Neutrophil-fibroblast crosstalk drives immunofibrosis in Crohn’s disease through IFNα pathway

Introduction Crohn’s disease (CD) is characterized by chronic inflammation and intestinal fibrosis leading to lifelong complications. However, the disease pathogenesis remains elusive, and the therapeutic options are limited. Here, we investigated the interaction between neutrophils and intestinal fibroblasts in the development of CD immunofibrosis, a disease mechanism predisposing to inflammatory and fibrotic complications. Methods Peripheral neutrophils, enriched neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies from CD, ulcerative colitis (UC) patients, and healthy individuals (HI), were studied. Transcriptome analysis of neutrophils, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed. Results Compared to UC, PIFs from CD patients, independently to the presence of strictures, displayed a distinct pro-fibrotic phenotype characterized by negative Krüppellike Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. In both UC and CD, PIFs-derived IL-8 acted as a culprit chemoattractant for neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs induced a CD-like phenotype in HI PIFs, suggesting their fibrotic plasticity. High IFNa in serum and IFΝ-responsive signature in peripheral neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum stimulated the release of fibrogenic eNETs from neutrophils in an IFNa-dependent manner, suggesting the priming role of IFNa in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, both serum IFNa levels and mRNA levels of key IFN signaling components in neutrophils were wellcorrelated with CD severity. Conclusions This study reveals the important role of the IFNa/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and putative therapeutic targets.


INTRODUCTION
Crohn's disease (CD) is characterized by chronic relapsing inflammation that, in contrast to ulcerative colitis (UC), may lead to intestinal fibrosis causing lifelong disabling illness with a large impact on the quality of life and the healthcare systems (1,2). Therefore, understanding the mechanisms of initiation and propagation of intestinal fibrosis in CD is crucial to provide knowledge for diagnosis and better care of patients (3,4).
Cellular plasticity is fundamental to human immunity and, as recently recognized, a key aspect of neutrophil biology (5,6). Recent studies suggest that, circulating neutrophils, as adaptation to the different environmental conditions, undergo transcriptional reprogramming that allows them to acquire disease-specific phenotypes and commit their cell-fate plasticity upon their entrance to the site of tissue inflammation attracted by tissue-derived chemotactic factors (6). Activated neutrophils release a plethora of antimicrobial and proinflammatory mediators on extracellular vesicles and traps that could dictate their diverse functional role in different diseases (7)(8)(9).
In line with these, previous studies indicated that activated neutrophils through the release of neutrophil extracellular traps (NETs) may play a pro-fibrotic role by promoting the differentiation and activation of human fibroblasts (10)(11)(12). More recently, our group suggested that NETs and downregulation of transcriptional factor Krüppel-like Factor 2 (KLF2) in human lung fibroblasts are linked with the inflammatory environment of COVID-19 that leads to immunofibrosis (13). Whether neutrophils exert immunofibrotic effects on intestinal fibroblasts and how these cells interact with each other in the inflammatory environment of CD leading to tissue damage is largely unknown yet (4,14).
This study provides a new understanding of the mechanisms involved in CD immunofibrosis through neutrophils/NETs functional plasticity. We identified that, in contrast to UC, neutrophils of CD are primed by IFNα/JAK signaling to commit an active role on intestinal fibroblasts, thus inducing their distinct fibrotic phenotype. The production of IL-8 by intestinal fibroblasts sustains their mutual interaction with neutrophils. Translating these findings, the levels of key IFN type I pathway components are positively correlated with disease severity in CD patients promising novel diagnostic and therapeutic targets.
Next, to decipher the way that neutrophils are attracted to the intestinal environment, we investigated whether fibroblasts produce known chemoattractant factors. Thus, we found that IL-8 (CXCL8) was the chemokine with significantly higher levels in supernatants of cultured PIFs obtained from UC and CD compared to HI (Fig. 2C). Increased IL-8 expression was also observed in isolated PIFs both at mRNA and protein levels, as well as in fibroblasts of intestinal sections ( Fig. 2D-F). No significant alterations were observed in the levels of other cytokines (Fig. S2).
Prompted by these results and in view that IL-8 has a distinct target specificity for neutrophils (15), we next performed a chemotactic assay that indicated increased migratory capacity of neutrophils when stimulated by the IL-8-rich supernatant of UC and CD PIFs, an effect that was inhibited after IL-8 neutralization (Fig. 2G).
These data suggest that both UC and CD fibroblasts express functional IL-8 as a key neutrophil chemoattractant factor of the intestinal environment. However, it warrants further investigation if neutrophils in each disease have differential functional properties during their crosstalk with fibroblasts.

Healthy PIFs acquire a CD-like fibrotic phenotype after treatment with ex-vivo enriched neutrophil extracellular traps (eNETs) from CD patients
To investigate the hypothesis that neutrophils of CD, UC patients, or HI have distinct functional properties in their crosstalk with fibroblasts, we obtained mixtures consisting of ex-vivo NETs enriched with the supernatant formed during the isolation procedure of NETs, to preserve the total inflammatory environment including structures of DNA scaffold and cellular extracts (eNETs). Moreover, eNETs were collected after in-vitro stimulation of HI neutrophils with PMA, a chemical inducer of NETosis, and used as a non-disease specific stimulus.
Next, we used these neutrophilic mixtures for stimulations on HI PIFs to assess KLF2 and CCN2 mRNA and protein levels, as well as collagen release. We found that HI PIFs stimulated with CD eNETs acquired a CD-like KLF2 (-), CCN2 (++), and collagen (++) fibrotic phenotype (Fig. 3A,B, G-K). This effect was not observed in stimulations with HI eNETs (Fig. 3C, G-K). In contrast, UC eNETs drive fibroblasts towards the UC-like phenotype, indicating KLF2 (+), CCN2 (-), and collagen (-) cells (Fig. 3D, G-K). Moreover, PMA generated mixtures that led fibroblasts towards KLF2 (+), CCN2 (+), and collagen (+) phenotype, indicative of mild fibrotic activity (Fig. 3E, G-K). The fact that the serum of CD patients alone was unable to transform the phenotype of HI fibroblasts to acquire fibrotic function, further supports the key role of neutrophils in the activation of fibroblasts ( Fig. 3F-J).
Taken together, CD neutrophils probably acquire different plasticity compared to UC neutrophils, being able to transform healthy PIFs to a CD-like fibrotic phenotype, demonstrating a functional role in their crosstalk with intestinal fibroblasts.

Transcriptome analysis of peripheral blood neutrophils unravels distinct pathways in Crohn's disease and ulcerative colitis
To provide some mechanistic explanation for the differential plasticity observed in IBD neutrophils, we sought to compare the transcriptome of peripheral blood neutrophils isolated from CD and UC patients. Our analysis identified 849 significantly upregulated and 789 downregulated genes in CD neutrophils, whereas 1421 genes were found upregulated and 1110 downregulated in UC neutrophils, relative to control neutrophils purified from healthy individuals (Fig. 4A). Interestingly, the two datasets exhibited a substantial overlap since 66% of upregulated and 57% of downregulated genes in CD were also significantly regulated in UC neutrophils (Fig. 4A, Fig.  S3). Bioinformatics analysis using the GeneCodis4 web-based tool revealed that common upregulated DEGs clustered mainly in immune-related pathways, including neutrophil degranulation, class I MHC mediated processing and presentation, and Toll-like receptor cascades, while translation, mRNA and rRNA processing were amongst the top downregulated processes (Fig. 4B). Despite the commonly regulated DEGs and pathways, we found several genes and processes uniquely regulated in each disease (Fig. 4B, Fig. S3). More specifically, interferon signaling was the top pathway selectively upregulated in Crohn's ( Fig. 4B), while the majority of unique upregulated genes in UC were involved in the neutrophil degranulation pathway. Regarding the unique downregulated genes, these clustered mainly to translation-and post-translational modification-related pathways in CD neutrophils, while the respective DEGs in UC neutrophils belonged to the pathways of chromatin organization and apoptotic process among others (Fig. 4B).
Gene set enrichment analysis was also performed, using the Reactome and Hallmark gene sets collections of the Human Molecular Signatures Database, to reveal enriched signatures in our datasets, independently verifying the overrepresentation of interferon signaling and neutrophil degranulation in CD and UC neutrophils, respectively (Fig. 4C). GSEA analysis also highlighted the increased expression of DEGs involved in autophagy and oxidative phosphorylation in UC, compared to CD neutrophils, consistent with the previously reported increased NETotic potential in UC (16).
Focusing on the interferon signaling pathway, 53 DEGs were significantly regulated in CD neutrophils, including several target genes, intracellular mediators, enzymes, and receptors ( Fig.  4D). Of those, 47 were up-and only six were down-regulated. On the other hand, only 14 out of 53 aforementioned genes were significantly upregulated in UC, while three major interferoninduced genes (OAS2, OAS3, and OASL) were downregulated (Fig. 4D, Fig. S3E). Moreover, RT-qPCR analysis in neutrophils from an independent group of CD and UC patients, confirmed that mRNA levels of the key IFN-signaling genes STAT1 and STAT2 followed the same expression pattern with transcriptome analysis (Fig. S4A). Hence, the molecular signature that distinguishes CD from UC neutrophils is positively correlated with interferon signaling activation.
Next, we sought to measure the levels of interferons, together with several other cytokines in the sera of patients with CD and UC. Although both IFNα2 and IFNγ were found elevated in the sera of IBD patients, in comparison to healthy individuals, only IFNα2 levels were significantly increased in CD over UC patients (Fig. 4E, Fig. S4B). Thus, elevated IFNα2 levels in the serum possibly account for the interferon fingerprint characterizing the transcriptome of peripheral neutrophils in CD.

IFNα primes neutrophils of CD patients to acquire fibrotic partnership
Based on the findings above, we hypothesized that IFNα may signal in neutrophils committing them to acquire a fibrotic partnership in their crosstalk with fibroblasts. To this end, we stimulated in-vitro HI neutrophils with serum from CD patients to produce eNETs (CD serum-generated eNETs), which were subsequently used for the treatment of HI primary fibroblasts.
Collectively, our data so far suggest that in CD, priming of circulating neutrophils by IFNα is essential to commit their fibrotic effect over intestinal fibroblasts and their plasticity is expressed through eNETs.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Neutrophil-dependent fibrosis in CD is eliminated by dismantling the DNA scaffold of eNETs or by inhibiting JAK signaling in fibroblasts
Prompted by the above-mentioned findings indicating that IFNα primes neutrophils to acquire a fibrotic role through eNETs and since previous results showed that JAK/STAT signaling in fibroblasts is involved in COVID-19 immunofibrosis (13), we tried to diminish the fibrotic transformation of fibroblasts either by dismantling the DNA scaffold of eNETs or by inhibiting JAK signaling in these cells.
Treatment of CD eNETs with DNAse I or pretreatment of HI fibroblasts with baricitinib inhibited the transformation of the KLF2 (++), CCN2 (-), collagen (-) HI fibroblasts towards the KLF2 (-), CCN2 (++), collagen (++) CD-like phenotype ( Fig. 6A-D, F-I). This effect was also observed with the plant-derived polyphenol tannic acid, a potent inducer of KLF2 expression (13,17), suggesting a potential role of KLF2 in the fibrotic process ( Fig. 6E-I). Next, we investigated whether JAK inhibition could reverse to normal the fibrotic phenotype of the already affected primary fibroblasts of CD. The KLF2 (-), CCN2 (++), collagen (++) phenotype of active CD primary fibroblasts was not altered following their treatment with baricitinib, suggesting that JAK inhibition should be performed early, before acquiring the CD phenotype ( Fig. S6A-E).
In conclusion, neutrophil-fibroblast crosstalk in CD is disrupted by targeting neutrophil DNA scaffold or/and JAK signaling in fibroblasts. This neutrophil-driven fibrotic process seems to be an early event in the pathogenesis of CD immunofibrosis.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Levels of interferon-signaling components are positively correlated with the severity of CD
To further support our findings, we sought to examine whether serum IFNα levels and transcriptomic alterations of IFN-signaling-related genes in peripheral neutrophils are associated with the severity of CD, as reflected by the classical disease activity index, CDAI (18).
We observed that IFNα2 levels in the serum of CD patients were correlated with the disease severity since individuals with higher CDAI scores were also characterized by higher concentrations of IFNα2 (Fig. 7A). In contrast to CD, in UC no correlation was observed between IFNα levels and Mayo DAI score, a common indicator of disease activity (19) (Fig. 7A).
We also noticed that the transcriptomic alterations observed in peripheral blood neutrophils of IBD patients were also correlated with the CDAI and Mayo DAI scores ( Fig. S7A-B). The whole set of DEGs followed an expression pattern related to the disease severity ( Fig. S7A-B), an observation that was prominent for interferon signaling components in CD as well (Fig. 7B). Indeed, the mRNA expression of key components of the interferon signaling pathway, namely AIM2, JAK2, STAT1, and STAT2, followed faithfully the CDAI score since it was increased relative to disease severity (Fig. 7C). The expression of several interferon-signaling components was also correlated with serum IFNα2 levels (Fig. S7C).
Taken together, serum IFNα and/or mRNA levels of interferon-signaling components in neutrophils emerge as candidate diagnostic/prognostic biomarkers for CD.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023.

DISCUSSION
In this study, we describe a novel mechanistic link between neutrophils and fibroblasts that characterizes immunofibrosis of CD. In contrast to UC, neutrophils of CD patients, exhibit profibrotic properties by activating intestinal fibroblasts leading to increased collagen release. This type of neutrophil plasticity is primed by IFNα signaling and expressed by the NET-enriched inflammatory environment. Moreover, neutrophils may be attracted in the intestinal environment by IL-8 secreted from activated fibroblasts sustaining this fibrotic loop of neutrophil-fibroblast interaction. Further supporting this mechanistic basis, the expression levels of key IFNα pathway components in serum and neutrophils are well correlated with the clinical activity of CD patients.
Growing evidence today implies that the development of tissue fibrosis involves the complex interplay between immune and stromal cells. However, in CD, cell-cell interactions and functions are incompletely understood, while most of the studies focus mainly on lymphocytes and/or other mononuclear immune cells, while the role of neutrophils remains obscure (14).
Here, we showed that neutrophils in active CD and UC may be recruited at the site of tissue damage by IL-8 produced by intestinal fibroblasts. Earlier clinical studies have indicated increased IL-8 levels in the intestinal mucosa of active IBD patients (20, 21). This study additional characterizes fibroblasts as a source of IL-8 in the tissue environment of IBDs suggesting a functional, chemoattractant, effect on peripheral neutrophils. Which mediator(s) initially trigger IL-8 expression in fibroblasts, as well as the putative role of the gut microbiota dysbiosis, needs to be clarified in future studies. Similar triggers might also induce IL-8 production from connective tissue mesenchymal cells in IBD extraintestinal manifestations, such as arthritis. In support of this, it has been shown previously, that NETs from rheumatoid arthritis patients can stimulate the production of IL-8 from fibroblast-like synoviocytes (10). Of note, although fibroblast-derived IL-8 appears to be implicated in neutrophil recruitment in both IBDs, we observed a differential intestinal distribution of neutrophils. In CD, dense neutrophil infiltrations were observed in proximity to fibrotic areas, implying a fibrotic role for recruited neutrophils. This finding combined by the differences in plasticity of neutrophils between the two IBDs, could explain the fibrotic phenotype of CD.
Today, the traditional concept that neutrophils comprise terminally differentiated cells with limited plasticity and highly conserved function has been critically revised (5,6). Several studies support that during inflammatory conditions, the transition of mature neutrophils from bone marrow to the bloodstream is accompanied by changes at the transcriptional level that enable the acquisition of distinct functions within the affected tissues (5,6). Previous clinical and experimental studies suggested that in the context of different inflammatory disorders, neutrophils may acquire differential plasticity which may be reflected by the NETs that they release (9,10). In this context, our group and others have indicated that activated neutrophils may acquire an immunofibrotic role through the release of NETs. These fibrogenic NETs were able to activate human fibroblasts inducing their proliferation, differentiation to myofibroblasts and immunogenicity (10)(11)(12).
Our functional studies indicated that NET-enriched extracellular mediators (eNETs) ex-vivo isolated from peripheral neutrophils of CD patients, and not the CD serum directly, were able to transform healthy intestinal fibroblasts toward the distinct CD phenotype characterized by negative KLF2 and high CCN2 expression, leading to collagen production. Similar immunofibrotic phenotype of lung fibroblasts has been also described in severe COVID-19 (13). However, eNETs from peripheral neutrophils of UC patients did not have a similar potential, suggesting differential neutrophil/NETs plasticity on fibroblasts between the two IBDs.
To identify possible molecular pathways and targets that may drive this plasticity, we applied whole transcriptome analysis in peripheral blood neutrophils from IBD patients. Importantly, although a substantial overlap of the DEGs between the two intestinal diseases was observed, peripheral neutrophils of CD were selectively characterized by an IFN-responsive signature. On the other hand, in UC, upregulated genes were enriched for pathways related to neutrophil degranulation, autophagy and oxidative phosphorylation, confirming our previous mechanistic studies which demonstrated the key role of autophagy-mediated NET formation in UC (16). Recently, top upregulated severity genes in the colonic mucosa of UC patients have been also found to be involved in innate immunity and neutrophil degranulation (22). Previous comparative transcriptome studies between UC and CD have yielded heterogeneous results mostly being nontargeted, performed in whole blood or mucosal tissue (23)(24)(25). Despite this, a mostly neutrophillike signature has been proposed for the whole blood of IBD patients, while the most significant signal within CD ileal mucosa with deep ulcers was for granulocytes, further favoring the role of neutrophils in active IBD (23,25). At the time of this writing, emerging single-cell transcriptome data in mucosal samples reveal the heterogeneity of tissue neutrophils in IBD patients. Noteworthy, A. Garrido-Trigo et al report that, in contrast to UC, the majority of intestinal neutrophils in CD display a marked IFN-response signature (26). This is in agreement with, and further supports, our transcriptome findings in peripheral neutrophils of CD.
Based on the findings indicating that IFN signature may distinguish CD from UC neutrophils, and multiplex cytokine analysis showing that IFNα, and not IFNγ, serum levels were significantly increased in CD compared to UC, it was reasonable to assume that IFNα may prime peripheral neutrophils of CD patients to exert their fibrotic effect on fibroblasts. In line with this, functional studies indicated that CD serum induces the generation of highly fibrogenic eNETs in an IFNαdependent manner. Furthermore, neutrophils primed with IFNα, during the production of PMAgenerated eNETs, enhanced their fibrotic plasticity. This further supports an additional key role for IFNα in the immunofibrotic plasticity of neutrophils, which is expressed via extracellular DNA structures. A large body of evidence has demonstrated that the integrity of DNA scaffold is important for NETs function (10,11,27). How the architecture of NETs and their proteins implicated in the immunofibrotic plasticity of neutrophils is an intriguing question that needs to be subjected to further investigation in the future.
Several studies have indicated that loss-of-function mutations in nucleotide-binding oligomerization domain 2 (NOD2) have been associated with CD (28). Of interest, it has been recently indicated, that NOD2 activation in hematopoietic cells protected mice from TLR9induced exacerbation of DSS-induced colitis by downregulating IFNα responses (29). Although several immune and non-immune cells may contribute, the exact sources of IFNα in the systemic circulation of CD remain to be identified. It has been previously shown that mature neutrophils in systemic lupus erythematosus (SLE) are primed in-vivo by type I IFNs to release NETs upon exposure to SLE-derived autoantibodies, indicating plasmacytoid dendritic cells as a major source of IFNα (30).
Fibrosis leads to abnormal tissue remodeling complicating several chronic inflammatory diseases, while early prevention of fibrosis remains a significant unmet medical need (31). Our mechanistic studies suggest that the interplay between neutrophils/eNETs and fibroblasts in CD is an early immunofibrotic event, that may be disrupted by using various pharmaceutical agents, such as anti-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint IL-8 (32) to interrupt the intestinal migration of pro-fibrotic neutrophils, recombinant DNAse to dismantle the chromatin scaffold of fibrogenic eNETs (33) or inhibitors of JAK signaling (34) both in peripheral neutrophils and intestinal fibroblasts to prevent their activation. Recent experience in severe COVID-19 patients indicates that combining different immunomodulatory therapies may be beneficial for complex fibrotic diseases such as CD (13,33). Lately, JAK inhibition has been approved as a new treatment for moderate-to-severe CD patients, further supporting the translational impact of our study (34).
In several cases, the assessment of disease activity or the discrimination between UC and CD is problematic, mainly based on invasive interventions such as ileocolonoscopy and intestinal biopsies (1). Linking our findings with clinical practice, we found that levels of IFNα in serum or/and mRNA expression of selective IFN-signaling-related components in peripheral neutrophils could be surrogate markers of CD activity, positively correlated with the CDAI, a well-established disease severity index which includes endoscopic findings. Recently, colonic tissue biopsies of CD or UC patients with active and remitted disease phases were characterized by the enhanced and reduced expression of IFN-stimulated genes, respectively (29). Here, we suggest that targeted analysis of peripheral blood neutrophils may be a more specific, easier, and less costly diagnostic approach.
In conclusion, this study unravels the role of IFNα/JAK signaling in the plasticity of neutrophils/NETs during their crosstalk with intestinal fibroblasts, eventually leading to immunofibrosis of CD. The IFNα/neutrophil/fibroblast pathway provides novel candidate targets for the design of future diagnostic and therapeutic strategies in CD.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Patients and sampling
The study was conducted in the First Department of Internal Medicine and the Gastroenterology-Hepatology Unit, at the University Hospital of Alexandroupolis. In total 26 treatment-naïve CD (16 male/10 female; mean age 35.3±15.0 years), 32 UC patients (24 male/8 female; mean age 52.2±19.1 years) and 18 healthy individuals including 4 who underwent preventive screening colonoscopy (HI; 12 male/6 female; mean age 39.1±12.6 years), were recruited. The diagnosis of UC and CD was according to standard clinical, endoscopic, radiological, and histological criteria (1,35). Clinical severity and disease behavior scores such as Mayo disease activity index (Mayo-DAI) (19), Crohn's disease activity index (CDAI) (18) and Montreal score (36) were evaluated in both UC and CD patients by two independent, expert gastroenterologists.
Neutrophils, serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies were collected. Baseline clinical characteristics of the patients and samples used in experimental procedures are depicted in Supplementary Tables S1-3.
All study participants provided written informed consent in accordance with the principles expressed in the Declaration of Helsinki. Patients' records were anonymized and de-identified prior to analysis to ensure anonymity and confidentiality. The study protocol was approved by the Scientific and Ethics Committee of the University Hospital of Alexandroupolis (Approval Number 803/23-09-2019).

Isolation of peripheral blood neutrophils and serum
Peripheral heparinized blood and serum were collected as previously described (16). Detailed methods are included in the Supplementary Materials and Methods.

Isolation, culture, and characterization of human primary intestinal fibroblasts
Detailed methods are included in the Supplementary Materials and Methods.

Generation and collection of enriched-neutrophil extracellular traps (eNETs)
A total of 1.5 × 10 6 neutrophils isolated from UC, CD patients and HI were re-suspended in Roswell Park Memorial Institute (RPMI) medium (21875; Thermo Fisher Scientific; Carlsbad, SA, USA) supplemented with 2% FBS (10082147; Thermo Fischer Scientific), and cultured at 5% CO2, at 37 o C, for 3.5 hours, based on the standard isolation protocol to generate ex-vivo NET structures. Similarly, healthy neutrophils were also incubated in-vitro in the presence of 5% serum from UC, CD patients and HI, or phorbol 12-myristate 13-acetate (PMA) (40 ng/ml; Sigma-Aldrich, St Louis, MO, USA), a generic inducer of NET release, and cultured in the aforementioned conditions. After the incubation period we performed a vigorous agitation for 5 minutes, to detach NET structures and this medium was collected. We omit the washing step, which was caried out during the classic method of NETs isolation (37)(38)(39) in order to collect more inflammatory mediators than NETs (10,40,41). This mixture of enriched NETs is hereafter . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint referred to as "eNETs". Aliquots of eNETs were stored at -80 o C until analyzed. Both ex-vivo and in-vitro generated eNETs were further used in stimulation studies in PIFs at a final concentration of 20%.
The concentrations and time points used to test neutrophil function were optimized before the experiments. All materials used were endotoxin-free, as determined by a Limulus amebocyte assay (E8029; Sigma-Aldrich).

Stimulation and inhibition studies in cultured cells
PIFs or peripheral blood neutrophils were seeded into 6-well culture plates (≈0.8-1 x 10 6 cells/well for fibroblasts, ≈1.5 x 10 6 cells/well for neutrophils; Corning Incorporated) in complete DMEM and RPMI medium respectively.

RNA isolation, cDNA synthesis and RT-qPCR
Detailed methods are included in the Supplementary Materials and Methods.

RNA sequencing and bioinformatics analysis
One μg of total RNA was used for the preparation of cDNA libraries, as previously described (44,45). Sequencing was performed in a single-end manner at the Greek Genome Center, using the NovaSeq 6000 SP 100c kit (Illumina, 20028401), generating 100 bp long reads.
Raw sequence data were uploaded to the Galaxy web platform (46), and standard tools of the public server "usegalaxy.org" were used for subsequent analysis, as previously described (45). HISAT2 (v2.2.1+galaxy1) was applied for the alignment of trimmed reads to the human GRCh37/hg19 genome assembly from the Genome Reference Consortium. Assessment of uniform read coverage for exclusion of 5'/3' bias and evaluation of RNA integrity at the transcript level . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint were performed, as previously reported (45). Moreover, absolute de-convolution of human immune cell types was applied in our datasets, according to the Shiny app, https://giannimonaco.shinyapps.io/ABIS (47), to ensure their enrichment in granulocytes gene expression signature (> 95%). Replicates with signatures enriched in contaminating lymphocytes (> 5%) were excluded from the analysis.
Pathway analysis was performed using the GeneCodis4 web-based tool (48). Cutoff values for differentially expressed genes were baseMean >30 and adjusted p-value (false discovery rate, FDR) <0.05. Gene set enrichment analysis (GSEA) was performed using the GSEA software (University of California, San Diego & Broad Institute, USA) (49). Briefly, normalized counts generated from DESeq2 (v2.11.40.7+galaxy2) and annotated gene sets from the Human Molecular Signatures Database (Human MSigDB v2023.1) were used as inputs. Gene sets were ranked by taking the -log10 (p-value) and signed as positive or negative based on the direction of fold change, followed by pre-ranked analysis using the default settings (1000 permutations, min, and max term size of 15 and 500, respectively).

In-cell ELISA (ICE assay, Cytoblot)
Details are included in the Supplementary Materials and Methods.

Immunofluorescence staining in human PIFs
Details are included in the Supplementary Materials and Methods.

Collagen measurement
Details are included in the Supplementary Materials and Methods.

Multiplex cytokine measurement
Details are included in the Supplementary Materials and Methods.

Immunohistochemistry (IHC-P), Masson's trichrome and Immunofluorescence (IF) staining in tissue sections.
Detailed methods are included in the Supplementary Materials and Methods.

In-vitro transwell migration assay (chemotaxis assay)
Details are included in the Supplementary Materials and Methods.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Statistical analysis
Statistical analysis was performed with the GraphPad Prism software (version 9.0, San Diego, CA, USA), using the nonparametric Mann-Whitney U test for comparison of continuous variables between two groups and the nonparametric Kruskal-Wallis test, followed by Dunn's test, to compare more than two groups. Data are expressed as the mean ± standard error of the mean (SEM). Simple linear regression was used to assess the relationship between two variables. The level of significance was set to *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Figures S1 to S6
Tables S1 to S4 References (13, 33, 37, 45, 50-55)  . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Fig. 2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Fig. 3 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.      . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Fig. 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) preprint
The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Fig. 7 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.   (AIM2, JAK2, STAT1, and STAT2), as determined by RNA-Seq analysis of CD neutrophils, versus disease severity in CD. Simple linear regression was used in all panels to assess the relationship between the studied variables. CD, Crohn's disease; CDAI, Crohn's disease activity index (R, remission, < 150; M, mild to moderate, 150-220; S, moderate to severe, > 220); HI, healthy individuals; RNA-Seq, RNA-Sequencing; UC, ulcerative colitis.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Isolation of peripheral blood neutrophils and serum
Peripheral heparinized blood was collected and circulating neutrophils were isolated by Histopaque double-gradient density centrifugation (1.119,1 g/mL and 1.077,1 g/mL, Sigma-Aldrich, St Louis, MO, USA) at 700 ×g, for 35 min at 20-25 o C. Then, cells were washed once with phosphate-buffered saline (PBS-1×, Biosera) and centrifuged at 200 ×g for 12 min before being cultured. Neutrophils were adjusted to desired concentration and used within 2 hours after isolation. Cell purity was estimated ≥98%.
To collect serum, venous blood from either HI or patients with CD and/or UC was collected in appropriate BD Vacutainer® Plus Plastic Serum tubes (Becton, Dickinson, and Company) and centrifuged at 500 ×g for 15 min. Serum samples were stored at -80°C until analyzed.

Isolation, culture, and characterization of human primary intestinal fibroblasts
Human primary intestinal fibroblasts (PIFs) were isolated from colonic and/or terminal ileum biopsies obtained by colonoscopy from HI, active UC and CD patients, as described above, based on a standard lab protocol (50,51). PIFs in passages 3-6 were cultured in DMEM (31885-023; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; 10082147; Thermo Fischer Scientific), 200 U/mL antibiotic/antimycotic solution (15240062; Thermo Fisher Scientific), 1% non-essential amino acids (11140-035; Thermo Fisher Scientific) at 37 °C, 5% CO2. To verify their phenotype, cells were stained using antibodies against alpha-smooth muscle actin (α-SMA), vimentin, and desmin (52). PIFs were also plated in 6-well culture plates (≈0.8-1 x 10 6 cells/well; Corning Incorporated) in complete DMEM to collect their supernatant. Once cells were 60% to 70% confluent, the medium was removed, cells were rinsed in PBS, and fresh lowserum DMEM (2% FBS) medium was added for 24 hours. After 24 hours, the medium was collected, centrifuged and the supernatant was transferred to a new tube. Aliquots of the supernatant were stored at −80°C until use.

RNA isolation, cDNA synthesis and RT-qPCR
RNA was extracted from PIFs and peripheral blood neutrophils in TRIzol reagent following the manufacturer's protocol (15596026; Thermo Fischer Scientific). Equal amounts of RNA were used to synthesize cDNA for Real-time qPCR (RT-qPCR) analysis or to prepare cDNA libraries for RNA-Sequencing. cDNA for RT-qPCR was generated using PrimeScript TM RT Reagent Kit (RR037A; Takara) according to the manufacturer's instructions. RT-qPCR for Krüppel-like Factor 2 (KLF2), Interleukin-8 (IL8), Signal transducer and activator of transcription 1 (STAT1), Signal transducer and activator of transcription 2 (STAT2) or/and Cellular Communication Network Factor 2 (CCN2) was performed by a KAPA SYBR FAST qPCR Master Mix (2X) (KK4602; KAPA Biosystems). To normalize the expression of the above-mentioned genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. Details regarding the primer pairs and conditions of RT-qPCR are given in Supplementary Table S4. The data were quantified and analyzed using the 2 -ΔΔCt mathematical model.

In-cell ELISA (ICE assay, Cytoblot)
To study the intracellular protein levels of CCN2 or KLF2, PIFs were cultured in 96-well highbinding microplates (655061; Thermo Fisher Scientific) in the presence of distinct stimuli for 4 h, as previously described (13,37). Briefly, goat anti-CCN2 (2 μg/mL; sc14939; Santa Cruz Biotechnology Inc) or mouse anti-KLF2 (6 μg/mL; MA5-24300; Thermo Fisher Scientific) were . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint used as primary monoclonal antibodies. Horseradish peroxidase-conjugated donkey anti-goat IgG (1:1000 dilution, HAF109; R&D Systems) or donkey anti-mouse IgG (1:1000 dilution, HAF018; R&D Systems) were used as secondary antibodies. Absorbance was measured at 620 nm. The corrections were performed by subtracting the signal of the wells incubated in the absence of primary antibody.
Quantification and determination of mean fluorescence intensity (MFI) from fluorescence microscopy images using confocal microscopy was performed with ImageJ software (ImageJ for Windows, Version 1.53k).

Collagen measurement
The soluble collagen types (I-V) were determined using a Sircol Collagen Assay Kit. Briefly, PIFs were cultured in 6-well plates (Corning Inc.), treated with various agents and the culture supernatant was collected after 24 h, the time point at which the highest collagen production was observed. Cell debris was removed by centrifugation and the resulting supernatant was incubated overnight, at 4 o C, with isolation and concentration reagent, provided with the kit. Τhe assay was therefore conducted according to the manufacturer's protocol (CLRS1000; Biocolor), as previously described (13,53).

Multiplex cytokine measurement
The levels of multiple inflammatory cytokines were measured in serum and PIFs supernatants using the Human Inflammation Panel 1, LEGENDplex™ Multi-Analyte Flow Assay Kit (740809; Biolegend) in a CyFlow Cube 8 flow cytometer (Sysmex Partec, Germany), according to the manufacturer's instructions (45).

Immunohistochemistry (IHC-P), Masson's trichrome and Immunofluorescence (IF) staining in tissue sections.
To evaluate the presence of neutrophils in intestinal tissue biopsies, cross sections of 4 μm thickness, were stained using a mouse anti-human neutrophil elastase monoclonal antibody (NE; 1:50 dilution; clone NP57; DAKO). Serial cross sections, obtained from the same intestinal biopsies were further stained with Masson's trichrome to identify the presence of intestinal . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

In-vitro transwell migration assay (chemotaxis assay)
To evaluate neutrophil chemotaxis, low-serum complete DMEM medium suitable for the growth of intestinal fibroblasts (served as blank) and/or supernatant from HI, UC and CD PIFs were added to the lower chamber of a 24-well QCM TM chemotaxis 3μm cell migration assay (ECM505; Merck Millipore), as described in the manufacturer's instructions (55). In brief, freshly isolated human peripheral blood neutrophils (0,2-2 x 10 6 cells/mL) were seeded in the upper well in low-serum complete DMEM. After 2.5 hours of incubation at 37 o C and 5% CO2, migrating cells were quantified by measuring fluorescence using a Perkin Elmer fluorescence plate reader (Enspire, Waltham, MA, USA), set at excitation/emission wavelengths of 480/520 nm. Four independent experiments were performed, using neutrophils from a different healthy donor each time, in which mean fluorescence intensity was evaluated. As an inhibitor of neutrophil chemotaxis, a mouse anti-IL-8/CXCL8 monoclonal antibody with neutralizing effect (0,3 μg/mL; MAB208-100; R&D systems) was used, while an IgG rabbit polyclonal antibody (0,3 μg/mL; ab171870; Abcam) was used as a control in neutralization experiments.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Fig. S3
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Figure S3. Representative differentially expressed genes in peripheral blood neutrophils isolated from patients with IBD and HI. Graphs of representative DEGs (baseMean >30 and FDR <0.05), specifically upregulated in CD (A) or UC (B), commonly upregulated (C) or downregulated (D), or showing a reverse expression pattern (E), following RNA-Seq analysis of peripheral blood neutrophils from patients with CD (n=18) or UC (n=24). DEGs were identified following comparison with neutrophils isolated from healthy individuals (n=18). CD, Crohn's disease; DEGs, differentially expressed genes; HI, healthy individuals; UC, ulcerative colitis.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint

Fig. S7
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Figure S7. Transcriptomic alterations in peripheral blood neutrophils correlate with disease severity in IBD patients. (A, B) Heatmaps depicting the relative expression of all DEGs (baseMean > 30 and FDR < 0.05), as determined by RNA-Seq analysis of neutrophils isolated from healthy individuals (n=18) and CD (n=18), A) or UC patients (n=24), B). CDAI and Mayo DAI were used for assessing disease activity in CD and UC, respectively. (C) Correlation plots of the mRNA expression of key interferon signaling components, as determined by RNA-Seq analysis of CD neutrophils, versus serum IFNα2 levels. Simple linear regression was used to assess the relationship between normalized counts and IFNα2 levels. IBD, inflammatory bowel disease; FDR, false discovery rate; CD, Crohn's disease; ); UC, ulcerative colitis; CDAI, Crohn's disease activity index (R, remission, < 150; M, mild to moderate, 150-220; S, moderate to severe, > 220); HI, healthy individuals; Mayo DAI, Mayo score disease activity index (Mi, mild, 3-5; Mo, moderate, 6-10; S, severe, 11-12).
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 11, 2023. ; https://doi.org/10.1101/2023.09.08.23295281 doi: medRxiv preprint Table S4. Sequence of Primers and real-time RT-PCR conditions 1 Oligonucleotide primers were designed by Beacon Designer™ ver. 4.0.