18 h after CLP, but it was difficult to detect any stimulation, likely because the cells were already highly activated. Therefore, we collected PLF cells 3 h after CLP, because they were less activated. The coverslips were stained with anti-Histone H3 (citrulline R2\u0026thinsp;+\u0026thinsp;R8\u0026thinsp;+\u0026thinsp;R17) antibody (Abcam) and Hoechst 33342 (Thermo Fisher Scientific), see also Supplemental Methods. 30 images were obtained using a confocal microscope (Zeiss LSM780, Zeiss) from different areas from 2\u0026ndash;3 slides in each group. These images were analyzed for % NETs formed and NET extension.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eThe results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of means (SEM). The normality of the data distribution was visually checked using histograms. Differences between two groups were analyzed using unpaired Welch\u0026rsquo;s t-tests and differences among three or more groups were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test or two- way ANOVA followed by Š\u0026iacute;d\u0026aacute;k test. Mouse survival was depicted with Kaplan\u0026ndash;Meier curves (log-rank test). All statistical calculations were performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered a statistically significant.\u003c/p\u003e\u003cp\u003eFurther details are provided in Supplemental Methods.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests, as this work was funded entirely by the National Institutes of Health (Z01 DK43403).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eThis research was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH). The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.N., N.H., P.Y., and R.S. conceived and designed research; Y.N., D.G., N.H., and X.H. performed experiments; Y.N., D.G., and N.H. analyzed data; Y.N., P.Y., and R.S. interpreted results of experiments; Y.N., P.Y., and R.S. prepared figures; Y.N., D.G., N.H., P.Y., and R.S. drafted the manuscript; Y.N., D.G., N.H., P.Y., and R.S. edited and revised the manuscript; all authors approved the final version of manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH). The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eUnderlying data are published on Mendeley Data (Reserved DOI 10.17632/y2tbj7h96t.1).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePeerapornratana, S. et al. Acute kidney injury from sepsis: current concepts, epidemiology, pathophysiology, prevention and treatment. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e, 1083\u0026ndash;1099 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBagshaw, S. M. et al. 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Innate Immun.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 181\u0026ndash;193 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7474386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7474386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere are no specific treatments for Sepsis-associated acute kidney injury (SAKI). We previously reported that \u003cem\u003eIl-17a\u003c/em\u003e-knockout mice had dramatically improved survival after cecal ligation and puncture (CLP). Neutrophil extracellular traps (NETs) induce IL-17A, which causes harm in some diseases, but this pathway is poorly understood in sepsis. We found that knockout of \u003cem\u003ePad4\u003c/em\u003e (Peptidyl Arginine Deiminase 4), an enzyme essential for NET formation, improved survival and AKI, and suppressed neutrophil infiltration into remote organs, involving a peritoneal IL-17A/distant organ CXCL-1/CXCL-2 pathway after CLP. NETs were detected in the peritoneal cavity, and not in plasma or distant organs. Adoptive transfer of peritoneal NETs restored the IL-17A/CXCL-1/CXCL-2 pathway in \u003cem\u003ePad4\u003c/em\u003eKO mice, leading to neutrophil infiltration and damge to remote organs. These results revealed a pathway from peritoneal NET formation to remote organ injury/inflammation via production of IL-17A at the infectious site and distant organ CXCL-1/CXCL-2. While NETs promoted intraperitoneal IL-17A production, we also showed that conversely, peritoneal IL-17A or CXCL-1/CXCL-2 promoted intraperitoneal NET formation after CLP. This peritoneal vicious cycle that includes NET formation, IL-17A, CXCL-1/CXCL-2 that may amplify organ injury in sepsis. Breaking this vicious cycle by inhibiting NET formation and/or IL-17A might be a promising therapeutic target for sepsis treatment.\u003c/p\u003e","manuscriptTitle":"Peritoneal Neutrophil Extracellular Traps contribute to septic AKI via peritoneal IL-17A and distant organ CXCL-1/ CXCL-2 pathway in abdominal sepsis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 14:03:34","doi":"10.21203/rs.3.rs-7474386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T14:47:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T07:59:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-02T21:38:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205629070213443051381881599114988255107","date":"2025-10-02T13:27:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170375506818881451986089424029358950871","date":"2025-10-02T13:26:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236696082598657250471188886122862647008","date":"2025-09-17T13:20:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8686374043179578740271299611364801627","date":"2025-09-15T15:13:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T13:06:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-15T13:02:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-11T10:46:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T19:37:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-09T19:33:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"76ec23cb-88c2-49aa-ace6-8f8cc13bb2e1","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54923011,"name":"Health sciences/Diseases"},{"id":54923012,"name":"Biological sciences/Immunology"},{"id":54923013,"name":"Health sciences/Medical research"},{"id":54923014,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-02-02T16:10:31+00:00","versionOfRecord":{"articleIdentity":"rs-7474386","link":"https://doi.org/10.1038/s41598-025-34770-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-27 15:57:59","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-09-23 14:03:34","video":"","vorDoi":"10.1038/s41598-025-34770-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34770-1","workflowStages":[]},"version":"v1","identity":"rs-7474386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7474386","identity":"rs-7474386","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}