Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis

Gut Microbes ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kgmi20 Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis Ainize Peña-Cearra, Janire Castelo, Jose Luis Lavín, Monika Gonzalez-Lopez, Miguel Angel Pascual-Itoiz, Miguel Fuertes, Virginia Gutiérrez de Juan, Laura Bárcena, Itziar Martín-Ruiz, Aize Pellón, Iratxe Seoane, Diego Barriales, Ainhoa Palacios, Asier Fullaondo, Iago Rodríguez-Lago, María L. MartinezChantar, Ana Mª Aransay, Hector Rodriguez, Juan Anguita & Leticia Abecia To cite this article: Ainize Peña-Cearra, Janire Castelo, Jose Luis Lavín, Monika Gonzalez-Lopez, Miguel Angel Pascual-Itoiz, Miguel Fuertes, Virginia Gutiérrez de Juan, Laura Bárcena, Itziar Martín-Ruiz, Aize Pellón, Iratxe Seoane, Diego Barriales, Ainhoa Palacios, Asier Fullaondo, Iago Rodríguez-Lago, María L. Martinez-Chantar, Ana Mª Aransay, Hector Rodriguez, Juan Anguita & Leticia Abecia (2023) Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis, Gut Microbes, 15:2, 2266626, DOI: 10.1080/19490976.2023.2266626 To link to this article: https://doi.org/10.1080/19490976.2023.2266626 © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. View supplementary material Published online: 16 Oct 2023. Submit your article to this journal Article views: 2734 View related articles View Crossmark data Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kgmi20 Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis Ainize Peña-Cearra a,b *, Janire Castelo a , Jose Luis Lavín a,c , Monika Gonzalez-Lopez a , Miguel Angel PascualItoiz a , Miguel Fuertes d , Virginia Gutiérrez de Juan a , Laura Bárcena a , Itziar Martín-Ruiz a , Aize Pellón a *, Iratxe Seoane a , Diego Barriales a , Ainhoa Palacios a , Asier Fullaondo e , Iago Rodríguez-Lago f , María L. Martinez-Chantar a,g , Ana Mª Aransay a,g , Hector Rodriguez a , Juan Anguita a,h , and Leticia Abecia a,b a CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Spain; b Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Bilbao, Spain; c Applied Mathematics Department, NEIKERBasque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Derio, Spain; d Animal Health Department, NEIKER-Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Derio, Spain; e Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Bilbao, Spain; f Department of Gastroenterology, Hospital de Galdakao, Galdakao, Spain; g CIBERehd, ISCIII, Madrid, Spain; h Ikerbasque, Basque Foundation for Science, Bilbao, Spain ABSTRACT Anti-TNF therapy can induce and maintain a remission status during intestinal bowel disease. However, up to 30% of patients do not respond to this therapy by mechanisms that are unknown. Here, we show that the absence of MCJ, a natural inhibitor of the respiratory chain Complex I, induces gut microbiota changes that are critical determinants of the lack of response in a murine model of DSS-induced inflammation. First, we found that MCJ expression is restricted to macrophages in human colonic tissue. Therefore, we demonstrate by transcriptomic analysis of colon macrophages from DSS-induced mice that MCJ-deficiency is linked to the expression of genes belonging to the FcγR signaling pathway and contains an anti-TNF refractory gene signature identified in ulcerative colitis patients. The gut microbial composition changes observed upon DSS treatment in the MCJ-deficient mice revealed the increased presence of specific colitogenic members, including Ruminococcus gnavus and Oscillospira, which could be associated with the non-response to TNF inhibitors. Further, we show that the presence of a microbiota associated resistance to treatment is dominant and transmissible to responsive individuals. Collectively, our findings underscore the critical role played by macrophage mitochondrial function in the gut ecological niche that can substantially affect not only the severity of inflammation but also the ability to successfully respond to current therapies. ARTICLE HISTORY Received 8 March 2023 Revised 22 September 2023 Accepted 29 September 2023 KEYWORDS IBD; microbiota; complex I; mitochondriopathy; FcγR signaling; anti-TNF therapy CONTACT Juan Anguita [email protected] CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Spain; Leticia Abecia [email protected] Department of Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Bilbao, Spain *Present address: Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Science, King’s College London, SE1 9RT London, UK. Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2266626. GUT MICROBES 2023, VOL. 15, NO. 2, 2266626 https://doi.org/10.1080/19490976.2023.2266626 © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Introduction Ulcerative colitis (UC) is a chronic, heterogeneous and severe inflammatory disease that primarily affects the gastrointestinal tract. Although its etiology remains unknown, evidence suggests a complex interplay between microbiota, the immune system, host genetics and environmental factors. Whilst there is no cure for UC, anti-tumor necrosis factor (TNF) agents are the most effective to induce and maintain a remission status in intestinal bowel disease (IBD) patients as they target the excessive production of TNF, a key proinflammatory cytokine produced mainly by activated immune cells, improving the quality of life of patients. However, there are IBD patients that are refractory to anti-TNF treatment and they can be categorized into two groups; those who never respond to anti-TNF induction therapy (primary failure), and 50% of those who initially respond to anti-TNF treatment but subsequently lose response overtime (secondary failure). 1 Furthermore, the treatment poses risks as serious adverse effects including severe infections have been reported. 2 Therefore, the identification of key factors that could allow clinicians to identify patients who will respond to anti-TNF therapy seems a priority to promote personalized treatment to IBD patients. Recently, a study of the UC mucosal transcriptome revealed a decrease in mitochondrial electron transport chain Complex I activity, which was found to correlate with disease severity and treatment response in both adult and pediatric patients. 3 In addition, Mottawea et al. stated that host mitochondria-microbiota crosstalk was disturbed in IBD. 4 Consequently, mitochondrial dysfunction and microbial composition might play an important role in the response to therapy. Therefore, microbial-host mitochondria interactions seem to be a milestone in the development of effective therapies for UC patients. Aberrant immune responses against bacteria are extensively described in the pathogenesis of UC. During UC, macrophages are activated by translocated pathogenic bacteria and are associated with the increased proinflammatory status characteristic of the disease. 5 In this regard, methylationcontrolled J protein (MCJ, Dnajc15), a natural negative regulator of mitochondrial respiration that inhibits complex I activity of the electron transport chain regulating oxidative phosphorylation and ATP production, 6,7 has been linked to macrophages. Deficiency of MCJ in bone marrowderived macrophages leads to the upregulation of the tumor necrosis factor α-converting enzyme (TACE) inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP-3), which inhibits TNF shedding from the plasma membrane. 8 As macrophages are pivotal for coordinating processes in the gut and a significant infiltration is produced during colitis, we investigated the expression levels of both genes in colon samples from UC patients reporting MCJ downregulation and TIMP3 upregulation. 9 These results led us to study the impact of MCJ deficiency in acute 9 and chronic 10 experimental colitis and are in line with parameters disrupted during IBD activity in individuals suffering microbial dysbiosis. 11 In addition, we have rencently reported that the influence of MCJ deficiency on microbial composition has a detrimental effect on the severity of UC. 12 Because of the intimate relationship that is reported to exist between mitochondrial dysfunction and the efficacy of current therapies, particularly anti-TNF treatments, as well as the role played by macrophages during UC, we have now investigated the effect of mitochondrial dysfunction on the colon response to anti-TNF therapy. Our results show that the mitochondrial electron transport chain Complex I activity is determinant on the response to anti-TNF therapy. Importantly, our results link mitochondrial dysfunction with a microbiota composition leading to a lack of therapeutic efficacy that is dominant and transferable to otherwise responsive individuals. These data highlight the relevance of the physiological response of intestinal macrophages to maintain a homeostatic ecological niche that affects both the severity of gut inflammation and the response to current therapies. Results MCJ deficiency prevents a protective response to anti-TNF agents during experimental colitis Mitochondria dysfunction, including the reduction of complex I activity in active patients, 3 has been 2A. PEÑA-CEARRA ET AL. related to disease severity and a refractory response to treatment during UC. We have shown that the absence of the complex I negative regulator, MCJ, results in increased DSS-induced pathology. 9 In order to evaluate the impact of mitochondrial dysfunction on the responsiveness to anti-TNF therapy during the disease, we evaluated the response of MCJ-deficient mice to Infliximab (IFX, antiTNF) treatment. MCJ deficiency diminished the therapeutic efficacy of the infliximab biosimilar, as evidenced by the absence of a significant improvement in body weight (Figure 1(a)) and histological scores (Figure 1(b)), in contrast to WT mice that responded successfully to the treatment. Moreover, TACE levels within the colon only increased in treated WT mice independently of TNF (Figure 1(c)) and infiltrated macrophage numbers that did not vary (Figure S1a,b). TACE activity is responsible for the final release of mature TNF protein from cells. MPO levels were significantly increased (p ≤ .05) in both MCJ KO groups compared to WT groups independently of treatment (Figure S1c). Transcriptional analysis of colon macrophages from MCJ-deficient mice shows overlapping signatures to a refractory response to treatment. We have shown that MCJ is expressed weakly in the intestinal tissue both in mice and humans, 9 and that macrophages are, together with CD8 T cells, the cells of the immune system with the highest expression levels of the protein. 6,8 We, therefore, determined the expression of MCJ in inflamed and healthy colon tissue from IBD patients by immunofluorescence staining. The transmembrane scavenger receptor CD163 was also used to identify the macrophage population within the tissue. MCJ was readily detected in both diseased (Figure 2(a)) and adjacent, healthy colon tissue in colocalization with CD163. These data indicate that the impact of MCJ in IBD patients is primarily associated with macrophages. We then examined the role of MCJ in the immune response during the disease. We a b c WT MCJ-KO IFXIFX+ WT MCJ KO WT MCJ KO IFXIFX+ 0 10 20 30 40 ** *** IFXIFX+ 0 100 200 300 * ** Figure 1. In vivo anti-TNF therapeutic response. WT and MCJ-KO mice were treated the first 6 d with DSS and infliximab (IFX) was administrated orally from day 3 to day 6, followed by 3 d of recovery period. All groups are positive for DSS. (a) Weight loss (%) (n = 8 mice per group at minimum). Statistical differences (P value < .05) were observed between WT IFXand WT IFX+ from day 3 onward (asterisks below WT IFX+ line) and between MCJ KO IFX+ and WT IFX+ at day 3 and from day 5 onward (asterisks above WT IFXline). (b) Histological scores and representative images of colon tissue (scale bar size, 100 µm). (c) TACE activity in colonic protein extracts (Rfu/mg protein). For statistical analysis two-way ANOVA was performed. Within boxplots, asterisks above boxes versus control genotype (IFX+ versus IFX-) and asterisks above line versus different genotypes in the same experimental group. (Wt_ifxn and MCJ KO_IFXn: DSS positive and IFX negative; WT_IFXp and MCJ KO_IFXp: DSS and IFX positive). GUT MICROBES 3 performed a transcriptome analysis of colon macrophages under DSS-induced colitis. Principal component analysis showed changes according to the disease status of the animals (Figure 2(b)). The analysis of the 50 most regulated genes showed different patterns of expression associated with the presence/absence of MCJ. MCJ-deficient macrophages expressed 305 upregulated genes compared to WT controls. Among them, there were several confirmed susceptibility-related genes for UC such as Irf5, Tnfrsf9, Fcgr2b Slc11a1, Itgal, Gpr65, Cd40, and Lsp1, suggesting a relevant ab c MCJ KO Mice Up (305) MCJ KO Mice Down (34) UC gene signature Up (3600) UC gene signature Down (1696) Motif Pvalue SpiB 1e-16 Erg 1e-9 Elf5 1e-8 Runx 1e-6 d e MCJ KO DSS+ WT DSS+ MCJ KO DSSWT DSSMCJ KO Mice Up (305) Anti-TNF (50) Corticosteroid (115) Srgn Clec4e Ptgs2 Trem1 Clec4d Fpr2 Gpr84 Il1b Osm Chronic inflammation MCJ CD163 MCJ-CD163 MCJ CD163 MCJ-CD163 Healthy adjacent Figure 2. Transcriptomic analysis of intestinal tissue macrophages. (a) Immunofluorescence of MCJ (red) and CD163 (green) in inflamed and healthy adjacent colon tissue from IBD patients. Colocalization is represented in yellow. (b) Principal component analysis showing differences between WT and MCJ-deficient DSS positive colon macrophages´ transcriptomes. (c) HOMER identified several transcription factors enriched in DSS-induced MCJ-KO colon macrophages. (d) venn diagram representing 339 differentially expressed genes found in colon macrophages due to MCJ-KO and a human core rectal UC gene expression signature consisting of 5296 genes. 3 Out of 305 genes upregulated in colitis induced MCJ-deficient mice colon macrophages, 183 were shared with the human UC gene signature. (e) venn diagram showing shared genes between upregulated genes due to MCJ-KO in DSS-induced mice colon macrophages and patients refractory to anti-TNF and corticosteroid treatments. 4A. PEÑA-CEARRA ET AL. role of mitochondria dysfunction in immune cells such as macrophages during the course of the disease. The HOMER package identified a set of transcription factors putatively responsible for the expression changes in macrophages from DSSinduced colitis animals. 13 The analysis of the genes upregulated in MCJ-deficient macrophages compared to WT mice identified several transcription factors (Figure 2(c), Table S2). Two of them (SpiB and Elf5) are related to Fcgr2b expression, while Runx is involved in Irf5 expression and Erg in the expression of Slc11a1 and Tnfrsf9. Moreover, Elf5 and SpiB possess motifs that regulate Il1b expression, while Erg showed a motif that regulates the expression of Tnf, Il10 and Tgfbi. On the other hand, Erg and Runx may regulate Cxcl3 expression and therefore be involved in leucocyte recruitment. In order to discern whether transcriptional patterns associated with mitochondrial dysfunction in murine colon macrophages are related to the disease in humans, UC transcriptomes from human rectal samples were compared to experimental colitis macrophage transcriptomes. Out of the 305 upregulated genes in macrophages isolated from the colons of MCJ-deficient mice with induced experimental colitis compared to DSS+ controls (WT), 183 were shared between both cohorts (mouse model and patients) (Figure 2(d), Table S3), suggesting a critical role for mitochondrial function during disease progression. A subset of genes obtained from transcriptional analyses were linked to treatment response and shifts in microbial composition. In this regard, the 305 upregulated genes due to MCJ-deficiency in macrophages from experimental colitis mice were compared to the list of genes proposed to identify patients that are refractory to TNF blockade, 14 and corticosteroids. 3 Strikingly, 18 genes were shared with these 2 gene signatures (Figure 2(e)), in which Trem1 and Osm, connected to decreased responsiveness to anti-TNF therapy, were shared between the three gene signatures. Besides, the high expression of activating Fcgr1 and Fcgr3 found in inflammatory MCJdeficient macrophages, which are cell surface glycoproteins that bind to the Fc portion of IgG antibodies, and the high levels of colonic IgG found (Figure 3(f)), suggested that the MCJ-deficient genotype might be associated with resistance to TNF blockade. Functional annotation enrichment analysis using ToppGene, ToppCluster, and ClueGO mapped groups of related genes to immunological processes. 3 Overview of the ClueGo-derived immune system-related pathways (Figure 3(a,b)) comparing gene expression based on MCJ deficiency during DSS-induced colitis showed 46% enrichment for the Fc gamma receptor (FCGR) signaling pathway, followed by myeloid cell (13,1%), macrophage (6,7%) and dendritic cell activation (6,7%), regulation of adaptive immune response (6,7%) and leukocyte differentiation (6,7%). The P values for the top specific biological processes and pathways were obtained as an output from ToppGene. A more detailed ToppCluster pathways analysis output showed genes related to immunological and biological processes (Figure 3(c,d)). MCJ-deficient mice presented higher expression of genes involved in immune system activation and therefore, higher production of cytokines and chemokines, and higher transendothelial migration of leukocytes. Importantly, IgA production was upregulated in MCJ-deficient colitis mice. Furthermore, the study of microbiota–host interactions along the DSS-induced colitis period showed distinct immune response kinetics. Live bacteria and immunoglobulin G (IgG)-coated fecal bacteria were quantified by flow cytometry. Starting at day 2 after the initiation of DSS treatment, the percentage of live bacteria was similar in all experimental groups (Figure 3(e)). MCJ-deficient DSS-induced mice exhibited higher IgG-coated fecal bacteria than WT mice increasing gradually until day 8, being significantly higher from day 6 (p = 0.022) to day 8 (p = 0.011) (Figure 3(f)). These data might indicate earlier microbial-host cross-talk and a faster activation of the immune system. Overall, these data suggest that MCJ modifies the kinetics of the immune response. We then sought to find correlations between genes expressed in colon macrophages and specific microbial operational taxonomic units (OTUs). We analyzed the microbiota composition in MCJdeficient and WT mice, both under homeostatic and DSS-induced pathology. As expected, 9 MCJ deficiency resulted in significant changes in microbiota composition (Figure S2(a,b)). Figure 4 shows the 50 strongest correlations between genes differentially expressed due to MCJ deficiency in macrophages infiltrated during colon inflammation and OTUs present in the 4 GUT MICROBES 5 b c a d FcγR signalling pathway (46,7%) Myeloid dendritic cell activation (6.7%) Macrophage activation (6.7%) Alpha-beta T cell proliferation (6.7%) Positive regulation of leukocyte differentiation (6.7%) Regulation of adaptative immune responses (6.7%) TLR7 signalling pathway (6.7%) Myeloid cell activation involved in immune responses (13.1%) Up Down Regulation of microtubule polymerization (50%) Chaperone cofactor-dependent refolding (50%) NOD-like receptors. Intracellular sensors of PAMPs Mitochondrial respiration Cytokines Cell adhesion and migration Immune system ef 02468 0 50 100 150 Days % Live bacteria * DSS 02468 0 20 40 60 Days % IgG * * DSS WT DSS+ MCJ KO DSS+ WT DSSMCJ KO DSSFigure 3. Functional annotation enrichment analysis of inflamed intestinal macrophages according to MCJ levels. RNA-seq data analysis shows 305 upregulated and 34 downregulated genes in MCJ-KO mice treated with DSS compared to control colitis mice (FDR <0.05 and fold change ≥1.5). ClueGO charts related to immunological function reveals (a) upregulation of FCγR signaling pathway (46,7%) in MCJ-KO colitis mice compared to WT colitis (Log2 > 2, P value < 0.05) and (b) downregulation of chaperone cofactor refolding (50%) and microtubule polymerization (50%). Detailed functional annotation enrichment analyses of (c) 305 upregulated and (c) 34 downregulated genes in MCJ-KO colitis mice using ToppGene, ToppCluster and Cytoscape are represented. (c) Pathways related to upregulated genes; immune system (purple), cytokines (orange), cell adhesion and migration (yellow) and other pathways (green). (d) Biological processes enriched by downregulated genes; mitochondrial respiration (blue), cytokines (orange), NOD-like receptors, the intracellular sensors of PAMPS (purple) and heat acclimation (red). (e) Fecal live bacteria percentage (n = 3) and (f) fecal IgG-coated bacteria percentage obtained by flow cytometer (n = 3). For statistical analysis two-way ANOVA was used. 6A. PEÑA-CEARRA ET AL. experimental groups. Those included associations between Oscillospira and Ruminococcus gnavus, often increased with disease activity, and Cst7 that may play a role in immune regulation through the hematopoietic system. Akkermansia muciniphila presence, often regarded as an immunomodulator, correlated with Jak2 expression. Jak2 is involved in the production of key pro-inflammatory cytokines and consequently, Jak inhibitors are currently being investigated as therapeutic agents for UC. g__Streptococcus g__Lactobacillus s__Lysinibacillus boronitolerans g__Pseudomonas s__Bacteroides acidifaciens g__Oscillospira o__Clostridiales f__Ruminococcaceae s__Ruminococcus gnavus f__S24-7 s__Akkermansia muciniphila f__S24-7 s__Akkermansia muciniphila g__Bacteroides o__Clostridiales g__Corynebacterium s__Akkermansia muciniphila o__Clostridiaceae f__Coriobacteriaceae g__Lactobacillus f__Enterococcaceae g__Achromobacter f__Clostridiaceae o__RF32 f__Enterococcaceae f__Rikenellaceae f__S24-7 f__Clostridiaceae s__Akkermansia muciniphila o__Clostridiales s__Ruminococcus gnavus o__Clostridiales g__Allobaculum s__Bacteroides acidifaciens f__S24-7 f__Clostridiaceae g__Bacteroides o__Clostridiales f__Rikenellaceae s__Bacteroides acidifaciens f__Lachnospiraceae f__S24-7 g__Enterococcus o__Clostridiales g__Bacteroides g__Odoribacter o__Clostridiales g__Sutterella Figure 4. Heatmap of Spearman´s rank correlation coefficients. Bacterial abundances from the 4 experimental groups that were not treated with antibiotic and genes modified by the level of MCJ in colon macrophages upon intestinal inflammation (DSS+) were used. To find associations hierarchical all against all association (HAIIA) was performed. A. muciniphila correlates with Jak2 and R. gnavus with Cst7. The numbers indicate the highest correlation between bacteria and gene expression, having number 1 the highest correlation. Red color illustrates positive correlation and blue negative. In the x axis each column shows a different gene and in the y axis each row shows different bacterial species; Firmicutes (black font), Proteobacteria (red), bacteroidetes (blue), actinobacteria (green) and Verrucomicrobia (purple). GUT MICROBES 7 significance was assessed by two-way ANOVA statistical test determined as *P value < 0.05, **P value < 0.01, ***P value < 0.001, and ****P value < 0.0001. Significance is represented by an asterisk or asterisks upside the box to indicate differences within the same genotype in different experimental conditions, infliximab negative (IFX-) versus infliximab positive (IFX+), and cohoused infliximab positive (Coh) versus alone infliximab positive (Alone). Asterisks above the lines indicate differences between the different genotypes in the same experimental group. Abbreviations DSS Dextran sulfate sodium FcγR Fc gamma receptor HRP Horseradish Peroxidase IBD Inflammatory bowel disease IFX Infliximab MCJ Methylation-controlled J protein OTU Operational taxonomic unit SCFA Short-chain fatty acid TACE Tumor necrosis factor α-converting enzyme TIMP3 Tissue inhibitor of metalloproteinase 3 TNF Tumour necrosis factor UC Ulcerative colitis Acknowledgments We thank Estibaliz Atondo for technical support. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This work was supported by grant [RTI2018-096494-B-100 and PID2021-124328OBI00 to JA] from the Spanish Ministry of Economy and Competitiveness co-financed with FEDER funds, the V Grant from GETECCU-MSD (Grupo Español de Trabajo en Enfermedad de Crohn y Colitis ulcerosa to LA), Basque Government project for health [number 2015111117 to LA] and Research Committee from OSI Barrualde-Galdakao (2018-2-2 to IRL and LA). APC was a fellow of the University of the Basque Country (UPV/EHU) and is currently a postdoctoral fellow funded by the Basque Government. Support was provided by the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek Programs) and the Innovation Technology Department of Bizkaia County. CIC bioGUNE thanks MINECO for the Severo Ochoa Excellence Accreditation [SEV-2016-0644]. ORCID Ainize Peña-Cearra http://orcid.org/0000-0003-3855-6664 Janire Castelo http://orcid.org/0000-0002-9628-8242 Monika Gonzalez-Lopez http://orcid.org/0000-0003-15060332 Miguel Fuertes http://orcid.org/0000-0001-5454-0078 Itziar Martín-Ruiz http://orcid.org/0000-0002-9915-8101 Aize Pellón http://orcid.org/0000-0003-0415-6566 Ainhoa Palacios http://orcid.org/0000-0001-8945-5260 Asier Fullaondo http://orcid.org/0000-0001-5387-4762 Iago Rodríguez-Lago http://orcid.org/0000-0003-11334578 María L. Martinez-Chantar http://orcid.org/0000-00026446-9911 Ana Mª Aransay http://orcid.org/0000-0002-8271-612X Juan Anguita http://orcid.org/0000-0003-2061-7182 Leticia Abecia http://orcid.org/0000-0003-4097-8903 Authors’ contributions Conception and design of the study (LA), data collection (APC, JC, MAPI, VGJ, AP, DB, APa and LA), data analysis (APC, JLL, MGL, MAPI, MF, IS, AMA, JLL and LA), drafting the manuscript (APC, JA and LA), manuscript revision (HR and MLMC), statistical analysis (APC, JLL and LA), obtained funding (AF, IRL, JA, and LA), and technical support (LB and IMR). All authors approved the final version for publication. Data availability statement Raw sequences used for metagenomics analysis were made available at European Nucleotide Archive (ENA www.ebi.ac. uk/ena) under the project number PRJEB33422 for dysbiosis and PRJEB41595 for infliximab experiment. Raw sequences used to perform the transcriptomic analysis were uploaded to GEO (Gene Expression Omnibus) database under project accession code GSE135033 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?&acc=GSE135033). Ethics approval Animal protocols were approved by the Animal Research Ethics Board of CIC bioGUNE (Spain; permit number CBBA-0615). Collection of colon samples from IBD patients were approved by the Clinical Research Ethics Board of Euskadi (CEIC-E; code 16-12). References 1. Gisbert JP, Chaparro M. Primary failure to an anti-TNF agent in inflammatory bowel disease: switch (to a second anti-TNF agent) or swap (for another mechanism of action)? J Clin Med. 2021;10(22):5318. doi:10.3390/jcm10225318. 14 A. PEÑA-CEARRA ET AL. 2. Gerriets V, Goyal A, Khaddour K. Tumor necrosis factor inhibitors. Florida: StatPearls. Treasure Island (FL); 2022. 3. Haberman Y, Karns R, Dexheimer PJ, Schirmer M, Somekh J, Jurickova I, Braun T, Novak E, Bauman L, Collins MH, et al. Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nat Commun. 2019;10(1):38. doi:10.1038/ s41467-018-07841-3. 4. Mottawea W, Chiang CK, Muhlbauer M, Starr AE, Butcher J, Abujamel T, Deeke SA, Brandel A, Zhou H, Shokralla S, et al. Altered intestinal microbiota–host mitochondria crosstalk in new onset Crohn’s disease. Nat Commun. 2016;7(1):13419. doi:10.1038/ncomms13419. 5. Peloquin JM, Goel G, Villablanca EJ, Xavier RJ. Mechanisms of pediatric inflammatory bowel disease. Annu Rev Immunol. 2016;34(1):31–64. doi:10.1146/ annurev-immunol-032414-112151. 6. Hatle KM, Gummadidala P, Navasa N, Bernardo E, Dodge J, Silverstrim B, Fortner K, Burg E, Suratt BT, Hammer J, et al. MCJ/DnaJC15, an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations. Mol Cell Biol. 2013;33:2302–2314. doi:10.1128/MCB.00189-13. 7. Schusdziarra C, Blamowska M, Azem A, Hell K. Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria. Hum Mol Genet. 2013;22:1348–1357. doi:10.1093/hmg/dds541. 8. Navasa N, Martin I, Iglesias-Pedraz JM, Beraza N, Atondo E, Izadi H, Ayaz F, Fernandez-Alvarez S, Hatle K, Som A, Dienz, O., et al. Regulation of oxidative stress by methylation-controlled J protein controls macrophage responses to inflammatory insults. J Infect Dis. 2015;211(1):135–145. doi:10.1093/infdis/jiu389. 9. Pascual-Itoiz MA, Pena-Cearra A, Martin-Ruiz I, Lavin JL, Simo C, Rodriguez H, Atondo E, Flores JM, Carreras-Gonzalez A, Tomas-Cortazar J, et al. The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis. Sci Rep. 2020;10(1):572. doi:10.1038/ s41598-019-57348-0. 10. Pena-Cearra A, Pascual-Itoiz MA, Lavin JL, Fuertes M, Martin-Ruiz I, Castelo J, Palacios A, Barriales D, Fullaondo A, Aransay AM, et al. Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitis. Sci Rep. 2022;12(1):9977. doi:10.1038/s41598022-13480-y. 11. Lloyd-Price J, Arze C, Ananthakrishnan AN, Schirmer M, Avila-Pacheco J, Poon TW, Andrews E, Ajami NJ, Bonham KS, Brislawn CJ, et al. Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature. 2019;569(7758):655–662. doi:10.1038/ s41586-019-1237-9. 12. Peña-Cearra A, Song, D, Castelo, J, Palacios, A, Lavin, JL, Azkargorta, M, Elortza, F, Fuertes, M, Pascual-Itoiz, MA, Barriales, D, Martin-Ruiz, I, Fullaondo, A, Aransay, AM, Rodriguez, H, Palm, NW, Anguita, J, Abecia, L. Mitochondrial dysfunction promotes microbial composition that negatively impacts on ulcerative colitis development and progression. npj Biofilms and Microbes. 2023;9(74):0709–2023. doi:10.1038/s41522023-00443-y37805634. 13. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010;38 (4):576–589. doi:10.1016/j.molcel.2010.05.004. 14. West NR, Hegazy AN, Owens BMJ, Bullers SJ, Linggi B, Buonocore S, Coccia M, Gortz D, This S, Stockenhuber K, et al. Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor–neutralizing therapy in patients with inflammatory bowel disease. Nat Med. 2017;23(5):579–589. doi:10.1038/nm.4307. 15. Czarnewski P, Parigi SM, Sorini C, Diaz OE, Das S, Gagliani N, Villablanca EJ. Conserved transcriptomic profile between mouse and human colitis allows unsupervised patient stratification. Nat Commun. 2019;10 (1):2892. doi:10.1038/s41467-019-10769-x. 16. Smith PD, Janoff EN, Mosteller-Barnum M, Merger M, Orenstein JM, Kearney JF, Graham MF. Isolation and purification of CD14-negative mucosal macrophages from normal human small intestine. J Immunol Methods. 1997;202(1):1–11. doi:10.1016/s0022-1759(96) 00204-9. 17. Castro-Dopico T, Dennison TW, Ferdinand JR, Mathews RJ, Fleming A, Clift D, Stewart BJ, Jing C, Strongili K, Labzin LI, et al. Anti-commensal IgG drives intestinal inflammation and type 17 immunity in ulcerative colitis. Immunity. 2019;50(4):1099–1114 e1010. doi:10.1016/j.immuni.2019.02.006. 18. Ohtsuka Y, Lee J, Stamm DS, Sanderson IR. MIP-2 secreted by epithelial cells increases neutrophil and lymphocyte recruitment in the mouse intestine. Gut. 2001;49(4):526–533. doi:10.1136/gut.49.4.526. 19. Hall AB, Yassour M, Sauk J, Garner A, Jiang X, Arthur T, Lagoudas GK, Vatanen T, Fornelos N, Wilson R, et al. A novel Ruminococcus gnavus clade enriched in inflammatory bowel disease patients. Genome Med. 2017;9(1):103. doi:10.1186/s13073-0170490-5. 20. Dovrolis N, Michalopoulos G, Theodoropoulos GE, Arvanitidis K, Kolios G, Sechi LA, Eliopoulos AG, Gazouli M. The interplay between mucosal microbiota composition and host gene-expression is linked with infliximab response in inflammatory bowel diseases. Microorganisms. 2020;8(3):438. doi:10.3390/ microorganisms8030438. 21. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS, Mackay F, Artis D, et al. Regulation of inflammatory responses by gut GUT MICROBES 15 microbiota and chemoattractant receptor GPR43. Nature. 2009;461(7268):1282–1286. doi:10.1038/ nature08530. 22. Zhai R, Xue X, Zhang L, Yang X, Zhao L, Zhang C. Strain-specific anti-inflammatory properties of two Akkermansia muciniphila strains on chronic colitis in mice. Front Cell Infect Microbiol. 2019;9:239. doi:10. 3389/fcimb.2019.00239. 23. Png CW, Linden SK, Gilshenan KS, Zoetendal EG, McSweeney CS, Sly LI, McGuckin MA, Florin TH. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am J Gastroenterol. 2010;105(11):2420–2428. doi:10.1038/ajg.2010.281. 24. Wang B, Chen X, Chen Z, Xiao H, Dong J, Li Y, Zeng X, Liu J, Wan G, Fan S, et al. Stable colonization of Akkermansia muciniphila educates host intestinal microecology and immunity to battle against inflammatory intestinal diseases. Experimental & Molecular Medicine. 2023;55(1):55–68. doi:10.1038/s12276-022-00911-z. 25. Koelink PJ, Bloemendaal FM, Li B, Westera L, Vogels EWM, van Roest M, Gloudemans AK, van ’t Wout AB, Korf H, Vermeire S, et al. Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling. Gut. 2020;69(6):1053–1063. doi:10. 1136/gutjnl-2019-318264. 26. Rajca S, Grondin V, Louis E, Vernier-Massouille G, Grimaud JC, Bouhnik Y, Laharie D, Dupas JL, Pillant H, Picon L, et al. Alterations in the intestinal microbiome (dysbiosis) as a predictor of relapse after infliximab withdrawal in Crohn’s disease. Inflamm Bowel Dis. 2014;20:978–986. doi:10.1097/MIB. 0000000000000036. 27. Camuesco D, Comalada M, Rodriguez-Cabezas ME, Nieto A, Lorente MD, Concha A, Zarzuelo A, Galvez J. The intestinal anti-inflammatory effect of quercitrin is associated with an inhibition in iNOS expression. Br J Pharmacol. 2004;143(7):908–918. doi:10.1038/sj.bjp.0705941. 28. Lopetuso LR, Petito V, Cufino V, Arena V, Stigliano E, Gerardi V, Gaetani E, Poscia A, Amato A, Cammarota G, et al. Locally injected infliximab ameliorates murine DSS colitis: differences in serum and intestinal levels of drug between healthy and colitic mice. Dig Liver Dis. 2013;45:1017–1021. doi:10.1016/j.dld.2013.06.007. 29. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29 (1):15–21. doi:10.1093/bioinformatics/bts635. 30. Liao Y, Smyth GK, Shi W. The subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013;41:e108. doi:10.1093/nar/ gkt214. 31. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186/ s13059-014-0550-8. 32. Huang da W, BT S, RA L. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57. doi:10.1038/ nprot.2008.211. 33. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 2009;37(Web Server):W305–311. doi:10.1093/nar/gkp427. 34. Kaimal V, Bardes EE, Tabar SC, Jegga AG, Aronow BJ. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems. Nucleic Acids Res. 2010;38:W96–102. doi:10.1093/nar/gkq418. 35. Saito R, Smoot ME, Ono K, Ruscheinski J, Wang PL, Lotia S, Pico AR, Bader GD, Ideker T. A travel guide to Cytoscape plugins. Nat Methods. 2012;9 (11):1069–1076. doi:10.1038/nmeth.2212. 36. Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pages F, Trajanoski Z, Galon J. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009;25 (8):1091–1093. doi:10.1093/bioinformatics/btp101. 37. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using Real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–408. doi:10.1006/meth.2001.1262. 38. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5):335–336. doi:10.1038/nmeth.f.303. 39. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26 (19):2460–2461. doi:10.1093/bioinformatics/btq461. 40. McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, Andersen GL, Knight R, Hugenholtz P. An improved greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 2012;6 (3):610–618. doi:10.1038/ismej.2011.139. 16 A. PEÑA-CEARRA ET AL.