Endothelial cell responses in sepsis are attenuated by targeting truncated procalcitonin

Domain: biology
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Sepsis is associated with hypotension, vascular leakage, vasoplegia and microvascular dysfunction. Therefore, the endothelium is a target for sepsis therapies. Since truncated procalcitonin exerts vascular activity, we here evaluated the efficacy of targeting procalcitonin for vascular integrity and sepsis outcomes. Sepsis up-regulated >2000 genes involved in pro-inflammatory responses while similar numbers of genes involving cell growth and maintenance were down-regulated. Transcriptomic changes in endothelial cells diminished by >50% by anti-procalcitonin antibodies and this was functionally associated with preserved vascular barrier integrity in lungs and intestines, reduced sepsis-induced vasoplegia, preserved endothelial nitric oxide bioavailability, improved organ integrity and reduced sepsis severity in mice. Mechanistically, procalcitonin neutralization was associated with reduced signaling of the interleukin-17 pathway. We here show sepsis induces substantial changes to the endothelial transcriptome and vascular integrity and neutralizing procalcitonin is a suitable means to preserve endothelial homeostasis at a transcriptomic and functional level that could translate into organ protection during sepsis.

Introduction
Sepsis is common and associated with high mortality1. The presence of (mostly) bacterial specimen in the bloodstream leads to a dysregulated immune response that affects vascular function and organ integrity. Despite numerous efforts in identifying immune signatures and patient phenotypes, the primary care bundle for patients with sepsis is still limited to timely delivery of antibiotics and treatment of hypotension2. Hypotension is a cardinal symptom of patients with sepsis that mirrors a severe, ubiquitous vascular barrier defect resulting in fluid extravasation into tissues and intravascular hypovolemia. Hypovolemia and hypotension critically affect organ perfusion and endanger for the often detected sepsis-induced multiple organ dysfunction syndrome (MODS). At the level of the microcirculation, interstitial edema compromises capillary perfusion and tissue oxygenation that further aggravates cellular injury and organ dysfunction.

This importance of the vasculature in maintaining macro-hemodynamics, microcirculatory function and thus organ integrity makes the endothelium an attractive target for limiting sepsis-induced complications and death3,4. Loss of endothelial integrity and function are crucial players during the development of sepsis-associated organ dysfunction5–7. However, little is known about the characteristics of the endothelium in sepsis and the possible impact of therapeutic strategies on endothelial profiles during severe systemic inflammation.

Procalcitonin is a 116 amino acid peptide hormone with mere ubiquitous expression in inflamed tissue8. During systemic inflammation, procalcitonin blood concentrations rapidly increase and decrease upon sufficient focus control (i.e., reduction of bacteremia due to antibiotic delivery). This dynamic makes procalcitonin a sufficient biomarker for the clinical workup of sepsis patients9. Until a few years ago, the biological role of procalcitonin itself was unclear. Then, we discovered that procalcitonin exerts direct effects on the endothelium following activation by cleavage of two N-terminal amino acids10. These procalcitonin isoforms have also been found in humans and under inflammatory conditions found in equal parts11,12. Following binding to the calcitonin receptor-like receptor (CRLR) in complex with the receptor activity modifying protein 1 (RAMP1), procalcitonin resulted in phosphorylation of endothelial adherens junction proteins thus inducing loss of barrier function that perpetuated the above mentioned mechanisms of sepsis-induced organ dysfunction. In turn, inhibition of procalcitonin activation conserved vascular integrity and reduced interstitial edema in septic mice and resulted in augmented microcirculation, reduced hypovolemia and vasopressor requirements in patients with systemic inflammation. These results suggested that targeting procalcitonin could be a means to directly preserve vascular function during sepsis.

So far, most therapeutic strategies failed to improve sepsis outcome13,14, including antagonizing IL-115, TNF alpha16 or application of hydrocortisone17. Therapeutic strategies for the treatment of septic patients may be successful when a punctual therapeutic intervention induces secondary effects that inhibit the vicious circle of tissue ischemia, injury and further immune activation during systemic inflammation. We here focused on the endothelium as a therapeutic target in sepsis and performed an in-depth characterization of the endothelial transcriptome in septic mice. We then evaluated the overall effects of antagonizing hyperprocalcitonemia in septic mice on endothelial transcriptomic profiles by synthesis of antibodies specifically targeting activated murine procalcitonin. Finally, we evaluated antibody-induced effects on endothelial transcriptomic profiles for relevance for the occurrence of vasoplegia and capillary leakage, the hallmarks of vascular dysfunction in patients on intensive care units.

Results
Transcriptome of murine endothelial cells in sepsis
To evaluate the changes induced by sepsis at the transcriptomic level in murine endothelial cells, we subjected wild type mice to the translational model of polymicrobial sepsis due to cecal puncture and peritonitis. As the lung is the most susceptible organ to sepsis-induced tissue injury18–20, we particularly focused on the evaluation of pulmonary endothelial cells (Fig. 1a). RNA sequencing of pulmonary endothelial cells 18 h after cecal puncture revealed that sepsis induced up- and down regulation of various genes in the endothelium (Fig. 1b). Principal component analysis of n=5 mice/group revealed close clustering of septic mice in contrast to both sham-operated and untreated mice (Fig. 1c). The most regulated genes are highlighted in Fig. 1d,e. Further characterization of up- and down-regulated pathways showed that endothelial cells upregulate various pathways associated with inflammation, in particular cytokine production and interaction with immune cells (Fig. 1f). In contrast, pathways associated with cell growth, angiogenesis and assembly of cellular junctions were down-regulated (Fig. 1g). In addition we subjected mice to procalcitonin injection and analyzed VEGF gene expression in freshly isolated murine pulmonary endothelial cells 18 h after injection. According to gene expression analysis, we found procalcitonin induced a reduction in VEGFa and VEGFc expression (Fig. 1h).

Human gene expression analysis
In response to septic patients plasma, human pulmonary microvascular endothelial cells show higher expression of Calca, the gene encoding procalcitonin (PCT). This effect was abolished by procalcitonin antibodies, indicating that procalcitonin itself can perpetuate Calca expression and that endothelial cells are a source of procalcitonin. In contrast, Calcrl and Ramp1 remained unchanged in endothelial cells (Fig. 1i-k). In line with recent literature21, we found VEGF mRNA level was downregulated in human pulmonary endothelial cells when exposed to septic patient plasma with hyperprocalcitonemia (Fig. 1l). We then related our findings to results previously obtained in humans. After 8 h of LPS exposure, 416 unique genes were regulated, 243 up- and 173 down. 331 of these DEGs were also annotated in our datasets. Of these LPS-DEGs, 169 overlapped with DEGs from the contrast of CLP versus controls and 118 overlapped to the DEGs from the contrast CLP plus AB to controls. These results showed that our findings were very similar to the human LPS-induction study. More importantly, AB-treatment suppressed these responses. We then looked in detail at the top 50 up-regulated DEGs from the 8h LPS study. 36 of the LPS-DEGs were also annotated in our dataset. As shown in Fig. 1m, almost all LPS-DEGs genes were also up-regulated in our study, and all of these DEGs (except one) showed reduced expression in CLP plus AB treated samples. Again, these findings showed that our results were very similar to findings in LPS-treated human cells, and demonstrated an effect of AB treatment.

Neutralizing PCT with an antibody targeting truncated PCT
A hallmark of sepsis is capillary leakage due to endothelial barrier breakdown which results from downregulation and phosphorylation of adherens junctions such as vascular endothelial cadherin (VE-cadherin) at tyrosine 685. With the plasma from mice previously subjected to transcriptomic analysis, we first verified the opening-effect of adherens junctions due to VE-cadherin phosphorylation induction in murine endothelial cells (from 1.0 to 1.9±0.3-fold, P<0.05, n=5/group, Fig. 2a,b). As we had previously identified procalcitonin to be a potent mediator of VE-cadherin disassembly, we next confirmed hyperprocalcitonemia in septic mice (increase from 1.2±0.2 ng/mL in sham-operated to 9.5±0.4 ng/mL in septic animals 18 h after sepsis induction, P<0.001, n=5–18 mice/group, Fig. 2c). Procalcitonin exists in two forms, a full-length, 116 amino acid variant isoform and a truncated variant isoform, where dipeptidyl-peptidase 4 (DPP4) cleaves two amino acid at procalcitonins N-terminus. Since only the truncated variant isoform exerts activity on the vasculature10, we designed a polyclonal antibody targeting the truncated N-terminus of procalcitonin (Fig. 2d). Using recombinant synthesized procalcitonin variant isoforms, we then confirmed antibodies targeting procalcitonins N-terminus of the truncated variant isoform sufficiently inhibit procalcitonin-induced VE-cadherin phosphorylation at tyrosine 685 (Fig. 2e). In addition the antibody antagonized loss of endothelial barrier function following application of septic mouse plasma on endothelial monolayers in vitro (from 1.7±0.2-fold to 1.0±0.03-fold, P<0.01, n=4–8 mice/group, Fig. 2f).

Neutralizing PCT ameliorates endothelial activation
Having confirmed functional antagonization of procalcitonin effects by the neutralizing antibody in vitro, we evaluated the potency of neutralizing procalcitonin on modulating sepsis-induced changes to the endothelial transcriptome. Strikingly, antibody application was associated with a marked reduction of differentially expressed genes (DEGs) in septic animals (Fig. 3a). Antagonizing procalcitonin led to a reduction of the number of differentially regulated genes in septic mice endothelium by approximately 50% in accordance with the findings shown in Fig. 3A (Fig. 3b–d). For example, we found sepsis to induce the expression of the interleukin-17 (IL-17) a and f genes (Supplementary Fig. 3). KEGG-pathway analysis revealed antibody-mediated downregulation of IL-17 and the IL-17 signaling pathway genes (Fig. 3e). This finding was confirmed at the protein expression level. While sepsis induced a 100-fold increase in IL-17 (from 0.9±0.4 to 100.2±46.3 pg/mL, P<0.001, n=6 mice/group), neutralization of truncated procalcitonin (to 2.7±1.0 pg/mL, P<0.001), application of olcegepant, a procalcitonin receptor agonist during sepsis (to 3.4±2.4 pg/mL, P<0.001) as well as application of an inhibitor of dipeptidyl-peptidase 4 (DPP4) that inhibits the conversion of full-length to truncated procalcitonin (to 0.7±6.3 pg/mL, P<0.001) all nearly completely abolished the presence of IL-17 in septic mice plasma (Fig. 3f). Downregulation of various cytokines and inflammatory mediators in line with the genomic data was also observed (Fig. 3g).

Neutralizing PCT protects of endo- and epithelial barriers
We next aimed to dissect whether the reduced genomic activation and concentration of humoral mediators translates into the preservation of the vascular barrier integrity during sepsis. While septic mice exhibited fulminant pulmonary edema, extravasation of Evans blue-bound albumin (from 0.9±0.2 pulmo/plasma ratio to 2.8±0.4, P<0.05, n=5-14 mice/group) as well as fluid accumulation in the pulmonary interstitium (from 6.4±0.4 µm to 12.3±0.3 µm of alveolar thickening, indicative of interstitial pulmonary edema, P<0.001, n=6 mice/group) was diminished with antibody application (to 0.9±0.2 pulmo/plasma ratio, P<0.01 and 6.3±0.7 µm, P<0.001, respectively, Fig. 4a–c). Importantly, barrier protection was also evident in other organs such as the intestines, where application of the procalcitonin targeting antibody also reduced wet/dry ratios of murine guts (from 1.4±0.0 to 1.3±0.0, P<0.01, n=5–9 mice/group, Fig. 4d,e). Further, reduced edema formation in intestines in antibody-treated animals was associated with reduced bacterial translocation. While peritoneal lavage showed no difference between CLP groups (Supplementary Fig. 2), less CFUs/mL tissue homogenate were found in the liver and the blood (22173±16188 vs. 76289±20713 in the liver, P<0.05, n=12 mice/group and 3775±1367 vs. 46340±12304 per mL blood, P<0.001, n=12 mice/group Fig. 4f,g). Since vasoplegia is a common clinical phenomenon in patients with systemic inflammation, we evaluated the response of murine mesenteric resistance vessels from septic or control-treated, sham-operated mice to the vasoconstrictor phenylephrine. Vessels from septic mice exhibited a reduced response to the vasopressor (0.6±0.04-fold vasoconstriction compared to that seen in control-treated animals defined as 1.0, P<0.05, n=7-11 mice/group). In contrast, septic mice that had received truncated procalcitonin-neutralizing antibodies exhibited reduced vasoplegia (1.5±0.2-fold, P<0.001, Fig. 4h). Further, antibody-treated mice exhibited preserved bioavailability of nitric oxide (NO) of the endothelium by showing preserved response to vasodilation-inducing agents (response to carbamoylcholine, reduction to 73.7±4.6% vasoconstriction vs. 98.3±0.8% in control-treated septic animals, P<0.001, n=5–7, Fig. 4i,j).

Procalcitonin neutralization protects organ integrity
As a possible summation of the above described effects, we next evaluated measures of organ integrity in mice subjected to treatment with procalcitonin targeting antibodies with focus on liver and kidney. While sepsis induced severe damage to liver tissue and interstitial fluid accumulation, these adverse effects were diminished after application of procalcitonin-targeting antibodies (1.4±0.3 vs. 3.1±0.2 hepatic tissue damage score, P<0.01, n=9-11 and 1.0±0.0 vs. 1.1±0.0 wet/dry ratio, P<0.001, n=11, Fig. 5a–c). Further, antibody administration diminished acute tubular injury in murine kidneys (1.8±0.3 with control-IgG vs. 3.2±0.2 with antibody, P<0.01, n=7-13, Fig. 5d,e) and protected the integrity of the glomeruli (P<0.001, n=4-9, Fig. 5f-i). These results indicated that neutralizing procalcitonin with antibodies designed for targeting the truncated, active procalcitonin variant isoform, is a potent measure for organ protection during sepsis.

PCT neutralization improves murine health conditions
To evaluate whether reduced endothelial activation, capillary leakage and improved organ integrity result improved clinical wellbeing and health, we scored the general health condition, murine behavior and sepsis-related disease criteria hourly during the course of sepsis. Scores varied between antibody-treated mice and control-IgG-treated mice and indicated improved wellbeing, health and reduced sepsis-related disease severity in mice subjected to procalcitonin neutralization (2.55 vs. 3.55 at 14 h, P=0.024 until 4.75 vs. 6.6 at 18 h, P<0.001 for health score, 1.85 vs. 2.85 at 6 h, P=0.029 until 5.65 vs. 7.95 at 18 h, P<0.001 at 18 h for behavior score and 5.45 vs. 7 at 15 h, P=0.04 until 7.7 vs. 12.6 at 18 h, P<0.001 for sepsis score and 18.3 vs. 26.1 for the cumulative scores at 18 h, P<0.001, Fig. 6a–d). Based on an endpoint defined as 20 in either score category, we calculated predicted survival that showed a significant advantage for mice treated with procalcitonin-targeting antibodies. Of note, these effects of the procalcitonin antibody were not observed with inhibitors targeting the procalcitonin receptor complex (BIBN4096, Fig. 6e,f).

Methods
Murine sepsis model
Animal experiments were approved by the governmental ethical board at the Animal Care and Use Committee of North Rhine Westphalia, Germany, 81-02.04.2021.A396. Female and male C57Bl/6J mice (#000664, The Jackson Laboratory) were housed in the central animal facility under pathogen-free conditions with standard diet food and water ad libitum and in a 12 h light/dark cycle. The animal facility was maintained at 23 ± 2°C. 10-12 week-old mice were used for polymicrobial sepsis induction by cecal ligation and puncture performed by one experienced researcher to avoid technical variations (3–20 animal per group, depending on experiment). Briefly, laparotomy was performed following the ligation of the distal cecum (14 mm) and two punctures of the cecum using a 20 G cannula. After replacement of the cecum into the abdomen, the incision was closed. Mice in the sham operated control group underwent the same surgical procedure without ligation nor puncture of the cecum10. Following CLP surgery, the mice displayed lethargy and reduced mobility, showed no interest in their surroundings, and did not engage in social behaviors such as sniffing or grooming. These observations are consistent with the typical clinical signs of sepsis. During the course of sepsis, mice were scored for general condition (fur and outer aspect, mouse grimace scale, food and water intake), behavior (spontaneous movement, reactions) and criteria related to sepsis (outer aspect, indications for dehydration, diarrhea, shivering, vigilance) after surgery, 6 h, 12 h and every consecutive hour until 18 h. Each score ranged between 0 and 20 points. A score of 20 in one category was considered the humane endpoint used for calculation of predicted survival.

Patient Recruitment
Between February and November 2022, we recruited 19 adult patients from the intensive care units of the University Hospital Münster who met the Sepsis-3 definition criteria and exhibited hyperprocalcitonemia (serum concentration >0.5 ng/mL). The recruitment was based on defined criteria and was performed without any bias (study protocol: https://clinicaltrials.gov/study/NCT05703802). Written informed consent was obtained from patients or their legal representatives. The study received approval from the Ethics Committee of the University Hospital Münster (ID: 2019-494-f-S) and was registered on ClinicalTrials.gov (registration number: NCT05703802, principal investigator: S. Kintrup, MD). Patients were excluded if they had immunological disorders, were taking immunosuppressive agents other than hydrocortisone, had viral or fungal sepsis, or had a terminal pre-existing disease. The study population consisted of 19 participants with a mean age of 69.5±2.9 years. Of these, 12 were male and 7 were female. The mean body weight was 84.4±5.9 kg, and the mean body mass index (BMI) was 28.2±1.7 kg/m². The average body surface area (BSA) was 2.0±0.1 m². Upon recruitment, 15 mL of serum were collected from each patient using two serum gel monovettes. The samples were centrifuged, and the serum was stored at -80°C until analysis. Data on hemodynamics, laboratory parameters, and drug dosing on the day of blood sampling were retrieved from the patient data management system (Quantitative Sentinel, GE). SOFA score variables were assessed immediately after blood collection. Total PCT levels were measured in all patients using an electrochemiluminescence immunoassay on a cobas e801 analyzer (Roche Diagnostics GmbH) in the central laboratory of the University Hospital Münster.

Isolation of endothelial cells and transcriptome analysis
For isolation of endothelial cells33, murine lungs were excised 18 h after sepsis induction. Dissociation of lung tissue into single-cell suspensions for subsequent cell separations was performed using the Miltenyi lung dissociation kit and standard protocol (130-095-927, Miltenyi Biotec). Lungs were dissected and collected in a C-tube containing Enzyme A and D and Buffer S. The preinstalled lung dissociation protocol 37C_m_LDK_1 on the Miltenyi Octo Dissociator with heaters was used immediately for obtaining single cell culture. Endothelial cells were isolated from suspensions using MACS Technology (Miltenyi Biotec). Cells were resuspended in PEB (Phosphate Buffered Saline, 2 mM EDTA, 0.5% bovine serum albumine) buffer following depletion of CD45+ cells by using CD45-coupled microbeads. After 15 min of incubation, cells were placed on a LS column in a magnetic separator and rinsed with PEB buffer three times. The CD45- fraction was collected and CD31 antibody-coupled beads were used for magnetic sorting of endothelial cells to obtain a pure cell culture. For RNA sequencing, RNA was isolated immediately. For permeability analysis, cells were seeded on transwell inserts (0.4 µm pore size, Corning) and incubated for 96 h until confluence. Purity and viability of isolated pulmonary endothelial cells were verified by flow cytometry analysis (Supplementary Fig. 1, BD Accuri C6 Plus). Following RNA isolation using RNeasy Mini Kit (Quiagen), RNA was subjected to next generation sequencing. Sequencing was performed at the Core Facility Genomics of the Medical Faculty Münster. Quality and integrity of total RNA was controlled on Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). Poly A+ RNA were purified from 100 ng total RNA using Poly(A) mRNA Magnetic Isolation module Kit (NEB E7490L, New England Biolabs). The RNA Sequencing library was prepared with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs). The libraries were sequenced on Illumina NextSeq 2000 using NextSeq 2000 P2 Reagent Kit (238 cycles, paired end reads 2×111 bp) with an average of 26.7 M reads per RNA sample. Reads were quality checked with package FastQC (version 0.11.4), and then processed by two different pipelines resulting in Figs. 1 and 3. For data in Fig. 3 reads were trimmed using Trimgalore (version 0.4.4) with default settings. Reads were mapped to mouse genome annotation mm11 (ENSMBL Mus_musculus.GRCm39, release 104) using STAR (version 2.5.2b)34 with default settings. Further analyses and visualizations of data was performed using the R software package (version 4.2.1)35. Mapped reads were counted using RsubRead (version 1.32.4)36. Raw counts were normalized and log2 transformed using function rlogTransformation from the DESeq2 package (version 1.16.1)37 and from the limma package (version 3.58.1)38 and an increment was added to the normalized values to make all values positive. For identification of differentially expressed genes (DEGs), DEseq2 was used with the model design=~group and then contrasting the groups. Differentially expressed genes (DEGs) were identified using a multiple-testing adjusted p value of <0.05 and exhibiting more than a 2-fold (log2=1) difference in expression levels. For Fig. 1: Reads were aligned, and quantified using integrated counting in the STAR package. Differential gene expression analysis was then performed using the limma package (version 3.58.1)38. As the data for Fig. 1 originated from two batches of sequencing a model design=~group+batch was applied. VENN diagrams were generated with the function vennPlot39. Functional analyses of DEGs were performed using the R software package clusterProfiler (version 4.10.1)40. Heatmaps were generated with the function heatmap2 of package gplots (version 3.1.3; https://github.com/talgalili/gplots) and package pheatmap (version 1.0.12.)41. KEGG pathway views were generated using package pathview, version 1.36.142. We then related our findings to results previously obtained in a human system. Since there was no data set available of endothelial cells exposed to the septic condition in vivo, we correlated our genomic data to gene expression dataset obtained from endothelial cells exposed to LPS. We searched the EndoDB database (https://endotheliomics.shinyapps.io/endodb/) with the term ‘lung’. We obtained 29 hits, 13 were from human. Of these, 12 were from freshly isolated cells or primary cell cultures. No dataset was found for sepsis-treated lung endothelial cells. One micro-array dataset reported gene expression changes after treatment of primary endothelial cells with LPS (E-GEOD-5883) at 4 h, 8 h and 24 h post treatment and corresponding untreated controls, which we found appropriate for comparison. We downloaded the respective data from the GEO expression database (GSE588343) and compared it our findings. Downloaded raw data was quantile normalized and log2 transformed and differentially expressed probe sets (DEPs) were determined using the limma package44,45 with the model.matrix(~0+group), and then contrasting groups. DEPs were identified using a multiple-testing adjusted p value of <0.05 and exhibiting more than a 2-fold (|log2|>1) difference in expression levels. 643 probe sets were differentially expressed at 4 h (treatment versus respective control), 827 DEPs at 8 h, and 195 DEPs at 24 h. We chose the 8 h treatment for our comparisons since it showed the strongest response.

Culture of human and murine endothelial cells, permeability assay
Human pulmonary endothelial cells (PromoCell) were cultured in cell culture media and exposed to serum from human sepsis patients diluted 1:1 in serum-free media. Murine endothelial cells were isolated from murine lungs using magnetic-bead-conjugated CD31-antibodies and brought into culture. Following several additional purification steps, cells were exposed to plasma of septic mice (10% with complete cell culture media) or recombinant procalcitonin (Abcam) as indicated. Endothelial cells were incubated with 1 µg/mL of the antibody targeting truncated procalcitonin and respective IgG control for two h following stimulation with septic mouse plasma diluted 1:1 in serum-free cell culture media and application of 1 mg/mL 70 kDa fluorescein isothiocyanate (FITC-) labeled dextran (Sigma Aldrich). After two h, fluorescence was measured at 520 nm using a plate reader (Synergy Mx, Biotek) in samples taken from lower chambers.

Western blot
Following stimulation of endothelial cells and lysing with homburg lysis buffer, protein concentration was determined using PierceTM BCA Protein Assay Kit (ThermoFisher). Protein loading was adjusted to 20 µg and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed for protein separation. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 3% bovine serum albumin in tris buffered saline with tween 20 following incubation with primary antibodies targeting phosphorylated VE-cadherin at tyrosine 685 (abcam, ab119785, 1:1000), total VE-cadherin (Santa Cruz Biotechnology, sc9989, 1:200) and actin (Thermofisher, MA5-15739, 1:10000) at 4°C overnight. Horseradish peroxidase (HRP)-linked secondary antibodies, anti-rabbit IgG or anti-mouse IgG (Cell Signaling, 7074S, 7076S, 1:1000, 1:5000, respectively), were incubated at room temperature for 1 h. ECLTM Prime Western Blotting System (GE Healthcare RPN2232) was used for protein detection.

Quantitative real-time PCR (qPCR)
Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, 74134). Complementary DNA (cDNA) was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4374966). Gene expression was quantified by real-time PCR using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, 4444557) and TaqMan™ Gene Expression Assays (ThermoFisher Scientific). For mouse samples, the following assays were used: Vegfa (Thermofisher, Mm00437306_m1), Vegfc (Thermofisher, Mm00437310_m1), IL17a (Thermofisher, Mm00439618_m1) IL17f (Thermofisher, Mm00521423_m1) and Gapdh (Thermofisher, Mm99999915_g1). For human samples following human TaqMan™ Gene Expression Assays were used: Calca (Thermofisher, Hs01100741_m1), Vegfa (Thermofisher, Hs00900055_m1), Calcrl (Thermofisher, Hs00907738_m1), Ramp1 (Thermofisher, Hs00195288_m1), and Gapdh (Thermofisher, Hs02786624_g1).

Recombinant procalcitonin variant isoforms, procalcitonin plasma concentrations and in vivo interference with the procalcitonins signaling pathway
Procalcitonin variant isoforms were synthesized recombinantly in E. coli as previously described10: Procalcitonin (PCT) was PCR-amplified from Calca cDNA in a pPCT-ScriptAmpSK(+) vector (Dharmacon MHS6278-202856628) using Q5 polymerase (New England Biolabs). FLAG-tagged variants were generated with primers introducing an NcoI site at the 5′ end and a HindIII site plus His-tag at the 3′ end. PCR products were cloned into pET28a(+) using NcoI-HF and HindIII-HF, transformed into BL21 (C2987H) E. coli, and sequence-verified. For expression, cultures were grown to OD600≈0.7 and induced with 500 µM IPTG for 4 h. Cells were lysed after centrifugation, and soluble fractions were purified on HIS-Select HF Nickel Affinity Gel (Sigma). Proteins were eluted with 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, and the FLAG tag was removed by enterokinase (4 U, 12 h). Endogenous procalcitonin was detected by murine ELISA (Cloud-Clone). For inhibition of the procalcitonin pathway in vivo, 100 µg/kg olcegepant or 5 mg/kg sitagliptin were applied intravenously for CRLR/RAMP1 blockade or DPP4 inhibition10.

Generation of a polyclonal antibody targeting murine truncated procalcitonin
Polyclonal antisera against mouse procalcitonin were raised by immunizing rabbits with the peptide LRSILESSPGMATLSC (equal to the truncated procalcitonin N-terminus amino acid sequence) coupled to KLH. For affinity purification of procalcitonin-specific antibodies from rabbit sera the peptide used for immunization was coupled to SulfoLink resin (ThermoFisher Scientific) (Supplementary Fig. 1). Procalcitonin targeting antibody was injected intravenously to 100-fold excess of the mean procalcitonin concentration detected in mice (10 ng/mL), i.e., 1 µg/mL of blood volume, or IgG control six h after sepsis induction.

Cytokine multiplex analysis
Cytokine multiplex analysis in murine plasma withdrawn 18 h after sepsis induction was performed according to the manufacturer’s instructions (Cytokine & Chemokine 36-Plex Mouse ProcartaPlexTM Panel 1A including IL-17a, ThermoFisher Scientific) to analyze humoral characteristics of the ongoing inflammation. The microplate was analyzed in a Luminex analysis device.

Miles assay
10 mg/kg body weight Evans Blue (Sigma) were injected 17.5 h after sepsis induction by cecal ligation and puncture and sham surgery46. 30 min later, mice were sacrificed and murine lungs and blood were harvested. Lungs were homogenized and dissolved in formamide following incubation at 60°C for 18 h to extract extravasated Evans blue dye from tissue. Lungs were centrifuged and supernatant was used for further analysis. Absorbance of lung-supernatant and plasma was measured at both 620 nm and 740 nm using a spectrometer. The degree of albumin extravasation was calculated as quotient of Evans Blue content tissue to plasma. Wet/dry ratio indicating edema formation was analyzed in murine liver and intestines after 7 days of incubation at 37°C and weight measurement on a precision balance.

Assessment of bacterial colony-forming units
Agar plates (Carl Roth) were produced under sterile conditions. Peritoneal lavage was performed by injection of 1 mL Dulbecco’s Phosphate Buffered Saline (DPBS). Murine lungs, liver, kidney and spleen were dissolved in 1 mL DPBS and homogenized under sterile conditions. 30 µl whole blood was diluted with equal amounts of thioglycolate. 50 µl of the dilution were plated on agar plates. 50 µl of the peritoneal lavage and 10 µl of the tissue homogenates were plated. Colony forming unit counting was performed after 24 h of incubation at 37°C.

Histology
Murine lungs, liver and kidney were removed, kept in 4% formaldehyde at 4°C, embedded with parraffin and 6 µm thick sections were stained with hematoxylin-eosin. Five representative pictures per mouse were taken using Lionheart FX microscope (BioTek, Software Gen5 Version 3.05) by a blinded observer. Alveolar septal thickening was analyzed by measurement of 10 representative septa using ImageJ software (Version 1.51r, NIH). Histopathological changes in murine liver was scored based on the criteria congestion, edema, infiltration of immune cells and necrosis47. Tubular injury of murine kidney was quantified by presence or absence of epithelial necrosis, loss of brush border, cast formation and tubular dilatation. Glomerular tuft, Bowmans space and capsule size were measured using the ImageJ freehand selection tool.

Pressure myography
For the assessment of vasomotor function, third-order mesenteric resistance arteries were dissected 18 h after sepsis induction and pulled on glass cannulas in a vessel chamber (Living Systems Instrumentation) in calcium-free buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, 1 mM EGTA)48. Experiments were performed in calcium-containing buffer (Ca-free buffer without EGTA including 2 mM CaCl2). After development of a stable myogenic tone at 80 mmHg, vessels were exposed to 10 nM (R)-(−)-phenylephrine hydrochloride (Phe, Tocris) for maximum constriction. Relaxation response to increasing concentrations of carbamoylcholine (10−9 to 10−4 M, Tocris) was assessed using digital video edge detection.

Statistical analysis
Data are presented as mean values with standard error of mean. To test for normal distribution, the Shapiro Wilk-Test was used. Statistical significance was determined by using One- or Two-way analysis of variance (ANOVA) followed by correction for multiple testing applying the Bonferroni method. Effect was estimated by difference in means and 95% confidence interval and P values ≤0.05 were considered to be statistically significant. Survival curves were analysed using log rank test. All analyzes were performed using Graph Pad Prism Software 7 and 10 (GraphPad, USA) or SPSS Statistics 14.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "NatComm Review\nThe present article investigates the effect of polymicrobial sepsis on endothelial cell responses in relation to procalcitonin\nsignaling. In this regard, the authors had shown in a previous study that vascular leakage, vasoplegia and microvascular\ndysfunction are regulated by procalcitonin in sepsis. To induce acute sepsis, in the current study the authors subjected WT\nmice to the Cecal Ligation and Puncture (CLP) procedure. Subsequently, the authors conducted next-generation RNA\nsequencing on endothelial cells derived from murine lungs following the induction of polymicrobial sepsis, with the objective\nof assessing transcriptomic changes. The presented RNA sequencing data indicates the expression of numerous genes that\nare differentially expressed, including expression of the Calca gene, encoding procalcitonin, and Il17a.\nThe study is well executed and written. However, the authors' principal objective is to examine the role of procalcitonin\nduring sepsis, a topic that has already been extensively investigated. A harmful role of procalcitonin in sepsis has been\ndemonstrated in multiple studies in a variety of different organisms, including mice, rats, and pigs (e.g., Baranowsky et al.,\n2021; Nylén et al., 1998; Wagner et al., 2002; Tavares et al., 2014). Although the described antibodies in the current study\nappear to be of novel design, the studies referenced above have already explored the therapeutic potential of antagonizing\nor neutralizing procalcitonin. Unfortunately, in the current study the key experiment, namely survival rates in mice treated\nwith the novel Procalcitonin antibodies, is not reported. Although the RNA seq data of vascular endothelial cells with/without\nanti-procalcitonin treatment are interesting, the functional impact of procalcitonin on vascular integrity during sepsis has\nbeen reported by the same group before (Brabenec 2022). Also, the proposed role of Il17A as a downstream mediator of\nprocalcitonin has already been described in the study by Baranowsky et al. 2021. Together, the study provides only limited\nconceptual advance and novelty, and, in my opinion, is thus better suited for a more specialized journal\nNylén ES, Whang KL, Steinwald PM et al. Procalcitonin increases mortality and procalcitonin recognizing antiserum\nimproves mortality in an experimental model of sepsis. Crit Care Med 1998; 26:1001–1006.\nWagner KE, Martinez JM, Vath SD et al. Early immunoneutralization of calcitonin\nprecursors attenuates the adverse physiologic response to sepsis in pigs. Crit Care\nMed 2002; 30:2313–2321.\nTavares E, Maldonado R, Miñano FJ. Immunoneutralization of endogenous aminoprocalcitonin attenuates sepsis-induced\nacute lung injury and mortality in rats. Am J Pathol 2014; 184:3069-83.\nBaranowsky A, Appelt J, Kleber C, Lange T, Ludewig P, Jahn D, Pandey P, Keller D, Rose T, Schetler D, Braumüller S,\nHuber-Lang M, Tsitsilonis S, Yorgan T, Frosch KH, Amling M, Schinke T, Keller J. Procalcitonin Exerts a Mediator Role in\nSeptic Shock Through the Calcitonin Gene-Related Peptide Receptor. Crit Care Med. 2021 Jan 1;49(1):e41-e52.\nBrabenec L, Müller M, Hellenthal KEM, Karsten OS, Pryvalov H, Otto M, Holthenrich A, Matos ALL, Weiss R, Kintrup S,\nHessler M, Dell'Aquila A, Thomas K, Naß J, Margraf A, Nottebaum AF, Rossaint J, Zarbock A, Vestweber D, Gerke V,\nWagner NM. Targeting Procalcitonin Protects Vascular Barrier Integrity. Am J Respir Crit Care Med. 2022 Aug\n15;206(4):488-500.", "reviewer_2": "Calcitonin is a polypeptide hormone, which is made up of 32 amino acids and is produced mainly by parafollicular cells (C\ncells) in the thyroid gland, secreted by cells of the glandular ultimobranchial bodies. The overall effect of calcitonin is to\nlower the concentration of calcium in the blood when it has risen to an above the normal level. Procalcitonin, a protein that\nconsists of 116 amino acids, is the peptide precursor of calcitonin. Multiple studies have shown that procalcitonin is involved\nin the pathogenesis of infections, and that it can be a useful diagnostic marker for infections such as bacterial pneumonia,\nbacterial sepsis and septic shock, meningitis, infectious endocarditis, pancreatitis, and urinary tract infections. Secretion of\nprocalcitonin is stimulated by IL-1β and tumor necrosis factor–α in patients with bacterial infections and by interferon-γ in\npatients with viral infections. The authors previously reported that procalcitonin induced endothelial barrier disruption\n(Wagner et al. Anesth Alalg 2017). Furthermore, their recent study has shown that targeting the procalcitonin receptor during\nsepsis-induced hyperprocalcitonemia can significantly reduce capillary leakage (Brabenec et al. Am J Respir Crit Care Med\n2022). In this study, the authors demonstrated that more than half of transcriptomic changes in endothelial cells were\nreduced by anti-procalcitonin antibodies and this was functionally associated with preserved vascular integrity in lungs and\nintestines, reduced sepsis-induced vasoplegia, preserved endothelial NO bioavailability, and organ integrity during sepsis.\nThey also found that neutralizing procalcitonin exerted protection of pulmonary endothelial and epithelial barriers in mice\nwith sepsis-induced sepsis. While this study includes interesting findings, I have a number of concerns for the authors to\nconsider.\nMajor comments are summarized below.\n1) The disruption of endothelial integrity and functioning plays a crucial role in the development of sepsis-associated organ\ndysfunction. In regards to this, there are a number of excellent review articles. In the Introduction, the authors should cite\nthese reviews.\n2) The authors describe that “so far, most therapeutic strategies failed to improve sepsis outcome (page 4, line 101). Without\na doubt, despite advances in overall medical care, sepsis continues to be a complex clinical entity with an unacceptably\nhigh mortality rate. The authors should provide several appropriate references for this.\n3) The authors describe that the lung is the most susceptible organ to sepsis-induced tissue injury (page12, line 276-277).\nThe authors should provide an appropriate reference pointing out that the respiratory system is the most affected organ of the\nbody and the lung dysfunction is the first step in the development of multiple organ failure in septic patients.\n4) The gene encoding procalcitonin (Calca) was among the top up-regulated genes, while the gene encoding the\nprocalcitonin receptor (Calcrl) was among the top down-regulated genes (page 12, line 283-285). It would be easy to\nunderstand if RT-PCR analysis showing gene expressions for Calca and Calcrl in pulmonary endothelial cells from sham-\noperated and CLP mice is visually provided.\n5) The authors found that pathways associated with cell growth, angiogenesis, and assembly of cellular junctions were\ndown-regulated (Fig. 1G) (page12, line 288-289). In this regard, VEGF mRNA level was significantly down-regulated in lung\ntissues from mice with CLP-induced sepsis (Tomita et al. Naunyn-Schmiedeberg’s Arch Pharmacol 2020). This report may be\nconsidered by the Authors.\n6) Kaplan-Meier survival curves examining whether neutralizing procalcitonin can improve mice subjected to CLP should be\nprovided.\n7) In conclusion, the authors state that antagonizing the procalcitonin signaling pathway would display a precision medicine\nstrategy effective in those identified with particular hyperprocalcitonemia. What percentage are septic patients with\nhyperprocalcitonemia occupy in the whole septic patients?", "reviewer_3": "The authors utilize multi-omic techniques and some well-designed murine animal model experiments to determine the\nimpact of neutralizing a procalcitonin transcript isoform in a murine model of sepsis. The experiments are very well\nperformed and the study is very elegant. However, as the authors state in the introduction, no therapeutic discoveries have\nbeen made that can treat the sepsis syndrome. This is primarily related to the fact that most studies have been performed in\nrodent models. Unfortunately, the findings in mice rarely translate in humans. Therefore, it is important for the authors to\nprovide some evidence that a similar pathway related to procalcitonin inhibition improved endothelial function in a human\nmodel such as primary cell culture, lung organoid, etc.\n1. As the authors stated – the primary benefit of neutralizing procalcitonin, was the impact on endothelial and epithelial\nbarrier integrity. The experiments are well performed and described. However, one finding is that the inhibition of the\nprocalcitonin isoform also improves intestinal barrier function. Their results demonstrate that there are decreased CFU’s in\nall organ tissues they looked at. It seems to me that a potential rational for the reduced CFU’s throughout the periphery could\nbe related to the improved integrity of intestinal barrier function. Simply put, the bugs have less ability to cross past the gut\ntherefore many of the inflammatory responses are tamped down. Clarification on this point is important to understand the\nimpact of the potential therapeutic - is it improved endothelial barrier function in lung tissues, or the reduced bacterial load\nbecause the bugs can’t cross?\n2. In the methods, the authors state that human endothelial cell cultures were performed for permeability assays. However, the\nmethods and results do not include any information about the human endothelial culture. This is unfortunate, as a human\nendothelial model that recapitulates the mouse findings would tremendously improve the impact of this study. Considering\nthat the study utilizes plasma of septic mice, plasma from septic patients on human endothelial cell cultures could similarly\nbe performed and would greatly enhance this study.\nSimilarly, it would be great if the authors could perform a similar RNAseq analysis, or at least target 5 -10 of the key genes\nidentified in the mouse study, using the primary airway endothelial cell model and plasma challenge.\n3. The recombinant procalcitonin variant study: This is an excellent component of the study and suggests that antagonizing\nthis isoform could have potential therapeutic benefit. That said, there are a couple of areas that need to be clarified. First, in\nthe methods the authors refer to the transcript isoforms as variants. I am not certain this is the correct terminology as that has\nme thinking about SNP variants. It would probably be easier for the reader to follow if you refer to it as a splice isoform or\ntranscript isoform consistently throughout the manuscript.\nMore importantly, though, how does this isoform relate to human biology? I assume this isoform is also found in humans. A\ndescription of the human isoform would be helpful – how evolutionarily conserved are these isoforms in mice to humans?\nEvidence that the isoform can be found in human endothelial cells is necessary to demonstrate that the pathway is\nconserved.\nDoes the polyclonal antibody that was developed also antagonize the human isoform and have similar results? It would be\nhelpful to know if the antibody developed has cross reactivity or if a new antibody needs to be developed.\nThe authors also discuss olcegepant and inhibition of DPP4 as abolishing the IL-17 mediated response. Anything that can\nconfirm these results in a human model would be helpful to understand the potential translational impact of inhibiting this\ntranscriptional isoform.\nMinor concerns – The paragraphs are overly long. Particularly in the discussion. Breaking these up would make the\nmanuscript easier to read.\nLine 417 – custom is spelled incorrectly.", "reviewer_4": "Brabenec et al. performed bulk RNA sequencing of lung endothelial cells isolated from a mouse model of sepsis and\nidentified transcriptomic changes that describe the impact of sepsis pathology on this critical population. This team has\npreviously reported that a truncated form of procalcitonin disrupts vascular integrity using in vitro and in vivo studies that\nwere supported with human clinical data from subjects that included those taking inhibitors of the procalcitonin activator\nDPP4. In new data reported here, the authors use a procalcitonin neutralizing antibody that was generated to test if inhibition\nblocks changes in the endothelial transcriptome as well as accompanying vascular defects and organ damage. This work\naddresses an important area of investigation. New host-based therapies are needed to improve sepsis outcomes. Beyond\nsupportive care and antimicrobials there are few options available for clinicians to treat sepsis and septic shock. Below are\nsome comments for clarifications to strengthen the paper prior to recommendation for publication.\nThe authors previously reported a role for procalcitonin in vascular pathology using a variety of experimental and\nobservational approaches. This included mouse models of sepsis and various inhibition strategies to prove that\nprocalcitonin is more than just a biomarker. In this study, the key new results are the transcriptomic evaluation of the lung\nendothelial population with respect to procalcitonin and expanded assessment of the protective role of procalcitonin\ninhibition on organ systems beyond the lung.\n1. Because the transcriptomic analysis is a proposed major contribution of this study, there remain some questions that\nwould be helpful to fully understand the data. The authors use enzymatic dissociation of the lung followed by CD45+\ndepletion and CD31 enrichment to generate “pure” endothelial populations. There are a number of detailed protocols\n(Conchinha NV 2021) that describe this procedure. Can the authors provide more detail in the methods on their protocol\nsince it is provided “in brief”. Similarly, is there data available to show that the isolated population is pure?\n2. Also, in the methods there is a 96h expansion of the isolated endothelial cells. Is the RNAseq performed after this 96h\nexpansion? It is not clear as written especially since the main text on line 278 indicates that the RNAseq was done 18h after\ncecal puncture. This is an important detail since in vitro expansion of endothelial cells can cause phenotypic drift as\nmeasured by transcriptomics (Cleuren ACA 2019).\n3. The generation of the antibody against the activated procalcitonin is used as a tool to neutralize the effects of procalcitonin\non the endothelium. Although information is supplied in the supplement on the purified antibody using surface coating and\ndata is shown that the truncated procalcitonin can be blocked experimentally, do the authors have data showing the\nspecificity of the antibody? Either following overexpression and by Western blot showing the full blot or dose dependent\ninhibition in vitro of tyrosine phosphorylation on cadherin?\n4. On line 319 the authors claim that the procalcitonin antibody “reduced the expression of almost all DEGs during sepsis”.\nOn line 419 the authors indicate that the antibody cut the number of genes in half. Figure 3D shows significant heterogeneity\nacross the individual animals tested. Can the authors clarify the interpretation of how this is described and discussed? This\nis a key point since this can be interpreted that activated procalcitonin is the primary driver of lung endothelial pathology at\nthe genomic level. This data could be strengthened by assessing impacts on endothelial transcriptomes following a\ncomplementary inhibition such as those shown in 3F for IL-17a protein.\n5. The authors and other groups have previously shown a causal link or correlation with vascular pathology and\nprocalcitonin in multiple systems and models including sepsis. Therefore the gene expression analysis following\nprocalcitonin seems to be the major new contribution here and could be a valuable dataset to understand therapeutic\nintervention in sepsis and translational potential. This data modality could also be a bridge to linking the CLP model of\nsepsis to any human pathology. Therefore, a major deficit of the study and a missed opportunity is the lack of any\ntranscriptomic comparison of the data generated here to publicly available human datasets. It would be valuable to include\nan analysis of the genes regulated by procalcitonin inhibition to those human paradigms (endoDB). In addition, although the\nauthors acknowledge in the discussion that single cell RNAseq of lung has shown that it is one of the least heterogenous\norgans with respect to endothelial cell diversity (Kalucka 2020), this study would be much more impactful if a single cell\nanalysis was performed to understand the action of procalcitonin on the endothelium. If this is out of scope for this study, it\nwould be important to discuss how some of the specific genes regulated in this study compare or align to those specific to\nendothelial subtypes in Kalucka 2020.\nMinor points:\nOn line 276 is there a reference that can be added to support the point that the “lung is the most susceptible organ” to sepsis\ninduced injury?\nIn Figure 2E in column 4 of the Western Blot, is it correct that it is “trunc-PCT” alone, or is this with IgG as well?"}, "author_response": "Point-by-Point-Response\nREVIEWER COMMENTS\nDear Reviewers,\nwe very much appreciate the opportunity to substantially revise our manuscript and,\nimportantly, extent its translational relevance and mechanistic insights. In response to\nto your comments, we used septic patients plasma to dissect the effects on gene\nexpression profiles of human endothelial cells and further specify the importance of the\nprocalcitonin antibody mechanistically at the molecular level. We also correlated the\ngenomic data obtained in the vasculature of septic mice with publicly available human\ndatasets exposed to sepsis-like conditions to verify the translational relevance of our\nfindings to the human system. Manuscript and figures were substantially revised and\nthe majority of your comments addressed by additional experiments added to the\nrevised version of the manuscript.\nBased on the extensive experiments conducted for the manuscript, we gathered\nadditional expertise and added two additional authors on the revised version of the\nmanuscript. All authors have read and approved the final version of the manuscript.\nWe regard our findings of relevance to a broad readership of physicians, basic\nscientists and clinician scientists. We would thus appreciate a re-evaluation of our work\nfor publication in Nature Communication.\nReviewer #1 (Remarks to the Author):\nC1: The present article investigates the effect of polymicrobial sepsis on endothelial\ncell responses in relation to procalcitonin signaling. In this regard, the authors had\nshown in a previous study that vascular leakage, vasoplegia and microvascular\ndysfunction are regulated by procalcitonin in sepsis. To induce acute sepsis, in the\ncurrent study the authors subjected WT mice to the Cecal Ligation and Puncture (CLP)\nprocedure. Subsequently, the authors conducted next-generation RNA sequencing on\nendothelial cells derived from murine lungs following the induction of polymicrobial\nsepsis, with the objective of assessing transcriptomic changes. The presented RNA\nsequencing data indicates the expression of numerous genes that are differentially\nexpressed, including expression of the Calca gene, encoding procalcitonin, and Il17a.\nThe study is well executed and written. However, the authors' principal objective is to\nexamine the role of procalcitonin during sepsis, a topic that has already been\nextensively investigated. A harmful role of procalcitonin in sepsis has been\ndemonstrated in multiple studies in a variety of different organisms, including mice,\nrats, and pigs (e.g., Baranowsky et al., 2021; Nylén et al., 1998; Wagner et al., 2002;\nTavares et al., 2014). Although the described antibodies in the current study appear to\nbe of novel design, the studies referenced above have already explored the therapeutic\npotential of antagonizing or neutralizing procalcitonin. Unfortunately, in the current\nstudy the key experiment, namely survival rates in mice treated with the novel\nProcalcitonin antibodies, is not reported.\nAlthough the RNA seq data of vascular endothelial cells with/without anti-procalcitonin\ntreatment are interesting, the functional impact of procalcitonin on vascular integrity\nduring sepsis has been reported by the same group before (Brabenec 2022). Also, the\nproposed role of Il17A as a downstream mediator of procalcitonin has already been\ndescribed in the study by Baranowsky et al. 2021. Together, the study provides only\nlimited conceptual advance and novelty, and, in my opinion, is thus better suited for a\nmore specialized journal.\nR1->C1. We thank the reviewer for his remarks. The goal of translational research\nadvances is to identify novel therapeutic strategies for the benefit of patients. This is of\ninterest to both basic scientists as well as clinicians. Sepsis is a disease with high\nincidence and high and unchanged mortality rates over the past decades, making\nsepsis a substantial burden to the health care system and society. The organ\ndysfunction induced by sepsis is undisputedly connected to a loss of vascular integrity\n(for example, Joffre J et al., Am J Resp Crit Care Med, 2020). However and although\nurgently needed and a promising therapeutic opportunity, the impact of therapeutic\nstrategies targeting the endothelium in sepsis has not been systematically dissected\nso far.\nWe here present the first endothelial transcriptome in the clinically most relevant\ndisease model of a polymicrobial sepsis at a time point where most patients with sepsis\nare diagnosed. The transcriptomic data provides extensive insights in the pathways\nup- and down-regulated in the vasculature at a time point, where mice exhibit sepsis\nsymptoms and elevated levels of procalcitonin. Procalcitonin is one of the most widely\nused biomarkers in sepsis, however, we were the first to describe a molecular pathway\ninduced by activated procalcitonin that directly mediates loss of vascular barrier\nfunction, the most critical hallmark in the pathophysiology of septic patients. However,\nthe verification of the use of targeting procalcitonin actions on the endothelium was\nmissing.\nWe here verify antagonizing procalcitonin massively cuts down the number of\nregulated genes in the septic endothelium, ameliorates loss of vascular barrier function\nwhich results in robust organ protection during sepsis. We thus assume our results of\nrelevance to a wide audience, including basic science researcher and clinician scientist\nas well as physicians and thus the broad audience of Nature Communications.\nUnfortunately, survival experiments are not in accordance with the local authorities.\nHowever, we present robust data showing that organ integrity is preserved with regards\nto various aspects in organs most affected by lethal sepsis such as the lung, the kidney\nand the liver.\nReviewer #2 (Remarks to the Author):\nC2.0: Calcitonin is a polypeptide hormone, which is made up of 32 amino acids and is\nproduced mainly by parafollicular cells (C cells) in the thyroid gland, secreted by cells\nof the glandular ultimobranchial bodies. The overall effect of calcitonin is to lower the\nconcentration of calcium in the blood when it has risen to an above the normal level.\nProcalcitonin, a protein that consists of 116 amino acids, is the peptide precursor of\ncalcitonin. Multiple studies have shown that procalcitonin is involved in the\npathogenesis of infections, and that it can be a useful diagnostic marker for infections\nsuch as bacterial pneumonia, bacterial sepsis and septic shock, meningitis, infectious\nendocarditis, pancreatitis, and urinary tract infections. Secretion of procalcitonin is\nstimulated by IL-1β and tumor necrosis factor–α in patients with bacterial infections\nand by interferon-γ in patients with viral infections. The authors previously reported that\nprocalcitonin induced endothelial barrier disruption (Wagner et al. Anesth Alalg 2017).\nFurthermore, their recent study has shown that targeting the procalcitonin receptor\nduring sepsis-induced hyperprocalcitonemia can significantly reduce capillary leakage\n(Brabenec et al. Am J Respir Crit Care Med 2022).\nIn this study, the authors demonstrated that more than half of transcriptomic changes\nin endothelial cells were reduced by anti-procalcitonin antibodies and this was\nfunctionally associated with preserved vascular integrity in lungs and intestines,\nreduced sepsis-induced vasoplegia, preserved endothelial NO bioavailability, and\norgan integrity during sepsis. They also found that neutralizing procalcitonin exerted\nprotection of pulmonary endothelial and epithelial barriers in mice with sepsis-induced\nsepsis. While this study includes interesting findings, I have a number of concerns for\nthe authors to consider. Major comments are summarized below.\nR2.0: We thank the Reviewer for his important remarks.\nC2.1: The disruption of endothelial integrity and functioning plays a crucial role in the\ndevelopment of sepsis-associated organ dysfunction. In regards to this, there are a\nnumber of excellent review articles. In the Introduction, the authors should cite these\nreviews.\nR2.1: We appreciate the reviewer’s insightful comment. We fully agree that the loss of\nendothelial cell integrity and function represents a critical hallmark in the pathogenesis\nof sepsis-associated organ dysfunction. To further emphasize this point, we have\nincorporated additional references into our manuscript (page 3) that highlight the role\nof endothelial dysfunction in sepsis (reference No. 5, 6 and 7 of the revised\nmanuscript):\n- WL Lee, AS Slutsky. Sepsis and endothelial permeability, N Engl J Med, 2010\n- Ince, Can; Mayeux, Philip R.; Nguyen, Trung; Gomez, Hernando; Kellum, John A.;\nOspina-Tascón, Gustavo A.; Hernandez, Glenn; Murray, Patrick; De Backer, Daniel on\nbehalf of the ADQI XIV Workgroup. The Endothelium in Sepsis. SHOCK 45(3):p 259-\n270, March 2016. | DOI: 10.1097/SHK.0000000000000473\n- Joffre J, Hellman J, Ince C, Ait-Oufella H. Endothelial Responses in Sepsis. Am J\nRespir Crit Care Med. 2020;202(3):361-370. doi:10.1164/rccm.201910-1911TR\nThese reviews provide comprehensive insights into the mechanisms of endothelial\ndysfunction in sepsis and reinforce its pivotal role in organ failure.\nC2.2) The authors describe that “so far, most therapeutic strategies failed to improve\nsepsis outcome (page 4, line 101). Without a doubt, despite advances in overall\nmedical care, sepsis continues to be a complex clinical entity with an unacceptably\nhigh mortality rate. The authors should provide several appropriate references for this.\nR2.2: According to the reviewers suggestion, we added several examples of studies\nthat showed that most targeted approaches in sepsis failed (i.e. antagonizing IL-1,\nTNFalpha or application of hydrocortison (references No. 15, 16, and 17 of the revised\nmanuscript).\nC2.3) The authors describe that the lung is the most susceptible organ to sepsis-\ninduced tissue injury (page12, line 276-277). The authors should provide an\nappropriate reference pointing out that the respiratory system is the most affected\norgan of the body and the lung dysfunction is the first step in the development of\nmultiple organ failure in septic patients.\nR2.3: We appreciate the reviewer’s comment and added a supporting reference that\nhas dissected the massive sepsis-induced destruction on the lungs (References No.\n34-36).\nC2.4) The gene encoding procalcitonin (Calca) was among the top up-regulated\ngenes, while the gene encoding the procalcitonin receptor (Calcrl) was among the top\ndown-regulated genes (page 12, line 283-285). It would be easy to understand if RT-\nPCR analysis showing gene expressions for Calca and Calcrl in pulmonary endothelial\ncells from sham-operated and CLP mice is visually provided.\nR2.4: We thank the reviewer for this important point. To clarify the dynamics of Calca\nexpression in endothelial cells during sepsis and add to the translational importance of\nthe data, we exposed human pulmonary microvascular endothelial cells to plasma from\nseptic patients exhibiting procalcitonin levels between 4 and 70ng/mL. Here, we could\nshow a robust induction of Calca. Importantly, this effect was abolished by procalcitonin\nantibodies, indicating that procalcitonin itself can perpetuate its expression and that\nendothelial cells are a source of procalcitonin during sepsis. Instead, we exhibited no\nregulation of the procalcitonin receptor encoding gene Calcrl. interestingly, we also\nobserved a trend towards a downregulation of the receptor activity modifying protein 1\nencoding gene (Ramp1) in human endothelial cells exposed to septic patients plasma.\nRAMP1 is required for the CRLR to function as the procalcitonin receptor. We included\nthis data in Figure 1 of the revised version of the manuscript.\nC2.5) The authors found that pathways associated with cell growth, angiogenesis, and\nassembly of cellular junctions were down-regulated (Fig. 1G) (page12, line 288-289).\nIn this regard, VEGF mRNA level was significantly down-regulated in lung tissues from\nmice with CLP-induced sepsis (Tomita et al. Naunyn-Schmiedeberg’s Arch Pharmacol 2020).\nThis report may be considered by the Authors.\nR2.5: We thank the reviewer for this important point. To strengthen our results\ncomparing it to previously published reports and to add to the translational relevance\nof the data, we analyzed VEGF mRNA expression in human pulmonary endothelial\ncells exposed to plasma from septic patients. We exhibited a downregulation of VEGF\nmRNA under exposition with septic patients plasma (Figure 1L).\nWe also subjected mice to procalcitonin injection and analyzed endothelial gene\nexpression in pulmonary endothelial cells isolated after 18h. We here also found PCT\ninduces a trend towards a reduction in VEGFa and c mRNA expression (Figure 1H).\nWe also included the report from Tomita and collegues as a reference into our\nmanuscript (reference number 37).\nC2.6) Kaplan-Meier survival curves examining whether neutralizing procalcitonin can\nimprove mice subjected to CLP should be provided.\nR2.6: Very unfortunately, the local authorities do not permit survival experiments.\nC2.7) In conclusion, the authors state that antagonizing the procalcitonin signaling\npathway would display a precision medicine strategy effective in those identified with\nparticular hyperprocalcitonemia. What percentage are septic patients with\nhyperprocalcitonemia occupy in the whole septic patients?\nR2.7: We thank the reviewer for his remark. Approximately 80% of all septic patients\nexhibit hyperprocalcitonemia (i.e. above 0.5ng/mL concentration in serum).\nReviewer #3 (Remarks to the Author):\nC3.0: The authors utilize multi-omic techniques and some well-designed murine animal\nmodel experiments to determine the impact of neutralizing a procalcitonin transcript\nisoform in a murine model of sepsis. The experiments are very well performed and the\nstudy is very elegant. However, as the authors state in the introduction, no therapeutic\ndiscoveries have been made that can treat the sepsis syndrome. This is primarily\nrelated to the fact that most studies have been performed in rodent models.\nUnfortunately, the findings in mice rarely translate in humans. Therefore, it is important\nfor the authors to provide some evidence that a similar pathway related to procalcitonin\ninhibition improved endothelial function in a human model such as primary cell culture,\nlung organoid, etc.\nR3.0: We sincerely thank the reviewer for the positive remarks and acknowledge that\nmany previous attempts to translate findings into the human model have been\nunsuccessful. However, in a prior study, we investigated the effects of inhibiting the\nprocalcitonin pathway in patients undergoing on-pump cardiac surgery, a population\nknown to exhibit elevated procalcitonin levels postoperatively, which correlate with\ncapillary leakage, microcirculatory dysfunction and poorer outcomes.\nOur findings demonstrated that cardiac surgery patients receiving sitagliptin, a DPP4\ninhibitor that suppresses procalcitonin activation, required less intraoperative fluid\nadministration and norepinephrine to maintain comparable mean arterial pressure—\nsuggesting improved vascular integrity. Additionally, these patients exhibited a higher\nproportion of perfused vessels and an increased microvascular flow index, both\nindicative of improved microcirculatory function and overall outcomes (Brabenec L et\nal., Targeting procalcitonin protects vascular barrier integrity, Am J Resp Crit Care Med\n2022, https://doi.org/10.1164/rccm.202201-0054OC).\nThese findings underline the potential of targeting the procalcitonin pathway in a\nclinical setting, highlighting its promise as a therapeutic approach in sepsis—not only\nin animal models but also in human patients (Brabenec et al., Am J Resp Crit Care\nMed, 2022)\nC3.1. As the authors stated – the primary benefit of neutralizing procalcitonin, was the\nimpact on endothelial and epithelial barrier integrity. The experiments are well\nperformed and described. However, one finding is that the inhibition of the procalcitonin\nisoform also improves intestinal barrier function. Their results demonstrate that there\nare decreased CFU’s in all organ tissues they looked at. It seems to me that a potential\nrational for the reduced CFU’s throughout the periphery could be related to the\nimproved integrity of intestinal barrier function. Simply put, the bugs have less ability\nto cross past the gut therefore many of the inflammatory responses are tamped down.\nClarification on this point is important to understand the impact of the potential\ntherapeutic - is it improved endothelial barrier function in lung tissues, or the reduced\nbacterial load because the bugs can’t cross?\nR3.1 Our hypothesis is, that procalcitonin directly affects barrier stability. According to\nthe reviewers suggestions, we now began to dissected the role of procalcitonin on\nendothelial vs. epithelial barrier integrity and found that – contrary to the endothelial\nbarrier – procalcitonin did not affect epithelial integrity in an in vitro assay of intestinal\nepithelial cells. These evaluations strengthen the notion that procalcitonins action is\nspecifically exerted on the endothelium and that this is crucial for the reduction in\nbacterial translocation during sepsis.\nUsing Caco-2 cells in a two-chamber transwell system, the trans-epithelial resistance\n(TER) through the cell monolayer was measured to evaluate monolayer integrity. Six\nhours after exposure, TER was similar in the PCT, Sita and PCT+Sita groups and did\nnot differ from the control group (DMEM + 10% FBS). These findings show that none\nof the substance(s) alters the integrity of the Caco-2 monolayer in vitro and suggest\nthat PCT does not affect gut epithelial barriers.\nFigure 2: Caco-2 cells barrier integrity in response to\nexposure of procalcitonin. TER-values across Caco-2\ncells seeded on transwell inserts. TER values after 6 h\nincubation of 10 ng/ml PCT, 1 µM Sita or PCT+Sita did not\ndiffer significantly from control (DMEM + 10% FBS) and\nbetween groups. N = 4, n = 29 PCT, n = 28 PCT+Sita, n =\nnot significant.\nC3.2.1 In the methods, the authors state that human endothelial cell cultures were\nperformed for permeability assays. However, the methods and results do not include\nany information about the human endothelial culture. This is unfortunate, as a human\nendothelial model that recapitulates the mouse findings would tremendously improve\nthe impact of this study. Considering that the study utilizes plasma of septic mice,\nplasma from septic patients on human endothelial cell cultures could similarly be\nperformed and would greatly enhance this study.\nR3.2.1: We thank the reviewer for this comment. In a prior study, that included human\ndata (please also see answer 3.0), we investigated the effects of human procalcitonin\nand the inhibition of the procalcitonin pathway in human endothelial cells. In this study\nwe were able to demonstrate that procalcitonin is the driver of loss of endothelial barrier\nintegrity (Brabenec L et al., Targeting procalcitonin protects vascular barrier integrity,\nAm J Resp Crit Care Med, 2022, https://doi.org/10.1164/rccm.202201-0054OC). We\nalso showed that patients with hyperprocalcitonemia in the setting of postoperative\nsterile systemic inflammation exhibit capillary leakage, microcirculatory dysfunction\nand increased fluid demands due to hypovolemia.\nIn response to the reviewers comment, we now extended this data further by including\ndata on gene expression exerted by procalcitonin in human endothelial cells as well as\na newly conducted biostatistical analysis correlating our findings in the murine septic\nendothelium to a human dataset obtained from EndoDB in Figure 1M (exposure of\nhuman endothelial cells to LPS) in the revised manuscript. In addition, we also\nconducted experiments analyzing septic patients plasmas effect on human endothelial\ncells and show that procalcitonin antagonization (with a polyclonal antibody specifically\ntargeting human truncated procalcitonin) can specifically reduce septic patients plasma\nmolecular effects on endothelial barrier disruption in human endothelial cells:\nFigure 3: A,B VE-Cadherin phosphorylation is increased in human endothelial cells\nupon stimulation with septic patients serum. C Increased endothelial cell permeability\nafter septic human serum stimulation is reduced by an antibody targeting\nprocalcitonins N-terminus that resulted in a reduction of VE-Cadherin phosphorylation\nin response to septic patients plasma (D, E). F Schematic overview.\nThese results are now provided in the supplemental figures of the revised version of\nthe manuscript (Supplemental Figure II C-H).\nC3.2.2 Similarly, it would be great if the authors could perform a similar RNAseq\nanalysis, or at least target 5 -10 of the key genes identified in the mouse study, using\nthe primary airway endothelial cell model and plasma challenge.\nR3.2.2 We thank the reviewer for his suggestion and performed gene expression\nanalysis of Calca, Calcrl, Ramp1, VEGFa and VEGFc as well as biostatistical analysis\nof a LPS induced sepsis human dataset to meet his concerns. The results are added\nto the revised manuscript in Figure 1.\nC3.3.1 The recombinant procalcitonin variant study: This is an excellent component of\nthe study and suggests that antagonizing this isoform could have potential therapeutic\nbenefit. That said, there are a couple of areas that need to be clarified. First, in\nthe methods the authors refer to the transcript isoforms as variants. I am not certain this is\nthe correct terminology as that has me thinking about SNP variants. It would probably\nbe easier for the reader to follow if you refer to it as a splice isoform or transcript isoform\nconsistently throughout the manuscript.\nR3.3.1 In response to the reviewers remark, we exchanged the term isoform to variant\nisoform.\nC3.3.2 More importantly, though, how does this isoform relate to human biology? I\nassume this isoform is also found in humans. A description of the human isoform would\nbe helpful – how evolutionarily conserved are these isoforms in mice to humans?\nEvidence that the isoform can be found in human endothelial cells is necessary to\ndemonstrate that the pathway is conserved.\nR3.3.2: Procalcitonin isoforms have also been found in humans. Under inflammatory\nconditions, they can be found at equal parts in humans. We added the literature\naccordingly to the revised version of the manuscript (page 5, references number 11\nand 12).\nC3.3.3 Does the polyclonal antibody that was developed also antagonize the human\nisoform and have similar results? It would be helpful to know if the antibody developed\nhas cross reactivity or if a new antibody needs to be developed.\nR3.3.3 Human and murine procalcitonin exhibit partially different amino acid\nsequences and we had developed antibodies specifically targeting human truncated\nprocalcitonin to antagonize endogenous human procalcitonin, for example in human\npatients plasma. For these experiments, recombinantly made truncated procalcitonin\nwas used for evaluation of specificity (Brabenec et al., Am J Resp Crit Care Med,\n2022).\nC3.3.4 The authors also discuss olcegepant and inhibition of DPP4 as abolishing the\nIL-17 mediated response. Anything that can confirm these results in a human model\nwould be helpful to understand the potential translational impact of inhibiting this\ntranscriptional isoform.\nR3.3.4: In human patients showing hyperprocalcitonemia postoperatively, we have\ndetected upregulation of IL-17 expression in CD4+ cells (see below). This is also\nconfirmed by other authors showing that DPP4 inhibition led to a shift in Th cell\nphenotype and subsequently lowered IL-17a expression (Pinheiro MM, Stoppa CL,\nValduga CJ, et al. Sitagliptin inhibit human lymphocytes proliferation and Th1/Th17\ndifferentiation in vitro. Eur J Pharm Sci. 2017;100:17-24.\ndoi:10.1016/j.ejps.2016.12.040).\n2EN\nhB\nS-\n2EN\nhB\nS-\nIL-17\n10 10\n0 0\n-10 -10\n-20 -20\n-20 -10 0 10 Bh-SNE1 -20 -10 0 10 Bh-SNE1\nFigure 4: Left: Clusters within the CD4+ panel and their reactions under diabetic\nconditions compared to non-diabetic control patients, n= 6 vs. 7, bh- and Phenograph-\nalgorithm. Right: IL-17-expression within the clusters\nMinor concerns –\nC3.4: The paragraphs are overly long. Particularly in the discussion. Breaking these\nup would make the manuscript easier to read.\nR3.4: In response to the reviewers comment, we shortened the paragraphs of the\ndiscussion in the revised version of the manuscript.\nC3.5: Line 417 – custom is spelled incorrectly.\nR3.5: We thank the reviewer for noticing the mistake in spelling and corrected it\naccordingly.\nReviewer #4 (Remarks to the Author):\nC4.0 Brabenec et al. performed bulk RNA sequencing of lung endothelial cells isolated\nfrom a mouse model of sepsis and identified transcriptomic changes that describe the\nimpact of sepsis pathology on this critical population. This team has previously reported\nthat a truncated form of procalcitonin disrupts vascular integrity using in vitro and in\nvivo studies that were supported with human clinical data from subjects that included\nthose taking inhibitors of the procalcitonin activator DPP4. In new data reported here,\nthe authors use a procalcitonin neutralizing antibody that was generated to test if\ninhibition blocks changes in the endothelial transcriptome as well as accompanying\nvascular defects and organ damage. This work addresses an important area of\ninvestigation. New host-based therapies are needed to improve sepsis outcomes.\nBeyond supportive care and antimicrobials there are few options available for clinicians\nto treat sepsis and septic shock. Below are some comments for clarifications to\nstrengthen the paper prior to recommendation for publication.\nThe authors previously reported a role for procalcitonin in vascular pathology using a\nvariety of experimental and observational approaches. This included mouse models of\nsepsis and various inhibition strategies to prove that\nprocalcitonin is more than just a biomarker. In this study, the key new results are the\ntranscriptomic evaluation of the lung endothelial population with respect to procalcitonin\nand expanded assessment of the protective role of procalcitonin\ninhibition on organ systems beyond the lung.\nR4.0: We thank the reviewer for valuable and encouraging feedback.\nC4.1. Because the transcriptomic analysis is a proposed major contribution of this\nstudy, there remain some questions that would be helpful to fully understand the data.\nThe authors use enzymatic dissociation of the lung followed by CD45+ depletion and\nCD31 enrichment to generate “pure” endothelial populations. There are a number of\ndetailed protocols (Conchinha NV 2021) that describe this procedure. Can the authors\nprovide more detail in the methods on their protocol since it is provided “in brief”.\nSimilarly, is there data available to show that the isolated population is pure?\nR4.1: We thank the reviewer for this important remark. In response to the comment,\nwe now describe our protocol in the methods section in full detail and included the\nreference in our protocol. In addition, we added our data on flow cytometry-based\nverification of endothelial cell purification in Supplemental Figure I A.\nC4.2. Also, in the methods there is a 96h expansion of the isolated endothelial cells. Is\nthe RNAseq performed after this 96h expansion? It is not clear as written especially\nsince the main text on line 278 indicates that the RNAseq was done 18h after\ncecal puncture. This is an important detail since in vitro expansion of endothelial cells can\ncause phenotypic drift as measured by transcriptomics (Cleuren ACA 2019).\nR4.2 We thank the author for this important remark. RNA was isolated immediately\nafter isolation of endothelial cells from murine lungs. This is now clearly described in\nthe revised methods section of the manuscript (page 7-8). The 96h expansion was only\nnecessary for permeability assays.\nC4.3. The generation of the antibody against the activated procalcitonin is used as a\ntool to neutralize the effects of procalcitonin on the endothelium. Although information\nis supplied in the supplement on the purified antibody using surface coating and data\nis shown that the truncated procalcitonin can be blocked experimentally, do the authors\nhave data showing the specificity of the antibody? Either following overexpression and\nby Western blot showing the full blot or dose dependent inhibition in vitro of tyrosine\nphosphorylation on cadherin?\nR4.3: In response to comment 3.2.1 of reviewer #3 we now show that the procalcitonin\ntargeting the truncated procalcitonin isoform specifically blocks human sepsis plasma-\ninduced VE-cadherin phosphorylation.\nC4.4. On line 319 the authors claim that the procalcitonin antibody “reduced the\nexpression of almost all DEGs during sepsis”. On line 419 the authors indicate that the\nantibody cut the number of genes in half. Figure 3D shows significant heterogeneity\nacross the individual animals tested. Can the authors clarify the interpretation of how\nthis is described and discussed? This is a key point since this can be interpreted that\nactivated procalcitonin is the primary driver of lung endothelial pathology at the\ngenomic level. This data could be strengthened by assessing impacts on endothelial\ntranscriptomes following a complementary inhibition such as those shown in 3F for IL-\n17a protein.\nR4.4: In response to this important reviewer remark, we rephrased the claim in the\nmanuscript to “antagonizing procalcitonin led to a reduction of the number of\ndifferentially regulated genes in septic mice endothelium by approximately 50%” in\naccordance with the findings shown in Figure 3A-D (page 18 of the revised version of\nthe manuscript).\nC4.5. The authors and other groups have previously shown a causal link or correlation\nwith vascular pathology and procalcitonin in multiple systems and models including\nsepsis. Therefore the gene expression analysis following procalcitonin seems to be the\nmajor new contribution here and could be a valuable dataset to understand therapeutic\nintervention in sepsis and translational potential. This data modality could also be a\nbridge to linking the CLP model of sepsis to any human pathology. Therefore, a major\ndeficit of the study and a missed opportunity is the lack of any transcriptomic\ncomparison of the data generated here to publicly available human datasets. It would\nbe valuable to include an analysis of the genes regulated by procalcitonin inhibition to\nthose human paradigms (endoDB). In addition, although the authors acknowledge in\nthe discussion that single cell RNAseq of lung has shown that it is one of the least\nheterogenous organs with respect to endothelial cell diversity (Kalucka 2020), this\nstudy would be much more impactful if a single cell analysis was performed to\nunderstand the action of procalcitonin on the endothelium. If this is out of scope for this\nstudy, it would be important to discuss how some of the specific genes regulated in\nthis study compare or align to those specific to endothelial subtypes in Kalucka 2020.\nR4.5: We appreciate the reviewer's critique and agree that incorporating a comparison\nto a human dataset from EndoDB would significantly enhance our results. To address\nthe reviewers remarks, we related our findings to results obtained in human endothelial\ncells exposed to LPS (no data was found for sepsis) and added our findings to the\nManuscript (Figure 1M). For this, we searched the EndoDB database\n(https://endotheliomics.shinyapps.io/endodb/) with the term ‘lung’. We obtained 29 hits,\n13 were from human. Of these, 12 were from freshly isolated cells or primary cell\ncultures. No dataset was found for sepsis-treated lung endothelial cells. One micro-\narray dataset reported gene expression changes after treatment of primary endothelial\ncells with LPS (E-GEOD-5883) at 4h, 8h and 24h post treatment and corresponding\nuntreated controls, which we found appropriate for comparison. We downloaded the\nrespective data from the GEO expression database (GSE5883,\nhttps://www.ncbi.nlm.nih.gov/geo/),and compared it our findings.\nIn human endothelial cells exposed to LPS, 643 probests were differentially expressed\nat 4h (treatment versus respective control), 827 DEPs at 8h, and 195 DEPs at 24h. We\nchose the 8h treatment for our comparison since it showed the strongest response. At\n8h treatment, 416 unique genes (genes on the array were represented by multiple\nprobsets) were regulated, 243 up- and 173 down-regulated. 331 of these DEGs were\nalso annotated in our datasets. Of these LPS-DEGs, 169 overlapped with DEGs from\nthe contrast of CLP versus controls and 118 overlapped to the DEGs from the contrast\nCLP plus AB to controls. These results showed that our findings were very similar to\nthe human LPS-induction study.\nWe then looked in detail at the top 50 up-regulated DEGs from the 8h LPS study. 36\nof the LPS-DEGs were also annotated in our dataset. As shown in Figure 1M almost\nall LPS-DEGs genes were also up-regulated in our study, and all of these DEGs\n(except one) showed reduced expression in CLP plus AB treated samples. Again,\nthese findings showed that our results were very similar to findings in LPS-treated\nhuman cells.\nMinor points:\nC4.6 On line 276 is there a reference that can be added to support the point that the\n“lung is the most susceptible organ” to sepsis induced injury?\nR4.6 A reference supporting this point was added to the revised version of the\nmanuscript (reference No. X), please also see our response to comment 2.3 of\nreviewer #2.\nC4.7In Figure 2E in column 4 of the Western Blot, is it correct that it is “trunc-PCT”\nalone, or is this with IgG as well?\nR4.7: We have added the missing information to our figure, noting that the cells were\nalso treated with control IgG."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have revised the manuscript and figures, and included additional experimental data, in an effort to enhance the\noverall quality, clarity and translational value of the article under review. These efforts are recognised and appreciated.\nNevertheless, I believe that my primary and most substantial concerns regarding the manuscript have largely gone\nunaddressed. The role of procalcitonin in the pathophysiology of sepsis has been extensively characterised in the literature,\nparticularly with regard to the inhibition of Calcrl-signalling by the antagonist BIBN4096, which has been shown to improve\nsurvival in mouse models. Furthermore, it has been convincingly demonstrated that the observed survival benefit of Calcrl-\nsignalling antagonism is mechanistically mediated via the induction of IL-17A, as reported by Baranowsky et al. (2021). It is\nimportant to note that while this proposed therapeutic intervention was found to be promising in small animal models, it failed\ntranslation in a more clinically relevant large animal model of porcine polymicrobial sepsis, as documented by Messerer et\nal. (2022). Consequently, the novel, clinically applicable information that is provided by the current manuscript remains\nunclear, especially due to the absence of a clearly defined clinical outcome measure. If the authors wanted to claim that their\nstudy provides meaningful novel insight of clinical relevance, they would have to provide compelling comparative data\ndemonstrating that the truncated version of their novel procalcitonin antibody is significantly superior to the known CALCR\nantagonist BIBN4096. In the absence of such evidence, it is challenging to substantiate the claim of novelty or clinical\ntranslatability. In summary, I believe that the present manuscript makes only a negligible contribution to the current\nunderstanding of the role of procalcitonin in sepsis and falls short of providing substantial translational or clinical value and\nshould therefore be published in a more specialized journal", "reviewer_2": "I appreciate that the authors have made a diligent revision of the manuscript.\nIn the revised version, the authors have addressed all my comments appropriately and further enhanced the manuscript.\nThe manuscript has been improved at a reasonably significant level.", "reviewer_3": "The authors have addressed all my comments.", "reviewer_4": "R4.1: Thank you for the added data.\nR4.2: Thank you for the clarification\nR4.3: Thank you for the added data\nR4.4: Thank you for the clarification\nR4.5: I thank the authors for the addition of the human transcriptomics analysis.\nR4.6: Thank you for the added references.\nR4.7: Thank you for the correction.\nMinor Comments:\nI have noted a number of spelling corrections needed in the text and figure legends. Please do a final check.\nThe addition of the human cohort and human in vitro studies strengthens the manuscript. Please ensure that references to\nplasma and serum are used correctly throughout the report. The legends and methods indicate that serum was collected\nfrom the human subjects, but it seems that in the discussion of the results you indicate that plasma was used. Please double\ncheck. Also, can you indicate in the methods more specifically how experiments were performed with mouse or human\nserum (plasma) with respect to percent of human or mouse serum added to the cell culture media? Was FBS still included in\nthese experiments?"}, "author_response": "Point-by-point-response to the reviewers comments\nDear reviewers,\nwe very much appreciate your effort to again revise our manuscript. Please find a point-by-\npoint response to all of your comments below. Based on your comments, we added additional\ndata to the manuscript to address your concerns and conducted the important corrections.\nPlease find all changes highlighted in the tracked-changes version oft he manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have revised the manuscript and figures, and included additional experimental\ndata, in an effort to enhance the overall quality, clarity and translational value of the article\nunder review. These efforts are recognised and appreciated. Nevertheless, I believe that my\nprimary and most substantial concerns regarding the manuscript have largely gone\nunaddressed. The role of procalcitonin in the pathophysiology of sepsis has been extensively\ncharacterised in the literature, particularly with regard to the inhibition of Calcrl-signalling by\nthe antagonist BIBN4096, which has been shown to improve survival in mouse models.\nFurthermore, it has been convincingly demonstrated that the observed survival benefit of\nCalcrl-signalling antagonism is mechanistically mediated via the induction of IL-17A, as\nreported by Baranowsky et al. (2021). It is important to note that while this proposed\ntherapeutic intervention was found to be promising in small animal models, it failed translation\nin a more clinically relevant large animal model of porcine polymicrobial sepsis, as documented\nby Messerer et al. (2022). Consequently, the novel, clinically applicable information that is\nprovided by the current manuscript remains unclear, especially due to the absence of a clearly\ndefined clinical outcome measure. If the authors wanted to claim that their study provides\nmeaningful novel insight of clinical relevance, they would have to provide compelling\ncomparative data demonstrating that the truncated version of their novel procalcitonin antibody\nis significantly superior to the known CALCR antagonist BIBN4096. In the absence of such\nevidence, it is challenging to substantiate the claim of novelty or clinical translatability. In\nsummary, I believe that the present manuscript makes only a negligible contribution to the\ncurrent understanding of the role of procalcitonin in sepsis and falls short of providing\nsubstantial translational or clinical value and should therefore be published in a more\nspecialized journal.\nResponse: We thank the reviewer for raising the important point regarding the effect of the\nprocalcitonin antibody on clinical outcome measures and the probability of survival. Based on\nyour comments, we analyzed all scoring of health status, behavior and sepsis criteria that we\nwere demanded to conduct on an hourly basis throughout all mouse experiments based on\nthe animal protocol approved by the authorities. We provide this data as Figure 6 in the revised\nversion of the manuscript and a detailed description of the scoring system in the Methods. This\ndata shows that the health status, behavior and sepsis criteria are significantly improved\nalready during early courses of the disease and continue to diverge showing beneficial effects\nof the anti-procalcitonin antibody (Figure 6a-c). The overall score at 18h post sepsis-induction\nequally verifies the beneficial effect of the antibody (Figure 6d).\nAs a score of 20 in any category was defined as the humane endpoint for mice in these\nexperiments, we conducted a Kaplan-Meier-analysis plotting the probability of survival. Please\nfind the results included in the revised version of the manuscript as Figure 6e. This data shows\na significant difference between septic animals treated with control-IgG and septic mice treated\nwith the procalcitonin-targeting antibody.\nTo address the reviewers concerns regarding the comparability with the CRLR/RAMP1-\ninhibitor BIBN4096, we plottet the data regarding the reach of the humane endpoint of a score\nof 20 in any category during the course of sepsis from a previous study on the use of BIBN4096\nin septic mice. Interestingly, we can clearly show that the beneficial effect of the antibody\nspecifically targeting truncated procalcitonin shows superior effects on murine survival during\nsepsis compared to BIBN4096 that did now show a significant benefit vs. vehicle-control (Fig.\n6f).\nBIBN4096 is a substance clinically licensed as an anti-migraine treatment known to exert\npossible side effects (for example, potentially liver toxicity). CGRP exerts various actions on\nthe vasculature and has recently been shown to ameliorate sepsis-induced intestinal injury\n(Ning W et al., 2023, https://doi.org/10.1016/j.intimp.2023.109747). These aspects could\nrender the inhibition of this pathway potentially not beneficial during sepsis. In contrast, the\nprocalcitonin antibody appears to not exert adverse effects but ameliorate sepsis severity and\nbeneficially influence murine outcomes. This underlines the novelty of the presented strategy\nusing procalcitonin-targeting antibodies to inhibit the procalcitonin signaling pathway.\nReviewer #2 (Remarks to the Author):\nI appreciate that the authors have made a diligent revision of the manuscript. In the revised\nversion, the authors have addressed all my comments appropriately and further enhanced\nthe manuscript. The manuscript has been improved at a reasonably significant level.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my comments.\nReviewer #4 (Remarks to the Author):\nR4.1: Thank you for the added data.\nR4.2: Thank you for the clarification\nR4.3: Thank you for the added data\nR4.4: Thank you for the clarification\nR4.5: I thank the authors for the addition of the human transcriptomics analysis.\nR4.6: Thank you for the added references.\nR4.7: Thank you for the correction.\nMinor Comments:\nI have noted a number of spelling corrections needed in the text and figure legends. Please\ndo a final check.\nResponse: Thank you very much for this remark, we corrected multiple spelling errors\nthroughout the manuscript.\nThe addition of the human cohort and human in vitro studies strengthens the manuscript.\nPlease ensure that references to plasma and serum are used correctly throughout the report.\nThe legends and methods indicate that serum was collected from the human subjects, but it\nseems that in the discussion of the results you indicate that plasma was used. Please double\ncheck. Also, can you indicate in the methods more specifically how experiments were\nperformed with mouse or human serum (plasma) with respect to percent of human or mouse\nserum added to the cell culture media? Was FBS still included in these experiments?\nResponse: Thank you very much for your comment. We revised the use of the words serum\nand plasma carefully throughout the manuscript – for procalcitonin concentrations in mice,\nplasma was used. For human samples, serum was used. We also specified the Methods\nregarding the in vitro experiments in the revised version of the manuscript. For these\nexperiments, we used plasma from septic or control mice diluted 1:1 in serum-free cell culture\nmedia (see Methods in the revised version of the manuscript)."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The manuscript has been strengthened by the Kaplan-Meier survival curves showing that neutralizing procalcitonin, but not\nBIBN4096, was able to improve mice after CLP surgery. Please mention that log rank test or Gehan-Breslow-Wilcoxon test\nwas used for comparing Kaplan-Meier survival curves in the Statistical analysis section or in the legend of Fig. 6."}, "author_response": "Point-by-point response to reviewers comments\nDear reviewer,\nwe very much appreciate your effort to again revise our manuscript. Please find a point-\nby-point response to all of your comments below. Based on your comments, we revised\nour manuscript to address your concerns and conducted the important corrections.\nPlease find all changes highlighted in the tracked-changes version of the manuscript.\nReviewer #2 (Remarks to the Author):\nComment 1: The manuscript has been strengthened by the Kaplan-Meier survival curves\nshowing that neutralizing procalcitonin, but not BIBN4096, was able to improve mice after\nCLP surgery. Please mention that log rank test or Gehan-Breslow-Wilcoxon test was used\nfor comparing Kaplan-Meier survival curves in the Statistical analysis section or in the\nlegend of Fig. 6.\nResponse 1: We sincerely thank the reviewer for drawing our attention to the omission\nof statistical information. We have analysed the survival data using the log-rank test and\nhave included this information in the revised manuscript, both in the Statistical Analysis\nsection and in the legend of Figure 6.\nComment 2: In addition, there are still open questions regarding the CLP model's\nmultifactorial determinants of its reproducibility. The CLP model can be modified to\nproduce varying degrees of severity, depending on the cecum ligation site and the number\nof punctures made. Thus, variation in the number and size of the puncture(s) can be used\nto modulate sepsis severity, inflammatory response, and mortality rate. Accordingly, the\nauthors should clearly mention how many centimeters from the cecum tip they tightly\nligated.\nResponse 2: We thank the reviewer for this insightful comment and fully agree that\nvariability can occur in the CLP model due to its multifactorial determinants. As the\nreviewer correctly points out, we also took particular care to minimize such variability in\nour experiments. Specifically, we ligated the cecum 14 mm from the cecal tip in each\nmouse, and to further reduce technical variation, all CLP surgeries were performed by\none experienced researcher blinded to the experimental group to avoid technical\nvariations. We added this information to our manuscript.\nIn addition, we added supplemental figure II, showing that bacterial colony forming units\nfrom peritoneal lavages show no significant differences in bacterial load in CLP groups.\nWe think this information is crucial showing that the insult of sepsis induction did not vary\nin mean between groups (see below).\nComment 3: Also, the authors may describe that their mice after CLP surgery were all\nfatal (although, in this study, CLP mice appears to be nearly fatal, and it is a bit concerning\nthat there is an apparent difference between the CLP+IgG group (Fig. 6e) and the\nCLP+DMSO group (Fig. 6f)). For instance, in our laboratory, all mice subjected to CLP\nwithout treatment die within 48 hours. Moreover, we have only one surgeon for CLP\nsurgery in one study to avoid technical variations.\nResponse 3: Regarding the reviewer’s concern about the apparent difference between\nthe CLP+IgG and CLP+DMSO groups, we have now plotted the survival curves together\nand found no statistically significant differences between these groups. As noted, CLP-\ntreated animals are typically fatal within 48 hours; however, in our study, the observation\nperiod was limited to the first 18 hours post-surgery. As mentioned above, all CLP\nsurgeries were performed by a single experienced researcher to minimize variability.\nComment 4: Finally, the authors may describe behavioral characteristics of CLP mice.\nThus, mice after CLP surgery were all lethargic, showed lack of interest in their\nenvironment, displayed piloerection, and had crusty exudates around their eyes. These\npoints would provide more evidence that great care was taken to minimize variability in\ndisease severity.\nResponse 4: In terms of behavioral characteristics, following CLP surgery, the mice\ndisplayed lethargy and reduced mobility, showed no interest in their surroundings, and\ndid not engage in social behaviors such as sniffing or grooming. These observations are\nconsistent with the typical clinical signs of sepsis. We added these observations to our\nmanuscript."}, {"round": 4, "reviewer_comments": {"reviewer_2": "All the points raised by this reviewer have been addressed satisfactorily."}, "author_response": ""}]

Decision letter