TYPE Original Research PUBLISHED 21 September 2022 DOI 10.3389/fimmu.2022.966184 Experimental and genetic OPEN ACCESS evidence for the impact EDITED BY Peter Hasselblatt, University Hospital Freiburg, Germany of CD5 and CD6 expression REVIEWED BY Lena Sophie Mayer, and variation in inflammatory University of Freiburg Medical Center, Germany Anna-Maria Globig, bowel disease Salk Institute for Biological Studies, United States Wolfgang Reindl, Sergi Casadó-Llombart 1, Marı́a Velasco-de Andrés 1, University of Heidelberg, Germany Cristina Català 1, Alejandra Leyton-Pereira 1, *CORRESPONDENCE Rebeca Gutiérrez-Cózar 2, Belén Suárez 3, Noelia Armiger 1, Francisco Lozano flozano@clinic.cat Esther Carreras 1, Miriam Esteller 4,5,6, Elena Ricart 4,5,6, SPECIALTY SECTION Ingrid Ordás 4,5,6, Javier P. Gisbert 6,7, Marı́a Chaparro 6,7, This article was submitted to Autoimmune and Autoinflammatory Marı́a Esteve 6,8, Lucı́a Márquez 9, David Busquets 10, Disorders, Eva Iglesias 11,12, Esther Garcı́a-Planella 13, a section of the journal Frontiers in Immunology Marı́a Dolores Martı́n-Arranz 14, Juliane Lohmann 15, RECEIVED 10 June 2022 C. Korcan Ayata 16, Jan Hendrik Niess 16,17, Pablo Engel 1,2, ACCEPTED 31 August 2022 PUBLISHED 21 September 2022 Julián Panés 4,5,6, Azucena Salas 2,4,6, Eugeni Domènech 6,18, CITATION Francisco Lozano 1,2,3* and ENEIDA Project of GETECCU Casadó-Llombart S, 1 Immunoreceptors del Sistema Innat i Adaptatiu, Institut d’Investigacions Biomèdiques August Pi i Velasco-de Andrés M, Català C, Sunyer (IDIBAPS), Barcelona, Spain, 2 Departament de Biomedicina, Facultat de Medicina, Universitat Leyton-Pereira A, Gutiérrez-Cózar R, de Barcelona, Barcelona, Spain, 3 Servei d’Immunologia, Centre de Diagnòstic Biomèdic, Hospital Suárez B, Armiger N, Carreras E, Clı´nic de Barcelona, Barcelona, Spain, 4 Inflammatory Bowel Disease Group, Institut d’Investigacions Esteller M, Ricart E, Ordás I, Gisbert JP, Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 5 Inflammatory Bowel Disease Unit, Chaparro M, Esteve M, Márquez L, Gastroenterology Department, Hospital Clı´nic de Barcelona, Barcelona, Spain, 6 Centro de Busquets D, Iglesias E, Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Garcı´a-Planella E, Martı´n-Arranz MD, Spain, 7 Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Lohmann J, Ayata CK, Niess JH, Sanitaria Princesa (IIS-IP), Universidad Autónoma de Madrid (UAM), Madrid, Spain, Engel P, Panés J, Salas A, 8 Gastroenterology Department, Hospital Universitari Mútua Terrassa, Terrassa, Spain, Domènech E, Lozano F and ENEIDA Project of GETECCU (2022) 9 Gastroenterology Department, Hospital del Mar and Institut Hospital del Mar Investigacions Experimental and genetic evidence for Mèdiques, Barcelona, Spain, 10Department of Gastroenterology, Hospital Universitari de Girona Dr the impact of CD5 and CD6 Josep Trueta, Girona, Spain, 11 Department of Gastroenterology, Hospital Universitario Reina Sofı´a, expression and variation in Córdoba, Spain, 12 Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, inflammatory bowel disease. Spain, 13 Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, Front. Immunol. 13:966184. 14 Department of Gastroenterology, and Innate Immunity Group, IdiPAZ Institute for Health doi: 10.3389/fimmu.2022.966184 Research, La Paz Hospital, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain, 15 Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany, 16 Department of Biomedicine, University of Basel, Basel, Switzerland, 17 University Center for Gastrointestinal and Liver Diseases, St. Clara Hospital and University Hospital, Basel, Switzerland, 18 Gastroenterology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co- receptors involved in fine-tuning intracellular signals delivered upon antigen- specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune- Frontiers in Immunology 01 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)- induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T- rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD’s clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders. KEYWORDS Crohn’s disease, inflammatory bowel disease, ulcerative colitis, CD5, CD6 Introduction region devoid of catalytic activity but well adapted for phosphorylation and association with downstream signaling Inflammatory bowel diseases (IBD) are a group of chronic effectors. Importantly, CD5 and CD6 are physically associated inflammatory conditions of the gastrointestinal tract, including with the T cell receptor complex (TCR) with which co-localize at ulcerative colitis (UC) and Crohn’s disease (CD). The precise the center of the immunological synapse (6), providing etiology of IBDs remains unknown, though their relation inhibitory (CD5) and activating/inhibitory (CD6) signals (7). to multiple and diverse genetic, immunological and This is likely achieved through interaction with endogenous environmental factors is accepted. Genome-wide association counter-receptors such as CD166/activated leukocyte cell studies (GWAS) have identified immune-related genes adhesion molecule (ALCAM) (8), Galectins 1 and 3 (9), and associated to susceptibility and/or clinical manifestations that CD318/CUB domain-containing protein 1 (CDCP-1) (10) for point to an inappropriate regulation of innate and/or adaptive CD6, and still ill-defined ligands (CD72, IgVh framework, immune responses in IBD (1). However, these polymorphisms gp200, gp40-80, gp150, IL-6 and CD5 itself) for CD5 (11–18). alone do not account for IBD heritability, suggesting that other Both molecules also act as pattern-recognition receptors (PRRs) environmental, epigenetic and genetic factors, including rare for microbial-associated molecular patterns (MAMPs), where variants, must be in place (1). CD5 interacts with fungal (b-glucan) (19), viral (hepatitis C CD5 and CD6 are paralogs sharing homology in tissue virus) (20), and parasitic (E. granulosus teguments) structures expression patterns, structure and function (2–4). They encode (21), while CD6 does it with bacterial (lipopolysaccharide, signal-transducing surface co-receptors expressed on all T and lipoteichoic acid and peptidoglycan) (22), viral (gp120 HIV-1) B1a cells and involved in the fine tuning of intracellular (23), and parasitic (E. granulosus tegument) structures (21). This activation signals delivered upon specific antigen recognition dual role of CD5 and CD6 as both immunomodulatory and by lymphocyte’s clonotypic receptors (5). Both CD5 and CD6 microbial PRR receptors is supported by pre-clinical models of receptors are composed of an extracellular region encompassing infection, autoimmunity and cancer involving Cd5- and Cd6- three tandem scavenger receptor cysteine-rich (SRCR) domain deficient mouse lines, as well as infusion of wild-type mice with repeats, a transmembrane region, and a cytoplasmic soluble CD5 and CD6 proteins (24–26). Frontiers in Immunology 02 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 To date, no CD5 or CD6 deficiencies have been reported in background weighing >20 g. Body weight and disease activity humans. However, functionally relevant single nucleotide index (DAI) were monitored every day. DAI was scored as polymorphisms (SNPs) of CD5 and CD6 have been identified, follows: rectal bleeding (absent=0, present=1), animal motility which act as susceptibility or disease modifier markers in (normal=0, reluctant=1, hunched=2), fur appearance (normal=0, autoimmune and neoplastic processes. Allelic combinations of ruffled=1, spiky=2) and body weight loss (none=0, 0-5%=1, 5-10% the CD5 rs2241002 and rs2229177 SNPs resulting in hyper- =2, 10-15%=3, >15%=4). At day 8, mice were euthanized by reactivity to TCR stimulation are associated to more severe cervical dislocation for collection of blood and organ samples. systemic lupus erythematosus (SLE) forms, but predict better Colons were measured and weighted, and terminal pieces were prognosis in chronic lymphocytic leukemia (CLL) and collected for histology and RNA extraction. EDTA-anticoagulated melanoma (27–29). Moreover, GWAS have involved CD5 blood was centrifuged in heparinized capillaries for 30 min at 1000 (rs2229177) in rheumatoid arthritis susceptibility (30). xg and hematocrit was calculated as the length of packed red Regarding CD6, the rs12360861, rs17824933 and rs11230563 blood cells (RBC) divided by the total blood length (RBC + serum) SNPs are revealed as disease modifiers in psoriasis, and as multiplied by 100. For RBC count, blood was diluted in PBS and susceptibility markers in multiple sclerosis (MS) and Behç et’s RBC were counted with a hemocytometer. For microbiological disease (31–34). Also, GWAS and meta-analyses have associated analyses, mesenteric lymph nodes (mLN) and liver were collected the CD6 rs11230563 SNP to IBD susceptibility (35, 36). under sterile conditions and disaggregated through a 40 mm nylon However, its role as a disease modifier in IBD, and the mesh for overnight (o/n) seeding at 37 °C on Columbia agar plates involvement of other neighboring SNPs from the CD6 and with 5% sheep blood (Becton-Dickinson) and colony forming unit CD5 genes, and from the functionally related CD166/ALCAM (cfu) counting. Pieces of ~2 mm were cut from the terminal colon gene, are still unknown. of mice and submerged in RNA later (Sigma) o/n at 4 °C before The present work explores the consequence of CD5 and CD6 being stored dry at -80 °C for further RNA analysis or fixed in PBS expression and variation in experimental and clinical IBD. To containing 4% paraformaldehyde during 48 h for histological this end, we first analyzed the impact of Cd5 and Cd6 deficiency studies. RNA was extracted using the TRIzol® Reagent (Life on dextran sulphate sodium (DSS)-induced colitis, an Technologies) and the PureLink™ RNA Mini Kit (Ambion, Life experimental model of human IBD (37). Subsequent clinical Technologies) following manufacturer’s instructions, with the aid association studies assessed the impact of CD5 and CD6 of a QIAGEN TissueLyser. RNA was quantified and variations on different clinically relevant manifestations and retrotranscribed into cDNA by using the High-capacity cDNA therapeutic requirements of CD and UC. Kit (Life Technologies). Cytokine mRNA levels were assessed by real-time quantitative PCR (RT-qPCR) with the TaqMan™ Fast Universal PCR Master Mix No AmpErase™ UNG (Life Materials and methods Technologies) using a 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA, US) and the following Mice FAM gene expression assays: Mm01179194_m1 (Cd3e), Mm00435532_m1 (Pdcd1), Mm00432423_m1 (Cd79a), Mm Cd5-deficient (Cd5 -/- ) mice backcrossed to C57BL/6 01337324_g1 (Ncr1), Mm00447885_m1 (Klrc1), Mm00 background were provided by Chander Raman (University of 447885_m1 (Mpo), Mm01324470_m1 (Lcn2), Mm00440502 Alabama at Birmingham) (38). Cd6-deficient (Cd6-/-) C57BL/6 _m1 (Nos2), Mm00801778_m1 (Ifng), Mm00439619_m1 mice were obtained through a development agreement with the (Il17a), Mm00445259_m1 (Il4), Mm00439614_m1 (Il10), Mm0 Knock-Out Mouse Project Repository (KOMP), an international 0444241_m1 (Il22), Mm00443260_g1 (Tnf), Mm00434228_m1 consortium promoted by the National Institutes of Health (NIH; (Il1b), Mm00446190_m1 (Il6), Mm01178820_m1 https://www.komp.org) (39). Wild-type C57BL/6 mice from (Tgfb1), Mm00441259_g1 (Ccl3), and Mm04207460_m1 (Cxcl1), Charles River Laboratories (France) were bred in our animal Mm00450960_m1 (Tbx21), Mm01261022_m1 (Rorc), facility. All mouse procedures were approved by the Animal Mm00484683_m1 (Gata3) and Mm00475162_m1 (Foxp3), all Experimentation Ethical Committee from University of Barcelona. from Thermo Fisher Scientific. Relative cytokine mRNA expression normalized to Gapdh (Mm99999915_g1) expression was calculated as 2 -DDCt , where DDCt = (CT Gene of interest sample − CT GAPDH DSS-induced mouse colitis model sample) − (CTGene of interest basal − CTGAPDH basal). For histological analysis, fixed samples were included in Colitis was induced by administration of 2% (w/v) 36-50 kDa paraffin. Three micrometer tissue sections were obtained and DSS (MP Biomedicals) in drinking water for 5 days to 11- to 19- stained with hematoxylin-eosin. Histology was scored by two week-old wild-type, Cd5-/- and Cd6-/- female mice of C57BL/6 independent evaluators according to the following parameters: Frontiers in Immunology 03 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 degree of inflammation (0-3), goblet cell loss (0-2), abnormal or Genotyping Master Mix and TaqMan probes for CD5 hyperproliferative crypts (0-3), abscesses (0-1), architectural (rs2229177, rs2241002), CD6 (rs12360861, rs17824933, damage (0-2), transmural damage (0-3). Images were obtained rs11230563), and CD166/ALCAM (rs6437585) (all from with an Eclipse 50i microscope, using a Pan Fluor 10x/0.30 Thermo Fisher), following manufacturer’s instructions. objective and a Digital Sight DS-5M camera, all from Nikon. Genotyping failure rate was lower than 0.02 for all SNPs. For immunohistochemical analysis, paraffin-embedded 5 mm tissue sections were immersed in xylene and dehydrated in ethanol. After antigen retrieval, tissue sections were blocked with PBS Definitions 5% FBS. For myeloperoxidase (MPO) chromogenic immunohistochemistry assay, primary goat anti-mouse MPO Location (terminal ileum, colon, ileocolon, and upper polyclonal antibody (R&D Systems) was incubated at 4°C gastro-intestinal) and behavior (nonstricturing and overnight. Then, endogenous peroxidase activity was blocked nonpenetrating, stricturing, and penetrating) of CD were using PBS 0.3% H2O2 solution for 10 min at room temperature classified according to the Montreal classification (41). For and peroxidase-labelled rabbit anti-goat IgG secondary antibody statistical analysis of location, a value of 1 was assigned to (SIGMA) was incubated for 1h at room temperature. Tissue patients with colonic disease, 2 to patients with ileocolonic sections were stained using 3,3’-diaminobenzidine (DAB; disease and 3 to patients with ileal disease, independently of SIGMA) and then hematoxylin staining was performed following upper gastro-intestinal tract involvement. Upper gastro- standard protocols. Sections were mounted with DPX and intestinal tract involvement (presence vs. absence) was visualized at 20x magnifications using a NIKON e600 microscope. assessed independently of distal ileal and colonic involvement. For CD3ϵ and IgM immunofluorescence assay, endogenous For statistical analysis of extent in UC patients, a value of 0 was biotin was blocked with the Avidin/Biotin blocking kit SP-2001 assigned to patients with ulcerative proctitis (Montreal (VectorLabs) following manufacturer’s indications. Then, classification E1) and a value of 1 was assigned to patients primary antibodies rabbit anti-mouse CD3ϵ (Cell signaling, with left-sided UC or extensive UC (Montreal classification E2 D4V8L; dilution 1/100) and FITC-labelled goat anti-mouse and E3). Prognosis was calculated as previously described: IgM (Southern Biotech; dilution 1/200) were incubated at 4 °C patients not requiring any immunomodulatory nor surgical overnight. Biotin-labelled secondary donkey anti-rabbit IgG treatment during at least 4 years of follow-up from diagnosis antibody (Jackson Immunoresearch, dilution 1/200) was were classified as “good prognosis” while patients requiring two incubated for 1 h at room temperature. Finally, A555-labelled or more immunomodulatory treatments and/or two or more streptavidin (Roche, dilution 1/200) was incubated for 20 min at abdominal surgeries were described as “poor prognosis” (42). room temperature and samples were mounted with mounting medium (PBS 80% glycerol). Samples were visualized at 10 and 20x magnifications using a NIKON e600 microscope. Statistical analyses Statistical analysis in patient/donor cohort studies was DNA samples from patients and controls performed with R 3.6.0 (R Foundation for Statistical Computing, Vienna, Austria), with the packages ‘SNPassoc’, Genomic DNA samples from CD (n=1352) and UC patients ‘survival’, and ‘haplo.stats’ available at the Comprehensive R (n=1013) were retrieved from the ENEIDA biobank upon Archive Network (CRAN) repository. The ‘association’ function approval from the Spanish Working Group on CD and UC included in the ‘SNPassoc’ package was used to assess linkage (GETECCU) (40). Control genomic DNA samples from between each SNP and desired clinical variables with generalized volunteer donors of the Blood and Tissue Bank (BST) of the linear models. For each analysis, 4 models were generated Generalitat de Catalunya (n=604) were purified by using the (codominant, dominant, recessive, log-additive), and the MagNA Pure 96 DNA and Viral NA Large Volume Kit (Roche model with lowest Akaike information criterion (AIC) was Diagnostics, Rotkreuz, Switzerland) and the High-throughput chosen. Analyzed variables were age of onset (calculated date robotic workstation MagNa Pure 96 (Roche Diagnostics). The of diagnosis − date of birth), peripheral arthritis/arthralgia, study was approved by the Ethical Committee of Clinical ankylosing spondylitis, sacroiliitis, sclerosing cholangitis, Research of the Hospital Clı́ nic de Barcelona. cutaneous manifestations (pyoderma gangrenosum or erythema nodosum), ocular manifestations (uveitis or iritis), requirement of biological treatments and prognosis. SNP genotyping Additionally, location of the disease, presence of stenosis, presence of fistulae and perianal disease were included in the Genomic DNA samples (20 ng) were subjected to RT-PCR CD cohort and extent of the disease was included in the UC in a LightCycler® 480 Instrument (Roche) using TaqMan cohort. In all analyses, sex and persistent tobacco consumption Frontiers in Immunology 04 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 were included as co-variants. To generate stenosis-free survival responsible for the differences observed in disease severity. and fistulae-free survival curves, time between enrolment and Histological analyses showed noticeable crypt architectural complication (patients with stenosis or fistulae) or between distortion in colon samples from both Cd6+/+ and Cd6-/- mice, enrolment and last follow-up (patients without stenosis or with no significant differences between their histology scores fistulae) was calculated. Cox proportional hazards regression (Figure 2D). Immunohistochemical analyses of the colonic was used to assess the linkage between each SNP and stenosis- mucosa composition revealed no significant differences in free survival or fistulae-free survival. Association of SNPs with terms of granulocyte (MPO+), T cell (CD3ϵ+) and B cell susceptibility to CD, UC or combined (IBD) was assessed with (IgM+) infiltrates (Figure 2E). Gene expression analyses of a the ‘association’ function by comparing each cohort with the wide panel of pro-/anti-inflammatory cytokine and chemokine control cohort. P values were corrected for false discovery rate and transcription factors revealed decreased expression of Ifng, (FDR) with the ‘p.adjust’ function (q values). The ‘haplo.glm’ Cd3e, Ncr1 and Gata3 together with increased expression of Il6 function included in the ‘haplo.stats’ package was used to assess and Cxcl1 in Cd6-/- mice with regard to controls (Figure 3A). A linkage between haplotypes and binary clinical variables with trend towards increased expression of Lcn2 was also observed generalized linear models, and odds ratio (OR) and confidence (Figure 3A). No differences were observed regarding expression intervals (CI) for these associations were obtained with the of Tgfb1, Tnf, Il1b, Il17a, Il10, Il22, Tbx21, Rorc, Foxp3, Ccl3, ‘haplo.cc’ function. Klrc1, Pdcd1, Cd79a, Mpo and Nos2 (Supplementary Figure 1). In the study of animal models, normality of data was Expression of Il4, a target of GATA3, was also analyzed but it assessed with the D’Agostino & Pearson normality test. When was undetectable in a high proportion of samples. Additional data was normally distributed, differences were assessed by t- analyses showed significantly increased Il17a/Ifng ratio and a tests, otherwise Mann-Whitney tests were performed. In similar trend for Rorc/Gata3 but no differences in the Tbx21/ multiple comparisons, P values were corrected for false Gata3 ratio in Cd6-/- mice (Figure 3B). discovery rate. CD5 and CD6 variants impact clinical Results expression of IBD Cd5 and Cd6 deficiency modulate DSS- CD (n=1352) and UC (n=1013) patients from the ENEIDA induced colitis registry and volunteer blood donor controls (n=604) were genotyped for functionally relevant CD5 (rs2229177, The putative role of CD5 and CD6 lymphocyte co-receptors rs2241002), CD6 (rs12360861, rs11230563, rs17824933), and in the pathophysiology of IBD was first explored by subjecting CD166/ALCAM (rs6437585) SNPs. All SNPs were in Hardy- Cd5-/- and Cd6-/- mice to the DSS-induced colitis. Cd5-/- mice Weinberg equilibrium, except for the rs2241002 in the CD showed a less aggressive disease than Cd5 +/+ controls cohort (p=0.0276). Description of the study SNPs and cohorts (Figure 1A), as deduced from lower body weight loss and DAI, are shown in Tables 1, 2. in agreement with a previously published result (43). In contrast, No association was found for any of the SNPs analyzed with Cd6-/- mice showed an exacerbated phenotype with regard to disease susceptibility following comparisons of controls with the Cd6+/+ controls. Accordingly, Cd6-/- mice presented a higher CD and UC cohorts, either separately (CD vs. controls, UC vs. body weight loss (Figure 1B, left) and a higher DAI (Figure 1B, controls) or together (IBD vs. controls). Next, the effect of the right), which was seasonally influenced, as observed in spring/ CD5, CD6 and CD166/ALCAM gene variants on different summer and autumn/winter variations (Figure 1C). clinically relevant parameters of CD (age at diagnosis, Contrary to the Cd5 -/- case, the lack of published behavior, location, perianal disease and prognosis) and UC information of Cd6-/- mice on the DSS-induced colitis model (age at diagnosis, extent and prognosis) was assessed. A encouraged a deeper evaluation of different experimental significant association was found for the CD5 rs2241002CC parameters at the end of disease follow-up (day 8). No genotype with preferential ileal location in the CD cohort differences were observed in colon length, weight or weight/ (Table 3). Because CD location can influence the risk of length ratio relative to Cd6+/+ controls (Figure 2A). As illustrated developing stenosis and fistulae, association between the SNP in Figure 2B, Cd6-/- mice presented increased hematocrit and stenosis-free and fistulae-free survival was tested, but no consistent with higher diarrhea-induced fluid loss, and a trend significant results were found. Association between CD5 SNPs to lower RBC counts together with increased mean corpuscular and upper-gastrointestinal (GI) tract affectation was also not volume (MCV) consistent with moderate rectal bleeding and significant. Haplotypic analyses showed increased need of erythroblast production, respectively (44). No differences in cfu biologic therapies in CD patients carrying the CD5 rs2241002C count were observed in mLN and liver (Figure 2C), arguing rs2229177T haplotype compared with those carrying the most against differential bacterial translocation to draining organs as common rs2241002C rs2229177C haplotype (Table 4). Similarly, Frontiers in Immunology 05 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 A B C FIGURE 1 DSS-induced colitis in Cd5-/- and Cd6-/- mice vs. wild-type controls. (A) Percentage of basal body weight (left) and DAI (right) of Cd5-/- mice vs. Cd5+/+ controls. Data combined from two independent experiments is shown. (B) Percentage of basal body weight (left) and DAI (right) of Cd6-/- mice vs. Cd6+/+ controls in spring/summer (between April and September). Basal body weight data are combined from four independent experiments, while DAI data are combined from two independent experiments. (C) Percentage of basal body weight (left) and DAI (right) of Cd6-/- mice vs. Cd6+/+ controls in autumn/winter (between October and February). Basal body weight data are combined from four independent experiments, while DAI data are come from a single experiment. Mean ± SD values are depicted. Statistical differences were assessed by multiple t-tests (one per day) controlled with the FDR approach. *, q<0.01. UC patients carrying the CD5 rs2241002T rs2229177T haplotype Figure 4, the CD6 rs17824933GG genotype was significantly had a worse prognosis than those carrying the rs2241002C associated with shorter fistula-free survival (HR = 1.56, 95% rs2229177C haplotype (Table 4). CI 1.01–12.42, p = 0.046). No significant association was found Regarding CD6 SNPs associations, the rs17824933 GG between CD6 SNPs and upper-GI tract affectation. The CD6 genotype was associated with preferential ileal location in CD rs17824933GG genotype was also associated with higher extent patients (Table 3). This led us to also test association between (left or extensive colitis) in UC patients (Table 5). The CD6 this SNP and stenosis-free and fistulae-free survival. As seen in minor rs12360861A allele showed association with better Frontiers in Immunology 06 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 A C B D E FIGURE 2 Monitoring of DSS-induced colitis parameters from Cd6-/- mice vs. Cd6+/+ controls at day 8 post-induction. (A) Dot plot showing colon length, weight and weight to length ratio of Cd6-/- (n=17) and Cd6+/+ control (n=17) mice. Mean ± SD values are depicted. Statistical differences were assessed by t-test. (B) Hematocrit, RBC count and mean corpuscular volume (MCV) at day 8 from Cd6-/- (n=8) and Cd6+/+ (n=8). Mean ± SD values are depicted. Statistical differences were assessed by t-test. **, p<0.01. (C) Analysis of microbial translocation into mesenteric lymph nodes (mLN; top) and liver (bottom) from the same mice as in (B) Depicted are mean ± SD of cfu/mg. Statistical differences were assessed by Mann-Whitney tests. (D) Histology score (mean ± SD, left) and representative haematoxylin-eosin stains from DSS-treated Cd6+/+ (center) and Cd6-/- (right) mice. Scale bar: 200 mm. Statistical differences were assessed by t-test. (E) Immunohistochemical analyses of the terminal colon in DSS-treated mice. Percentage of MPO, CD3ϵ and IgM-stained tissue (mean ± SD) from colon sections. Statistical differences were assessed by t-test. prognosis in CD patients (Table 5). Regarding the appearance of SNP, which has been reported to influence CD166/ALCAM extra-intestinal manifestations, logistic regression analyses transcriptional activity and MS risk (45, 46). showed a significant association of homo- or heterozygous combinations of the CD6 rs17824933G allele with lower risk of ankylosing spondylitis in the whole cohort of IBD Discussion patients (Table 5). No statistical association was observed with any of the We provide experimental and clinical evidence for the clinical parameters analyzed for CD166/ALCAM rs6437585 involvement of CD5 and CD6 expression and variation in Frontiers in Immunology 07 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 A B FIGURE 3 mRNA expression in colons from Cd6-/- vs. Cd6+/+ mice at day 8 post DSS-induced colitis. (A) Relative mRNA expression of different transcripts from colon samples. Depicted are mean ± SD of mRNA fold increase (DSS/basal). (B) Fold increase ratio of indicated mRNA transcripts from colon samples. Ratios were calculated by dividing the fold increase of the following transcripts: Il17a, Ifng, Rorc, Gata3, Tbx21 and Gata3. Statistical differences were assessed by Mann-Whitney tests and corrected for FDR. *, q< 0.1. IBD. Previous GWAS and meta-analysis studies have identified and signal the presence of microbial components via PRRs, the CD6 locus (SNP rs11230563) as a susceptibility marker in which belong to different structural families such as lectin C- CD and UC, thus supporting its contribution to IBD type, leucine-rich repeats (LRR), immunoglobulin (Ig), or etiopathogenesis (35, 36). Here, we used genetically modified scavenger receptor cysteine-rich (SRCR) domains (48). Both mice and candidate gene-driven association analyses to clinical CD5 and CD6 are lymphocytic members of the SRCR traits and prognosis with functionally relevant SNPs from the superfamily, expressed by all T cells and the B1a subset CD5 and CD6 paralogs, as well as from CD166/ALCAM. responsible for production of polyreactive natural IgM Etiopathogenic factors for IBD include host genetic antibodies (4, 49). CD5 and CD6 are also represented in susceptibility, dysregulated immune response, intestinal certain immune cells subsets present in mucosal barriers such dysbiosis, and impairment of intestinal epithelial barrier as regulatory T (Treg) and B (B1a, Breg) cells, certain function. Under normal circumstances, there is continuous macrophage and dendritic cells and innate lymphoid cells crosstalk between gut microbiota and the immune system, (NK, iNKT, ILCs) (50–52). CD166/ALCAM, the best where gut microbiota modulates the host’s innate and adaptive characterized CD6 ligand, is also found in the gastrointestinal immunity and vice versa (47). Gut microbiota is in close contact epithelial tract (53). Increased expression of both CD6 and with the intestinal barrier, consisting of an epithelial cell layer CD166/ALCAM has been reported in inflamed mucosa from and a variety of immune cells of hematopoietic origin. Cells from IBD patients, a fact that is attributed to higher CD6-expressing T the intestinal barrier (both epithelial and hematopoietic) sense cell infiltration rather than surface CD6 expression levels (54). Frontiers in Immunology 08 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 This may relate to quantitative trait loci studies in which the with differential expression of certain mRNA transcripts. This rs11230584 SNP in the intergenic region between CD5 and CD6 included decreased expression of Ifng —the prototypical Th1 modulates expression of both genes in IBD patients but not in cytokine— and Gata3 —the master regulator of Th2 healthy controls (55). Taken together, their tissue and cell differentiation—, no differences in Il17a and Il10 expression, and expression pattern, microbial recognition properties and ability increased expression of Il6 and Cxcl1 —a cytokine and a to modulate lymphocyte activation/differentiation and cell chemokine involved in the Th17 function, all this pointing to a adhesion provide the basis for considering both CD5 and CD6 somehow misbalanced Th1/Th2/Th17 response. Additionally, as contributors to IBD pathogenesis. reduced mRNA expression of Ncr1 —coding for NKp46, one of The observation that both CD5- and CD6-deficient mice the NK triggering receptors— and a trend towards increased Lcn2 differ in their response to DSS-induced colitis further supports —coding for lipocalin-2, also named NGAL, a neutrophil their involvement in IBD. In Cd5-/- mice, attenuated DSS- secondary granule marker— in colons of Cd6-/- mice undergoing induced colitis was observed in agreement with a previous DSS-induced colitis was observed. These findings would fit with report (43). The mechanism underlying such attenuated colitis the observation that i) decreased NK cell activity would lead to has already been explored and attributed to increased increased granulocyte infiltrate in DSS-induced colitis (63), and ii) suppressive function of Treg cells from Cd5-/- mice (43), a fact increased lipocalin-2 expression would act as marker and a counter that was not confirmed by others (56). An alternative reactor of colonic inflammation (64, 65). mechanism could be the increased activation-induced cell Given the multifaceted nature of CD6, the above-mentioned death (AICD) in Cd5-/- effector T cells as a result of inhibitory mechanistic findings for exacerbated symptoms during DSS role assigned to the CD5 receptor (7, 57). Further evidence for colitis in Cd6-/- mice do not exclude other possibilities such as CD5 expression involvement in IBD comes from recent report decreased Treg functionality in Cd6-/- mice (59), a cell subset showing that inducible Cd5-deficient mice in the autoimmune- known for its role in mucosal protection during DSS-induced prone non-obese diabetic (NOD) background undergo colitis (66). Another possibility could be the deficient production exacerbated DSS-induced colitis by modifying T cell effector of natural antibodies, as found in Cd6-/- mice from DBA-1 function (58). background (60), and confirmed by us in the C57BL/6 Regarding Cd6-/- mice, no analysis of DSS-induced colitis background Cd6-/- mice used here (67). Natural antibodies are has been brought forward, in spite of reports of Cd6-/- mice an innate component of humoral immunity and have a behavior in several other immune-related inflammatory disease protective role in IBD (68, 69). models (i.e., intestinal ischemia-reperfusion, bovine or avian In humans, no CD5 or CD6 deficiencies have been reported. type II collagen-induced arthritis, chronic graft-versus-host Functionally relevant CD5 or CD6 SNPs previously described act disease-induced lupus-like, imiquimod-induced psoriasis-like as susceptibility or disease modifier markers for immune-related skin inflammation, experimental autoimmune encephalitis, disorders. Regarding CD5, the rs2241002 and rs2229177 SNPs and autoimmune uveitis) (10, 34, 39, 59-62). CD6 deficiency cause nonsynonymous substitutions at the extracellular results in attenuated or exacerbated phenotypes according to (Pro224>Leu) and cytoplasmic (Ala471>Val) regions, mouse background and experimental models responsive to respectively, which are relevant to CD5-mediated signal different underlying mechanisms (e.g., increased AICD or transduction (70). Thus, homozygous carriers of the ancestral defective Treg function). This puzzling situation has been Pro224-Ala471 (rs2241002C and rs2229177C) CD5 haplotype are unveiled by CD6 receptor’s multitask signalosome with hyper-reactive to TCR/CD3 cross-linking, and present more opposite functions in T cell activation (7). CD6 multifaceted severe clinical forms of SLE (27) but better CLL and role accounts for past difficulties in classifying it as a co- melanoma prognosis (28, 29). Regarding the functionality of inhibitory or -stimulatory receptor. CD6 SNPs, the intronic rs17824933G allele identified as a Here we observed that Cd6-/- mice exhibit an increased body susceptibility marker for MS causes over-expression of a CD6 weight loss and DAI upon DSS-colitis induction, in conjunction isoform devoid of the CD166/ALCAM-binding domain TABLE 1 Summary of the CD5, CD6 and CD166/ALCAM SNPs analyzed in the present study. Gene SNP ID Location Major/Minor allele Effect CD5 rs2241002 Exon 5 C>T Pro224>Leu rs2229177 Exon 10 C>T Ala471>Val CD6 rs17824933 Intron 1 C>G CD6Dd3 rs11230563 Exon 4 C>G Arg225>Trp rs12360861 Exon 5 G>A Ala271>Thr CD166/ALCAM rs6437585 5’ UTR C>T ↑Transcription Frontiers in Immunology 09 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 TABLE 2 Clinical characteristics of the study cohorts. Parameter CD (n=1352) UC (n=1013) IBD (n=2365) Sex Male 661 (48.9%) 530 (52.3%) 1191 (50.3%) Female 691 (51.1%) 483 (47.7%) 1174 (49.6%) Ethnicity Caucasian 1173 856 2029 Arab 13 10 23 Asian 6 3 9 African 5 3 8 Jew 4 1 5 Romani 3 3 6 Other 11 6 17 Smoking* 380 (28.1%) 108 (10.7%) 488 (20.6%) Age at diagnosis (years) 29.7 (22.4, 41.2) 35.2 (26.8, 47.8) 32.0 (23.7, 44.2) Follow-up (years) 12.0 (7.4, 19.2) 12.4 (7.4, 19.2) 12.2 (7.4, 19.2) Extra-intestinal manifestations Peripheral arthritis 287 (12.1%) Ankylosing spondylitis 73 (3.1%) Sacroiliitis 68 (2.9%) Sclerosing cholangitis 22 (0.9%) Cutaneous 158 (6.7%) Ocular 56 (2.4%) Location Colonic 234 (17.3%) Ileocolonic 631 (46.7%) Ileal 355 (26.3%) Phenotype Stricturing 342 (25.3%) Penetrating 251 (18.6%) Perianal disease 361 (26.7%) Extent Proctitis 153 (15.1%) Left or extensive colitis 828 (81.7%) Biological treatments 809 (59.8%) 282 (27.8%) 1091 (46.1%) Prognosis Good 137 (10.1%) 441 (43.5%) 578 (24.4%) Poor 577 (42.7%) 232 (22.9%) 809 (34.2%) (*) persistent habit at the last follow-up. Number of patients is shown for categorical parameters. Median and interquartile range is shown for numerical parameters. (CD6Dd3) concomitant with diminished proliferation and long- severity parameters in CD (requirement of biological term activation of CD4+ T cells (71, 72). Also, the MS-protective treatments) and UC (poor prognosis) patients. The haplotype involving the CD6 rs11230563C and rs2074225C SNPs rs2229177T variant involves the substitution of ancestral results in higher surface CD6 expression on several lymphocyte Ala471 for Val, which results in increased CD5 inhibitory subsets (CD4+ and CD8+ naïve T, and NKT cells) (31). capacity (27, 70). This can turn activated lymphocytes less In our genetic analysis the CD5 rs2241002 SNP, which sensitive to AICD and more damaging, thus making more causes a nonsynonymous substitution in the extracellular intensive therapies necessary. SRCR domain 2 of CD5 (Pro224>Leu), showed association Analysis of CD6 SNPs showed association of the with CD location. Further analyses showed association of CD5 rs17824933 G allele with preferred ileal CD location and haplotypes containing the cytoplasmic rs2229177T variant with increased UC extent. These results consolidate the damaging Frontiers in Immunology 10 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 TABLE 3 Linear regression analysis of CD5 rs2241002 and CD6 rs17824933 SNPs association with CD location. Corrected for sex and smoking. SNP Model Genotype Colonic =1 Ileo-colonic =2 Ileal =3 mean s. e. Difference of means (95% CI) q value CD5 Dominant C/C 119 391 228 2.148 0.025 0.005 rs2241002 C/T-T/T 103 206 105 2.005 0.035 -0.142 (-0.224,-0.059) CD6 Recessive C/C-C/G 215 557 302 2.081 0.021 0.022 rs17824933 G/G 8 38 31 2.299 0.074 0.218 (0.060, 0.377) Variable “location” is codified as: colonic=1, ileocolonic=2, ileal=3. p value corrected for FDR. s. e.: standard error. effect of the rs17824933G allele in inflammatory diseases, as environmental factors is illustrated by seasonal onset and suggested from its association with more aggressive forms of exacerbation patterns in IBD patients (75). We have observed psoriasis and with MS susceptibility (34, 46). Patients with left- seasonal variations also for Cd6-/- mice regarding susceptibility to sided or extensive UC also tend to need more aggressive DSS-induced colitis. More precisely, the exacerbated DSS-induced therapies and are at higher risk of developing colorectal cancer colitis phenotype of Cd6-/- mice manifested during the spring/ (73). A relatively short follow-up (median 12.37 years; Q1 7.43 summer but not the autumn/winter season (Figure 1C), years; Q3 19.21 years) may underlie the lack of significant reminiscent of other mouse models of human diseases (i.e., EAE) differences observed for this SNP regarding prognosis (76). Though incompletely understood, it has been proposed that (Supplementary Table 1). seasonal variations might be regulated by endogenous circannual The CD6 rs17824933G allele was further associated with lower rhythms, since they are found even when the animals are subjected risk of ankylosing spondylitis in the whole IBD cohort. This result to a constant, controlled environment, and genetically regulated appears to contradict the above-mentioned deleterious (77). Seasonality in Cd5-/- mice could be neither confirmed nor contribution of this variant in UC, as well as in psoriasis and denied, since the two experiments performed were carried out in MS. However, this variant also showed association with a more the summer season (July and September). However, recent ileal location of CD. Articular extra-intestinal manifestations of unpublished results from a collaborative study show seasonality IBD are more common in patients with colonic disease than in phenomena in Cd5-/- mice upon mannan-induced psoriatic those with small-bowel disease (74). Thus, preferential ileal arthritis induction (Merino R and Merino J, University of location in CD patients may account for the association of Cantabria, Spain). rs17824933G with lower ankylosing spondylitis risk. The main strengths of our study are the use of a large patient The study also showed association of the CD6 rs12360861 cohort, which together with the experimental mouse model SNP with prognosis in CD patients but not susceptibility, in highlights a role for CD5 and CD6 in IBD. We also agreement with a major genetic contribution to prognosis from acknowledge some caveats in our study. Separate breeding of loci distinct from those driving disease susceptibility, applicable Cd5-/- or Cd6-/- mice and their wild-type counterparts can be a in this case (42). source of confusion, which we minimized by periodic colony As stated above, genetic susceptibility is only one of the known refresh, the use of high sample sizes, and a large number of factors in IBD etiopathogenesis. The importance played by other repetitions. We measured mRNA expression as a proxy for TABLE 4 Logistic regression analysis of CD5 haplotype association with biological therapy requirement in CD (top half) and to prognosis in UC (bottom half). Haplotype % in CD patients Biological p value OR (95% CI) rs2241002 rs2229177 % no % yes C C 43.4 45.3 42.2 C T 35.9 33.4 37.5 0.048 1.20 (1.00, 1.44) T T 17.0 17.4 16.7 0.811 1.02 (0.83, 1.27) T C 3.7 3.8 3.7 0.861 1.05 (0.64, 1.72) Haplotype % in UC patients Prognosis p value OR (95% CI) rs2241002 rs2229177 % good % poor C C 43.3 45.2 39.5 C T 37.1 37.1 37.2 0.345 1.14 (0.87, 1.49) T T 14.9 13.7 17.0 0.048 1.42 (1.00, 2.02) T C 4.7 3.9 6.3 0.097 1.78 (0.90, 3.51) Frontiers in Immunology 11 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 A B FIGURE 4 Stenosis and fistulae in CD patients according to rs17824933. Stenosis-free survival (A) and fistulae-free survival (B) of CD patients carrying different CD6 rs17824933 genotypes. Statistical differences were assessed by the Cox proportional hazards model. (A) In the stenosis-free survival analysis hazard ratio (HR) comparing GG and CC genotypes was 1.28, (95% CI 0.85–1.92), p = 0.230, and HR comparing CG and CC genotypes was 1.05, (95% CI 0.84–1.31), p = 0.671. (B) In the fistulae-free survival analysis HR comparing GG and CC genotypes was 1.56, (95% CI 1.01–12.42), p = 0.046, and HR comparing CG and CC genotypes was 1.19, (95% CI 0.92–1.55), p = 0.195. TABLE 5 Logistic regression analysis of CD6 SNP association with CD prognosis (top), UC extent (middle), and ankylosing spondylitis in IBD patients (bottom), corrected for sex and smoking. SNP Model Genotype Good prognosis (%) Poor prognosis (%) OR (95% CI) q value rs12360861 Log-additive A alleles (0, 1, 2) 127 (18.9) 544 (81.1) 0.62 (0.45, 0.86) 0.027 SNP Model Genotype Proctitis (%) Left/extensive colitis (%) OR (95% CI) q value rs17824933 Recessive C/C-C/G 151 (98.7) 758 (93.0) 0.010 G/G 2 (1.3) 57 (7.0) 5.68 (1.37, 23.48) SNP Model Genotype No ankylosing spondylitis (%) Ankylosing spondylitis (%) OR (95% CI) q value rs17824933 Dominant C/C 456 (54.1) 51 (71.8) 0.016 C/G-G/G 387 (45.9) 20 (28.2) 0.45 (0.27, 0.78) protein expression, but correlation between mRNA and protein Data availability statement levels is limited. Therefore, further protein expression assays (e.g.: flow cytometry) will be needed to ascertain the molecular The original contributions presented in the study are mechanisms underlying differences between Cd6-/- and their included in the article/Supplementary Material. Further wild-type counterparts. Similarly, further molecular inquiries can be directed to the corresponding author/s mechanisms driving clinical SNP associations in IBD patients are to be identified. In conclusion, our findings support a role for the CD5 and Ethics statement CD6 lymphocyte receptors in the pathophysiology of IBD and hint at their potential in patient stratification and as therapeutic The studies involving human participants were reviewed and targets. The latter is particularly valid for CD6, where the approved by Ethical Committee of Clinical Research of the humanized anti-CD6 mAb Itolizumab currently represents a Hospital Clı́ nic de Barcelona. The patients/participants therapeutic option in several immune-mediated disorders (78), provided their written informed consent to participate in this since could modulate the activity of the T cell subsets (i.e., Th1 study. The animal study was reviewed and approved by Comitè and Th17) involved in their pathogeny (79). Ètic d’Experimentació Animal, Universitat de Barcelona, Spain. Frontiers in Immunology 12 frontiersin.org Casadó-Llombart et al. 10.3389/fimmu.2022.966184 Contributing GETECCU members Immunological Societies-Immunology Letters (EFIS-IL) and Erasmus+ from the European Union, respectively. The ENEIDA Alfredo J. Lucendo, Gastroenterology Department, Hospital registry of GETECCU is supported by Biogen, Janssen, Takeda General de Tomelloso, IIS-IP, Ciudad Real, Spain, and Centro de and Pfizer. The funders were not involved in the study design, Investigació n Biomé dica en Red de Enfermedades Hepá ticas y collection, analysis, interpretation of data, the writing of this Digestivas (CIBERehd). Jordi Guardiola, Hospital Universitari article or the decision to submit it for publication. de Bellvitge (IDIBELL), l’Hospitalet de Llobregat, Spain. Xavier Calvet, Servei d’Aparell Digestiu, Hospital Universitari Parc Taulı́ , Departament de Medicina, Universitat Autònoma de Acknowledgments Barcelona, Sabadell, Spain, and CIBERehd. Lorenzo Olivá n, Hospital San Jorge, Huesca, Spain. Marta Piqueras, We thank Belchin Kostov for statistical analysis support, Gastroenterology Department, Consorci Sanitari de Terrassa, Marcos Isamat for manuscript editing and critical comments, Barcelona, Spain. and Silvia Ariño for technical help with RT-PCR and immunohistochemistry quantification and interpretation. We are indebted to GETECCU and the IDIBAPS Biobank for Author contributions clinical data and sample procurement. Conceptualization: FL, AS, SC-L, and JP. Mouse studies: SC-L, MV-dA, CC, NA, AL-P, RG-C, EC, MiE, JL, KA, JHN, PE, Conflict of interest and AS. Genetic studies: SC-L, MV-dA, CC, NA, AL-P, BS, EC, and JL. Sample and clinical information collection: ER, IO, JPG, FL is founder and ad-honorem scientific advisor of Sepsia MaE, LM, DB, EI, EG-P, MDM-A, AJL, JG, XC, LO, MP, and JP. Therapeutics. The remaining authors declare that the research Statistical analyses and figures: SC-L, EC, and JL. Writing was conducted in the absence of any commercial or financial original draft: SC-L and FL. All authors read, critically revised relationships that could be construed as a potential conflict and approved the final version of the manuscript. of interest. Funding Publisher’s note This work was supported by Spanish Ministerio de Economı́ a y Competitividad (MINECO, SAF2016-80535-R) and Ministerio All claims expressed in this article are solely those of the de Ciencia e Innovació n (MCIN/AEI/10.13039/501100011033, authors and do not necessarily represent those of their affiliated PID2019-106658RB-I0 0), c o-financed by Europ ean organizations, or those of the publisher, the editors and the Development Regional Fund “A way to achieve Europe” ERDF, reviewers. Any product that may be evaluated in this article, or and Agència de Gestió d’Ajuts Universitaris i de Recerca from claim that may be made by its manufacturer, is not guaranteed Generalitat de Catalunya (2017/SGR/1582). SC-L, MV-dA, CC, or endorsed by the publisher. AL-P, and EC are recipients of fellowships from Spanish Ministerio de Educació n, Cultura y Deporte (FPU15/02897), Spanish MINECO (BES-2014-069237 and BES-2017-082107), Supplementary material Chilean Agencia Nacional de Investigació n y Desarrollo (2018- 72190154), and European Community Seventh Framework The Supplementary Material for this article can be found Program (FP7/2007/2013; 229673), respectively. SC-L and JL are online at: https://www.frontiersin.org/articles/10.3389/ recipients of short-term fellowships from European Federation of fimmu.2022.966184/full#supplementary-material References 1. Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inflammatory 3. Padilla O, Calvo J, Vilà JM, Arman M, Gimferrer I, Places L, et al. Genomic bowel disease. Nature (2011) 474:307–17. doi: 10.1038/nature10209 organization of the human CD5 gene. Immunogenetics (2000) 51:993–1001. 2. Lecomte O, Bock JB, Birren BW, Vollrath D, Parnes JR. 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