Recurrent Clostridioides difficile Infection Is Associated With Impaired T Helper Type 17 Immunity to C difficile Toxin B

Clostridioides difficile is a Gram-positive, toxin-producing bacteria that major cause of hospital-acquired infection.1Clements A.C. et al.Lancet Infect Dis. 2010; 10: 395-404Abstract Full Text PDF PubMed Scopus (138) Google Scholar During C infection (CDI), the bacterial toxins toxin A (TcdA) and B (TcdB) are produced, which intestinal epithelial injury inflammation symptoms including diarrhea abdominal pain.2Aktories K. al.Annu Rev Microbiol. 2017; 71: 281-307Crossref (85) First-line CDI treatment typically vancomycin or metronidazole, antibiotics kill vegetative forms eliminate production. However, in 25%–35% patients, commensals fail to effectively repopulate colon, allowing antibiotic-resistant spores germinate recurrent disease.1Clements Despite prevalence CDI, immune response this pathogen poorly characterized. Most previous studies have focused on analysis antitoxin antibodies, do not correlate with protection from CDI.3Wullt M. al.Clin Vaccine Immunol. 2012; 19: 1552-1554Crossref (22) We hypothesized CD4+ T-cell TcdA TcdB after natural may be new way characterize how pathogen-specific immunity shapes disease susceptibility severity. specifically aimed identify distinguishing features patients primary versus compared healthy control individuals no clinical history CDI. Study protocols were approved by research ethics boards University British Columbia (H15-01682, H09-01238, H18-02553) Vancouver Coastal Health Authority (V15-01682). All participants ?18 years old provided written informed consent. Cohort demographics shown Supplementary Table 1, inclusion/exclusion criteria Methods. Patients new-onset those experiencing their first known episode; had experienced >2 recurrences. The OX40 assay was performed as described.4Cook L. al.Cell Mol Gastroenterol Hepatol. 2020; 9: 485-506Abstract (4) antibodies used CD3-BV785, CD8-APCFire750, CD39-FITC, CXCR3-BV421, CCR6-APC, CCR4-BV605 (BioLegend) CD4-AlexaFluor700, CD25-PECy7, CD134(OX40)-PE, Integrin ?7-PECy5 (Becton, Dickinson Company). 4-laser LSRII FortessaX20 flow cytometer Company) for data acquisition, using application settings. Assay cutoffs >0.02% (mean ± 3 standard deviations unstimulated wells) T cells at least 20 gate. Analysis FlowJo software, version 10.6. (Treestar Inc). See Methods additional details. To quantify toxin-specific cells, we assay,4Cook detects induced coexpression CD25 44 hours incubation antigen. Pediacel (Sanofi Pasteur Ltd), childhood pentavalent vaccine, positive antigen (Figure 1A). confirm antigen-specific enrichment within CD25+OX40+ CD25–OX40– population, sorted TcdB-stimulated assays, only proliferating restimulation 1B). majority detectable TcdB-, but TcdA-, specific cells. tended higher frequencies TcdB- TcdA-specific increase significant There differences Pediacel-specific between groups 1C), indicating TcdA- TcdB-specific antigen- disease-specific. found correlation Age, Treatment Systemic antibiotics, Leukocyte count, serum Albumin, Serum creatinine measure renal function (ATLAS) score cohort 1D). cohort, frequency and, greater extent, significantly moderate/severe mild 1E). epitopes, peptide pool 15mers overlapping 12 amino acids C-terminal combined repetitive oligopeptides (CROPs) domain (TcdBCROPS) tetramer-guided mapping HLA-DRB1?07:01–recurrent peptides (5?-3?): 37, VGFVTINDKVFYFSDSGIIE (amino [aa] 2119–2138); 38, KVFYFSDSGIIESGVQNIDD (aa2127–2146); 64, FTDEYIAATGSVIIDGEEYY (aa2335–2354) detected n = 2 (Supplementary Figure 1C) represent immunogenic regions TcdB. Enzyme-linked immunosorbent assays IgG IgA TcdA, TcdB, TcdBCROPS, target neutralizing antibodies.5Chen P. al.Nat Struct Biol. 2019; 26: 712-719Crossref (12) levels, anti-TcdB levels both patient Antibody did severity (data shown) next quantified proportions helper (Th) Th1, Th2, Th17, Th17.1 enriched Th17 proportion reduced recurrent, new-onset, 1F). This unique observed cell phenotype CD25+OX40+CD4+ restimulated phorbol myristate acetate/ionomycin, showing cell–associated cytokines interleukin (IL) 10, IL17A, IL17F, IL22 CD25–OX40–CD4+ Similarly, vitro expansion, expressed marker CD161 secreted report here T-cell, antibody, responses distinguished positively correlated finding echoes what has been Th other microbes, pathogenic commensal.4Cook Scholar,6Becattini S. al.Science. 2015; 347: 400-406Crossref (202) Scholar,7Gaffen S.L. Moutsopoulos N.M. Sci 5: eaau4594Crossref importance IL17 gut against infections well-described,7Gaffen although there also roles described CDI,8Saleh M.M. Host Microbe. 25: 756-765Abstract (27) highlighting need further work into protective effects Although present most individuals, increased repeated more severe infection. Thus, rather than potentially useful biomarker In conclusion, study provides mechanistic insight develops colonization suggests effective requires development authors wish thank Drs Rebecca Gomez, I-Ting Chow, Eddie A. James Benaroya Research Institute Virginia Mason, Seattle, assistance epitope experiments; Xiaojiao Wang, Hannah Peters, Laura Oliveira, Torey Lau, Regan Mah Columbia, Vancouver, Canada, experiments, recruitment, critical revision manuscript; Dr. Simon Hirota Calgary growing difficile. Cook, PhD (Conceptualization: Supporting; Formal analysis: Lead; Funding acquisition: Investigation: Methodology: Writing – original draft: Lead); William D. Rees, MSc (Formal review & editing: Supporting); May Q. Wong, BSc W. Kwok, Data curation: Megan Levings, Equal; Supervision: Equal); Theodore Steiner, MD Resources: Equal). Inclusion laboratory-confirmed diagnosed tcdB PCR result presence (?3 loose watery bowel movements per day ?2 consecutive days), antibiotic treatment, lack spontaneous resolution. Exclusion all cohorts HIV infection, receipt intravenous immunoglobulin rituximab 6 months, hemorrhagic disorder precluding phlebotomy, pregnancy.•New-onset cohort: incidence receiving physician-prescribed metronidazole vancomycin, samples collected 5 days diagnosis.•Recurrent assessment and/or ?1 episode unprovoked recurrence (return months successful without antibiotics).•Healthy group: diagnosis compromise, chronic diseases/conditions. medical obtained participants. received phone follow-ups every 14 45 assess (initial cessation diarrhea, followed completion therapy stool test difficile). For classification, given an ATLAS published criteria.1Hernandez-Garcia R. Garza-Gonzalez E. Miller al.Application evaluating Clostridium teaching hospitals Mexico.Braz J 399-402Crossref (11) Individuals classified having if they never required hospitalization. If hospitalization administration fluids during episode, defined moderate disease, multiple hospitalizations, disease. Peripheral blood 9-mL sodium heparin vacutainers Company [BD]) transported ambient temperature. set up whole 4 collection, plasma aliquots stored –80°C. Staphylococcal enterotoxin (Sigma-Aldrich) 1 ?g/mL. Pediacel, vaccine containing components pertussis diphtheria tetanus toxoids, inactivated poliomyelitis Haemophilus influenzae conjugate Ltd, Lyon, France) 1/40 dilution. toxoids (formaldehyde inactivated, List Biological Laboratories Inc) 10 ?g/mL ?g/mL, respectively. Soluble anti-CD3 (clone OKT3, UBC Lab) 0.25 Tetanus toxoid (Enzo Life Sciences, C. ATCC strain 9689 grown YT agar plates anaerobically 48–72 37°C; then colonies brain heart infusion broth 37°C anaerobic chamber 24–48 hours. Genomic DNA extracted previously described,2Sim J.H. Anikst V. Lohith al.Optimized protocol simple extraction high-quality genomic whole-genome sequencing.J Clin 53: 2329-2331Crossref (10) TcdBCROPS amplified following primers: 5?-GGTTCGAACTATTCACTAATCACTAATTGAGC-3? 5?-CTCGGATCCTGAAGAAAATAAGGTGTCACAAG-3?. 6xHis tag Escherichia coli BL21(DE3)PLysS purified metal affinity chromatography described.3Donnelly M.A. Steiner T.S. Two nonadjacent enteroaggregative flagellin activation toll-like receptor 5.J Biol Chem. 2002; 277: 40456-40461Abstract (117) Quantification anti-TcdA, IgG, enzyme-linked (ELISA), described,4Cook Lisko D.J. Wong M.Q. al.Analysis flagellin-specific adaptive reveals links dysbiosis inflammatory disease.Cell (8) coated Inc (Campbell) 1/100 dilution Pediacel. Levels anti-TcdBCROPS measured ELISA made pooled our high-titer included each generate relative arbitrary units cross-assay standardization. Cell supernatants stimulation ng/mL 12-myristate 13-acetate (PMA) 500 ionomycin. Concentrations 13-plex cytokine bead array kit according manufacturer’s directions. Cytokine acquired 3-laser Cytoflex (Beckman Coulter) analyzed FCAP v3 software (BD). Approximately 30 mL peripheral centrifuged 500g 15 minutes, buffy coat layer mixed Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific), plated well 6-well tissue culture plate (BD) stimulated 40–48 isolated RosetteSep cocktail (STEMCELL Technologies) live CD4+CD25+OX40+ FACSAria IIu sorter >95% purity. Sorted expanded autologous antigen-presenting (or soluble anti-CD3) 200 U/mL IL2 (Proleukin). Antigen-presenting mononuclear negative isolation CD3+ EasySep (Miltenyi Biotec), irradiated 25 Gy, × 105 cells/well U-bottom 96-well plates; 20,000 added, incubated (5% CO2). days. Culture media X-VIVO (Lonza) supplemented 5% heat-inactivated human (NorthBio Inc), 1% Glutamax (Invitrogen), penicillin-streptomycin (Invitrogen). proliferation staining ?mol/L Proliferation Dye eF450 Epitope described5Archila L.L. Kwok W.W. Tetramer-guided mapping: rapid approach HLA-restricted epitopes composite allergens.Methods 1592: 199-209Crossref (9) HLA-DRB–restricted CROPS (UniProt identifier: P18177, aa1831–2366) library 20mers (total 66 peptides, Mimotopes). MP63, influenza (flu) epitope, tetramer.6Ge X. Tan Bollyky P.L. al.Assessment seasonal virus-specific CD4 2009 pandemic H1N1 swine-origin virus.J Virol. 84: 3312-3319Crossref (91) Briefly, pools well. After days, stained patient-specific HLA-DRB1 tetramers loaded mix pool. Cultures tetramer individual peptides. Statistical analyses Mann-Whitney U or, paired samples, Wilcoxon signed rank test. ?3 Kruskal-Wallis 1-way variance (ANOVA) Friedman ANOVA Dunn comparison posttest. P values considered when <.05. Prism, 8 (GraphPad Software statistical analyses. Error bars median interquartile range; ?P ? .05, ??P .01, ???P .001, .0001; ns indicates significant. NA, applicable.