# Immunization reveals a key role of Enterobacterial ClpB protein in the development of host anorexia

Benjamin Thomas, Nadia Andrea Andreani, Emilie Lahaye, Lisa Wallart, Karthi Duraisamy, Benjamin Lefranc, Jérôme Leprince, Sophie Bernard, Philippe Chan, Jean-Luc Do Rego, et al.

► To cite this version:

Benjamin Thomas, Nadia Andrea Andreani, Emilie Lahaye, Lisa Wallart, Karthi Duraisamy, et al. Immunization reveals a key role of Enterobacterial ClpB protein in the development of host anorexia. Brain, Behavior, and Immunity, 2026, 131, pp.106152. 10.1016/j.bbi.2025.106152. hal-05353691

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# Immunization reveals a key role of Enterobacterial ClpB protein in the development of host anorexia

Benjamin Thomas<sup>a</sup>, Nadia Andrea Andreani<sup>b,c</sup>, Emilie Lahaye<sup>a</sup>, Lisa Wallart<sup>a</sup>, Karthi Duraisamy<sup>a</sup>, Benjamin Lefranc<sup>a</sup>, Jérôme Leprince<sup>a</sup>, Sophie Bernard<sup>d</sup>, Philippe Chan<sup>d</sup>, Jean-Luc do Rego<sup>e</sup>, Jean-Claude do Rego<sup>e</sup>, Astrid Dempfle<sup>f</sup>, John F. Baines<sup>b,c</sup>, Sergueï O. Fetissov<sup>a,w ID</sup>

## ARTICLE INFO

**Keywords:**

Gut microbiota-brain axis

Adaptive immunity

Autoantibodies

Enterobacteriaceae

ClpB

Appetite

Neuropeptides

Animal models

## ABSTRACT

Anorexia nervosa (AN) is a psychiatric disease of unknown etiology. Recently, caseinolytic protease B (ClpB), produced by *Enterobacteriaceae*, was identified as an antigen mimetic of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide. Here, we studied the role of ClpB in the development of activity-based anorexia (ABA), a rodent model of AN. In two series of ClpB immunization experiments, we show that when ClpB was lowered by antibodies, mice were protected against ABA, showing lean mass preservation, an increase in food intake, and reduced feeding anticipatory activity. On the contrary, if antibody production was accompanied by an increase of ClpB, ABA was aggravated. A key role of anti-ClpB IgG in ABA protection was confirmed in ClpB-tolerized mice and by IgG inhibiting ClpB- and α-MSH- activation of melanocortin 4 receptor. ClpB immunization influenced *clpB* DNA abundance in ABA mice without affecting fecal microbiota diversity and energy content. These data support a key role of Enterobacterial ClpB as well as its control by the immune system in host anorexia and provide background for ClpB immunoneutralization as a putative biotherapy of AN.

## 1. Introduction

Anorexia nervosa (AN) is a psychiatric disease of unknown etiology still lacking efficient therapy (Himmerich et al., 2023). Pathophysiology of AN involves the interaction of several risk factors, including genetic background, psychological stress, and weight-loss behavior such as severe dieting accompanied by intense physical activity (Bulik et al., 2021; Himmerich et al., 2019). Recently, gut microbiota appeared as another possible environmental risk factor involved in the onset and/or

maintenance of AN. Indeed, dysbiosis is a common feature of patients with AN (Fan et al., 2023; Fouladi et al., 2022; Prochazkova et al., 2021). Importantly, a longitudinal study showing that members of gut microbiota can be predictive of disease prognosis (Andreani et al., 2024), as well as some fecal microbiota transplantation experiments from patients with AN to mice (Fan et al., 2023; Hata et al., 2019), point to a causative role of gut bacteria in AN pathophysiology. While the molecular mechanisms linking gut bacteria to the brain and behavior need clarification, identification of specific microbial metabolites

**Abbreviations:** α-MSH, α-melanocyte-stimulating hormone; ABA, activity-based anorexia; AN, anorexia nervosa; ASV, amplicon sequence variant; ClpB, casein-olytic protease B; CFA, complete Freund's adjuvant; E. coli, *Escherichia coli*; FAA, feeding anticipatory activity; IFA, incomplete Freund's adjuvant; IgG, immunoglobulins G; IP, intraperitoneal; MC4R, melanocortin receptor type 4; SPR, surface plasmon resonance; unc., unclassified.

* Corresponding author.

E-mail address: Serguei.Fetissov@univ-rouen.fr (S.O. Fetissov).

<footnote><sup>a</sup> Regulatory Peptides - Energy Metabolism and Motivated Behavior Team, Neuroendocrine, Endocrine and Germinal Differentiation and Communication Laboratory, Inserm UMR1239, University of Rouen Normandie, Rouen, France</footnote>

<footnote><sup>b</sup> Guest Group Evolutionary Medicine, Max Planck Institute for Evolutionary Biology, Plönen, Germany</footnote>

<footnote><sup>c</sup> Section of Evolutionary Medicine, Institute for Experimental Medicine, University of Kiel, Kiel, Germany</footnote>

<footnote><sup>d</sup> Imaging Platform PRIMACEN, University of Rouen Normandie, INSERM US51, CNRS UAR 2026, HeRacLeS, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France</footnote>

<footnote><sup>e</sup> Animal Behavioral Platform SCAC, University of Rouen Normandie, INSERM US51, CNRS UAR 2026, HeRacLeS, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France</footnote>

<footnote><sup>f</sup> Institut for Medical Informatics and Statistics, Schleswig-Holstein University Hospital, University of Kiel, Kiel, Germany</footnote>
appears as a promising strategy for the development of therapeutic targets in neuropsychiatric diseases (Foster, 2022).

Recently, a pathophysiological model has been proposed that implicates bacterial caseinolytic protease B (ClpB) in the origin of AN (Fetissov and Hökfelt, 2019). ClpB is a 96 kDa chaperon protein produced by the *Enterobacteriaceae* family of gut bacteria, which was identified as an antigen-mimetic of α-melanocyte-stimulating hormone (α-MSH) (Tennoune et al., 2014). α-MSH is an anorexigenic neuropeptide involved in the central regulation of appetite and body weight via the activation of the melanocortin receptor type 4 (MC4R) (Cone, 2005). MC4R is also expressed in the gut enteroendocrine cells and vagal afferents, supporting a role of peripheral α-MSH-like ligands in signaling satiety (Panaro et al., 2014; Wan et al., 2008). Therefore, the α-MSH/MC4R system has been identified as a valid pharmacological target for the pathological conditions of altered appetite and body weight (Yeo et al., 2021; Zhu et al., 2020).

The model implicating ClpB in AN is based on both direct anorexigenic action of this bacterial protein as well as on its antigenic properties to stimulate α-MSH cross-reactive immunoglobulins G (IgG), which play a role of the α-MSH carrier protein in the blood (Dominique et al., 2021; Legrand et al., 2020; Seitz et al., 2024; Tennoune et al., 2014). Relevance of α-MSH-reactive IgG to AN was shown by correlations of their plasma levels with psychopathological traits in patients with AN and by patients' IgG enhancement of MC4R activation (Fetissov et al., 2005; Lucas et al., 2019). Moreover, a recent study in adolescents with AN revealed low serum levels of anti-ClpB IgG (Thomas et al., 2025). Taken together, these data justify ClpB as a molecular target in AN; however, the experimental evidence is still missing.

In the present study, we aimed to obtain a proof of concept that ClpB underlies host anorexia using ClpB immunization as an experimental approach. For this purpose, we exploited the activity-based anorexia (ABA) phenomenon in rodents, an established animal model of AN (Carrera et al., 2014). We hypothesized that immunization-induced anti-ClpB IgG before exposure to an ABA protocol may protect mice from ABA by counteracting the anorexigenic effects of both ClpB and α-MSH. Two independent experiments were performed consecutively, because in the 1st experiment (Exp #1), immunization failed to produce ClpB neutralizing IgG; therefore, the dosage of antigen was changed in the 2nd experiment (Exp #2). Furthermore, in Exp #2, as a control for the role of immunization-induced anti-ClpB IgG, one group of mice was orally tolerized by ClpB before immunization. Effects of ClpB immunization on body weight, body composition, food intake, and meal pattern, as well as physical activity during the ABA protocol, were measured. To determine the molecular pathways underlying the behavioral results, plasma levels and affinities of IgG binding ClpB and α-MSH, as well as plasma concentration of both molecules, were analyzed. Effects of immunization-induced IgG on ClpB- and α-MSH activation of MC4R were assayed *in vitro*. Feeding-regulatory neuropeptide mRNA expression was measured in the hypothalamus. We coupled our analyses with the observation of the fecal microbiome to test whether any of the observed behavioral effects could be associated with changes in the gut microbiome composition, fecal *clpB* DNA, and fecal energy content. Here we present the data from both experiments showing that ClpB immunization impacts both behavioral and biochemical parameters of mice exposed to the ABA protocols, depending on properties of ClpB IgG and, importantly, that production of IgG leading to ClpB immunoneutralization efficiently protects against the development of anorectic phenotype.

## 2. Results

### 2.1. Experimental design

To test our hypothesis that immunoneutralization of ClpB may protect mice from developing ABA, in Exp #1, mice were immunized via a single intraperitoneal (IP) injection of 50 µg of recombinant *Escherichia coli* (E. coli)

96 kDa ClpB protein in 100 µL of complete Freund's adjuvant (CFA). The immunization dose was selected based on the previous study (Tennoune et al., 2014). Two weeks later, immunized mice received a boost of ClpB (50 µg) in incomplete Freund's adjuvant (IFA). Control mice received an IP 0.9 % NaCl solution. One week after the boost, all mice were placed into individual BioDaq food and drink intake monitor cages equipped with a running wheel and exposed to an ABA protocol or were kept with *ad libitum* (ad lib) food access for 12 days. As such, four groups of mice (n = 8, in each group): ClpB-immunized (imm) ad lib, ClpB-imm ABA, NaCl ad lib, and NaCl ABA were studied with several measurements taken at three time points: T0, T1 & T2, i.e., before immunization as well as before and after ABA, respectively (Fig. 1A).

Although immunized mice in Exp #1 developed a high titer of anti-ClpB IgG, this was not accompanied by a reduction in ClpB levels in the blood. Instead, an enhancing effect on ClpB and α-MSH was observed, which was probably due to increased affinity of IgG for both ClpB and α-MSH, i.e., increasing α-MSH carrier properties (Seitz et al., 2024). Therefore, in the 2nd experiment (Exp #2), we empirically selected a twice lower dose of ClpB (25 µg) and CFA (50 µl) for immunization. In Exp #2, non-immunized control mice received a 0.9 % NaCl solution, while an additional control group received CFA alone to assess adjuvants (Adj)-induced non-specific immune stimulation. Moreover, before ClpB immunization, one group of mice was tolerized (tol) by oral intake of recombinant ClpB protein (25 µg/ml) in drinking water for 10 days. One week after the immunization boost, all mice were placed into individual BioDaq cages and exposed to an ABA protocol for 11 days. As such, in Exp #2, four groups of mice (n = 8, in each group) were studied: NaCl, Adj, ClpB-imm, and ClpB-imm/tol (Fig. 1B).

### 2.2. Plasma anti-ClpB and α-MSH-reactive antibodies

Since the properties of anti-ClpB antibodies were the key determinant of different behavioral and biochemical outcomes in both experiments, the corresponding data from Exp #1 and Exp #2 are shown on the parallel left and right panels, respectively, to facilitate the comparison. Mice immunized in Exp #1 with a higher dose of ClpB (50 µg) displayed strong elevation of plasma anti-ClpB IgG levels in both ad lib and ABA groups (Fig. 1C). Due to ClpB molecular mimicry with α-MSH, ClpB immunization also increased plasma levels of α-MSH-reactive IgG (Fig. 1D). Increased plasma levels of anti-ClpB and α-MSH-reactive IgG were also found in Exp #2 in mice immunized with a lower dose of ClpB (25 µg); however, such an increase was less pronounced than in Exp #1 (Fig. 1E, F). Paired analysis of plasma anti-ClpB and α-MSH-binding antibodies from T0 to T2 also confirmed the efficiency of ClpB immunization in both experiments (Fig. S1A-D). Importantly, mice orally tolerized with ClpB showed diminished anti-ClpB and α-MSH-reactive IgG responses to ClpB immunization (Fig. 1E, F), resulting in increased ratios of ClpB/ClpB IgG (Fig. S1E), i.e., confirming the efficiency of tolerization.

The surface plasmon resonance (SPR) analysis of affinity kinetics of plasma-extracted IgG binding to ClpB and α-MSH revealed opposite patterns between the two experiments. In Exp #1, ClpB-immunized mice displayed lower $K_D$, i.e., increased IgG affinity for both ClpB and α-MSH (Fig. 1G, H). An increase in affinity was mainly due to a lower dissociation rate (Fig. S1F-K). In contrast, in Exp #2, the ClpB-imm group showed lower IgG affinity for ClpB and α-MSH (Fig. 1I, J), without significant differences in the association or dissociation rates (Fig. S1H-M). Moreover, no significant changes in IgG affinity for either molecule were observed in ClpB-tolerized mice (Fig. 1I, J).

### 2.3. Plasma ClpB protein and α-MSH peptide

Remarkably, in Exp #1, ClpB-immunized mice displayed increased plasma concentrations of bacterial ClpB protein, which was found in both ad lib and ABA groups (Fig. 1K). Moreover, ClpB immunization also
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Experimental design and plasma ClpB and α-MSH peptides and antibodies. In this and subsequent figures, data are displayed with Exp #1 panels on the left and Exp #2 panels on the right, delineated by a dashed line. In Exp #1, two groups were fed Ad lib and two were exposed to ABA as indicated in each bar graph on the left and right, respectively, separated by a dashed line. In Exp #2, all groups were exposed to ABA. Experimental design, for the details see the main text (A, B). All measurements shown in this figure are from terminal plasma samples (T2). Plasma levels of anti-ClpB IgG (C, E) and α-MSH-reactive IgG (D, F). Affinity (dissociation at equilibrium constants, K_D) of plasma-extracted IgG for ClpB (G, I) and α-MSH (H, J). Plasma concentration of ClpB (K, M) and α-MSH (L, N). Exp #1, Students t-test \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. Exp #2, ANOVA with Tukey's post-test \*p < 0.05, \*\*p < 0.01.</figcaption></figure>

increased plasma α-MSH concentrations in ABA mice (Fig. 1L). In contrast, in Exp #2, ClpB-immunized mice did not show an elevation of plasma ClpB levels as compared to NaCl or Adj controls. Moreover, ClpB levels in immunized mice were lower than in ClpB-tolerized mice, supporting the role of immunization-induced anti-ClpB IgG in the control of plasma ClpB (Fig. 1M). No significant differences in plasma α-MSH peptide levels were found between groups in Exp #2 (Fig. 1N). Thus, while immunization resulted in the production of ClpB- and α-MSH-enhancing antibodies in Exp #1, in Exp #2, ClpB immunization-induced antibodies prevented an increase in ClpB.

## 2.4. Body weight and body composition

In Exp #1, ClpB-immunized ABA mice showed a trend of a more

pronounced weight loss at the beginning of food restriction as compared to the NaCl ABA group, reaching about 20 % weight loss by the end of the experiment (Fig. 2A). ClpB immunization did not modify body weight gain in ad lib-fed mice (Fig. 2A and Fig. S2A). Remarkably, in Exp #2, in which all mice were exposed to an ABA protocol, the ClpB-immunized group lost significantly less body weight than the other groups (Fig. 2B and Fig. S2B). By the end of the experiment, the ClpB-imm group lost only about 10 % of body weight, while NaCl controls lost about 20 % and two other groups showed intermediate results (Fig. 2B).

Moreover, mortality (1 mouse/group) during ABA protocols was observed in the ClpB-imm group in Exp #1 and in all groups other than ClpB-imm in Exp #2. Thus, while ClpB immunization aggravated body weight loss during ABA in Exp #1, in Exp #2 it efficiently protected
Exp #1

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

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

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Body weight and body composition. Percentage of body weight changes from T1 to T2 (A, B). Body composition for lean (C, D) and fat mass (E,F) at T1 and T2. B, 2-way RM ANOVA with Bonferroni post-tests, ClpB imm vs. NaCl, \*p < 0.05; \*\*p < 0.01 and \*\*\*p < 0.001, and ClpB imm vs. Adj \#p < 0.05. C, D, ANOVA with Tukey's post-tests, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001. F, Kruskal-Wallis with Dunn's post-test, \*p < 0.05.</figcaption></figure>

against body weight loss and mortality. Furthermore, the protective effect was abolished in ClpB-tolerized mice, highlighting a key role of immunization-induced anti-ClpB antibodies in ABA protection.

Body composition was analyzed using a nuclear magnetic resonance imager at T1 and T2, i.e., before and at the end of the ABA protocols in both experiments, and also in ad lib-fed mice in Exp #1. In the latter groups, no significant changes in either lean or fat mass were noticed (Fig. 2C, E). A decrease in lean mass was observed in all groups of mice exposed to ABA; however, such a decrease in the ClpB-imm group in Exp #2 was not significant when compared to other groups (Fig. 2C, D). A loss of fat mass was also present in the NaCl ABA group in Exp #2, while it was not significant in other groups (Fig. 2E, F). Thus, ClpB immunization in Exp #2 protected against loss of both lean and fat mass during

food restriction. Moreover, in Exp #2, body weight as well as both lean and fat mass correlated positively with plasma levels of anti-ClpB IgG (Pearson's r = 0.42, p = 0.01; r = 0.39, p = 0.02, and r = 0.59, p = 0.0003, respectively), further supporting the protective role of these antibodies against weight loss (Fig. S2C-E).

Exp #2

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

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

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

## 2.5. Feeding behavior

In Exp #1, the cumulative food intake during feeding restriction in both ABA groups tended to be lower than in the ad lib-fed mice, but it did not reach significance (Fig. 3A). Nevertheless, mean daily food intake during this period was lower in ClpB-imm ABA mice vs. ad lib controls (p < 0.01), while such a tendency (p = 0.06) was observed in
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Food intake and feeding pattern. Cumulative food intake from T1 to T2 in Exp #1 (A) and Exp #2 (C). Mean daily food intake in Exp#1 (B). Food intake (D), meal size (E), and meal number (H) during 3 h of food access in Exp #1 and 4 h of food access in Exp #2 (F, G, and J, respectively). Food intake during the 1st hour of food access in Exp #1 (I) and in Exp #2 (K). B, Student's t-test \*\*p < 0.01. C, 2-way RM ANOVA with Bonferroni post-tests, \*p < 0.05, ClpB imm vs. NaCl. I, Student's t-test; \*p < 0.05. F, G, J, K, ANOVA with Tukey's post-tests, \*p < 0.05.</figcaption></figure>

the NaCl ABA group (Fig. 3B). Such marginal differences between ad lib-fed and ABA mice were due to an adaptation to the restrictive feeding schedule by ABA animals, which consumed about 3 times more food during 3 h of food access than ad lib-fed mice ate at the same time (Fig. 3D). Accordingly, during this 3 h-period, larger meals were consumed by both groups of ABA mice, in approximately 2 meals (Fig. 3E, H). However, the amount of food consumed during the 1st hour of food provision was lower in the ClpB-immunized group (Fig. 3I).

In contrast to Exp #1, in Exp #2 the cumulative food intake was higher in the ClpB-imm group (Fig. 3C). Moreover, increased food intake was observed in the same group during the 4 h-restriction period (Fig. 3F). This increase was mainly due to increased meal number since the meal size did not change (Fig. 3G, J). Nevertheless, a tendency of higher food intake during the 1st hour of food provision, corresponding to the 1st meal, was evident in the ClpB-imm group (Fig. 3K). Food intake and feeding patterns in ClpB-tolerized mice did not significantly

differ from the NaCl control group, supporting a key role of anti-ClpB antibody production in increased food intake (Fig. 3F, G, J, K). Daily water intake was not significantly different between ABA groups, but it was lower in ClpB-immunized mice under ad lib conditions (Fig. S2F, G).

## 2.6. Physical activity

Food restriction with regularly scheduled food access in rodents is characterized by the phenomenon of feeding anticipatory activity (FAA), e.g., by increased wheel running during 3 h before food provision. Such an increase in physical activity is considered a maladaptation to food restriction, causing increased energy loss during energy deficit, a typical feature of AN (Schaumberg et al., 2024). As expected, FAA was increased in both groups of ABA mice in Exp #1 as compared to the ad lib-fed groups (Fig. 4A, B). While ClpB immunization did not change FAA in ad lib mice, which was expectantly low, ClpB-imm ABA mice
displayed an earlier increase in FAA, starting during the first days of restriction and resulting in higher mean FAA levels during the 3 h food access period (Fig. 4A, B). In contrast, in Exp #2, the ClpB-imm group showed a tendency of lower FAA from the beginning of food restriction, resulting in a significantly lower FAA throughout the 4 h food access period (Fig. 4C, D). Notably, such a decrease in FAA was not present in ClpB-immunized mice that were orally tolerized by ClpB, highlighting a role of anti-ClpB antibodies in FFA changes (Fig. 4D). Concerning the daily physical activity, no significant effects of ClpB immunization on the total running distance were found in either experiment (Fig. 4E, F, G, H), suggesting that changes in food intake, but not physical activity, were the main factor impacting body weight and composition in ClpB-immunized ABA mice. In the Ad lib groups, ClpB-immunized mice showed a tendency for increased total daily physical activity (Fig. 4E).

## 2.7. Melanocortin receptor type 4 (MC4R) activation

In Exp #2, we tested whether ClpB immunization-induced IgG may modulate MC4R activation *in vitro* by ClpB and α-MSH as compared to IgG from non-immunized controls by using the β-arrestin recruitment TANGO assay in MC4R expressing cells (Fig. 5A). Before this experiment, using the same assay, we found that the full 96 kDa *E. coli* ClpB protein activated human MC4R with a potency similar to α-MSH (Fig. 5B, C). However, the α-MSH-like 14-amino acids fragment of *E. coli* ClpB (535–548) induced only a weak MC4R activation (Fig. 5B, C), with EC₅₀ significantly lower than that of either α-MSH or full ClpB protein

(Kruskal-Wallis test p = 0.0004, Dunn's post-tests, p < 0.001).

Next, ClpB and α-MSH preincubated at their EC₅₀ concentrations with plasma-extracted total IgG of mice from Exp #2 were applied to MC4R-expressing cells. We found that IgG from ClpB-immunized mice inhibited MC4R activation by both α-MSH and ClpB in comparison to IgG from non-immunized controls and ClpB-imm/tol groups (Fig. 5D, E).

## 2.8. Feeding regulatory neuropeptide mRNA expression

Hypothalamic expression of feeding regulatory peptide mRNA was analyzed in postmortem brain tissue. In Exp #1, mRNA levels of orexi- genic neuropeptide Y (NPY) were increased in the ABA mice as compared to ad lib-fed mice (Fig. 5F). In contrast, in Exp #2, a tendency for lower NPY mRNA levels was present in the ClpB-imm group (Fig. 5H). Increased mRNA levels of melanin-concentrating hormone (MCH), another orexigenic neuropeptide, were found in ClpB-imm ABA mice as compared to non-immunized controls in Exp #1, but in Exp #2, MCH mRNA changes were not significant (Fig. 5G, I). We did not find group differences in mRNA levels of proopiomelanocortin (POMC), the α-MSH precursor, in either experiment (Fig. 5J, L). However, mRNA levels of oxytocin, another anorexigenic neuropeptide, were lower in the ClpB-imm group in Exp #2, while they tended to be higher in ABA mice in Exp #1 (Fig. 5K, M). No significant group differences were observed for mRNA levels of some other feeding regulatory neuropeptides (Fig. S3).

<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Physical activity. Feeding anticipatory activity (FAA) in the running wheel during 3 h before food provision in the Exp #1 (A, B) and Exp #2 (C, D), shown as a daily dynamic of the running distance (A, C) and as its mean levels (B, D). Total daily running distance dynamics (E, G) and its daily means (F, H). A, Mann-Whitney test, \*p < 0.05. B, D, Student's t-test, \*p < 0.05.</figcaption></figure>
A
TANGO MC4R activation assay

<figure><img src="image_12.png" /></figure>

C

<table><thead><tr><td>Peptide</td><td>EC<sub>50</sub> (M)</td></tr></thead><tbody><tr><td>α-MSH</td><td>6,687.10<sup>-7</sup></td></tr><tr><td><i>E. coli</i> ClpB 96 kDa</td><td>5,071.10<sup>-7</sup></td></tr><tr><td><i>E. coli</i> ClpB (535-548)</td><td>0,3762</td></tr></tbody></table>

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

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

<figure><img src="image_15.png" /><figcaption>**Fig. 5.** Melanocortin receptor activation and hypothalamic neuropeptide mRNA. Schematic illustration of the TANGO MC4R activation assay used in the study (A). MC4R activation by α-MSH, *E. coli* ClpB full 96 kDa protein, and ClpB (535–548) peptide fragment (B). Half maximal effective concentration (EC₅₀) of MC4R activation by each tested ligand in B (C). Effects of plasma-extracted IgG on MC4R activation by α-MSH (D) or ClpB (E). Hypothalamic expression of mRNA for orexigenic NPY (F, H) and MCH (G, I) and anorexigenic POMC (J, L) and oxytocin (K, M). RLU, relative luminescence units. D, E, H, M, ANOVA with Tukey's post-tests, \*p < 0.05, \*\*\*p < 0.0001, F, Student's t-test \*\*\*p < 0.0001.</figcaption></figure>

2.9. Gut microbiota composition

To test whether the ClpB immunization-induced behavioral changes are associated with changes in gut microbiota composition, we analyzed

the fecal microbiome at T0 and T2 for both experiments. Overall, the sequencing of 121 samples resulted in 5,779,965 (47,768.31 ± 17,246.00) reads (Table S1; raw sequencing data are available at the Sequence Read Archive (SRA) website of NCBI, project number:
PRJNA122315). The Amplicon Sequence Variant (ASV) table summarizing the microbial composition across all samples is reported in Table S2.

About α-diversity, in Exp #1, we observed no significant changes in the Chao 1 index (a measure of total richness) when comparing all

groups to the baseline samples (Fig. 6A, *p* >0.05). However, exposure of mice to ABA significantly reduced community evenness as measured by the Shannon index (Fig. 6C). Similarly, in Exp #2, we observed no change in the Chao 1 index (Fig. 6B), but a reduction in the Shannon index, which is significant in Adj and ClpB-imm groups (Fig. 6D) and

<figure><img src="image_16.png" /><figcaption>**Fig. 6.** Fecal microbiota diversity. As a measure of α-diversity, the Chao1 (A, B) and Shannon (C, D) indices illustrate the richness and evenness of microbial communities, respectively. As a measure of β-diversity, Principal Coordinate Analysis (PCoA) based on the Bray-Curtis dissimilarity index (E, F) shows the clustering of microbial community compositions between baseline and post-treatment groups, with ellipses representing 95 % confidence intervals. Each symbol represents an individual sample. Significant differences between T0 are indicated by asterisks (Linear model; \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001).</figcaption></figure>
shows a trend in ClpB-imm/tol group ($p = 0.07$).

Analysis of β-diversity, based on the Bray-Curtis dissimilarity index, indicates that both time (characterized by changes from co- to single-housing) as well as ABA have a significant effect in Exp #1 (Fig. 6E-F). More specifically, time accounts for 22.7 % of the microbiome variation across the samples, while ABA about 9 % of this variation. Comparable results were obtained in Exp #2, with a large effect of time on β-diversity (explaining 30.8 % of microbiome variability) and a smaller effect of ABA (5.7 % of variation). Further, higher similarity between NaCl ABA and ClpB-imm ABA animals, as well as a significantly higher dissimilarity to ClpB-imm ABA mice, is observed in comparison to NaCl ad lib mice in Exp #1. In contrast, in Exp #2, the NaCl and Adj groups were significantly more distinct from ClpB-immunized and ClpB-imm/tol groups, which shared a more similar microbiome (Fig. S4).

In Exp #1, a comparison of core species and genera revealed 106 ASVs to significantly differ in abundance ($p < 0.05$) compared to the NaCl ad lib group, but only a few remained after p-value adjustment for multiple comparisons (Fig. S5A). At the genus level, the ClpB-imm ABA group had more abundant unclassified (unc.) Clostridia, unc. Prevotellaceae, Candidatus Saccharimonas and Alloprevotella, and less abundant Ligilactobacillus (Fig. S5B).

In Exp #2, only 11 core ASVs were associated with at least one of the tested treatments compared to the NaCl ABA group, but only one of these remains significant after p-value correction, unc. Lachnospiraceae in ClpB-immunized mice (Fig. S5C). Since ClpB is produced by members of the Enterobacteriaceae family, we looked if it was affected by immunization and ABA. Overall, only 5 ASVs were annotated as belonging to the Enterobacteriaceae family in both experiments, but they were not sufficiently frequent and/or abundant for statistical comparison between groups.

## 2.10. Fecal clpB DNA content

ClpB protein in plasma originates from the ClpB-producing gut Enterobacteria, as illustrated by electron microscopy of an E. coli bacterial cell (Fig. 7A and Fig. S6). Thus, to determine the effects of ClpB immunization on bacterial clpB gene abundance, fecal clpB DNA content was compared between the groups at the end of the experiment. In Exp #1, ClpB immunization was characterized by a relative increase in clpB DNA abundance, which was particularly prominent in ABA mice (Fig. 7B). In contrast, in Exp #2, ClpB-immunized mice had lower clpB DNA levels than NaCl controls and ClpB-imm/tol mice (Fig. 7C).

## 2.11. Fecal energy content

Fecal energy content may contribute to host energy metabolism, and it was analyzed at the T1 and T2 time-points, i.e., before and at the end of ABA, and also in ad lib-fed mice. In Exp #1, higher energy was present in ClpB-immunized vs. non-immunized combined groups of mice at T1, i.e., during ad libitum food intake in all mice (Fig. 7D). This difference was mainly due to a surprisingly low energy content in some animals from the NaCl control group, which was not seen at the later T2 period (Fig. 7E). Moreover, an inverse relation was found between the ad libitum-fed groups 12 days later (Fig. 7E). Exposure of mice to the ABA protocol in Exp #1 reduced fecal energy, without a significant effect of ClpB immunization (Fig. 7E). In Exp #2, no significant group differences in fecal energy content were found either before or after the exposure to ABA (Fig. 7F, G).

In Exp #1, fecal energy content correlated with the abundances of several bacterial taxa. The most frequent findings are positive associations with ASVs annotated as unc. Muribaculaceae and unc. Alistipes as well as negative associations with unc. Lachnospiraceae and unc. Roseburia (Fig. 7H). Positive correlations are also present at the genus level with Parassuterella and unc. Lactobacillaceae, and with Erysipelatoclostridiaceae and Sutterellaceae at the family level (Table S3). No significant associations are observed in Exp #2.

## 3. Discussion

The results of our study support the view of the Enterobacterial ClpB protein as a key anorexigenic signal from gut microbiota underlying the mechanisms of host anorexia via modulation of feeding and locomotor behavior. Importantly, we demonstrate that ClpB immunoneutralization efficiently protects mice from ABA, owing to immunization-induced anti-ClpB IgG antibodies that lower plasma ClpB and inhibit ClpB- and α-MSH- activation of MC4R. We show that the binding properties of anti-ClpB antibodies underlie the modulation of ClpB- and α-MSH-anorexigenic signaling leading to its enhancement or inhibition. This modulatory effect of IgG supports, on one hand, a pathophysiological model of AN, in which immune complexes of IgG with α-MSH contribute to an increased anorexigenic drive. On the other hand, it suggests a potential avenue for etiological therapeutic intervention through ClpB neutralization (Fetissov and Hökfelt, 2019). Indeed, in Exp #1, ClpB immunization produced a high titer of anti-ClpB IgG with increased affinity, leading to increased plasma ClpB and α-MSH and increased clpB DNA in feces. These changes lead to aggravation of ABA by decreasing food intake and increasing FAA, resulting in acceleration of body weight loss. Notably, IgG with increased affinity for ClpB and α-MSH were also observed in patients with AN (Seitz et al., 2024; Thomas et al., 2025), further supporting the pathophysiological role of such IgG in AN.

In contrast, after immunization with a lower dose of ClpB in Exp #2, a production of anti-ClpB IgG characterized by a lower affinity for ClpB and α-MSH prevented ClpB increase in plasma and lowered clpB DNA in feces. Such changes were accompanied by protection against ABA by increasing food intake and decreasing FAA, leading to the better preservation of lean mass and body weight. Table 1 summarizes the differences of the ClpB immunization-induced effects, highlighting opposite directions of behavioral and biochemical changes between Exp #1 and Exp #2.

To our knowledge, these results represent one of the most effective preventive treatments of ABA in rodents, comparable to a recently demonstrated pharmacological stimulation of NPY/agouti-related protein (AgRP) neurons (Lv et al., 2023). We can tentatively explain the opposite *in vivo* effects between immunizations of different doses by targeting only the main immunogenic epitopes of ClpB by immunization with its lower dose and multiple epitopes with its higher dose. Since ClpB contains an α-MSH-like epitope, it is possible that in mice immunized with a higher dose, IgG can bind to other than α-MSH epitopes, forming IC with α-MSH-like epitope available for molecular interaction with MC4R underlying its anorexigenic action. On the contrary, an antibody stimulated by a lower dose block α-MSH-like epitope, protecting against MC4R activation, which was indeed confirmed in our *in vitro* study. Different isotypes of IgG may also contribute to different physiological responses to immunization.

A key role of immunization-induced anti-ClpB IgG in behavioral and molecular changes during ABA was confirmed in mice that were orally tolerized against ClpB before immunization. Tolerization diminished anti-ClpB IgG production and, hence, abolished immunization-induced protection against ABA. Oral tolerization with an antigen is an established approach to reduce anti-antigen antibody production following immunization with the same antigen (Hardet et al., 2016).

Regarding the molecular mechanisms of ABA protection by anti-ClpB IgG, we show that IgG from ClpB-immunized mice inhibits ClpB activation of MC4R *in vitro*. Importantly, ClpB-blocking IgG also inhibits α-MSH activation of MC4R, indicating that such IgG binds to the α-MSH-like fragment of ClpB, and hence, may interfere with both gut bacteri-derived ClpB and with the endogenous α-MSH. Considering relatively low levels of IgG in the brain, it is likely that the tissue targets of anti-ClpB IgG are mainly located outside the blood-brain barrier. Such targets, relevant to AN, may include enteroendocrine L-cells expressing MC4R whose activation by α-MSH-like ligands stimulates secretion of satietogenic PYY (Panaro et al., 2014). Of relevance, both fasting and post-prandial elevated PYY plasma levels are consistent findings in
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(caption on next page)
**Fig. 7.** Fecal *clpB* DNA and energy content. Electron microscopy of ClpB molecules (black dots) inside an *E. coli* bacterial cell (A) with an insert a' showing ClpB labeling by gold particles (arrows). Relative abundance of *clpB* DNA in feces collected at the end of the experiment (B, C). Fecal energy content at T1 (D, F) and T2 (E, G). Bubble plot reporting associations (Spearman's correlations) between fecal energy content and individual ASVs in all groups combined from Exp #1 (H). Mean abundance is indicated by circle size B, D, E, Student's t-test, ***p < 0.001, ****p < 0.0001. C, ANOVA, with Tukey's post-test, ***p < 0.001, ****p < 0.0001. H, B-H-adjusted p-values, *p < 0.05.

patients with AN (Muhammed et al., 2025). Vagal afferents in the gut expressing MC4R may serve as another peripheral target for IgG to modulate intestinal satiety signaling (Gautron et al., 2010). The presence of IgG in gut mucosa was documented, where it plays a protective role interacting with gut commensals (Chen et al., 2020). The relevance of these tissue targets should be tested in future studies, e.g., to validate the therapeutic potential of monoclonal ClpB antibodies.

Thus, ClpB activation of MC4R validates ClpB as a gut bacteri-
derived melanocortin-like signal that can participate in the host regu-
lation of appetite and body weight, promoting negative energy balance.
Dose-dependent activation of MC4R by ClpB may also underline
different behavioral responses between the two experiments, with
higher ClpB plasma levels contributing to negative energy balance.
Future studies should better define the pharmacological profile of ClpB
as a ligand of MC receptors. This finding is further corroborated by the
data showing that ClpB concentrations in the blood of healthy subjects
correlate negatively with BMI-SDS (Thomas et al., 2025). We also show
here that an α-MSH-like fragment of ClpB (535–548) was only weakly
active on MC4R, indicating that for higher α-MSH-like activity, a larger
ClpB fragment is needed that preserves the α-MSH-like conformational
homology of the full 96 kDa ClpB. These results agree with the previous
data showing low affinity of the E.coli ClpB (535–548) fragment binding
to MC receptors (Ericson et al., 2015).

Neuropeptide Y (NPY) is a key orexigenic signal in the brain, while the arcuate NPY/AgRP neurons coordinate nutritional status with several physiological functions, including physical activity (Miletta et al., 2020; Qi et al., 2023; Sutton Hickey et al., 2023; Zhang et al., 2019). Expectedly, in response to starvation in Exp #1, hypothalamic NPY mRNA levels were elevated in ABA mice. In contrast, in Exp #2, lower NPY mRNA in ClpB-immunized mice should reflect a weaker metabolic drive to stimulate NPY expression because of a mild loss of body weight. Moreover, low mRNA levels of anorexigenic oxytocin should contribute to increased food intake in these mice (Lawson, 2017). Oxytocin-producing neurons in the paraventricular nucleus are a downstream target of NPY/AgRP neurons in the neural circuit for hunger (Atasoy et al., 2012). Although POMC mRNA levels were not affected in the present study, it cannot be ruled out that anti-ClpB IgG could also diminish ClpB electrical activation of these anorexigenic neurons (Breton et al., 2016).

Our study suggests that immunoneutralization of Enterobacterial
ClpB can be an etiologic treatment for AN. ClpB is a disaggregation
chaperon protein induced in bacteria by various stressors (Mogk et al.,
2003a). Here we demonstrate the moonlighting properties of ClpB by
the activation of mammalian MC4R associated with host anorexigenic
response. Such a response may be advantageous in some acute situa-
tions, e.g., during intestinal inflammation accompanied by Enterobac-
teriaceae overgrowth (Moreira de Gouveia et al., 2024). However, in the
long term, an increase of ClpB-producing bacteria and/or deficient im-
mune control of ClpB may facilitate food-restrictive behavior contrib-
uting to AN pathophysiology (Thomas et al., 2025). Some, but not all,
cross-sectional studies of the gut microbiota composition in AN indeed
reported a relative increase of Enterobacteriaceae (Borgo et al., 2017;
Hanachi et al., 2019; Million et al., 2013). Importantly, a longitudinal
study in patients with AN revealed that an increase in Enterobacteriaceae
abundance was associated with a worse evolution of the disease
(Andreani et al., 2024). Moreover, in vitro modelling of a dietary intake
in AN results in an increase of Enterobacteriaceae in the gut microbial
community (Liu et al., 2024).

Targeting specific proteins produced by common gut bacteria may,

thus, represent a new biotherapeutic approach in neuropsychiatric dis-
orders, which fits with an emerging concept of the gut microbiota-brain
axis in health and disease (O'Riordan et al., 2025; Schneider et al.,
2024). Here, we report that ClpB immunization does not significantly
alter gut microbiota diversity, but modulates the abundance of *clpB* DNA
in feces. The exact mechanism underlying this modulation is currently
unknown, but it points to the ability of the immune system to selectively
regulate gut bacterial gene abundance. A role of antibody-mediated
intestinal complement and IgA in targeting selective bacteria cannot
be excluded (Rollenske et al., 2021; Wu et al., 2024). The limited dif-
ferential abundance of specific bacterial taxa observed in the gut
microbiota of ClpB-immunized mice, whether associated with ABA
aggravation or protection, does not allow for attributing these changes
to the direct effects of ClpB immunization. Moreover, since these
changes did not involve ClpB-producing *Enterobacteriaceae*, they may
reflect alterations in nutritional status. However, since only a few ASVs
of enterobacteria were detected, it is possible that our approach was not
sensitive enough. Nevertheless, the low abundance of enterobacteria in
mice and also in humans does not apparently prevent the well-
detectable levels of the enterobacterial ClpB protein in the blood.
While changes of the relative abundance of such bacteria should influ-
ence ClpB levels in the long-term, some other mechanisms, including the
immune control, can be important regulators of ClpB effects on the host.
Of interest, the *clpB* gene function in humans is inversely related to their
body mass index, and patients with obesity have low *clpB* gene count in
their fecal microbiota (Arnoriaga-Rodríguez et al., 2020; Legrand et al.,
2020).

The present microbiome results do not contradict the overall patterns
in studies of patients with AN (Zhao et al., 2024). The changes of
microbiota composition in ABA mice confirm the impact of starvation
and malnutrition on gut microbiome (Blanton et al., 2016). Gut micro-
biota depletion studies in mice exposed to ABA reported altered hypo-
thalamic feeding-regulatory neuropeptide expression, supporting the
role of gut bacteria in the homeostatic regulation of body weight
(Roubalová et al., 2024). In our study, we further confirm this idea by
showing a link between fecal energy content and the state of starvation.
Indeed, while low fecal energy was associated with obesity and inter-
preted as facilitating energy harvest by the host (Turnbaugh et al.,
2006); in the condition of ABA, a decrease in fecal energy may represent
an adaptive response for host survival. Indeed, an increase of fecal en-
ergy content was observed after refeeding in patients with AN (Reed
et al., 2025). Although we did not find any effects of ClpB immunization
on fecal energy content in ABA mice, i.e., it was not involved in the ABA
protection, we could observe some variation in *ad lib*-fed mice. ClpB-
immunized ad lib mice had higher fecal energy, which can be tenta-
tively associated with their higher physical activity, i.e., fitness-
promoting. Nevertheless, the overall functional significance of fecal
energy content for the body's energy balance regulation remains pres-
ently unclear and needs further research. Some correlations between
bacterial taxon abundances and fecal energy content found in our study
are noteworthy. For instance, *Alistipes*, which correlates positively with
fecal energy, was previously associated with a negative long-term
prognosis in patients with AN (Andreani et al., 2024). In contrast,
*Roseburia* and *Lachnospiraceae* negatively associate with fecal energy.
The relative abundance of these taxa is typically low in AN (Scala et al.,
2025), possibly contributing to deficient energy harvest by the host, e.g.,
in the form of adenosine triphosphate (Breton et al., 2019).
<caption>**Table 1**</caption>

Summary of changes of the main behavioral and biochemical parameters in ClpB-immunized mice exposed to ABA in Exp #1 and Exp #2. FAA, feeding anticipatory activity. Both statistically significant and non-significant changes are shown.

<table><thead><tr><th>ClpB-immunized mice – ABA</th><th>Exp #1 (ClpB imm. 50 µg)</th><th>Exp #2 (ClpB imm. 25 µg)</th></tr></thead><tbody><tr><td>Body weight</td><td>~20 % decrease</td><td>~10 % decrease</td></tr><tr><td>Lean mass</td><td>~21 % decrease</td><td>~7.5 % decrease</td></tr><tr><td>Fat mass</td><td>~34 % decrease</td><td>~21 % decrease</td></tr><tr><td>Food intake</td><td>~12 % decrease</td><td>~42 % increase</td></tr><tr><td>FAA</td><td>~X4 times increase</td><td>~X4 times decrease</td></tr><tr><td>Plasma ClpB</td><td>~X2.7 times increase</td><td>~X1.5 times increase</td></tr><tr><td>Plasma α-MSH</td><td>~X1.4 times increase</td><td>~X1.6 times decrease</td></tr><tr><td>Plasma ClpB IgG</td><td>~X40 times increase</td><td>~X2.5 times increase</td></tr><tr><td>Plasma α-MSH IgG</td><td>~X5 times increase</td><td>~X1.5 times increase</td></tr><tr><td>ClpB IgG affinity</td><td>~X3.6 times increase</td><td>~X5.6 times decrease</td></tr><tr><td>α-MSH IgG affinity</td><td>~X2.5 times increase</td><td>~X10.9 times decrease</td></tr><tr><td>Fecal clpB DNA</td><td>~X1.4 times increase</td><td>~X1.2 times decrease</td></tr></tbody></table>

## 4. Study limitations

The study was performed in male mice, which are more susceptible to ABA (Achamrah et al., 2017), while in human AN, females are more frequently affected. Thus, although ClpB immunoneutralization provides a proof of concept for ABA protection, the possibility of sex-different responses to ClpB immunization cannot be excluded. In fact, sex differences of gut microbiota composition and their role in immune signaling and gut-brain communication are well-documented (Elderman et al., 2018; Shobeiri et al., 2022). Although food restriction was shown to increase clpB DNA and ClpB protein in both sexes, female rats had higher ClpB concentrations in the ileum and colon but lower in the upper gut vs. male rats (Breton et al., 2020). Another limitation of our study is the low detection of Enterobacteriaceae in the mouse gut microbiota. Given that ClpB-producing bacteria belong to this family, the under-representation of their ASVs has limited our ability to fully assess the impact of ClpB immunization on ClpB producers. Future studies of the microbiome in both animal models and humans using deeper metagenomic profiling (e.g., shotgun) may help to better clarify the dynamics of ClpB-producing species of gut bacteria. Keeping animals in individual cages appears as a stressful factor affecting gut microbiota composition, limiting the detection of ClpB immunization effect; studies of grouped animals may be needed.

## 5. Conclusion and perspectives

The study provided experimental evidence of the key role of enterobacterial ClpB in the development of host anorexia. This role is mediated by ClpB's ability to activate MC4R and by the immune system producing anti-ClpB antibodies, which, depending on their binding properties, modulate MC4R activation by ClpB and α-MSH and regulate feeding behavior. As such, insufficient control of ClpB by antibodies leads to aggravation of ABA. On the contrary, production of IgG leading to ClpB immunoneutralization efficiently protects against the development of ABA. The latter results suggest a potential clinical utility of ClpB immunoneutralization in patients with AN. For instance, in vitro and in vivo preclinical studies in both male and female animals can be designed to validate the utility of a monoclonal ClpB antibody as a putative bio-therapy of AN, taking into account possible toxicity risks of inhibiting the endogenous α-MSH. In fact, from one side, such antibodies should be efficient to block the anorexigenic and lipolytic effects of α-MSH, but from another side, should diminish other physiological α-MSH effects, which should be preserved, e.g., anti-inflammatory and melanocyte-stimulating. In relation to such effects, further studies should also verify the effects of ClpB and anti-ClpB antibodies on MC5R and MC1R.

# 6. Materials and methods

## 6.1. Animals, ClpB immunization, and ABA protocols

Animal care and experimentation complied with both French and European Community regulations (European Community Council Directive of November 24, 1986 (86:609) and was approved by the Normandy Regional Ethics Committee (n. 8690). C57BI/6J 7-week-old male mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). They were housed under an inverted 12-h light–dark cycle (dark phase: 10:00 AM – 10:00 PM) at 23 °C in individually ventilated standard holding cages for mice (Tecniplast, Buguggiate, Italy), 4–5 animals per cage with ad libitum access to a pelleted standard diet (Envigo, Paris, France) and drinking water. For the schematic illustration of experimental design, see Fig. 1A, B.

In Exp #1 (Fig. 1A), after 10 days of acclimation to housing conditions, blood samples were taken by tail puncture just before immunization, and 1–2 fecal pellets were collected from each mouse to analyze microbiota composition (T0). All mice were randomized into 2 groups: ClpB-immunized and NaCl control (n = 18, in each) according to their body weight (NaCl, 25.8 ± 1.16 g, ClpB-imm, 25.4 ± 1.65 g). Then, the ClpB-immunized group received an intraperitoneal (IP) injection of 50 µg of recombinant Escherichia coli (E. coli) ClpB protein custom produced in E. coli by SinoBiological (Beijing, P.R. China), diluted in 100 µl of sterile 0.9 % NaCl solution together with 100 µL of complete Freund's adjuvant (CFA) from Sigma Aldrich (L'Isle-d'Abeau Chesnes, Saint-Quentin-Fallavier, France). Before use, the recombinant ClpB protein quality was verified and confirmed by mass spectrometry in the PIS-SARO proteomic platform (Rouen University, France). The control group received IP 200 µL of 0.9 % NaCl only. After injection, mice were returned to their holding cages. 2 weeks after the initial immunization, the ClpB-immunized group received a boost with 50 µg of ClpB protein in 100 µl of 0.9 % NaCl and with 100 µl of incomplete Freund's adjuvant (IFA, Sigma Aldrich), while the control group received 200 µL of 0.9 % NaCl only, both by IP injection. 1 week after the boost, one mouse with the lowest body weight from each holding cage was eliminated from the experiment. This was done to avoid the influence of a non-specific proinflammatory effect of immunization on body weight, considering that mice with the lowest body weight are less resistant to ABA. Each group was further divided into 2 subgroups exposed to the ABA protocol or fed ad libitum (n = 8 in each), according to their body weight, with similar mean values among the groups (NaCl ad lib, 25.53 ± 1.16 g, ClpB-imm ad lib, 25.59 ± 1.14 g, NaCl ABA, 26.06 ± 1.34 g, and ClpB-imm ABA, 26.49 ± 2.20 g). Then, mice were placed individually into the BioDaq food and drink intake monitor cages equipped with a gate to the feeder (Research Diets, Inc., New Brunswick, NJ, USA) and a freely accessible activity wheel (TSE Systems, Bad Homburg, Germany). Food intake, meal patterns, and wheel activity were continuously recorded. The meal was defined as a non-feeding activity for at least 300 sec. Daily water intake was measured manually by weighing water bottles.

Mice were allowed to acclimate themselves to the BioDaq cages for 5 days with ad libitum access to food and water, and body composition was measured using a nuclear magnetic resonance instrument MiniSpec LF110 (Bruker, Woissembourg, France). During this procedure, feces were collected from each cage for the analysis of energy content (T1) as described below in detail. Then, two groups of mice, ClpB-immunized and NaCl controls, were exposed to an ABA protocol, while two other groups continued ad libitum access to food and a running wheel. The ABA protocol consisted of a gradual decrease of time per 24 h when food was accessible: 6 h-D0, 5 h-D1, 4 h-D2, and 3 h- starting from D3 until the end of the experiment (Fig. 3C). Access to food was given at 10:00 AM, i. e., at the onset of the inverted dark phase. At D11 (T2), a second analysis of body composition was performed using MiniSpec LF110 in all mice, and feces from each cage were collected and frozen for energy content measurement. At the end of the experiment, 1–2 feces from each mouse were collected for the analysis of microbiota composition, and mice
were killed by decapitation, trunk blood was collected and stored on ice before plasma separation by centrifugation (2500 rpm, 20 min at 4 °C). Brains were taken and the hypothalami dissected. All tissue samples were stored on dry ice before placement in a -80 °C freezer until further assay.

In Exp #2 (Fig. 1B), after 8 days of acclimation to housing conditions, blood samples from all mice were taken by tail puncture, and 1-2 fecal pellets were collected from each mouse to analyze microbiota composition (T0). Mice were randomized into 4 groups (n = 8 in each group): 0.9 % NaCl controls (NaCl), adjuvant (Adj), ClpB-immunized (ClpB imm), and ClpB-immunized after tolerization (ClpB imm/tol) according to their body weight (NaCl, 22.08 ± 0.39 g, Adj, 22.12 ± 0.58 g, ClpB-imm, 22.2 ± 0.78 g, and ClpB-imm/tol, 22.48 ± 0.83 g). The tolerized group received 25 µg/ml of recombinant *Escherichia coli* (*E. coli*) ClpB protein in drinking tap water for 10 days. After 10 days, two groups, including the one with ClpB oral tolerization, were IP immunized with 25 µg of recombinant ClpB protein diluted in 50 µL of sterile 0.9 % NaCl solution together with 50 µL of CFA. The non-immunized control group received IP 100 µL of 0.9 % NaCl only. The adjuvant group received IP 50 µL of 0.9 % NaCl together with 50 µL of CFA. After injection, mice were returned to their holding cages. 2 weeks after the initial immunization, the ClpB-immunized group received a boost with 25 µg of ClpB protein in 50 µL of 0.9 % NaCl and with 50 µL of IFA IP, while the control group received 100 µL of 0.9 % NaCl only, and the adjuvant group received 50 µL of IFA in 50 µL of 0.9 % NaCl. 1 week after the boost, mice were placed individually into the BioDaq food and drink intake monitor cages equipped with a gate to the feeder and a freely accessible activity wheel. Food intake, meal patterns, and wheel activity were continuously recorded. The meal was defined as a non-feeding activity for at least 300 sec. Daily water intake was measured manually by weighing water bottles. Mice were allowed to acclimate themselves to the BioDaq cages for 5 days with *ad libitum* access to food and water. Then, body composition was measured in all mice using MiniSpec LF110. During this procedure, feces were collected from each cage for the analysis of energy content (T1) as described below in detail. Starting from the next day, all mice were exposed to an ABA protocol. The ABA protocol consisted of a gradual decrease of time per 24 h when food was accessible: 6 h-D1, 5 h-D2, and 4 h from D3 until the end of the experiment (Fig. 3C). In this experiment, duration of a minimal food access period was limited to 4 h to increase the time window for modulation of food intake by the treatments. The ABA protocol was stopped after 11 days due to mortality and low body weight in some mice in the NaCl control group. Access to food was given at 10:00 AM, i.e., at the onset of the inverted dark phase. At D10 (T2), a second analysis of body composition was performed using MiniSpec LF110 in all mice, and feces from individual cages were collected and frozen for energy content measurement. On the last day of the experiment, 1-2 feces from each mouse were collected for the analysis of microbiota composition, and mice were killed by decapitation. Trunk blood was collected and stored on ice before plasma separation by centrifugation (2500 rpm, 20 min at 4 °C). Brains were taken and the hypothalami dissected. All tissue samples were kept on dry ice before storage in a -80 °C freezer until assay.

## 6.2. ClpB assay

For the analysis of plasma ClpB concentration, we have developed an enzyme-linked immunosorbent assay (ELISA). Anti-ClpB polyclonal rabbit IgG (custom produced by SinoBiological) was used for ClpB protein capture and was coated on 96-well ELISA plates (96-well Maxisorp plates, Nunc, Thermo Fisher Scientific), 100 µL per well, diluted at 0.1 µg/mL in coating buffer (0.5 M Na₂CO₃ and 0.5 M NaHCO₃ buffer, pH 9.6) overnight at 4 °C. Mouse plasma samples were diluted to 1:10 and added to the ELISA plate for 2 h at room temperature (RT) under mild agitation. After washing x3 in 0.05 M phosphate-buffered saline (PBS), mouse monoclonal anti-ClpB IgG (custom produced by SinoBiological) was added as a detection antibody at 1/2000e in PBS for 90 min

at RT with mild agitation. After washes x3 in 0.05 M PBS, anti-mouse IgG conjugated with alkaline phosphatase as a revelation antibody was added at 1/2000 in PBS for 90 min at RT with mild agitation. After washing x3 in 0.05 M PBS, *para*-nitrophenylphosphate (pNPP) was added at 1 mg/mL in 200 µL of revelation buffer (glycine 0.1 M; ZnCl₂ 1 mM; MgCl₂-6H₂O 1 mM, pH 10.4) for 30 min and protected from light. The reaction was stopped by adding 3 N NaOH solution, and the chromogenic reaction was read at 405 nm by the Infinite F200PRO spectrophotometer (Tecan, Männedorf, Switzerland). All samples were measured in duplicates, and their mean values were compared to the standard dilutions of the recombinant *E. coli* ClpB protein (custom-produced by SinoBiological). The development of ClpB ELISA, including specificity assays and showing a detection threshold at the picomolar range, was recently described in detail (Thomas et al., 2025).

## 6.3. α-MSH assay

α-MSH concentrations were assayed in mouse plasma using an α-MSH enzymatic immunoassay (EIA) kit (EK-043-01) according to the manufacturer's instructions (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). In brief, serum samples were acidified and peptides extracted using a C-18 column and then diluted in the "assay buffer" provided in the kit. All assays were performed in duplicates.

## 6.4. Anti-ClpB- and α-MSH-reactive antibody assay

Plasma levels of anti-ClpB and α-MSH-reactive IgG were measured using ELISA according to a published protocol (Fetissov, 2011). Briefly, α-MSH peptide (Bachem, Bubendorf, Switzerland) or recombinant ClpB (Sino Biologicals) was coated onto 96-well Maxisorp plates (Nunc, Rochester, NY, USA) using 100 µL and a concentration of 2 µg/ml in 0.5 M Na₂CO₃ and 0.5 M NaHCO₃ buffer, pH 9.6, for 48 h at 4 °C. The plates were washed (3x) in PBS with 0.05 % Tween 200, pH 7.4, and then incubated for 3 h at 37 °C with 100 µL of mouse plasma diluted at 1:400 in PBS, pH 7.4. The plates were washed (3x) and incubated with 100 µL of alkaline phosphatase-conjugated antibodies in PBS (1:2000, Sigma). Following washing (3x), 100 µL of p-NPP solution (Sigma) was added as a substrate. After 30 min of incubation at RT, the reaction was stopped by adding 3 N NaOH. The optical density (OD) was determined at 405 nm using a microplate reader, Infinite F200PRO. Blank OD values resulting from the reading of plates without the addition of plasma samples were subtracted from the sample OD values. Each determination was done in duplicate. The variation between duplicate values was lower than 5 %.

## 6.5. Affinity kinetics of anti-ClpB IgG

Total IgG was extracted from mouse plasma using MelonGel® Purification Kit (LifeTechnologies, Carlsbad, CA, USA). Briefly, 200 µL of plasma (diluted 1:4 in purification buffer) was incubated in the mini-spin columns containing 500 µL of MelonGel® purification support for 5 min at RT. To collect purified IgG, columns were centrifuged for 30 s at 5000 rpm and the collected samples were lyophilized (Bioblock Scientific, Illkirch, France) for 48 h, resuspended in HBS-EP + buffer (Cytiva, Marlborough, MA, USA). IgG concentrations were measured using NanoDrop 2000C (ThermoFisher Scientific, Waltham, MA, USA). Affinity kinetics of serum-extracted IgG for ClpB were analyzed by bio-specific interaction assay (BIA) based on surface plasmon resonance (SPR) phenomenon on a BIAcore T200 instrument (Cytiva), according to a previously published protocol (Legrand et al., 2014). ClpB protein (SinoBiological) was diluted to 0.1 mg/mL in 10 mM sodium acetate buffer, pH 4.5 (Cytiva), and was covalently coupled on the CM5 sensor chip (Cytiva). A multi-cycle affinity kinetic analysis method was run with five serial dilutions of purified IgG: 336, 168, 84, 42, 21 nM in HBS-EP + buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.003 mM EDTA and 0.05 % surfactant P20, Cytiva), each cycle included 120 s of analyte
injection and 600 s of dissociation with a 30 µL/min flow speed at 4 °C. Between sample injections, the binding surface was regenerated with 10 mM glycine-HCl buffer, pH 2.5, resulting in a return of the baseline level of the sensorgram. Affinity kinetic data were analyzed using the T200 Evaluation 4.1.1 program (Cytiva) and fitted with Langmuir's 1:1 model after blank values subtraction.

## 6.6. Neuropeptide mRNA assay

To analyze hypothalamic gene expression of appetite-regulating peptides, tissue RNA was extracted using a NucleoSpin RNA mini kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Before the RNA extraction, the entire hypothalamus tissue block was lysed in a buffer supplemented with β-mercaptoethanol. Complementary DNA (cDNA) was synthesized using SensiFAST™ cDNA Synthesis Kit with 1 µg of RNA material per sample. Quantitative polymerase chain reaction (qPCR) was performed with QuantStudio 12 K Flex (Applied Biosystems, Waltham, MA, USA) in a 384-well plate using SYBR™ Master Mix Fast (Fisher Scientific, Illkrisch, France). 384-well plates were filled using the Bravo automated liquid handling platform (Agilent, Santa Clara, CA, USA), with 3.8 µL of master mix and 1.2 µL of diluted cDNA (1/10⁶). Primers listed in the Table below were used at 300 nM. Relative to the NaCl group, mRNA expression was analyzed using three internal controls, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), and peptidylprolyl isomerase A (Ppia).

Table Primers used in the study.

## 6.7. Peptide synthesis

Acetylated and amidated α-MSH-like 14-amino acids fragment of *E. coli* ClpB (535–548) (Ac-EIAEVLARWTGIPV-NH2) was synthesized by Fmoc solid phase methodology on a Liberty microwave-assisted automated peptide synthesizer (CEM, Saclay, France) using the standard manufacturer's procedures at 0.1 mmol scale. All Fmoc-amino acids (0.5 mmol, 5 eq.) were coupled on a Rink amide resin, by *in situ* activation with HBTU (0.5 mmol, 5 eq.) and DIEA (1 mmol, 10 eq.) before Fmoc removal with 20 % piperidine in DMF. After completion of the chain assembly, the peptide was deprotected and cleaved from the resin by adding 10 mL of an ice-cold mixture of TFA/TIS/H₂O (9.5:0.25:0.25, v/v/v) and agitating for 3 h at RT. After precipitation in TBME followed by centrifugation (4500 rpm, 15 min), the crude peptide was purified by reversed-phase HPLC (Gilson, Villiers le Bel, France) on a 22 x 250 mm Vydac 218TP1022 C₁₈ column (Avantor, Rosny-sous-Bois, France) using a linear gradient (20–60 % over 45 min) of acetonitrile/TFA (99.9:0.1) at a flow rate of 10 mL/min. The peptide was then characterized by MALDI-TOF mass spectrometry on an ultraflxExtreme (Bruker, Strasbourg, France) in the reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix. Analytical RP-HPLC, performed on a 4.6 x 250 mm Vydac 218TP54 C₁₈ column, indicated that the purity of the peptides was > 99.9 %.

## 6.8. Melanocortin receptor type 4 activation in vitro

Melanocortin receptor type 4 (MC4R) activation was assayed by the PRESTO-Tango G-protein-coupled receptors β-arrestin recruitment method. The following protocol was adapted from a previous study (Duraisamy et al., 2024). HTLA cells were maintained in DMEM supplemented with 10 % FBS, 2 µg/mL puromycin, and 100 µg/mL hygromycin B in a humidified atmosphere at 37 °C in 5 % CO₂. Cells were stably transfected with 2 µg Tango-MC4R construct using lipofectamine 2000 reagent and selected with 500 µg/mL of geneticin. On day 1, cells were plated on a white 96-well plate at the rate of 2x10⁴ cells per well in 90 µL of supplemented DMEM. 6 h later, 10X compounds including recombinant ClpB protein (SinoBiological), α-MSH-like 14-amino acids fragment of *E. coli* ClpB (535–548) synthesized as described above, and α-MSH peptide (Bachem), were added at the rate of 10 µL/well, in triplicate. On day 2, the medium and drugs were slowly removed from the wells (aspiration), and 50 µL of 1/20X Bright-Glo luciferase substrate solution (Promega Corporation, Madison, WI, USA, Cat. No. E2620) was added to each well. After 2 min, luminescence was counted with an Infinite F200Pro plate reader (Tecan, Männedorf, Switzerland). Relative luminescence units (RLU) results were exported into Excel sheets, and GraphPad Prism 9 was used for data analysis. To measure constitutive basal activity, no ligand was added. Peptides and proteins were assayed 8 times in a dose-response manner to determine EC₅₀. Immune complexes formed by α-MSH or ClpB with IgG purified from mouse plasma were assayed 3 times by adding 0.05 mg of IgG and 10⁻⁶,⁵M of α-MSH and ClpB per well.

## 6.9. Fecal clpB DNA measurement

Fecal clpB DNA levels were measured by qPCR. Total DNA was extracted from the mice feces using a QIAamp Fast DNA stool kit (QIAGEN Valencia, CA, USA) and quantified with a Nanodrop 2000c spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The qPCR mix included 5 µL of SYBR Green Master (Fisher Scientific), 0.5 µM each of the forward and reverse primers, DNA from samples (7 ng/µL), and water to give a total volume of 10 µL. Previously validated (Tennoune et al., 2014) *E. coli* ClpB primer sequences have been used: Forward, 5'-GCAGCTCGAAGGCAAAACTA-3', and reverse 5'-ACCGCTTCGTTCTGACCAAT-3'. The primers were purchased from Invitrogen (Cergy-Pontoise, France). A three-step PCR was performed for 40 cycles. The samples were denatured at 95 °C for 10 min, annealed at 60 °C for 2 min, and extended at 95 °C for 15 s. Results are shown as 1/CT values.

## 6.10. Fecal energy content measurement

The fecal energy content was measured using a Global Standards Combustion Calorimeter (IKA, Staufen, Germany), using adiabatic 22 °C operating mode, with a minimum of two replicates. The day before

<table><thead><tr><th>Symbol</th><th>Name</th><th>Forward (5'-3')</th><th>Reverse (5'-3')</th></tr></thead><tbody><tr><td><i>Gapdh</i></td><td>Glyceraldehyde-3-phosphate dehydrogenase</td><td>CATGGCCTTCCGTGTTTCCTA</td><td>CCTGCTTCACCACCTTCTTGA</td></tr><tr><td><i>Npy</i></td><td>Neuropeptide Y</td><td>GTGGATCTCTTCTCTCACAG</td><td>CTTGTTACCTAGCATCGTG</td></tr><tr><td><i>Pomc</i></td><td>Proopiomelanocortin</td><td>CTCCTGCTTCAGACCTCCATAGA</td><td>GGATGCAAGCCAGCAGGTT</td></tr><tr><td><i>Ppia</i></td><td>Peptidylprolyl isomerase A</td><td>CCACTGTCGCTTTTCGCCGC</td><td>TGCAAACAGCTCGAAGGAGACGC</td></tr><tr><td><i>Oxt</i></td><td>Oxytocin</td><td>GACCCTGAGTCTGCCTTCTC</td><td>AAGGTATTCCCAGAAAGTGGGC</td></tr><tr><td><i>Bdnf</i></td><td>Brain-derived neurotrophic factor</td><td>GCGCCCATGAAAGAAGTAAA</td><td>TCGTCAGACCTCTCGAACCT</td></tr><tr><td><i>Crh</i></td><td>Corticotropin-releasing hormone</td><td>TCTGCGGGAAGTCTTGGAAA</td><td>TCCTGTTGCTGTGAGCTTGCT</td></tr><tr><td><i>Pmch</i></td><td>Promelanin-concentrating hormone</td><td>GAGTCCACACAGGAAAAGAGAGAAA</td><td>CATACACCTGAGCATGTCAAAATCT</td></tr><tr><td><i>Gal</i></td><td><i>Galanin</i></td><td>GCCCACATGCCATTGACA</td><td>TTGTAACTCCCTCTTGCCCTGTGA</td></tr><tr><td><i>Qrfp</i></td><td>Pyroglutamylated RFamide peptide</td><td>ACATCACTCTGCCGTCCTTACC</td><td>GTGCCCATGATGTCCACACTAG</td></tr><tr><td><i>Hrct</i></td><td>Hypocretin</td><td>TGTTCCTGCCGTCTCTACGAA</td><td>TGGTTACCGTTGGCCTGAA</td></tr><tr><td><i>Actb</i></td><td>β-actin,</td><td>AGGTCATCACTATTGGCAACGA</td><td>CACAGGATTCATACCCAAGAAG</td></tr></tbody></table>
analysis, the feces were removed from the -80 °C freezer and trans-
ferred to a -20 °C freezer for 4 h. Then, the samples were transferred to
4 °C for another 4 h. Finally, the feces were spread overnight at RT.
Benzoic acid was used to calibrate the bomb calorimeter, and the
measurements were carried out with 150 to 250 mg of each sample.

6.11. Fecal microbiota composition analysis

Fecal microbial composition was investigated at T0 and T2 time
points. Total DNA was extracted from one or two fecal pellets using the
QIAamp PowerFecal Pro DNA Kit (Qiagen). The V3-V4 region of the 16S
rRNA gene was amplified using 515F-806R primers, forward:
GTGCCAGCMGCCGCGGTAA and reverse: GGACTACHVGGGTWTC-
TAAT, with a dual barcoding approach (Caporaso et al., 2011). The
resulting library was sequenced on an Illumina Miseq sequencer
(300PE). During the demultiplexing process, only barcodes without
mismatches were allowed (Casava, Illumina). Raw 16S rRNA gene
sequencing data were processed using the DADA2 pipeline, imple-
mented in the DADA2 R package, version 1.16.0 (Callahan et al., 2016).
Specifically, the first 20 bases were trimmed from the 5' end of forward
or reverse read sequences, truncating the 290 bases from the 3' end of
forward or reverse sequences. The reads were also truncated at the first
instance of a quality score ≤ 3. An abundance table of 16S rRNA
amplicon sequence variants (ASVs) was generated, and the taxonomic
annotation of ASVs was obtained using the SILVA 138 database (Quast
et al., 2013) (Table S1). To normalize sequencing depth, the samples
were randomly sub-sampled to 9,000 sequences.

6.12. Electron microscopy of ClpB in E. coli

To illustrate the presence of ClpB in *Escherichia coli* (*E. coli*), the following strains were used: a laboratory strain *E. coli* K12, its derivative *E. coli* K12 ClpB mutant strain (ΔClpB) as a negative control, and another derivative transfected with ClpB -yellow fluorescent protein (YFP) as a positive control. The ClpB-modified strains were kindly provided by Axel Mogk from the University of Heidelberg, Germany (Mogk et al., 2003b). Free-cell bacterial cultures were performed in 250 mL Erlenmeyer flasks containing 50 mL of Luria-Bertani broth (LB, Difco). For the ΔClpB strain, kanamycin and spectinomycin were added at a final concentration of 30 µg/mL and 50 µg/mL, respectively. For the ClpB-YFP-producing strain, kanamycin, spectinomycin, and chloramphenicol were added at a final concentration of 30 µg/mL, 50 µg/mL, and 10 µg/mL, respectively. Flasks were inoculated with fresh overnight preculture and incubated for 24 h at 37 °C under mild shaking. Bacteria were recovered by centrifugation (5.000 g, 30 min, 4 °C, 5804 R centrifuge, Eppendorf, Hamburg, Germany). The pellets were washed three times with 10 ml of Dulbecco's phosphate buffer saline (DPBS, Sigma).

Bacterial pellets were fixed with a high-pressure freezer (HPM100,
Leica) and transferred to a freeze-substitution automate (AFS2, Leica).
Samples were substituted in anhydrous acetone with 0.5% uranyl ace-
tate at –90 °C for 96 h. The temperature was gradually raised to –60 °C,
–30 °C, and –15 °C using a gradient of +2 °C/h and stabilized during
12 h for each temperature. Then, samples were rinsed with anhydrous
ethanol before infiltration in a mixture of London Resin White (LRW)
and anhydrous ethanol (25%, 50%, 75%, 100%). Finally, bacteria
were infiltrated in a mixture of LRW with 0.5% benzoin methyl ether
(UV catalyst), before polymerization into the AFS2 apparatus at –15 °C
under UV light for 48 h.

Ultrathin sections (90 nm; ultramicrotome UC7, Leica) of bacteria
were collected onto formvar-carbon-coated nickel grids. For the
immunogold labeling, sections were blocked in TBS (20 mM Tris base;
200 mM NaCl; pH 7.2) + 0.2 % of bovine serum albumin (BSA), then
incubated with E. coli ClpB rabbit antibody (SinoBiological) 1/1000 in
TBS buffer + 0.2 % BSA + 1/30 normal goat serum) for 2 h at 25 °C.
After washing in TBS + 0.2 % BSA, grids were incubated for 1 h at 25 °C
with a goat anti-rabbit antibody conjugated to 10 nm gold particles (1/

20 in TBS + 0.2 % BSA, Aurion). After washing in TBS + 0.2 % BSA,
grids were stained using 0.5 % uranyl acetate in methanol and Reynold's
lead citrate solution. Sections were observed in a TALOS F200C G2
transmission electron microscope operating at 200 kV, with a Ceta 16 M
camera (ThermoFisher).

6.13. Statistical analysis

Data were analyzed, and the graphs were plotted using GraphPad
Prism 9.0. (GraphPad Software Inc., San Diego, CA). Individual groups
were compared with the Student's *t*-test, and the changes in the same
groups were compared with the paired *t*-test. Multiple groups were
compared with ANOVA and Tukey's post-test or by Kruskal-Wallis with
Dunn's post-test, according to normality analyzed by the Kolmogorov-
Smirnov's test. Dynamics of changes in body weight were compared
using 2-way RM-ANOVA and the Bonferroni post-test. Data are shown as
means ± standard error of means (SEM), and for all tests, *p* < 0.05 was
considered statistically significant. The microbiome data were analyzed
with the phyloseq and vegan packages in R (version 1.32.0) to estimate
α-diversity using the Shannon and Chao1 indexes and β-diversity using
the Bray-Curtis distance. Statistical test of the α-diversity was assessed
with a linear model using lme4 and lmerTest in R (Bates et al., 2015;
Kuznetsova et al., 2017). To account for repeated measures, mouseID
was included as a random intercept. β-diversity differences were
assessed with a permutational analysis of variance (PERMANOVA) test
on Bray-Curtis distance using the adonis function in the vegan package
and stratifying for mouseID to account for repeated measures. To test the
effect of intervention only, a PERMANOVA test was performed only of
fecal samples collected at the end of the experiment, and p-values were
corrected for a false discovery rate using the Benjamini-Hochberg (B-H)
method. Core microbiome was identified as taxa found in at least 25% of
the samples with a relative abundance higher than 1 %. Differentially
abundant core ASV and genera at the end of the experiment in fecal
samples from the 3 ABA groups were calculated with MaAslin2 (Mallick
et al., 2021). Correlation of taxa to energy content was performed using
Spearman's correlation with B-H correction for multiple testing.

CRedit authorship contribution statement

**Benjamin Thomas:** Writing – review & editing, Investigation, Data curation. **Nadia Andrea Andreani:** Writing – review & editing, Methodology, Investigation, Data curation. **Emilie Lahaye:** Investigation. **Lisa Wallart:** Investigation. **Karthi Duraisamy:** Methodology, Investigation. **Benjamin Lefranc:** Methodology. **Jérôme Leprince:** Methodology, Funding acquisition. **Sophie Bernard:** Methodology, Investigation. **Philippe Chan:** Methodology, Investigation. **Jean-Luc do Rego:** Methodology. **Jean-Claude do Rego:** Methodology. **Astrid Dempfle:** Methodology. **John F. Baines:** Methodology, Funding acquisition. **Sergueï O. Fetissov:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

Acknowledgments

This project was performed in the frame of the ERANET Neuron
MiGBAN project (01EW1906A-01EW1906B) "Microbiome Gut-Brain
Axis in Anorexia Nervosa", funded by the European Union (EU), the
German Ministry for Education and Research (BMBF), and the French
Agence Nationale de la Recherche (ANR); we thank the MiGBAN coor-
dinator Prof. Beate Herpertz-Dahlmann, RWTH University Aachen,
Germany, and other members of the consortium for their role in funding
acquisition. S.O.F. was also supported by the Translational Microbiota
Program (PTM-2) of Inserm, France, and J.F.B. by Deutsche For-
schungsgemeinschaft (DFG; BA 2863/14-1, project number
509492174). The authors thank the PISSARO proteomic platform,
Rouen University, France, for ClpB quality check, Biocodex, France, for
providing the Biacore T200 instrument, Prof. Sahil Adriouch, Inserm,
France, for his expert opinion about the tolerization protocol, and Mrs
Fatima Leon, SCAC platform, for technical assistance. PRIMACEN was
supported by the University of Rouen Normandy, Inserm, CNRS, IRIB,
CPER InfraBioSanté: the Région Normandie, French Ministry of Higher
Education, Research and Innovation, and the European Regional
Development Fund (ERDF) for the cryoTEM-CLEM project, the GIS
IBiSA, and the national infrastructure France-BioImaging, supported by
the French National Research Agency (ANR-10-INBS-04).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbi.2025.106152.

Data availability

Data will be made available on request.

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