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Author Correction: A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation.
Tavernier SJ, Athanasopoulos V, Verloo P, Behrens G, Staal J, Bogaert DJ, Naesens L, De Bruyne M, Van Gassen S, Parthoens E, Ellyard J, Cappello J, Morris LX, Van Gorp H, Van Isterdael G, Saeys Y, Lamkanfi M, Schelstraete P, Dehoorne J, Bordon V, Van Coster R, Lambrecht BN, Menten B, Beyaert R, Vinuesa CG, Heissmeyer V, Dullaers M, Haerynck F. Tavernier SJ, et al. Nat Commun. 2019 Nov 20;10(1):5337. doi: 10.1038/s41467-019-13379-9. Nat Commun. 2019. PMID: 31745085 Free PMC article.

Abstract

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

👁 Fig. 1

Fig. 1

Identification of a nonsense R688* mutation in RC3H1 in a consanguineous family. a Family pedigree indicating the index patient (V:2) with an arrow, the consanguineous link (double line) between the index patient’s parents and reported medical conditions as indicated in the legend. b Sanger sequencing of complementary DNA from selected individuals and control. c Graphical representation of Roquin-1 protein structure with indication of the R688* mutation. RING: Really Interesting New Gene zinc finger motif. ROQ: roquin-family RNA binding domain. Zinc finger: CCCH zinc finger motif. Coiled Coil: Coiled coil domain. d Immunoblot analysis of Roquin-1, its paralog Roquin-2, their cleavage products and the truncated R688* mutant in healthy controls (HC), the R688* proband and both parents. β-Tubulin is used as a loading control. NS: nonspecific band, SLE: systemic lupus erythematosus, SS: Sjögren’s syndrome. Source data are provided as a Source Data file

👁 Fig. 2

Fig. 2

Analysis of the R688* proband peripheral blood mononuclear cells (PBMCs) reveals immune dysregulation. a FlowSOM tree of concatenated 29-parameter cytometry data of PBMCs obtained from seven HCs and the R688* proband. b Normalized data of the relative contribution of R688* proband PBMCs to each immune cell cluster. Percentage of R688* immune cells was normalized into a Z score based on HC mean and SD. Each cluster with a Z score > 2 (red) or <−2 (blue) was considered as a relevant immune cell population. Color of cluster number corresponds with panel a. c Clusters with a Z score > 2 (red) or <−2 (blue) were plotted onto FlowSOM tree. Color of cluster number corresponds with panel a. d, e Phenotypic description of overrepresented (d) and underrepresented (e) clusters in the R688* proband. Histograms depict expression profile of surface markers of given clusters (colored) compared with relevant immune cell populations (black). f Histogram representing ICOS expression on CD4⁺ T cells of a HC and R688* proband. Mean fluorescence is given. g Scatter dot plot of geometric mean fluorescence (gMFI) of ICOS in T cell subsets of HCs (n = 4) or proband. N: naive; EM: effector memory; Tfh: T follicular helper cell. h Histogram of OX40 expression on CD4⁺ T cells of a representative HC and R688* proband. Mean fluorescence is given. i Scatter dot plot of gMFI of OX40 in T cell subsets of HCs (n = 4) or R688* proband. Data shown in (a–i) are representative for two independent experiments. Source data are provided as a Source Data file

👁 Fig. 3

Fig. 3

T cells and monocytes contribute to hypercytokinemia in the R688*/R688* proband. a Serum concentration of the cytokines IL-1β, IL-1RA, IL-6, IL-10, IL-17A, IL-18, IFNγ, CXCL9, and TNF in HCs (n = 4) and proband (two biological replicates) or HCs (n = 3) and proband (one biological replicate) in the case of the cytokine IL-17A. Mean and SEM are depicted. b, c ELISA of TNF and IFNγ produced by in vitro PMA/ionomycin stimulated CD4⁺ T cells (b) or CD8⁺ T cells (c) of HCs (n = 4) and proband. d ELISA of TNF produced by monocytes of HCs (N = 4) or proband treated in vitro overnight with LPS. e RT-qPCR quantifying TNF and IFNG transcripts in PMA/ionomycin stimulated T cells in absence or presence of mepazine pretreatment (20′). Cells were sampled 1 h after stimulation. Data was normalized using the housekeeping genes HPRT and GAPDH. HCs (n = 6). *p < 0.05 (paired t-test). R688* proband (n = 2). Data shown are accumulated from two independent experiments (a, e) or representative for two independent experiments (b–d). Source data are provided as a Source Data file

👁 Fig. 4

Fig. 4

Sanroque mice recapitulate some features of the R688* variant phenotype and develop severe hyperinflammation upon challenge. a Serum concentrations of cytokines TNF, IFNγ, IL-17A, CXCL9, IL-10, IL-6, IL-2 in sanroque mice (n = 4) and control littermates (n = 5). *p < 0.05 (unpaired t-test). b Number of blood neutrophils and lymphocytes in sanroque mice (n = 8) and control littermates (n = 8). ***0.001 < p < 0.0001 (unpaired t-test). c Number of platelets in sanroque mice (n = 11) and control littermates (n = 10) **p < 0.01 (unpaired t-test). d Concentration of serum soluble CD25 (sCD25) in sanroque mice (n = 9) and control littermates (n = 8). ***0.001 < p < 0.0001 (unpaired t-test). e Serum concentration of the liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) in sanroque mice (n = 6) and littermate controls (n = 12). ****p < 0.001 and **p < 0.01 (unpaired t-test). f Number of splenic immune cell subsets in sanroque chimeras (n = 11) and control chimeras (n = 6). *p < 0.05 and **p < 0.01 (unpaired t-test). g Percentage of splenic regulatory T cells (Treg) and T follicular helper cells (Tfh) in sanroque (n = 11) and control (n = 6) chimeras. *p < 0.05 and **p < 0.01 (unpaired t-test). h Contour plot of CD4⁺ and CD8⁺ T cell differentiation in sanroque and control chimeras. EM: effector memory; CM: central memory; N: naive. i Immunophenotyping of liver derived CD45⁺ cells in sanroque (n = 5) and control chimeric mice (n = 5). *p < 0.05 and **p < 0.01 (unpaired t-test). j Serum concentrations of TNF and IL-10 and k body weight of sanroque and control chimeras treated with 50 μg ODN-1826 CpG or vehicle control every 2 days for 8 days. *p < 0.05, **p < 0.01, ***0.001 < p < 0.0001, ****p < 0.001 (one-way ANOVA with Dunnett’s multiple comparisons test). Data shown are accumulated from three independent experiments (d), two experiments (a–c, e, f, j, k), or representative for two experiments (g–i). When applicable, mean and/or SEM are depicted. Source data are provided as a Source Data file

👁 Fig. 5

Fig. 5

Sanroque BM chimeras reveal cytokine driven immune dysregulation blocked by chemical JAK1/2 inhibition. a Percentage of CD4⁺ T cells in mixed CD45.2^control/CD45.1 (n = 7) and CD45.2^sanroque/CD45.1 bone marrow chimeric mice (n = 7). ***0.001 < p < 0.0001 (unpaired t-test). b Contour plot of CD4⁺ T cell differentiation in mixed bone marrow chimeras. EM: effector memory; CM: central memory; N: naive. c Percentage of regulatory T cells (Treg) in mixed CD45.2^control/CD45.1 (n = 7) and CD45.2^sanroque/CD45.1 chimeras (n = 7). ***0.001 < p < 0.0001 (unpaired t-test). d Percentage of CD8⁺ T cells in mixed CD45.2^control/CD45.1 (n = 7) and CD45.2^sanroque/CD45.1 chimeric mice (n = 7). ***0.001 < p < 0.0001 (unpaired t-test). e Contour plot of CD8⁺ T cell differentiation in mixed bone marrow chimeras. f Percentage of B cells in mixed bone marrow chimeras (n = 7). **p < 0.01 (unpaired t-test). g Contour plot of NK-cell maturation. Scatter dot plot of CD11b⁺ NK cells in mixed CD45.2^control/CD45.1 (n = 7) and CD45.2^sanroque/CD45.1 bone marrow chimeric mice (n = 7). **p < 0.01 (unpaired t-test). h, i Percentage of monocytes and neutrophils in mixed CD45.2^control/CD45.1 (n = 7) and CD45.2^sanroque/CD45.1 bone marrow chimeric mice (n = 7). j Spleen weight in control and sanroque bone marrow chimeric mice treated with ruxolitinib (RXL) or vehicle. n^{Ctrl chimera vehicle} = 3; n^{sanroque chimera vehicle} = 4; n^{Ctrl chimera RXL} = 3; n^{sanroque chimera RXL} = 3. **p < 0.01 (unpaired t-test). k Number of splenic monocytes, neutrophils and eosinophils in control and sanroque bone marrow chimeric mice treated with ruxolitinib (RXL) or vehicle. n^{Ctrl chimera vehicle} = 3; n^{sanroque chimera vehicle} = 4; n^{Ctrl chimera RXL} = 3; n^{sanroque chimera RXL} = 3. **p < 0.01; ****p < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test). l Serum concentration of TNF, IFNγ and CXCL9; median expression of CD64 on monocytes. n^{Ctrl chimera vehicle} = 3; n^{sanroque chimera vehicle} = 4; n^{Ctrl chimera RXL} = 3; n^{sanroque chimera RXL} = 3. *p < 0.05; **p < 0.01; ***0.001 < p < 0.0001; ****p < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test). Data shown are representative of two independent experiments (a–i), accumulated from two independent experiments (j, k) or one experiment (l). When applicable, mean and/or SEM are depicted. Source data are provided as a Source Data file

👁 Fig. 6

Fig. 6

Impaired P-body colocalization, CNOT1 interaction and ICOS mRNA degradation in the presence of Roquin-1 R688*. a HEK293T cells were transiently transfected with V5 tagged Roquin-1 (V5-FLRoquin-1) or V5-R688* Roquin-1 and subsequently stained with anti-V5 and anti-Edc4 (P-body marker). Nuclei were revealed using Hoechst. Scale bar = 10 μM. b Correlation analysis of Edc4 and Roquin-1 comparing V5-FLRoquin-1 (n = 26 cells) and V5-R688* Roquin-1 (n = 26 cells) transfected HEK293T cells. tPCC: tresholded Pearson correlation coefficient; CCM1/2: Manders coefficient1/2. Mean and standard deviation are plotted. *p < 0.05 and ****p < 0.0001 (unpaired t-test). c HEK293T cells were transiently transfected with a combination of wild-type Roquin-1 fused with GFP and V5 tagged wild type or R688* mutant Roquin-1. Slides were subsequently stained with anti-V5, anti-Edc4 and Hoechst. Scale bar = 10 μM. d, e Analysis of correlation between Edc4 and GFP fused Roquin-1 (d) and V5 tagged Roquin-1 (e), respectively. Analysis is based on at least 24 cells/group and mean and standard deviations are plotted. *p < 0.05; ***0.001 < p < 0.0001; ****p < 0.0001 (unpaired t-test). f HEK293T cells were transiently transfected with V5 tagged Roquin-1 (V5-FLRoquin-1) or V5-R688* Roquin-1. After immunoprecipitation with anti-V5 or control IgG coupled to Dynabeads Protein G, endogenous Edc4 and CNOT1 and overexpressed Roquin-1 variants were revealed by immunoblot analysis. β-Tubulin serves as a loading control. g ICOS mRNA transcripts upon actinomycin D treatment at given time points in R688*/R688* T cells. Data was normalized using the housekeeping genes HPRT and GAPDH. HCs (n = 11), R688*/R688* (n = 3). *p < 0.05 (unpaired t-test). Mean and SEM are depicted. h Poly(A) tail length measured for Icos mRNA in murine Rc3h1-2^fl/fl; CD4-CreERT2; rtTA CD4⁺ T cells retrovirally transduced with GFP, GFP fused WT Roquin-1 or R687* Roquin-1. Bar graph represents ratios of Poly(A) tailed Icos mRNA over de(A) Icos mRNA. Data shown are representative of 2 (f, h), 3 (a–e) or accumulated data of three experiments (g). Source data are provided as a Source Data file

👁 Fig. 7

Fig. 7

The murine equivalent Roquin-1 R687* fails to regulate proinflammatory cytokines TNFα, IL-2 and IL-17A. a Contour plots showing ICOS expression in 4-OHT treated murine Rc3h1-2^fl/fl; CD4-CreERT2; rtTA CD4⁺ T cells retrovirally transduced with GFP fused Roquin-1 variants. Representative histograms depict ICOS levels in GFP⁺ T cells expressing various Roquin-1 variants. b Scatter dot plot representing mean ICOS fluorescence in GFP⁺ T cells (n = 9). c Nonlinear regression of ICOS expression in murine T cells transfected with GFP fused Roquin-1 variants (n = 3). Mean and SEM are depicted. d, e Scatter dot plot representing mean Ox40 (d) and CTLA4 (e) fluorescence in GFP⁺ T cells (n = 9). **p < 0.01; ***0.001 < p < 0.0001; ****p < 0.0001 (one-way ANOVA with Tukey’s correction). f Contour plots showing TNF production 4-OHT treated murine Rc3h1-2^fl/fl; CD4-CreERT2; rtTA CD4⁺ T cells retrovirally transduced with GFP fused Roquin-1 variants and treated with PMA/ionomycin for 2 h and incubated for an additional 2 h after brefeldin A supplementation. Representative histograms depict TNF expression in stimulated GFP⁺ T cells. g Scatter dot plot representing mean TNF fluorescence in GFP⁺ T cells transduced with various Roquin-1 variants (n = 9). h Nonlinear regression of TNF fluorescence in murine T cells transduced with GFP fused Roquin-1 variants (n = 3). Mean and SEM are depicted. i, j Scatter dot plot representing mean IL-2 (h) and IL-17A (i) fluorescence in GFP⁺ T cells (n = 8). *p < 0.05; **p < 0.01; ***0^.001 < p < 0.0001 (one-way ANOVA with Tukey’s correction). Data shown are representative for four independent experiments (a, f), accumulated data from two independent experiments (c, h) or four independent experiments (b, d, e, g, i, j). Source data are provided as a Source Data file

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URL: https://pubmed.ncbi.nlm.nih.gov/31636267/

⇱ A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation - PubMed

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