March 1, 2013;
Ancient T-independence of mucosal IgX/A: gut microbiota unaffected by larval thymectomy in Xenopus laevis.
Many studies address the influence of the gut
microbiome on the immune system
, but few dissect the effect of T cells on gut
microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut
microbiota with and without the influence of T lymphocytes. Pyrosequencing of 16S ribosomal RNA genes was used to assess the relative abundance of bacterial groups present in the stomach
, small and large intestine
. Clostridiaceae was the most abundant family throughout the gut
, while Bacteroidaceae, Enterobacteriaceae, and Flavobacteriaceae also were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX
are not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX
based upon phylogeny, genomic synteny, and function.
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Figure 2. Rarefaction analysis of 16S rRNA gene sequences obtained from frog gastrointestinal contentThe analysis was performed on a randomly selected subset of 1800 sequences per sample. Lines represent the average of each sample type. A. stomach = orange, large intestine = red, and small intestine = blue. B. normal (no surgery) = blue and thymectomized = red. C. frog N011 = red, N321 = blue, T258 = green, T606 = yellow, T339 = brown, and N571 = orange.
Figure 3. Bacterial families in the gastrointestinal tract of Xenopus laevisFamilial distribution is shown with different colors from individual samples, grouped by thymus status from each of the three sampled positions in the gastrointestinal tract. Families with at least 1% representation in any sample are listed at the right. Complete taxonomic data is in Supplemental Table 2. Clostridiaceae were the predominant family, and no differences in bacterial groups between normal and thymectomized frogs were observed.
Figure 4. Principal Coordinates Analysis (PCoA) of unweighted UniFrac distances of 16S rRNA gene sequencingThe analysis was performed on a randomly selected subset of 1800 sequences per sample. A. Stomach (blue circles) samples separated from small (green square) and large (red triangle) intestine, indicative of distinct flora. B. No such separation was seen between non-thymectomized (blue circle) and thymectomized (red square) samples.
Figure 5. Amphibian IgX is orthologous to IgA of birds and mammalsNeighbor joining phylogenetic tree of the constant regions of diverse tetrapod immunoglobulin heavy chains, with the fish mucosal isotype IgZ/T included as an outgroup. Numbers at nodes show bootstrap support for each bifurcation after 1000 replications. The alignment used to build this is available as Supplemental Figure 2.
Figure 6. Thymectomy does not retard induction of mucosal IgX responseELISA for the IgX isotype on supernatant of lymphocytes cultured from spleen or intestine of frogs immunized to DNP-KLH. Units of the Y axes are absorbance at 450nm. A. Oral (PO) gives a significantly greater total IgX response in the gut than intracoelomic (IC) immunization (p=0.025, marked by *), but no significant difference was seen with thymectomized frogs. No significance was seen monitoring neither antigen-specific IgX in the gut (B.) nor total IgX in the spleen (C.) D. Antigen specific IgX actually increased from spleen cells with thymectomy (p=0.013, marked by*).
Figure 7. Model of immunoglobulin natural history with mucosal IgX/A emerging in early tetrapodsA. Simplified phylogeny of vertebrates showing approximate emergence times of heavy chain isotypes. B. This analysis suggests that the isotype previously described as IgX in amphibians may be orthologous to IgA of warm blooded vertebrates (model adapted from M. Flajnik’s chapter of Fundamental Immunology, W. Paul editor). In addition to the isotypes extant in man, the immunoglobulin light chain-less IgNAR of cartilaginous fish and the mucosal IgZ/T of bony fish is also shown.
Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE, IgG and IgA from the marsupial Monodelphis domestica.