Host immunomodulatory lipids created by symbionts from dietary amino acids

Abstract

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation¹. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.

Extended Figure 1.

Molecular structures of prototypic NKT agonist KRN7000, OCH and a representative B. fragilis-derived aGC (SB2217).

Extended Figure 2.. LC-MS profile of BfaGCs.

(A) Representative extracted ion chromatograms (XICs) of C₃₂–C₃₆ BfaGCs. (B) C₃₄ BfaGCs are the major component of B. fragilis glycosphingolipids (N=5).

Extended Figure 3.. LC-MS/MS assignment of C₃₄ BfaGC structural variants.

(A) The MS/MS-XIC of total C₃₄ BfaGCs (762→698) shows that BfaGCs are isobaric mixtures separated by RP-HPLC. (B, C) MS/MS-XICs of C₃₄ BfaGCs reveal co-eluting chemical homologues. Two isobaric species with aliphatic chains of C₁₇/C₁₇ (B) and C₁₈/C₁₆ (C) were assigned MS/MS fingerprints of 490 and 504, respectively. (D) MS/MS fingerprints of three peaks show a distinct difference in relative intensity between MS/MS fragments of 490 (C₁₇/C₁₇) and 504 (C₁₈/C₁₆), implying that the latter two peaks are a mixture of chain-length homologues. Chromatograms and spectra represent triplicate observations. (E-H) MS/MS spectra of the most abundant peaks of (E) C₃₂, (F) C₃₃, (G) C₃₅ and (H) C₃₆ BfaGCs. MS/MS fingerprint of 462–518 indicates lengths of sphinganine and acyl chains. Spectra are representative of triplicate observation.

Extended Figure 4.

Chemical structures of 23 synthetic BfaGCs. (SB2201-SB2223)

Extended Figure 5.. BCAA dictates branching of BfaGCs by direct incorporation in vivo.

(A–E) Ratios among differently branched C₃₄ BfaGCs (MS1 XIC=762.57, as [M+HCOO^-]) are clearly different for B. fragilis grown in rich medium (A) and B. fragilis grown in minimal medium (B). Supplementation with individual BCAAs (C–E) on defined medium increases production of branched-chain (both dibranched and monobranched) BfaGCs. (F–H) MS/MS fingerprints confirm the incorporation of leucine and isoleucine into the C₁₇/C₁₇ ceramide backbone (via C5 branched acyl-CoA) and of valine into the C₁₈/C₁₆ backbone (via C4 branched acyl-CoA). Chromatograms and spectra are representative of triplicate observations. (I) An MS/MS-XIC of d₃- and d₆-C₃₄ BfaGC shows that deuterium-labeled leucine is actively incorporated into BfaGC. (J-K) MS/MS pattern shows distinctive differences between gut luminal BfaGC (M+3 isotopolog) in (J) presence or (K) absence of d3-leucine, showing MS2 fragments in presence of d3-leucine reflect inclusion of deuterium-labeled leucine in the structure. Chromatograms and spectra are representative results of four mice.

Extended Figure 6.. Genetic study of B. fragilis Bcat orthologue (BF9343–3671).

(A) Confirmation of the target gene deletion by PCR. (B) The knockout strain (BF9343-Δ3671) shows comparable growth pattern to isogenic WT strain (grown in duplicate per group), and a complemented strain of KO strain with empty vector shows same pattern to BF9343–3671 complemented strain. (C) BF9343-Δ3671 complementation can recover the production of di-branched C17/C17 BfaGC production to wild-type level. (D) WT and mutant strain (N=5 for each group) can colonize mouse in comparable density. All results represent of two independent experiments with similar trend. For gel source data, see Supplementary Figure 1.

Extended Figure 7.. Structure-specific actions of BfaGCs.

(A) NKT cell–APC co-culture assays show that branching of sphinganine chain is, but 3’-OH group is not, critical for IL-2 inducing activity. Results are shown in duplicate and represent three independent experiment sets with similar trend (p=0.017 for 100nM and p=0.026 for 1000nM). (B-C) When injected intraperitoneally (N=5 per group, one sample in OCH group in panel C was lost), unlike Th1- or Th2-skewed prototypic ligands such as KRN7000 or OCH, SB2217 only weakly induce IFN-γ and did not induce IL-4 in vivo. (D-F) SB2217 weakly induced expression of co-stimulatory molecules such as CD86, CD40 and CD80 in splenic DCs, where SB2219 did not (N=5 per group).

Extended Figure 8.. Transcriptomic landscape of splenic NKT cells in responses to agonists.

(A) A heatmap shown with the Euclidean distances between different treatment groups. (B) Transcriptomic profile comparison of SB2217, SB2219 and OCH. (C) Pathway enrichment analysis of SB2217 reveals increased expression of immunoregulatory pathways in NKT cells when compared to vehicle or SB2219.

Extended Figure 9.. Comparison between SB2217 and SB2219 in mCD1d-BfaGC-2C12 complexes.

(A) 2Fo-Fc electron density map contoured at a 0.8σ level of the BfaGCs within each ternary complex. (B) Fo-Fc electron density map (in brown) contoured at a 2.2σ level of the BfaGCs and spacer lipids within each ternary complex. SB2217 is shown as blue and SB2219 is shown as green; Spacer lipids are shown as black sticks. (C) Superimposition of the headgroups of BfaGCs and KRN7000 (PDB code: 6BNK). (D) 2C12 TCR molecular interactions with SB2217 (in blue). mCD1d and CDR loops are colored as in Figure 4A. Hydrogen bonds are shown as red dashed lines. (E-F) The mCD1d–SB2217 complex shows higher affinity to 2C12 TCR than the mCD1d–SB2219 complex. (E) Each SPR datapoint is mean of techincal duplicate and K_D values (mean±SD) were calculated from two independent results, using a single-site binding model with K_D as a shared variable. (F) The sensorgrams are results of single experiment.

Extended Figure 10.. BfaGC profile in human microbiota-associated mice.

(A) BfaGC and B. fragilis abundance shows positive correlation in B. fragilis-gavaged HMB mice. Results are from longitudinally collected samples (2, 3 and 7 days after B. fragilis oral introduction) from five mice (total N=15). (B) BfaGC (C₁₇/C₁₇ dibranched and monobranched) are identified from neonatal (p14) GI contents. Chromatogram and spectrum represent seven samples.

Figure 1.. Chemical structure assignment of chain-length and branching variation in BfaGCs.

(A) Retrosynthetic analysis of BfaGC structural homologues. BfaGCs with all possible chain lengths and terminal branches in both fatty acyl chains (blue block with R group) and sphingoid chains (red block with R’ group) were designed and prepared. (B) An MS/MS spectrum mirror plot of synthetic and biogenic BfaGCs shows essentially identical spectra. (C) Co-injection analyses of B. fragilis lipid extract with synthetic BfaGCs assigned the structures of major bacterial glycosphingolipids (C34 BfaGCs). Chromatograms and spectra represent three samples.

Figure 2.. BfaGC branching is dictated by host dietary BCAAs, and loss of BCAA utilization in B. fragilis impairs its ability to modulate host colonic NKT cells.

(A) Host dietary BCAA is directly incorporated into BfaGCs produced by B. fragilis in the large intestine. The MS/MS fingerprint (m/z = 493) of d6-C34 BfaGC confirms d3-leucine incorporation into both sphinganine and fatty acyl chains. The spectrum represent four samples. (B) The gut luminal BfaGC branching of B. fragilis–monocolonized mice is directly associated with the BCAA availability in the diet (N=11 for baseline and N=10 for d7/d14 groups, p<0.0001 for both comparisons). (C) BF9343-Δ3671 monocolonization exhibits significantly less branched C34 BfaGCs (N=4 for each group, p=0.0079 for dibranched and p=0.00043 for monobranched). (D) BF9343-Δ3671 monocolonization at birth show dysregulated colonic NKT cell levels (p=0.0050).

Figure 3.. BfaGCs have distinct immunomodulatory signaling and actions.

(A) APC-NKT cell coculture with 21 synthetic BfaGCs showed a clear difference in IL-2 inducing efficacy, dependent on their sphinganine branching. Representative structures of BfaGCs with branched-chain sphinganine (SB2217) and with straight-chain sphinganine (SB2219) are shown. Results represents three independent experiments with similar trend. (B-C) When given prophylactically (N=5 for vehicle-treated and N=6 for SB2217-treated group), SB2217 can protect the host from NKT-mediated colitis, shown as less loss of weight (B, p=0.038) and less severe tissue histopathology score (C, p=0.015). Results represent three independent sets with similar trend. (D-E) Transcriptomic profile of splenic NKT cells from SB2217-treated group (mean of N=3) showed a distinct gene expression pattern. A volcano plot (D) and a heat map analysis (E) of differentially expressed, potential immunomodulatory genes with SB2217 treatment.

Figure 4.. Crystal structure of 2C12 NKT cell receptor–mCD1d–BfaGC ternary complexes showed conserved and distinct molecular interactions of BfaGCs with mCD1d and the 2C12 TCR.

(A) Cartoon representation of 2C12 TCR–mCD1d–BfaGC ternary complex crystal structures. The mCD1d and β2m are colored wheat and brown, respectively, while the 2C12 TCR α- and β-chains are colored purple and violet, respectively. CDR loops are colored as follows: CDR1α, blue; CDR2α, lime green; CDR3α, cyan; CDR1β, yellow; CDR2β, orange; CDR3β, pink. The lipid antigens are represented as sticks: SB2217, blue; SB2219, green. (B) Molecular interactions of SB2217 with mCD1d. Hydrogen bonds are shown as red dashed lines. (C) Superimposition of the sphinganine chains of branched-chain SB2217 and unbranched SB2219. (D) Comparison of interactions with mCD1d, with superimposition of the α1 and α2 domains of the crystal structures of 2C12 TCR–mCD1d–SB2217 (wheat) and 2C12 TCR–mCD1d–KRN7000 (gray) (PDB code: 6BNK). KRN7000 and SB2217 are shown as black and blue sticks, respectively.