ORIGINAL RESEARCH published: 14 May 2021 doi: 10.3389/fimmu.2021.671239 Patients With Common Variable Immunodeficiency (CVID) Show Higher Gut Bacterial Diversity and Levels of Low-Abundance Genes Than the Healthy Housemates Juraj Bosa´ k 1, Matej Lexa 2, Kristy´na Fiedorova´ 3,4, Darshak C. Gadara 5, Lenka Micenkova´ 5, Zdenek Spacil 5, Jirˇ´ı Litzman 4,6, Toma´ sˇ Freiberger 3,4 and David Sˇ majs 1* Edited by: Antonio Condino-Neto, 1 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia, 2 Faculty of Informatics, Masaryk University, University of São Paulo, Brazil Brno, Czechia, 3 Centre for Cardiovascular Surgery and Transplantation, Brno, Czechia, 4 Department of Clinical Immunology and Allergology, Faculty of Medicine, Masaryk University, Brno, Czechia, 5 RECETOX Center, Faculty of Science, Masaryk Reviewed by: University, Brno, Czechia, 6 Department of Clinical Immunology and Allergology, St. Anne’s University Hospital in Brno, Roshini Sarah Abraham, Brno, Czechia Nationwide Children’s Hospital, United States Vassilios Lougaris, Common variable immunodeficiency (CVID) is a clinically and genetically heterogeneous University of Brescia, Italy disorder with inadequate antibody responses and low levels of immunoglobulins including *Correspondence: David Sˇ majs IgA that is involved in the maintenance of the intestinal homeostasis. In this study, we [email protected] analyzed the taxonomical and functional metagenome of the fecal microbiota and stool metabolome in a cohort of six CVID patients without gastroenterological symptomatology Specialty section: and their healthy housemates. The fecal microbiome of CVID patients contained higher This article was submitted to Primary Immunodeficiencies, numbers of bacterial species and altered abundance of thirty-four species. Hungatella a section of the journal hathewayi was frequent in CVID microbiome and absent in controls. Moreover, the CVID Frontiers in Immunology metagenome was enriched for low-abundance genes likely encoding nonessential Received: 23 February 2021 Accepted: 26 April 2021 functions, such as bacterial motility and metabolism of aromatic compounds. Published: 14 May 2021 Metabolomics revealed dysregulation in several metabolic pathways, mostly associated Citation: with decreased levels of adenosine in CVID patients. Identified features have been ´ ´ BosakJ,Lexa M, Fiedorova K, consistently associated with CVID diagnosis across the patients with various Gadara DC, Micenkova´ L, Spacil Z, Litzman J, Freiberger T and Sˇ majs D immunological characteristics, length of treatment, and age. Taken together, this initial (2021) Patients With Common Variable study revealed expansion of bacterial diversity in the host immunodeficient conditions and Immunodeficiency (CVID) Show Higher Gut Bacterial Diversity and suggested several bacterial species and metabolites, which have potential to be Levels of Low-Abundance Genes diagnostic and/or prognostic CVID markers in the future. Than the Healthy Housemates. Front. Immunol. 12:671239. Keywords: common variable immunodeficiency, CVID, microbiome, metagenome, metabolome, doi: 10.3389/fimmu.2021.671239 Hungatella hathewayi Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 671239 Bosa´ k et al. Metagenome Changes of CVID Patients INTRODUCTION of CVID patients (13). Here, fecal microbiomes of CVID patients were characterized using metagenome sequencing of CVID patients The human gut microbiome is a complex microbial ecosystem and healthy partners sharing the same households. The use of significantly contributing to the host physiology. A number of paired samples minimalized influence of environmental factors on factors including host genetics, age, diet, and antibiotic exposure microbiome composition. Moreover, the effect of CVID-related can change the composition of the gut microbiota. Microbial complications on microbiome was eliminated by exclusion of dysbioses have been associated with various metabolic (obesity, patients with severe CVID phenotypes (e.g., enteropathy, chronic diabetes) and intestinal (inflammatory bowel diseases, colorectal diarrhea) from this study. cancer) diseases, with diseases of the cardiovascular system, liver The stool samples were collected between 2016 and 2017 from or kidney, and also with psychiatric disorders. Since the gut patients being treated at the St. Anne's University Hospital in microbiota modulates the host immune system, a microbial Brno (Czech Republic) and their household members were used dysbiosis was found in immune conditions including allergy, as paired healthy controls. All participants were Caucasians born asthma, atherosclerosis, and multiple sclerosis. The gut and living in the Czech Republic. The healthy controls provided microbiome and its role in human health and disease has been a self-report questionnaire and the patients fulfilled CVID-ICON recently extensively reviewed (1–4). diagnostic criteria (16). The pairs for this study were randomly Common variable immunodeficiency (CVID) is the most selected from the set of 8 participants in our study Fiedorová common primary immunodeficiency with a frequency of 1:25,000 et al. (13) after exclusion of immunosuppression treatment and to 1:50,000 in the human population worldwide. CVID includes CVID phenotypes with gastrointestinal complications. clinically and genetically heterogeneous disorders characterized by a Information about participants is shown in Tables 1 and S1. defect in B cell differentiation leading to inadequate antibody All human clinical samples were collected after patients/healthy responses and low levels of immunoglobulin G (IgG) and IgA, volunteers gave written informed consent regarding their and, inconsistently, also IgM. As a result of this deficient antibody participation in the study. All data used in the study were production, most patients suffer from recurrent respiratory and anonymized and the study was approved by the Ethic gastrointestinal infections and from noninfectious autoimmune or Committee of the Faculty of Medicine, Masaryk University inflammatory complications. This, together with clinical (Protocol no. 37/2016). heterogeneity, are the main reasons why CVID is frequently misdiagnosed and generally underdiagnosed. The current therapy Stool Collection and DNA Extraction is based on administration of IgG and, in some patients, In this study, the genomic DNA was freshly isolated from the immunosuppression, in order to manage the infectious and frozen stool aliquots originated from our previous study (13). autoimmune complications, respectively [reviewed in (5–9)]. Collection of stool samples and extraction of DNA were Since IgA is the dominant mucosal immunoglobulin maintaining performed as described previously Fiedorováet al. (13). Briefly, intestinal homeostasis (10), CVID is expected to impact the gut stool samples were self-collected using a sterile container, microbiome. Indeed, several studies showed changes in bacterial according to the standardized International Human composition of the gut associated with CVID and with severity of Microbiome Standards (IHMS) protocol SOP 03 and delivered the disease (11–15). While previous studies analyzed CVID to the laboratory in 24 hours; where were aliquoted (200 mg), microbiome using sequencing of 16S rRNA gene, we performed frozen, and stored at -80°C. From fecal aliqoutes, DNA was metagenome deep-sequencing for identification of differences among extracted using the current standard operating procedure bacterial species and genetic functions. Moreover, metagenome (protocol Q, International Human Microbiome Consortium) findings have been combined with analysis of stool metabolites. with minor modifications described in Fiedorováet al. (13). Effects of CVID complications (e.g., chronic diarrhea) and DNA eluates were stored at −20°C until processing. environment have been minimized by enrollment of a cohort of six CVID patients without gastroenterological symptomatology and their Metagenomic Sequencing healthy housemates. This initial study revealed, for the first time, an The preparation of DNA libraries and whole genome sequencing expansion of gut bacterial diversity in the immunodeficient were performed in Novogene Co., Ltd. (Hong Kong) with conditions and suggested several bacterial species and metabolites requested 100 Gb data output per each sample. Briefly, after as potential markers for CVID. Since the differences associated with quality control of extracted DNA, 300 bp fragments were CVID have been identified across the patients with various prepared by sonication and the DNA libraries were ® ™ immunological characteristics, various age, and various lengths of constructed using NEBNext Ultra II DNA Library Prep treatment, the identified metagenome and metabolome changes are (New England Biolabs, USA). The libraries were diluted to 2 relevanttodiagnosisratherthanparticularsymptomsorpatients’ age. ng/mL and their quality (>3 nM) was verified by qPCR. The libraries were sequenced using HiSeq platform (Illumina, USA) MATERIALS AND METHODS with paired-end strategy (150 bp). On average, metagenomic sequencing resulted in 365,613,222 Study Design, Recruitment of Participants, reads and 109.69×109 bases per sample (Table S2). and Ethical Approval The data are available under BioProject ID: PRJNA666684 This study extends our previous study, where 16S rRNA gene (NCBI database; https://dataview.ncbi.nlm.nih.gov/ analysis was used for characterization of microbiome composition object/PRJNA666684). Frontiers in Immunology