# Hematopoietic MyD88 orchestrates the control of gut colonization by segmented filamentous bacteria

Marie Cherrier, Teck Hui Teo, Renan Oliveira Corrêa, Marion Picard, Aurélie Couesnon, Corinne Lebreton, Francesco Carbone, Cécile Masson, Pamela Schnupf, Nadine Cerf-Bensussan, et al.

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## ► To cite this version:

Marie Cherrier, Teck Hui Teo, Renan Oliveira Corrêa, Marion Picard, Aurélie Couesnon, et al. Hematopoietic MyD88 orchestrates the control of gut colonization by segmented filamentous bacteria. Mucosal Immunology, 2025, 18, pp.717 - 729. 10.1016/j.mucimm.2025.03.002. hal-05193143

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Submitted on 30 Jul 2025

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# Hematopoietic MyD88 orchestrates the control of gut colonization by segmented filamentous bacteria

Marie Cherrier<sup>a</sup>, Teck Hui Teo<sup>b,c</sup>, Renan Oliveira Corrêa<sup>a</sup>, Marion Picard<sup>a</sup>, Aurélie Couesnon<sup>a</sup>, Corinne Lebreton<sup>a</sup>, Francesco Carbone<sup>d</sup>, Cécile Masson<sup>e</sup>, Pamela Schnupf<sup>b</sup>, Nadine Cerf-Bensussan<sup>a</sup>, Valérie Gaboriau-Routhiau<sup>a,f,*</sup>

## ARTICLE INFO

### Keywords:

Host-microbiota interactions

Gnotobiology

Innate immunity

SFB

MyD88

IL-22

## ABSTRACT

**Summary:** Host-microbiota cooperation is critical for successful intestinal homeostasis. The commensal segmented filamentous bacteria (SFB) are crucial for orchestrating the post-natal maturation of the host gut immune system and establishing a healthy state of physiological inflammation, which largely depends on their intimate attachment to the ileal mucosa. However, the signaling pathways used by SFB to induce gut immune responses and how such responses ultimately control SFB colonization remain controversial. Using gnotobiotic approaches, we showed that SFB load is controlled by complex interactions involving the gut microbiota and the host immune system. Therefore, to clearly determine the role of host immune responses induced by SFB in directly controlling their growth, immunodeficient mice monocolonized with SFB were used. Here, we show that in the absence of a complex microbiota, the humoral immune response is dispensable to control SFB growth in the jejunum and ileum, shortly and later after colonization. In contrast, MyD88 signaling in myeloid cells is critical for licensing interleukin (IL)-22 production by type 3 innate lymphoid cells (ILC3) and CD4⁺ T cells, which ultimately limits SFB expansion. Thus, by revisiting the hierarchy of immune mechanisms that directly control SFB growth, our results emphasize the necessary and sufficient role of a hematopoietic MyD88/IL-22 axis.

## Introduction

The mammalian intestine is colonized by a highly complex community of symbiotic microbes, which rely on host resources for their growth. In turn, symbiotic microbes influence multiple host functions that contribute to the success of the association. They can especially invigorate a robust state of physiological inflammation at the gut barrier that enables their containment in the gut lumen, thereby limiting tissue damage and systemic translocation detrimental to their host, while enhancing host protection against pathogens.¹ Studies in mice have revealed that among the hundreds of bacterial species that constitute the gut microbiota, segmented filamentous bacteria (SFB) – Type I Clostridia-related spore-forming commensal bacteria common in many

vertebrate species² – are unique in their capacity to recapitulate the maturation of gut homeostatic innate and adaptive immune responses that lead to the healthy state of physiological inflammation induced by a complex microbiota after birth.³,⁴ Thus, although microbiota-derived stimuli of maternal origin may, in conventionally raised mice, influence the early immune development of fetuses and newborns,¹,⁵ post-natal maturation of the gut immune barrier starts mainly after weaning, coinciding with the outgrowth of SFB in the mouse ileum.⁶ SFB notably trigger the activation of interleukin-22 (IL-22) production by type 3 innate lymphoid cells (ILC3),⁷,⁸ the induction of antimicrobial peptides and reactive oxygen species (ROS) by the gut epithelium,⁹ the expansion and maturation of gut lymphoid tissues,¹⁰ and the development of strong immunoglobulin A (IgA) and T cell responses, among

<footnote><sup>a</sup> Université Paris Cité, Imagine Institute, INSERM UMR1163, Laboratory of Intestinal Immunity, 75015 Paris, France</footnote>

<footnote><sup>b</sup> Université Paris Cité, INSERM UMR-S1151, CNRS UMR-S8253, Institut Necker Enfants Malades, F-75015 Paris, France</footnote>

<footnote><sup>c</sup> A*STAR Infectious Diseases Labs (A*STAR ID Labs), Agency for Science, Technology and Research, Singapore</footnote>

<footnote><sup>d</sup> Université Paris Cité, Imagine Institute, INSERM UMR 1163, Labtech Single-Cell@Imagine, 75015 Paris, France</footnote>

<footnote><sup>e</sup> Université Paris Cité, Imagine Institute, Structure Fédérative de Recherche Necker, Bioinformatics Core Facility, 75015 Paris, France</footnote>

<footnote><sup>f</sup> Université Paris-Saclay, INRAe, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France</footnote>

<footnote>* Corresponding author at: Institut Imagine, Laboratory of Intestinal Immunity, 24 Boulevard du Montparnasse, 75015 Paris, France.</footnote>

<footnote>E-mail address: valerie.gaboriau-routhiau@inserm.fr (V. Gaboriau-Routhiau).</footnote>
which the conspicuous intestinal T helper 17 (Th17) response is considered as a hallmark of gut colonization by the complex microbiota.<sup>11,12,13,14,15</sup>

In contrast with most, if not all, commensal bacteria, SFB grow in intimate contact with the ileal mucosa.<sup>2,11,16</sup> This attachment is required for the SFB-mediated induction of adaptive homeostatic immune responses,<sup>17,18</sup> suggesting that strict regulatory mechanisms must control SFB colonization in their ecological niche. Additionally, in immunocompetent hosts, expansion of SFB is transient, starting around weaning time and progressively declining after a few weeks, a phenomenon also observed in germ-free mice monocolonized with SFB,<sup>6,13,19</sup> suggesting that immune responses induced by SFB can, in turn, restrict their growth and thereby prevent excessive inflammation. Yet, the respective contribution of the different arms of the host immune response elicited by SFB remains debated. While several studies suggested a role for adaptive immune responses,<sup>20,21,22,23,24,25</sup> others involved epithelial release of antimicrobial peptides induced through epithelial myeloid differentiation primary response 88 (MyD88)-dependent signaling and IL-22 production by ILC3.<sup>26,27,28</sup> Of note, most studies have been performed in conventionally raised mice, precluding the discrimination between direct effects of host immune responses induced by SFB and indirect effects mediated by members of the microbiota, either on host responses or on SFB. Indeed, variability in microbiota composition associated with husbandry conditions<sup>29</sup> may influence bacterial inter-species competition. Here, taking advantage of mice monocolonized with SFB, we delineate the direct role of host innate and adaptive immune responses induced by SFB in controlling SFB expansion and provide evidence that while humoral adaptive immune responses *per se* are dispensable, MyD88 signaling in hematopoietic cells is critical for SFB to initiate a self-limiting immune response largely dependent on IL-22 production by ILC3 and CD4<sup>+</sup> T cells. However, in the absence of adaptive immune cells, ILC3s were able to control SFB growth, suggesting a redundancy of the adaptive IL-22 immune response.

## Results

*Complex microbiota differentially influences control of SFB growth in immunocompromised mice*

To determine whether the context of a complex intestinal microbiota may impact the control of SFB expansion independently of host immune responses, germ-free mice either wild-type (WT) or immunocompromised were colonized, in two independent experiments, with complex fecal SFB-positive microbiota from conventional C57BL/6 WT mice originating from two different vendors and housed in separate rooms in our animal facilities (thereafter referred to as microbiota-1 and microbiota-2). Immunocompromised mice lacked either immunoglobulins (IgH-, IgM-/- mice, referred to as JH-/- mice) or MyD88 and TRIF (TIR-domain-containing adaptor inducing interferon-β), two key components of innate immune signaling cascades (Myd88xTrif-/- mice, referred to as MT-/- mice). SFB were quantified in intestinal biopsies using targeted quantitative PCR specific for SFB 16S rDNA, on day 8 (d8) post-colonization to analyze the early control of SFB, presumably by innate immune mechanisms, and later on day 21 (d21) when SFB-induced adaptive immune responses are established.<sup>10,13</sup> SFB levels in the jejunum and the ileum were similar in JH-/- and WT mice colonized with microbiota-1, at both time points (Fig. 1A). In contrast, a significant SFB overgrowth was observed in the jejunum of JH-/- mice colonized with microbiota-2 on d8 and d21 post-colonization, while overgrowth in the ileum reached significance only on d8 (Fig. 1B). Regarding MT-/- mice, an SFB overgrowth was detected in the jejunum on d8 after colonization with microbiota-1 or microbiota-2, but only in the ileum of mice colonized with microbiota-2 (Fig. 1A-1B). Differences in colonization profiles between MT-/- and WT mice colonized with microbiota-2 were maintained on d21. Altogether, these data indicated that a complex gut microbiota significantly influences the fitness of SFB in

A

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B

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Complex microbiota differentially impact SFB growth in the context of immunodeficiency. SFB quantification in jejunal (open symbols) and ileal (full symbols) biopsies from ex-germ-free wild-type (WT) and immunodeficient JH<sup>-/-</sup> or Myd88xTrif<sup>-/-</sup> (MT<sup>-/-</sup>) mice conventionalized at adult age with complex SFB-positive fecal microbiota from WT conventional mice. Two independent assays using mice from different origins have been performed: microbiota-1 (A) and microbiota-2 (B). Quantification was performed on day 8 (d8) (black symbols) and day 21 (d21) (grey symbols) post-colonization by qPCR with primers specific for SFB 16S rDNA gene. Data represent individual values and medians. n.d: not done. Statistical significance between two groups was determined by the nonparametric Mann-Whitney test.
\*p < 0.05, \*\*p < 0.01 and \*\*\*p < 0.001.</figcaption></figure>

their ecological niche and thereby plays a critical role in the control of SFB expansion along the small intestine of immunocompromised hosts, supporting the need for alternative approaches to determine whether and how immune responses induced by SFB may directly control their growth.

*MyD88 signaling in hematopoietic cells is critical to control SFB growth*

To address these questions, SFB colonization was monitored in germ-free WT, MT-/-, JH-/- but also Rag1-/- mice, lacking B and T cell adaptive immune responses, monocolonized with SFB by oral gavage.
SFB levels were significantly higher in the jejunum and ileum of MT⁻/⁻ mice compared to WT mice, at both d8 and d21 post-colonization (Fig. 2A), although colonization was significantly higher in the ileum – the main SFB niche – in both models. In this line, SFB growth was significantly attenuated in the jejunum of MT⁻/⁻ mice on d21. Surprisingly, and at odds with former observations reporting a role for adaptive immunity in the control of SFB colonization,²⁰,²¹,²²,²⁴,²⁵ SFB levels were not increased in Rag1⁻/⁻ mice and were even significantly reduced in the ileum compared to WT mice at both time points (Fig. 2A). Accordingly, microscopic observation of tissue sections showed a higher density of SFB filaments attached to ileal epithelial cells in MT⁻/⁻ compared to WT and Rag1⁻/⁻ mice at both d8 (Fig. 2B) and d21 post-colonization (Supplementary Fig. 1A). Consistent with the observations made in Rag1⁻/⁻ mice, SFB levels detected in the jejunum and ileum of JH⁻/⁻ mice were similar to WT (Fig. 2A). Furthermore, the absence of both B and T cells or only B cells (Rag1⁻/⁻ and JH⁻/⁻ background, respectively) did not result in SFB overgrowth after 8 weeks of colonization (d56) (Supplementary Fig. 1B), suggesting that adaptive immunity plays a redundant role in controlling SFB colonization even in the long term and that immunoglobulins *per se*, and in particular IgA, do not play a critical role. Altogether, these observations indicated that in monocolonized mice, only MyD88- and/or Trif-dependent signaling were indispensable to control SFB growth.

To address the respective role of MyD88 and TRIF deficiency on SFB colonization, germ-free *Myd88*⁻/⁻ and *Trif*⁻/⁻ mice were colonized with SFB. SFB levels were similar in the jejunum and the ileum of *Myd88*⁻/⁻ and *MT*⁻/⁻ mice at d8 post-colonization and significantly higher than those measured in *Trif*⁻/⁻ and WT mice (Fig. 2A), indicating a non-redundant impact of MyD88-dependent signaling on the control of SFB growth along the small intestine. SFB overgrowth in the ileum of *Myd88*⁻/⁻ mice was confirmed at d21 and d56 post-colonization (Fig. 2A and Supplementary Fig. 1B), suggesting a long-lasting effect of MyD88 deficiency. It has been suggested that intestinal IgA production may require MyD88-dependent signals.<sup>30,31</sup> However, numbers of *lamina propria* IgA-secreting cells, expression of polymeric immunoglobulin receptor (Pigr) and fecal IgA concentrations during SFB colonization were comparable in *MT*⁻/⁻, *Myd88*⁻/⁻ and WT monocolonized mice (Supplementary Fig. 2), suggesting that, consistent with our observations in JH⁻/⁻ mice, SFB overgrowth in MyD88-deficient monocolonized mice is unlikely to result from an impaired local IgA response.

To further define the role of MyD88 in the epithelial and/or hematopoietic compartment on the control of SFB growth, we generated germ-free bone marrow chimeras where mice specifically lack MyD88 in the hematopoietic compartment (*Myd88*⁻/⁻→WT) or express MyD88 only in the hematopoietic compartment (WT→*Myd88*⁻/⁻). After 7–8 weeks of reconstitution, mice were colonized with SFB for 8 days. SFB quantification in intestinal biopsies (jejunum and ileum) showed comparable levels of SFB in WT → *Myd88*⁻/⁻ and WT → WT mice, but significantly higher levels in both *Myd88*⁻/⁻→WT and *Myd88*⁻/⁻→*Myd88*⁻/⁻ mice (Fig. 2C), indicating that MyD88 expression in the hematopoietic compartment was necessary and sufficient to control SFB growth along the small intestine, and raising questions on the nature of innate immune cell subset(s) affected by the lack of MyD88 that may be necessary to restrict SFB expansion.

*MyD88 deficiency profoundly affects the composition and functions of intestinal mononuclear phagocytes*

Multiple subsets of mucosal mononuclear phagocytes orchestrate immune responses to intestinal microbiota.<sup>32,33,34</sup> We therefore addressed whether and how MyD88 deficiency affects the number, composition and function of gut resident mononuclear phagocytes. A drastic reduction in the numbers of CD64⁺ CX3CR1⁺ CD11c⁺ monocyte-derived macrophages and CD103⁺ CD11b⁺ CD8⁻ conventional dendritic cells (cDCs) was observed in the ileum *lamina propria* of *Myd88*⁻/⁻ compared to WT and *Rag1*⁻/⁻ mice monocolonized with SFB for 8 days,

while CD103⁺ CD11b⁻ CD8⁺ cDCs were only mildly reduced (Supplementary Fig. 3), pointing to an important role of MyD88 in the recruitment and/or survival of intestinal mononuclear phagocytes.

To gain further insight into how MyD88 deficiency may affect the function of intestinal myeloid cells, lineage-negative CD11c⁺ MHCII⁺ cDCs and monocyte-derived macrophages were isolated and sorted from the whole small intestine *lamina propria* of *Myd88*⁻/⁻ and WT mice, either monocolonized with SFB for 8 days or kept germ-free, and their transcriptome was analyzed at the single-cell level using 10X Genomics droplet-based technology. Based on dimensional reduction of unsupervised clustering analysis, using uniform manifold approximation and projection (UMAP) of the expression of signature genes, cluster annotation identified twelve cell subsets including cDCs and monocyte-derived phagocytes (Fig. 3A and Supplementary Fig. 4A). Among cDCs, we could discriminate CD103⁺ and CD103⁻ type 2 cDCs (cDC2), proliferating and migrating cDC2, the recently described crypt patches and isolated lymphoid follicles-associated DCs (later referred to as CP-ILFs DCs),<sup>35</sup> CD8⁺ cDC1, proliferating and migrating CD8⁺ cDC1. Monocyte-derived phagocytes could be sub-divided into two main populations of maturing monocytes and resident macrophages. The remaining cell subsets were identified as contaminating B and T cells based on transcriptome analysis. All subsets were found in each condition (Fig. 3B and Supplementary Fig. 4B), indicating that MyD88 deficiency did not prevent the development of any myeloid population in the small intestine. The frequency of resident macrophages was however severely decreased in *Myd88*⁻/⁻ versus WT mice, in favor of CD103⁺ cDC2, and this whether mice were germ-free or monocolonized with SFB (Fig. 3B), overall indicating a prominent role of MyD88 on macrophage development in the intestine of germ-free mice before any exposure to SFB.

Strikingly, global gene expression analysis in myeloid cells showed that samples primarily segregated according to mouse genotype rather than colonization status (Fig. 3C). Thus, most differentially expressed genes between WT and *Myd88*⁻/⁻ monocolonized mice were already differentially expressed in their germ-free counterparts (Supplementary Fig. 4C-4D), overall indicating that MyD88 plays a dominant role in the transcriptional signature of intestinal myeloid cells regardless of SFB colonization. To get further insight into the function(s) of myeloid cells altered in the absence of MyD88, gene expression profiles were compared between SFB-monocolonized mice (Fig. 3D). KEGG pathways associated with the 200 most differentially expressed genes were identified as TNF, NF-κB, Toll- and NOD-like receptor, IL-17 and C-type lectin receptor signaling (Fig. 3E), with genes such as *Il1b*, *Tnf*, *Nfkb1*, *Nfkbia*, *Il6*, *Cxcl1* and *Cxcl2* being associated with several of these pathways (Fig. 3F). Altogether, these results suggested that the MyD88-NF-κB axis may be key for many functions of mononuclear phagocytes, possibly through the regulation of IL-1β, IL-6, IL-23 and TNF expression. To further identify myeloid cell subsets that maybe most profoundly affected by MyD88 deficiency, we analyzed gene expression using UMAP projections. *Il1b* expression was detected in most mononuclear phagocyte clusters isolated from WT mice and was globally reduced in MyD88-deficient cells, except in CP-ILFs DCs (Fig. 3G). In contrast, in keeping with the recent observation that IL-23 producing DCs are mostly confined to CPs and ILFs in the gut (Supplementary Fig. 4E),<sup>36</sup> *Il23a* transcripts, as well as *Il6* transcripts, were highly expressed in CP-ILFs DCs and, to a much lesser extent, in CD103⁺ cDC2 or CD8⁺ cDC1 from WT mice, but they were severely reduced in MyD88-deficient cells, especially in CP-ILFs DCs (Fig. 3G). Taken together, these results highlight that some CP-ILFs DCs critical functions are under the control of MyD88. The strong reduction in *Il1b*, *Il6* and *Il23a* gene expression, observed at the single cell level in myeloid cells from MyD88-deficient mice, was confirmed by RT-qPCR in hematopoietic cells isolated from the small intestine of SFB-monocolonized *Myd88*⁻/⁻ mice compared to WT and *Rag1*⁻/⁻ mice (Fig. 3H). A similar result was observed in monocolonized *Myd88*⁻/⁻→WT and *Myd88*⁻/⁻→*Myd88*⁻/⁻ chimeric mice compared to their WT→WT and WT→*Myd88*⁻/⁻ respective
<figure><img src="image_5.png" /><figcaption>**Fig. 2. MyD88 controls SFB growth in monocolonized mice.** (A) SFB quantification in jejunal and ileal biopsies from wild-type (WT) and immunodeficient *Myd88xTrif*⁻/⁻ (MT⁻/⁻), *Myd88*⁻/⁻, *Trif*⁻/⁻, *Rag1*⁻/⁻ or JH⁻/⁻ mice monocolonized with SFB for 8 (upper panels) or 21 days (lower panels) by qPCR. Individual values pooled from two or five independent experiments and medians are shown. (B) SFB detection by immunohistofluorescence in intestinal biopsies from jejunum and terminal ileum of WT, MT⁻/⁻ and *Rag1*⁻/⁻ mice monocolonized with SFB for 8 days. Nuclei and SFB are stained with DAPI (white) and actin with Phalloidin (red). Scale bars = 20 µm, or 5 µm in insert magnification. (C) SFB quantification in jejunal and ileal biopsies from bone marrow chimeric mice monocolonized with SFB for 8 days. Bone marrow cells from WT or *Myd88*⁻/⁻ germ-free donors were injected into *Myd88*⁻/⁻ (*Myd88*⁻/⁻) or WT (→WT) recipients. Individual values pooled from two independent experiments and medians are shown. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001 between two groups, determined by a nonparametric Mann-Whitney test.</figcaption></figure>
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(caption on next page)
**Fig. 3.** MyD88 deficiency profoundly impacts small intestine *lamina propria* mononuclear phagocytes composition and functions. (A-B) Unsupervised clustering of sorted viable Lin⁻ CD11c⁺ MHCII⁺/⁻ cells isolated from the small intestine *lamina propria* of WT and *Myd88⁻/⁻* deficient mice, either germ-free (GF_WT and GF_MyD) or monocolonized with SFB for 8 days (SFB_WT and SFB_MyD). (A) Data are represented as merged UMAP and clusters are annotated based on the expression of signature genes. (B) Cell composition as percentage of cluster in sample in each experimental condition. (C) Heatmap of pseudo bulk gene expression analysis in myeloid cells sorted from GF or SFB-monocolonized *Myd88⁻/⁻* and WT mice, showing most differentially expressed genes and sample segregation based on log2(Fold change) and Pvalue < 0.0001. (D) Volcano plot showing differentially expressed genes in sorted myeloid cells from SFB_MyD versus SFB_WT mice, distributed according to log2(Fold change) and -log10(Pvalue). Among downregulated genes in SFB_MyD compared to SFB_WT (blue dots), those labeled in red are associated with KEGG pathways listed in (E). (E) Bubble plot showing KEGG pathways for which the top 200 most differentially expressed genes in SFB_MyD versus SFB_WT have been found to be enriched, using Legacy Mouse KEGG pathway 2019 in EnrichR and according to combined score and -log10(Pvalue). (F) Chord plot showing the overlap in KEGG pathways associated with some downregulated genes identified in (D). (G) UMAP projection of Il1b, Il6 and Il23a Log2 expression in sorted myeloid cells from SFB_MyD and SFB_WT mice. (H) Relative mRNA expression of marker genes in cells isolated from the small intestine *lamina propria* of WT, *Myd88⁻/⁻* and *Rag1⁻/⁻* mice monocolonized with SFB for 8 days. Data represent individual values and medians. Data are pooled from two independent experiments.
*p < 0.05, **p < 0.01 and ***p < 0.001 between two groups, calculated by a nonparametric Mann-Whitney test.

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**Fig. 4.** Neutrophil depletion does not prevent control of SFB colonization. All WT, *Myd88⁻/⁻* and *Rag1⁻/⁻* mice were monocolonized with SFB for 8 days. (A) UMAP projection of Log2 normalized expression of *Cxcl1* and *Cxcl2* in sorted myeloid cells from SFB_MyD and SFB_WT mice. (B) Relative mRNA expression of marker genes in cells isolated from the small intestine *lamina propria*. (C) Representative and summary plots of the frequency and number of neutrophils isolated from ileal *lamina propria* of WT and *Myd88⁻/⁻* mice. (D) SFB quantification in ileal biopsies of neutrophil depleted (α-Ly6G) and isotype control antibody-treated (isotype) WT mice, on day 8 post-colonization with SFB. Data in (B) and (D) show individual values from two independent repeats and medians, and in (C) values representative of two independent experiments. *p* < 0.05 or otherwise exact p values between two groups, determined by a nonparametric Mann-Whitney test.
controls (Supplementary Fig. 4F), further supporting the critical role of MyD88 in controlling the expression of these genes in the hematopoietic compartment and the potential impact of these cytokines in the control of SFB.

Additionally, and in line with their role on the expression of CXCL1,³⁷ the combined reduction of NF-κB, IL-1β and TNFα gene expression identified in myeloid cells from *Myd88*⁻/⁻ mice by single cell RNA sequencing may account for the severely impaired expression of *Cxcl1* (Fig. 3D). Overall, these results indicated that the lack of MyD88 severely impairs the development and functions of mononuclear phagocytes in the intestine, and may preclude their ability to initiate, promote and/or maintain innate and adaptive immune responses upon SFB colonization. Therefore, and in keeping with the redundant role of adaptive immune responses in the control of SFB growth identified in monocolonized mice, we next investigated whether and how MyD88-deficient mononuclear phagocytes may compromise the recruitment and/or activation of innate responses and, as a result, the control of SFB expansion.

Neutrophil recruitment is impaired by MyD88 deficiency but dispensable for the control of SFB growth in monocolonized mice

As mentioned above, transcriptome analysis of mononuclear phagocytes showed a drastic decrease in *Cxcl1* and *Cxcl2* expression in *Myd88*⁻/⁻ compared to WT SFB-monocolonized mice. This decrease was observed in multiple mononuclear phagocyte subsets (Fig. 4A) as well as in hematopoietic cells isolated from the small intestine of SFB-monocolonized *Myd88*⁻/⁻, *Myd88*⁻/⁻ → WT and *Myd88*⁻/⁻ → *Myd88*⁻/⁻ mice compared to WT controls, on d8 post-colonization (Fig. 4B and Supplementary Fig. 4F). CXCL1 and CXCL2 are key chemokines for the recruitment of neutrophils, which play protective roles at mucosal barriers. Interestingly, in conventionally raised mice, colonization with SFB-containing microbiota induced CXCL1 and CXCL2, which could drive CXCR2-dependent recruitment of neutrophils that, in turn, participated in limiting SFB expansion.²³ Consistent with these results, the frequency and number of neutrophils were markedly reduced in the small intestine *lamina propria* of MyD88-deficient mice compared to WT on d8 post-colonization with SFB (Fig. 4C). To examine the role of neutrophils in the control of SFB, WT germ-free mice were depleted in neutrophils using anti-Ly6G antibodies³⁸ one day before colonization with SFB and daily until d8 post-colonization (Supplementary Fig. 5). SFB quantification in ileal biopsies showed comparable SFB levels in neutrophil-depleted and isotype-treated mice (Fig. 4D), ruling out a critical role for the MyD88-dependent recruitment of neutrophils in the control of SFB growth.

Critical role of MyD88-dependent IL-22 production in the coordinated control of SFB growth in monocolonized mice

In SPF mice, IL-22 production by ILC3s and CD4⁺ T cells strongly relies on myeloid cell-derived cytokines such as IL-23 and IL-1β,³⁹,⁴⁰ and is significantly induced by members of the gut microbiota such as SFB.⁸,¹⁴,²⁸ Accordingly, we showed that IL-22-producing ILC3s, and in particular the NKp46⁺ subset, although present in WT germ-free mice, rapidly increased after SFB colonization, whereas IL-22 production by CD4⁺ T cells was dependent on SFB colonization, presumably reflecting the activation of SFB-specific T cells (Supplementary Fig. 6A-6B). The increased IL-22 response to SFB colonization was not associated with increased expression of the aryl hydrocarbon receptor (Ahr) and the AhR target genes AhR repressor (Ahrr) and cytochrome P450 1A1 enzyme (Cyp1a1) (Supplementary Fig. 6C), suggesting that IL-22 induction by SFB may be independent of the AhR signaling pathway. In addition, IL-22 has emerged as a key mediator in maintaining gut homeostasis and shaping the gut microbiota equilibrium,⁷,²⁴ suggesting that IL-22 may play a critical role in controlling host-SFB interactions. In line with the reduced expression of *Il23a* and *Il1b* in myeloid cells from MyD88-

deficient mice (Fig. 3G-3H and Supplementary Fig. 4F), *Il22* mRNA expression was significantly reduced in intestinal hematopoietic cells of *Myd88*⁻/⁻ mice monocolonized with SFB compared to WT mice (Fig. 5A and Supplementary Fig. 7A). Accordingly, the expression of IL-22 controlled genes, such as the antimicrobial C-type lectins *Reg3b/g*,²⁴,²⁸ fucosyltransferase 2 (*Fut2*)⁷ and serum amyloid A 1 and 2 (*Saa1/2*),⁹ was significantly reduced in *Myd88*⁻/⁻ mice (Fig. 5A). Flow cytometry analysis of small intestine *lamina propria* cells further showed that MyD88 deficiency did not affect the frequency and number of ILC3s and CD4⁺ T cells (Fig. 5B-5E) but significantly reduced IL-22 producing ILC3s, notably NKp46⁺ ILC3s, and CD4⁺ T cells (Fig. 5C-5D-5F and Supplementary Fig. 7B-7C). The parallel reduction in the number and frequency of IL-17-secreting CD4⁺ T cells in *Myd88*⁻/⁻ mice (Supplementary Fig. 7D-7E) was in line with the decreased *Il6* expression in intestinal hematopoietic and myeloid cells (Fig. 3G-3H and Supplementary Fig. 4F) and with its important role, in combination with IL-23 and IL-1, in Th17 differentiation.⁸,⁴¹,⁴² Overall, these data indicate that the ability to produce IL-22 in response to SFB colonization is impaired in both ILC3 and CD4⁺ T cells in the absence of MyD88. Conversely, and in line with observations pointing at the over-activation of ILC3 in mice lacking adaptive immunity,²⁴ the expression of *Il23a*, *Il1b* and *Il22* was increased in *Rag1*⁻/⁻ mice compared to *Myd88*⁻/⁻ and WT mice (Fig. 3H and 5A). In addition, the frequency and number of IL-22-producing RORγt⁺ ILCs in the small intestine *lamina propria* of *Rag1*⁻/⁻ mice were similar to those of WT mice (Fig. 5C-5D), while showing lower SFB loads compared to WT mice (Fig. 2). Altogether, these results suggested, in line with results in conventionally raised mice,²⁴,²⁸ that IL-22 derived from either ILC3s or CD4⁺ T cells may be key to restrict SFB growth in monocolonized mice. Consistent with this hypothesis, our results in JH⁻/⁻ mice monocolonized with SFB for 8 days showed a significantly reduced frequency and number of IL-22-producing ILC3s compared to WT mice, but a strong induction of IL-22-producing CD4⁺ T (Supplementary Fig. 8), while no defect in the control of SFB colonization was observed in these mice (Fig. 2). Taken together, these results strongly suggest that adaptive IL-22 can compensate for the absence of its innate counterpart in the control of SFB growth. To further investigate the role of IL-22 in controlling SFB growth, germ-free *Rag2xIl2rgc*⁻/⁻ mice (referred to as *Ragyc*⁻/⁻ mice thereafter), which lack both an adaptive and innate source of IL-22, and *Il22*⁻/⁻ mice were colonized with SFB. Contrasting with results in *Rag1*⁻/⁻ mice, SFB load was markedly increased in the small intestine of *Ragyc*⁻/⁻ monocolonized mice compared to WT mice (Fig. 5G), as well as in the ileum of *Il22*⁻/⁻ mice (Fig. 5H). Consistent with IL-22 deficiency, ⁹,²⁸ *Il22*⁻/⁻ SFB-monocolonized mice failed to express Reg3β and Reg3γ in ileal biopsies and showed a significantly reduced expression of the ROS- and nitrogen species-based defense marker genes *Duox2*, *Duoxa2* and *Nos2* when compared to WT mice (Supplementary Fig. 9). Of note, an even more drastic decrease in gene expression was observed in *Ragyc*⁻/⁻ monocolonized mice. In contrast, mRNA expression of *Il17a* was not altered in *Il22*⁻/⁻ mice despite significantly impaired expression of both *Saa1* and *Saa2* (Supplementary Fig. 9).⁸ Altogether, these data indicated that in SFB-monocolonized mice, MyD88-dependent expression of IL-1β and/or IL-23 by myeloid cells licenses IL-22 production by ILC3, which is critical to restrict SFB growth in the small intestine in the absence of T cell-derived IL-22.

Given the possible role of IL-1 in the induction of IL-22 secretion by ILCs,³⁹ the key role of MyD88 in IL-1-dependent signaling pathway and the down regulation of associated genes, such as *Il1b*, *Il1a* and *Il1r*, in myeloid cells from SFB-monocolonized *Myd88*⁻/⁻ mice (Fig. 3), we next sought to evaluate the effects of a reduced activity in IL-1 signaling on IL-22 response and subsequent control of SFB growth. Germ-free WT mice were treated, or not, with the recombinant IL-1 receptor (IL-1R) antagonist Anakinra one day before colonization with SFB and daily until 8 days post-colonization. *Il23a* and *Il22*, as well as *Il1b*, mRNA expression was similar in intestinal hematopoietic cells from treated and non-treated mice (Fig. 6A). Accordingly, neither the frequency nor the
<figure><img src="image_17.png" /><figcaption>**Fig. 5. IL-22 is critical to control SFB growth in monocolonized mice.** (A) Relative mRNA expression of *Il22* in hematopoietic cells isolated from the small intestine *lamina propria* and of IL-22-related marker genes in ileal biopsies from WT, *Myd88*<sup>-/-</sup> and *Rag1*<sup>-/-</sup> mice monocolonized with SFB for 8 days. (B-F) IL-22 intracellular expression analyzed by flow cytometry in viable small intestine *lamina propria* CD45<sup>+</sup> CD3e<sup>-</sup> RORgt<sup>+</sup> ILC3s (B-C), RORgt<sup>+</sup> NKp46<sup>+</sup> ILC3s (D) and CD4<sup>+</sup> T cells (E-F). Frequencies and absolute numbers for each cell subset are shown. (G) SFB quantification in jejunal and ileal biopsies from WT and immunodeficient *Rag1c<sup>-/-</sup>* mice monocolonized with SFB for 8 days (d8, full symbols) or 21 days (d21, open symbols) by qPCR. (H) SFB quantification in ileal biopsies from WT and immunodeficient *Il22*<sup>-/-</sup> mice monocolonized with SFB for 8 days. Data represent individual values and medians. Data in (A) to (F) and in (H) are pooled from at least two independent experiments. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001 between two groups, determined by a nonparametric Mann-Whitney test.</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Fig. 6.** IL-1/IL-1R signaling is largely dispensable to the control of SFB growth. Germ-free wild-type mice were treated (Anakinra) or not (Non treated) with the recombinant IL-1R antagonist Anakinra the day before colonization with SFB and daily for 8 days post-colonization. Analyses were performed on day 8. (A) Relative mRNA expression of innate immune marker genes in hematopoietic cells isolated from the small intestine *lamina propria*. (B-D) Frequencies and absolute numbers of viable CD45<sup>+</sup> small intestine *lamina propria* CD3ε<sup>-</sup> RORgt<sup>+</sup> ILCs (B) and CD4<sup>+</sup> T cells (C-D) producing IL-22 or IL-17A, analyzed by flow cytometry. (E) SFB quantification by qPCR in jejunal and ileal biopsies. Data represent individual values and medians. \*p < 0.05 or otherwise exact p values between two groups, determined by a nonparametric Mann-Whitney test.</figcaption></figure>

number of IL-22-producing ILC3s and of CD4⁺ T cells secreting either IL-22 or IL-17A were modified (Fig. 6B-6C-6D). Finally, SFB levels were similar in the jejunum of treated and non-treated mice while higher levels in the ileum of treated mice did not reach statistical significance (Fig. 6E). Altogether, these results suggested that IL-1/IL-1R signaling is dispensable for the regulation of SFB growth but may contribute to, in combination with and independently of IL-22.

## Discussion

To date, how SFB and their host directly interact to control SFB expansion, and finally contribute to maintain a symbiotic relationship with the host, remains poorly understood. Although results from the literature suggested several immune candidate mechanisms, their exact contribution remained difficult to delineate as many studies have been performed in conventionally raised mice, thus making difficult to dissociate a direct impact of SFB on the host immune responses from indirect effects dependent on other members of the microbiota. Here,
revisiting the mechanisms that control intestinal colonization by SFB, we show that the respective contribution of the adaptive and innate immune responses is determined by the presence of a complex microbiota. Indeed, in immunodeficient mice colonized with a complex microbiota containing SFB, our results indicate that the control of SFB expansion critically depends on the origin of the intestinal microbiota rather than the host immune status, supporting recent reports highlighting the key role of bacterial interactions on host immune responses and on the maintenance of intestinal microbial equilibrium.<sup>43,44,45</sup> Additionally, they suggest that the impact of immune responses on SFB described in conventional mice may be indirect, possibly via the modulation of interspecies competitions. In contrast, in SFB-monocolonized mice, while humoral adaptive immune responses are dispensable for the control of SFB growth, MyD88 signaling is critical for SFB to initiate a pivotal IL-22 production and, to a lesser extent, IL-1R-mediated signals that may cooperate with IL-22 to restrict their growth. While both innate and adaptive sources of IL-22 can take over in an immunocompetent host monocolonized with SFB, in the absence of adaptive immunity, overactivated ILC3s remain a critical source of IL-22 to rescue control of SFB. However, sustained STAT3 activation in ILC3 and intestinal epithelial cells of immunodeficient mice may interfere with metabolic processes such as lipid transport, thereby putting the host at risk.<sup>24</sup>

Strikingly, our data indicate that a MyD88-dependent circuit in hematopoietic cells is crucial for the control of SFB, while MyD88 in epithelial cells appears redundant. These results contrast with observations suggesting that epithelial MyD88 may be central to promote host-bacterial mutualism<sup>26,46</sup> but are consistent with more recent data showing that epithelial SFB attachment over circadian rhythms stimulates a MyD88-dependent signal in myeloid cells resulting in activation of ILC3<sup>47</sup> by mechanisms that remain to be elucidated.

Transcriptome analysis of intestinal myeloid cells pointed at a critical role for MyD88 in their functions, including their capacity to prime naive T cells (CD40, CCR7) and to express key cytokines known to sustain both innate and adaptive immune responses induced by SFB, especially T cell differentiation towards Th17 cell fate (IL-6, IL-23, SAA1/2), a result further supported by the significantly reduced frequency of intestinal T cells producing IL-17 or IL-22 in *Myd88<sup>-/-</sup>* SFB-monocolonized mice. We therefore demonstrate that Th17 responses induced by the microbiota, notably SFB, are partially dependent on MyD88 signaling in the hematopoietic compartment, as previously suggested.<sup>42,48</sup> Despite their strong dependency on SFB colonization, the role of Th17 cells in the control of host-SFB homeostasis remains debated. Contrasting with studies reporting an SFB overgrowth in the feces of AhR-deficient mice despite intact Th17 responses,<sup>49,50</sup> some reports identified a critical role for IL-17/IL-17R signaling in regulating SFB expansion by promoting the expression of antimicrobial factors and polymeric immunoglobulin receptor,<sup>22</sup> or by promoting the recruitment of neutrophils<sup>23</sup> in the ileum of conventional mice. However, here we show that depletion of neutrophils does not prevent the control of SFB expansion along the small intestine in monocolonized mice, demonstrating that neutrophils are, *per se*, dispensable for the control of SFB growth. Moreover, we observed that SFB colonization in *JH<sup>-/-</sup>* monocolonized mice was not different from that in WT mice over time, and that SFB overgrowth in the small intestine of *Myd88<sup>-/-</sup>* monocolonized mice was not associated with a reduced number of IgA secreting cells in the *lamina propria* or any reduction in the amount of SIgA in the feces, illustrating that MyD88 does not control SFB expansion through the induction of IgA responses. Of note, as suggested by our results in *JH<sup>-/-</sup>* conventionalized mice, defective control of SFB expansion described in IgA-deficient conventional mice<sup>21,24,30</sup> may result from an indirect effect relayed by isolated members of a dysbiotic microbiota. However, other studies reported a loss in the control of SFB colonization in monocolonized IgA-deficient mice,<sup>20,25</sup> indicating that further investigations are needed to decipher the role of IgA in the regulation of host-SFB interactions. The final output and conclusions that may be drawn about the role of IgA in controlling SFB growth may also depend on the SFB

inoculation in germ-free mice (gavage or co-housing) and the timing of colonization (from birth, at weaning or in adults), which may not be comparable between studies.

In the absence of a T cell-dependent source of IL-22, we show that MyD88-dependent stimulation of an ILC3/IL-22 circuit is critical for the direct control of SFB growth, possibly through ROS and antimicrobial peptides (Supplementary Fig. 9).<sup>9,27,28</sup> However, whether and how these effector molecules could, alone or in combination, directly control SFB colonization remains to be established. In that line, contrasting observations have been reported on the role of Reg3γ in the control of SFB growth. While an initial study suggested a regulatory role,<sup>26</sup> a recent report showed that Reg3γ expression by epithelial cells is dependent on SFB adhesion but does not control SFB load, whereas Reg3γ prevents colonization of the mucus layer by other members of the microbiota.<sup>47</sup>

IL-23 seems to be central to activate IL-22 production by ILC3 and CD4<sup>+</sup> T cells, but which myeloid cells play a critical role in the initiation and regulation of this circuit also remains unclear. A study using IL-23 reporter mice recently identified cDC2s localized in intestinal lymphoid tissues, reminiscent of CP-ILFs DCs described by Guendel et al.,<sup>35</sup> as the primary source of IL-23.<sup>36</sup> Although the role of CP-ILFs DCs cannot be formally demonstrated in the absence of specific markers and, consequently, of specific depletion model, our data showing the unique capacity of these cells to express *Il23a* provide a new line of evidence suggesting that CP-ILFs DCs may be key candidates to control IL-22 response upon SFB colonization.

Overall, our results in monocolonized mice allowed us to establish a precise hierarchy between the immune mechanisms that control SFB growth in the mouse gut and identified a critical role for MyD88-mediated immune responses in monocolonization. However, several questions remain to be elucidated. We show that MyD88 deficiency has a profound effect on the functions of mononuclear phagocytes in the small intestine of germ-free mice, which may preempt the response to colonization, making it difficult to delineate the respective roles of MyD88 in cell differentiation prior to colonization and/or in response to SFB exposure. Thus, the absence of MyD88 may indirectly, independently of bacterial colonization, prevent the expression of receptors and/or ligands necessary to sense SFB and subsequently activate IL-23 production. Alternatively, but not exclusively, SFB may need to directly stimulate MyD88-dependent pathway, especially in myeloid cells, to amplify the production of IL-22 by ILCs while activation of SFB specific T cells producing IL-22 may require MyD88-dependent expression of CD40 and CCR7 by migrating cDC2s. Although our results plead against a key role of IL-1 during SFB colonization, a role for other MyD88-dependent signaling pathway(s) cannot be excluded. Future studies are therefore needed to define how SFB are sensed in the small intestine and whether and how they can signal via MyD88 in hematopoietic cells to ignite the IL-23/IL-22 axis.

## Material and methods

### Mice

Germ-free C57BL/6 mice, either wild-type (WT) or immunodeficient (*Igh-J<sup>-/-</sup>*, *Rag1<sup>-/-</sup>*, *Myd88xTrif<sup>-/-</sup>*, *Myd88<sup>-/-</sup>*, *Trif<sup>-/-</sup>)*, were bred at the IMAGINE Institute facilities. Germ-free *Rag2xIl2rgc<sup>-/-</sup>* and *Il22<sup>-/-</sup>* mice were kindly provided by A. Macpherson (Bern University, Switzerland) and B. Ryffel (CNRS Orléans, France), respectively. Germ-free and gnotobiotic mice were maintained in plastic isolators and fed *ad libitum* on a commercial diet (R03-40; UAR) sterilized by γ-irradiation (40 kGy) and autoclaved water.

To generate bone marrow chimeras, germ-free WT and *Myd88<sup>-/-</sup>* mice were intraperitoneally (i.p.) injected with 15 mg/kg Busulfan for 4 consecutive days (day 1 to day 4), then with 100 mg/kg Cyclophosphamide on days 5 and 6. On day 8, mice were intravenously injected with 5.10<sup>6</sup> freshly prepared bone marrow cells – from femur and tibia bones – from WT or *Myd88<sup>-/-</sup>* germ-free donors. After 7–8 weeks,
efficacy of bone marrow reconstitution was controlled by B and T cell analysis by flow cytometry on blood sample. At sacrifice, efficacy of chimerism was controlled by MyD88-specific quantitative PCR on *lamina propria* isolated cells.

Neutrophil depletion protocol, adapted from,³⁸ was initiated 24 h prior to SFB colonization. Mice were i.p. injected with 50 µg anti-Ly6G (clone 1A8; Bioxcell) or isotype control (clone 2A3; Bioxcell) daily and, on alternate days, with 100 µg anti-rat kappa light chain (clone MAR18.5; Bioxcell). Efficiency of neutrophil depletion was controlled by flow cytometry on *lamina propria* isolated cells on day 8 post-colonization.

For IL-1/IL-1R signalization blocking, germ-free WT mice were i.p. injected with 20 mg/kg recombinant IL-1R antagonist Anakinra (Kine-ret®) or similar volume of PBS, the day before colonization with SFB and daily until day 8 post-colonization.

Experiments were carried out using age and gender matched groups. All animal procedures were performed according to European guidelines and have been approved by the French governmental authorities (ethic protocol #9132).

## Colonization experiments and SFB quantification

Colonization of 8–10 week-old germ-free mice was performed by gavage, twice at a 24 hr-interval, with 0.3 mL caecal homogenate from either SFB-monocolonized or conventional mice. Gnotobiotic mice were sacrificed on days 8, 21 and 56 post-colonization. For SFB quantification, intestinal biopsies were lysed using a ribolyzer FastPrep® and Lysing Matrix B beads (MP Biomedical) and DNA was extracted according to a method adapted from.⁵¹ SFB levels were monitored by qPCR using 16S rDNA primer pairs specific for SFB (see *Supplemental Information*) and SYBR-Green PCR master mix, on a CFX384TM Real-time system (Bio-Rad). SFB quantification was performed using a comparative threshold cycle (Ct) method (2⁻ΔCt) with reference to a standard curve generated with quantified plasmid DNA where SFB 16S rDNA gene has been cloned in PCR-blunt II vector (ThermoFisher) according to suppliers' instructions.

## Lamina propria cells isolation

After excision of Peyer's patches, ileum or whole small intestine *lamina propria* cells were isolated as previously described.¹³ Once washed in FACS buffer (PBS-1 % FCS), isolated cells were counted using 123 countebeads counting beads (Invitrogen) and either lysed in RLTplus buffer, prepared for *ex vivo* stimulation or directly stained for flow cytometry analysis and/or cell sorting.

## Flow cytometry and analysis of cytokine production

Purified cells were resuspended in DMEM-Glutamax (Gibco) supplemented with 10 % FCS, 1 mM Hepes, 0.02 mM Folic acid, 0.67 mM L-Arginine and 0.27 mM L-Asparagine (all from Sigma), plated in 96-well plates (1.10⁶ cells/mL) and cultured for 4 h with recombinant murine IL-23 (50 ng/mL, R&D Systems), phorbol 12-myristate 13-acetate (PMA; 100 ng/mL), ionomycin (1 mg/mL) and Brefeldin A (10 µg/mL) (all from Sigma). After washing in FACS buffer, cells were stained for phenotyping and cytokine detection. Intracellular transcription factor and cytokine staining was performed using the Foxp3/transcription factor staining buffer set (eBioscience) or the CytoFix/CytoPerm staining buffer set (BD Bioscience), respectively, according to manufacturers' instructions. Multiparameter analysis was performed on a Canto II (BD Biosciences) and analyzed with FlowJo software (V.10.8). Dead cells and doublets were excluded from all analysis. See *Supplemental Information* for antibodies.

## Myeloid cell sorting and single-cell RNA sequencing analysis

Single cell suspensions were isolated from a pool of 4 mice in each experimental condition, i.e. WT and *Myd88*⁻/⁻ mice, either germ-free or monocolonized with SFB for 8 days. B and T cells were excluded as lineage (Lin) positive cells based on the expression of B220, CD19, CD3ε, TCRβ and TCRγ. Lin⁻ CD11c⁺ MHCII⁺ CX3CR1⁺/⁻ cells were sorted among live CD45⁺ singlets. Ten thousand cells from each sample were encapsulated into droplets using 10X Chromium GEM technology. Single-cell RNA sequencing (scRNA-seq) libraries were prepared by our in-house scRNA-seq facility using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) and were sequenced using Nova-Seq6000.

Sequencing reads were aligned to the mouse transcriptome (mm10-2020-A) using Cell Ranger Pipeline 7.0.1 (10X Genomics). Quality control, data integration and downstream analyses were performed using Seurat v4.1.1. Cells with less than 500 detected genes and more than 20 % mitochondrial gene expression or less than 5 % ribosomal genes were excluded from analyses. Data from each sample were normalized and scaled using the LogNorm method, and batch effect between samples was corrected using Seurat's FindIntegratedAnchors and the CCA reduction. Principal component (PC) analysis on the 3000 most variable genes was computed and UMAP generated using the 30 most significant PCs. Clustering was performed using Seurat's FindClusters function. Cell type labels were assigned to the clusters from resolution 1 based on a manually curated list of marker genes. All clusters were annotated and 21,944 cells were kept for further analysis. To evaluate the expression of the 12 surface proteins, the antibody-derived tags count for the cells retained after filtering were loaded in the integrated Seurat object and normalized using the CLR normalization method. Differential expression analysis was performed on clusters using the FindMarkers function of Seurat on the RNA assay with default parameters (Wilcoxon testing with Bonferroni correction). From the resulting list, genes with adjusted p-values < 0.05 were selected as significant. For the visualization, a combination of ggplot2 (v3.3.6) and Scpubr (v0.4.0) was used. KEGG enrichment analysis was performed using EnrichR⁵² while Bubble plot, Volcano plot, Chord Plot and Heat-maps were generated using SRplot.⁵³

## Quantitation of mouse gene expression by real-time PCR

Ileal biopsies were lysed in RLTplus buffer using a ribolyzer Fast-Prep® and Lysing Matrix D beads (MP Biomedical). Total RNA was extracted from ileal biopsies or *lamina propria* isolated cells using the RNeasy Mini Plus Kit (Qiagen) according to manufacturer's instructions. After cDNA synthesis, qRT-PCR was performed using either TaqMan gene expression assays (AppliedBiosystems) or SYBR-Green technology (see Supplemental Informations for reagents and oligonucleotides). The relative gene expression levels were determined by the 2⁻ΔCt method with reference to HPRT expression.

## Western Blot protein quantification

Ileal biopsies were lysed in RIPA buffer (Sigma) supplemented with cComplete protease inhibitors (Roche). Protein concentration was measured by Bradford protein assay (Bio-Rad). Twenty micrograms of proteins in Laemmli buffer (Bio-Rad) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked with 5 % milk protein in TBST (0.5 % Tween, Bio-Rad), and probed with rat biotinylated anti-Reg3β or rabbit anti-Reg3γ primary antibodies. The membranes were washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies. Proteins were visualized using ChemiDoc apparatus with Imageviewer software.
Quantification of immunoglobulins and immunoglobulin-secreting cells

Quantification of total IgA in fecal samples by ELISA and assessment of immunoglobulin-secreting cells (Ig-SCs) in the small intestine *lamina propria* by ELISPOT were performed as previously described.<sup>10,13</sup>

Immunofluorescence

Jejunal and terminal ileum biopsies were fixed for 2 h at 4 °C in 4 % paraformaldehyde (Sigma), incubated in PBS-30 % sucrose overnight at 4 °C, then embedded in agarose. Vibratome tissue sections, 100 µm thick, were stained with DAPI and Phalloidin 568 (Invitrogen). Slides were examined under a SP8-SMD microscope (Leica) to detect SFB.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8. Nonparametric Mann-Whitney tests were used for two-group comparison. Differences were considered statistically significant for *p* < 0.05.

CRediT authorship contribution statement

**Marie Cherrier:** Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. **Teck Hui Teo:** Writing – review & editing, Investigation. **Renan Oliveira Corrêa:** Writing – review & editing, Investigation. **Marion Picard:** Writing – review & editing, Investigation. **Aurélie Couesnon:** Writing – review & editing, Investigation. **Corinne Lebreton:** Writing – review & editing, Investigation. **Francesco Carbone:** Writing – review & editing, Formal analysis, Data curation. **Cécile Masson:** Writing – review & editing, Formal analysis. **Pamela Schnupf:** Writing – review & editing, Investigation. **Nadine Cerf-Bensussan:** Writing – original draft, Funding acquisition, Conceptualization. **Valérie Gaboriau-Routhiau:** Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

Funding

This work was supported by institutional funding from INSERM and INRAe, by grants from the INSERM transversal program Microbiota, the LabEx IBΕΙD ANR-10-LABX-0062, the Agence Nationale de la Recherche ANR-20-CE15-0016 and ANR-23-CE15-0018-01 (to V.G.R), the ERC Advanced Grant IMMUNOBIOTA-339407 (to N.C.B) and by Fondation Princesse Grace de Monaco. Institut Imagine is supported by the Investissement d'Avenir Grant ANR-10-IAHU-01. T.H.T and R.O.C benefited from fundings from a Singapore A\*STAR post-doctorate fellowship and the ANR-20-CE15-0016, respectively. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank A. Macpherson and B. Ryffel for providing germ-free mice, members of the gnotobiology and Single Cell platforms at Institut Image, especially E. Panafieu and J. Ziga, for their technical help, and G. Eberl and H. Lelouard for critical reading of the manuscript. We acknowledge the Structure Fédérative de Recherche Necker for providing access to the Cellular Imaging, Cytometry and Bioinformatic Plateforms.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mucimm.2025.03.002.

References

1. Ansaldo E, Farley TK, Belkaid Y. Control of immunity by the microbiota. *Annu Rev Immunol.* 2021;39:449–479. https://doi.org/10.1146/annurev-immunol-093019-112348.

2. Ericsson AC, Hagan CE, Davis DJ, Franklin CL. Segmented filamentous bacteria: commensal microbes with potential effects on research. *Comp Med.* 2014;64:90–98.

3. Schnupf P, Gaboriau-Routhiau V, Sansonetti PJ, Cerf-Bensussan N. Segmented filamentous bacteria, Th17 inducers and helpers in a hostile world. *Curr Opin Microbiol.* 2017;35:100–109. https://doi.org/10.1016/j.mib.2017.03.004.

4. Schnupf P, Gaboriau-Routhiau V, Cerf-Bensussan N. Host interactions with Segmented Filamentous Bacteria: an unusual trade-off that drives the post-natal maturation of the gut immune system. *Semin Immunol.* 2013;25:342–351. https://doi.org/10.1016/j.ssim.2013.09.001.

5. Kalbermatter C, Fernandez Trigo N, Christensen S, Ganal-Vonarburg SC. Maternal microbiota, early life colonization and breast milk drive immune development in the newborn. *Front Immunol.* 2021;12, 683022. https://doi.org/10.3389/fimmu.2021.683022.

6. Davis CP, Savage DC. Habitat, succession, attachment, and morphology of segmented, filamentous microbes indigenous to the murine gastrointestinal tract. *Infect Immun.* 1974;10:948–956. https://doi.org/10.1128/iai.10.4.948-956.1974.

7. Goto Y, Obata T, Kunisawa J, et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. *Science.* 2014;345, 1254009. https://doi.org/10.1126/science.1254009.

8. Sano T, Huang W, Hall JA, et al. An IL-23R/IL-22 circuit regulates epithelial serum amyloid a to promote local effector Th17 responses. *Cell.* 2015;163:381–393. https://doi.org/10.1016/j.cell.2015.08.061.

9. Grasberger H, Gao J, Nagao-Kitamoto H, et al. Increased expression of DUOX2 is an epithelial response to mucosal dysbiosis required for immune homeostasis in mouse intestine. *Gastroenterology.* 2015;149:1849–1859. https://doi.org/10.1053/j.gastro.2015.07.062.

10. Lécuyer E, Rakotobe S, Lengliné-Garnier H, et al. Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce Gut IgA and specific T helper 17 cell responses. *Immunity.* 2014;40:608–620. https://doi.org/10.1016/j.immuni.2014.03.009.

11. Umesaki Y, Okada Y, Matsumoto S, Imaoka A, Setoyama H. Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse. *Microbiol Immunol.* 1995;39:555–562. https://doi.org/10.1111/j.1348-0421.1995.tb02242.x.

12. Talham GL, Jiang HQ, Bos NA, Cebra JJ. Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. *Infect Immun.* 1999;67:1992–2000. https://doi.org/10.1128/IAI.67.4.1992-2000.1999.

13. Gaboriau-Routhiau V, Rakotobe S, Lécuyer E, et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. *Immunity.* 2009;31:677–689. https://doi.org/10.1016/j.immuni.2009.08.020.

14. Ivanov II, Atarashi K, Manel N, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. *Cell.* 2009;139:485–498. https://doi.org/10.1016/j.cell.2009.09.033.

15. Brockmann L, Tran A, Huang Y, et al. Intestinal microbiota-specific Th17 cells possess regulatory properties and suppress effector T cells via c-MAF and IL-10. *Immunity.* 2023;56:2719–2735.e7. https://doi.org/10.1016/j.immuni.2023.11.003.

16. Schnupf P, Gaboriau-Routhiau V, Gros M, et al. Growth and host interaction of mouse segmented filamentous bacteria in vitro. *Nature.* 2015;520:99–103. https://doi.org/10.1038/nature14027.

17. Atarashi K, Tanoue T, Ando M, et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. *Cell.* 2015;163:367–380. https://doi.org/10.1016/j.cell.2015.08.058.

18. Ladinsky MS, Araujo LP, Zhang X, et al. Endocytosis of commensal antigens by intestinal epithelial cells regulates mucosal T cell homeostasis. *Science.* 2019;363. https://doi.org/10.1126/science.aat4042.

19. Snel J, Hermsen CC, Smits HJ, et al. Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice. *Can J Microbiol.* 1998;44:1177–1182. https://doi.org/10.1139/cjm-44-12-1177.

20. Jiang HQ, Bos NA, Cebra JJ. Timing, localization, and persistence of colonization by segmented filamentous bacteria in the neonatal mouse gut depend on immune status of mothers and pups. *Infect Immun.* 2001;69:3611–3617. https://doi.org/10.1128/IAI.69.3611-3617.2001.

21. Suzuki K, Meek B, Doi Y, et al. Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. *Proc Natl Acad Sci U S A.* 2004;101:1981–1986. https://doi.org/10.1073/pnas.0307317101.

22. Kumar P, Monin L, Castillo P, et al. Intestinal interleukin-17 receptor signaling mediates reciprocal control of the gut microbiota and autoimmune inflammation. *Immunity.* 2016;44:659–671. https://doi.org/10.1016/j.immuni.2016.02.007.

23. Flannigan KL, Ngo VL, Geem D, et al. IL-17A-mediated neutrophil recruitment limits expansion of segmented filamentous bacteria. *Mucosal Immunol.* 2017;10:673–684. https://doi.org/10.1038/mi.2016.80.
24. Mao K, Baptista AP, Tamoutounour S, et al. Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism. *Nature*. 2018;554: 255-259. https://doi.org/10.1038/nature25437.

25. Earley ZM, Lisicka W, Sifakis JJ, et al. GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology. *Immunity*. 2023;56:43-57.e10. https://doi.org/10.1016/j.immuni.2022.12.009.

26. Vaishnava S, Yamamoto M, Severson KM, et al. The antibacterial lectin RegIIgamma promotes the spatial segregation of microbiota and host in the intestine. *Science*. 2011;334:255-258. https://doi.org/10.1126/science.1209791.

27. Upadhyay V, Poroyko V, Kim T, et al. Lymphotoxin regulates commensal responses to enable diet-induced obesity. *Nat Immunol*. 2012;13:947-953. https://doi.org/10.1038/ni.2403.

28. Shih V-F-S, Cox J, Kljavin NM, et al. Homeostatic IL-23 receptor signaling limits Th17 response through IL-22-mediated containment of commensal microbiota. *Proc Natl Acad Sci U S A*. 2014;111:13942-13947. https://doi.org/10.1073/pnas.1323852111.

29. Ubeda C, Lipuma L, Gobourne A, et al. Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice. *J Exp Med*. 2012;209:1445-1456. https://doi.org/10.1084/jem.20120504.

30. Wang S, Charbonnier L-M, Noval Rivas M, et al. MyD88 adaptor-dependent microbial sensing by regulatory T cells promotes mucosal tolerance and enforces commensalism. *Immunity*. 2015;43:289-303. https://doi.org/10.1016/j.immuni.2015.06.014.

31. Kubinak JL, Petersen C, Stephens WZ, et al. MyD88 signaling in T cells directs IgA-mediated control of the microbiota to promote health. *Cell Host Microbe*. 2015;17: 153-163. https://doi.org/10.1016/j.chom.2014.12.009.

32. Niess JH, Adler G. Enteric flora expands gut lamina propria CX3CR1+ dendritic cells supporting inflammatory immune responses under normal and inflammatory conditions. *J Immunol*. 2010;184:2026-2037. https://doi.org/10.4049/jimmunol.0901936.

33. Persson EK, Uronen-Hansson H, Semmrich M, et al. IRF4 transcription-factor-dependent CD103(+)-CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. *Immunity*. 2013;38:958-969. https://doi.org/10.1016/j.immuni.2013.03.009.

34. Panea C, Farkas AM, Goto Y, et al. Intestinal monocyte-derived macrophages control commensal-specific Th17 responses. *Cell Rep*. 2015;12:1314-1324. https://doi.org/10.1016/j.celrep.2015.07.040.

35. Guendel F, Kofoed-Branzk M, Gronke K, et al. Group 3 innate lymphoid cells program a distinct subset of IL-22BP-producing dendritic cells demarcating solitary intestinal lymphoid tissues. *Immunity*. 2020;53:1015-1032.e8. https://doi.org/10.1016/j.immuni.2020.10.012.

36. Ohara D, Takeuchi Y, Watanabe H, et al. Notch2 with retinoic acid license IL-23 expression by intestinal EpCAM+ DCIR2+ cDC2s in mice. *J Exp Med*. 2024;221, e20230923. https://doi.org/10.1084/jem.20230923.

37. Korbecki J, Barczak K, Gutowska I, Chlubek D, Baranowska-Bosiacka I. CXCL1: Gene, promoter, regulation of expression, mRNA stability, regulation of activity in the intercellular space. *Int J Mol Sci*. 2022;23:792. https://doi.org/10.3390/ijms23020792.

38. Boivin G, Faget J, Ancey P-B, et al. Durable and controlled depletion of neutrophils in mice. *Nat Commun*. 2020;11:2762. https://doi.org/10.1038/s41467-020-16596-9.

39. Reynders A, Yessaad N, Vu Manh T-P, et al. Identity, regulation and in vivo function of gut NKp46+RORγt+ and NKp46+RORγt- lymphoid cells. *EMBO J*. 2011;30: 2934-2947. https://doi.org/10.1038/emboj.2011.201.

40. Basu R, O'Quinn DB, Silberger DJ, et al. Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria. *Immunity*. 2012;37: 1061-1075. https://doi.org/10.1016/j.immuni.2012.08.024.

41. Atarashi K, Nishimura J, Shima T, et al. ATP drives lamina propria T(H)17 cell differentiation. *Nature*. 2008;455:808-812. https://doi.org/10.1038/nature07240.

42. Shaw MH, Kamada N, Kim Y-G, Núñez G. Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. *J Exp Med*. 2012;209:251-258. https://doi.org/10.1084/jem.20111703.

43. Gould AL, Zhang V, Lamberti L, et al. Microbiome interactions shape host fitness. *Proc Natl Acad Sci U S A*. 2018;115:E11951-E11960. https://doi.org/10.1073/pnas.1809349115.

44. Rice TA, Bielecka AA, Nguyen MT, et al. Interspecies commensal interactions have nonlinear impacts on host immunity. *Cell Host Microbe*. 2022;30:988-1002.e6. https://doi.org/10.1016/j.chom.2022.05.004.

45. Kawano Y, Edwards M, Huang Y, et al. Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome. *Cell*. 2022; 185:3501-3519.e20. https://doi.org/10.1016/j.cell.2022.08.005.

46. Frantz AL, Rogier EW, Weber CR, et al. Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides. *Mucosal Immunol*. 2012;5:501-512. https://doi.org/10.1038/mi.2012.23.

47. Brooks JF, Behrendt CL, Ruhn KA, et al. The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock. *Cell*. 2021;184:4154-4167.e12. https://doi.org/10.1016/j.cell.2021.07.001.

48. Schenten D, Nish SA, Yu S, et al. Signaling through the adaptor molecule MyD88 in CD4+ T cells is required to overcome suppression by regulatory T cells. *Immunity*. 2014;40:78-90. https://doi.org/10.1016/j.immuni.2013.10.023.

49. Qiu J, Guo X, Chen Z-M-E, et al. Group 3 innate lymphoid cells inhibit T-cell-mediated intestinal inflammation through aryl hydrocarbon receptor signaling and regulation of microflora. *Immunity*. 2013;39:386-399. https://doi.org/10.1016/j.immuni.2013.08.002.

50. Murray IA, Nichols RG, Zhang L, Patterson AD, Perdew GH. Expression of the aryl hydrocarbon receptor contributes to the establishment of intestinal microbial community structure in mice. *Sci Rep*. 2016;6:33969. https://doi.org/10.1038/srep33969.

51. Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R. Molecular microbial diversity of an anaerobic digester as determined by small-subunit rDNA sequence analysis. *Appl Environ Microbiol*. 1997;63:2802-2813. https://doi.org/10.1128/aem.63.7.2802-2813.1997.

52. Chen, E.Y., Tan, C.M., Kou, Y., Duan, Q., Wang, Z., Meirelles, G.V., Clark, N.R., and Ma'ayan, A. (2013). Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128. 10.1186/1471-2105-14-128.

53. Tang D, Chen M, Huang X, et al. SRplot: A free online platform for data visualization and graphing. *PLoS One*. 2023;18, e0294236. https://doi.org/10.1371/journal.pone.0294236.