Proteoglycofili are glycosaminoglycan-containing fibrillar
components released by neutrophils to neutralize
bacteria

Antonin André, Marina Barroso, Jurate Skerniskyte, Lorine Debande, Vanessa Paul, Nathan Broussaudier, Ahmad Sabbah, Jules Tritschler, Mélina Siegwald, Caroline Ridley, et al.

► To cite this version:

Antonin André, Marina Barroso, Jurate Skerniskyte, Lorine Debande, Vanessa Paul, et al. Proteoglycofili are glycosaminoglycan-containing fibrillar components released by neutrophils to neutralize bacteria. Cell Reports, 2025, 44 (11), pp.116541. 10.1016/j.celrep.2025.116541. hal-05400000

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Cell Reports

# Proteoglycofili are glycosaminoglycan-containing fibrillar components released by neutrophils to neutralize bacteria

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Antonin C. André, Marina V. Barroso, Jurate Skerniskyte, ..., Romain R. Vivès, David J. Thornton, Benoit S. Marteyn

## Correspondence

marteyn@unistra.fr

## In brief

Alternative to the formation of NETs, André et al. describe here that viable neutrophils may release a fibrillar component named proteoglycofili or PGF upon stimulation. PGF formation is associated with neutrophil degranulation. GAGs were identified as central components supporting PGF fibrillar structure and antimicrobial activity.

## Highlights

* Neutrophil PGF are composed of granular proteins, GAGs, granule proteins, and cytokines

* PGF are distinct from NETs; they are released by viable neutrophils and do not contain DNA

* PGF release is stimulated under anoxia or upon *Shigella* infection

* PGF target *Shigella* surface, degrade virulence factors, and block bacterial growth

<figure><img src="image_3.png" /></figure>
Article

# Proteoglycofili are glycosaminoglycan-containing fibrillar components released by neutrophils to neutralize bacteria

Antonin C. André,<fnref n="1" />,<fnref n="2" />,<fnref n="13" /> Marina V. Barroso,<fnref n="1" />,<fnref n="13" /> Jurate Skerniskyte,<fnref n="1" />,<fnref n="12" /> Lorine Debande,<fnref n="1" /> Vanessa Paul,<fnref n="1" />

Nathan Broussaudier,<fnref n="1" /> Ahmad Sabbah,<fnref n="1" /> Jules Tritschler,<fnref n="1" /> Mélina Siegwald,<fnref n="1" /> Caroline Ridley,<fnref n="3" /> Isabelle Svahn,<fnref n="4" />

Amber D. Bowler,<fnref n="5" /> Valérie Demais,<fnref n="6" /> Katia Boniface,<fnref n="7" /> Stéphane Rigaud,<fnref n="8" /> Jean-Yves Tinevez,<fnref n="8" /> Philippe J. Sansonetti,<fnref n="2" />

Freddy Radtke,<fnref n="5" /> Romain R. Vivès,<fnref n="9" /> David J. Thornton,<fnref n="3" /> and Benoit S. Marteyn<fnref n="1" />,<fnref n="10" />,<fnref n="11" />,<fnref n="14" />,*

## SUMMARY

Here, we uncover that neutrophils release a fibrillar complex, named proteoglycofili (PGF), under hypoxic conditions or upon bacterial infection, using *Shigella* as a model. PGF are preferentially released by neutrophils over neutrophil extracellular traps upon bacterial infection. PGF are released by living neutrophils, do not contain DNA, are mainly composed of granule proteins, and contain cytokines. We reveal that the fibrillar structure of PGF is sustained by two glycosaminoglycans (GAGs), hyaluronic acid, and chondroitin sulfate, released by neutrophils. We demonstrate that PGF are potent antimicrobials, which degrade virulence factors of *Shigella* and block its growth, similar to *E. coli* or *Salmonella*. GAGs are essential for PGF antimicrobial activity both *in vitro* and *in vivo*. Beyond bacterial infections, and reported in a mouse model of colon cancer, our results strongly suggest that GAGs may be released by neutrophils in a broad range of inflammatory diseases.

## INTRODUCTION

Neutrophils are essential players of the antimicrobial host response and modulate the adaptive immune response in a wide range of diseases. Historically, neutrophils were mainly known for their capacity to detect and kill pathogens by internalization or through the secretion of potent antimicrobial compounds, including proteins stored in pre-formed granules (α, β1, β2, and γ), or reactive oxygen species (ROS). It has long been considered that antimicrobials released by neutrophils are mainly soluble and freely diffuse within inflammatory or infectious tissues. Recently, it was reported that neutrophils release heterogeneous and non-soluble antimicrobial complexes such

as neutrophil extracellular traps (NETs),<fnref n="1" /> exosomes,<fnref n="2" /> LC3-dependent extracellular vesicles,<fnref n="3" /> and elongated neutrophil-derived structures (ENDS).<fnref n="4" /> Little is known about the impact of pathological microenvironments on the ability of neutrophils to release these non-soluble complexes. As we and others have reported that hypoxia was induced in many inflammatory or infectious contexts,<fnref n="5" /> we challenged the capacity of neutrophils to produce non-soluble complexes in these conditions.<fnref n="6" /> Neutrophils are glycolytic cells, which are well-adapted to low-oxygen environments, in which their survival is extended *in vitro*.<fnref n="7" /> Hypoxia has been shown to induce neutrophil degranulation<fnref n="8" /> and to reduce both neutrophil ROS production and ROS-dependent NET formation.<fnref n="9" />,<fnref n="10" />

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l'ARN, CNRS UPR9002, 67000 Strasbourg, France</footnote>

<footnote n="2">²Institut Pasteur, Université Paris Cité, Inserm U1202, Unité Pathogénie Microbienne Moléculaire, 75015 Paris, France</footnote>

<footnote n="3">³Wellcome Centre for Cell-Matrix Research and the Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK</footnote>

<footnote n="4">⁴University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UAR 3420, US 4, 33000 Bordeaux, France</footnote>

<footnote n="5">⁵Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland</footnote>

<footnote n="6">⁶Plateforme Imagerie In Vitro, CNRS UAR 3156, University of Strasbourg, Strasbourg, France</footnote>

<footnote n="7">⁷University of Bordeaux, CNRS, Immuno ConcEpT, UMR 5164, 33000 Bordeaux, France</footnote>

<footnote n="8">⁸Institut Pasteur, Université Paris Cité, Image Analysis Hub, 75015 Paris, France</footnote>

<footnote n="9">⁹Université de Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France</footnote>

<footnote n="10">¹⁰University of Strasbourg Institute for Advanced Study (USIAS), Strasbourg, France</footnote>

<footnote n="11">¹¹Institut Pasteur, Université de Paris, Inserm U1225, Unité de Pathogenèse des Infections Vasculaires, 75015 Paris, France</footnote>

<footnote n="12">¹²Present address: Life Sciences Center, Institute of Biosciences, Vilnius University, Vilnius, Lithuania</footnote>

<footnote n="13">¹³These authors contributed equally</footnote>

<footnote n="14">¹⁴Lead contact</footnote>

<footnote>*Correspondence: marteyn@unistra.fr
https://doi.org/10.1016/j.celrep.2025.116541</footnote>
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_10.png" /></figure>

(legend on next page)
Analyzing complexes released by neutrophils under low-oxygen or infectious conditions (using *Shigella* as a model<sup>11</sup>) allowed the identification of a non-soluble and fibrillar complex released by viable neutrophils, hereafter named proteoglycofili (PGF). PGF was confirmed to be DNA-free and to contain granule proteins at high yield. This extracellular fibrillar component was identified using adapted neutrophil purification and culture methods under anoxic conditions<sup>7</sup> and specific markers (e.g., Myelotracker<sup>12</sup>) developed in our team. Unexpectedly, two glycosaminoglycans (GAGs), hyaluronic acid (HA), and chondroitin sulfate, were found to enter the composition of PGF. The GAG family is composed of heparin, heparan sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), and HA. In the 1950s–60s, various GAGs, including HA, CS-like substances, or HS, were detected in total neutrophil extracts.<sup>13–15</sup> However, the ability of neutrophils to release GAGs has not been reported until this study. The contribution of HA and CS to PGF fibrillar structure and function was further deciphered here in the context of *Shigella* infection.

## RESULTS

### Neutrophils release PGF during shigellosis and under anoxia

PGF was observed in the colonic mucosa of guinea pigs infected by *Shigella*,⁵ and appeared as DNA-free fibrillar structures stained with Myelotracker (or MUB₄₀, a lactoferrin-specific marker¹²) (Figure 1A). PGF were detected in the vicinity of neutrophils, recruited at *Shigella* foci of infection (Figure 1A). Conversely, the release of NETs, as previously reported in vivo in the context of *Shigella* infection,¹ neutrophil exosomes, LC3-dependent extracellular vesicles, or ENDs were not observed in these samples, using these markers. This result suggested that PGF were released by neutrophils, as they contain lactoferrin (specifically stored in neutrophil β1, β2 granules), and were not detected in healthy tissues (Figure 1A). As we previously showed that an infectious hypoxia was induced by *Shigella*,¹¹ we hypothesized that PGF release may be promoted either by hypoxia or *Shigella* infection.

To address this hypothesis and characterize PGF in more detail, human neutrophils were purified and cultured in vitro under anoxic conditions (Figure 1B), which extends neutrophil viability and maintains their basal state¹⁶ (Figures 1C and S1A). We confirmed that in these conditions, viable neutrophils release a fibrillar material composed of carbohydrate-containing macromolecules, as detected by Alcian blue and periodic acid-Schiff's (PAS) staining (Figure 1D); the amount of PGF released by neutrophils increased over the culture time (Figure S1B).

PGF release was observed on glass- or plastic-bottom devices, without coating. Based on their composition and fibrillar ultrastructure, these extracellular non-soluble components were named PGF. It is important to note that the integrity of PGF, like other mucoidal biomaterials, was optimally preserved upon fixation with a Carnoy's solution, compared to other fixative solutions such as paraformaldehyde (Figure S1C). Using scanning electron microscopy (SEM), we determined that the mean diameter of individual PGF fibers released by neutrophils was 43.9 ± 20.3 nm and may associate with each other to form much thicker PGF filaments (>100 nm) (Figure 1E).

We confirmed that neutrophils secreting PGF have multilobed nuclei (Figure 1F), and the presence of lactoferrin within PGF strongly suggested that neutrophil degranulation occurred upon PGF release. These results were consistent with previous reports showing that neutrophil degranulation was induced under low-oxygen conditions.⁸ Furthermore, it appears that PGF fibers may differ from NETs as they are released by viable neutrophils, and no DNA was detected by DAPI staining (Figure 1F). We further aimed to determine PGF biochemical properties and overall composition (proteins and cytokines) to define their function.

### PGF are mainly composed of granule proteins and do not contain DNA

To proceed, PGF were purified using differential centrifugation of anoxic neutrophil cultures. We confirmed by SEM the fibrillar organization of purified PGF (Figure 2A); we further reported their stability in a wide range of pH and in the presence of high salt (CaCl₂ and KCl) concentrations (Figure S2A).

As neutrophil activation is associated with cytokine release, we hypothesized that PGF may contain a cocktail of cytokines. By screening a set of neutrophil cytokines, we demonstrated that a high yield of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-8 were detected in PGF (Figure 2B). This result suggests that PGF may serve as platforms for local accumulation of cytokines to promote cytokine gradient formation.

In parallel, we demonstrated by tandem mass spectrometry that PGF were mainly composed of granule proteins (Figure S2B); the most abundant ones were myeloperoxidase, elastase, lactoferrin, cathelicidin, and albumin (Figure 2C). The presence of these proteins within purified PGF was confirmed by western blot analysis, using both SDS-PAGE and Ag-PAGE, the latter allowing the migration of high molecular weight components, such as PGF (Figure 2D). The presence of these additional granule proteins within PGF released by neutrophils was confirmed in situ by immunofluorescence (Figure 2E). As expected, these granule proteins were found to be accumulated

**Figure 1. Neutrophils release PGF during shigellosis and under anoxia**

(A) Detection of PGF in vivo with Myelotracker-Cy3<sup>12</sup> (lactoferrin, red) within the guinea pig colonic mucosa infected by *Shigella* pGFP (green). DNA was stained with DAPI (blue). White boxes indicate neutrophils (non-infected) or PGF DNA-free fibrillar structures (+*Shigella*). Scale bars, 10 µm.

(B) Method of purification and culture of neutrophils under anoxia.

(C) Viability of neutrophils releasing PGF under anoxia (PI-neutrophils, n = 3, mean ± SD, “ns” indicates *p* > 0.05 [Student's *t* test], see also Figure S1A).

(D) PGF released by neutrophils stained with Alcian blue or PAS after fixation with a Carnoy's solution, (18-h culture, –O₂). Scale bars, 100 µm.

(E) SEM imaging of neutrophils secreting PGF. Scale bars, 300 nm. Diameter of PGF individual fibers (n = 377).

(F) Neutrophils releasing PGF (4-h culture, –O₂) fixed in a Carnoy's solution and stained with Myelotracker-Cy5 (magenta) and DAPI (blue). Arrowheads indicate PGF fibers. Scale bars, 40 µm.
<figure><img src="image_11.png" /><figcaption>Figure 2. PGF protein moiety is mainly composed of granule proteins and contains a cytokine cocktail</figcaption></figure>

(A) SEM imaging of purified PGF. Scale bars, 200 nm.

(B) PGF cytokine content (mean ± SD, n = 3).

(legend continued on next page)
within PGF in the vicinity of *Shigella* hypoxic foci of infection *in vivo* (Figure 2F). Considering the abundant release of granule proteins upon PGF release by neutrophils, we hypothesized that transcription of the corresponding genes may be activated. However, we did not observe such a regulation by RNA sequencing (RNA-seq) analysis *in vitro* during PGF secretion (Figure S2C).

In conclusion, these results identify granule proteins as major components of the PGF protein moiety; in addition, they strongly suggest that all types of neutrophil granules were mobilized during PGF formation. Additional and preliminary results suggest the presence of lipids (labeled with Bodipy) and actin (labeled with phalloidin) in the vicinity of PGF, if not associated with (Figure S2D), which will have to be further investigated in subsequent studies.

Among other non-soluble complexes, which have been previously shown to be released by neutrophils upon activation, PGF structure is clearly distinct from exosomes, LC3-dependent extracellular vesicles, or ENDS. PGF share the fibrillar structure of NETs, and we confirmed, using complementary approaches, that these structures were distinct. Using SEM, we confirmed that upon PGF secretion, the integrity of neutrophils was maintained, as opposed to the disintegration of the plasma membrane observed upon NET formation (Figure 3A). In contrast to PMA-induced NETs, we confirmed that no extracellular DNA was detected within PGF (Figures 3B and 3C), while Myelotracker labeled both (Figure 3C), as confirmed by quantitative imaging analysis (Figure 3C). Contrary to NETs,¹ PGF were not susceptible to DNase I but were efficiently disrupted by proteinase K, as expected (Figure S3). In conclusion, although they share a similar fibrillar structure, PGF are distinct from NETs, as they do not contain DNA and are released from viable neutrophils.

**Shigella** induces neutrophil PGF release, which binds to its surface, blocks its growth, and degrades its virulence factors

As PGF was preferentially released by neutrophils within the *Shigella* foci of infection (Figures 1A and 2F), we further evaluated its impact on *Shigella* survival and virulence. First, we confirmed that *Shigella* may stimulate neutrophils for PGF release *in vitro*, by performing infections in –O₂ and +O₂; indeed, neutrophils infected with *Shigella* for short periods of time (1 or 2 h) released PGF at high yields (Figures 4A, S4A, and S4B). Moreover, a direct interaction between *Shigella* and PGF was observed upon neutrophil infection (Figures 4A, S4A, and S4B) and was confirmed by 3D-reconstitution imaging (Figure 4B). We could additionally visualize that extracellular bacteria may be engulfed within PGF fibers (Figure 4B). We confirmed here *in vitro* that PGF release was mostly promoted over NET formation upon *Shigella* infection in all the tested conditions (Figure 4C), consistent with the results obtained *in vivo* (Figure 1A). Interestingly, we could also demonstrate that upon infection of neutrophils, *Shigella* remained mainly extracellular,

as only a limited number of bacteria were phagocytized. In addition, most extracellular bacteria were tagged by PGF (PGF+, Figure 4D). These results strongly support the importance of PGF in the neutrophil response to *Shigella* infection, compared to other neutrophil antimicrobial functions.

We first confirmed that PGF has a proteolytic activity using casein as a target; importantly, we showed that PGF did not degrade important human proteins involved in the host antimicrobial responses, such as lactoferrin, and the heavy and light chains of IgG and IgA (Figure 4E). Based on these results and the ability to directly target *Shigella* cell surface, we hypothesized that PGF could target *Shigella* virulence components. The pathogenicity of *Shigella* relies on the expression of secretion systems. The invasion of epithelial cells by *Shigella* is mediated by an oxygen-dependent type 3 secretion system (T3SS).¹⁷ *Shigella* may additionally export virulence factors belonging to the type 5 secretion system (T5SS) during its dissemination within infected tissues.¹⁸,¹⁹ Here, we demonstrated that PGF degraded all tested *Shigella* T3SS virulence factors (IpaB, IpaC, and IpaD) or T5SS virulence factors (e.g., IcsA), including secreted protease (SepA, Pic, and SigA) (Figure 4F). On a limited number of targets (SepA and Pic), we confirmed that the PGF proteolytic activity was abolished in the presence of a protease inhibitor cocktail or upon heat inactivation (Figure S5A). These results are consistent with a previous study showing that *Shigella* virulence factors were degraded by elastase,²⁰ a component of PGF, which may contribute to the PGF proteolytic activity.

In addition to the degradation of *Shigella* virulence factors, we demonstrated that purified PGF blocked the growth of *S. flexneri* 5a *in vitro* (Figure 4G). This property was not restricted to this *Shigella* strain, as the same results were obtained on other *Shigella* species, such as *S. flexneri* 2a and *S. sonnei* (Figure S5B), and other bacteria, such as *E. coli* K12 or *S. typhimurium* (Figure 4G). We confirmed that the antimicrobial activity of PGF was maintained to the same extent regardless of the pO₂, using *S. flexneri* 5a as a target (Figure S5C). Using *E. coli* K12 as a target, we reported that the antimicrobial activity of PGF was surprisingly not altered by heat-inactivation (Figure S5D), in contrast to its proteolytic activity (Figure S5A). This result suggests that, in addition to granule proteins, other components were essential for PGF antimicrobial activity. In the last section of this study, we further aimed to identify these additional components, which may additionally sustain the PGF fibrillar structure.

**PGF contains two GAGs: HA and CS sulfate**

As PGF fibers were stained with polysaccharide markers (Figure 1D), we hypothesized that GAGs may be these yet unidentified components of PGF, as the intracellular production of GAGs by neutrophils has been shown in several old studies,¹⁴,¹⁵,²¹ while their precise location remained discussed (granules or lysosomes).²² The importance of GAGs in neutrophil function has not been investigated further since then and their

<footnote>(C–F) Purified PGF protein content identified by MS (C) (see also Figure S2B) and confirmed on SDS-agarose gel and SDS-PAGE gel (D) by Coomassie staining and western blot using specific antibodies. (E) Neutrophil-secreting PGF was fixed with a Carnoy's solution and analyzed by immunofluorescence imaging using specific antibodies. (F) Immunodetection of the most abundant PGF proteins within *S. flexneri* 5a pGFP (green) foci of infection in guinea pig colonic mucosa. Samples were additionally stained with DAPI (blue) and Myelotracker-Cy3 (lactoferrin, red). Scale bars, 10 µm.</footnote>
<figure><img src="image_12.png" /><figcaption>Figure 3. PGF are distinct from NETs</figcaption></figure>

(A) Comparative morphological analysis of neutrophils secreting PGF (4-h culture, -O₂ conditions) and neutrophils releasing NETs (4-h culture, +O₂ conditions, +PMA) by SEM at ×4,000, ×10,000, and ×25,000 magnifications. Scale bars, 1 µm.

(B) Comparative detection of DNA in PGF and NETs in neutrophil cultures (cell vs. supernatant [sup.]) in conditions described in (A).

(C) Detection of DNA in NETs and PGF by immunofluorescence imaging (Myelotracker-Cy5, lactoferrin [magenta], and DAPI [blue]). Scale bars, 40 µm, combined with quantitative image analysis.

secretion has not been reported. We could confirm this hypoth-
esis as we demonstrated using analytical methods that two
GAGs, namely hyaluronic acid (HA) and chondroitin sulfate
(CS), were components of purified PGF (Figure 5A); concerning
CS, only non-sulfated (non-S) and mono-sulfated (4S/6S) chon-
droitin were detected (Figure 5A). We confirmed that HA and CS,
but not heparan sulfate (HS) or keratan sulfate (KS), were immu-
nodetected in PGF on Ag-PAGE (Figure S6A). These results were
confirmed by immunofluorescence, showing the colocalization
of both HA and CS together with lactoferrin in PGF released by
neutrophils (Figure 5B). Preliminary observations suggested
the presence of actin in the vicinity of PGF, potentially associated
with HA and CS (Figure S6B).

GAGs are essential for PGF antimicrobial activity and
fibrillar structure

We further aimed to decipher the importance of HA and CS in
PGF fibrillar structure and antimicrobial activity. To proceed,
specific GAG-targeting enzymes were used. Bovine hyaluroni-
dase type I-S (Hyal. type I-S) cleaves β-N-acetylhexosamine-

[1-4]-glycosidic bonds in both HA and CS (Figure 5C),
hyaluronidase from Streptomyces hyalurolyticus (Hyal. Strep.)
cleaves β-N-acetylhexosamine-[1-3]-glycosidic bonds in HA
only (Figure S6C), and hyaluronidase 4 (Hyal. 4) cleaves β-N-ace-
tylhexosamine-[1-3]-glycosidic bonds in CS only (Figure S6C).

We demonstrated that the degradation of both HA and CS with
Hyal. type I-S abolished PGF antimicrobial activity, using
S. flexneri 5a as a target (Figure 5D). A similar result was obtained
by the combined action of Hyal. 4 and Hyal. Strep., as expected
(Figures S6D and S6E). Targeting HA only (+Hyal. Strep.) or CS
only (+Hyal. 4) was not sufficient to abolish PGF antimicrobial ac-
tivity (Figures S6D and S6E). Similar and consistent results were
obtained using E. coli K12 as a PGF target (Figures S7A and
S7B), confirming the essential contribution of HA and CS to
PGF antimicrobial activity. As control, we demonstrated that
the degradation of the PGF protein moiety with proteinase K
abolished PGF antimicrobial activity (Figures S6D, S6E, and
S7A-S7D), suggesting that interactions between GAGs and pro-
teins composing PGF are essential for their antimicrobial activity.
As a control, we confirmed that DNase I treatment did not alter
<figure><img src="image_13.png" /><figcaption>**Figure 4. Neutrophil PGF bind to the extracellular *Shigella* surface and dampen its virulence**</figcaption></figure>

(A) Induction of PGF secretion by *S. flexneri* 5a infection (1 h, —O₂, see also Figures S4A and S4B).

(B) Direct contact between PGF and *Shigella* infection (1 h, —O₂) by 3D segmentation. DAPI (blue), *Shigella* (green), and PGF (magenta). Scale bars, 30 µm (left) and 10 µm (right).

(C) Proportion of PGF and NETs among extracellular fibrillar structures released by neutrophils upon *Shigella* infection (1 or 2 h, —O₂ or +O₂) (n = 3).

(D) Proportion of intra- or extracellular *Shigella* upon neutrophil infection. (1 or 2 h, —O₂ or +O₂), including scoring of extracellular bacteria coating by PGF (PGF+) (n = 3).

(E) Absence of degradation of purified human lactoferrin and immunoglobulins (IgG and IgA heavy/light chains) by PGF, Coomassie staining.

(F) Degradation of *Shigella* T3SS and T5SS virulence factors by PGF (overnight incubation, 37°C), Coomassie staining (SepA, Pic, SigA), or western blotting (IpaB, IpaC, IpaD, and IcsA).

(G) Inhibition of *S. flexneri* 5a, *E. coli* K12, and *S. typhimurium* growth by PGF (M9 agar pads). Scale bars, 5 µm. Quantification of bacterial cultures' confluence (n = 3, mean ± SD, *p* < 0.05, **p* < 0.01, and ***p* < 0.001 (Student's t test).
<figure><img src="image_14.png" /><figcaption>Figure 5. Neutrophil PGF contain two glycosaminoglycans, which are essential for their fibrillar structure and antimicrobial activity</figcaption></figure>

(A) Quantification of HA and CS in purified PGF by ELISA (mean ± SD, *n* = 3) and RPIP-HPLC (mean ± SD, *n* = 3), respectively.

(B) HA (green) and CS (magenta) were immunodetected with specific antibodies together with lactoferrin (Myelotracker-Cy3, red) and DNA (DAPI, blue) on neutrophils secreting PGF (Carnoy's fixation). Scale bars, 20 µm.

(legend continued on next page)
PGF antimicrobial activity using both S. *flexneri* 5a and E. *coli* as targets (Figures 5D, S7A, and S7B).

We further aimed at defining the impact of GAG-targeting enzymes on PGF ultrastructure, as we hypothesized that GAGs were responsible for PGF fibrillar structure, based on their intrinsic capacity to oligomerize in long linear oligosaccharide chains. Indeed, we demonstrated that, in the presence of Hyal. type I-S, PGF released by neutrophils were disrupted and no longer fibrillar (Figure 5E). As expected, a similar result was obtained by the combined action of Hyal. 4 and Hyal. Strep. (Figure S7C). These results confirmed the central contribution of GAGs to PGF fibrillar structure. It is noteworthy that the degradation of HA only (+Hyal. Strep.) or CS only (+Hyal. 4) was insufficient to fully disrupt the PGF structure (Figure S7C). We confirmed that PGF were not susceptible to DNase I, as expected (Figure 5A), while proteinase K disrupted the protein moiety of PGF (Figure S7C). Together, these results emphasize the importance of HA and CS, which are essential to PGF fibrillar structure and contribute to their antimicrobial activity.

## PGF GAGs target *Shigella* upon infection, in vitro and in vivo

As we reported that *Shigella* infection induced neutrophil PGF release and its subsequent binding to the extracellular bacterial surface (Figures 4A-4D, S4A, and S4B), we specifically deciphered the fate of PGF GAGs in this process. We first demonstrated that, upon infection, the surface of most extracellular *Shigella* was coated with both HA and CS released by neutrophils *in vitro* (Figures 6A, 6B, and S8A, regardless of the fixation method). This result was strengthened by transmission electron microscopy (TEM) combined with immunogold staining of HA or CS, which was confirmed on extracellular bacteria (Figures 6C and 6D). We further aimed at deciphering intracellular trafficking pathways involved in the *Shigella*-mediated HA and CS release. However, none of the tested specific inhibitors (PI3K, p38, or Akt inhibitors) blocked PGF release and extracellular bacteria coating with HA or CS (Figure S8B). It must be noticed that we could visualize by TEM the formation of the compartmentalized reservoir of CS and HA within the cytoplasm of activated neutrophils (Figures 6C and 6D). These compartments were relatively small, under basal conditions (Figures 6C and 6D, non-infected), although both their size and their GAG content significantly increased upon *Shigella* infection (Figures 6C and 6D, +*Shigella*). Further investigations will be required to fully characterize these compartments and the dynamics of their formation, beyond these initial observations. Together, these results confirmed that both PGF GAGs (HA and CS) directly target the extracellular *Shigella* surface.

*In vivo*, using the guinea pig model of shigellosis, we confirmed that most, if not all, extracellular *Shigella* were coated with HA and CS (Figures 6E and 6F), consistent with results obtained *in vitro* (Figures 6A and 6B). In addition, we could

report the presence of HA and CS within DNA-free PGF fibrillar structures, in the vicinity of *Shigella* foci of infection (Figures 6E and 6F), confirming our preliminary results (Figure 1A). The length and size of PGF filaments detected *in vivo* were reduced compared to those released by purified neutrophils *in vitro*, although the colocalization of lactoferrin, HA, and CS was confirmed in both. These results shed new light on the adaptation of neutrophil antimicrobial activity under low-oxygen conditions, encountered within *Shigella* foci of infection.¹¹ suggesting that neutrophils may limit *Shigella* dissemination by directly targeting bacteria extracellularly.

To confirm the importance of GAGs in PGF antimicrobial activity on *Shigella* *in vivo*, the impact of Hyal. type I-S was assessed during *Shigella* infection. Consistent with the results obtained *in vitro* (Figure 5D), we confirmed that treatment with Hyal. type I-S increased the severity of shigellosis symptoms together with an increased destruction of the colonic mucosa by *Shigella* (Figures 6G and 6H). No significant adverse effect of the Hyal. type I-S treatment was observed on their colonic mucosa structural organization and integrity (Figures 6G and 6H). These results confirm that GAGs released by neutrophils within PGF are essential to control *Shigella* infection *in vivo*.

## Neutrophils release PGF within murine colon inflammatory tumors

Beyond the validation of the importance of GAG-containing PGF in the host response to bacterial infection, in this last section, we further aimed to extend the study of this fibrillar compound to other inflammatory models. We proceeded in an inflammatory colon tumor mouse model (Apc<sup>lox/lox</sup> Cdx2- CreERT2<sup>fl/fl</sup>) in which neutrophil infiltration was previously characterized and associated with T cell suppression.²³ We first confirmed *in vitro* that mouse neutrophils released PGF, similarly to human neutrophils, and that HA and CS composed mouse neutrophil PGF (Figure 7A). We demonstrated that DNA-free PGF structures, co-stained with lactoferrin-, HA-, and CS-specific antibodies, were abundantly detected in the vicinity of neutrophils and accumulated within the inflammatory colon tumor (Figures 7B and 7C). These results confirmed the relevance of neutrophil PGF release in this model of inflammatory disease and strongly suggested that it may be of great importance in a wide range of inflammatory diseases, which will have to be defined further.

## DISCUSSION

In this study, we report that neutrophils release at least two members of the GAG family, HA and CS. We show that these GAGs are essential for the structure and antimicrobial activity of fibrillar structures named PGF. PGF are released by living neutrophils; low-oxygen conditions or bacterial infections have been identified as triggers of PGF release. Changes in PGF shape or

<footnote>(C) Cleavage sites of hyaluronidase bovine type I-S (Hyal. type I-S - orange), within HA and CS oligomers.</footnote>

<footnote>(D) Blocking of PGF-mediated inhibition of S. *flexneri* 5a growth upon treatment with Hyal. type I-S, not DNase I (n = 3, mean ± SD, ***p < 0.001 and “ns” indicates *p* > 0.05 [Student's *t* test]).</footnote>

<footnote>(E) Human neutrophils secreting PGF (–O₂, 4 h) were post-treated for 1 h with Hyal. type I-S or DNase I. Arrowheads indicate intact PGF. Samples were stained with Myelotracker-Cy3 (lactoferrin, red)-, α-HA (green)-, α-CS (magenta)-specific antibodies, and DAPI (DNA-blue). Scale bars, 50 µm.</footnote>
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

(legend on next page)
length may be observed in the context of *Shigella* infection (in vitro versus in vivo); their identification was validated by a triple-staining using lactoferrin-, HA-, and CS-specific antibodies or markers and negative staining with DAPI. The results presented in this princeps study, obtained using various models, in vitro or in vivo, confirm that PGF are distinct from NET, although it may not be excluded that PGF release may be related to NET formation, as they share some characteristics. Further investigations will be required to clarify this fundamental question.

In the context of the infectious hypoxia caused by *Shigella*,⁵,¹¹ we identified an oxygen-independent antimicrobial weapon deployed by neutrophils, which significantly limits the dissemination of *Shigella* in the colonic mucosa. It will be interesting to further study the interaction of *Shigella* and PGF over time, at different steps of the infectious process, as changes in PGF composition, shape, or length may be observed together with changes in the tissue oxygenation level. In addition, it will be of great interest to investigate whether the LC3 decoration of extracellular bacteria previously reported by our team²⁴ is related to PGF release.

Beyond their detection in the context of *Shigella* infection, we confirmed the release of GAGs by neutrophils *in vivo* in a murine model of colon cancer, and we anticipate that they will be reported in the future in a wide range of inflammatory diseases. It will also be interesting to investigate the presence of GAGs in other components released by neutrophils, such as NETs,¹ exosomes,² LC3-dependent extracellular vesicles,³ or elongated neutrophil-derived structures (ENDS),⁴ which were all only recently identified. Indeed, the cocktails of proteins composing PGF, NETs, and these various extracellular vesicles are comparable and mainly composed of granule proteins (e.g., MPO, lactoferrin, MMP9, albumin, among others).

Further investigation of GAGs and granule proteins is required to better understand their immunomodulatory role upon release. It is expected that the GAG-dependent fibrillar structure of PGF may contribute to the accumulation of degranulated proteins within inflammatory sites, hence increasing their concentration locally. This may be relevant for TNF-α, IL-6, and IL-8, which were detected in purified PGF. Further investigations will be required to define their association with GAGs in PGF, as GAGs have long been known to interact with high affinity with chemokines such as IL-8, CXCL1, CXCL5²⁵,²⁶ suggesting that PGF may play a central role in the formation of cytokine concentration gradients *in vivo*, favoring the chemoattraction of other immune cells

to inflammatory sites. The immunomodulatory functions of GAGs in PGF need to be explored further, since they are anticipated to activate many immune cells expressing the HA-receptor CD44.²⁷,²⁸ Further investigations will also be required to decipher the intracellular mechanisms involved, since compartmentalized stocks of GAGs were also found in the neutrophil cytoplasm in the present study.

Overall, and beyond this initial study, it will be important to better characterize stimulatory conditions leading to PGF release in various inflammatory diseases. We identified $pO_2$ as a potential factor influencing the release of PGF (in vitro and in vivo), suggesting that PGF will be preferentially released during tumor, infectious, or inflammatory hypoxia.¹¹,²⁹ This is most likely because hypoxia induces neutrophil degranulation⁸ and reduces both neutrophil ROS production and ROS-dependent NET formation.⁹,¹⁰ However, additional context-specific stimulatory signals may be further identified. Based on the results presented in this study, we anticipate that new inflammatory markers and new drugs targeting neutrophil-released products will be developed.

## Limitations of the study

Although many methods have been optimized to visualize and characterize GAG-containing PGF, several potential caveats remain. First, the structural organization of PGF fibers was characterized on fixed samples. Fixation preserves some components but may alter others; here, Carnoy's fixative solution may damage lipids composing PGF. Alternative fixative solutions may be used to address this point, especially to further improve microscopy imaging resolution and colocalization studies. Second, the PGF purification methods are based on differential centrifugations, as previously applied to purify NETs.¹ This method was also applied at higher centrifugation speed to purify exosomes,² LC3-dependent extracellular vesicles,³ and ENDS.⁴ Therefore, cross-contamination between these secreted components may occur. It may be further envisaged that they may be physiologically associated during neutrophil activation.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Benoit S. Marteyn (marteyn@unistra.fr).

<caption>**Figure 6. Neutrophil HA and CS target Shigella and are essential to limit tissue destruction**</caption>

(A) Accumulation of neutrophil CS (red, top panel) and HA (red, bottom panel) on *S. flexneri* 5a (green) surface (white squares). MOI 10, 1-h infection, Carnoy's fixation. DNA was stained with DAPI (blue). Scale bars, 20 µm.

(B) Proportion of extracellular and intracellular bacteria; extracellular HA+ and CS+ bacteria scoring (n = 3).

(C) Immunodetection of HA and CS by TEM immunogold staining using specific antibodies (anti-HA and anti-CS) in neutrophils under basal conditions (non-infected) or upon 1-h *Shigella* infection under anoxic conditions (+*Shigella*). "i.c." indicates intracellular compartment and "b." indicates bacteria. Scale bars, 1 µm.

(D) Quantification of the number of HA- or CS-containing vacuoles per neutrophil with or without *Shigella* infection.\*p < 0.01 and \*\*\*p < 0.0001 (Student's t test).

(E) Detection of PGF- and GAG-coated *in vivo* within *S. flexneri* 5a (green) infectious sites. Samples were stained with Myelotracker-Cy5 (lactoferrin, magenta)-, α-HA (red)-, and α-CS (yellow)-specific antibodies. DNA was stained with DAPI (blue). Arrowheads indicate PGF; asterisk (*) indicates HA- and CS-coated bacteria. Scale bars, 20 µm.

(F) Proportion of extracellular and intracellular bacteria; extracellular HA+ and CS+ bacteria scoring (n = 3).

(G and H) Impact of local Hyal. type I-S treatment (degrading both HA and CS) on shigellosis symptoms and guinea pig colonic mucosa thickness. Scale bars, 300 µm. Mean ± SD, n = 3, \*p < 0.01 and \*\*p < 0.001 (Student's t test).
<figure><img src="image_22.png" /><figcaption>**Figure 7.** GAG-containing PGF are released by neutrophils in mouse colonic tumors</figcaption></figure>

(A) GAG-containing PGF released by mouse neutrophils (4-h culture in -O₂ conditions. Upon Carnoy's fixation, PGF were stained with Myelotracker-Cy3 (lactoferrin, red)-, α-HA (green)-, α-CS (magenta)-specific antibodies, and DAPI (DNA-blue). Scale bars, 50 µM.

(B and C) Detection of PGF in neutrophil-infiltrated mouse colonic tumors (Apc<sup>lox/lox</sup> Cdx2-CreERT2<sup>Tg/+</sup>). (B) Healthy colon and colon tumors were stained with hematoxylin and eosin for morphological analysis. Scale bars, 200 mm. (C) For immunofluorescence analysis, healthy colon and colon tumors were co-stained with Myelotracker-Cy5 (lactoferrin, magenta)-, α-HA (red)-, and a-CS (yellow)-specific antibodies to visualize neutrophils and released PGF (indicated with white arrowheads). Scale bars, 50 µm.

**Materials availability**

* There are restrictions to the availability of Myelotracker-Cy3 and Myelotracker-Cy5, which are distributed by Bertin Technologies.

* There are restrictions to the availability of purified PGF because of the lack of an external centralized repository for their distribution and our need to maintain the stock. We are glad to share purified PGF with reasonable compensation from the requestor for the processing and shipping.

* All stable reagents generated in this study are available from the [lead contact](#) without restriction.

**Data and code availability**

* All data reported in this paper will be shared by the [lead contact](#) upon request.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the [lead contact](#) upon request.

**ACKNOWLEDGMENTS**

The authors sincerely acknowledge the support of the Biological Mass Spectrometry core facility at the University of Manchester; the Interdisciplinary Research Institute of Grenoble (IRIG, CEA); and the Bordeaux Imaging Center, a member of the FranceBiolmaging national infrastructure.

A.C.A. was granted a PhD fellowship from the University of Paris; L.D., A.S., and J.T. were granted a PhD fellowship from the University of Strasbourg. We acknowledge the support from the Biological Mass Spectrometry core facility at the University of Manchester. I.S. acknowledges the integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work was supported by the Agence Nationale de la Recherche ANR grant (grant nos. ANR-17-CE15-0012, ANR-21-CE15-0039 [NEUTROX], ANR-22-CE15-0026 [DIFOX], and ANR-22-CE15-0041-01 [NEUTROGAG]). It has also benefited from the support provided by the University for Advanced Study (USIAS) for a fellowship, within the French national program "Investment for the future" (IdEx-UNISTRA) (B.S.M.). This work was also supported by a grant from the Medical Research Council (grant no. MR/R002800/1) (D.J.T.). The Wellcome Trust Center for Cell-Matrix Research, University of Manchester (D.J.T.), is supported by core funding from the Wellcome Trust (grant no. 088785/Z/09/Z). This work was also supported by the CNRS and the GDR GAG (GDR 3739), the "Investissements d'avenir" program Glyco@Alps (grant no. ANR-15-IDEX-02), and the Agence Nationale de la Recherche (grant no. ANR-17-CE11-0040). Electron microscopy imaging was performed on the Bordeaux Imaging Center, a member of the FranceBiolmaging national infrastructure, funded by the ANR (grant no. ANR-10-INBS-04) and a European Research Council grant (ERC-2013-3395679 [DECRYPT] to P.J.S.). In memory of Dr. Fabrizia Stavru (1978-2021) and Spencer Shorte (1966-2025), who greatly supported this project in the early stages.
## AUTHOR CONTRIBUTIONS

B.S.M. designed the study and supervised the experiments. A.C.A. and M.V.B. carried out most of the experiments, performing the cell biology and biochemical experiments and acquiring and analyzing images. M.V.B., M.S., L.D., V.P., N.B., and A.S. performed the microbiology experiments. B.S.M. performed the animal experiments. M.V.B., J.S., J.T., and R.R.V. performed GAG detection experiments. K.B. performed cytokine detection in PGF. C.R. and D.J.T. performed the mass spectrometry experiments. I.S. performed the scanning electron microscopy experiments. A.D.B. and F.R. developed the murine colon cancer model. V.D. performed the transmission electron microscopy immunogold experiments. S.R. and J.-Y.T. performed quantitative imaging analysis. R.R.V. performed PGF glycosaminoglycan analysis. P.J.S. analyzed data and discussed the findings. B.S.M., A.C.A., and M.V.B. wrote the manuscript. All authors edited and approved the manuscript.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

No generative AI or AI-assisted technologies were used in this manuscript.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Human cell culture

* Blood collection and mouse neutrophils purification

* Guinea pig model of shigellosis

* Bacterial strains and culture

* Colon cancer mouse model

* METHOD DETAILS

* Production and purification of PGF

* Tandem mass spectrometry

* Neutrophil glycosaminoglycans (GAGs) analysis

* Antibodies and fluorescent dyes

* Flow cytometry

* Electrophoresis and western blot

* RNAseq analysis

* Neutrophil infection with Shigella

* Bacterial growth assay

* Purification of *Shigella* virulence factors and lcsA-containing membrane fractionation

* PGF proteolytic activity

* Microscopy

* PGF vs. NETs comparison by quantitative imaging analysis

* 3D image visualization and contact assessment

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116541.

Received: July 19, 2024

Revised: July 17, 2025

Accepted: October 21, 2025

Published: November 13, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>Anti-Lactoferrin (rabbit polyclonal)</td><td>Abcam</td><td>Cat#ab77780; RRID: AB_2192495</td></tr><tr><td>Anti-Elastase (rabbit polyclonal)</td><td>Abcam</td><td>Cat#ab68672; RRID: AB_1658852</td></tr><tr><td>Anti-Cathelicidin (rabbit polyclonal)</td><td>Abcam</td><td>Cat#ab93357; RRID: AB_10561919</td></tr><tr><td>Anti-Myeloperoxidase (rabbit monoclonal)</td><td>Dako</td><td>Cat#A0398; RRID: AB_2315082</td></tr><tr><td>Anti-Hyaluronic acid (sheep polyclonal)</td><td>Biorad</td><td>Cat#5029-9990; RRID: AB_619714</td></tr><tr><td>Anti-Chondroitin sulfate (mouse monoclonal)</td><td>Merck</td><td>Cat#C8035; RRID: AB_10563638</td></tr><tr><td>Anti-Heparan sulfate (mouse monoclonal F58-10E4)</td><td>Amsbio</td><td>Cat#370255-1; RRID: AB_10013644</td></tr><tr><td>Anti-Keratan sulfate (mouse monoclonal 373E1)</td><td>Amsbio</td><td>Cat#AMS.PRPG-KS-M01</td></tr><tr><td>Anti-Shigella 5a LPS (mouse polyclonal)</td><td>This paper – Phalipon et al.<sup>36</sup></td><td>N/A</td></tr><tr><td>Anti-IpaB (mouse monoclonal H16)</td><td>Gift from Dr. Armelle Phalipon (Institut Pasteur)</td><td>N/A</td></tr><tr><td>Anti-IpaC (mouse monoclonal K24)</td><td>Gift from Dr. Armelle Phalipon (Institut Pasteur)</td><td>N/A</td></tr><tr><td>Anti-IpaD (rabbit polyclonal)</td><td>Gift from Dr. Claude Parsot (Institut Pasteur)</td><td>N/A</td></tr><tr><td>Anti-IcsA (rabbit polyclonal)</td><td>Gift from Dr. Claude Parsot (Institut Pasteur)</td><td>N/A</td></tr><tr><td>Anti-rabbit secondary Ab Alexa 488</td><td>Invitrogen</td><td>Cat#A32731; RRID: AB_2633280</td></tr><tr><td>Anti-rabbit secondary Ab Alexa 568</td><td>Invitrogen</td><td>Cat#32723; RRID: AB_2633275</td></tr><tr><td>Anti-mouse secondary Ab Alexa 488</td><td>Invitrogen</td><td>Cat#A11029; RRID: AB_2534098</td></tr><tr><td>Anti-mouse secondary Ab Alexa 568</td><td>Invitrogen</td><td>Cat#A11031; RRID: AB_144696</td></tr><tr><td>Anti-mouse secondary Ab Alexa 532</td><td>Invitrogen</td><td>Cat#A11002; RRID: AB_2534069</td></tr><tr><td>Anti-mouse secondary Ab Alexa 647</td><td>Invitrogen</td><td>Cat#32720; RRID: AB_2633273</td></tr><tr><td>Anti-sheep secondary Ab Alexa 488</td><td>Abcam</td><td>Cat#ab150181; RRID: AB_2687946</td></tr><tr><td>Anti-sheep secondary Ab Alexa 568</td><td>Abcam</td><td>Cat#ab175713; RRID: AB_2864497</td></tr><tr><td>Anti-rabbit HRP-conjugated secondary Ab</td><td>Jackson Immuno</td><td>Cat#111-035-003; RRID: AB_2313567</td></tr><tr><td>Anti-mouse HRP-conjugated secondary Ab</td><td>Jackson Immuno</td><td>Cat#115-035-003; RRID: AB_10015289</td></tr><tr><td>Anti-sheep HRP-conjugated secondary Ab</td><td>Abcam</td><td>Cat#ab97130; RRID: AB_10675311</td></tr><tr><td>CD235a MicroBeads (Anti-Glycophorin A microbeads)</td><td>Miltenyi Biotec</td><td>130-050-501<br>RRID:SCR_020326</td></tr></tbody></table>

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<figure><img src="image_23.png" /></figure>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Bodipy 493/503</td><td>Thermo Fisher</td><td>Cat#D3922; RRID: AB_2533939</td></tr><tr><td>Phalloidin Alexa Fluor 647</td><td>Thermo Fisher</td><td>Cat#A22287; RRID: AB_2620155</td></tr><tr><td>Goat anti-rabbit F(ab')<sub>2</sub> – nanogold fragment</td><td>Aurion</td><td>Cat#25360; RRID: AB_2314410</td></tr></tbody></table>

Bacterial and virus strains

<table><tbody><tr><td><i>E. coli</i> K12 MG1655</td><td>Lab stock</td><td>N/A</td></tr><tr><td><i>E. coli</i> HB101</td><td>Lab stock</td><td>N/A</td></tr><tr><td><i>E. coli</i> HB101 pZK15 (pUC19-SepA)</td><td>Claude Parsot (Institut Pasteur)</td><td>N/A</td></tr><tr><td><i>E. coli</i> HB101pPic (pACYC184-Pic)</td><td>Claude Parsot (Institut Pasteur)</td><td>N/A</td></tr><tr><td><i>Shigella flexneri</i> 5a (M90T)</td><td>Lab stock</td><td>N/A</td></tr><tr><td><i>Shigella flexneri</i> 5a pGFP</td><td>This study (pFpV25.1 plasmid)</td><td>N/A</td></tr><tr><td><i>Shigella flexneri</i> 2a</td><td>Lab stock</td><td>N/A</td></tr><tr><td><i>Shigella sonnei</i></td><td>Lab stock</td><td>N/A</td></tr><tr><td><i>Salmonella enterica</i> serovar Typhimurium (WT)</td><td>Lab stock</td><td>N/A</td></tr></tbody></table>

Biological samples

<table><tbody><tr><td>Human peripheral blood</td><td>ICAReB Pasteur Institute/EFS Aquitaine-Limousin/EFS Strasbourg</td><td>Authorization DC No.2008-68/No. AC 2013-2025/n°ALC/PIL/DIR/AJR/FO/606</td></tr><tr><td>Mouse blood (C57BL/6)</td><td>Chronobiotron (CELPHEDIA)</td><td>APAFIS#29175-2021010711306157v1</td></tr><tr><td>Colon tissue from infected guinea pigs</td><td>This study</td><td>N/A</td></tr></tbody></table>

Chemicals, peptides, and recombinant proteins

<table><tbody><tr><td>Mouse Neutrophil Isolation Kit</td><td>Miltenyi Biotec</td><td>Cat#130-097-658<br>RRID:SCR_020326</td></tr><tr><td>Red Blood Cell Lysis Solution</td><td>Miltenyi Biotec</td><td>Cat#130-094-183<br>RRID:SCR_020326</td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>Cat#17-0891-01</td></tr><tr><td>DNase I</td><td>Merck</td><td>Cat#4716728001</td></tr><tr><td>Proteinase K</td><td>Merck</td><td>Cat#RPROTK-RO</td></tr><tr><td>Hyaluronidase type I-S (from bovine testes)</td><td>Merck</td><td>Cat#H3506</td></tr><tr><td>Hyaluronidase (from Streptomyces hyalurolyticus)</td><td>Sigma-Aldrich</td><td>Cat#H1136</td></tr><tr><td>Recombinant human HYAL4</td><td>R&amp;D Systems</td><td>Cat#6904-GH</td></tr><tr><td>Carnoy's fixative (ethanol/chloroform/acetic acid)</td><td>Prepared in the lab</td><td>N/A</td></tr><tr><td>Alcian blue</td><td>Merck</td><td>Cat#B8438</td></tr><tr><td>Periodic Acid Schiff (PAS) reagents</td><td>Merck</td><td>Cat#P7875; Cat#3952016</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen</td><td>Cat#P36930<br>RRID:SCR_015961</td></tr><tr><td>Chondroitinase ABC</td><td>Sigma-Aldrich</td><td>Cat#C3667</td></tr><tr><td>Heparinase I, II, III</td><td>Grampian Enzymes</td><td>By request</td></tr><tr><td>OCT Compound</td><td>Sakura Finetek</td><td>Cat#4583</td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>Cat#T5648</td></tr><tr><td>Sunflower oil</td><td>Skerniskyte et al.<sup>24</sup></td><td>N/A</td></tr><tr><td>Haematoxylin</td><td>Sigma-Aldrich</td><td>Cat#H3136</td></tr><tr><td>Eosin 1%</td><td>Sigma-Aldrich</td><td>Cat#E6003</td></tr><tr><td>DAPI</td><td>Merck</td><td>Cat#D9542<br>AB_2307445</td></tr><tr><td>RI_Myelotracker-Cy3</td><td>Anderson et al.<sup>12,35</sup></td><td>N/A</td></tr><tr><td>RI_Myelotracker-Cy5</td><td>Anderson et al.<sup>12,35</sup></td><td>N/A</td></tr><tr><td>Eukitt mounting medium</td><td>Sigma-Aldrich</td><td>Cat#03989</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Propidium iodide (PI)</td><td>Merck</td><td>Cat#P4170</td></tr><tr><td>InstantBlue Coomassie</td><td>Abcam</td><td>Cat#119211</td></tr><tr><td>ECL Prime Western Blotting Detection Reagent</td><td>GE Health care</td><td>Cat#RPN2236</td></tr><tr><td>RNA Lysis buffer (RLT Buffer)</td><td>Qiagen</td><td>Cat#79216</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>Cat#74104</td></tr><tr><td>Wortmannin (PI3K inhibitor)</td><td>Merck</td><td>Cat#681675</td></tr><tr><td>SB202190 (p38 inhibitor)</td><td>Merck</td><td>Cat#559388</td></tr><tr><td>Akt inhibitor VIII</td><td>Merck</td><td>Cat#124018</td></tr><tr><td>Complete Mini Protease Inhibitor Cocktail</td><td>Merck</td><td>Cat#11836153001</td></tr><tr><td>Human IgA</td><td>Merck</td><td>Cat#14036</td></tr><tr><td>Lactoferrin</td><td>Merck</td><td>Cat#L1294</td></tr><tr><td>Human IgG</td><td>Merck</td><td>Cat#14506</td></tr><tr><td>Casein</td><td>Merck</td><td>Cat#C3400</td></tr><tr><td>Protein-A Gold 6 nm</td><td>Aurion</td><td>Cat#806.111</td></tr><tr><td>Ultrasmall nanogold F(ab')<sub>2</sub> fragments anti-rabbit</td><td>Aurion</td><td>Cat#800.166</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>ZipTip C18</td><td>Millipore</td><td>Cat#ZTC18S096</td></tr><tr><td>Hyaluronan DuoSet ELISA Kit</td><td>R&D Systems</td><td>Cat#DY3614-05</td></tr><tr><td>LEGENDplex Human Essential Immune Response Panel</td><td>BioLegend</td><td>Cat#740932</td></tr></table>

Deposited data

<table><tr><td>SwissProt protein sequence database</td><td>UniProt</td><td>RRID:SCR_002380</td></tr><tr><td>Mass Spectrometry data</td><td>ProteomeXchange</td><td>PXD068719</td></tr><tr><td>RNAseq data</td><td>GEO accession number</td><td>GSE308441</td></tr></table>

Experimental models: Cell lines

<table><tr><td>Primary human neutrophils</td><td>Purified from peripheral blood (see above)</td><td>N/A</td></tr><tr><td>Mouse neutrophils (genotype C57BL/6J)</td><td>Purified from blood (see above)</td><td>N/A</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>C57BL/6 mice (genotype C57BL/6J)</td><td>Chronobiotron (CELPHEDIA)</td><td>RRID:IMSR_JAX:000664</td></tr><tr><td>Hartley guinea pigs (&lt;150g)</td><td>Charles River</td><td>RRID:RGD_2308852</td></tr><tr><td>Ap clox/lox Cdx2-CreERT2 Tg/+ (A-mice)</td><td>EPFL Skerniskyte et al.<sup>24</sup></td><td>RRID:MGI:5650180 (CreERT2)</td></tr><tr><td>Ap clox/lox Cdx2-CreERT2 +/+ (Control mice)</td><td>EPFL Skerniskyte et al.<sup>24</sup></td><td>RRID:MGI:5650180 (CreERT2)</td></tr></table>

Recombinant DNA

<table><tr><td>Plasmid pFpV25.1 (GFP-expressing plasmid)</td><td>Marteyn et al.<sup>17</sup></td><td>N/A</td></tr><tr><td>Plasmid pUC19-SepA (pZK15)</td><td>Claude Parsot (Institut Pasteur)</td><td>N/A</td></tr><tr><td>Plasmid pACYC184-Pic (pPic)</td><td>Claude Parsot (Institut Pasteur) - REF</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>Mascot search engine</td><td>Matrix Science</td><td>RRID:SCR_014322</td></tr><tr><td>Scaffold Proteome Software</td><td>Proteome Software Inc</td><td>RRID:SCR_014345</td></tr><tr><td>HPLC fluorescence detection software</td><td>Manufacturer-specific</td><td>N/A</td></tr><tr><td>CellQuest Pro</td><td>BD Biosciences</td><td>N/A</td></tr><tr><td>FlowJo</td><td>FlowJo LLC</td><td>N/A</td></tr><tr><td>LEGENDplex Software</td><td>BioLegend</td><td>Version 8.0</td></tr><tr><td>Leica Application Suite X (LAS X)</td><td>Leica Microsystems</td><td>N/A</td></tr><tr><td>Fiji (ImageJ 2.1.0/1.53j)</td><td>NIH/ImageJ</td><td>RRID:SCR_002285</td></tr></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>DESeq2</td><td>Bioconductor</td><td>RRID:SCR_015687</td></tr><tr><td>Otsu Thresholding Algorithm</td><td>Fiji/ImageJ plugin</td><td>N/A</td></tr><tr><td>Difference of Gaussian filter (DoG)</td><td>Fiji/ImageJ plugin</td><td>N/A</td></tr><tr><td>Imaris v9.6</td><td>Bitplane</td><td>RRID:SCR_007370</td></tr><tr><td>GraphPad Prism v10</td><td>GraphPad</td><td>RRID:SCR_002798</td></tr></tbody></table>

Other

<table><tbody><tr><td>Anoxic chamber (DG250)</td><td>Don Whitley Scientific</td><td>DG250</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Model-specific</td></tr><tr><td>UltiMate 3000 RSLC system</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>LTQ Velos Pro mass spectrometer</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>Centricon 3 kDa cutoff filters</td><td>Merck</td><td>UFC800324</td></tr><tr><td>RPIP-HPLC system with C18 column</td><td>Agilent</td><td>N/A</td></tr><tr><td>Cryostat CM-3050</td><td>Leica</td><td>CM3050</td></tr><tr><td>Vivaspin 10 kDa MWCO centrifugal unit</td><td>Sartorius</td><td>VS0101</td></tr><tr><td>FACSCalibur flow cytometer</td><td>BD Biosciences</td><td>N/A</td></tr><tr><td>FACS Canto II flow cytometer</td><td>BD Biosciences</td><td>N/A</td></tr><tr><td>VacuGene XL Vaccum Blotting Unit</td><td>GE Healthcare</td><td>N/A</td></tr><tr><td>Mini TransBlot Electrophoretic Transfer Cell</td><td>BioRad</td><td>N/A</td></tr><tr><td>Axioscope 2 microscope</td><td>Zeiss (Germany)</td><td>N/A</td></tr><tr><td>SP8 Confocal Microscope</td><td>Leica</td><td>N/A</td></tr><tr><td>GeminiSEM 300 FESEM</td><td>Zeiss</td><td>N/A</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>N/A</td></tr><tr><td>Hitachi H7500 transmission electron microscope</td><td>Hitachi</td><td>N/A</td></tr></tbody></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Human cell culture

Blood collection and neutrophil purification. All participants gave written, informed consent in accordance with the Declaration of Helsinki principles. Peripheral human blood was collected from healthy patients at the ICAREB service of the Pasteur Institute (authorization DC No.2008-68), the Etablissement Français du Sang (EFS) Aquitaine-Limousin (authorization DC No. AC 2013-2025) and the Etablissement Français du Sang (EFS) de Strasbourg (authorization n°ALC/PIL/DIR/AJR/FO/606). The work on human blood was authorized by national committees (IRB and CNIL).

Blood samples were rapidly transferred into an anoxic chamber (DG250, Don Whitley) for human neutrophil anoxic purification, using oxygen-free media, as previously described.¹⁶ Briefly, whole blood samples were centrifuged at 600 × g for 20 min. Platelet rich plasma (PRP) was collected and centrifuged at 2650 × g for 20 min to form platelet poor plasma (PPP). Blood cells were resuspended in NaCl (0.9%) and dextran sulfate (0.72%). After 30 min sedimentation, the neutrophil-containing upper layer was centrifuged at 300 × g for 10 min. Pelleted cells were resuspended in 1 mL PPP and were subsequently separated on a 42% Percoll-plasma (GE Healthcare) gradient by centrifugation at 800 × g for 20 min. Neutrophils were collected from the pellet with remaining red blood cells, which were removed using CD235a (glycophorin) microbeads (Miltenyi Biotec). Purified neutrophils were centrifuged at 300 × g for 10 min. Pelleted cells were resuspended in PPP or RPMI 1640 culture medium supplemented with 10 mM HEPES and 3mM glucose,⁷ according to experimental settings.

Blood collection and mouse neutrophils purification

Blood was collected from C57BL/6 mice by cardiac puncture on anesthetized mice. Mice were sacrificed after the procedure. Neutrophils were purified by Mouse Neutrophil Isolation Kit (Miltenyi Biotec, 130-097-658) according to the manufacturer's recommendation in hypoxic conditions. Red blood cells were lysed using Red Blood Cell Lysis Solution (Miltenyi Biotec, 130-094-183) according to the manufacturer's recommendation. The experimental procedures were authorized by the Ministry of Higher Education, Research, and Innovation Ethics Committee (auth. n°APAFIS#29175-2021010711306157v1). Experiments with mice were performed in animal facilities at the Chronobiotron, an animal facility part of the National Research Infrastructure CELPHEDIA (Creation, Breeding, Phenotyping, Distribution and Archiving of model organisms). All experimental procedures were approved by the Chronobiotron Ethics Committee (auth. n°APAFIS#46786 2023111011373972_v14).
## Guinea pig model of shigellosis

Young guinea pigs (Hartley, <150g) were previously infected intrarectally with 10¹⁰ CFU exponentially grown S. flexneri 5a pGFP to evaluate acute infection (8h p.i).¹¹ In this study, ascorbate-deficient guinea pigs were challenged intrarectally with 10¹⁰ CFU exponentially grown S. flexneri 5a pGFP and animals were sacrificed 48h p.i. In this animal model of shigellosis, infection symptoms are more severe and colonization of the colonic mucosa by Shigella is more prolonged.³⁰ After animals were sacrificed, infected colons were collected and fixed in 3.3% paraformaldehyde (PFA) for 2 h (or Carnoy's solution). Fixed colon samples were washed in PBS, incubated at 4°C in PBS containing 15% sucrose for 90 min, followed by incubation in PBS with 30% sucrose overnight. Samples were frozen in OCT (Sakura) on dry ice. 7 µm sections were obtained using a cryostat CM-3050 (Leica).

**Immunofluorescent labeling.** The tissues were washed and permeabilized with PBS-Triton 0.1% and stained with primary antibodies in the presence of 1% BSA overnight at 4°C, washed three times in PBS-Triton 0.1%, prior to incubation with secondary antibodies for 1h at RT. After three washes in PBS-Triton 0.1%, three washes in PBS and three washes in water, tissues were mounted with 10µL ProLongGold (Invitrogen) and allowed to dry overnight at RT.

**Haematoxylin and eosin staining.** For a better analysis of the tissue morphology, the samples were dehydrated, stained with haematoxylin (30 s) and 1% eosin (1 min), rehydrated, and after drying, the tissues were mounted with Eukitt mounting medium.

## Bacterial strains and culture

Escherichia coli (K12 MG1655 or HB101) strains were cultured in Lysogeny broth (LB) at 37°C. Shigella flexneri 5a (M90T), Shigella flexneri 2a, Shigella sonnei and Salmonella typhimurium wild-type strains were grown in Tryptic soy broth (TSB, 211768, ThermoFisher Scientific) medium at 37°C. Shigella flexneri 5a pGFP was obtained upon the transformation of the pFpV25.1 plasmid.¹⁷

E. coli HB101 strain was transformed with pUC19-SepA (pZK15)³¹ (supplied by Dr. Claude Parsot, Institut Pasteur) or pACYC184-Pic (pPic)³² for the expression and the purification of SepA and Pic (Serine Protease Autotransporters of Enterobacteriaceae or SPATEs).

## Colon cancer mouse model

For tumor studies, Apc<sup>lox/lox</sup> Cdx2-CreERT2<sup>Tg/+</sup> (A-mice) and Apc<sup>lox/lox</sup> Cdx2-CreERT2<sup>+/+</sup> (Ctrl-mice) received a single i.p. injection with tamoxifen in sunflower oil (25 mg/kg) to induce colon-constrained Apc-inactivation, as previously described.²³ Tumors were allowed to develop for 8–12 weeks. After euthanasia, the cecum and proximal colon of the mice were resected, opened vertically, cleaned, and rinsed with PBS. Three to 5 mm strips of colon were cut, swiss-rolled in a cassette, and fixed overnight in 4% PFA and/or in Carnoy's solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 2 h at 4°C. The tissues were dehydrated and then embedded in paraffin. Tumorigenesis experiments were conducted at EPFL, Lausanne, Switzerland as outlined in licenses VD3035.1 and VD3823 under the regulations imposed by the Veterinary Authorities of the Canton of Vaud. Mice were housed in the conventional facility in cages furnished with enrichment such as houses, toys, and tissue, and were supplied chow and water *ad libitum*. The facility is maintained at a temperature of 23°C ± 1° and undergoes 12-h light/dark cycles.

**Haematoxylin and eosin staining.** For a better analysis of the tissue morphology, the samples were dehydrated, stained with haematoxylin (30 s) and 1% eosin (1 min), rehydrated, and after drying, the tissues were mounted with Eukitt mounting medium.

## METHOD DETAILS

### Production and purification of PGF

Neutrophils were allowed to secrete PGF upon 4h or 18h culture at 37°C in RPMI1640 medium (ref. 32404-014, ThermoFisher Scientific) supplemented with 10mM HEPES and 3mM glucose (2 × 10⁶ cells/mL). When indicated, after 4h culture, the following enzymes were added for 1h at 37°C prior fixation in a Carnoy's solution: DNase I (ref. 4716728001, Merck), Proteinase K (ref. RPROTK-RO, Merck), Hyaluronidase type I-S from bovine testes (ref H3506, Merck), Hyaluronidase from Streptomyces hyalurolyticus (ref. H1136, Sigma-Aldrich) and Recombinant human hyaluronidase 4/HYAL4 (ref. 6904-GH, R&D systems).

For high-yield production of PGF, neutrophils were cultured in 75 cm² polystyrene culture flasks (ThermoFisher Scientific) under anoxic conditions (DG250 workstation, Don Withley). After the indicated culture time, neutrophil cultures were collected and centrifuged at 300 × g for 5 min to pellet cells. PGF were collected from culture supernatant by ultracentrifugation (46.000 × g for 50 min). Pelleted PGF were resuspended in 1mL PBS, as previously described for NETs purification.³³ Samples were sonicated before storage at -20°C.

For imaging analysis, neutrophils were allowed to secrete PGF onto glass coverslips in polypropylene 24-well plates (ref. 290-8324-03F, Evergreen Scientific). At each indicated time point, neutrophils were fixed in Carnoy's solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 1 h at room temperature. Sample fixation in 3.3% PFA was tested but did not maintain the structural integrity of PGF. Samples were subsequently washed in PBS prior to staining with Alcian blue (ref. B8438, Merck) or Periodic Acid Schiff (PAS) (refs. P7875 and 3952016, Merck). For immunofluorescent labeling, coverslips were carefully washed and cells permeabilized with 0.1% PBS-Triton and stained with primary antibodies for 1h at room temperature (RT), washed three times in 0.1% PBS-Triton, prior to incubation with secondary antibodies for 1h at RT. After three washes in 0.1% PBS-Triton, three washes in PBS, and three washes in water, coverslips were mounted with 10µL ProLongGold (Invitrogen) and allowed to dry overnight at RT.
For electron microscopy imaging, fixed samples were rinsed twice in 0.1M phosphate buffer and dehydrated in a series of increasing ethanol concentrations (30–100%). Samples were dried via critical point drying (Leica EM CPD300, Austria). Afterward cover glasses were mounted onto specifics stubs and coated with platinum, using a sputter coater (Q150T, Quorum Technologies, Kent, UK).

## Tandem mass spectrometry

Prior to tandem mass spectrometry samples were prepared via reduction, alkylation, and trypsin digestion, as previously described.¹² After treatment with trypsin, digested peptides were transferred to a 10 kDa MWCO Vivaspin centrifugal concentrating column and centrifuged at 4.500 × g until the whole sample volume had been recovered. Digested tryptic peptides were then acidified to pH 2.0 using 1% (v/v) trifluoroacetic acid, and subsequently purified using C18 ZipTips (Millipore, Durham, UK). Using an UltiMate 3000 Rapid Separation LC coupled to an LTQ Velos Pro mass spectrometer liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Thermo Fisher Scientific, Waltham, USA) was performed on peptide containing samples. Firstly, a pre-column of 20 mm × 180 µm was used to concentrate sample peptides. A 75 mm × 250 mm 1.7 µM ethylene bridged hybrid C18 analytical column (Waters, Manchester, UK) was then used to separate peptides in a gradient from 99% solution A (0.1% formic acid in ddH₂O) and 1% solution B (0.1% formic acid in acetonitrile) to 25% solution B. Peptides were then automatically selected for fragmentation via a process of data dependent analysis. “The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD068719”.

The MASCOT search engine was used to analyze the processed mass spectrometry data. Data were searched against the SwissProt database using the following parameters: 1.2 Da mass accuracy for parent ions, 0.6 Da mass accuracy for fragment ions, one missed cleavage site was allowed for, 2+ and 3+ ions were selected, carbamidomethyl-cysteine was selected as a fixed modification, and methionine oxidation as a variable modification. The data generated from MASCOT was then analyzed in a semi-quantitative manner using Scaffold Proteome software (Proteome Software, Portland, USA), allowing for unique peptide counts of identified proteins to be determined.

## Neutrophil glycosaminoglycans (GAGs) analysis

**Hyaluronic acid.** Hyaluronan was quantified in PGF samples using a specific enzyme-linked immunosorbent assay, the commercial Hyaluronan DuoSet ELISA Kit (catalog number: DY3614-05). Samples were run in triplicate with a detection limit of 90 ng/mL, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

**Chondroitin sulfate.** GAG extraction and compositional analysis was performed as described previously.³⁴ Here, PGF were digested with 0.2mg/mL proteinase K (Merck) for 1h at 37°C. Potential GAG/protein complexes were dissociated by incubation with 2M NaCl (30 min at room temperature); protein denaturation was achieved by heating samples (100°C for 10 min). After centrifugation, supernatants were recovered, and concentrated to 50–100 µL in 50 mM Tris-HCl pH 7.5 supplemented with 50 mM NaCl and 2 mM CaCl₂ (GAG digestion buffer) using a 3kDa cutoff Centricon (Merck).

GAGs were then exhaustively digested into disaccharides by incubating samples for 48 h at 37°C, with either chondroitinase ABC (Sigma-Aldrich, 100 mU) for CS/DS analysis, or with a mix of Heparinase I, II and III (Grampian enzymes, 10 mU each) for HS analysis. Compositional analyses were performed by RPIP-HPLC, as previously described.³⁴ Samples were applied to a C18 reverse phase column equilibrated in a H₂O/acetonitrile (8.5%) buffer supplemented with 1.2 mM of ion-pairing tetra-N-butylammonium hydrogen sulfate (TBAHS), then resolved using a multi-step NaCl gradient calibrated with authentic GAG disaccharide standards (Iduron). Online post-column disaccharide derivatization was achieved by the addition of 2-cyanoacetamide (0.25%) in NaOH (0.5%), followed by fluorescence detection (excitation 346 nm, emission 410 nm).

## Antibodies and fluorescent dyes

Lactoferrin was labeled with a rabbit polyclonal antibody (ref. ab77780, Abcam) (1:500 dilution for WB and 1:100 for IF) or with Rl-Myelotracker-cy3 or RI-Myelotracker-Cy5, as indicated, used at 1 µg.mL⁻¹¹,³⁵ (contact the corresponding author for more details). DNA was labeled with 4',6-Diamidino-2-phenylindole (DAPI, ref. D9542, Merck) and was used at 1 µg/mL. Elastase was detected with a rabbit polyclonal antibody (ref. ab68672, Abcam) (1:500 final dilution for WB and 1:100 final dilution for IF). Cathelicidin was detected with a rabbit polyclonal antibody (ref. ab93357, Abcam) (1:1000 final dilution for WB and 1:200 for IF). Myeloperoxidase was detected with a rabbit monoclonal antibody (ref. A0398, Dako) (1:500 final dilution for WB and 1:200 final dilution for IF). Hyaluronic acid was detected with a sheep polyclonal antibody (ref. 5029-9990, Biorad) (1:500 dilution for WB and 1:100 for IF). Chondroitin sulfate was detected with a mouse monoclonal antibody (ref. C8035, Merck) (1:200 final dilution for WB and 1:100 for IF). Heparan sulfate was detected with a mouse monoclonal antibody (clone F58-10E4, ref. 370255-1, Amsbio) (1:100 final dilution for WB). Keratan sulfate was detected with a mouse monoclonal antibody (clone 373E1, ref: AMS.PRPG-KS-M01, Amsbio) (1:100 final dilution for WB). Murine anti-Shigella 5a lipopolysaccharide polyclonal antibody was used for immunofluorescent labeling (1:500 final dilution).³⁶ IpaB was detected with a mouse monoclonal antibody (clone H16) kindly supplied by Dr. Armelle Phalipon, Institut Pasteur (1:1.000 final dilution for WB). IpaC was detected with a mouse monoclonal antibody (clone K24) kindly supplied by Dr. Armelle Phalipon, Institut Pasteur (1:1.000 final dilution for WB). IpaD and IcsA were detected with rabbit polyclonal antibodies kindly supplied by Dr. Claude Parsot, Institut Pasteur (1:10.000 final dilutions for WB). Phalloidin-AlexaFluor647 (1:1000; ref A22287 ThermoFisher Scientific) was used to label actin, and lipid vesicles were labeled with BODIPY (1:1000; ref. D3922 ThermoFisher Scientific). For immunofluorescence staining, rabbit and mouse secondary antibodies conjugated with Alexa 488 and Alexa 568 fluorophores were used
(refs. A32731 and A32723, and refs. A11036 and A11031 respectively, Invitrogen) (1:1.000 final dilution). Mouse secondary antibodies conjugated with Alexa 532 and Alexa 647 (ref. A11002 and A32720 respectively, Invitrogen) were also used. Sheep secondary antibodies conjugated with an Alexa 488 or Alexa 568 were used alternatively (refs. ab150181 or ab175713 respectively, Abcam). For western-blot analyses, rabbit and mouse secondary antibodies conjugated with horseradish peroxidase were used (ref. 115-035-003 and 111-035-003, Jackson Immuno Research) (1:10.000 final dilution) or a sheep secondary antibody conjugated with horseradish peroxidase was used (ref. ab97130, Abcam) (1:10.000 final dilution).

## Flow cytometry

Cell viability was determined by staining with 0.01% propidium iodide (PI, ref. P4170, Merck) in PBS +2 mM EDTA for 15 min at RT. PI only penetrates and stains the DNA of non-viable cells. Fluorescence was measured using a FACSCalibur (BD Bioscience) flow cytometer, recording at least 10,000 events. Data were analyzed with CellQuest Pro software (BD Biosciences) and were quantified using the FlowJo software (FlowJo, LLC).

For the determination of PGF chemokine concentration, purified PGF were analyzed using LEGENDplex HU Essential Immune Response Panel (BioLegend), according to the manufacturer's instructions, and data were acquired with a FACS Canto II instrument (BD Biosciences, San Jose, CA). Analyses were performed using Legendplex software 8.0.

## Electrophoresis and western blot

**SDS-agarose gel.** To separate high molecular weight protein complexes (as described previously³⁷), samples were loaded on an 0.8% agarose gel (containing 0.1% SDS). Sample migration occurred at 60V for 3h. The gel was then reduced for 15 min in a 20× SSC (Saline Sodium Citrate) buffer with 10 mM DTT and washed in a 20× SSC buffer without DTT. Protein transfer onto a PVDF membrane was achieved under vacuum (GE Healthcare, VacuGene XL Vacuum Blotting Unit) at 50 mbar for 1h30.

**SDS-PAGE gel.** 4–12% SDS-PAGE gradient gel (NuPAGE; ref NO0322PK2, ThermoFisher Scientific). Migration occurred at 200V for 25 min. When indicated, gels were stained with InstantBlue Coomassie (ref. ab119211, Abcam) or proteins were transferred onto a PVDF membrane (at 350 mA for 180 min) (Mini TransBlot Electrophoretic Transfer Cell, BioRad).

**Agarose gel.** The presence of DNA in cells and in purified PGF was determined by migrating samples on 1% agarose gel in the presence of propidium iodide, followed by UV revelation (Molecular Imager, BioRad).

**Western blot.** PVDF membranes were saturated with a solution of Tris-buffered saline with Tween 20 (TBST) solution (ref. 91414, Merck) containing 5% milk for 1h and incubated overnight with primary antibodies in TBST. Membranes were washed with TBST three times (5 min) and incubated with a secondary antibody for 1h in TBST containing 1% milk. After three additional washes (5 min), antibody binding was detected with chemiluminescence (ECL Prime Western Blotting Detection Reagent, GE Healthcare).

## RNAseq analysis

Neutrophil mRNAs were purified from 5 × 10⁷ neutrophils either freshly purified or cultured for 18h in anoxia in RPMI 1640 medium supplemented with 10 mM HEPES and 3 mM glucose. Cells were washed once with PBS and centrifuged for 5 min at 300 × g. Pelleted cells were lysed in 350 µL RLT buffer (Qiagen). Lysates were resuspended with a needle to increase the lysis efficiency. TRIzol (3 volumes) was added to samples followed by vortex and short spin. 1/5 of the volume of chloroform was added and samples were vortexed and incubated for 3 min at room temperature prior to transferring into PLG heavy tubes. Samples were centrifuged for 15 min at 12.000 × g (4°C) and RNA-containing aqueous solution was transferred into a new tube. RNA was purified using the RNeasy Mini Kit (Qiagen) and quantified with a nanodrop, and the quality was assessed with Tapestation. RNA samples were analyzed and sequenced by the GENOM'IC platform (Institut Cochin, Paris, France); RNA sequences were analyzed with the Deseq2 method. Results are averaged from three biological triplicates for each condition. RNAseq data have been deposited to GEO with the accession number GSE308441 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE308441).

## Neutrophil infection with Shigella

Neutrophils were seeded (1 × 10⁶ cells/well) onto coverslips in RPMI 1640 medium supplemented with 10 mM HEPES and 3 mM glucose in 24-well plates. Cells were infected at a multiplicity of infection (MOI) of 20 with S. flexneri 5a pGFP grown until exponential phase. Bacteria were spun onto cells by centrifugation at 300 × g for 10 min. After 1h, 2h and 4h incubation at 37°C, samples were fixed with Carnoy's solution for 1h at room temperature. For immunofluorescent labeling, coverslips were washed and permeabilized with PBS-Triton 0.1% and stained with primary antibodies for 1h at room temperature (RT), washed three times in PBS-Triton 0.1%, prior incubation with secondary antibodies for 1h at RT. After three washes in PBS-Triton 0.1%, three washes in PBS and three washes in water, coverslips were mounted with 10µL ProLongGold (Invitrogen) and allowed to dry overnight at RT.

When indicated, commercial inhibitors have been tested on their ability to modulate neutrophil PGF release. PI3K inhibitor (100 nM wortmannin, ref. 681675, Merck), p38 inhibitor (10 mM SB202190, ref. 559388, Merck), Akt inhibitor (2.6 mM Akt inhibitor VIII, ref. 124018, Merck).

## Bacterial growth assay

Overnight bacterial cultures were subcultured in fresh M9 medium (1:100 dilution) at 37°C until a OD₅₀₀ of 0.3 was reached. 1 mL bacterial suspension was centrifuged at 8.500 × g for 3 min; pelleted bacteria were resuspended in 500 µL PBS. 1 µL bacterial
suspension was deposited onto a 1% M9-agar pad and covered with a square coverslip. Agar pads were incubated at 37°C to allow bacterial growth, which was assessed by widefield microscopy at t = 0, t = 4h and t = 18h. When indicated, bacterial growth was performed in an anoxic chamber (-O₂ condition). To evaluate the impact of PGF on bacterial growth, when indicated, 5 µg of purified PGF were deposited onto the agar pad after the deposition of the bacterial suspension. When indicated, PGF heat-inactivation may be performed (80°C, 10 min); the addition of a protease inhibitor cocktail may also be done following manufacturer instructions (Merck Complete, Mini Protease Inhibitor Cocktail). Purified PGF was treated with the following enzymes for 1h at 37°C, when indicated: Hyaluronidase type I-S from bovine testes (ref H3506, Merck), Hyaluronidase from *Streptomyces hyalurolyticus* (ref. H1136, Sigma-Aldrich), Recombinant human hyaluronidase 4/HYAL4 (ref. 6904-GH, R&D systems), DNase I (ref. 4716728001, Merck) and Proteinase K (ref. RPROTK-RO, Merck).

## Purification of Shigella virulence factors and IcsA-containing membrane fractionation

SPATES. *E. coli* HB101 pSepA an *E. coli* HB101 pPic were grown overnight in LB medium with ampicillin (100 µg/mL) and subcultured in 500 mL fresh LB medium with ampicillin (100 µg/mL) at 37°C. SigA was purified from *S. sonnei* culture medium. Cultures were centrifuged for 20 min at 800 × g and the supernatant was filtered through a 0.22 µm diameter filter (TPP Filtermax rapid bottle filter unit, ref. Z760900, Merck). Filtrated culture media were precipitated in 70% ammonium sulfate (ref. A4418, Merck). Precipitates were pelleted by centrifugation (7 500 × g, 30 min) and subsequently resuspended in 10 mL cold PBS. Samples were further dialyzed in cold PBS and concentrated by centrifugation on 100kDa filter (Sartorius Vivaspin 20 Centrifugal Concentrator). Protein samples were stored at -20°C, as described previously.

Ipas. Purification of IpaB, IpaC and IpaD was achieved as described previously.¹⁷,³⁸ S. flexneri 5a was cultured at 37°C in 50 mL TSB culture medium until OD₆₀₀ = 0.4 was reached. The bacterial culture was centrifuged at 3.000 × g for 5 min and pelleted bacteria were washed twice in PBS and further resuspended in 200 µL PBS. Ipas secretion was induced by the addition of 0.05% Congo red (ref. 6277, Merck). After 30 min at 37°C samples were centrifuged at 17.000 × g for 1 min. Ipas-containing supernatants were collected and stored at -20°C.

IcsA-containing membranes. An overnight culture of S. flexneri 5a was diluted at 1:100 in 200 mL of fresh TSB medium and incubated at 37°C until an OD₆₀₀ = 0.4 was reached. The bacterial culture was centrifuged at 17,000 × g for 20 min and pellets were resuspended in 1mL PBS. Samples were sonicated (4 cycles of 30 s) and ultracentrifuged at 120,000 × g for 1h30 at 4°C. IcsA-containing pelleted membranes were resuspended in 1mL PBS and stored at -20°C.

## PGF proteolytic activity

To assess PGF proteolytic activity, purified PGF (10µg) were incubated in PBS overnight at 37°C with 2 µg commercial purified proteins (lactoferrin (ref. L1294, Merck), IgA (ref. I4036, Merck), IgG (ref. I4506, Merck) and casein (ref. C3400, Merck)) or 10 µg *Shigella* bacterial virulence factors (SepA, Pic, IcsA and IpaB/C/D). Samples were separated by electrophoresis on 4–12% SDS-PAGE gradient gel (NuPAGE; Invitrogen). As indicated, proteins were stained with Coomassie (InstantBlue Coomassie Stain, Abcam) or analyzed by western blot. As a negative control, PGF proteolytic activity inactivation was achieved by heating (80°C, 10 min), or by the addition of a protease inhibitor cocktail (Merck Complete, Mini Protease Inhibitor Cocktail) following manufacturer instructions.

## Microscopy

*Transmitted light microscopy.* Bacteria (grown on agar pads) and neutrophil producing PGF (stained with Alcian blue or PAS) were imaged with Axioscope 2 light microscope (Zeiss, Germany) equipped with an Optikam Pro6 digital Camera (Optika, Italy), using a 100× (Plan-NEOFLUAR 100X 1.3NA Ph3 oil) and a 40× oil objective (Plan-NEOFLUAR 100× 1.3NA oil) respectively.

*Fluorescence microscopy.* Immunolabeled neutrophil secreting PGF, NETs, colon cancer mouse tissue, and guinea pig tissues were imaged with an SP8 confocal microscope (Leica) using a 40× oil 1.3NA objective and the Leica Application Suite X (LAS X) software. Images were analyzed with the Fiji software (ImageJ version 2.1.0/1.53j).

*Scanning Electron microscopy.* Neutrophil secreting PGF or releasing NETs were observed at 3.5 kV, in “high vacuum” mode, with a GeminiSEM 300 FESEM (ZEISS Germany).

*Transmission electron microscopy and immunogold labeling.* For immunogold labeling, neutrophils were fixed on coverslips with PFA (4%) and glutaraldehyde (0.1%) in PBS at pH 7.4 for 20 min), further incubated with Triton ×100 (0.05%) in PBS for 1 h, and washed in PBS supplemented with BSA (2%), 5 × 10 min. Samples were incubated overnight at 4°C with anti-CS (1:500) and anti-HA (1:500) in PBS with supplemented with BSA (0.1%). After washing in PBS (2% BSA), samples were incubated with ultrasmall nanogold F(ab')2 fragments of goat anti-rabbit or protein-A Gold 6nm (1:50 in PBS with 0.1% BSA; Aurion) for 1hour. After several washes in PBS, samples were postfixed in PBS supplemented with glutaraldehyde (2.5%) and washed with PBS and with distilled water. Ultrasmall gold particles were then silver enhanced using the R-Gent SE-EM kit (Aurion) for 35 min. Immunogold-labeled neutrophils were postfixed in OsO4 (0.5%) in aqua bidestillata for 15 min. Samples were ultimately dehydrated in graded ethanol series and embedded in Embed 812 (EMS). The ultrathin sections were cut with an ultramicrotome (Leica), stained with uranyl acetate [1% (w/v) in 50% ethanol], and examined with a Hitachi H7500 transmission electron microscope equipped with an Advanced Microscopy Techniques/Hamamatsu digital camera.
## PGF vs. NETs comparison by quantitative imaging analysis

In order to make a distinction between PGF and NETs by imaging quantitative analysis, fixed samples were stained with Myelotracker-Cy5 and DAPI and imaged with a confocal microscope (see microscopy section above). Both Myelotracker and DAPI signals were normalized using the 1st and 99.9th percentile as minimum and maximum limits. The cell body region of interest (ROIcell) was computed using the Myelotracker channel. First, a Difference of Gaussian filter with corresponding sigma s1 = 4 and s2 = 16 for PGF and s1 = 5 and s2 = 20 for NETs was applied. Then, a threshold using Otsu method is used, followed by a morphological binary dilation with a structural element of radius r = 10 for PGF and r = 2 for NETs. The region of interest was then used to remove any cell related intensity for both Myelotracker and DAPI channels. The remaining structure in the image, corresponding to the extracellular polymeric structures, was then detected by thresholding the Myelotracker channel minus the cell areas with the Otsu method. Both Myelotracker and DAPI intensities were collected for each pixel belonging to the polymeric structures. The distribution of Myelotracker and DAPI signal intensities was displayed for both PGF and NETs. A linear regression was used to compare the relationship between Myelotracker and DAPI signal intensities on both samples. A significant difference between PGF (0.0751 ± 0.009) and NETs (0.361 ± 0.009) was observed.

## 3D image visualization and contact assessment

Imaris (v9.6, Bitplane) was used to generate the visualization and contact assessment of Figure 5B. The nuclei, *Shigella* and PGF filaments were segmented with the surface tool. The oblique slicer tool was used on the PGF surfaces to observe *Shigella* bacteria contacting with PGF filaments.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical significance was determined using GraphPad Prism v10. Pairwise comparisons were determined using a Student's unpaired t test. For multiple comparisons, one-way or two-way ANOVA tests were used, as indicated in the figure legend.