# SOD mimics delivered to the gut using lactic acid bacteria mitigate the colitis symptoms in a mouse model of Inflammatory Bowel Diseases

Gabrielle Schanne<sup>a,b</sup>, Amandine Vincent<sup>a</sup>, Florian Chain<sup>c</sup>, Pauline Ruffié<sup>c</sup>, Célia Carbonne<sup>c</sup>, Elodie Quévrain<sup>a,b</sup>, Emilie Mathieu<sup>a</sup>, Alice Balfourier<sup>a</sup>, Luis G. Bermúdez-Humarán<sup>c</sup>, Philippe Langella<sup>c</sup>, Sophie Thenet<sup>b,d,e</sup>, Véronique Carrière<sup>b,e</sup>, Nassim Hammoudi<sup>f</sup>, Magali Svreck<sup>b</sup>, Sylvie Demignot<sup>b,d</sup>, Philippe Seksik<sup>b,e,g</sup>, Clotilde Policar<sup>a*</sup>, Nicolas Delsuc<sup>a*</sup>

email: nicolas.delsuc@ens.psl.eu

## Highlights

- SOD mimics (SODm) can be loaded in *Lactiplantibacillus plantarum* strains to create chemically modified bacteria (CMB) able to deliver SODm in digestive tract

- Loaded bacteria possess an increased anti-superoxide activity

- Orally administrated CMB significantly decrease colitis symptoms in mice

- CMB complement for the SOD2 in inflamed colonic homogenates

- CMB are potential antioxidant probiotics for inflammatory bowel diseases (IBD)

## Graphical abstract

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

<footnote><sup>a</sup> Laboratoire Chimie Physique et Chimie du Vivant, CPCV, UMR8228, Département de chimie, Ecole Normale Supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France</footnote>

<footnote><sup>b</sup> Centre de Recherche Saint Antoine, INSERM, UMRS 938, Microbiota, intestine and inflammation team, 184 Rue du Faubourg SaintAntoine, 75012 Paris, France</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.</footnote>

<footnote><sup>d</sup> EPHE, PSL university, 75014 Paris, France</footnote>

<footnote><sup>e</sup> Paris Center for Microbiome Medicine (PaCeMM) FHU, APHP, F-75012 Paris, France</footnote>

<footnote><sup>f</sup> Department of Gastroenterology, Hôpital Saint-Louis, AP-HP, INSERM U1160, Université Paris Cité, Paris, France</footnote>

<footnote><sup>g</sup> Gastroenterology Department, Saint-Antoine Hospital, Sorbonne Université, APHP, Paris, France</footnote>
**Abstract**

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis represent a global health issue as a prevalence of 1% is expected in the western world by the end of this decade. These idiseases are associated with a high oxidative stress that induces inflammatory pathways and severely damages the gut tissues. IBD patients suffer from antioxidant defenses weakening, through, for instance, an impaired activity of superoxide dismutases (SOD) — that catalyze the dismutation of superoxide — or other endogenous antioxidant enzymes including catalase and glutathione peroxidase. Manganese complexes mimicking SOD activity have shown beneficial effects on cells and murine models of IBD. However, efficient SOD mimics are often manganese complexes that can suffer from decoordination and thus inactivation in acidic stomachal pH. In order to improve their delivery in the gut after oral administration, two SOD mimics **Mn1** and **Mn1C** were loaded into lactic acid bacteria that serve as delivery vectors. When orally administrated to mice suffering from a colitis, these chemically modified bacteria (CMB) showed protective effects on the global health status of mice. In addition, they have shown beneficial effects on lipocalin-2 content and intestinal permeability. Interestingly, mRNA SOD2 content in colon homogenates was significantly decreased upon mice feeding with CMB loaded with **Mn1C**, suggesting that the beneficial effects observed may be due to the release of the SOD mimic in the gut that complement for this enzyme. These CMB represent new efficient chemically modified antioxidant probiotics for IBD treatment.

**Key words**

superoxide dismutase mimic and mimetic; manganese complexes; inflammatory bowel diseases; lactic acid bacteria; DNBS induced colitis

**Abbreviations:**

Cat catalase

CD Crohn's disease

CFU colony forming unit

CMB chemically modified bacteria

DNBS 2,4-dinitrobenzene sulfonic acid

COX-2 cyclooxygenase 2

DSS dextran sodium sulfate

EPR electron paramagnetic resonance

Hepes (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

GPx glutathione peroxidase

GRAS generally recognized as safe

IBD inflammatory bowel diseases

ICP-MS inductively-coupled mass spectrometry

IL-8 interleukine 8

IP intraperitoneal injection

IR intrarectal injection

LAB lactic acid bacteria

LCN2 Lipocalin-2

LOX lipoxygenase

MPO myeloperoxidase

MRS De Man, Rogosa and Sharpe medium

NLRs NOD-like receptors

NOS2 nitric oxide synthase 2

NOX NADPH oxidase

OD optical density

qPCR quantitative polymerase chain reaction

QPS qualified presumption of safety

ROS reactive oxygen species

RNS reactive nitrogen species

SOD superoxide dismutase

SODm superoxide dismutase mimic

TBP TATA-box binding protein

TLRs Toll-like receptors

UC ulcerative colitis
Introduction

Inflammatory Bowel Diseases (IBD) are idiopathic diseases which include ulcerative colitis (UC) and Crohn's disease (CD) affecting worldwide about 6.8 millions people [1] and with an expected prevalence of 1% in 2030 in the western world [2–4]. These figures establish IBD as a global health issue. UC and CD can cause diarrhea, rectal bleeding, anemia, weight loss and abdominal pains [5]. They are linked to an alteration of the sensing of gut microbiota and environmental factors in genetically predisposed individuals that lead to aberrant and chronic immune responses. For now, no curative therapy exists to treat IBD and patients mostly receive drugs aiming at controlling inflammation in order to alleviate the symptoms and/or to prevent the flare-ups. The main treatments include corticosteroids and immunomodulators comprising small molecules and monoclonal antibodies targeting specific inflammatory pathways or cytokines [6]. However, these expensive drugs are not always efficient, can induce weak response and adverse effects. Eventually, surgery, and particularly intestinal resection, is often advocated to reduce symptoms or following complications [7]. IBD are associated with leukocytes infiltration whose activation in the gut leads to the production of an excessive amount of reactive oxygen species (ROS) and nitrogen species (RNS) establishing an oxidative stress situation [8,9]. ROS sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families activates signaling pathways triggering the immune pro-inflammatory responses [10]. In turn, the activation of TLRs or NLRs receptors was also shown to stimulate the production of ROS by mitochondria and NOXs enzymes. In addition, inflammation mediates the overexpression of other ROS-producing enzymes such as lipoxygenases (LOX), myeloperoxidase (MPO), and inducible isoforms of nitric oxide (NO) synthase (NOS2) [11], thus contributing to a sustained vicious circle [12–14]. Importantly, it has been shown that IBD are associated with antioxidant defenses weakening. For instance, the content and/or activity of superoxide dismutases (SOD) — that catalyze the dismutation of the $ROS O_2^{•−}$ — was shown significantly reduced in plasma [15–17] or in tissues homogenates from resection specimen of IBD patients as compared to healthy subjects [18]. The activity of other endogenous antioxidant enzymes including catalase (CAT) and glutathione peroxidase (GPx) were also shown to be imbalanced in IBD patients mucosal tissue [19]. In this context, the management of oxidative stress has thus been considered as an interesting therapeutic alternative to existing treatments [14,20]. Antioxidant metalloenzymes have been efficiently assayed on animal models of IBD [21–23] or even on patients [24,25], showing the validity of an anti-ROS strategy. However, the use of proteins for therapeutics is generally associated with major drawbacks, including low cellular penetration, short half-life, and immunogenicity [26]. To overcome these shortcomings, low molecular weight redox active complexes able to reproduce the superoxide dismutase activity, called SOD mimics (SODm), or SOD mimetics have been investigated [27–30]. Two manganese complexes SODm, one involving a penta-aza-macrocycle as ligand (M40403) [31] and one a salan (Mn1, Figure 1) [32] have been assayed in rodent models of IBD and showed protective effects when they were administrated by an intraperitoneal injection or by gavage, respectively. In both cases, the complexes were administrated in a basic bicarbonate buffer to reduce metal decoordination that occurs with such metal complexes in acidic media. In particular, after oral gavage, the complex has to pass through
the stomach (low pH 1-2) to reach the inflammation site, namely the intestine. To improve the therapeutic action of SODm *in vivo* by protecting the complex from decoordination, a protective delivery vector is required. Various types of colonic delivery technologies have been developed, including stimuli-dependent delivery vectors, that sometimes require the combination of several triggers to deliver the active principle [33]. This is particularly interesting to selectively deliver a payload in a specific part of the gastro intestinal tract. Herein, we propose a new approach to deliver two SODm in the gut, **Mn1** [32,34] and a more inert complex due to the rigidication of the ligand **Mn1C** (Figure 1) [35], using food grade bacteria. For this purpose, one lactic acid bacterium strain (LAB), *Lactiplantibacillus plantarum* MD007 strain, has been loaded with the different SODm leading to chemically modified bacteria (CMB). CMB were administrated by gavage to mice suffering from a DNBS-induced colitis. **Mn1C** CMB showed markedly beneficial effect on mice colitis. This was observed either on their global health status or on selected inflammation markers.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** SOD mimics bioinspired from the MnSOD active site used in this study.</figcaption></figure>

## Material and methods

**Instruments.** Inductively Coupled Plasma – Mass Spectrometry (ICP-MS): the quantification of ⁵⁵Mn isotope content was performed using an Agilent 7700 Series ICP-MS system with the following parameters: RF power: 1550 W; sampling depth: 10 mm; helium flow rate: 5 mL.min⁻¹ measurement was replicated five times with 100 sweeps per replicate, and an integration time/mass of 1s.

**SOD mimics synthesis.** The ligands to prepare **Mn1** and **Mn1C** complexes were synthesized as previously described [32,35,36]. The corresponding complexes were prepared by mixing the ligands and MnCl₂ in a ratio 1:1.3 in Hepes ((2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) 0.1 M (pH 7.5) and let for 2 hours at ambient temperature to afford a complete complexation of the ligands.

**Bacteria cultures.** *Lactiplantibacillus plantarum* with a defective alanine racemase gene Alr-mutants MD007 (derived from *Lactiplantibacillus plantarum* WCFS1 or under the former name *L. plantarum* NCIMB8826) was grown in MRS containing D-alanine (400 µg/mL) at 37°C.

An overnight culture was used to inoculate fresh medium in order to get OD600 of 0.2, and cells were grown to an OD600 of 0.7 (corresponding to an exponential phase) and were washed three times with D-alanine free MRS medium. The washed bacteria were incubated in medium without D-alanine for 3h at 37°C before SOD mimic loading. This may fragilize their cell wall and favor SOD mimic release in the intestinal lumen.

**Bacteria loading with SOD mimics.** After removal of the culture medium supernatant, bacteria were incubated with Hepes (0.1 M, pH 7.4) only, or with the tested compounds (MnCl₂, **Mn1** or **Mn1C**) at the desired concentration (0.4 mM being the finally selected concentration) in the same buffer for two or twenty-four hours, at 37 °C. It must be noticed
that Mn1C, Mn1 and MnCl₂ solutions were prepared extemporaneously to prevent possible degradation and manganese oxidation reactions. After cultures centrifugation and washing with Hepes (0.1 M, pH 7.4), the bacteria were resuspended in Hepes 0.1 M containing 16% glycerol v:v to reach a concentration about 10⁹ bacteria per 200 µL. This volume corresponds to the usual volume administrated by gavage to mice every day. The bacteria were stored at -80°C in 2 mL aliquots to avoid freeze-thaw cycles. Samples dedicated to mice gavage were warmed up to room temperature before being administrated to mice.

**Bacteria viability.** The overnight culture and the frozen vials were controlled by CFU (colony forming unit) test by diluting the preparation in PBS with serial 10-fold dilution and plating the dilutions on the corresponding medium. The plates were incubated at 37°C.

**Quantification of manganese in bacterium lysates by ICP-MS.** For ICP-MS analysis, 50 µL of bacterial aliquots (ca. 2.5 10⁸ bacteria) were poured into 1 mL of a 2% HNO₃ aqueous solution and incubated for 1h, to lyse cells and free Mn from all coordination sites. Samples were then filtrated on 0.2 µm sized filters to get rid of bacteria debris. The solution was diluted 100 times in a 2% HNO₃ aqueous solution and analyzed. A calibration curve was established using a commercial Mn standard. A range of concentration going from zero to 100 ppb was generally used for calibration.

**Determination of SOD activity in bacteria lysates using the Fridovich assay.** Aliquots of loaded and non-loaded bacteria (200 µL at approx. 5 10⁹ CFU/mL) were submitted to mechanical lysis: bacterial samples were mixed with glass beads (0.1 mm diameter) and ceramic beads (1.4 mm diameter) and submitted to 3 agitation cycles in a Precellys (Bertin, France) at 6 800 RPM. Then, 3 identical aliquots (obtained after incubation in the same condition) were pooled and centrifuged at 12000 rcf for 10 min. The protein content of the supernatant were measured using Bicinchoninic acid assay (BCA assay, Uptima-Interchim, Montluçon, France) following instructions from the manufacturer. Then, supernatants were used to measure SOD activity by the McCord and Fridovich assay. In this assay, superoxide was generated with the xanthine/xanthine oxidase system. XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) was used as UV-Vis marker as it reacts with superoxide with a k = 2.9 ± 0.1 10⁴ M⁻¹s⁻¹ to give a formazan derivative that possesses a absorption maximum at 470 nm. The kinetic measurements were consequently conducted at 470 nm using the following procedure: to a solution of xanthine (200 µM) and XTT (100 µM) in HEPES (50 mM, pH 7.4), a solution of xanthine oxidase (20 mg mL⁻¹) was added. The solution was quickly mixed and the measurement of the absorbance started (giving a first slope P₁). The volume of xanthine oxidase added was chosen according to the value of the slope P₁ of formazan formation that must be kept between 0.025 and 0.030 min⁻¹. After 1.5 min, a known volume of bacterial lysate supernatant was added to the cuvette, the solution was mixed and formazan formation was measured between 3 and 4 min (slope P₂). The experiment was repeated for various volumes (from 10 to 50 µL) of bacterial lysate giving a set of collinear points when plotting the ratio (P₁-P₂)/P₂ versus the protein content in the cuvette. The IC₅₀ value corresponds to the concentration at P₂ = P₁/2, meaning that the superoxide flow has been reduced by 50%. The IC₅₀ reported are mean ± SEM of 4 samples.

**Animals experiments.** Male C57BL/6 mice (6 weeks old) were purchased from Janvier (France) and maintained at the animal care facilities of the National Institute of Agricultural and Environmental Research (INRAE, Jouy-en-Josas) under specific pathogen-free conditions
(SPF) conditions. Mice were housed under standard conditions for a minimum of 1 week before experimentation for their acclimatation. All animal studies were conducted in accredited research facilities and approved by local ethics committees in addition to the French government (authorization n°: 16744-201807061805486_v2).

**Assays with CMB.** The gavage with empty or loaded bacteria were started the day before inflammation induction by DNBS (D-1) to ensure that bacteria are already present in the colon. At D0, mice were anesthetized with an intra-peritoneal injection of a mix of ketamine (75 mg/kg, Imalgene, Boehringer Ingelheim Animal Health, Lyon, France) and xylazine (9 mg/kg, Rompun, KVP, Kiel, Germany). DNBS (3 mg/mouse in 30% ethanol/PBS) was administrated by an intrarectal injection using a catheter at about 3.5 cm into the colon. One group, non-colitis control, received only ethanol/PBS and is called “Healthy control”. Bacteria (≈ $10^9$ bacteria/200 µL), loaded bacteria (CMB) or Hepes were daily intragastrically administrated to mice for 4 or 5 days. The amount of CMB given to the mice was determined in such a way that all mice receive the same amount of total manganese, fixed at 150 nmol per dose, which corresponds to a dose ranging from $1x10^9$ and $2x10^9$ bacteria. The amount of bacteria given to the DNBS MD007 control group was determined to correspond to the maximal number of bacteria given to the groups treated with CMB. Mice were supervised all along the experiment with special attention. The last day (D3 or D4), mice were euthanized by cervical dislocation. The studied groups are named as follows: control healthy group without bacteria gavage (Healthy group), colitis group without bacteria that received Hepes buffer instead (DNBS Hepes), a colitis group that received empty bacteria (DNBS MD007 control) and 2 colitis groups gavaged with CMB: DNBS MD007 **Mn1** and DNBS MD007 **Mn1C**. Each group was composed of 8 mice. The assay was repeated three or four times independently meaning that, in total, 24 or 32 mice for each condition were used.

**Evaluation of colitis severity.**

- Post-euthanasia mice dissection and determination of macroscopic scores

The abdominal cavity of euthanized mice was opened and the colon was removed, opened longitudinally and washed from feces. The macroscopic score was then immediately assessed. This score evaluates the state of the colon by taking into account: thickening of colon wall, presence of ulcers, hyperemia, adhesion to other intra-abdominal tissues and consistency of colon content (indicator of transit anomalies). The colon was then longitudinally separated in two pieces. The right part was laterally separated in 4 parts intended to: from distal extremity to proximal one 1) the assay of myeloperoxidase activity, 2) the quantification of SOD2 gene transcription (stored in RNase free buffer (RNAlater) for an improved conservation of RNA) and 3) the determination of histologic scores (tissues fixed in 10% formalin and then stored in 70% ethanol until analysis). All the colon samples, except those for histology assessment, were stored at -80°C.

- Histological scores determination

For histological assessment, the colon sample fixed in formalin was embedded in paraffin. Five-micrometer sections were stained with hematoxylin and 0.2% eosin and examined blindly according to the Ameho criteria. The most inflamed part of each section was blindly assessed using the Mouse Colitis Histology Index (MCHI) as described previously [37]. Briefly, goblet cell loss, crypt density, hyperplasia and submucosal infiltrate (weighting
respectively 1, 2, 2 and 3) were assessed resulting in a score from 0 (not inflamed) to 22 (highly inflamed).

- Intestinal permeability assay

Three hours and a half before their euthanasia, mice received by gavage fluorescein conjugated to dextran (FITC-dextran 3000-5000 Da, 12 mg per mouse). Just before mice euthanasia, blood was collected from the submandibular vein. Blood samples were centrifuged at 2000 g for 10 minutes at 20°C and the fluorescence intensity (λ<sub>Exc</sub> = 488 λ<sub>Em</sub> = 520 nm, TECAN plate reader) was measured in the recovered plasma. The FITC-dextran concentration was determined from a standard curve obtained by serial dilution of FITC-dextran.

- Measurement of myeloperoxidase activity

Myeloperoxidase (MPO) activity, used as a marker of neutrophilic infiltration, was assayed as previously described [38]. Briefly, one-centimeter length of the distal colon was recovered and homogenized (50 mg/mL) in ice-cold 50 mM potassium phosphate buffer (pH 6) containing 5% hexadecyl trimethyl ammonium bromide (Sigma-Aldrich) as source of bromide. Hydrogen peroxide ($H_2O_2$) was added to an O-dianisidine solution right before use, and aliquots of colon extract were added. This results in the catalytic conversion of $H_2O_2$ and Br- into hypobromous acid (BrOH) and water and oxidation of O-dianisidine. The colorimetric reaction was followed by measuring the absorbance with a spectrophotometer using standard as reference (myeloperoxidase ref. 475911, Sigma-Aldrich). MPO activity was expressed as units per milligram of wet tissue (1 unit = reaction with 1 mM of $H_2O_2$ in 1 minute at room temperature).

- Lipocalin 2 (LCN2) concentration determination

For determination of plasma LCN2 concentration, centrifuged blood samples (2000 g for 10 minutes at 20°C) were used. A commercially available sandwich enzyme-linked immunosorbent assay (Mouse Lipocalin-2/NGAL DuoSetELISA; R&D Systems Europe, Abingdon, United Kingdom) was used according to the manufacturer's instructions. For determination of colon LCN2 concentrations, colon supernatants prepared to measure MPO activity were used with the same kit as for plasma.

- Quantification of SOD2 gene transcription

Total RNA were extracted from colon homogenates by using a Qiagen RNeasy kit according to the manufacturer's instructions. β-mercaptoethanol was used as reducing agent for the irreversible denaturation of RNases enzymes. Reverse transcription was then performed to produce complementary DNA with oligo dT<sub>12-18</sub> and super script II reverse transcriptase enzyme (Invitrogen). SOD 2 gene expression was quantified by quantitative PCR on a StepOne Real-time PCR system using Takyon rox Sybr mastermix DTTP blue (Eurogentec). The following primer pair was 5'-ATTAACGCGCAGATCATGCA-3' (forward) and 5'-TGTCCCCCACCATTGAACTT-3' (reverse) and the annealing temperature was set to 60°C. The SOD2 levels were normalized with that of housekeeping GAPDH (forward 5'-AGGTCGGTGTGAACGGATTTG-3'; reverse 5'-TGTAGACCATGTAGTTGAGGTCA-3') and TATA-box binding protein, TBP (forward 5'-ACCGTGAATCTTGGCTGTAAAC-3'; reverse 5'-GCAGCAAATCGCTTGGGATTA-3') genes, which are known to be not impacted in inflammatory conditions [39].
**Statistical analysis.** Statistical analysis was completed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Results are presented as dot plots with means ± SEM. The p-values were calculated using the non-parametric Mann-Whitney test (one-tailed test) as the data did not show a Gaussian distribution according to the Anderson-Darling normality test. Outliers found using the Prism software were discarded. The mean ranks of each column were compared to that of the DNBS Hepes control; each comparison stands alone. ****: p < 0.0001, ****: p < 0.001, **: p < 0.01, and *: p < 0.05 versus DNBS Hepes control, and ns means non-significant. A p value of less than 0.05 was considered significant. To compare the clinical scores (macroscopiques and histo-pathological scores) across treatment groups, we performed generalized linear model (glm), where the clinical score of each individual mouse is following a binomial distribution of size parameter set to the number of sub-clinical scores. The glm was fitted using the glm() function from the stats R package and the clinical group of each mouse was used as a covariable. Statistical significance of the treatment group was assessed using a Wald's test, implemented by the summary() function.

**Results and discussion**

Lactic acid bacteria (LAB) are microorganisms of particular interest as a mean of drug delivery, since (i) they are generally recognized as safe (GRAS status, FDA, USA) with qualified presumption of safety (QPS, EFSA, Europe), (ii) they are known to survive in a large range of pH including the acidic pH of the gastrointestinal tract and (iii) they can be modified to increase their delivery efficiency. Consequently, LAB are ideal strains to develop chemically modified bacteria (CMB) as probiotics possessing antioxidant properties.

In order to develop and validate LAB as efficient delivery vector for SOD mimics, the LAB strain has to fulfill the following criteria: (i) it must be neutral toward the colitis (neither improve nor degrade mice health status) so it does not interfere with SOD mimics potential effects, (ii) it must allow SODm loading and release, (iii) it must resist to a freeze-thaw cycle to be used on demand and (iv) it may possess a high content of endogenous Mn²⁺ to increase Mn complexes SODm stability by shifting the equilibrium towards the complex formation in presence of an excess of Mn²⁺. Considering these important features, we have selected the D-alanine auxotrophic strain of *L. plantarum* MD007 strain. Indeed, *L. plantarum* strains are known to be rich in manganese, with an intracellular concentration reaching 30 mM [40] (to be compared with a micromolar concentration in *E. coli* for instance [41]). Mn²⁺ is one of the most bioavailable metal ion [42]. Consequently, this high Mn²⁺ content may reduce intracellular decoordination of the expected Mn complexes SODm. In addition, MD007 strain auxotrophic properties could induce bacterial lysis and thus may favor SODm release in D-alanine-deprived conditions. Finally, D-alanine deprivation will prevent also MD007 dissemination in the environment.

**1. MD007 strain as a neutral vector for SODm delivery**

Loading of the bacteria was performed using the SODm **Mn1**. SODm loading concentrations were optimized in order to reach the highest intracellular **Mn1** content as possible without inducing bacteria death, even after a freeze-thaw cycle. To note, **Mn1** content was not evaluated strictly speaking but the total manganese content was measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS); as a first approximation, Mn accumulation being associated with **Mn1** loading. Also, the quantities measured are Mn
contents associated with bacteria, i.e. inside the bacteria, they also may include Mn adsorbed onto the membranes. After optimization of the loading conditions (incubation concentration, time... without inducing bacteria death), a 2-hour incubation of **Mn1** at 0.4 mM at 37°C was selected (Figure S1). The same condition was used with MnCl₂ (2-hour incubation of MnCl₂ at 0.4 mM at 37°C) to prepare MnCl₂-loaded bacteria (Figure S1). In order to ascertain that loaded bacteria possess improved anti-superoxide and thus antioxidant activities, SOD activity of bacterial lysates was measured using the McCord and Fridovich assay [43–45]. In this assay, a superoxide flow is generated by the xanthine/xanthine oxidase system and the kinetics of the reaction of superoxide with a UV/vis marker (v₁), here XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), is evaluated spectrophotometrically. By addition of a SOD mimic reacting with superoxide faster than the UV-vis marker, a reduction in v₁ defined above is recorded. This assay allows determining IC₅₀, i.e. concentration at which v₁ is divided by two. In this assay, the lower the IC₅₀, the better. As shown in Table 1, in the assay conditions, the IC₅₀ of non-loaded bacteria was too high to be measured. In contrast, bacteria loaded with **Mn1** showed SOD-like activities. Bacteria loaded with MnCl₂ also exhibited a SOD activity, which may be attributed to the formation of manganese complexes involving endogenous metabolites (peptides, nucleosides...) forming catalytic antioxidants as shown in gamma radiation resistant *D. radiodurans* strain for instance [46,47]. Finally, **Mn1**-loaded bacteria showed a higher IC₅₀ than MnCl₂-loaded bacteria, which may be due to a lower Mn content (due to lower penetration of **Mn1** compared to MnCl₂) as measured by ICP-MS (Figure S1B).

<table><caption>**Table 1.** SOD activity of the bacteria lysates obtained after incubation with **Mn1**, **Mn1C** or MnCl₂. SOD activity was measured using the McCord and Fridovich assay. IC₅₀ are given according to the protein content of each sample measured using the BCA assay. For non-incubated bacteria (MD007 control) the IC₅₀ was too high to be measured in the conditions used.</caption><thead><tr><th>Bacteria lysate</th><th>IC<sub>50</sub> mg mL<sup>-1</sup><br>(lysates protein content)</th></tr></thead><tbody><tr><td>MD007 control</td><td>&gt; 0.046</td></tr><tr><td>MD007 Mn1</td><td>0.032 ± 0.005</td></tr><tr><td>MD007 Mn1C</td><td>0.025 ± 0.006</td></tr><tr><td>MD007 MnCl<sub>2</sub></td><td>0.025 ± 0.004</td></tr></tbody></table>

Mice were gavaged with the different bacterial suspensions with a volume adjusted to correspond to 150 nmol of manganese. Importantly, the bacteria were administrated one day before colitis induction to ensure the presence of the bacteria in the gut at the beginning of the assay.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Evaluation of global health status of mice subjected or not ("Healthy control") to DNBS injection and treated with Hepes or with bacteria (DNBS MD007 control) loaded with **Mn1** (DNBS MD007 Mn1) or MnCl₂ (DNBS MD007 MnCl₂). A/ Weight monitoring over time. The weights are expressed as a percentage of each mouse initial weight at day 0. Curves represent the average of 32 mice (4 independent assays with 8 mice per condition) for the conditions DNBS Hepes, DNBS MD007 control and DNBS MD007 **Mn1**. For the condition "Healthy control" and DNBS MD007 MnCl₂, 3 assays have been performed (24 mice). The number of remaining mice at the end of the assay (not died or pre-euthanized before the end of the three-day assay) are indicated on the figure. B/ Area under the curve of weight monitoring between day 0 to 3 (D0-D3). C/ Macroscopic scores of mice subjected to DNBS injection and determined on the euthanasia day (day 4 for three assays). Data represent the mean ± SEM for all mice of each group. \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001 and \*\*\*\* p < 0.0001 vs DNBS Hepes.</figcaption></figure>

Mice body weights were monitored during the experiment (Figure 2A and B) and macroscopic scores were evaluated at the euthanasia day (Figure 2C). The results presented in Figure 2 showed that MD007 bacteria are neutral toward the DNBS induced colitis since no significant effect was observed on the weight and macroscopic score for mice receiving the non-loaded strain (DNBS MD007 control condition). Interestingly, on the one hand, **Mn1** loaded MD007 bacteria seemed to mitigate weight loss (Figure 2A and B) even if non-significative statistical results were observed (p value = 0.2271 for B in Figure 2). On the other hand, a clear protective effect of **Mn1** CMB was observed on macroscopic scores (p < 0.01 for DNBS MD007 **Mn1** vs DNBS Hepes, Figure 2C) indicating better overall colon health. Bacteria loaded with MnCl₂ are deleterious since this treatment seems to deteriorate mice health (Figure 2). This supports the fact that the metal complexes (**Mn1**) are the efficient active principles and the catalytic antioxidants formed between metabolites and MnCl₂ are not stable enough to exert a protective effect.

**2. CMB improvement using a more inert Mn complex as SOD mimic.**

We have recently shown that rigidifying the ligand by incorporating a cyclohexyl-diamine (Figure 1) is efficient to increase the resistance of the resulting complex (**Mn1C**) toward metal exchange that can occur in cells [35]. Indeed, it is of importance to limit these metal exchanges in cells that lead to the formation of non-active metal complexes [48]. **Mn1C** has shown improved anti-inflammatory and antioxidant activities in comparison to **Mn1** on an intestine epithelial cellular model of inflammation (HT29-MD2 cells, intestine epithelial cells HT29 stably transfected to overexpress MD2 protein [32,35,49]). **Mn1C** was thus assayed to improve the therapeutic effect of CMB. **Mn1C** was loaded in MD007 bacteria in the same
conditions as **Mn1**. Interestingly, in these conditions, Mn content in **Mn1C** incubated bacteria was higher than in bacteria incubated with **Mn1**, which may be due to its higher lipophilicity that can favor cell penetration (Figure S1) [35]. The same feature was also observed in HT-29-MD2 cells [35]. More importantly, the IC₅₀ of **Mn1C**-loaded bacteria (Table 1) is lower than the IC₅₀ of **Mn1**-loaded bacteria, confirming the expected improvement of their antioxidant properties.

## 2.1. CMB improve global mice health status.

As in the previous experiment, untreated mice subjected to DNBS injection (Hepes condition, Figure 3A) lost weight (about 16% of their initial at day 2) and started to recover at day 3. As observed previously in Figure 2, the mice gavage with MD007-internalized **Mn1** seems to limit the weight loss of mice and to fasten recovery (Figure 3A and B). Very interestingly, the group that received **Mn1C** loaded bacteria showed the same effects but in a much larger extent (Figure 3A MD007 **Mn1C** condition, green line). To note, the group that received **Mn1C** CMB is the only group where no mice had to be euthanized before the end of the three-day assay because of a too large body weight loss.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Evaluation of global health status of mice subjected to DNBS injection and treated with Hepes or with bacteria (MD007) loaded with the indicated SODms. A/ Weight monitoring over time. The weights are expressed as a percentage of each mouse initial weight at day 0. Curves represent the average of approximately 24 mice. The number of remaining mice at the end of the assay (not dead or pre-euthanized before the end of the 3D-assay) are indicated on the figure. B/ Area under the curve of weight monitoring between day 0 to 3 (D0-D3). Data represent the mean ± SEM collected during three independent assays (8 mice/assay/condition). C/ Macroscopic scores of mice subjected to DNBS injection and determined on the euthanasia day. \* p < 0.05 vs DNBS Hepes control.</figcaption></figure>

Area under the weight curve calculated between day 0 to 3 (Figure 3B) showed a significant beneficial effect in the **Mn1C** loaded bacteria group. Consistently, macroscopic scores in this group were significantly decreased (Figure 3C) and colon were found longer than in the DNBS Hepes group (Figure S2A). **Mn1** CMB also improved the colitis macroscopic scores as shown previously (Figure 2C). To note, the colon length was also found longer for the empty bacteria control group than for the DNBS Hepes group. However, it is a rare colitis parameter for which empty bacteria showed beneficial effects, suggesting that this parameter is not representative of the overall neutral effect of the non-loaded bacteria. Altogether, these data suggest that the bacteria loaded with the more inert SOD mimic (**Mn1C**) are more potent than **Mn1** to limit the colitis effects.

## 2.2. Anti-inflammatory effects of CMB.
Increasing evidence suggests that intestinal barrier disturbances such as among others mucus alterations, defective antimicrobial peptide production and increased permeability may be crucial in the pathogenesis of IBD [50,51], and IBD is somehow considered as an impaired barrier disease [52]. It has been shown that DNBS treated mice exhibit increased gut permeability measured by the paracellular passage of a FITC-dextran tracer, which is associated by the reduced expression of junction proteins Claudin-4, Occludin, F11r, ZO-1 and E-Cadherin [53]. After intragastrical administration of FITC-dextran, lower levels of labeled dextran were quantified in the plasma samples of mice treated with **Mn1C** CMB compared to DNBS Hepes mice (Figure 4A). This demonstrates the efficacy of MD007-vectorized **Mn1C** to attenuate significantly the DNBS-induced intestinal hyperpermeability.

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

**Figure 4.** Influence of the CMB treatment on several parameters characterizing the colitis induced by DNBS intrarectal injection. A/ Quantification in blood of FITC-dextran 3.5 h after oral gavage, as a marker of the intestinal barrier permeability. B/ Lipocalin-2 concentration measured in the colon. C/ Lipocalin-2 concentration measured in serum. D/ Levels of mRNA SOD2 in colon samples of mice suffering from DNBS-induced colitis. Average on 24 mice for each condition (3 independent assays with 8 mice per condition) are presented excepted for D/ for which the measures have been performed during 2 assays (16 mice per condition). Data represent the mean ± SEM for two or three independent assays. Unless noted, \* p < 0.05, \*\* p < 0.01 and \*\*\* p < 0.0001 vs DNBS Hepes control.

IBD are very often associated with the infiltration of immune cells secreting the heme myeloperoxidase (MPO). We have thus measured MPO activity in the colon, which did not reveal significative impact of any treatment (Figure S3A). The same result was obtained on the total histological scores (Figure S3B-F). However, even if not statistically significative,
mice that received vectorized **Mn1C** seemed to show a slightly lower MPO activity (Figure S3A, P value: 0.3251) as well as lower total histo-pathological scores (Figure S3B, P value 0.2747) as compared to the colitis group (DNBS Hepes conditions). This trend is observed for the **Mn1C** group only.

As a component of the innate immune system, lipocalin-2 (LCN2), also called neutrophil gelatinase-associated lipocalin (NGAL), is a siderophore binding protein that prevents bacteria from acquisition of iron and therefore limits bacterial growth [54]. High LCN2 expression by neutrophils and gut epithelial cells has been demonstrated in colonic biopsies from inflamed areas of patients with IBD [55] and more recently, serum LCN2 has been proposed as a disease activity marker [56]. Very interestingly, treatment of DNBS-injured mice by the **Mn1C** CMB decreases significantly the LCN2 level both in colon and in serum, suggesting a reduction of colitis severity in these conditions whereas **Mn1** CMB showed a significative effect only in colon samples (Figure 4B and C). Such a LCN2 content decrease was not observed in mice stools (Figure S2B).

Taking into account that LCN-2 is strongly expressed by both epithelial cells and immune cells, including neutrophils [55] while MPO is expressed only by immune cells, in particular neutrophils [57], the results suggest that the beneficial effects of the SOD mimics are more important on the intestinal epithelium itself than on the surrounding environments (lamina propria and intestinal lumen) where immune cells are present in inflamed conditions [58]. This is consistent with the fact that SOD mimics were administrated by gavage, thus reaching and acting on the intestinal epithelium before diffusion and action on the underlying tissue. Furthermore, for each of the beneficial results obtained, that of Mn1C was consistently better than that of **Mn1**, which may be due to the higher inertness towards the release of Mn(II) of **Mn1C** in comparison with **Mn1** [35].

## 2.3. **Mn1C** CMB effect on SOD2 expression

Using the previously mentioned cellular model of inflammation HT29-MD2, we have shown that a 6-hour LPS stimulation induces a strong overexpression of the MnSOD (SOD2) enzyme as a feedback response against oxidative stress [32]. Interestingly, after LPS challenge and upon incubation with SOD mimics such as **Mn1** and **Mn1C**, altogether with a reduced inflammation (quantified by IL-8 and Cox2), the MnSOD overexpression was strongly limited [32,35] indicating that these SODm are able to complement the MnSOD by exerting their superoxide dismutase-like activity in cells, which limits the oxidative stress. To see whether such an effect can also be observed in colon *in vivo*, mRNA SOD2 content was measured in tissue homogenates by quantitative PCR. As shown in Figure 4D, mRNA SOD2 level was significantly decreased upon mice feeding with empty bacteria. This was also the case for CMB loaded with **Mn1C** but in a much larger extent. Consistently with what observed on cultured epithelial cells, this suggests that the SODm **Mn1C** is present in the gut and is able to complement the endogenous SOD2 enzyme, thus requiring less SOD2 production by intestinal cells. These results may represent an indirect proof that the anti-inflammatory activity observed for **Mn1C** CMB may be attributed to the anti-superoxide effect of **Mn1C**.

Altogether, from all parameters measured, **Mn1C** CMB appeared to be the most efficient treatment to reduce the inflammation in this DNBS colitis model.
**Conclusion**

In this study, we have demonstrated that the administration of a D-alanine auxotrophic strain of *L. plantarum* loaded with a Mn complex mimicking SOD activity **Mn1C** significantly alleviates the severity of a colitis induced by DNBS in mice. The beneficial effect of CMB has been observed on several parameters including body weight loss, macroscopic scores, intestinal barrier permeability and lipocalin-2 content in serum and in colon. In addition, this anti-inflammatory effect is associated with a decrease of MnSOD expression in colonic tissue, suggesting that it is linked to the antioxidant properties of the SODm loaded into the CMB that has been released in the gut. This study paves the way toward the development of new efficient chemically modified antioxidant probiotics for IBD treatment.

**Financial support**

This work was supported by Association François Aupetit, ANR (ANR-15-CE07-0027 project MAGIC; ANR-16-CE07-0025 Project Metallopezyme; ANR 20-CE07-0039-01 projet CATMAN; ANR-21-CE18-0053 projet MOBIDIC, ANR-22-PEBI-0003 PEPR. CNRS and the MITI program (Bactman project) is acknowledged for financial supports.

**Acknowledgments**

We thank ENS-PSL, Sorbonne university and CNRS for recurrent funding, the program *Interface pour le Vivant* (Sorbonne university) for G. Schanne's PhD fellowship, B. Goetz and P. Legeay for help with ICP-MS, L. Brot, and E. Tauziet for their guidance and help with cell culture and cell assays.

This work has benefited from the facilities and expertise of @BRIDGe (Université Paris-619 Saclay, INRAE, AgroParisTech, GABI, 78350 Jouy-en-Josas, France) for histology tissue preparation. We wish to thank the staff of the INRAE Infectiology of Fishes and Rodents Facility (IERP-UE907, Jouy-en-Josas Research Center, France) in which animal experiments have been performed. IERP Facility belongs to the National Distributed Research Infrastructure for the Control of Animal and Zoonotic Emerging Infectious Diseases through In Vivo Investigation.

**Disclosure statement**

The authors report there are no competing interests to declare.

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