Human TGF-β1 deficiency causes severe inflammatory bowel disease and encephalopathy

Abstract

Transforming growth factor (TGF)-β1 (encoded by TGFB1) is the prototypic member of the TGF-β family of 33 proteins that orchestrate embryogenesis, development and tissue homeostasis^1,2. Following its discovery ³ , enormous interest and numerous controversies have emerged about the role of TGF-β in coordinating the balance of pro- and anti-oncogenic properties^4,5, pro- and anti-inflammatory effects ⁶ , or pro- and anti-fibrinogenic characteristics ⁷ . Here we describe three individuals from two pedigrees with biallelic loss-of-function mutations in the TGFB1 gene who presented with severe infantile inflammatory bowel disease (IBD) and central nervous system (CNS) disease associated with epilepsy, brain atrophy and posterior leukoencephalopathy. The proteins encoded by the mutated TGFB1 alleles were characterized by impaired secretion, function or stability of the TGF-β1-LAP complex, which is suggestive of perturbed bioavailability of TGF-β1. Our study shows that TGF-β1 has a critical and nonredundant role in the development and homeostasis of intestinal immunity and the CNS in humans.

Fig. 1|. Identification of a biallelic TGFB1 mutation in patient 1 with very early-onset inflammatory bowel disease and global neurological defects.

a, Pedigree of patient 1 (P1, A.II-1) born to a nonconsanguineous Malaysian family (family A). b, Images showing severe perianal disease with purulent discharge (leftmost image) in P1, massive suppuration (second image from left) and superficial ulcerations and multiple pseudopolyps (third image from left) in the colon, as revealed by endoscopy, and crypt abscesses and inflammatory infiltrations of the epithelium with mucosal ulceration, as documented by histology (rightmost image; scale bar, 500 μm). c, MRI images of the brain indicating global brain atrophy and posterior periventricular leukencephalopathy in P1. Scale bars, 2 cm. d, Representative immunophenotypic analysis of CD4⁺CD25⁺CD127^lo T_reg cells, CCR6⁻CXCR3⁺ T_H1 and CCR6⁺CXCR3⁻ T_H 17 T cells and assessment of CD3⁺CD4⁺ T cell activation and proliferation in response to anti-CD3/anti-CD28 or specific antigens (DT/TT, diphtheria and tetanus toxoid). Immunophenotyping was performed in two independent experiments. e, CyTOF analysis of the composition of lamina propria mononuclear cells derived from two control (Ctrl) patients without IBD (uninflamed and inflamed), a patient with Crohn’s disease and P1. Left, clusters of CD45⁺ viSNE plots were manually gated and color-coded for various populations on the basis of similar marker expression. Right, graphical representations depicting percentages of the indicated immune cell populations. CyTOF analysis was performed once owing to limited availability of patient material.

Fig. 2|. Effects of TGFB1 mutations on the biosynthesis and bioavailability of TGF-β1.

a, Schematic illustration showing the distribution of identified alterations relative to the TGF-β1 structure depicting the N-terminal signal peptide (SP), the latency-associated peptide (LAP) and the C-terminal mature growth factor (TGF-β1). The mutations identified in patients, including a previously described gain-of-function mutation in TGFB1 causing CED, are depicted for the DNA and protein sequences. b, Structural visualization of the identified TGF-β1 alterations using the crystal structure of latent TGF-β1 (PDB accession 3RJR). The structure is depicted as a ribbon model with highlighted secondary structure. Color code: yellow and green, pro-domain dimer; blue, TGF-β1 dimer; magenta spheres, mutation sites. c, Detailed views of the altered sites (stick model, with altered side chains highlighted in magenta). d, Representative immunoblot (n = 3) for TGF-β1 levels in lysates and conditioned medium from HEK293T cells that stably overexpressed WT and mutant TGF-β1 variants. RFP, red fluorescent protein; R45C, TGF-β1 Arg45Cys; R110C, TGF-β1 Arg110Cys; R218C, TGF-β1 Arg218Cys; C387R, TGF-β1 Cys387Arg. e, ELISA determining the TGF-β1 levels in conditioned medium from HEK293T cells that stably overexpressed WT and mutant TGF-β1 variants± HCl treatment for the release of mature growth factor from latent complexes. Samples of ten biologically independent cell culture experiments were analyzed. Box-and-whisker plots: center line, median; box limits, upper and lower quartiles; whiskers, quartile range. P values (indicated in the graphs) were calculated using a two-tailed unpaired t test with Welch’s correction. f, SMAD-dependent luciferase reporter assays in HEK293T cells that were stimulated with conditioned medium from cells stably overexpressing WT or mutant TGF-β1. Samples of nine biologically independent cell culture experiments were analyzed. Box-and-whisker plots: center line, median; box limits, upper and lower quartiles; whiskers, quartile range; individual data points as overlays. P values were calculated using a two-tailed unpaired t test with Welch’s correction. g, Analysis of the re-association capacity of mutant TGF-β1 variants and LAP over time. Data shown represent the means± s.e.m. of 11 independent cell culture experiments. P values were calculated using two-way repeated-measures ANOVA with Dunnett’s correction for multiple comparisons. h, CyTOF analysis of SMAD2/3 phosphorylation (p-SMAD2/3) in lamina propria mononuclear cells derived from patients without IBD (uninflamed, inflamed), a patient with Crohn’s disease (CD) and P1 (A.II-1). Histogram plots of baseline p-SMAD2/3 levels (left) and median expression values (MEV) of p-SMAD2/3 (right) are shown for the indicated hematopoietic cell (CD45⁺, CD19⁺ or CD3⁺) populations. CyTOF analysis was performed once owing to limited availability of patient material.