Impaired Autophagy in Intestinal Epithelial Cells Alters GutMicrobiota and Host Immune Responses

Lingyu Yang,a Chao Liu,b Wenjing Zhao,a Chuan He,a Jinmei Ding,a Ronghua Dai,a Ke Xu,a Lu Xiao,a Lingxiao Luo,a

Shuyun Liu,a Wei Li,b He Menga

aSchool of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai Key Laboratory of VeterinaryBiotechnology, Shanghai, People's Republic of China

bState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy ofSciences, Beijing, People's Republic of China

ABSTRACT Establishing and maintaining beneficial interactions between the hostand associated gut microbiota are pivotal requirements for host health. Autophagyis an important catabolic recycling pathway that degrades long-lived proteins andsome organelles by lysosome to maintain cellular homeostasis. Although impairedautophagy is thought to be closely correlated with Crohn’s disease (CD), the func-tional role of autophagy in the maintenance of gut microbiota is poorly understood.As autophagy-related 5 (Atg5) is a key gene associated with the extension of thephagophoric membrane in autophagic vesicles, we established a gut-specific Atg5knockout mouse model, and we found that the disruption of autophagic flux in theintenstinal epithelium cells dramatically altered the composition of the gut mi-crobiota and reduced alpha diversity. Microbial function prediction indicated thatthe pathway allocated for infectious diseases was enriched in Atg5�/� mice.“Candidatus Arthromitus” and the Pasteurellaceae family were increased inAtg5�/� mice, whereas Akkermansia muciniphila and the Lachnospiraceae familywere reduced. Transcriptome analysis revealed that two key inflammatory boweldisease (IBD)-related transcription factors, RORC and TBX21, of host cells were up-regulated in Atg5�/� mice, thus elevating the Muc2-related immunological response.The findings suggest that intestinal autophagy plays a vital role in modulating thediversity and composition of gut microbiota.

IMPORTANCE The homeostasis of host-microbiota interactions is of great impor-tance to host health. Previous studies demonstrated that disruption of autophagywas linked to inflammatory bowel disease. However, the interaction mechanism ofgut microbiota regulated by autophagy was obscure. In an intestinal epithelium-specific autophagy-related 5 (Atg5) knockout mouse model, we observed a signifi-cant alteration and decreased diversity in the gut microbiota of Atg5-deficient micecompared with that of wild-type mice. Although the numbers of some organisms(e.g., Akkermansia muciniphila and members of the Lachnospiraceae family) associ-ated with the control of inflammation decreased, those of proinflammationory bac-teria (e.g., “Candidatus Arthromitus”) and potential pathogens (the Pasteurellaceaefamily) increased in Atg5�/� mice. Differential gene expression analysis revealed thattwo key genes, RORC and TBX21, involved in inflammatory bowel disease were up-regulated in Atg5�/� mice. Our study suggests that Atg5 deficiency results in an im-balance of the host-microbe interaction and deterioration of the gut microenviron-ment.

KEYWORDS gut microbiota, autophagy, Atg5, abnormal Paneth cell, host-microbeinteraction

Received 20 April 2018 Accepted 9 July 2018

Accepted manuscript posted online 13 July2018

Citation Yang L, Liu C, Zhao W, He C, Ding J,Dai R, Xu K, Xiao L, Luo L, Liu S, Li W, Meng H.2018. Impaired autophagy in intestinalepithelial cells alters gut microbiota and hostimmune responses. Appl Environ Microbiol84:e00880-18. https://doi.org/10.1128/AEM.00880-18.

Editor Andrew J. McBain, University ofManchester

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Wei Li,leways@ioz.ac.cn, or He Meng,menghe@sjtu.edu.cn.

L.Y., C.L., and W.Z. contributed equally to thisarticle.

MICROBIAL ECOLOGY

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The mammalian gastrointestinal tract is colonized by trillions of bacteria, which haveinteracted with the host over millions of years (1). Intestinal homeostasis involves

reciprocal communication between the host and gut microbiota and plays vital roles inthe host’s physiological metabolism and immune and nervous systems (2–4). A previ-ous review revealed that an imbalance in the composition of the gut microbiota wasassociated with intestinal diseases (5). Host-related genetic factors, especially thoseassociated with the immune system, shape the gut microbiota, as shown by manystudies which demonstrated that deficiencies in specific genes involved in immuneresponses played important roles in regulating the distribution of gut microbiota. Forexample, a mutation of nucleotide-binding oligomerization domain protein 1 influ-enced the structure and composition of gut microbiota due to the failure of pepti-doglycan recognition and enhanced systemic innate immunity (6). A deficiency inrecombination-activating gene 2 or deletion of immunoglobulin A affected gut micro-bial composition and led to increased abundance of segmented filamentous bacteria(SFB) (7). Research also demonstrated that specific microbes modulated the generationof some subclasses of immune cell populations and reestablished a new steady-stateimmune system. Colonization of the gut by SFB induced an increase in T helper 1 (TH1)and T helper 17 (TH17) (8) cells. Polysaccharide A of Bacteroides fragilis resulted ininterleukin 10 (IL-10) feedback by intestinal T cells, which restrained the accumulationof TH17 cells and potentially damaged the mucosal barrier (9). Although cross talkbetween the gut microbiota and host immune system is well known, the interactionmechanisms remain obscure.

Autophagy is a membrane-trafficking process that primarily involves lysosomaldegradation of cellular components, such as long-lived proteins and organelles. Au-tophagy is indispensable for cell growth, adaptation to stressful conditions, intracellularquality control, renovation during development and differentiation, and organellebiogenesis (10–13). The process of autophagy includes the initiation, nucleation, ex-pansion, maturation, and degradation; more than 40 autophagy-related (ATG) proteinshave been characterized during autophagy (14, 15). Nutrient, carbon, or nucleic acidstarvation and some physiological stress stimuli could trigger the induction of au-tophagy in many organisms (16, 17). Autophagy primarily serves as a modulator toprevent organisms from suffering due to diverse pathologies (18). For example, re-search reported that various autophagy-related genes, such as Atg16L1, Atg5, and Atg7,played important roles as intestinal immunological barriers in small intestinal Panethcells and that the disruption of these genes was correlated with Crohn’s disease (CD)(19). Research also reported that Atg7 deficiency influenced the composition andstructure of gut microbiota and that it was involved in inhibiting tumorigenesis (20).However, when the mutation was accompanied by Apc deletion, it contributed to theoccurrence of colorectal cancer (20). The structural composition of gut microbiota ofcolorectal cancer patients was imbalanced compared with that of a healthy population(21). The functional role of autophagy in interacting with the gut microbiota isunknown. Given the role of the interaction between the host and gut microbiota inregulating the physiological activities of the host, it is imperative that the potentialmechanism of interaction between the gut microbiota and autophagy is explored.

Previous research demonstrated that Atg5 was required for autophagosome forma-tion (22) and that it was necessary for maintaining the function of Paneth cells (19, 23).Antimicrobials secreted by Paneth cells were disseminated in the mucous layer andmucins, especially mucins produced by goblet cells, which, together with antimicrobi-als, prevented microbes from contacting the epithelial surface and being exposed toimmune cells directly by forming a physical and biochemical barrier (24). Other studiesreported that Atg5 contributed to antibiosis, especially by increasing susceptibility toinfection and controlling dissemination of Listeria monocytogenes, Mycobacterium tu-berculosis, and Salmonella (25–29). Thus, Atg5 was selected for further studies to explorethe mechanism of interaction between autophagy and the gut microbiota.

A previous study reported that the influence of Toll-like receptor deficiency on gutmicrobiota was weaker under homeostatic conditions than under long-term breeding

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(30). In our previous studies, we demonstrated that the host genotype markedlyinfluenced the composition of the gut microbiota after controlling for other factors,including the effects of diet and the environment (31–33). In the present study, all theexperimental animals were maintained in essentially identical environments, except fordifferences in host genotype. To that end, a gut-specific Atg5 knockout mouse modelwas established. Then we applied next-generation sequencing technology to identifythe composition of gut microbiota from the duodenum, jejunum-ileum, cecum, colon,and feces of C57BL/6J mice and C57BL/6J Atg5�/� mice. In addition, according to theanalysis of the transcriptome, we found that some components of the immune systemwere clearly increased in the gut of the C57BL/6J Atg5�/� mice. By integrating theseresults with findings regarding the gut microbiota, we aimed to explore the possiblemechanism of interaction between host autophagy and gut microbiota.

RESULTSEstablishment of an intestinal epithelium-specific Atg5 knockout mouse

model. To investigate the functional role of autophagy in intestinal disease, wespecifically knocked out Atg5 in the intestinal epithelium by crossing C57BL/6J micewith a floxed allele of Atg5 (34) with C57BL/6J PVillin-Cre mice. The latter, whichspecifically express Cre recombinase in the intestinal epithelium from the duodenum tothe cecum, were generated by the Nanjing Biomedical Research Institute of NanjingUniversity (T000142), which could specifically conditionally knock out the autophagygene Atg5 in the intestinal epithelium of the small intestine and colon. These intestinalepithelium-specific Atg5 knockout C57BL/6J mice were called Atg5�/� mice. To exam-ine whether the knockout was indeed successful, we first detected the protein level ofATG5 in intestinal epithelium homogenates from Atg5�/� and Atg5�/� mice by im-munoblotting and found that ATG5 was dramatically reduced in the intestinal epithe-lium of Atg5�/� mice compared with that of control groups (Fig. 1a). Consistent withthe function of Atg5 in autophagy (35), LC3-I accumulated in Atg5�/� mice, whereas themembrane-associated form, LC3-II, and the autophagic substrate, SQSTM1/p62, did not

FIG 1 Intestinal epithelium-specific knockout of Atg5 in mice. (a) Autophagic flux was disrupted in Atg5-deficient intestinal epithelia.The intestinal epithelia of Atg5�/� and Atg5�/� mice were extracted and analyzed by immunoblotting using anti-ATG5, anti-p62, andanti-LC3 antibodies. Actin served as a loading control. (b) LC3 punctate structures disappeared in Atg5-deficient intestinal epithelia.Immunofluorescence detection of LC3 (green) in Atg5�/� and Atg5�/� mouse intestinal epithelia is shown. The nucleus (blue) wascounterstained with DAPI. (c) Morphological abnormalities in Atg5-deficient Paneth cells, as shown by a histological examination ofthe intestines in Atg5�/� and Atg5�/� mice.

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(Fig. 1a). An immunofluorescence analysis of LC3 showed the disappearance of punc-tate structures (representing autophagosomes) in intestinal epithelial cells of Atg5�/�

mice (Fig. 1b). The results suggested that autophagic flux was destroyed in theintestinal epithelium of Atg5 knockout mice. Previous research reported that disruptionof autophagy in the intestinal epithelium caused abnormalities in Paneth cells associ-ated with CD (19, 20). Remarkably, Paneth cells of Atg5-deficient mice showed mor-phological abnormalities (Fig. 1c). These results indicated that we had generated anintestinal epithelium autophagy-deficient mouse model and that disruption of au-tophagy in the intestinal epithelium was responsible for the observed Paneth cellpathology.

Atg5 deficiency altered gut microbiota composition and structure. To study theeffect of knockout of Atg5 on the gut microbiota, we collected the intestinal contentsof the duodenum, jejunum-ileum, cecum, and colon, as well as feces, and analyzed themicrobiome by deep sequencing. A total of 9,342,844 filtered reads resulted fromhigh-throughput sequencing of 60 samples, and they were classified into differenttaxonomies and diversity analyses. Taxonomies that appeared in at least one-fourth of60 samples were regarded as common, and their relative abundance in samples wasemployed for further analyses. Phylogenetic dissimilarities between communities ofmicroorganisms among the microbiota samples were analyzed using UniFrac metrics(36). First, on the basis of the UniFrac metrics, the canonical analysis of principalcoordinates (CAP) showed separation among the samples from different intestinallocations (Fig. 2a). Samples derived from the large intestine (cecum, colon, and feces)clustered with each other, and samples from the small intestine (duodenum andjejunum-ileum) clustered together. The results of a correlation analysis indicated thatthe correlation coefficient was very high, over 0.85 (P � 0.01), among the largeintestinal segments but was less than 0.78 (P � 0.01) for the correlation between largeand small intestinal segments (see Table S3 in the supplemental material), suggestingthat the distribution of the microbiome in different intestinal segments was diverse. Inaddition, samples that originated from different genotypes clustered together (Fig. 2a).Atg5 status did influence the composition of gut microbiota, as confirmed by anonparametric permutational multivariate analysis, Adonis (P � 0.001; duodenum,jejunum-ileum, cecum, colon, and feces from Atg5�/� and Atg5�/� mice). We furtherconfirmed that samples from Atg5�/� and Atg5�/� mice were significantly differentusing analysis of similarities (ANOSIM) (P � 0.001, P � 0.001, P � 0.05, P � 0.001, andP � 0.05). Alpha diversity was evaluated by the Simpson and Shannon indices. TheSimpson and Shannon indices for colon and feces were significantly different (P � 0.05)for Atg5�/� and Atg5�/� mice (Fig. 3a), pointing to substantially decreased alphadiversity in Atg5�/� mice. A Venn diagram of operational taxonomic unit (OTU)numbers further confirmed the conclusion that reduced for diversity was caused byAtg5 deficiency (Fig. 3b). However, alpha diversity and OTU numbers in other intestinalsegments were similar in the two groups (P � 0.05). Thus, these results revealed thathost genotypes played a vital role in shaping the gut microbiome.

Comparisons of bacterial taxonomy in Atg5�/� and Atg5�/� mice. To furtherinvestigate the gut microbiota in autophagy-deficient mice, we compared the variationin bacterial taxa between Atg5�/� and Atg5�/� mice. Firmicutes, Proteobacteria, andBacteroidetes, which generally constituted more than 90% of the entire sequences ineach group, dominated the gut microbiome of mice. However, Cyanobacteria, Actino-bacteria, Tenericutes, and Verrucomicrobia were present as minor constituents (Fig. S1).Verrucomicrobia were significantly decreased in Atg5�/� mice (P � 0.05) (Fig. 2b).

Using linear discriminant analysis (LDA) effect size (LEfSe), an algorithm that canrobustly identify features that are significantly different among biological groups, weidentified significant biomarkers at various taxonomic levels. At the family level, Pas-teurellaceae were enriched in whole intestinal segments of Atg5�/� mice, especially inthe jejunum-ileum (LDA � 4.02) (Fig. 4a). Clostridiaceae (LDA � 4.08) also accumulatedin the jejunum-ileum in Atg5�/� mice (Fig. 4b). In the duodenum, Pseudomonadaceae

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(LDA � 4.74) and Ruminococcaceae (LDA � 4.12) were enriched in Atg5�/� and theAtg5�/� groups, respectively (Fig. 4c and d). In large intestinal segments (cecum, colon,and feces) of Atg5�/� mice, Lachnospiraceae (LDA � 4.40) and Verrucomicrobiaceae(LDA � 3.15) were present at significantly higher levels in Atg5�/� mice (Fig. 4e and f).In contrast, Lactobacillaceae (LDA � 3.86) levels were lower in the cecum of Atg5�/�

mice (Fig. 4g).

FIG 2 (a) Canonical analysis of principal coordinates based on unweighted UniFrac metrics; (b) relative abundance distribution ofVerrucomicrobia in Atg5�/� and Atg5�/� mice.

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At the genus level, the results showed that 23, 25, 33, 35, and 23 genera weresignificantly altered in the duodenum, jejunum-ileum, cecum, colon, and feces, respec-tively, of Atg5�/� mice compared with the respective anatomical sites in control mice(Table S4). Among these altered genera, 20 were identified to be the most alteredbiomarkers (LDA score � 3.5 and P � 0.05) (Table 1). Of these, we observed that withthe exception of the Sutterella and Veillonella genera, seven genera (Pseudomonas,Aggregatibacter, Klebsiella, “Candidatus Arthromitus,” Mannheimia, Gemella, and Strep-tococcus) were all enriched in the jejunum-ileum of Atg5�/� mice. In particular, theabundance of “Candidatus Arthromitus” was dramatically increased (LDA score � 4.09and P � 0.05) (Table 1). In the colons of Atg5�/� mice, the relative abundance of sevengenera (Acinetobacter, Akkermansia, Ochrobactrum, Brevibacterium, Ruminococcus, Sph-ingomonas and Meiothermus) was all significantly decreased. The abundance of Akker-mansia was decreased in large intestine segments of Atg5�/� mice, including thececum, colon, and feces (LDA score � 3.5 and P � 0.05), whereas Lactobacillus wasenriched in the cecum of Atg5�/� mice (LDA score � 4.36 and P � 0.05) (Table 1).

Next, we investigated the influence of autophagy deficiency at the species level. InAtg5�/� mice, 7, 10, 12, 14, and 13 species were significantly different from the controls

FIG 3 Comparison of alpha diversity. (a) Shannon and Simpson index comparisons of gut microbiota inthe colon and feces of Atg5�/� and Atg5�/� mice; (b) OTUs detected in the colon and feces of Atg5�/�

and Atg5�/� mice.

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in the duodenum, jejunum-ileum, cecum, colon, and feces (Fig. 5; see also Fig. S2),respectively. Interestingly, Akkermansia muciniphila was the most significant biomarker(LDA score � 4.4 and P � 0.05) and was remarkably enriched in the large intestinesegments of Atg5�/� mice (cecum, colon, and fecal samples). However, the levels of“Candidatus Arthromitus” spp. (LDA score � 4.2 and P � 0.05) were distinctly increasedin all the intestinal samples from Atg5�/� mice, except for the duodenum (Fig. 5; seealso Fig. S2). In addition, Aggregatibacter pneumotropica, the most significantly alteredspecies, was expanded throughout the whole intestine of Atg5�/� mice, particularly inthe jejunum-ileum (LDA score � 4.66 and P � 0.05) (Fig. 5; see also Fig. S2). Theseresults hinted that disruption of autophagy had a severe impact on the compositionand population structure of the gut microbiota.

Gene functional enrichment of gut microbiota. Functional prediction of micro-biota was performed using PICRUSt to predict differences in gene functional enrich-

FIG 4 Comparisons of the relative abundances of abundant bacterial families in Atg5�/� and Atg5�/� mice in different sites. *, P �0.05; **, P � 0.001.

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ment in different intestinal segments of Atg5�/� and Atg5�/� mice. After P valuecorrection, the results revealed that microbiota functions shifted remarkably in smallintestinal segments after the disruption of Atg5. In the duodenum, environmentaladaptation and nucleotide metabolism pathways were the most significant in Atg5�/�

mice (Fig. S3). Endocrine system and energy metabolism pathways were enriched in thejejunum-ileum of Atg5�/� mice. However, pathways of infectious diseases were en-riched in small intestinal segments (duodenum and jejunum-ileum) of Atg5�/� mice(Fig. 6a; see also Table S5), indicating that deletion of Atg5 may potentially expose thehost to attack by pathogenic bacteria. As shown in Fig. 4a, the Pasteurellaceae family,which contains a number of potential pathogens (37), was enriched in Atg5�/� mice,further supporting this hypothesis. Thus, autophagy-deficient mice may be susceptibleto attack by pathogenic bacteria.

Altered host immune response. In intestinal epithelium, autophagy exerts selec-tive influences on the cell biology and specialized regulatory properties of Paneth cells,which are critically important intestinal innate immune cells. Thus, we speculated thatdisruption of Atg5 might influence the immune response. As indicated by levels ofimmunofluorescence of LAMP1, cytoplasmic lysosomes were clearly decreased in Atg5-deficient Paneth cells (Fig. 6b). This finding was consistent with those of previousreports (19, 23). In addition to the decline in cytoplasmic lysozymes levels, we observedthat the host cell may be vulnerable to pathogenic infections. The latter was alsoindicated by microbial functionality predictions (Fig. 6a). In support of this idea, wefound that key protective molecules, including MUC2, originated from goblet cells. Inaddition, MUC2 was increased in Atg5-deficient intestinal epithelia based on immuno-histochemistry analysis (Fig. 6c). These findings suggested that the inner layer of theintestine was altered and that the immune response was overactivated in Atg5-deficient tissues.

TABLE 1 The most different genera (LDA score � 3.5) between Atg5�/� and Atg5�/�

mice in various intestinal segments

Gut position Genus High groupa LDA score P valueb

Duodenum Klebsiella Atg5�/� 4.558 0.013*Labrys Atg5�/� 3.948 0.000**Mannheimia Atg5�/� 3.885 0.009**Pseudomonas Atg5�/� 4.953 0.009**Serratia Atg5�/� 3.613 0.004**Meiothermus Atg5�/� 4.546 0.012*

Jejunum-ileum Aggregatibacter Atg5�/� 4.241 0.009**Klebsiella Atg5�/� 4.108 0.016*Mannheimia Atg5�/� 4.081 0.041*Pseudomonas Atg5�/� 4.661 0.021*Sutterella Atg5�/� 4.95 0.033*Veillonella Atg5�/� 3.728 0.000**“Candidatus Arthromitus” Atg5�/� 4.094 0.032*Gemella Atg5�/� 3.585 0.001**Streptococcus Atg5�/� 3.578 0.041*

Cecum Akkermansia Atg5�/� 3.541 0.001**Lactobacillus Atg5�/� 4.367 0.008**

Colon Acinetobacter Atg5�/� 4.091 0.013*Ochrobactrum Atg5�/� 3.764 0.013*Sphingomonas Atg5�/� 3.602 0.004**Akkermansia Atg5�/� 3.939 0.000**Brevibacterium Atg5�/� 3.667 0.001**Meiothermus Atg5�/� 3.577 0.000**Ruminococcus Atg5�/� 3.606 0.016*

Feces Akkermansia Atg5�/� 40136 0.001**Bacteroides Atg5�/� 3.625 0.043*

aThe line with the significantly greater relative abundance.b*, P � 0.05; **, P � 0.01.

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To further analyze global shifts in the host immune response, we performedtranscriptome sequencing to detect differentially expressed genes (DEGs). We usedDESeq to identify the DEGs. A total of 349 DEGs were identified (P � 0.05; fold change� 2) in either Atg5�/� or Atg5�/� mice (Table S6). A hierarchical-clustering heat mapwas used to cluster samples into groups based on Atg5 status (Fig. S4). Of the DEGs, 222genes were upregulated and 127 were downregulated in Atg5�/� mice (Table S6).Characterization of Gene Ontology Consortium (GO) and Kyoto Encyclopedia of Genesand Genomes (KEGG) annotations of the 349 DEGs by DAVID identified 40 DEGsassociated with the immune response. Furthermore, the expression of Defa5, a genethat encodes a protein that synthesizes an important antimicrobial peptide in Paneth

FIG 5 LEfSe identified species with significantly different abundances in different intestinal segments inAtg5�/� and Atg5�/� mice. Taxa enriched in Atg5�/� mice are shown with a positive linear discriminantanalysis (LDA) score (green), and taxa enriched in Atg5�/� mice are shown with a negative score (red).Only taxa meeting an LDA significant threshold of �2 and P value of �0.05 are shown.

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cells of the ileum, was markedly reduced in Atg5�/� mice (Fig. 7a). Interestingly, two

key transcription factors, RORC and TBX21, which are related to TH17 and TH1 cell

differentiation, respectively, and are known to be involved in the inflammatory boweldisease (IBD) pathway, were significantly elevated in Atg5�/� mice (Fig. 7a). In addition,the expression levels of CD5, CD6, CD7, and CD96 were all increased in Atg5-deficientmice (Fig. 7b). Some inflammation-associated genes, such as CCL5, TLE, and THEMIS,

FIG 6 (a) Comparisons of functional microbiota pathways in Atg5�/� and Atg5�/� mice. (b) The lysozyme distribution in Atg5-deficientintestinal epithelia was detected using immunofluorescence detection of LAMP1 (red) in Atg5�/� and Atg5�/� mice. The nucleus (blue)was counterstained with DAPI. (c) Immunohistochemistry detection of MUC2 in Atg5�/� and Atg5�/� mice.

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were also differentially expressed due to the loss of Atg5 (Fig. 7b). These resultssuggested that the immune response in the Atg5�/� mice was dramatically altered.

DISCUSSION

Evidence indicates that the imbalance between host genes and microbes is animportant contributor to disease occurrence (5, 38–40). Autophagy plays a protectiverole in combatting intestinal inflammation by maintaining the shape and function ofPaneth cells (19, 23). In this study, we used an intestinal epithelium-specific Atg5knockout mouse model to characterize an additional vital role of autophagy: theregulation of gut microbial communities. We observed strong alteration of the com-position of the gut microbiota and a reduction of alpha diversity in the colon ofAtg5�/� mice (Fig. 2), similar to previous findings for patients with CD (41). Many factorsregulate the gut microbiota, of which the host immune system is the major factoramong them. We found that dysfunction of Paneth cells led to decreased expression ofdefensins (Fig. 7a). Previous research reported that defensins played a vital role in the

FIG 7 (a) Comparisons of transduction-related Defa5, RORC, and TBX21 mRNAs, which differed in expression inAtg5�/� and Atg5�/� mice. (b) Heat map of 40 DEGs associated with the immune response in Atg5�/� and Atg5�/�

mice.

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composition of the microbiota (42) and that dysfunction or reduced expression ofdefensins was associated with greater susceptibility to enteric infection and the initi-ation of ileal CD (43). Research also showed that elevated MUC2 was associated withthe induction of high levels of reactive oxygen species production, which furtherstimulated endoplasmic reticulum stress and apoptosis in goblet cells and subse-quently damaged the intestinal mucus barrier function in some pathologies (44). In thisstudy, autophagy played an important role in maintaining Paneth cell morphology andfunction, as well as host intestinal health.

Many protective intestinal bacterial groups are involved in the maintenance of guthomeostasis (45, 46). Numerous IBD-related genes regulate antimicrobial defenses andintestinal immune homeostasis (47). Previous research demonstrated that the Nlrp12gene was associated with innate immunity and that basal colonic inflammation in-creased in Nlrp12-deficient mice, which resulted in a less diverse microbiome and areduction in the relative abundance of Lachnospiraceae (48). This finding was consistentwith that in the present study, in which the relative abundances of Lachnospiraceaeaeand Ruminococcaceae were reduced in the large intestine and duodenum, respectively,in Atg5�/� mice. A previous study showed that both these bacterial families werenegatively associated with the occurrence of CD disease (49). Although the relativeabundance of Lachnospiraceae is decreased in patients with IBD, limited information isavailable on how these protective microbes regulate the host immune system. Previousresearch reported that Lachnospiraceae and Ruminococcaceae were preferentially en-riched in mucosal folds (50) and that they engaged in cross talk with intestinal immunecells directly, suggesting that these microbes may serve as immunological regulators toprevent enteric pathogen adhesion and colonization. Bacteria belonging to the Lach-nospiraceae and Ruminococcaceae are mainly responsible for the production of butyrate(51), which enables dendritic cells to accelerate regulatory T (Treg) cell differentiation(52). Thus, the loss of bacteria belonging to these two families in Atg5�/� mice mightlead to a reduction in short-chain fatty acids, which could be associated with elevatedinflammation in Atg5�/� mice. In this study, the relative abundance of Pasteurellaceaewas significantly enriched in the jejunum-ileum of Atg5�/� mice (Fig. 4a). The Pasteu-rellaceae family comprises mainly Gram-negative bacteria and includes multiplepathogens. These bacteria possess an outer membrane consisting mainly of lipo-polysaccharides (37), which induce recruitment and activation of inflammatory cellsand proinflammatory cytokine production (53). In previous research, an increase inthe relative abundance of Pasteurellaceae was positively correlated with IBD (49). Inour study, according to the prediction analysis of microbial function, the pathwayallocated for infectious diseases was enriched in Atg5�/� mice (Fig. 6a). Takentogether, these results indicate that impaired autophagy may alter the colonizationpatterns of gut microbiota, thereby disrupting microbiota as a physical barrier tomaintain intestinal immune homeostasis.

A. muciniphila is a mucin-degrading bacterium which preferably inhabits the colonof humans and rodents (54). In this study, A. muciniphila was reduced in the largeintestine of Atg5�/� mice and was the most significant biomarker (Fig. 5). Previousstudies indicated that mice fed a high-fat diet and treated with A. muciniphila via theoral route had elevated levels of intestinal endocannabinoids and that these controlledinflammation, enhanced mucus layer thickness and the gut barrier, increased the abilityof the mucus layer to harvest energy from the diet, and increased gut peptide secretion(55, 56). A reduction in the relative abundance of A. muciniphila was linked to IBD (57)and obesity (56). Thus, the absence of A. muciniphila caused by Atg5 deficiency maycontribute to aggravating colonic inflammation and increase the risk of obesity.

In this study, “Candidatus Arthromitus” (also known as SFB) was another importantbiomarker regulated by autophagy. “Candidatus Arthromitus” was predominantly andpersistently expanded in Atg5�/� mice (Fig. 5). This phenomenon was consistent withfindings in a previous study, which demonstrated distinct expansion in the abundanceof SFB in the small intestines of mice lacking activation-induced cytidine deaminaseand immunoglobulin A (7, 58). In addition, treatment with antibiotics inhibited colo-

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nization by SFB (59). These results hint that loss of selective stressors, including immunepressure or antibiotics, may enable overcolonization of SFB in the intestine. SFB aretightly associated with the epithelium (60), and they play an important role in stimu-lating the mucosal immune system. Thus, autophagy might participate in the immuneresponse against SFB expansion. Colonization of the small intestine by SFB is sufficientto induce the appearance of TH17 cells (8, 61), which have a potential function in thepathogenesis of IBD (62) and autoimmune disease (61). Although there is no strongevidence demonstrating that SFB can induce IBD, SFB have been discovered at someinflammatory sites in IBD patients (63), suggesting that overexpansion of SFB may resultin excessive immune responses and further promote inflammation of the intestine.However, a previous study reported that SFB were not “Candidatus Arthromitus” but“Candidatus Savagella” (64). Thus, much attention should be focused on the classifica-tion of SFB and distinguishing the functions of “Candidatus Arthromitus” and “Candi-datus Savagella.” There are still some scientists who consider “Candidatus Arthromitus”to be SFB (65–67).

Atg5 deficiency alters the composition and diversity of the gut microbiota. Themicrobiome can also induce adaptive changes in host immunity. In this study, someimmune-associated genes were highly expressed in Atg5�/� mice, including two keyIBD-related transcription factors, RORC and TBX21. RORC is a key regulator of immunehomeostasis and promotes the development of TH17 cells (68). TH17 cells and theircytokines are linked to several autoimmune and inflammatory diseases, such as allergyand asthma, psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythem-atosus, and IBD (69). TBX21 has been characterized as the “master regulator” of TH1 celldevelopment (70). Excess activation of TH1 cells was also associated with autoimmunityand IBD (71). In addition, the chemokines CCL5, TLE1, and Themis were also elevatedin Atg5�/� mice in our study. Previous research reported that the composition of gutmicrobiota of NLRP6-deficient mice was altered and that the altered compositionstimulated epithelial cells to secrete the chemokine CCL5, which recruited immunecells, such as neutrophils, to initiate a chronic inflammatory response that can presentas IBD (72). Research also reported that TLE1 inhibited the activation of nuclearfactor-�B via NOD2 and that the dysfunction of this gene was associated with IBDpathogenesis (73). Themis was linked to the suppressive function of Treg cells and wasreported to be involved in intestinal inflammation. Based on the above-describedresults, we propose that the host intestine may be vulnerable to pathogen invasion andan excessive immune burden in Atg5-deficient mice.

Considering the cross talk that occurs between the host and the gut microbiotaduring autophagy, we propose that overcolonization by SFB may promote excessivedifferentiation of TH17 cells. In addition, decreases in butyrate-producing bacteria, suchas those belonging to the Lachnospiraceae and Ruminococcaceae, are related to limitedTreg cell differentiation. All the results obtained in this study indicated that autophagydeficiency in the intestinal epidermal cell may induce an imbalance in the ratio ofTH17/Treg cells. TH17 and Treg cells have adverse function in the development ofautoimmune and inflammatory diseases. TH17 cells can execute strong proinflamma-tory impacts and are highly pathogenic, leading to the development of inflammationand severe autoimmunity (69). Treg cells serve as commanders in the control ofself-tolerance and regulate overall immune responses against infections. After elimina-tion of invading pathogens, Treg cells inhibit an excessive immune response, therebyprotecting the host from the chronic immunopathology related to chronic infections(74). In a previous study, the balance between TH17 and Treg cells was disrupted, anda high TH17/Treg ratio was identified in patients with rheumatoid arthritis, whichfurther confirms the importance of immune homeostasis of TH17 and Treg cells (75).Taken together, these results indicate that imbalanced microbial communities inducedby autophagy deficiency may activate a persistent immune response and expose thehost to excessive immune activation, which may further enhance basal intestinalinflammation.

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MATERIALS AND METHODSAnimals and sample collection. Three C57BL/6J Atg5flox/flox female mice (RBRC02975) which were

purchased from the RIKEN BioResource Center (34) were mated with two C57BL/6J PVillin-Cre male micewhich were obtained from the Nanjing Biomedical Research Institute of Nanjing University (T000142) inorder to generate heterozygous (Atg5�/�) mice. A total of 12 littermates consisting of 7 C57BL/6J Atg5�/�

mice and 5 C57BL/6J Atg5�/� were generated via intercrossing of heterozygous (Atg5�/�) mice. Becausethe PVillin-Cre mice could specifically express Cre recombinase in the entire intestinal epithelium from theduodenum to the cecum (76), the Atg5 gene can be conditionally knocked out in the intestinalepithelium of the small intestine and colon of Atg5�/� mice. All mice were cohoused in the same roomand on the same shelf of a maximum-barrier, specific-pathogen-free facility at State Key Laboratory ofStem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China;a 12-h light-dark cycle was maintained. After 4 weeks, the 12 mice were randomly assigned to individualcages. The mice were fed with a mixed diet containing the standard diet, soybeans, peanuts, andsunflower seeds. After 1 month, fresh fecal samples were obtained directly from individual cages. Thenmice were euthanized immediately, and the intestinal contents of the duodenum, jejunum-ileum, cecum,and colon were collected within 20 min after euthanasia. All animal experiments were approved by theAnimal Research Panel of the Committee on Research Practice of the University of Chinese Academy ofSciences. In total, there were 60 gut content samples from 7 C57BL/6J Atg5�/� mice and 5 C57BL/6JAtg5�/� mice. Microbial genomic DNA was extracted from these contents (detailed information onsamples is provided in Table S1). Distal ileum tissues were collected from each individual mouse forhistological examination and RNA transcriptome sequencing.

Immunoblotting. Intestinal tissue was resuspended (homogenized) in cold RIPA buffer (25 mMTris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate)supplemented with 1 mM phenylmethylsulfonyl fluoride and a protein inhibitor cocktail (04693132001;Roche Diagnostics, Basel, Switzerland) for 30 min on ice. The homogenates were centrifuged at 12,000rpm for 10 min, and then the supernatant was transferred to a new tube. Next, 4� SDS loading bufferwas added to samples, which were then boiled and subjected to immunoblotting. Immunoblots werevisualized using the ODYSSEY Sa infrared imaging system (LI-COR Biosciences, USA). (Antibody informa-tion is given in Table S2.)

Immunohistochemistry and immunofluorescence. For tissue sections, the ileum was dissected andthen fixed and prepared as described previously (77). Serial 5-�m-thick sections were cut perpendicularto the crypt-surface epithelial cuff axis and parallel to the cephalocaudal axis. Sections were stained withhematoxylin and eosin (H&E). For a subset of immunohistochemical studies, sections were boiled for 15min in sodium citrate buffer for antigen retrieval. Next, 3% H2O2 was added to the sections to eliminateinternal peroxidase activity. After blocking with 5% bovine serum albumin (BSA; Sigma) for 30 min, eachsection was incubated with the primary antibody in 1% BSA at 4°C overnight, followed by staining withhorseradish peroxidase (HRP)-conjugated secondary antibody. Negative controls were prepared withoutthe primary antibody. Finally, the sections were stained with 3,3=-diaminobenzidine (DAB), and the nucleiwere stained with hematoxylin. Images were taken using a Nikon 80i inverted microscope with acharge-coupled device (CCD).

Paraffin sections were boiled for 15 min in the sodium citrate buffer for antigen retrieval. After blockingwith 5% BSA for 30 min, each section was incubated with the primary antibody in 1% BSA at 4°C overnight.After a washing with phosphate-buffered saline (PBS), the sample was incubated with fluorescein isothio-cyanate (FITC)- or tetramethyl rhodamine isocyanate-conjugated secondary antibody diluted in PBS for 1 h at37°C. Next, the slides were washed in PBS, and nuclei were stained with 4=,6-diamidino-2-phenylindole (DAPI).Images were taken immediately using an LSM 780 microscope (Zeiss).

Gut bacterial 16S rRNA gene sequencing. Microbial genomic DNA was extracted from feces andintestinal content samples using a TIANGEN DNA stool minikit (catalog number DP328) following themanufacturer’s recommendations. The V4 hypervariable regions of the 16S rRNA were PCR amplifiedfrom microbial genomic DNA, which was harvested from intestinal contents and fecal samples usingbarcoded fusion primers (forward primer, 5=-AYTGGGYDTAAAGNG-3=; reverse primer, 5=-TACNVGGGTATCTAATCC-3=). The PCR program was as follows: initial denaturation at 94°C for 5 min, 27 cycles of 94°Cdenaturation for 30 s, 50°C annealing for 30 s, and 72°C extension for 30 s, and then a final extension at72°C for 7 min. PCR products were purified using a Qiagen quick gel extraction kit (catalog number28706). Barcoded V4 PCR amplicons were sequenced by an Illumina Miseq platform. Amplification of theV4 regions of 16S rRNA genes and sequencing services were provided by Shanghai Personal Biotech-nology Co., Ltd. (Shanghai, China).

Analysis of classification and abundance. Sequences were filtered as in our previous study (31).Barcodes and sequencing primers were trimmed from sequencing reads. The trimmed and assembledsequences were uploaded to the QIIME and MG-RAST pipelines for further analysis. The trimmedsequence of each sample was compared to the RDP and M5RNA databases using the best hit classifi-cation option to classify the abundance in QIIME (78) (http://qiime.org/index.html) and MG-RAST (79)(http://metagenomics.anl.gov/), respectively. For QIIME, data on the OTU level were generated using theuclust script (http://qiime.org/scripts/pick_otus.html), and then according to these OTUs, QIIME auto-matically generated phylum to genus level data for different intestinal segments and genotypes.Simpson and Shannon indices were calculated by the mothur program. For statistical analysis, anunpaired Student’s t test was performed using GraphPad. LEfSe was applied to identify microbes fromdifferent taxa among lines using the default parameters (LDA score � 2; P � 0.05) (80). Correlationanalyses among different gut positions were carried out in Excel. For MG-RAST, data on the species levelwere generated, and parameter classification was done using cutoffs of 5 for the maximum E value, 97

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for the minimum percent identity, and 60 bp for the minimum alignment length. The 16S rRNAgene sequences used in this study were deposited in the MG-RAST server under the project name“Autophagy_gut microbiota.”

Functional annotation and prediction of microbiota. Microbial function was predicted usingPICRUSt (81). The OTUs were mapped to the gg13.5 database with 97% similarity by the QIIME command“pick_closed_otus.” The OTU abundance was normalized automatically using 16S rRNA gene copynumbers from known bacterial genomes in Integrated Microbial Genomes and Microbiomes (IMG/M[http://img.jgi.doe.gov/]). The predicted genes and their functions were aligned to the Kyoto Encyclo-pedia of Genes and Genomes (KEGG; http://www.kegg.jp/) database, and the differences betweengroups were compared with STAMP (http://kiwi.cs.dal.ca/Software/STAMP) (82). A two-sided Welch t testand the Benjamini-Hochberg false-discovery rate (FDR) (P � 0.05) correction were used in the two-groupanalysis.

Transcriptome sequencing of intestinal tissues. Six samples from 3 Atg5�/� and 3 Atg5�/� micewere used for transcriptome analysis. Ileal total RNAs were extracted using TRIzol (Invitrogen). Oligo(dT) beadsthat bind poly(A) were used to purify mRNA. After high-temperature fragmentation, 275-bp mRNAs wereobtained (TruSeq RNA sample prep kit; Illumina). Mouse mRNA was reverse transcribed to cDNA. Followingprocesses to repair ends, adenylate 3= ends, ligate adapters, purify ligation products, enrich DNA fragments,and validate the library and pool cDNAs, the DNA library was constructed for sequencing by the IlluminaMiseq platform. Reads were mapped to the mouse genome and were annotated. Reads per kilobase permillion mapped reads (RPKM) were calculated as the normalized abundance of each gene as follows: totalexon reads divided by the product of mapped reads (millions) and exon length (in kilobases). Differentiallyexpressed genes (fold change � 2, P � 0.05, and after Benjamini-Hochberg adjustment) were calculated byDESeq (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). Enrichment analysis wasperformed using DAVID (https://david.ncifcrf.gov/).

Accession number(s). The transcriptome sequencing data were deposited in the NCBI SRA databaseunder project number PRJNA396813 and accession number SRP115402.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00880-18.

SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 3, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 4, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 5, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 6, XLS file, 0.1 MB.SUPPLEMENTAL FILE 7, XLSX file, 0.1 MB.

ACKNOWLEDGMENTSWe thank Noboru Misushima for providing the Atg5 floxed mice and Yan Zhang

from Carilion Clinic, USA, for helping revise the paper.This study was supported by the National Science Foundation of China (grant no.

31572384 to H.M.) and the National Key Research and Development Program of China(grant no. 2017YFD0500506 to H.M.).

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