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An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome


Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion led to successful treatment with IL-1–blocking agents1. Herein we report a de novo missense mutation (c.1009A>T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4–transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.

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Figure 1: Severe flares in an individual with an NLRC4 mutation are consistent with MAS.
Figure 2: The NLRC4 mutation occurs de novo and affects a highly conserved area of the NBD.
Figure 3: Peripheral blood signatures differentiate NLRC4-MAS from healthy controls and patients with NOMID.
Figure 4: NLRC4-MAS monocytes and macrophages have increased inflammasome-related cytokine secretion, cell death and ASC aggregate formation.
Figure 5: Cells transduced with virus expressing mutant NLRC4 exhibit spontaneous inflammasome activity.
Figure 6: IL-1 receptor antagonist treatment normalized markers of systemic inflammation and enabled cessation of steroids, whereas serum IL-18 levels remained elevated.

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The authors are grateful to O. Navarro and G. Somers for assistance with data acquisition and to H. Convery and the patient and her family for assistance with research coordination. This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the US National Institutes of Health. S.W.C. was also supported by the Arthritis National Research Foundation.

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Authors and Affiliations



S.W.C. and R.G.-M. conceived the study. S.W.C., J.J.O., R.M.L. and R.G.-M. directed the study. G.A.M.S., H.K., D.C., N.P., Y.H., S.B. and R.M.L. coordinated patient care and obtained clinical samples. A.A.d.J. performed Sanger sequencing. A.A.d.J. and T.A.F. generated RNA sequencing libraries. S.W.C., A.A.d.J., S.R.B., A.V.V. and Z.D. analyzed genomic and transcriptional data. S.W.C. and A.B. performed serum cytokine experiments and analysis. M.A.D. performed structural analysis. S.W.C. and S.G. performed all stimulation experiments, with assistance from B.M. and Y.L. S.W.C., S.G. and K.J.M.Z. performed fluorescence microscopy, K.J.M.Z. performed the quantification. J.A.D. and A.G. provided reagents and critical direction. S.W.C., R.M.L. and R.G.-M. guided clinical assessment and intervention. S.W.C. and R.G.-M. wrote the manuscript with the assistance and final approval of all authors.

Corresponding authors

Correspondence to Scott W Canna or Raphaela Goldbach-Mansky.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Supporting clinical information.

(a) The patient’s growth chart. (b) Additional parameters associated with macrophage activation syndrome (MAS) reflecting all of the patient’s available laboratory data (see also Supplementary Table 1). Pink bars indicate severe disease flares, and the blue bar indicates testing done after IL-1 receptor antagonist treatment (anakinra). The dashed lines indicate the normal ranges. The dotted lines indicate the patient’s age in years. ESR, erythrocyte sedimentation rate; WBC, white blood cell count; ALT, alanine aminotransferase; LDH, lactate dehydrogenase. (c) Sonographic measurement of the patient’s spleen during a flare before the initiation of treatment with anakinra (left; z score = 3.95) and 3 months after the initiation of anakinra (right; z score = 1.3)32.

Source data

Supplementary Figure 2 Whole-exome sequencing identifies a de novo threonine-to-serine conversion in a highly conserved region of NLRC4 predicted to be pathogenic.

(a) Schematic of whole-exome sequence analysis. *, 105 samples processed in the same batch as the control for technical artifacts. GATK, Genome Analysis Toolkit; MAF, minor allele frequency. (b) Position of the affected patient’s T337S conversion in a highly conserved region of NLRC4. (c) Analysis of the frequency of the T337S mutation in the dbSNP and NHLBI Exome Sequencing Project (ESP)18 databases. The effect of the mutation was also predicted using Genomic Evolutionary Rate Profiling (GERP)17, MutationTaster14, Sorting Intolerant From Tolerant (SIFT)15 and Polymorphism Phenotyping, v2 (PolyPhen-2)16 analyses. (d) Sequence chromatographs of the patient and her parents showing the de novo c.1009A>T, p.Thr337Ser mutation in NLRC4.

Supplementary Figure 3 Serum cytokines distinguish NLRC4-MAS from NOMID and establish a cytokine signature.

(a) Cytokines associated with flares of primary hemophagocytic lymphohistiocytosis10 are not extensively elevated in NLRC4-MAS sera. (b) Cytokines with highly elevated levels in NLRC4-MAS patient samples but not in samples from NOMID patients or healthy controls. Data for pediatric and family controls are combined. *P < 0.01, **P < 0.001, ***P < 0.0001 for an unpaired two-tailed Student’s t test of all NOMID versus all NLRC4-MAS samples for a given cytokine. “Pre” and “post” represent samples taken before and after the initiation of IL-1 receptor antagonist (anakinra) therapy.

Source data

Supplementary Figure 4 Whole-blood transcriptional analyses suggest macrophage activation and compensatory upregulation of apoptosis and hematopoiesis distinct from NOMID.

Whole-blood RNA sequencing was performed (Online Methods). Functional gene lists were generated (Supplementary Table 2), and genes showing differential regulation in the NLRC4-MAS patient’s flare sample versus samples from healthy controls and NOMID patients were selected. The other samples included were drawn from the NLRC4-MAS patient before the initiation of IL-1 receptor antagonist treatment with inactive disease (pre), after IL-1Ra treatment (post) or from matched samples from seven NOMID patients with active disease before the initiation of IL-1Ra treatment (NOMID pre) or with inactive disease after the initiation of IL-1Ra treatment (NOMID post). Transcript levels were normalized to the average expression of the same transcript in five healthy pediatric controls (HC) and are expressed as the fold change (FC). Transcripts that are upregulated (UP) or downregulated (DN) in the flare sample are separated for NOMID patients for clarity. (a) Downregulation of NLRP1, NLRP3 and associated genes (MEFV, HSP90AB1 and SUGT1) and of NOD2 was seen in NLRC4-MAS but not in NOMID, whereas upregulation of NLRC4, AIM2 and NLRP6 was seen in both conditions. (b) Downregulation of various lymphocyte (CCL3, CCR3, CCR4 and CCR7) and neutrophil (IL8 and CXCR2) chemokines and chemokine receptors (with the exception of CXCR2) was observed in NLRC4-MAS but not in NOMID. (c) Upregulation of apoptotic factors (BCL2A1, BIK, BAD and LTB4R) and downregulation of antiapoptotic factors (TP53, BCL2 and CD40LG) were present in NLRC4-MAS but minimal in NOMID. Downregulation of the proapoptotic gene DIABLO was inconsistent with this paradigm and was not observed in NOMID. (d) Downregulation of genes associated with interferon production (STAT4) or signaling (IFNAR2, JAK1, STAT1 and STAT2) was observed in the flare sample. Only JAK3 upregulation was observed in both NLRC4-MAS and active NOMID. (e) Dysregulation of markers of classical (TLR5, IL7R, PTGS2 and HIF1A) and alternative (ARG1, DECTIN1 and IL1R2) macrophage activation. Upregulated genes were similarly regulated in NLRC4-MAS and NOMID.

Source data

Supplementary Figure 5 Absence of peripheral blood neutrophilia in NLRC4-MAS flare.

White blood cell differentials drawn on the same day as whole-blood RNA samples from the NLRC4-MAS patient. PMN, polymorphonuclear leukocytes.

Source data

Supplementary Figure 6 Increased secretion of inflammasome-related cytokines by NLRC4-MAS cells before IL-1 receptor antagonist treatment.

Monocytes and monocyte-derived macrophages were isolated from healthy controls and the NLRC4-MAS patient (Online Methods). Cells were stimulated, and secreted cytokines were measured by Luminex. Columns represent the mean and s.d. of technical duplicates. FLA-hi, flagellin (5 μg/ml); IC-FLA lo, liposomal flagellin for intracellular delivery (1 μg/ml); IC-FLA-hi, liposomal flagellin for intracellular delivery (5 μg/ml).

Source data

Supplementary Figure 7 NLRC4-MAS macrophages overproduce inflammasome-related cytokines at all time points.

Equal numbers of NLRC4-MAS (red bars) or healthy control (open bars) monocyte-derived macrophages were stimulated with 5 μg/ml Intracellular flagellin (IC-FLA) for the indicated times, and supernatants were assessed for cytokine production. Columns represent the mean and s.d. of technical duplicates.

Source data

Supplementary Figure 8 NLRC4-MAS and NOMID macrophages have comparable production of TNF-α and IL-10.

Monocytes and monocyte-derived macrophages were isolated from healthy controls, the NLRC4-MAS patient and an NOMID patient (Online Methods). Cells were stimulated as indicated, and secreted cytokines were measured by Luminex. (a) NLRC4-MAS monocytes from before and after treatment with IL-1 receptor antagonist were stimulated, and secreted cytokines were measured by Luminex. (b) Pre- and post-treatment monocyte-derived macrophages were stimulated, and secreted cytokines were measured by Luminex. Columns represent the mean and s.d. of technical duplicates. No appreciable secretion of IL-12p70 or IFNα2 was detected for any sample (data not shown). FLA-hi, flagellin (5 μg/ml); IC-FLA lo, liposomal flagellin for intracellular delivery (1 μg/ml); IC-FLA-hi (5 μg/ml).

Source data

Supplementary Figure 9 T337S NLRC4–transduced THP1 cells have a proliferative disadvantage.

THP1 cells were transduced with an empty vector, mutant NLRC4 or various dilutions of wild-type NLRC4 retrovirus. Equal amounts of plasmid construct were used to generate virus-containing supernatant. Mutant and wild-type constructs differed by 1 bp. Transduction efficiency was assessed at the indicated time points by flow cytometry for GFP expression. Median fluorescence intensities for the GFP-positive population are depicted.

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Supplementary Figures 1–9 and Supplementary Note. (PDF 4722 kb)

Supplementary Tables 1–3

Supplementary Tables 1–3. (XLSX 28 kb)

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Canna, S., de Jesus, A., Gouni, S. et al. An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nat Genet 46, 1140–1146 (2014).

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