Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis

Immunoglobulin A represents the most abundant Ig in gut, which plays a critical role in the maintenance of intestinal homeostasis (1). Secretory IgA (sIgA) is constitutively released into the lumen by intestinal epithelium, where it prevents the invading pathogens from binding to mucosal epithelial cells and neutralizes the toxins (2). There are two distinct manners to produce sIgA: T cell–independent and T cell–dependent mechanisms (3, 4). Under homeostasis, sIgA is dominantly produced through T cell–independent manner and displays lower affinity. Once invading enteric pathogens are recognized by the gut immune system, high-affinity IgA is produced via T cell–dependent manner. Follicular helper T (T_FH) cells in Peyer patches (PPs) and mesenteric lymphoid nodes play a critical role in T cell–dependent IgA production through facilitating the somatic hypermutation process in germinal center (GC) B cells (5, 6). Mice and human with defective IgA production displayed enhanced susceptibility to inflammatory bowel disease (1).

It has been well documented that the gut microbiota could shape hosts’ immune system to prevent inflammation (7, 8). The symbiotic bacteria promote immune tolerance, and the pathogenic ones trigger intestinal inflammation such as inflammatory bowel disease (9). sIgA has been reported to regulate the composition of bacteria and affects its motility, thus preventing the colonization of pathogenic bacteria and promoting the integrity of gut mucosal barrier (10). Meanwhile, the interaction between the microbiota and gut immune system could stimulate the secretion of IgA into lumen (11). Therefore, the symbiotic relationship between microbiota and intestinal immune system is essential to prevent inflammation and disorders in gut.

Activating transcription factor 3 (ATF3) is an early inducible gene in response to stress or environmental stimuli (12). ATF3 is a basic leucine zipper (bZip) transcription factor and it can activate or inhibit the transcription of its target genes through binding to the canonical ATF/CREB site or the similar AP-1 site (13). Regulation of immune responses by ATF3 has been reported in distinct immune cell types. ATF3 could restrict the inflammatory responses by suppressing the expression of TLR4 (14) or CCL4 in macrophage (15). The importance of ATF3 in inflammatory disorders including, but not limited to, asthma (16), sepsis (17), autoimmune diseases (18, 19) has been recognized. Recently, we and other group reported that ATF3 plays an important role in the maintenance of gut homeostasis. Deficiency of ATF3 significantly enhanced the susceptibility of chemically induced colitis in mice (20, 21). The defective generation of T_FH cells in PPs and the resulted impaired IgA production contributed to the aggravated colitis in ATF3-deficient mice (21). However, it remains to be clarified that whether the microbiota participates in the aggravated colitis observed in ATF3-deficient mice.

In this study, a significant difference in the composition and diversity of gut microbiota between ATF3-deficient and ATF3-sufficient mice was observed. Particularly, Prevotella was notably increased in ATF3-deficient mice. Adoptive transfer of T_FH cells or IgA⁺ B cells abrogated these differences. Further microbiota depletion and fecal transplantation experiments showed that microbiota from ATF3-deficient mice significantly aggravated colitis in mice. These observations revealed a critical role of microbiota in ATF3-mediated gut homeostasis.

Atf3^fl/fl mice, CD4^cre mice, and CD4^creAtf3^fl/fl mice were previously described (21). Atf3^−/− mice were kindly provided by Dr. T. Hai (Ohio State University, Columbus, OH). C57BL/6 mice were from the Laboratory Animal Center of Sun Yat-sen University. C57BL6/Ly5.1 (CD45.1)–congenic mice and Rag1^−/− mice were purchased from Shanghai Nanfang Research Center for Model Organisms (Shanghai, China). All mice were maintained on a C57BL/6 background and kept in specific pathogen–free facilities; sex- and age-matched littermates were used as controls. Experiments in this study were performed in accordance with the Institutional Animal Care and Use Committee of Sun Yat-sen University.

For dextran sulfate sodium (DSS)–induced colitis, mice were exposed to 2.5% (w/v) DSS (MP Biomedicals) in drinking water for 7 d, followed by 2 d with normal water. Mice were examined daily for body weight and disease activity index (DAI) to evaluate the severity of colitis. DAI and colitis score were recorded as previous reports (21, 22).

T cell–mediated colitis model was performed as previous report (23). Briefly, spleen and s.c. lymph nodes from 6 to 8 wk C57BL/6J mice were digested into single-cell suspension, RBCs were depleted, and CD4⁺ T cells were enriched using Mouse CD4 T Lymphocyte Enrichment Set (catalog no. 55813; BD Biosciences). Then CD4⁺ T cells were stained with anti-CD4, anti-CD25, and anti-CD45RB. A fraction of the CD4⁺CD25⁻CD45RB^hi population was sorted using an FACS Aria III (BD Biosciences). The purity of the sorted cells was higher than 98%, as evaluated by flow cytometric analysis. Rag1^−/− mice were then i.p. injected with 0.4 × 10⁶ naive CD4 T cells in 200 μl of sterile PBS. The severity of colitis was monitored weekly.

PPs were carefully dissected from the small intestine under a stereo microscope using fine scissors. For immunofluorescence staining, PPs were embedded in OCT. Frozen sections were prepared on microslides and stored at −70°C until use. For flow cytometric analysis and sorting, PPs were digested in RPMI-1640 medium (Invitrogen) containing 100 μg/ml Liberase TM and 50 μg/ml DNase I (Sigma-Aldrich) at 37°C on a roto-mixer. After 15 min, tissues were mixed using a 1-ml pipette. The supernatant was collected and resuspended in ice-cold MACS buffer (pH 7.4; PBS plus 2% FBS and 2 mM EDTA). The remaining tissues were digested again in fresh digestion buffer, and supernatant was collected after 15 min. Supernatants from the two steps were combined and passed through 70-μm filters, and cells were collected for further analysis.

For the depletion of intestinal microbiota, wild-type (WT) or Atf3^−/− mice were administered with an antibiotic mixture (a mixture of 1 g/l ampicillin, 0.5 g/l vancomycin, 1 g/l neomycin sulfate, and 1 g/l metronidazole [AVNM]) in drinking water for 4 wk. For Rag1^−/− mice, 2-wk treatment of AVNM was applied to deplete microbiota. The efficiency of depletion was evaluated by PCR detection of 16S rRNA gene copy numbers (total eubacteria) based on bacteria per gram feces (24, 25).

For fecal transplantation, the procedure started 12 h after the withdrawal of antibiotics. Fresh intestinal contents from WT or Atf3^−/− mice were collected, weighed, and suspended in sterile PBS (200 mg in 1 ml of PBS). The solution was vortexed for 10 s, followed by centrifuge at 800 × g for 3 min to remove the large particles. Then the supernatants were collected and transplanted into the AVNM-treated mice by gavage every 3 d with 200 μl per mouse. PCR detection of 16S rRNA genes was performed to evaluate the efficiency of fecal transplantation (26, 27). The recipients were then exposed to DSS to induce colitis. In T cell–mediated colitis model, Rag1^−/− mice were colonized with feces from either WT or Atf3^−/− mice every 3 d for 8 wk after depletion of intestinal microbiota.

For analysis of IgA-coated bacteria, we followed a previous study (28). Briefly, bacterial pellets were washed twice by resuspending in 500 μl of sterile PBS and spinning for 5 min at 8000 × g. The suspension was then blocked with 500 μl of sterile PBS containing 1% BSA for 15 min. Samples were spun at 8000 × g for 5 min and stained with rat anti-mouse IgA Ab (catalog no. 1040-20; Southern Biotech) for 30 min at 4°C. Pellets were washed twice in sterile PBS and analyzed on an LSR Fortessa Flow Cytometer (BD Biosciences).

Fecal DNA was extracted using Power Fecal DNA Isolation Kit (MO BIO). Amplification of the V1-V3 region of the 16S rRNA gene was performed as previously described (29). Briefly, each reaction mixture contains 50 ng of purified DNA, 0.2 mM dNTPs, 1.5 mM MgCl₂, 2.5 U of Platinum Taq DNA polymerase, 2.5 μl of 10× PCR buffer, and 0.5 μM of each primer designed to amplify the V1-V3: V1_27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and V3_534R (5′-GCATTACCGCGGCTG CTGG-3′). A unique 12-base Golay barcode (Ns) preceded the primers to allow sample identification, and one to eight additional nucleotides were placed in front of the barcode to offset the sequencing of the primers. Amplicons were purified using the QIAquick PCR Purification Kit (Qiagen). PCR products were quantified and pooled at equimolar amounts before ligation of Illumina barcodes and adaptors, according to Illumina TruSeq Sample Preparation protocol. The completed library was sequenced on an Illumina MiSeq platform following the Illumina-recommended procedures. For data analysis, reads were preprocessed, and quality control was performed by FastQC. QIIME (Version 1.9.1) was used to handle operational taxonomic unit (OTU), sequences with ≥97% similarity were assigned to the same OTUs, and the representative sequence for each OTU was screened for further taxonomy assignment using the Ribosomal Database Project (Release 11). Data analysis of α diversity and β diversity was performed on QIIME, the visualization was displayed by R software (Version 3.3.1).

Fecal genomic DNA was extracted by the Power Fecal DNA Isolation Kit (MO BIO). Bacterial genomic DNA was quantified by eubacteria-specific 16S primers. The abundance of the indicated bacteria was analyzed with a SYBR Premix ExTaq Kit (Takara Bio) at CFX connect Optics Module (Bio-Rad Laboratories). The relative abundance was calculated by comparing the cycle threshold values using the 2^{−ΔΔCycle threshold} method. The primer sequences for bacteria used were listed: Universal 16S (5′-ACTCCTACGGGAGGCAGCAGT-3′ and 5′-ATTACCGCGGCTGCTGGC-3′) (26), Lactobacillus (5′-TGGAAACAGRTGCTAATACCG-3′ and 5′-GTCCATTGTGGAA GATTCCC-3′) (30), Prevotella (5′-CACCAAGGCGACGATCA-3′ and 5′-GGATA ACGCCYGGACCT-3′) (31), Clostridium XIVa (5′-AAATGACGGTACCTGACT AA-3′ and 5′-CTTTGAGTTTCATTCTTGCGAA-3′) (31), Bacteroides spp (5′-CGAT GGATAGGGGTTCTGAGAGGA-3′ and 5′-GCTGGCACGGAGTTAGCCGA-3′) (31), Akkermansia (5′-CAGCACGTGAAGGTGGGGAC-3′, 5′-CCTTGCGGTTGGC TTCAGAT-3′) (31), Prevotellaceae (5′-CCAGCCAAGTAGCGTGCA-3′ and 5′-TG GACCTTCCGTATTACC-3′) (32), Prevotella copri (5′-CCGGACTCCTGCCCCTGCAA-3′ and 5′-GTTGCGCCAGGCACTGCGAT-3′) (26), Prevotella falsenii (5′-CGTGGACCAAA GTTATTTCGGTAGA-3′ and 5′-AAACAACCCCTCATTTCTCA-3′) (33), Prevotella intermedia (5′-AATACCCGATGTTGTCCACA-3′ and 5′-TTAGCCGGTCCTT ATTCGAA-3′) (34), and Prevotella ruminicola (5′-GAAAGTCGGATTAATGCTCTATGTTG-3′ and 5′-CATCCTA TAGCGGTA AACCTTTGG-3′) (35).

Single-cell suspensions were blocked with anti-CD16/CD32 before staining with fluorochrome-conjugated Abs. Staining of surface markers was performed in PBS containing 2% BSA. For intracellular staining, cells were fixed with the Transcription Factor Staining Buffer Set (eBioscience) after staining with Abs against surface markers. Staining of Abs against specific transcription factors was performed in 1× permeabilization buffer (eBioscience) for 1 h at room temperature. To detect cytokine production, cells were stimulated with 50 ng/ml PMA, 1 μg/ml ionomycin, and 2 μg/ml brefeldin A for 4 h at 37°C before analysis. The Abs used were listed below: CD4 (GK1.5; eBioscience), CD25 (PC61; BioLegend), CD45RB (C363-16A; BioLegend), CD45.1 (A20; BioLegend), CD45.2 (104; eBioscience), CXCR5-biotin (2G8 (RUO); BD Bioscience), streptavidin (eBioscience), PD-1 (J43; eBioscience), ICOS (7E.16G9; eBioscience), Bcl6 (K112-91; BD Bioscience), B220 (RA-3-6B2; BioLegend), IgA (1040-20; Southern Biotech), CD95 (Fas) (15A7; eBioscience), GL7 (GL-7; eBioscience), PNA (Sigma-Aldrich), CD138 (281-2; BioLegend), Foxp3 (FJK-16S; eBioscience), recombinant mouse IL-21R Fc chimera (596-MR; R&D Systems), R-PE–conjugated anti–human IgG (109-116-098; Jackson ImmunoResearch), Ki67 (16A8; BioLegend), and annexin V (556420; BD Bioscience). Fluorochrome-labeled cells were acquired on an LSR Fortessa Flow Cytometer (BD Biosciences). Data were analyzed with FlowJo V10.0.7 software (FlowJo, Ashland, Oregon).

The p values were calculated using unpaired two-tailed Student t tests in GraphPad Prism. A p value <0.05 is considered statistically significant. The p values are as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The following methods, including H&E staining, immunofluorescence staining, and ELISA, were performed as previously reported (21).

Consistent with previous report, whole-body knockout of ATF3 (Atf3^−/−) led to aggravated colitis when the mice were challenged with 2.5% DSS, as revealed by increased weight loss, DAI, colon shortening, as well as colitis score (Supplemental Fig. 1A–F). When mice were challenged with higher dose of DSS (3%), Atf3^−/− mice displayed a dramatically lower chances survival than WT control (Supplemental Fig. 1G). In agreement with the aggravated colitis, PPs from Atf3^−/− mice were severely shrunken in size (Supplemental Fig. 2A), and their cellularity was only approximately half of those from WT littermates (Supplemental Fig. 2B). Consistent with the previous report (21), the frequency of T_FH cells in PPs from Atf3^−/− mice, as well as their secretion of IL-21 cytokine, were pronouncedly reduced when compared with those of WT control (Supplemental Fig. 2C). No significant differences were observed in the level of regulatory T_FH cells (Supplemental Fig. 2D). PP T_FH cells from Atf3^−/− mice displayed lower expression of T_FH markers (including Bcl6, PD-1, and ICOS) when compared with ATF3-sufficient T_FH cells (Supplemental Fig. 2E). The reduction of PP T_FH cells in Atf3^−/− mice was not caused by the changes in cell proliferation or cell apoptosis, as represented by Ki-67 staining or annexin V staining, respectively (Supplemental Fig. 2F, 2G). As a consequence of the impaired T_FH cells, GC reaction was apparently diminished in PPs from Atf3^−/− mice, as evaluated by flow cytometric analysis of GC B cells and immunofluorescence staining of GC area (Supplemental Fig. 3A, 3B). The defects in T_FH/GC B axis subsequently reduced IgA⁺ B cells in PPs, as well as the levels of IgA⁺ plasma cells in lamina propria of the small intestine and colon in Atf3^−/− mice (Supplemental Fig. 3C–E). The lower amounts of intestinal IgA in Atf3^−/− mice were further confirmed by ELISA (Supplemental Fig. 3F). It is important to point out that under steady-state conditions PPs from ATF3-deficient mice also showed pronounced lower cellularity when compared with WT control (data not shown). The reduction of PP T_FH cells as well as diminished GC reaction and IgA production were also observed in Atf3^−/− mice in the absence of DSS (data not shown). Collectively, these observations indicate that whole-body knockout of ATF3 impaired the T_FH/GC B/IgA axis in gut.

Intestinal IgA plays a critical role in the regulation of the composition and diversity of microbiota, therefore promoting immune homeostasis in gut (36). We next sought to investigate whether the impaired T_FH/IgA axis in gut impacts intestinal microbiota in Atf3^−/− mice. It was found that the proportion of IgA-bound microbiota was substantially diminished in Atf3^−/− mice when compared with WT littermates (Fig. 1A). High-throughput 16S rRNA sequencing of fecal DNA showed that the diversity of gut microbiota of Atf3^−/− mice was significantly lower when compared with WT controls, as indicated by the numbers of OTU (Fig. 1B, 1C). Principal coordinates analysis of the β (between-sample) diversity revealed a distinct composition of microbial community between Atf3^−/− mice and WT controls (Fig. 1D). Further Shannon index showed that the richness and biodiversity of species were significantly reduced in Atf3^−/− mice (Fig. 1E). Evaluation of relative abundance of distinct phylum showed that Firmicutes was obviously increased, whereas Bacteriodetes was pronouncedly decreased in Atf3^−/− mice (Fig. 1F). Interestingly, colitogenic Prevotella (26) and Akkermansia (37) were much more abundant, whereas intestinal Lactobacillus (30) was less abundant in Atf3^−/− mice, as indicated by heatmap depicting (Fig. 1G), and percentage analysis (Fig. 1H), in genus level. These observations indicate that ATF3 deficiency causes dysbiosis of gut microbiota.

To confirm the changes in microbiota composition, quantitative PCR (qPCR) analysis of 16S rRNA genes were next performed. The relative abundance of colitogenic taxa Prevotella and Bacteriodes were remarkably elevated, whereas Lactobacillus taxa were dramatically decreased in Atf3^−/− mice when compared with WT littermates (Fig. 2A). DNA electrophoresis confirmed the dramatic changes of Prevotella and Lactobacillus in Atf3^−/− mice (Fig. 2B). Moreover, Prevotellaceae family, which Prevotella genus belongs to, was obviously upregulated in Atf3^−/− mice (Fig. 2C, 2D). Evaluation of the representative species of Prevotella genus showed that P. copri was clearly increased in Atf3^−/− mice when compared with WT control (Fig. 2E, 2F).

We next sought to determine whether the dysbiosis observed in Atf3^−/− mice was caused by the defective T_FH/IgA axis, intestinal microbiota from CD4^creAtf3^fl/fl mice, and Atf3^fl/fl littermates were evaluated. Consistently, flow cytometric analysis showed that the proportion of IgA-bound bacteria was significantly reduced in CD4^creAtf3^fl/fl mice when compared with Atf3^fl/fl control (Supplemental Fig. 4A). Further 16S rRNA sequencing of fecal DNA confirmed a clear distinction in microbiota composition between CD4^creAtf3^fl/fl and Atf3^fl/fl mice (Supplemental Fig. 4B–F). Prevotella taxa were consistently increased in CD4^creAtf3^fl/fl mice (Supplemental Fig. 4F). Further qPCR and DNA electrophoresis showed that Prevotella genus (Fig. 2G, 2H), Prevotellaceae family (Fig. 2I, 2J), and P. copri species (Fig. 2K, 2L), were significantly elevated in feces from CD4^creAtf3^fl/fl mice as compared with Atf3^fl/fl littermates. Collectively, these observations reveal a critical role of T cells in the intestinal dysbiosis observed in ATF3-deficient mice.

To determine the causal relationship between the impaired T_FH/IgA axis and intestinal dysbiosis in ATF3-deficient mice, adoptive transfer of T_FH cells or IgA⁺ B cells was performed as described previously (21). T_FH cells or IgA⁺ B cells from PPs of CD45.1⁺-congenic mice were adoptively transferred into CD4^creAtf3^fl/fl mice, followed by DSS treatment. Fecal samples were collected for qPCR and DNA electrophoresis analysis. Results showed that the abundance of Prevotella taxa was dramatically decreased after adoptive transfer of either T_FH cells (Fig. 3A, 3B) or IgA⁺ B cells (Fig. 3C, 3D) into CD4^creAtf3^fl/fl mice. These observations indicate that the defective T_FH-IgA axis contributes to the intestinal dysbiosis in the absence of ATF3.

To investigate whether the dysbiosis contributes to the aggravated colitis observed in ATF3-deficient mice, WT and Atf3^−/− mice were administered with a broad spectrum of antibiotics (including AVNM) in drinking water for 4 wk, followed by exposure to 2.5% DSS (w/v) (Fig. 4A). As expected, total bacteria in gut were apparently reduced after 4 wk of AVNM treatment in both WT and Atf3^−/− mice (Fig. 4B). Depletion of microbiota significantly alleviated the disease severity of colitis in Atf3^−/− mice, which were comparable to AVNM-treated WT littermates. The disease symptoms of colitis were represented by body weight loss, DAI, and shortened colon length (Fig. 4C–F). The alleviation of colitis was further confirmed by histological staining of colon tissues and the quantitated colitis score (Fig. 4G, 4H). Collectively, these data indicate that gut microbiota from Atf3^−/− mice aggravated colitis.

To further confirm the causal relationship between the gut dysbiosis and aggravated colitis in Atf3^−/− mice, fecal transplantation experiments were performed. WT and Atf3^−/− mice were initially treated with AVNM to deplete gut microbiota, followed by fecal transplantation from either WT or Atf3^−/− mice every 3 d for 3 wk. Mice were then subjected to DSS challenge (Fig. 5A). The abundance of total microbiota was examined to confirm the efficiency of AVNM treatment and fecal transplantation (Fig. 5B). Evaluation of colitis symptoms showed that the recipients receiving microbiota from Atf3^−/− mice displayed significantly higher susceptibility to colitis when compared with those received microbiota from WT controls (Fig. 5C–H). Moreover, transplantation of microbiota from WT mice clearly alleviated the colitis severity in Atf3^−/− recipients (Fig. 5C–H). These observations indicate that gut microbiota from Atf3^−/− mice enhance the susceptibility of colitis in mice.

The colitogenic effect of microbiota from Atf3^−/− mice on colitis was further alternatively verified in T cell–mediated colitis model. Rag1^−/− mice were administered with antibiotics for 2 wk, followed by fecal transplantation from either WT or Atf3^−/− mice for 8 wk. Two weeks after the initiation of fecal transplantation, mice were i.p. transferred with naive CD4⁺ T cells (CD4⁺CD25⁻CD45RB^hi) (Fig. 6A). The total abundance of gut microbiota was evaluated to confirm the efficiency of AVNM treatment and fecal transplantation (Fig. 6B). Consistently, it was found that Rag1^−/− mice colonized with Atf3^−/− feces displayed much severer symptoms of colitis, including sharper body weight loss (Fig. 6C), shorter colon length (Fig. 6D, 6E), as well as increased colonic inflammation severity indicated by histological staining and colon histology scores (Fig. 6F, 6G). Taken together, these observations suggest that the effect of microbiota from Atf3^−/− mice on colitis is a common finding.

IgA is the most abundant Ig in the gut. The interaction between IgA and commensal bacteria plays a critical role in the maintenance of gut mucosal homeostasis. Recently, several molecules were reported to mediate the interaction between IgA and microbiota in gut, including the inhibitory coreceptor PD-1 (38), innate adaptor MyD88 (28), and the ATP-gated ionotropic P2 × 7 receptor (39). Furthermore, the interaction between selected IgA and the diversified microbiota facilitated the expansion of Foxp3⁺ T cells, which, in turn, induced GCs reaction and IgA production by forming a symbiotic regulatory loop in the gut (40). In this study, we demonstrated that stress-inducible transcription factor ATF3 represents another molecule mediating the cross-talk between T_FH-dependent IgA production and microbiota in gut.

It was reported that IgA-bound bacteria were prone to be colitogenic, and therefore promoting the development of colitis in both adults and infants (36, 41). In this study, a dramatic reduction in the proportion of IgA-bound bacterial was observed in both Atf3^−/− and CD4^creAtf3^fl/fl mice when compared with their littermate controls. Further 16S rRNA sequencing analysis failed to show significant differences in the composition of IgA-bound microbiota between ATF3-deficient and ATF3-sufficient mice (data not shown). It is possible that the defective IgA production in ATF3-deficient mice leads to less pathogenic bacteria bound by IgA, and therefore facilitating their invasion into the gut mucosal barrier and eventually aggravating colitis.

The most enriched bacteria in gut were two phyla: Bacteroidetes and Firmicutes (42). Prevotella belongs to the Prevotellaceae family and represents one genus microbe of Bacteroidetes. Prevotella is more prevalent in non-Westernized population who prefer plant-rich diet (43). However, there were several reports indicating that Prevotella genus and its species P. copri participate in the pathogenesis of chronic inflammatory disorders such as colitis (44), rheumatoid arthritis (26), osteomyelitis (45), and ankylosing spondylitis (46). Animals colonized with P. copri showed higher susceptibility to DSS-induced colitis (26). Mice deficient in IL-17F were resistant to colitis and displayed reduced abundance in Prevotella (47). These investigations suggested that Prevotella may promote inflammation in gut. In this study, it was found that Prevotella genus, especially P. copri species, was remarkably elevated in ATF3-deficient mice when compared with littermate controls. This was consistent with the aggravated colitis in ATF3-deficient mice, indicating that the elevation of P. copri may drive murine colitis in the absence of ATF3. Depletion of P. copri in ATF3-deficient mice may be helpful to clarify this possibility.

Although the deficiency of ATF3 caused aggravated colitis in mice, there were no available reports in public databases regarding ATF3 mutations in patients suffering intestinal inflammation, including The Human Gene Mutation Database, Online Mendelian Inheritance in Man, and ClinVar of National Center for Biotechnology Information. We previously showed that ATF3 expression in colonic tissues from ulcerative colitis patients was negatively correlated with the severity of ulcerative colitis (21). The clinical significance of ATF3 in colitis and dysbiosis of gut microbiota, however, deserves further investigation.

In conclusion, this study reveals ATF3 as an important mediator of IgA-microbiota interaction in gut, which acts as a protective mechanism underlying intestinal homeostasis.

The authors have no financial conflicts of interest.