# AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by rfaP and ybaT deletion

G. Leccese, M. Chiara, I. Dusetti, D. Noviello, Elisabeth Billard, A. Bibi, G.

Conte, C. Consolandi, M. Vecchi, Mp Conte, et al.

<figure><img src="image_1.png" /></figure>

► To cite this version:

G. Leccese, M. Chiara, I. Dusetti, D. Noviello, Elisabeth Billard, et al. AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by rfaP and ybaT deletion. Gut microbes, 2024, 16 (1), pp.2380064. 10.1080/19490976.2024.2380064. hal-04868800

HAL Id: hal-04868800

https://hal.science/hal-04868800v1

Submitted on 8 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

# AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by *rfaP* and *ybaT* deletion

G. Leccese, M. Chiara, I. Dusetti, D. Noviello, E. Billard, A. Bibi, G. Conte, C. Consolandi, M. Vecchi, MP Conte, N. Barnich, F. Caprioli, F. Facciotti & M. Paroni

**To cite this article:** G. Leccese, M. Chiara, I. Dusetti, D. Noviello, E. Billard, A. Bibi, G. Conte, C. Consolandi, M. Vecchi, MP Conte, N. Barnich, F. Caprioli, F. Facciotti & M. Paroni (2024) AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by *rfaP* and *ybaT* deletion, Gut Microbes, 16:1, 2380064, DOI: 10.1080/19490976.2024.2380064

**To link to this article:** https://doi.org/10.1080/19490976.2024.2380064

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

© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.

<figure><img src="image_5.png" /></figure>

View supplementary material 🌐

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

Published online: 29 Jul 2024.

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

Submit your article to this journal 🌐

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

Article views: 1363

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

View related articles 🌐

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

CrossMark

View Crossmark data 🌐

<figure><img src="image_11.png" /></figure>

Citing articles: 2 View citing articles 🌐
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

# AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by rfaP and ybaT deletion

G. Leccese IDª, M. Chiara IDª, I. Dusetti IDª, D. Noviello IDᵇ.c, E. Billard IDᵈ, A. Bibiª, G. Conteª, C. Consolandiᵉ, M. Vecchiᵇ.c, MP Conte IDf, N. Barnich IDd†, F. Caprioli IDᵇ.c, F. Facciotti IDg, and M. Paroni IDª

ªDepartment of Biosciences, Università degli Studi di Milano, Milan, Italy; ᵇGastroenterology and Endoscopy Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; ᵈDepartment of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; ᵉM2iSH, UMR 1071 Inserm, INRAe USC 1382, CRNH, University of Clermont Auvergne, Clermont-Ferrand, France; ᵈInstitute of Biomedical Technologies, National Research Council (ITB-CNR), Segrate, Milan, Italy; ᵈDepartment of Public Health and Infectious Diseases, 'Sapienza' University of Rome, Rome, Italy; ᵉDepartment of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy

## ABSTRACT

Mucosal enrichment of the Adherent-Invasive *E. coli* (AIEC) pathotype and the expansion of pathogenic IFNγ-producing Th17 (pTh17) cells have been linked to Crohn's Disease (CD) pathogenesis. However, the molecular pathways underlying the AIEC-dependent pTh17 cell transdifferentiation in CD patients remain elusive. To this aim, we created and functionally screened a transposon AIEC mutant library of 10.058 mutants to identify the virulence determinants directly implicated in triggering IL-23 production and pTh17 cell generation. pTh17 cell transdifferentiation was assessed in functional assays by co-culturing AIEC-infected human dendritic cells (DCs) with autologous conventional Th17 (cTh17) cells isolated from blood of Healthy Donors (HD) or CD patients. AIEC triggered IL-23 hypersecretion and transdifferentiation of cTh17 into pTh17 cells selectively through the interaction with CD-derived DCs. Moreover, the chronic release of IL-23 by AIEC-colonized DCs required a continuous IL-23 neutralization to significantly reduce the AIEC-dependent pTh17 cell differentiation. The multi-step screenings of the AIEC mutant's library revealed that deletion of *ybaT* or *rfaP* efficiently hinder the IL-23 hypersecretion and hampered the AIEC-dependent skewing of protective cTh17 into pathogenic IFNγ-producing pTh17 cells. Overall, our findings indicate that *ybaT* (inner membrane transport protein) and *rfaP* (LPS-core heptose kinase) represent novel and attractive candidate targets to prevent chronic intestinal inflammation in CD.

## ARTICLE HISTORY

Received 14 May 2024
Revised 1 July 2024
Accepted 9 July 2024

## KEYWORDS

pathogenic Th17 cells;
Crohn's disease; AIEC; IL-23

<figure><img src="image_14.png" /></figure>

**CONTACT** M. Paroni moira.paroni@unimi.it Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, Milano 20133, Italy;
F. Facciotti federica.facciotti@unimib.it Department of Biotechnology and Bioscience University of Milano-Bicocca, Milan, Italy

†Deceased.

<figure><img src="image_15.png" /></figure>

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2380064
<figure><img src="image_16.png" /></figure>

# 1. Introduction

Crohn's disease (CD) is a chronic inflammatory and multifactorial non-communicable disease affecting the digestive tract, characterized by a dysregulated activation of the mucosal immune system in response to a dysbiotic gut microbiota.¹ Current therapies in CD mostly target effector immune responses and are characterized by a non-optimal net remission rate,² due to primary non-response or secondary loss of response.³ Microbiota-based therapies⁴,⁵ to restore normobiosis are still in an early stage of clinical investigation, and their development is currently limited by the knowledge gap regarding the activation of microbiota-specific immune cells.⁶

Intestinal inflammation in CD has been classically linked to an altered activation of tissue-resident memory ($T_{RM}$) Th1 and Th17 cells. However, while the protective role of Th1 in response to intracellular pathogens has been clearly established, the role of $T_{RM}$ Th17 cells in CD has long been debated. Clinical evidence and murine models of colitis reported both protective and pathogenic functions for intestinal Th17 cells. Colonization of germ-free mice with commensal bacteria results in the generation of intestinal-resident homeostatic conventional Th17 cells with a non-inflammatory cytokine profile and a protective function for the mucosal barrier ⁷⁻⁹. In contrast, intestinal infection of mice with bacterial pathogens⁷,¹⁰ or microbiota transplantation from CD patients¹¹ promotes the differentiation of IFNγ-producing Th17 cells with a pro-inflammatory profile and pathogenic functions. High IL-23 levels have been demonstrated to be necessary to shift Th17 cells toward a pathogenic IFNγ-producing profile in several experimental models of autoimmune and inflammatory conditions.¹²⁻¹⁴

Distinct Th17 cell populations with opposite functions have also been described in human,¹⁵,¹⁶ with a clear pathogenic role in several autoimmune diseases including CD.¹⁷,¹⁸ Enrichment of IFNγ/IL-17 co-producing Th17 cells with a pathogenic profile has been reported in the intestinal mucosa of CD patients.¹⁹⁻²¹ Moreover, in contrast to the failure of anti-IL-17 therapies,²² anti-IL-23p19

biologicals showed better clinical response and remission rates in CD patients.²³

We recently described the presence, induction, and pathogenic functions of a novel subset of $T_{RM}$ IFNγ-producing Th17 cells (pTh17), which are selectively increased in the intestinal tissues of CD patients, induced by high amount of IL-23, and strongly reduced following anti-IL-23 treatment.²⁴ Functionally, these pTh17 cells are specifically activated by Adherent-Invasive *E. coli* (AIEC) strains, thus sustaining the hypothesis that T cells over-activation in CD stems from a loss of tolerance to enteric microbiota.

Within the dysbiotic CD gut microbiota AIEC has been pointed out as one of the most concerning determinants in CD pathogenesis,²⁵ being highly prevalent in the ileal mucosa of CD patients²⁶,²⁷ and colitogenic in several murine models.²⁸ The virulence of AIEC strains known nowadays rely on their ability to adhere and invade intestinal epithelial cells (IECs),²⁹ and extensively survive within macrophages secreting high levels of tumor necrosis factor alpha (TNFα).³⁰ However, while the interplay between AIEC and intestinal epithelial cells or macrophages has been extensively characterized,³¹⁻³⁴ the molecular mechanisms behind the AIEC-antigen dependent activation of pTh17 cells are completely unknown.

Here, we combined microbial genetics approaches with functional immunological assays to identify specific AIEC determinants triggering the hypersecretion of IL-23 and the exaggerated pTh17 cell activation in CD patients. We demonstrated that AIEC directly promotes the transdifferentiation of pathogenic IFNγ-producing pTh17 cells from the precursors non-pathogenic Th17 cells. By screening a library of 10.058 mutants, we identified a restricted set of AIEC genes involved in triggering IL-23 hyperproduction by CD-derived dendritic cells (DCs).

Finally, we demonstrated that inhibiting AIEC pathways associated with LPS core biosynthesis (*rfaP*), and the inner membrane transport protein (*ybaT*), hampered the IL-23 overly secretion by DCs and strongly hinder the AIEC-dependent generation of pTh17 cells. This effect is similar to that observed with a continuous IL-23 neutralization,
<figure><img src="image_17.png" /></figure>

thus providing for the first time a rationale for
a functional targeting of the AIEC determinants
triggering pathogenic pTh17 cells in CD patients.

**2. Materials and methods**

**2.1. Patients**

Clinical characteristics of CD patients and HD included in this study are listed in **Table S1**. The HD cohort comprises 47 HD with no personal or family history of allergies or immune-mediated diseases. The CD cohort includes 44 patients with a confirmed diagnosis of CD for at least 6 months according to the current ECCO guidelines either in clinical remission or with a mild clinical activity according to the Harvey-Bradshaw index (HBI) enrolled in the IBD outpatient clinic of the Foundation IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy, during scheduled visits according to clinical practice. Subjects taking oral corticosteroids in the preceding 4 weeks, immunomodulators in the preceding 3 months, biologics in the preceding 12 weeks, or antibiotics in the preceding 4 weeks were excluded. The studies involving human participants were carried out in compliance with the Declaration of Helsinki protocols and were reviewed and approved by the Ethics commission (Milano Area B, code 128_2018bis).

**2.2. Bacterial strains and generation of transposon library of AIEC-LF82 mutants**

The AIEC bacterial strains used in this project are
listed in **Table S2**. The probiotic *E. coli* Nissle 1917
strain (EcN, Mutaflor; DMS 6601), belonging to the
same phylogroup of LF82, was used as non-patho-
genic control. Bacteria were grown in YESCA
medium,³⁵ unless otherwise indicated.

A Tn5 transposon library of 10.058 AIEC-LF82 kanamycin-resistant mutants was generated using EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit (Lucigen) following the manufacturer's protocol. LF82 mutants were grown in YESCA supplemented with kanamycin (50 µg/ml). The mutated genes in selected LF82 mutants were identified by rescue cloning according to the manufacturer's instructions and sequenced by Eurofins Sanger

Sequencing Services. For the identification of the
transposon insertion point, the sequencing results
were aligned on the LF82 genome (sequence ID:
CU651637) with BLAST program (NCBI).

LF82-Δ*fimH*, -Δ*fraP*, and -Δ*ybaT* isogenic deletion mutants were generated using the λ-Red recombination system<sup>36,37</sup> with the primers listed in **Table S3**. For subsequent infection experiments, antibiotic resistance was removed transforming mutants with pCP20 plasmid,<sup>36</sup> primers used to confirm target gene deletion are listed in **Table S3**.

**2.3. Human primary cells isolation and cultures**

Monocyte-derived dendritic cells (moDCs), mono-
cyte-derived macrophages (MDMs) and Human
intestinal epithelial HT29 cell line (IECs) were dif-
ferentiated and cultured as previously described.<sup>35</sup>
Th1 and Th17 cell subsets were sort purified
(FACSAria III, BD Biosciences) from peripheral
blood mononuclear cells (PBMC) of HD and CD
patients according to the expression of specific sur-
face markers combination<sup>24</sup>: Th1: CD4<sup>+</sup>IL-7
R<sup>+</sup>CD25<sup>low</sup>CCR6<sup>-</sup>CXCR3<sup>+</sup>;<sup>conventional</sup> cTh17
cells: CD4<sup>+</sup>IL-7 R<sup>+</sup>CD25<sup>low</sup>CCR6<sup>+</sup>CXCR3<sup>-</sup>CCR5<sup>-</sup>;
pathogenic pTh17 cells: CD4<sup>+</sup>IL-7
R<sup>+</sup>CD25<sup>low</sup>CCR6<sup>+</sup>CXCR3<sup>-</sup>CCR5<sup>+</sup> (gating strategy
reported in Figure S1b).

**2.4. Infection assays and antigen-specificity assays**

HD- and CD-derived MDMs or moDCs were
infected with 10⁷ CFU/ml of bacterial strain
(Multiplicity of infection-MOI 1:10). Bacterial pha-
gocytosis and intracellular persistence were evalu-
ated as previously described.³⁵ When stated 40 µg/
ml of neutralizing human anti-TLR4 (clone
W7C11; invivogen) was added to moDCs 1 h
before bacterial infection. HT29 cells were infected
with LF82 or LF82 mutants at the final concentra-
tion of 1.4 × 10⁶ CFU/well. At 24 h post infection,
supernatants of infected cells were collected and
stored at -20°C for cytokine quantification by
ELISA assay.

AIEC-dependent T-cell transdifferentiation into
IFNγ-producing Th17 cells (pTh17) was analyzed
by co-culturing 5 × 10⁴ autologous cTh17 or Th1
cells with LF82- or LF82 mutants-infected moDCs
or MDMs at a 5:1 ratio for 10 d in RPMI medium
supplemented with 2 µg/ml gentamycin and 20 U/ml of recombinant human IL-2 (rhIL-2). For proliferation assay, cTh17 cells were first labeled with 5 µM Carboxyfluorescein succinimidyl ester (CFSE; Biolegend) and proliferating cells (CFSE dye dilution) were analyzed by flow cytometry. Background proliferation induced by autologous unstimulated moDCs was subtracted to calculate antigen-specific proliferation. For AIEC-dependent pTh17 cell transdifferentiation, co-cultures of T cells with infected moDCs or MDMs were analyzed for intracellular cytokine expression after a further polyclonal stimulation with PMA/ Ionomycin as previously described.²⁴ After fixation and permeabilization, T cells were stained with anti-CD40L, anti-IL-17, and anti-IFNγ (Biolegend). Background activation of T cells stimulated with autologous uninfected moDCs or MDMs was subtracted to calculate antigen-dependent pTh17 cell transdifferentiation. In some experiments neutralizing anti-IL-23 (clone HNU2319; Invitrogen) and/or anti-IL-1β antibodies (clone CRM56; Invitrogen) were added together with cTh17 cells at T = 0 or every 48 h during the assay. Mouse IgG1k (clone P3.6.2.8.1; Invitrogen) has been used as isotype control. Acquisition was performed with FACSCanto II cytometer and data were analyzed using FlowJo software (BD Biosciences).

## 2.5. Functional screenings of AIEC-LF82 mutant library

For the first screening, the parental LF82 strain and LF82 mutants were individually grown in YESCA medium in 96 well plates O/N at 37°C in an orbital shaking. Bacterial concentration of LF82 and of each single mutant were evaluated in a microplate reader (SAFAS MP96) and, thereafter, the same bacterial concentration calculated on the correspondence of OD₆₀₀-CFU/ml for the parental LF82 strain was used to infect human cells with each single LF82 mutant strain resuspended in complete RPMI without antibiotics.

The amount of IL-23 (Invitrogen) and IL-1β (BioLegend) released by moDCs, as well as CCL20 (MIP-3α; R&D Systems) released by HT29 cells, after 24 h of infection with each single mutant (M) was quantified by ELISA assays. Cytokine

levels were normalized on the growth of each mutant strain (OD₆₀₀ of the O/N bacterial cultures) and compared with the LF82 reference strain using the following formula:

$$ \text{Norm C/GR} = \log_2 \left( \frac{C_M}{GR_M} \right) - \log_2 \left( \frac{C_{LF82}}{GR_{LF82}} \right) $$

where C is the cytokine concentration (OD₄₅₀) released in response to each single mutant (Cₘ) or to the parental LF82 strain (C<sub>LF82</sub>); GR is the corresponding growth rate (OD₆₀₀) of each mutant (GRₘ) or of the LF82 (GR<sub>LF82</sub>).

The top 1% mutants associated with a reduced IL-23 production were selected for subsequent screenings by statistical analysis of the values distribution. Mutant strains in essential genes or with a defective growth rate as compared to the parental LF82 strain were discarded.

From the second screening onward, to obtain a strict specific MOI of 1:10, selected mutants were individually normalized on the specific correspondence of OD₆₀₀-CFU/ml.

## 2.6. Phylogenetic distribution of selected genes across E. coli pathotypes and phylogroups

E. coli pathotypes and phylogroups were assigned according to the definitions of Clark et al.³⁸ Complete genome sequences, and corresponding genome annotations were retrieved from GenBank.³⁹ Gene presence/absence profiles were determined by protein sequence similarity searches based on blastp with default parameters⁴⁰; sequences derived from the reference assembly of the LF82 strain were used as a query. A gene was considered to be present in a reference genome assembly if/when the following criteria were fulfilled: level of identity with the query sequence >80%; the alignment covered at least 75% of the size of the query sequence.

## 2.7. Phenotypic characterization of AIEC-LF82 mutant strains

For assessing differential bacterial growth in a medium that mimic the phagolysosome vacuole content, O/N cultures of LF82 and LF82 mutants were diluted to an OD₆₀₀ = 0.02 in the Acid and Nutrient-poor medium (ANp)³³ and bacterial
<figure><img src="image_18.png" /></figure>

growth curves were evaluated for 16 h in a microplate reader (SAFAS MP96) which ensures shaking conditions and constant temperature. Biofilm formation (crystal violet assay),⁴¹ flagellar motility,⁴² Type 1 fimbriae expression (FimH, yeast aggregation assays)⁴² and curli (congo red assay)⁴³ were evaluated as previously described.

## 2.8. Statistical analysis

Statistical significance was assessed through paired or unpaired t-test for comparison of two groups (applying Mann-Whitney's or Welch's correction for non-normally distributed variables) or by one-way ANOVA for comparison of more than two groups (with Dunn's multiple comparisons for non-normally distributed variables). All experiments were performed at least 3 times. Statistics were performed with Prism software (version 9; GraphPad Software) and indicated in the respective figure legends.

# 3. Results

## 3.1. Interplay between AIEC and CD-derived dendritic cells promoted the overly secretion of IL-23 and transdifferentiation of pTh17 cells

We functionally evaluated the inflammatory response of AIEC-infected MDMs and moDCs isolated from the peripheral blood of CD patients, focusing on IL-23 and IL-1β secretion for their important polarizing role in pTh17 cell differentiation.¹⁴,⁴⁴⁻⁴⁶ The probiotic EcN strain, belonging to the same phylogroup of LF82, was used as nonpathogenic control (Figure 1).

LF82 persisted significantly more than EcN within both MDMs and moDCs at 24 h post infection, with a significantly higher intracellular bacterial load within moDCs (Figure 1a). At this time point, LF82 triggered hypersecretion of IL-23 exclusively by moDCs (Figure 1b) and promoted a significantly higher release of IL-1β by moDCs, but lower than IL-23, compared to MDMs (Figure 1c). Interestingly also EcN triggered the secretion of pro-inflammatory IL-23 and IL-1β. Instead, no difference in the secretion of TGF-β and IL-6, which have a more prominent role in the differentiation of naive CD4⁺ T cells to cTh17 cells,⁴⁷ was observed in response to LF82 or EcN

by moDCs or MDM derived from CD patients (Figures 1d,e).

LF82 and EcN strains persisted significantly more within CD-derived moDCs than in those derived from healthy donors (HD) (Figure 1f), like previous observations in CD-macrophages.⁴⁸ A dysregulated inflammatory response selectively in CD patients was confirmed by the secretion of IL-23, but not of IL-1β, by CD- compared to HD-moDCs (Figure 1g,h) and slightly higher levels of IL-6 and TGF-β (Figure S1a) in response to LF82 and EcN. Similar results, were confirmed upon DCs exposure to other AIEC clinical strains isolated from CD patients (Figures 1i-k). Indeed, although AIEC clinical isolates differentially persisted within DCs as compared to the reference LF82 strain (Figure 1i), they all promoted similar IL-1β amount from HD- and CD-derived DCs (Figure 1k) but a significant IL-23 overproduction selectively by CD-moDCs (Figure 1j).

Next, we assessed whether AIEC was directly implicated in the transdifferentiation of human pathogenic IFNγ-producing pTh17 cells, employing the well-established antigen-dependent functional assay (Figure 2a). Exposure of human conventional Th17 cells (cTh17), but not of Th1 cells (Figure S1b), to LF82-infected CD-moDCs induced a sustained co-production of IFNγ/IL-17 (Figures 2b,c and S1d), resembling ex vivo activated pTh17 cells (Figure S1c). In contrast, EcN-infected moDCs induced a phenotype more similar to unstimulated T cells, despite the high IL-23 levels released by EcN-stimulated CD-moDCs (Figure 2b and 1b). Moreover, precursors cTh17 cells shifted toward IFNγ-producing pTh17 cells exclusively upon exposure to LF82-infected moDCs derived from CD patients but not from HD (Figure S1e). Importantly, MDMs derived from CD patients did not secrete IL-23 in response to *E. coli* strains tested (Figure 1b) and failed to induce transdifferentiation of cTh17 cells into pTh17 cells (Figures 2d,e), thus indicating a more prominent role of dendritic cells in the AIEC-dependent pTh17 cell generation.

To demonstrate the functional roles of IL-23 and IL-1β in AIEC-dependent pTh17 cell generation we added neutralizing antibodies against IL-23 and/or IL-1β at different time points during the in vitro pTh17 transdifferentiation assay (Figure 2f). Single blockade of IL-23 or IL-1β at day 0 only slightly
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 1.** AIEC intracellular persistence within dendritic cells promotes dysregulated IL-23 hypersecretion selectively in CD patients. (a) Intracellular bacterial load of AIEC-LF82 and the probiotic Nissle 1917 (EcN) strains after 24 h of infection within monocyte-derived macrophages (MDMs, *n* = 6) or monocyte-derived dendritic cells (moDCs, *n* = 10) isolated from CD patients. (b) IL-23, (c) IL-1β, (d) IL-6, and (e) TGF-β secretion by CD-derived MDMs (*n* = 6) or CD-derived moDCs (*n* = 10) after 24 h of infection with LF82 or EcN strains was measured by ELISA. (f) Intracellular bacterial load of LF82 or EcN strains within moDCs isolated from CD patients (*n* = 10) or healthy donors (HD, *n* = 15), at 24 h post-infection. (g) IL-23 and (h) IL-1β secretion by CD-derived (*n* = 10) or HD-derived (*n* = 15) moDCs at 24 h post-infection with LF82 or EcN strains was measured by ELISA. (a-h) Statistical significance was calculated using Mann-Whitney test and reported as *p* < 0.05, **p** < 0.01, ***p** < 0.001. (i) Intracellular bacterial load of LF82 and other indicated clinical AIEC strains within moDCs isolated from CD patients (*n* = 3–4) or HD (*n* = 3–4), at 24 h post-infection. Horizontal lines represent the median of the intracellular bacterial load of LF82 within CD-moDCs (purple line) or within HD-moDCs (blue line); respectively. (j) IL-23 and (k) IL-1β production by CD-derived (*n* = 3–4) or HD-derived moDCs (*n* = 3–4) at 24 h post-infection with LF82 or other indicated clinical AIEC strains. Horizontal lines represent the median of IL-23 or IL-1β released by CD-moDCs (purple line) or by HD-moDCs (blue line), respectively, in response to LF82. (i-k) Statistical significance was calculated by comparing CD- and HD-derived moDCs infected with the indicated AIEC strains using Mann-Whitney test, and reported as: *p* < 0.05, **p** < 0.01, ***p** < 0.001.</figcaption></figure>

reduced the IFNγ/IL-17 co-expression by cTh17 cells in response to LF82-stimulated DCs (Figure 2g-left graph). Conversely, a continuous IL-23 blocking by adding neutralizing antibodies every 48 h resulted in a sharp reduction of IFNγ/IL-17 co-expression by AIEC-exposed cTh17 cells (Figure 2g-right graph). No difference was observed between untreated cells and treated cells with isotype control every 48 h (data not shown).

Taken together, these results indicated that AIEC interacts differently with DCs derived

from CD or HD triggering a sustained IL-23 hypersecretion and the transdifferentiation of pTh17 cells from precursors protective cTh17 cells exclusively in CD patients.

## 3.2. Functional screening of a LF82 mutant library identified AIEC determinants specifically involved in triggering IL-23 hypersecretion by DCs

We then aimed at identifying the AIEC determinants triggering the transdifferentiation of cTh17
<figure><img src="image_21.png" /></figure>

a

<figure><img src="image_22.png" /></figure>

b

cTh17 + CD-derived moDCs (t=10 days)

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

c

<figure><img src="image_24.png" /></figure>

d

cTh17 + CD-derived MDMs (t=10 days)

<figure><img src="image_25.png" /></figure>

e

<figure><img src="image_26.png" /></figure>

f

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

cTh17 cell trans-differentiation (10 days)

<figure><img src="image_29.png" /></figure>

g

<figure><img src="image_30.png" /><figcaption>**Figure 2.** AIEC strongly promotes the transdifferentiation of conventional Th17 cells into pathogenic IFNγ-producing pTh17 cells selectively through the interaction with CD-derived DCs. (a) Experimental strategy to analyze the antigen-dependent transdifferentiation of conventional Th17 cells (cTh17) into pathogenic IFNγ-producing Th17 cells (pTh17 cells). Peripheral cTh17 cells sort-purified from blood of CD patients were stimulated with autologous moDCs (b-c; *n* = 6) or MDMs (d-e; *n* = 4) alone (unstimulated), or infected with AIEC-LF82, or EcN strains. After 10 d of co-culture, expression of IFNγ, IL-17 and co-expression of IFNγ/IL-17 was measured by</figcaption></figure>
<figure><img src="image_31.png" /></figure>

cells into pTh17 cells. We generated and function-
ally screened a library of 10.058 LF82 mutants,
carrying a single transposon insertion compared
to the parental LF82 strain, using the EZ-Tn5™
<R6Kγori/KAN-2>Tnp Transposome system
(Figure 3a). Having demonstrated the crucial
polarizing role of IL-23 as downstream signal dur-
ing antigen-dependent pTh17 cell activation, we
performed a multi-step screening assay using the
IL-23 levels released by AIEC-infected moDCs as
readout. The multi-step screening was consequent
of the technical impossibility to carry out the anti-
gen-specificity assay with 10 thousand mutants
directly on CD-derived immune cells (isolated
from a limited amount of peripheral blood).
Thus, in the first step, all mutants were individually
tested on buffy-coat derived HD-moDCs and those
displaying the highest reduction in IL-23 secretion
(under the 1st percentile, *p* < 0.01 as compared to
the parental LF82 strain), were selected for
the second step of screening (Figure 3b-left graph).

As comparative control, given the important
role of CCL-20 in recruiting DCs and Th17 cells
at the site of infection, we checked if AIEC mutants
also affected CCL-20 released by intestinal epithe-
lial HT29 cells (IECs).

Among 96 selected mutants inducing a significant
IL-23 reduction (Figure 3b-left graph), 5 mutants
(5.2%) also induced a significant reduction of IL-1β
secretion by HD-moDCs (Figure 3b-right graph),
while 10 distinct mutants (9.4%) also a reduced
CCL20 secretion by IECs compared to the parental
LF82 strain (Figure S2a) thus indicating that these
three inflammatory pathways are stimulated by dif-
ferent AIEC determinants.

As second screening, to exclude the selection of
false-positive mutants based on an underestimated
infection rate, a more stringent normalization was

applied for each single mutant, by evaluating the
correspondence between OD₆₀₀ and bacterial cell
count (CFU/ml) and the probiotic EcN strain was
used as reference threshold. Results revealed that,
when tested at the effective MOI of 1:10, 22 out of
the initial 96 LF82 mutants triggered the secretion
of similar or even significantly lower levels of IL-23
by HD-moDCs as compared to EcN (Figure 3c and
S2b). Again, among these 22 strains, 6 mutants also
triggered a significantly lower IL-1β secretion by
HD-moDCs, resembling the non-pathogenic
inflammatory response following EcN infection,
while 15 distinct mutants out of 96 induced only
a lower secretion of IL-1β but not of IL-23
(Figure 3c and S2c).

Next, as third screening we tested these 22
selected LF82 mutants on CD-moDCs. Results
showed that 13 out of 22 mutants stimulated
a lower secretion of IL-23 (Figure 3d-upper graph),
and 8 of them also lower levels of IL-1β by CD-
moDCs, compared to the parental LF82 strain
(Figure 3d-lower graph). To evaluate whether the
reduced pro-inflammatory response was associated
with a reduced bacterial survival within DCs, we
analyzed the intracellular bacterial load of LF82
mutants within HD- and CD-derived moDCs at
24 h post infection, as well as the bacterial growth
in an acid and nutrient-poor medium (ANp) which
mimics the phagocytic vacuole stress conditions.³³
LF82 mutants displayed a different proficiency in
persisting within MoDCs, both derived from HD
and CD patients (Figure 3e). No correlation was
observed between intracellular persistence within
moDCs and bacterial growth curve in the ANp
medium of mutants (Figure S3), thus indicating
that ANp formulation did not properly mimic the
DC intracellular environment. No correlation was
observed either between intracellular bacterial load

intracellular staining. (b and d) One representative experiment showing the gating strategy to identify antigen-dependent pTh17
transdifferentiation is shown. (c and e) Mean percentage of single expression of IFNγ+, IL-17+ and IFNγ+/IL-17+ co-expression on
CD40L+cTh17 cells after 10 d of co-culture with indicated stimuli were reported. Statistical significance was calculated using one-way
ANOVA and reported as *p* < 0.05, **p** < 0.01, ***p** < 0.001. (f) Experimental strategy to analyze the effect of single or continuous IL-
23 and/or IL-1β blocking on the AIEC-dependent transdifferentiation of cTh17 into pTh17 cells. (g) Fold-change values of the
percentages of single expression of IFNγ+, IL-17+ and IFNγ+/IL-17+ co-expression on peripheral CD40L+cTh17 cells after 10 d of co-
culture with autologous CD-derived moDCs infected with LF82 in the presence of indicated neutralizing antibodies added at t = 0 (left
graph) or every 48 h (right graph). Data are represented as mean percentage (fold-change over LF82) ± SEM of at least three
independent experiments. Statistical significance was calculated using one-way ANOVA and reported as *p* < 0.05, **p** < 0.01, ***p* <
0.001.
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Figure 3.** Immunological and functional screening of the AIEC-LF82 mutant library on HD-and CD-derived DCs. (a) Experimental design for generating and screening the AIEC-LF82 transposon insertion library and the subsequent selection of mutants as described in material and methods. (b) First screening: Distribution of growth-normalized IL-23 (left graph) and IL-1β (right graph) production, log scaled (normC/G), measured in the supernatants of HD-derived DCs after 24 h of infection with each single LF82 mutant in comparison to the parental LF82 strain taken as zero. Dots represent individual measures for each mutant, and the inferred probability</figcaption></figure>
<figure><img src="image_43.png" /></figure>

and IL-23 secretion (Figure 3f-left graph). Indeed, 4 out of the 13 mutants with an impaired ability to promote IL-23 secretion by CD-moDCs, survived within moDCs similarly to the parental LF82 strain (T74.A5, T75.E8, T82.D7, and T96.B8) (Figure 3e). Likewise, 3 out of 9 mutants that triggered a similar amount of IL-23 as LF82 did not persist within CD-moDCs (T31.B12, T52.H10, and T53.A8), thus confirming that the reduced IL-23 secretion by CD-moDCs was not due to a lower intracellular bacterial load at 24 h post infection. Conversely, IL-1β secretion was dependent on intracellular bacterial load exclusively in CD-moDCs (Figure 3f-right graph).

**3.3. Several AIEC determinants promoted the dysregulated IL-23 hypersecretion by CD-derived moDCs**

To identify the mutated genes in the final 13 selected LF82 mutants (Figure 3d-upper graph), and to analyze the effect of these mutations on the expression of the key AIEC pathogenic features, we performed an in depth phenotypic characterization. The DNA sequencing after rescue cloning revealed that the transposon insertions in these 13 selected LF82 mutants occurred within genes involved in a few specific pathways including

metabolic processes, stress response, LPS and capsule biosynthesis (Table 1, Figure S4).

Importantly, 2 out of 3 mutants in pathways involved in biofilm formation (T69.D10 and T74.A5), and 2 out of 3 mutants in pathways involved in the LPS core biosynthesis (T12.G9 and T45.G12) resulted mutated in the same gene, *yhjK* and *rfaP*, respectively, thus confirming the strength of our functional screenings.

Remarkably, except for *entD* and *kpsS*, which displayed relatively lower levels of conservation and a patchy phylogenetic distribution, all the genes identified by our screening were almost universally present across distinct *E. coli* pathotypes and phylogroups (Figure 4a). These 11 genes showed high levels of sequence conservation, with a pattern that was not dissimilar to the universal single copy marker genes *dnaJ* and *rpoB*. Similar considerations could be applied to *fimH*, one of the most studied AIEC virulence factors,²⁹,⁴⁹,⁵⁰ which was found to be associated with all the *E. coli* strains included in our analyses (Figure 4a) with the exclusion of EAEC and EAHEC pathotypes, as previously demonstrated.³⁹

Next, we analyzed whether mutations in these 11 genes impacted on the expression of key AIEC pathogenic features, including biofilm formation, motility, and fimbriae expression (Table 2, Figure

density function is represented at the top. Vertical dotted blue lines indicate the 1st and 99th percentile, respectively. Green dots outline the 96 selected LF82 mutants with a normC/G IL-23 value below the 1st percentile (**left graph**), and their corresponding IL-1β values (**right graph**), calculated with the normalization formula reported in the material and methods. Probability density functions were inferred using the density() function from the R stats package. Graphical representation of the data by ggplot2 R library. R version 4.2.1 was used. (c) Second screening: fold-change values, represented as heatmap, of the percentage of IL-23 and IL-1β (rows) of the 96 selected LF82 mutants (columns), in comparison to the parental LF82 strain taken as zero, tested on HD-moDCs (n = 3-5 independent experiments) after a strict normalization of each single mutant on the corresponding CFU/ml value. Statistical significance between LF82 and LF82 mutants was calculated using one-way ANOVA and reported as: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (d) Third screening: fold-change values of IL-23 (**upper graph**) and IL-1β (**lower graph**) of the 22 selected LF82 mutants after the second screening, in comparison to the parental LF82 strain taken as zero, tested on CD-moDCs (n = 5). Data are represented as mean percentage (fold-change over LF82) ± SEM of at least 3-5 independent experiments. Vertical dotted purple lines indicate the mean ± SEM of fold-change values for the probiotic EcN strain. Statistical significance was calculated using one-way ANOVA and reported as: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (e) Intracellular bacterial load (CFU/ml) of LF82 mutants within moDCs isolated from CD patients (n = 5; purple bars) or HD (blue bars) after 24 h of infection. Data are represented as mean ± SEM of at least 3-5 independent experiments. Horizontal lines indicate the mean of LF82 intracellular bacterial load within CD-derived (purple line) or HD-derived moDCs (blue line). LF82 mutants with an impaired ability to trigger IL-23 secretion by CD-moDCs are marked in green. Statistical significance between values of LF82 mutants and the parental LF82 strain was calculated using one-way ANOVA and reported as \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 (in black for the comparison among HD-moDCs, in purple for the comparison among CD-moDCs). Statistical significance between values of the same LF82 mutant in DCs derived from HD vs CD patients was calculated using unpaired t-test and reported as: \#p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (f) Correlation between intracellular persistence of LF82 mutants within HD- (white dots) or CD-moDCs (purple dots) at 24 h post-infection and fold changes in the percentage of IL-23 (**left graph**) and IL-1β (**right graph**) production of each LF82 mutant, as compared to the parental LF82 strain. Correlations were determined using Pearson correlation coefficients. R squares and *p* values for each correlation were reported in the graphs.
<figure><img src="image_44.png" /><figcaption>Table 1. Target gene of the EZ-Tn5-based transposon in LF82 mutant strains.</figcaption></figure>

<table><thead><tr><th>Mutant</th><th>Gene name - description</th><th>Pathway</th></tr></thead><tbody><tr><td>T6.E4</td><td><i>aceF</i></td><td>pyruvate dehydrogenase, E2 subunit</td></tr><tr><td>T77.A6</td><td><i>purA</i></td><td>adenylosuccinate synthetase</td></tr><tr><td>T82.C9</td><td><i>rhlB</i></td><td>ATP-dependent RNA helicase</td></tr><tr><td>T53.G6</td><td><i>entD</i></td><td>enterobactin synthetase component D</td></tr><tr><td>T59.F12</td><td><i>ybaT</i></td><td>Inner membrane transport protein</td></tr><tr><td>T75.E8</td><td><i>cpxA</i></td><td>sensor histidine kinase</td></tr><tr><td>T69.D10<br>T74.A5</td><td><i>yhjK</i></td><td>c-di-GMP phosphodiesterase</td></tr><tr><td>T82.D7</td><td><i>visC</i></td><td>2-octaprenylphenol 6-hydroxylase</td></tr><tr><td>T12.G9<br>T45.G12</td><td><i>rfaP</i></td><td>lipopolysaccharide core heptose(I) kinase</td></tr><tr><td>T95.A6</td><td><i>rfaG</i></td><td>lipopolysaccharide glucosyltransferase I</td></tr><tr><td>T96.B8</td><td><i>kpsS</i><br>(LF82_p466)</td><td>capsular polysaccharide biosynthesis protein</td></tr></tbody></table>

S5). Phenotypic characterization confirmed the higher proficiency of AIEC to form biofilm as compared to EcN (Table 2) and the distinctive expression of type 1 fimbriae (FimH) by AIEC³¹ (Figure S5A), despite the presence of *fimH* gene both in LF82 and EcN with a 98.67% of protein sequence identity level (Figure 4a). In contrast, both LF82 and EcN resulted curli-positive displaying the “rdar” (for red, dry, and rough) morphology on CR agar plates (Table 2, Figure S5b), consistently with the curli production by most of Enterobacteriaceae.⁴³

The phenotypic analysis of the 13 selected mutants displayed a positive correlation both between AIEC intracellular persistence and biofilm production (Figure 4b), and flagellar motility (Figure 4c). Instead, neither biofilm production nor flagellar motility correlated with IL-23 or IL-1β secretion by DCs (Figure 4d,e) thus indicating that AIEC adhesion determinants are not relevant for this type of inflammatory response by DCs. Notably, among mutants promoting the lowest IL-23 secretion by CD-moDCs, even lower than EcN (Figure 3D-upper graph), we found mutants in the LPS structure (Δ*rfaP* and Δ*rfaG*) and the Δ*purA* mutant, which did not produce curli fimbriae and displayed the highest reduction in FimH production compared to the parental strain LF82 (Table 2, Figure S5). These results, together with the evidence that several pathogenic *E. coli* strains trigger mucosal inflammation via fimbriae-

dependent TLR4 activation,⁵¹,⁵² led us to investigate whether also the IL-23 hypersecretion by CD-moDCs may be TLR4-dependent. Blockade of the TLR4 biological activity with neutralizing antibodies did not affect neither the LF82 phagocytosis/persistence within CD-moDCs (Figure 4f), nor IL-23/IL-1β secretion (Figure 4g). Similarly, also the deletion of *fimH*, a potent TLR4 agonist that promotes inflammation in an LPS-independent manner,⁵³ did not hamper LF82 internalization or persistence within CD-moDCs (Figure 4h) and neither triggered a lower inflammatory response (Figure 4i), thus indicating that fimbriae-LPS/TLR4-dependent signaling pathway does not play a major role in the AIEC intracellular persistence or in the inflammatory response of DCs to AIEC infection.

Taken together, these results indicated that AIEC determinants implicated in triggering IL-23 release and survival within DCs are different from AIEC adhesion determinants promoting adhesion and invasion of intestinal epithelial cells.²⁹,⁴²

### 3.4. Mutations in an inner membrane protein and in the LPS structure hamper the AIEC-dependent transdifferentiation of cTh17 into pathogenic IFNy-producing pTh17 cells

To functionally validate the link between the absence of specific AIEC determinants and the transdifferentiation of cTh17 into pTh17 cells, we
<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /><figcaption>**Figure 4.** Conservation of selected AIEC-gene targets among different *E. coli* pathotypes and phenotypic characterization of the LF82 mutant strains. (a) Heatmap showing presence and conservation (protein sequence identity) of AIEC-target genes (rows) of the 13 LF82 mutants with an impaired ability to stimulate IL-23 secretion by CD-moDCs, among different *E. coli* phylogroups and pathotypes (columns). Columns are organized first by pathotype, namely Adherent-Invasive (AIEC), Avian Pathogenic (APEC), Enteroaggregative (EAEC), Enteroaggregative Hemorrhagic (EAHEC), Enterohemorrhagic (EHEC), Enteropathogenic (EPEC), Enterotoxigenic (ETEC),</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>**Table 2.** Phenotypic characterization of the adhesion determinants production.</figcaption></figure>

<table><thead><tr><th rowspan="2">Strain</th><th rowspan="2"></th><th rowspan="2">Biofilm production<br>(Al<sup>a</sup>, mean ± SEM)</th><th rowspan="2">Flagellar Motility<br>(cm<sup>b</sup>, mean ± SEM)</th><th colspan="2">Fimbriae expression</th></tr><tr><th>Curli<br>(congo red<sup>c</sup>)</th><th>Type 1 fimbriae<br>(Yeast agglutination)</th></tr></thead><tbody><tr><td>LF82</td><td></td><td>0.737 ± 0.062</td><td>4.36 ± 0.22</td><td>+</td><td>1/64</td></tr><tr><td>EcN</td><td></td><td>0.080 ± 0.024<br>(p&lt;0.001) ***</td><td>2.07 ± 0.09<br>(p&lt;0.001) ***</td><td>+</td><td>1</td></tr><tr><td rowspan="3">Metabolic<br>Pathways<br>and Basic cell<br>Processes</td><td>ΔaceF<br>(T6.E4)</td><td>1.542 ± 0.2173<br>(p&lt;0.001) ***</td><td>1.40 ± 0.06<br>(p&lt;0.001) ***</td><td>+</td><td>1/32</td></tr><tr><td>ΔpurA<br>(T77.A6)</td><td>0.222 ± 0.037<br>(p=0.005) **</td><td>3.83 ± 0.03<br>(p=0.004) **</td><td>-</td><td>1/4</td></tr><tr><td>ΔrhlB<br>(T82.C9)</td><td>0.993 ± 0.199</td><td>3.37 ± 0.12<br>(p&lt;0.001) ***</td><td>+</td><td>1/16</td></tr><tr><td rowspan="3">Nutritional<br>and/or<br>Environmental<br>stress response</td><td>ΔentD<br>(T53.G6)</td><td>0.944 ± 0.127</td><td>3.77 ± 0.09<br>(p=0.001) **</td><td>+</td><td>1/64</td></tr><tr><td>ΔybaT<br>(T59.F12)</td><td>0.700 ± 0.072</td><td>3.73 ± 0.03<br>(p&lt;0.001) ***</td><td>+</td><td>1/32</td></tr><tr><td>ΔcpxA<br>(T75.E8)</td><td>1.116 ± 0.210<br>(p=0.04) *</td><td>3.47 ± 0.03<br>(p&lt;0.001) ***</td><td>+</td><td>1/32</td></tr><tr><td rowspan="3">Biofilm<br>formation</td><td>ΔyhjK<br>(T69.D10)</td><td>0.958 ± 0.186</td><td>2.90 ± 0.12<br>(p&lt;0.001) ***</td><td>+</td><td>1/16</td></tr><tr><td>ΔyhjK<br>(T74.A5)</td><td>0.778 ± 0.127</td><td>2.37 ± 0.27<br>(p&lt;0.001) ***</td><td>+</td><td>1/16</td></tr><tr><td>ΔvisC<br>(T82.D7)</td><td>1.086 ± 0.162</td><td>4.10 ± 0.12</td><td>+</td><td>1/32</td></tr><tr><td rowspan="3">LPS core<br>biosynthesis</td><td>ΔrfaP<br>(T12.G9)</td><td>0.257 ± 0.022<br>(p=0.008) **</td><td>1.23 ± 0.09<br>(p&lt;0.001) ***</td><td>-</td><td>1/16</td></tr><tr><td>ΔrfaP<br>(T45.G12)</td><td>0.282 ± 0.035<br>(p=0.01) *</td><td>1.23 ± 0.03<br>(p&lt;0.001) ***</td><td>-</td><td>1/8</td></tr><tr><td>ΔrfaG<br>(T95.A6)</td><td>0.147 ± 0.036<br>(p=0.001) **</td><td>0.67 ± 0.12<br>(p&lt;0.001) ***</td><td>-</td><td>1/8</td></tr><tr><td>Capsule<br>Biosynthesis</td><td>ΔkpsS<br>(T96.B8)</td><td>0.608 ± 0.052</td><td>3.97 ± 0.09<br>(p=0.03) *</td><td>+</td><td>1/32</td></tr></tbody></table>

Extraintestinal Pathogenic (ExPEC), Neonatal Meningitis (NMEC), Uropathogenic (UPEC), and then by phylogroup indicated by the colored squares. Identity levels are represented according to the color gradient indicated by the scale. White barred squares indicate the absence of relative gene in the analyzed bacterial strains. The *fimH*, one of the most studied AIEC virulence factors, is also included in this analysis for comparative purpose, while *rpoJ* and *dnaJ* are used to represent the identity levels and phylogenetic distribution of universal single copy marker genes. Graphical representation of the data by heatmap R library. R version 4.2.1 was used. Correlation between biofilm formation (b) or flagellar motility (c) of the 13 selected LF82 mutants and their intracellular bacterial load within HD- (white dots) or CD-moDCs (purple dots) at 24 h post-infection. Correlations were determined using Pearson correlation coefficients. R squares and *p* values for each correlation were reported in the graphs. Correlation between biofilm formation (d) or flagellar motility (e) and fold changes in the percentage of IL-23 (**left graphs**) or IL-1β (**right graphs**) production of each LF82 mutants, as compared to the parental LF82 strain, within HD- (white dots) or CD-moDCs (purple dots) at 24 h post-infection. Correlations were determined using Pearson correlation coefficients. R squares and *p* values for each correlation were reported in the graphs. (f) Intracellular bacterial load of LF82 within CD-moDCs after 2 h (phagocytosis) or after 24 h of infection (persistence), in the presence or absence of human-TLR4 neutralizing antibody (α-hTLR4; *n* = 3). (g) Secretion of IL-23 (**left graph**) and IL-1β (**right graph**) by CD-moDCs after 24 h of infection with LF82 in the presence or absence of α-hTLR4 neutralizing antibody (*n* = 3). (h) Intracellular bacterial load of LF82 or LF82-Δ*fimH* isogenic deletion mutant within CD-moDCs (*n* = 5) after 2 h (phagocytosis) or 24 h of infection (persistence). (i) Production of IL-23 (**left graph**) and IL-1β (**right graph**) by CD-moDCs after 24 h of infection with LF82 or LF82-Δ*fimH* isogenic deletion mutant (*n* = 5). (f-i) Data are represented as mean ± SEM of 3–5 independent experiments. Statistical significance was calculated using Mann-Whitney's test.

<footnote><sup>a</sup>Adhesion index, calculated as previously described.<sup>35</sup> Statistical significance was calculated using Mann-Whitney's test and reported as *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001.</footnote>

<footnote><sup>b</sup>Halo diameter (cm) in motility soft agar plates. Statistical significance was calculated using Mann-Whitney's test and reported as *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001.</footnote>

<footnote><sup>c</sup>+ indicates "rdar" (for red, dry, and rough) phenotype, – for white colonies on congo red (CR) agar plates.</footnote>
<figure><img src="image_48.png" /></figure>

screened our selected LF82 mutants with the anti-
gen-specific assay.

Our results revealed that ΔaceF (T6.E4), ΔpurA
(T77.A6), ΔentD (T53.G6), ΔyhjK (T74.A5; T69.
D10), and ΔkpsS (T96.B8) mutants, albeit exhibited
a significant reduction in IL-23 secretion by CD-
moDCs (Figure 3d-upper graph) and, except for
ΔyhjK and ΔkpsS, also a defective intracellular per-
sistence (Figure 3e), did not significantly impair the
IFNγ/IL-17 co-expression by cTh17 cells as com-
pared to LF82 (Figure 5a,b), thus not representing
the most interesting candidate targets.

On the contrary, six mutants significantly reduced the IFNγ/IL-17 co-expression and differently modulated the single expression of IL-17 or IFNγ by stimulated cTh17 cells compared to the parental LF82 strain (Figure 5a,b and S6a). Among them, ΔybaT (T59.F12) and in a lesser extend ΔrfaP (T12.G9 and T45.G12) mutants drastically impaired the skewing of cTh17 into pTh17 cells (Figure 5b), inducing similar IFNγ/IL-17 co-expression levels as observed in response to the probiotic EcN strain (Figure 2b), or after prolonged IL-23 neutralization (Figure 2g-right graph). None of these strains affected the viability, fitness, or activation of cTh17 cells (Figure S6b).

Finally, to exclude putative polar effects of trans-
poson insertion in our mutants, and to validate
whether the AIEC virulence determinants identified
through the screenings of our library were effective
candidate targets to hinder pTh17 cell transdifferen-
tiation, we generated individual isogenic deletion
mutants in rfaP and ybaT genes. Our data strongly
confirmed that the absence of rfaP or ybaT genes in
LF82 led to a compromised intracellular persistence
within CD-moDCs (Figure 5c), together with
a significant reduction in IL-23 secretion (Figure 5d)
and, especially, to a drastic impairment of pTh17 cell
generation (Figure 5e,f).

In summary, our findings indicated that the
transdifferentiation of pTh17 cell is strongly driven
in CD patients by some AIEC determinants, gen-
erated within antigen processing compartments of
DCs, and presented to cTh17 cells together with
very high IL-23 levels selectively secreted by AIEC-
infected DCs. Therefore, targeting the AIEC viru-
lence determinants specifically involved in trigger-
ing IL-23 signaling and in the harmful
transdifferentiation of pTh17 cells, as those

identified in this study, could represent
a promising and unprecedented therapeutic strat-
egy to prevent chronic intestinal inflammation in
CD patients.

4. Discussion

The chronic and progressive nature of the CD is
correlated with an exaggerated mucosal adaptive
inflammatory response against luminal bacterial
antigens, with the AIEC pathotype proposed as
one of the main triggers.²⁵,²⁶ Although the strong
association between AIEC and CD gut dysbiosis
has been extensively demonstrated,²⁵,⁵⁴ compara-
tive genomics analysis failed to identify
a characteristic genomic signature profile for
AIEC distinguishing it from other pathogenic or
commensal E. coli strains. As a result, AIEC cannot
be selectively targeted with the common microbial-
based or microbiota-targeted therapies.⁵⁵

An increasing body of evidence points to a crucial
and prominent role in the pathogenesis of CD for
IL-23-dependent activation of pathogenic intestinal
resident IFNγ-producing Th17 cells (pTh17 cells) in
CD patients.²⁴,⁵⁶ We recently demonstrated that
pathogenic intestinal resident IFNγ-producing
pTh17 cells isolated from CD, but not from UC
patients, are selectively activated by AIEC strains
and are specifically decreased following anti-IL-23
treatment.²⁴ This work paved the way in defining
a role for AIEC in triggering IL-23 secretion and
pTh17 cell activation without, however, elucidating
the underlying molecular mechanisms.

Most of the therapeutic strategies in CD are
based on systemic immunomodulation or targeting
pro-inflammatory cytokines involved in the activa-
tion of mucosal T~RM~ cells, such as antibodies
against TNFα, IL-12 and/or IL-23, or against integ-
rin α4β7.⁵⁷ In this context, however, the relation-
ship between AIEC and the dysregulated activation
of IL-23/pTh17 axis in CD patients has never been
fully characterized.

AIEC virulence is mainly defined at the pheno-
typic level for its ability to adhere and invade
intestinal epithelial cells (IECs), together with its
proficiency in persisting within macrophages.³⁰
Several AIEC virulence determinants have been
described to play a crucial role in these processes,
<figure><img src="image_49.png" /><figcaption>a</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_55.png" /></figure>

**Figure 5.** Deletion of the AIEC-determinants*bybaT* or *rfaP* drastically impaired the transdifferentiation of cTh17 cells into pTh17 cells. (a) One representative experiment out of four showing the gating strategy to identify antigen-dependent pTh17 transdifferentiation after 10 d of cTh17 cell stimulation with autologous moDCs infected with AIEC-LF82 or indicated LF82 mutant strains is shown. (b) Fold-change values of the percentages of IFNγ+/IL-17+ co-expression on peripheral CD40L+ cTh17 cells after 10 d of co-culture with autologous CD-moDCs infected with indicated mutants. Data are represented as mean percentage (fold-change over LF82) ± SEM of
<figure><img src="image_56.png" /></figure>

promoting a sustained inflammatory response, and thus contributing to the chronic intestinal inflammation in CD.⁴⁹,⁵⁰ For example, intramacrophage replication of AIEC strongly promotes mucosal inflammation through the secretion of very high TNFα levels.³⁰ Here, instead, we observed that AIEC intramacrophage persistence did not promote neither IL-23 nor very high levels of IL-1β, and, especially, that AIEC-infected macrophages completely failed to induce pTh17 cells transdifferentiation from protective cTh17 cells. These results are in line with the clinical evidence reporting that anti-TNFα drugs hamper AIEC replication within macrophages⁵⁸ but do not affect the IL-23/IL-17 axis.⁵⁹ In contrast, DCs derived from CD patients secreted very high IL-23 levels in response to AIEC strains and, importantly, also in response to the probiotic EcN strain compared to DCs isolated from healthy subjects. These results not only confirm the genetic predisposition of CD patients to an overwhelming mucosal inflammatory response¹ but also imply that some probiotics could be ineffective or even detrimental for CD patients.³⁵,⁶⁰ EcN strain harbors some key virulence genes commonly associated with pathogenic phenotypes in *E. coli*,⁶¹ including some LF82 genes with immunostimulatory potential.²⁴ Nevertheless, the EcN-DCs interaction did not promote pTh17 cell generation, suggesting that the expression of certain bacterial antigens leading to the transdifferentiation of pTh17 cells might be selectively processed within CD-derived DCs during AIEC infection.

In this work, we aimed to identify and functionally characterize the AIEC virulence determinants specifically implicated in pTh17 cell differentiation. By generating and screening a library of more than 10.000 AIEC transposon insertion mutants, we

demonstrated that the AIEC molecular determinants triggering the IL-23 hypersecretion by CD-DCs and promoting pTh17 cell generation, were distinct from those associated with the AIEC intracellular persistence and the exaggerated inflammatory response in macrophages and IECs. Indeed, among AIEC mutants defective in triggering IL-23 we did not find mutants in virulence genes commonly associated with AIEC pathogenicity like *ibeA*, *htrA*, *fimH*, *chiA*.³³,⁴⁹,⁵⁰ The screenings of our AIEC mutant library revealed instead that the AIEC-dependent IL-23 hypersecretion by CD-derived DCs was linked to a restricted subset of pathways, mainly involved in metabolic and stress response processes, biofilm formation, LPS and capsule biosynthesis. Interestingly, most of the genes identified by our screenings displayed a broad phylogenetic distribution within *E. coli* and are consistently found in the *E. coli* genomes belonging to different pathotypes and phylogroups, with levels of conservation comparable to those of some universal single copy number genes. Moreover, if this observation confirms that AIEC-dependent intestinal inflammation in CD relies on the complex interplay of several factors (i.e., genetic, environmental, and immunological) and not exclusively on the AIEC virulence determinants, it remains unclear why these genes do not confer a selective advantage in colonizing the ileal mucosa of CD patients and promoting chronic inflammation also to other *E. coli* pathotypes.

Several works propose a direct correlation between the expression of specific bacterial virulence determinants (i.e., biofilm formation, fimbriae/adhesins expression) and the AIEC proficiency to invade and persist within host cells like IECs and macrophages.⁵⁰,⁶² In contrast,

at least four independent experiments. Statistical significance was calculated using one-way ANOVA and reported as *p* < 0.05, **p** < 0.01, ***p** < 0.001. Intracellular bacterial load after 24 h of infection (c) and IL-23 production (d) by CD-moDCs infected with LF82, indicated LF82-isogenic deletion mutants, and probiotic EcN strain (n = 6). Data are represented as mean percentage (fold-change over LF82) ± SEM of six independent experiments. Statistical significance was calculated using one-way ANOVA and reported as *p* < 0.05, **p** < 0.01, ***p** < 0.001. (e) One representative experiment out of three, showing the gating strategy to identify antigen-dependent pTh17 transdifferentiation in response to LF82-isogenic deletion mutants, is shown. (f) Fold-change values of the percentages of single IFNγ⁺, IL-17⁺ expression and IFNγ⁺/IL-17⁺ co-expression on peripheral CD40L⁺cTh17 cells after 10 d of co-culture with autologous CD-derived moDCs infected with indicated LF82-isogenic deletion mutants. Data are represented as mean percentage (fold-change over LF82) ± SEM of at least three independent experiments. Statistical significance was calculated using one-way ANOVA and reported as *p* < 0.05, **p** < 0.01, ***p** < 0.001.
<figure><img src="image_57.png" /></figure>

our results clearly demonstrated that mutants
defective in persisting within DCs and triggering
lower IL-23 secretion displayed both a biofilm-,
curli-, and fimbriae-producing phenotype (i.e.,
ΔaceF, ΔrhlB) and a non-producing phenotype
(i.e., ΔpurA and LPS mutants). Moreover, dele-
tion in LF82 of the FimH adhesin, one of the best
characterized AIEC virulence factors,²⁹,⁴⁹,⁵⁰
whose CEACAM6 receptor is upregulated in
IECs of CD patients³¹ and expressed on myeloid
cells, did not affect neither the uptake and survi-
val of AIEC within DCs nor the inflammatory
response. Therefore, our data indicated that the
AIEC virulence determinants promoting inva-
sion, intracellular replication, and the inflamma-
tory response in IECs and macrophages are not
involved in the IL-23 hypersecretion and pTh17
cell generation which instead rely on the func-
tions of dendritic cells. Epithelial infiltrating DCs
can indeed directly detect bacteria across the
mucosal epithelium,⁶³ leading to the secretion of
high IL-23 levels and, especially in CD patients
where AIEC is highly enriched,²⁶ to pTh17 cell
transdifferentiation. Therefore, blocking the
interaction between AIEC and intestinal epithe-
lial cells may not prevent DC-dependent activa-
tion of AIEC-specific pTh17 cells.

Conversely, our results outlined a new crucial
point in the knowledge of AIEC virulence, defining
the AIEC determinants specifically involved in
triggering IL-23 hypersecretion and in priming
pTh17 cells in CD. Our functional assays identified
the LPS core heptose(I) kinase rfaP, and the inner
membrane transport protein ybaT, as two key
AIEC virulence determinants directly involved in
the skewing of pTh17 cells from protective cTh17
cells. Interestingly, 2 mutants were found in dis-
tinct genes of the rfa operon, ΔrfaP and ΔrfaG,
which promoted both a significant lower IL-23
secretion and a strong impairment in the LF82
intracellular persistence, but mutation in rfaP dis-
played a higher inhibition of pTh17 cell transdif-
ferentiation as compared to mutation in rfaG.
These differences, together with the different phe-
notype of ΔrfaP and ΔrfaG mutants compared to
the parental LF82 strain, both in terms of biofilm
production, motility, and fimbriae expression, are
most probably linked to the pleiotropic effects of

LPS-genes mutations on the outer membrane pro-
tein properties as previously described by Pagnout
and colleagues.⁶⁴ Indeed, we exclude that the lower
IL-23 secretion, and the impaired intracellular per-
sistence of ΔrfaP and ΔrfaG mutants, could be
exclusively attributed to a truncated LPS structure
since the TLR4 blockade – which has been pro-
posed moreover as a promising therapeutic
approach because extensively upregulated in
CD⁶⁵⁻ hampered neither the uptake/intracellular
persistence nor the inflammatory response of
infected CD-derived DCs.

In this regard, Guttsches and colleagues reported
that the truncated LPS structure of the probiotic
EcN strain only partially explains its loss of patho-
genicity, since LPS extracted from EcN has lower
immunomodulatory effects and anti-inflammatory
activity than an EcN lysate. Therefore, they pro-
posed that the EcN surface polysaccharide struc-
ture, along with other undefined bacterial
determinants, are crucial for the protective immu-
nomodulatory effects of this probiotic strain.⁶⁶

We speculate that the absence of *rfaP*, which leads
to a modification in the LPS inner core due to the
lack of phosphorylation of the first heptose added to
KDO2-lipid A structure, results in an altered struc-
ture and stability of the outer membrane.
Consequently, during the intracellular processing of
the *ΔrfaP* mutant, it is possible that the altered mem-
brane structure leads to the generation of different
AIEC antigens compared to those generated from the
parental AIEC LF82 strain, which fail to generate
pTh17 cell when loaded on MHC-II of DCs.

Similarly, mutations in *ybaT*, encoding an
amino acid permease involved in the glutamate-
dependent acid tolerance system within the gene
cluster implicated in copper (Cu) tolerance,
resulted in a compromised release of IL-23 and
abrogated pTh17 cell generation.

It is known that enteric pathogens, like AIEC,
respond to high Cu levels and low pH as those
encountered in the small intestine and within pha-
golysosomes through Cu efflux pumps. In other
pathogenic *E. coli* strains, deletion of *ybaT* leads
to loss of protection against acid stress and Cu
accumulation, resulting in a diminished ability of
bacteria to survive under these stress conditions.⁶⁷
Moreover, it has been demonstrated that the
<figure><img src="image_58.png" /></figure>

adaptive response of bacteria to acid stress leads to
changes in the membrane permeability and mod-
ification in the lipid bilayer.⁶⁸

Therefore, it is likely that the absence of *ybaT* in
the inner membrane of AIEC leads to an altered
membrane composition or stability, which in turn
promotes a different antigenic intracellular proces-
sing failing to generate AIEC antigens capable of
inducing the skewing of cTh17 to pTh17 cells.

Importantly, in contrast to some virulence fac-
tors like FimH which is absent in other pathogenic
*E. coli* strains, *YbaT* is ubiquitously found across all
*E. coli* pathotypes, also showing a narrow phyloge-
netic distribution across Enterobacteriales
(observed in 8/522 non-*E. coli* genomes according
to the orthoDB database)⁶⁹ and therefore, could
represent an ideal therapeutic target.

Although further studies will be needed to
specifically define which AIEC antigens are
altered or no longer generated during the intra-
cellular processing of these mutants, rfaP and
ybaT undoubtedly represent promising and
unprecedented candidate therapeutic targets to
prevent chronic intestinal inflammation in CD
patients. Indeed, our results demonstrated that
the targeting of ybaT and rfaP induced a drastic
reduction both in AIEC intracellular persistence
and IL-23 secretion by CD-DCs and, most impor-
tantly, inhibited the skewing of cTh17 cells
toward the pTh17 pathogenic phenotype without
affecting the viability and function of protective
Th17 cells. This point is of particular significance
if we consider that current therapeutic strategies
aimed to block IL-23 result in a high remission
rate in CD patients, but neither completely abol-
ish T~RM~ pTh17 cells²⁴ nor prevent further micro-
bial-dependent production of IL-23. Accordingly,
only a continuous IL-23 blockade significantly
reduced the in vitro AIEC-dependent pTh17 cell
differentiation. This result depends on the high
proficiency of AIEC in persisting for a long time
within DCs, and thus resulting in a chronic
hypersecretion of IL-23. In this regard, further
studies correlating mucosal AIEC abundance in
CD patients with clinical response to anti-IL-23
treatment could be of crucial importance for
understanding disease progression as well
response rates to therapies. Moreover, besides its
contributing role in the pTh17 cell induction and

in CD pathogenesis, IL-23 also plays important
homeostatic functions by regulating innate lym-
phoid cells (ILC) or enhancing intestinal barrier
mechanism by promoting the release of antimi-
crobial peptides.⁷⁰ Therefore, targeting upstream
the bacterial stimuli selectively triggering the IL-
23 overproduction could be a turning point in the
treatment of chronic intestinal inflammation
in CD.

In conclusion, here we identified two AIEC viru-
lence determinants directly implicated in triggering
IL-23 hypersecretion and IFNγ-producing pTh17
cell generation, two immunological pathways
highly dysregulated in CD patients. Particularly,
rfaP and ybaT represent paramount bacterial tar-
gets for the development of more efficient thera-
peutic strategies, which may have the great
advantage of selectively preventing the activation
of the IL-23/pTh17 axis leaving protective immune
cells unaffected.

## Acknowledgments

We thank Dr Claudia Bazzini (Flow cytometry facility – Department of Biosciences, University of Milan) for technical assistance and support with sorting experiments. The authors gratefully acknowledge the support of the APC central fund of the University of Milan.

Disclosure statement

No potential conflict of interest was reported by the
author(s).

Funding

This study was financially supported by Cariplo Foundation-Biomedical Research Conducted by Young Researchers (GR-2017-0816) to M.P, C.C., and F.C, by the Department of Biosciences - University of Milan (funding program for younger staff members “Linea 2”) to M.P.; and by University of Milan (SEED grant-funding program for interdepartmental projects) to F.C. and M.P.; and partly funded by Italy’s Ministry of Health and the current IRCCS research programme to F.C.

ORCID

G. Leccese ID http://orcid.org/0000-0003-2058-0009

M. Chiara ID http://orcid.org/0000-0003-3983-4961

I. Dusetti ID http://orcid.org/0009-0000-9220-1819
<figure><img src="image_59.png" /></figure>

D. Noviello ID http://orcid.org/0000-0002-3482-0109

E. Billard ID http://orcid.org/0000-0002-6066-4584

MP Conte ID http://orcid.org/0000-0003-0753-6003

N. Barnich ID http://orcid.org/0000-0001-9465-7844

F. Caprioli ID http://orcid.org/0000-0002-8077-8175

F. Facciotti ID http://orcid.org/0000-0002-2541-9428

M. Paroni ID http://orcid.org/0000-0001-8199-3128

## Contributors

D.N. and F.C. enrolled patients; G.L., M.C., I.D., A.B., G.C., and C.C. performed experiments; MP.C., N.B. and E. B. provided *E. coli* strains and gave important intellectual contributions, M.P., G.L., M.C., and I.D. analyzed and interpreted data; M.P., F.F., and F.C. initiated, designed, and supervised the study and wrote the manuscript; M.P., and F. C. obtained funding. M.V., and N.B. contributed to interpretation of the data and gave important intellectual contributions. All authors have read and agreed to the published version of the manuscript.

## Data and material availability

All the data required to evaluate the conclusions presented in this paper are included within the paper and supplementary material. All LF82 mutant strains used in the current study are available from the lead contact with a completed Materials Transfer Agreement.

## Dedication

This paper is dedicated to the memory of our dear colleague Nicolas Barnich who recently passed away. His untimely departure is a profound loss to the scientific community and to all who had the privilege of knowing him. Prof. Barnich's contributions to this work, and to the field at large, were invaluable. His dedication, intellectual rigor, and passion for research were evident in every project he undertook. He was not only a brilliant scientist, a dedicated and thorough collaborator, but also a generous mentor and a cherished friend. We will deeply miss him.”

## References

1. Chang JT. Pathophysiology of inflammatory bowel diseases. N Engl J Med. 2020;383(27):2652-2664. doi:10.1056/NEJMra2002697.

2. Na SY, Moon W. Perspectives on Current and novel treatments for inflammatory bowel disease. Gut Liver. 2019;13(6):604-616. doi:10.5009/gnl19019.

3. Paramsothy S, Rosenstein AK, Mehandru S, Colombel JF. The current state of the art for biological therapies and new small molecules in inflammatory

bowel disease. Mucosal Immunol. 2018;11 (6):1558-1570. doi:10.1038/s41385-018-0050-3.

4. Borody TJ, Dolai S, Gunaratne AW, Clancy RL. Targeting the microbiome in Crohn's disease. Expert Rev Clin Immunol. 2022;18(9):873-877. doi:10.1080/1744666X.2022.2093186.

5. Gowen R, Gamal A, Di Martino L, McCormick TS, Ghannoum MA. Modulating the microbiome for Crohn's disease treatment. Gastroenterology. 2023;164 (5):828-840. doi:10.1053/j.gastro.2023.01.017.

6. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014;157(1):121-141. doi:10.1016/j.cell.2014.03.011.

7. Omenetti S, Bussi C, Metidji A, Iseppon A, Lee S, Tolaini M, Li Y, Kelly G, Chakravarty P, Shoaie S, et al. The intestine harbors functionally distinct homeostatic tissue-resident and inflammatory Th17 cells. Immunity. 2019;51(1):77-89 e6. doi:10.1016/j.immuni.2019.05.004.

8. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009;139(3):485-498. doi:10.1016/j.cell.2009.09.033.

9. McGeachy MJ, McSorley SJ. Microbial-induced Th17: superhero or supervillain? J Immunol. 2012;189 (7):3285-3291. doi:10.4049/jimmunol.1201834.

10. Zielinski CE, Mele F, Aschenbrenner D, Jarrossay D, Ronchi F, Gattorno M, Monticelli S, Lanzavecchia A, Sallusto F. Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β. Nature. 2012;484(7395):514-518. doi:10.1038/nature10957.

11. Britton GJ, Contijoch EJ, Mogno I, Vennaro OH, Llewellyn SR, Ng R, Li Z, Mortha A, Merad M, Das A, et al. Microbiotas from humans with inflammatory bowel disease alter the balance of gut Th17 and RORγt+ regulatory T cells and exacerbate colitis in mice. Immunity. 2019;50(1):212-24 e4. doi:10.1016/j.immuni.2018.12.015.

12. Buckner JH, Harrison OJ. Th17 cells: from gut homeostasis to CNS pathogenesis. Trends Immunol. 2022;43 (3):167-169. doi:10.1016/j.it.2022.01.005.

13. Hue S, Ahern P, Buonocore S, Kullberg MC, Cua DJ, McKenzie BS, Powrie F, Maloy KJ. Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med. 2006;203(11):2473-2483. doi:10.1084/jem.20061099.

14. Ahern PP, Schiering C, Buonocore S, McGeachy MJ, Cua DJ, Maloy KJ, Powrie F. Interleukin-23 drives intestinal inflammation through direct activity on T cells. Immunity. 2010;33(2):279-288. doi:10.1016/j.immuni.2010.08.010.

15. Sallusto F, Zielinski CE, Lanzavecchia A. Human Th17 subsets. Eur J Immunol. 2012;42(9):2215-2220. doi:10.1002/eji.201242741.

16. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S,
<figure><img src="image_60.png" /></figure>

Frosali F, et al. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007;204(8):1849-1861. doi:10.1084/jem.20070663.

17. Bedoya SK, Lam B, Lau K, Larkin J 3rd. Th17 cells in immunity and autoimmunity. Clin Dev Immunol. 2013;2013:986789. doi:10.1155/2013/986789.

18. Kleinschek MA, Boniface K, Sadekova S, Grein J, Murphy EE, Turner SP, Raskin L, Desai B, Faubion WA, de Waal Malefyt R, et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J Exp Med. 2009;206(3):525-534. doi:10.1084/jem.20081712.

19. Hegazy AN, West NR, Stubbington MJT, Wendt E, Suijker KIM, Datsi A, This S, Danne C, Campion S, Duncan SH, et al. Circulating and tissue-resident CD4 (+) T cells with reactivity to intestinal microbiota are abundant in healthy individuals and function is altered during inflammation. Gastroenterology. 2017;153 (5):1320-37 e16. doi:10.1053/j.gastro.2017.07.047.

20. Calderon-Gomez E, Bassolas-Molina H, Mora-Buch R, Dotti I, Planell N, Esteller M, Gallego M, Martí M, Garcia-Martín C, Martínez-Torró C, et al. Commensal-specific CD4+ cells from patients with Crohn's disease have a T-Helper 17 inflammatory profile. Gastroenterology. 2016;151(3):489-500 e3. doi:10.1053/j.gastro.2016.05.050.

21. Nizzoli G, Burrello C, Cribiù FM, Lovati G, Ercoli G, Botti F, Trombetta E, Porretti L, Todoerti K, Neri A, et al. Pathogenicity of in vivo generated intestinal Th17 lymphocytes is IFNγ dependent. J Crohns Colitis. 2018;12(8):981-992. doi:10.1093/ecco-jcc/jjy051.

22. Hueber W, Sands BE, Lewitzky S, Vandemeulebroecke M, Reinisch W, Higgins PD, Wehkamp J, Feagan BG, Yao, MD, Karczewski M, et al. Secukinumab, a human anti-IL-17A monoclonal antibody, for moderate to severe Crohn's disease: unexpected results of a randomised, double-blind placebo-controlled trial. Gut. 2012;61:1693-1700. doi:10.1136/gutjnl-2011-301668.

23. D'Haens G, Panaccione R, Baert F, Bossuyt P, Colombel JF, Danese S, Dubinsky M, Feagan BG, Hisamatsu T, Lim A, et al. Risankizumab as induction therapy for Crohn's disease: results from the phase 3 ADVANCE and MOTIVATE induction trials. Lancet. 2022;399(10340):2015-2030. doi:10.1016/S0140-6736(22) 00467-6.

24. Paroni M, Leccese G, Ranzani V, Moschetti G, Chiara M, Perillo F, Ferri S, Clemente F, Noviello D, Conforti FS, et al. An intestinal Th17 subset is associated with inflammation in Crohn's disease and activated by adherent-invasive Escherichia coli (AIEC). J Crohns Colitis. 2023;17(12):1988-2001. doi:10.1093/ecco-jcc/jjad119.

25. Zheng L, Duan SL, Dai YC, Wu SC. Role of adherent invasive Escherichia coli in pathogenesis of inflammatory bowel disease. World J Clin Cases. 2022;10 (32):11671-11689. doi:10.12998/wjcc.v10.i32.11671.

26. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser A-L, Barnich N, Bringer M-A, Swidsinski A, Beaugerie L, Colombel J-F, et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology. 2004;127 (2):412-421. doi:10.1053/j.gastro.2004.04.061.

27. Martinez-Medina M, Aldeguer X, Lopez-Siles M, González-Huix F, López-Oliu C, Dahbi G, Blanco JE, Blanco J, Garcia-Gil JL, Darfeuille-Michaud A, et al. Molecular diversity of Escherichia coli in the human gut: new ecological evidence supporting the role of adherent-invasive *E. coli* (AIEC) in crohn's disease. Inflamm Bowel Dis. 2009;15(6):872-882. doi:10.1002/ ibd.20860.

28. Carvalho FA, Barnich N, Sivignon A, Darcha C, Chan CHF, Stanners CP, Darfeuille-Michaud A. Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM. J Exp Med. 2009;206(10):2179-2189. doi:10.1084/jem.20090741.

29. Boudeau J, Barnich N, Darfeuille-Michaud A. Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells. Mol Microbiol. 2001;39:1272-1284. doi:10.1111/j.1365-2958.2001.02315.x

30. Bringer MA, Billard E, Glasser AL, Colombel JF, Darfeuille-Michaud A. Replication of Crohn's disease-associated AIEC within macrophages is dependent on TNF-α secretion. Lab Invest. 2012 Mar. 92(3):411-419. doi:10.1038/labinvest.2011.156.

31. Barnich N, Carvalho FA, Glasser AL, Darcha C, Jantscheff P, Allez M, Peeters H, Bommelaer G, Desreumaux P, Colombel J-F, et al. CEACAM6 acts as a receptor for adherent-invasive *E. coli*, supporting ileal mucosa colonization in Crohn disease. J Clin Invest. 2007;117(6):1566-1574. doi:10.1172/JCI30504.

32. Cieza RJ, Hu J, Ross BN, Sbrana E, Torres AG. The IbeA invasin of adherent-invasive Escherichia coli mediates interaction with intestinal epithelia and macrophages. Infect Immun. 2015;83(5):1904-1918. doi:10.1128/IAI.03003-14.

33. Bringer MA, Barnich N, Glasser AL, Bardot O, Darfeuille-Michaud A. HtrA stress protein is involved in intramacrophagic replication of adherent and invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Infect Immun. 2005;73:712-721. doi:10.1128/IAI.73.2.712-721.2005.

34. Small CL, Reid-Yu SA, McPhee JB, Coombes BK. Persistent infection with Crohn's disease-associated adherent-invasive Escherichia coli leads to chronic inflammation and intestinal fibrosis. Nat Commun. 2013;4(1):1957. doi:10.1038/ncomms2957.

35. Leccese G, Bibi A, Mazza S, Facciotti F, Caprioli F, Landini P, Paroni M. Probiotic lactobacillus and bifidobacterium strains counteract adherent-invasive Escherichia coli (AIEC) virulence and hamper IL-23/ Th17 axis in ulcerative colitis, but not in Crohn's disease. Cells. 2020;9(8):1824. doi:10.3390/cells9081824.
<figure><img src="image_61.png" /></figure>

36. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000;97 (12):6640-6645. doi:10.1073/pnas.120163297.

37. Chaveroche MK, Ghigo JM, d'Enfert C. A rapid method for efficient gene replacement in the filamentous fungus aspergillus nidulans. Nucleic Acids Res. 2000;28(22): E97. doi:10.1093/nar/28.22.e97.

38. Clark JR, Maresso AM. Comparative pathogenomics of Escherichia coli: polyvalent vaccine target identification through virulome analysis. Infect Immun. 2021;89(8): e0011521. doi:10.1128/IAI.00115-21.

39. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL. GenBank. Nucleic Acids Res. 2005;33 (Database issue):D34-8. doi:10.1093/nar/gki063.

40. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinf. 2009;10(1):421. doi:10.1186/1471-2105-10-421.

41. Rossi E, Leccese G, Baldelli V, Bibi A, Scalone E, Camilloni C, Paroni M, Landini P. Inactivation of the pyrimidine biosynthesis pyrD gene negatively affects biofilm formation and virulence determinants in the Crohn's disease-associated adherent invasive Escherichia coli LF82 strain. Microorganisms. 2022;10 (3):537. doi:10.3390/microorganisms10030537.

42. Migliore F, Macchi R, Landini P, Paroni M. Phagocytosis and epithelial cell invasion by Crohn's disease-associated adherent-invasive Escherichia coli are inhibited by the anti-inflammatory drug 6-mercaptopurine. Front Microbiol. 2018;9:964. doi:10.3389/fmicb.2018.00964.

43. Smith DR, Price JE, Burby PE, Blanco LP, Chamberlain J, Chapman MR. The production of curli amyloid fibers is deeply integrated into the biology of Escherichia coli. Biomolecules. 2017;7(4):75. doi:10.3390/biom7040075.

44. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med. 2005;201(2):233-240. doi:10.1084/jem.20041257.

45. Duhen T, Campbell DJ. IL-1β promotes the differentiation of polyfunctional human CCR6+CXCR3+ Th1/17 cells that are specific for pathogenic and commensal microbes. J Immunol. 2014;193(1):120-129. doi:10.4049/jimmunol.1302734.

46. Stritesky GL, Yeh N, Kaplan MH. IL-23 promotes maintenance but not commitment to the Th17 lineage. J Immunol. 2008 Nov 1. 181(9):5948-5955. doi:10.4049/jimmunol.181.9.5948.

47. McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, Cua DJ. TGF-β and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain TH-17 cell-mediated pathology. Nat Immunol. 2007 Dec. 8(12):1390-1397. doi:10.1038/ni1539.

48. Buisson A, Douadi C, Ouchchane L, Goutte M, Hugot J-P, Dubois A, Minet-Quinard R, Bouvier D, Bommelaer G, Vazeille E, et al. Macrophages inability to mediate adherent-invasive *E. coli* replication is linked to autophagy in Crohn's disease patients. Cells. 2019;8 (11):1394. doi:10.3390/cells8111394.

49. Abdelhalim KA, Uzel A, Unal NG. The role of major virulence factors and pathogenicity of adherent-invasive Escherichia coli in patients with Crohn's disease. Prz Gastroenterol. 2020;15(4):279-288. doi:10.5114/pg.2020.93235.

50. Mansour S, Asrar T, Elhenawy W. The multifaceted virulence of adherent-invasive Escherichia coli. Gut Microbes. 2023;15(1):2172669. doi:10.1080/19490976. 2023;2172669.

51. Frendeus B, Wachtler C, Hedlund M, Fischer H, Samuelsson P, Svensson M, Svanborg C. Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation. Mol Microbiol. 2001;40(1):37-51. doi:10.1046/j.1365-2958.2001.02361.x.

52. Mossman KL, Mian MF, Lauzon NM, Gyles CL, Lichty B, Mackenzie R, Gill N, Ashkar AA. Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand. J Immunol. 2008;181(10):6702-6706. doi:10.4049/jimmunol.181.10.6702.

53. Chevalier G, Laveissiere A, Desachy G, Barnich N, Sivignon A, Maresca M, Nicoletti C, Di Pasquale E, Martinez-Medina M, Simpson KW, et al. Blockage of bacterial FimH prevents mucosal inflammation associated with Crohn's disease. Microbiome. 2021;9 (1):176. doi:10.1186/s40168-021-01135-5.

54. Palmela C, Chevarin C, Xu Z, Torres J, Sevrin G, Hirten R, Barnich N, Ng SC, Colombel J-F. Adherent-invasive Escherichia coli in inflammatory bowel disease. Gut. 2018;67(3):574-587. doi:10.1136/gutjnl-2017-314903.

55. Oka A, Sartor RB. Microbial-based and microbial-targeted therapies for inflammatory bowel diseases. Dig Dis Sci. 2020;65(3):757-788. doi:10.1007/s10620-020-06090-z.

56. Yokoi T, Murakami M, Kihara T, Seno S, Arase M, Wing JB, Søndergaard JN, Kuwahara R, Minagawa T, Oguro-Igashira E, et al. Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn's disease. Proc Natl Acad Sci USA. 2023;120(1): e2204269120. doi:10.1073/pnas.2204269120.

57. Song Y, Yuan M, Xu Y, Xu H. Tackling inflammatory bowel diseases: targeting proinflammatory cytokines and Lymphocyte Homing. Pharmaceuticals (Basel). 2022;15(9):1080. doi:10.3390/ph15091080.

58. Douadi C, Vazeille E, Chambon C, Hébraud M, Fargeas M, Dodel M, Coban D, Pereira B, Birer A, Sauvanet P, et al. Anti-TNF agents restrict adherent-invasive Escherichia coli replication within macrophages through modulation of chitinase 3-like 1 in patients with Crohn's disease. J Crohns Colitis. 2022;16(7):1140-1150. doi:10.1093/ecco-jcc/jjab236.
<figure><img src="image_62.png" /></figure>

59. Milanez FM, Saad CG, Viana VT, Moraes JCB, Périco GV, Sampaio-Barros PD, Goncalves CR, Bonfá E. IL-23/Th17 axis is not influenced by TNF-blocking agents in ankylosing spondylitis patients. Arthritis Res Ther. 2016;18(1):52. doi:10.1186/s13075-016-0949-6.

60. Jonkers D, Penders J, Masclee A, Pierik M. Probiotics in the management of inflammatory bowel disease: a systematic review of intervention studies in adult patients. Drugs. 2012;72(6):803-823. doi:10.2165/11632710-000000000-00000.

61. Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, Dobrindt U. Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain nissle 1917. J Bacteriol. 2004;186(16):5432-5441. doi:10.1128/JB.186.16.5432-5441.2004.

62. Prudent V, Demarre G, Vazeille E, Wery M, Quenech'du N, Ravet A, Dauverd - Girault J, van Dijk E, Bringer M-A, Descrimes M, et al. The Crohn's disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack. Commun Biol. 2021;4(1):627. doi:10.1038/s42003-021-02161-7.

63. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl J-P, Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2(4):361-367. doi:10.1038/86373.

64. Pagnout C, Sohm B, Razafitianamaharavo A, Caillet C, Offroy M, Leduc M, Gendre H, Jomini S, Beaussart A, Bauda P, et al. Pleiotropic effects of rfa-gene mutations on Escherichia coli envelope properties. Sci Rep. 2019;9(1):9696. doi:10.1038/s41598-019-46100-3.

65. Tam JSY, Coller JK, Hughes PA, Prestidge CA, Bowen JM. Toll-like receptor 4 (TLR4) antagonists as potential therapeutics for intestinal inflammation. Indian J Gastroenterol. 2021;40(1):5-21. doi:10.1007/s12664-020-01114-y.

66. Güttsches AK, Löseke S, Zähringer U, Sonnenborn U, Enders C, Gatermann S, Bufe A. Anti-inflammatory modulation of immune response by probiotic Escherichia coli Nissle 1917 in human blood mononuclear cells. Innate Immun. 2012;18(2):204-216. doi:10.1177/1753425910396251.

67. Djoko KY, Phan MD, Peters KM, Walker MJ, Schembri MA, McEwan AG. Interplay between tolerance mechanisms to copper and acid stress in Escherichia coli. Proc Natl Acad Sci USA. 2017;114(26):6818-6823. doi:10.1073/pnas.1620232114.

68. Boot IR, Cash P, O'Byrne C. Sensing and adapting to acid stress. Antonie Van Leeuwenhoek. 2002;81(1-4):33-42. doi:10.1023/a:1020565206835.

69. Kuznetsov D, Tegenfeldt F, Manni M, Seppey M, Berkeley M, Kriventseva E, Zdobnov EM. OrthoDB v11: annotation of orthologs in the widest sampling of organismal diversity. Nucleic Acids Res. 2023;51(D1):D445-D51. doi:10.1093/nar/gkac998.

70. Korta A, Kula J, Gomulka K. The role of IL-23 in the pathogenesis and therapy of inflammatory bowel disease. Int J Mol Sci. 2023;24(12):10172. doi:10.3390/ijms241210172.