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Parallelism of intestinal secretory IgA shapes functional microbial fitness

Abstract

Dimeric IgA secreted across mucous membranes in response to nonpathogenic taxa of the microbiota accounts for most antibody production in mammals. Diverse binding specificities can be detected within the polyclonal mucosal IgA antibody response1,2,3,4,5,6,7,8,9,10, but limited monoclonal hybridomas have been studied to relate antigen specificity or polyreactive binding to functional effects on microbial physiology in vivo11,12,13,14,15,16,17. Here we use recombinant dimeric monoclonal IgAs (mIgAs) to finely map the intestinal plasma cell response to microbial colonization with a single microorganism in mice. We identify a range of antigen-specific mIgA molecules targeting defined surface and nonsurface membrane antigens. Secretion of individual dimeric mIgAs targeting different antigens in vivo showed distinct alterations in the function and metabolism of intestinal bacteria, largely through specific binding. Even in cases in which the same microbial antigen is targeted, microbial metabolic alterations differed depending on IgA epitope specificity. By contrast, bacterial surface coating generally reduced motility and limited bile acid toxicity. The overall intestinal IgA response to a single microbe therefore contains parallel components with distinct effects on microbial carbon-source uptake, bacteriophage susceptibility, motility and membrane integrity.

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Fig. 1: Monoclonal dimeric IgA coats distinct bacterial subpopulations.
Fig. 2: In vivo mIgA reconstitution of antibody-deficient JH−/− mice provides a scalable model to study mSIgA function.
Fig. 3: Surface-binding mSIgA exerts antigen-dependent individual and generic functional alterations in intestinal bacteria.

Data availability

Raw sequence reads are deposited under BioProject PRJNA702008 and are associated with Fig. 3a, d and Extended Data Figs. 6a–d, h–k and 9b. Bacterial sequencing reads were annotated to the E. coli genome of strain MG1655 (U00096.2). GO terms and gene set members were extracted from the EcoCyc E. coli database at https://ecocyc.org. There are no restrictions on data availability. Source data are provided with this paper.

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Acknowledgements

The Clean Mouse Facility is supported by the Genaxen Foundation, Inselspital and the University of Bern. We thank P. Nicholson, I. Keller, F. Blank, B. Haenni and staff at the flow cytometry, sequencing and microscopy core facilities of the German Cancer Research Center and University of Bern. J. Gärtner, C. Winter, D. Foster, C. Busse and R. Murugan helped with monoclonal antibody production and single-cell Ig-gene sequencing. H. Waller, D. Kalbermatter, K. Lau, R. Glockshuber, B. Yilmaz, J. Leo, C. Piselli, M. Winterhalter and J. Stanisich provided reagents and support. S. Hapfelmeier (University of Bern), provided the smooth E. coli strain BW24599. J. Zimmermann, F. Ronchi, H. Li, S. Ganal-Vonarburg, I. D. Young and M. Felber (University of Bern) helped with in vivo experimentation. Grant support was obtained from the Swiss National Science Foundation (310030_179479 and Sinergia CRSII5_177164) and European Research Council (ERC advanced grant HHMM-Neonates project no.742195) to A.J.M.; and through the Swiss National Science Foundation (310030_184757) and Swiss Cancer League/Swiss Cancer Research (KFS-4958-02-2020) to S.v.G. T.R. was supported by a postdoctoral long-term EMBO fellowship (ALTF 1040-2018) and funding from the Bern University Research Foundation.

Author information

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Authors

Contributions

T.R. and A.J.M. conceived the project and designed and interpreted experiments. Experiments were carried out by T.R., with support from S.B. L.M. and S.v.G. performed glycan array analysis. J.L. performed LPS structure analysis and provided purified defined LPS samples. H.W. established and supervised the technology for monoclonal antibody selection, expression and analysis. T.R. and A.J.M. wrote the manuscript. All authors approved the manuscript.

Corresponding authors

Correspondence to Tim Rollenske or Andrew J. Macpherson.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Sidonia Fagarasan, Maria Rescigno and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Ig gene and antibody-binding characteristics of monoclonal antibodies derived from intestinal plasma cells of germ-free and transitorily HA107-colonized mice.

a, Strategy to obtain HA107-reactive monoclonal antibodies from lamina propria plasma cells after transient intragastric (i.g.) microbial priming of germ-free wild-type (WT) mice. b, Flow cytometric binding analysis of luminal polyclonal SIgA derived from donor mice (each line shows a dilution series from an individual mouse, n = 6) to intact live E. coli HA107, 21 days after transitory colonization. Red dashed line indicates upper limit of binding from germ-free controls. c, Fluorescence-activated cell sorting gate for isolation of IgA+ lamina propria plasma cells for single-cell Ig sequencing, analysis and monoclonal Ig expression. dg, Isotype distribution (d), clonal expansion (e) IGHV and IGKV gene combination (f) and number of IGHV somatic hypermutations (g) in single lamina propria plasma cell Ig gene repertoires of HA107-primed mice derived from six donor mice (df) or compared to germ-free controls obtained from six donor mice (g), red bar shows mean. n = 378 and 379 IGHV sequences derived from single plasma cells from germ-free or HA107-exposed animals, respectively (g). h, Cloning and recombinant antibody expression strategy for monoclonal human IgG1. i, Frequency of Igλ light-chain-expressing cells in Ig gene repertoires of HA107-primed mice compared to germ-free controls (n = 6 mice), red bar shows mean. j, k, Flow cytometric binding analysis of live intact HA107 bacteria surface-coating monoclonal IgG1 and negative control B8-030 (j) showing the frequency of surface-binding IgG1 in HA107-associated antibodies and germ-free controls used at 1 μg ml−1 within each animal (k). l, ELISA binding of monoclonal IgG1 derived from HA107-primed mice or germ-free controls used at 1 μg ml−1 to HA107 lysate. m, Clonal expansions of lysate- (grey fill) and surface-binding (dark fill) monoclonal IgG1: the frequencies of B cell clusters are shown separated within the overall populations. n, Numbers of IGHV somatic hypermutations in HA107-binding (n = 22) and nonbinding (n = 168) antibodies and their clonal members derived from HA107-primed mice, red bars show mean values. Statistics show two-sided Mann–Whitney U-test (g, n). Data are representative of two (b, c, j, l) independent experiments.

Extended Data Fig. 2 Antibody-binding characteristics of HA107-binding monoclonal dimeric IgA.

a, Cloning and recombinant antibody expression strategy for mouse dimeric IgA. b, c, f, Additional data showing further mIgAs that are not included in Fig. 1a–c due to space limitations. b, Individual monoclonal IgA binding to intact live HA107 bacteria by flow cytometry (top panels), or the HA107 lysate (bottom panels) by ELISA. Red dashed lines indicate cutoff for positivity. c, Flow cytometry analysis of mIgA binding of non-auxotrophic JM83 parental strain of HA107. d, Flow cytometric analysis showing distinctive nonreplicating, replicating and dying subpopulations of JM83 isolated from fresh faecal pellets of antibody-deficient mice ex vivo. e, Flow cytometric microscopy of faecal JM83 nonreplicating (top), replicating (middle) and dying (bottom) subpopulations stained with mIgA4250. f, Binding of individual monoclonal IgAs to faecal JM83 bacterial nonreplicating, replicating or dying subpopulations gated as in Extended Data Fig. 2d. mIgA clone numbers are indicated at the top of each column (b, c, f). Data are of one (e) or representative of two (bd, f) independent experiments.

Extended Data Fig. 3 Intestinal dimeric mIgAs bind discrete bacterial membrane fractions.

ad, ELISA binding of individual monoclonal dimeric IgAs ordered as in Fig. 1a–c (a, b) or Extended Data Fig. 2b, c, f (c, d) to HA107 membrane fraction (top), cytoplasmic fraction (middle) and purified ribosomes (bottom) (a, c) or intact outer membrane vesicles (b, d). mIgA clone numbers are indicated at the top of each column. Red dashed lines indicate upper limit of binding from negative control antibody mIgAmGO53. The insets within the mIgA4559 (a, b) and the mIgA4025 (c, d) column show control serum IgG binding from a mouse injected with 108 c.f.u. of E. coli HA107 14 days previously.

Extended Data Fig. 4 Intestinal dimeric mIgAs bind discrete membrane-associated antigens.

a, E. coli LPS core oligosaccharide structures (E. coli O86 (K12), W3110 (K12), O10 (R1), O100 (R2), and O111 (R3))37. K12, R1, R2 and R3 depict core type. R indicates nonstoichiometric substitutions; phosphate group (P); pyrophosphate group (PP); pyrophosphorylethanolamine (PPEtn). *Indicates heterogeneity of the core OS regarding terminal sugar residues. Blue indicates outer and black indicates inner core region. LA indicates lipid A. b, Binding of mIgA4053 to different structures of purified rough and smooth LPS shown in (a). c, Flow cytometric analysis of mIgA4308 coating of selective fimbrial (fim) gene E. coli deletion strains34 and the E. coli wild-type (BW25113). d, e, Flow cytometric binding analysis to E. coli wild-type (left) and single gene deletion strains (right) of mIgA4249 (d) and mIgA4250 (e). f, ELISA binding of indicated mIgA to lysates of E. coliΔenvZ, E. coliΔompC, or a series of targeted E. coli deletion strains, or the wild-type with normal OmpC expression (details in Supplementary Table 4). g, Electron micrograph of the outer membrane vesicle fraction of E. coli HA107. Scale bar, 200 nm. h, i, Flow cytometric binding analysis of mIgA4308 to smooth strain BW2459932 (h) and E. coli JM83 pre-incubated with and without α-methyl mannoside42 to block high mannose binding sites (i). j, Gene expression ratios assessed by RNA sequencing showing the 20 most upregulated genes from fractionated mIgA4308 coated compared to uncoated HA107 bacteria (left), fimbrial (fim) operon members are highlighted (right). k, ELISA binding of mIgA4219, mIgA4250 and mIgA4584 to purified recombinant OmpC protein. Each line represents the dilution series of an individual mIgA and the negative control mIgAmGO53 is shown in green. l, Surface plasmon resonance affinity measurements of OmpC-binding mIgA4250 (left) and mIgA4219 (right). Antibody affinities (KD) are shown in the upper left of each panel. Data were obtained from one (g, j, l) or are representative of two independent experiments (b-f, h, i, k).

Extended Data Fig. 5 IgA secretion and elimination in the in vivo mIgA reconstitution model.

a,b,c IgA concentration measurements of secretion and elimination after i.v. injection by ELISA following injection of monomeric (n = 2 mice) or dimeric mIgA in unmanipulated wild-type C57BL/6 (n = 2 mice) and dimeric mIgA in C57BL/6 mice (n = 3) following bile duct ligation (BDL). Serial measurements from serum (a), bile (b) or faeces (c), individual data points are shown. d, Decay of mIgA in serum after injection of different dimeric mIgAs (n = 3 for each condition; \(\bar{x}\) ± s.d.). e, c.f.u. in colonic/faecal fractions of JM83 at 10.5 h after reconstitution with the indicated mIgA species (\(\bar{x}\) ± s.d., mSIgA4250 n = 6, mSIgA4308 n = 3 and mSIgAB8-030 n = 5 faecal samples from individual mice).

Source Data

Extended Data Fig. 6 Functional consequences of reconstituted mSIgA in vivo and in vitro.

a, OmpC target gene expression from faecal JM83 bacterial RNA Sequencing analysis at 10.5 h following reconstitution with different mIgAs as shown on the x axis (\(\bar{x}\) ± s.d., mSIgA4250 n = 8, mSIgA4584 n = 10, mSIgA4219 n = 4 and mSIgAB8-030 n = 10 faecal samples from individual mice). b, ompF and lamB gene expression from faecal JM83 bacterial RNA sequencing at 10.5 h p.i. after mSIgA4250 reconstitution (\(\bar{x}\) ± s.d., mSIgA4250 n = 8 and mSIgAB8-030 n = 10 faecal samples from individual mice). c, d, Venn diagrams of downregulated (c) or upregulated genes (d) after reconstitution with different OmpC-binding mSIgAs at 10.5 h p.i. e, Flow cytometric binding analysis of mIgA-4250 to ompC-deficient strain BL21∆C (top) and BL21∆CF (bottom) with (right) and without (left) ectopic OmpC expression35,36. Numbers adjacent to gates indicate bound bacterial frequencies. f, [14C]Glucose uptake in BL21∆CF bacteria with or without pGOmpC complementation coated with indicated mIgA, (\(\bar{x}\) ± s.d., mSIgAB8-030, n = 5 with and 3 without pGOmpC, mSIgA4250 n = 6 with and 3 without pGOmpC). g, Protection from bacteriophage T4 infection of mIgA-4250-coated OmpC-deletion and complemented bacteria (\(\bar{x}\) ± s.d., n = 4 for each condition). h, Fimbrial operon members target gene expression from faecal JM83 bacterial RNASeq analysis at 10.5 h following reconstitution with different mIgAs as shown on the x axis (\(\bar{x}\) ± s.d., mSIgA4308 n = 5 and mSIgAB8-030 n = 10 faecal samples from individual mice). i, Differential gene expression of enriched GO term gene members in faecal JM83 bacteria after reconstitution with fimbriae (fimb.)-binding mSIgA4308 and negative control mSIgAB8-030. Numbers (right y axis) identify gene annotations in Supplementary Table 10. j, k, mSIgA target gene expression from faecal JM83 bacterial RNA sequencing 24 h following mIgA reconstitution, at a time when mSIgA had been almost completely shed from the animal via the faeces (\(\bar{x}\) ± s.d., individual data points are shown). Compare 10.5 h in a and h. Statistics show two-sided Wald test (a, b, h) and two-sided paired (f) and unpaired t-test (g), *P < 0.05. Data are from one experiment (k) or representative from two experiments (e) or pooled from two (f, g, j), four (a, b, h) and five (c, d, i) independent experiments.

Source Data

Extended Data Fig. 7 Bacterial aggregation of mIgA-4308.

a, OD of E. coli JM83 grown in the presence of mIgA (\(\bar{x}\) ± s.d., n = 4 for each condition, mIgA4308 P = 0.0054). b, c, Bacterial aggregates of fim-locked E. coli strain AAEC189[pSH2]33 after addition of mIgA or yeast mannan quantified (b) and as representative images (c). Box plot shows mean and 25–75th percentiles, whiskers show minimum and maximum value (b). mSIgA4308 n = 68, mSIgAB8-030 n = 70, Mannan n = 58 and mSIgA4250 n = 68. Bar corresponds to 10 μm. Statistics show two-sided paired t-test (a) and two-sided unpaired t-test (b). Data are representative from three experiments (c) or pooled from two (a) or three (b) independent experiments.

Source Data

Extended Data Fig. 8 Properties of polyreactive monoclonal antibodies derived from plasma cells of germ-free and HA107-primed mice.

a, ELISA binding of monoclonal IgG1 derived from plasma cells of germ-free (n = 85 monoclonal antibodies, top) and HA107-primed mice (n = 91 monoclonal antibodies, lower row) using standard polyreactivity measurement conditions27 to LPS O111:B4 (left panels), insulin (middle) and double-stranded (ds) DNA (bottom). Red dashed lines show cutoffs according to convention: red and green lines show positive and negative control antibodies mIgG1ED38 and mIgG1mGO53, respectively. b, c Polyreactive antibody frequencies among all tested antibodies ordered by donor mouse (b) or clonal frequencies for HA107-primed mice (c). d, e, Summary data of frequency of antibodies that are clonally-expanded among polyreactive (upper left) or nonreactive antibodies (upper right) or including HA107-binders (lower left and right respectively) among all expressed antibodies (d) and per HA107-primed donor animal (e). Number of antibodies per group is indicated (d). \(\bar{x}\) ± s.d., n = 3 mice for each condition (e). f, Frequency of polyreactive antibodies within HA107-binding (left) and nonbinding (right) antibodies. Number of antibodies per group is indicated. g, ELISA binding of mIgA4250 and mIgA4186 to LPS (left), insulin (middle) and double-stranded (ds) DNA (right). Red dashed lines show cutoffs according to the highest value of the mIgAmGO53 negative control, red and green lines show positive and negative control antibodies mIgAED38 and mIgAmGO53, respectively. h, Flow cytometric binding analysis of polyreactive (black) and nonpolyreactive (light grey) IgG1 and mIgA to faecal bacteria pooled from C57BL/6 SPF (n = 6) or RAG−/− (n = 2) animals (\(\bar{x}\) ± s.d.). Number of antibodies tested is indicated at the top of each group. Red and green symbols show polyreactive antibodies 4186 and 4250, respectively. i, Glycan array binding of mIgA4186, mIgA4250 and pooled SPF control serum (n = 3). j, Differential gene expression of enriched GO term gene members in faecal JM83 bacteria after reconstitution with polyreactive mSIgA4186 and negative control mSIgAB8-030. k, Differential gene expression of enriched GO terms in faecal JM83 and JW2203(ΔompC) bacteria after reconstitution with polyreactive mSIgA4186, mSIgA4250 and negative control mSIgAB8-030. Statistics show two-sided Fisher’s Exact (d, f), two-sided unpaired t-test (e) and two-sided Mann–Whitney U-test (h) + Padj < 0.1. Data are obtained from one (h, k), representative from two (g, i) or three (a) and pooled from five (j, k) independent experiments.

Extended Data Fig. 9 Generic functional effects of surface-coating mSIgA.

a, Flow cytometric live/dead analysis to faecal E. coli JM83 with (left) or without (right) preincubation with bile acids. b, DE flagellar motility genes after indicated mSIgA reconstitution (P values mSIgA4308: flgM P = 0.038, flhA P = 0.036, flhB P = 0.044, fliF P = 0.035, fliN P = 0.048, fliO P = 0.015, fliR P = 0.002, fliT P = 0.003, fliZ P = 0.037, fliI P = 0.008, fliL P = 0.028, fliP P = 0.042; mSIgA4081: fliR P = 0.010, fliI P = 0.035, fliP P = 0.013; mSIgA4250: flgA P = 0.028, flgB P = 0.025, flgC P = 0.014, flgD P = 0.042, flgF P = 0.019, flgG P = 0.049, flhC P < 0.001, flhD P = 0.030, fliA P = 0.019, fliE P = 0.025). Data are representative from two (a) or five (b) independent experiments.

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Rollenske, T., Burkhalter, S., Muerner, L. et al. Parallelism of intestinal secretory IgA shapes functional microbial fitness. Nature 598, 657–661 (2021). https://doi.org/10.1038/s41586-021-03973-7

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