Mutation of NLRC4 causes a syndrome of enterocolitis and autoinflammation

Upon detection of pathogen-associated molecular patterns, innate immune receptors initiate inflammatory responses. These receptors include cytoplasmic NOD-like receptors (NLRs), whose stimulation recruits and proteolytically activates caspase-1 within the inflammasome, a multi-protein complex. Caspase-1 mediates the production of interleukin-1 family cytokines (IL1FCs), leading to fever, and inflammatory cell death (pyroptosis)1,2. Mutations that constitutively activate these pathways underlie several autoinflammatory diseases with diverse clinical features3. We describe a family with a previously unreported syndrome featuring neonatal-onset enterocolitis, periodic fever, and fatal/near-fatal episodes of autoinflammation caused by a de novo gain-of-function mutation (p.V341A) in the HD1 domain of NLRC4 that co-segregates with disease. Mutant NLRC4 causes constitutive Interleukin-1 family cytokine production and macrophage cell death. Infected patient macrophages are polarized toward pyroptosis and exhibit abnormal staining for inflammasome components. These findings describe and reveal the cause of a life-threatening but treatable autoinflammatory disease that underscores the divergent roles of the NLRC4 inflammasome.

Upon detection of pathogen-associated molecular patterns, innate immune receptors initiate inflammatory responses. These receptors include cytoplasmic NOD-like receptors (NLRs), whose stimulation recruits and proteolytically activates caspase-1 within the inflammasome, a multiprotein complex. Caspase-1 mediates the production of interleukin-1 family cytokines (IL1FCs), leading to fever, and inflammatory cell death (pyroptosis) 1,2 . Mutations that constitutively activate these pathways underlie several autoinflammatory diseases with diverse clinical features 3 . We describe a family with a previously unreported syndrome featuring neonatal-onset enterocolitis, periodic fever, and fatal/near-fatal episodes of autoinflammation caused by a de novo gain-offunction mutation (p.V341A) in the HD1 domain of NLRC4 that co-segregates with disease. Mutant NLRC4 causes constitutive Interleukin-1 family cytokine production and macrophage cell death. Infected patient macrophages are polarized toward pyroptosis and exhibit abnormal staining for inflammasome components. These findings describe and reveal the cause of a life-threatening but treatable autoinflammatory disease that underscores the divergent roles of the NLRC4 inflammasome.
He gradually improved and was discharged after 9 weeks, remaining on cyclosporine; serum ferritin normalized but IL-18 remained markedly elevated (Fig. 1d,e). He subsequently reported a lifelong history of periodic fevers (>40 °C) provoked by physical and emotional stressors. During infancy he had an extended hospitalization for fever, vomiting, non-bloody diarrhea and failure to thrive; no specific diagnosis was made. His gastrointestinal symptoms resolved by one year. In adulthood erythematous plaques and joint pains accompanied fevers; sero-negative psoriatic arthritis was diagnosed.
The father's family history revealed healthy parents and two additional offspring, one without illness and a five-year-old half-brother (III.2) of the deceased infant (III.3) who also had periodic fevers (range 38.9 -40 °C) beginning on day three of life after circumcision. A more severe febrile episode associated with vomiting, non-hemolytic anemia and acute renal failure occurred at 6-weeks of age (Supplementary Table 1). Later his fevers were induced by over-exertion and accompanied by abdominal pain. A duodenal biopsy in the first year revealed villous blunting and intraepithelial lymphocytes (Fig. 1b, lower panel). Inflammatory markers including ferritin (516-856 ng/ml), C-reactive protein, soluble IL-2R and plasma IL-18 (11,520 to 24,129 pg/ml) were persistently elevated (Fig. 1d,e). NK cells, normal in number, were dysfunctional by chromium release assays (Supplementary Table  1). Clinical signs of chronic inflammation included short stature (< 3 rd percentile for height and weight) and recurrent myalgias.
During the index case's acute illness, the possibility of a novel genetic syndrome was considered, leading to exome sequencing of the index case and his parents (see Methods). Clincial features suggesting hemophagocytic lymphohistiocytosis led to examination of genes implicated in this syndrome 11 ; no rare variants were identified (Supplementary Table 2). Upon the father's illness, 34 novel protein-altering variants (absent in dbSNP, 1000 genomes, NHLBI and Yale exome databases) shared by the index case and his father were identified, including six occurring at positions invariant among orthologs (Supplementary Table 3). While none of these altered genes causing known inflammatory diseases, one was in NLRC4, which encodes a core inflammasome protein. This p.Val341Ala variant occurs within helical domain 1 (HD1), which provides a 'lid' to the ADP nucleotide-binding domain (NBD) in the crystal structure of inactive NLRC4 (Fig. 2). Ligand binding normally opens this structure, leading to exchange of ATP for ADP, promoting oligomerization and inflammasome assembly 12 . Gain-of-function mutations in the NBD of the related protein NLRP3 cause constitutive NLRP3 inflammasome assembly, resulting in production of IL-1β, fever and a spectrum of autoinflammatory disorders, the cryopyrinopathies [13][14][15] . These diseases are clinically distinct from the disease in our family as the cryopyrinopathies lack gastrointestinal pathology 16 .
Evaluation of NLRC4 V341A in the extended family demonstrated that it occurred de novo in the affected father, and co-segregated with the inflammatory disease (Fig. 1a). None of the other five novel variants at conserved positions showed co-segregation with disease or de novo mutation. The finding of a de novo mutation in NLRC4, that co-segregates with a consistent clinical syndrome and biomarkers of inflammasome activation, provides strong evidence that NLRC4 V341A causes this syndrome (syndrome of enterocolitis and autoinflammation associated with mutation in NLRC4; SCAN4).
We measured cell death-associated LDH release in the same 18-hour culture supernatants. SCAN4 macrophages released more LDH than healthy control macrophages (12.3% versus 4.7% P<0.0001) (Fig. 4b). Addition of Z-YVAD-FMK, which inhibits the catalytic site of cleaved caspase-1, significantly reduced IL-1 family secretion, but did not reduce cell death (Supplementary Fig. 3a,b). Thus NLRC4 V341A is gain-of-function, eliminating the requirement for "signal 2" for activation of caspase-1 and production of IL1FCs. NLRC4 V341A also promotes pyroptosis independent of caspase-1 cytokine processing.
We next infected LPS-primed healthy control or patient macrophages with either of two flagellated, TTSS-positive pathogens, S. typhimurium (strain SL1344) or P. aeruginosa (strain PAKΔSTY), thus providing both 'signal 1' and 'signal 2' provocation. As anticipated, LPS-primed healthy control macrophages secreted abundant IL1FCs and initiated pyroptosis upon infection (Supplementary Fig. 4a,b). Responses were reduced when infected with P. aeruginosa strain PAKΔpopD, which lacks a functional TTSS (Supplementary Fig. 4a,b). In contrast, SCAN4 macrophages secreted significantly less IL1FCs upon infection with pathogenic strains, yet showed more cell death than healthy control macrophages (Supplementary Fig. 4a,b).
These findings describe a previously unreported Mendelian autoinflammatory syndrome featuring periodic fever, neonatal-onset enterocolitis and high levels of IL1FCs, and demonstrate its causation by a gain-of-function mutation in NLRC4. Like NLRP3 cryopyrinopathies 14 , SCAN4 is associated with constitutive activation of caspase-1 and production of IL1FCs. In the inhibited, ADP-bound state, Val341 of NLRC4 makes van der Waals contacts with side chains of an adjacent helix in the HD1 domain, comprising the 'lid' on the nucleotide-binding site. The decreased hydrophobicity of the Ala341 mutation may reduce this interaction, allowing more movement of helix α12 and promoting ATP for ADP exchange, either by favoring the open conformation of NLRC4, or by disrupting the stabilizing interaction of His443 with the beta-phosphate of ADP (Fig. 2). Either possibility would promote ligand-independent activation of NLRC4. It is compelling that Canna et al. have identified an independent de novo mutation in NLRC4, p.Thr337Ser 19 . The identification of two de novo mutations in close proximity in the same gene that segregate with a novel clinical phenotype provides strong support for a causal relationship of the mutations to disease pathogenesis. SCAN4 is distinctive from NLRP3 cryopyrinopathies in its association with neonatal-onset enterocolitis. This may relate to NLRC4 being highly expressed in intestinal macrophages while NLRP3 is not 20 . It is interesting that the marked enterocolitis of each surviving SCAN4 patient resolved by one year of age. We speculate that this chronic inflammatory state may be exacerbated in the infant gut by constant "signal 1" provocation from newly acquired symbionts. As host-microbe interactions mature, a less pro-inflammatory microflora may account for reduced gut inflammation 21 .
IL-1β -targeted drugs are approved for treatment of NLRP3 cryopyrinopathies [22][23][24] . We expect IL-1β blockade will be similarly effective in SCAN4 patients. Although both surviving members in our family have presently declined interictal therapy, the complementary report by Canna et al provides evidence for efficacy of IL-1 receptor blockade 19 .
SCAN4 macrophages show high IL1FC secretion and increased cell death with "signal 1" despite the absence of "signal 2"; addition of "signal 2" frequently produces multiple ASC foci with increased cell death, despite blunted IL1FC secretion. One possible explanation is that mutant NLRC4 promotes traditional inflammasome assembly in the absence of "signal 2" provocation but intracellular ligand binding promotes formation of ASC foci that lack activated caspase-1, resulting in smaller structures with impaired ability to produce cytokines. This proposal is supported by mice with mutated inflammasome components 6,9,10 which demonstrate defective cytokine processing yet intact pyroptosis . Modulating the balance between cytokine production and pyroptosis may determine the distinct states of subclinical autoinflammation, periodic fever, and fatal or near-fatal autoinflammation seen in SCAN4 patients.

Research Subjects
The study protocol was approved by the Yale Human Research Protection Program. Informed consent was provided by all participants or their legal guardians. Clinical data was abstracted from medical records. Tissues from biopsy and autopsy specimens were labeled using standard hematoxylin and eosin staining protocols or by immunohistochemical staining with an anti-CD163 antibody (Abcam).

Genetic analysis
DNA was prepared from venous blood samples of the index case and kindred members. Exome sequencing of the index case and his parents was performed by capture on the NimbleGen 2.1 Exome reagent followed by 74 base paired end sequencing on the Illumina platform to high coverage (each targeted base was read by a mean of more than u80 independent reads in each subject) as previously described26. Sequences were aligned to NCBI Build 36 of the human genome and SNV and indel calls were assigned quality scores (QS) using SAMtools and annotated for novelty (using Yale, 1000 genomes, and NHLBI exome databases), for impact on encoded proteins, and for conservation of variant position as previously described26. Variants were sought in genes implicated in hemophagocytic lymphohistiocytosis; none were identified (Supplementary Table 2). Thirty-four proteinaltering variants that were absent in dbSNP, 1000 genomes, NHLBI and Yale exome databases that were shared by the index case and affected father were identified (Supplementary Table 3) and evaluated. Only one was in a gene known to play a role in activation of the innate immune system (NLRC4). Variants in NLRC4 and the other 5 genes in which novel variants that occurred at completely conserved positions (ALK, DCC, FBXO4, KIF13B, and SLC7A6OS) were confirmed by PCR amplification followed by Sanger sequencing and transmission through the complete pedigree was evaluated. The NLRC4 variant proved to be de novo in the affected father and perfectly cosegregated with the autoinflammatory syndrome in the pedigree, while the others were all transmitted from an unaffected grandparent of the index case and did not co-segregate with disease. The NLRC4 p.Val341Ala mutation has been deposited into the National Center for Biotechnology Information's ClinVar database (ClinVar accession #, SCV000172282)

Functional studies of monocyte derived macrophages
CD14 + monocytes were purified from peripheral blood mononuclear cells of SCAN4 patients and healthy donor controls using anti-human CD14 magnetic beads (Miltenyi). Cells from the two living SCAN4 patients were used for all functional studies whereas the number and relatedness of healthy donors used varied from experiment to experiment and was based upon same-day availability. Monocytes were differentiated to macrophages in RPMI containing 10% FBS and M-CSF (10ng/ml) over 7 days 27 . 2 × 10 5 macrophages were cultured for 18 hours in culture media containing LPS (1ng/ml) with or without Z-YVAD-FMK (Enzo Life Sciences) at 0.1-0.5μM concentrations. Culture supernatants were collected and cells washed and re-cultured with LPS free media before infection with P. aeruginosa or S. typhimurium. PAKΔSTY and SL1344 are flagellated strains that are type 3 secretion system (T3SS)-positive. PAKΔpopD is a Fla+ strain that does not express a functional T3SS. Construction and characterization of bacterial strains has been previously described 6,28 . Infected macrophage culture supernatants were collected after one hour. Secreted IL-1β and IL-18 were measured by ELISA (Millipore and MBL, respectively) in both the 18-hour and one-hour culture supernatants. Cell-free LDH was measured according to manufacturer's protocol (Takara). LDH release in supernatants was normalized to LDH released from macrophages lysed with Triton X-100 (0.1%).

Immunofluoresence microscopy of infected macrophages
2×10 5 macrophages, differentiated as above, were plated on glass coverslips. Cells were incubated with 2 μM biotin-YVAD-CMK for 30 minutes prior to infection with P. aeruginosa PAKΔSTY or ΔpopD or S. typhimurium SL1344 at a multiplicity of infection of 20 bacteria/cell for 1 hour. Cell were fixed with paraformaldehyde (4%), blocked with 1% fish scale gelatin (Sigma) in PBS + 0.1% TX-100, and stained with rabbit anti-ASC (AL177; AdipoGen) and 4',6-Diamidino-2-phenylindole (Sigma). A streptavidin-Alexa Fluor 488 conjugate (Invitrogen) and anti-rabbit Alexa Fluor 594 antibody (Life Technologies) were used for secondary stainings. Macrophages were visually inspected for immunofluorescence using an Axiovert 200M microscope. Manual enumeration of ASC + and YVAD + macrophages was performed over 20 representative fields at 20x magnification. DAPI staining of bacterial DNA was used to confirm macrophage infection. High detail magnification for phased images at 60X and 100X was performed on a Nikon Eclipse TE2000-S microscope.  The V341A amino acid substitution is positioned within the HD1 domain of NLRC4. (a) A schematic representation of the NLRC4 protein with individual domains colored as follows: CARD in black, NBD in blue, HD1 in cyan, WHD in pink, HD2 in green, LRR in lilac. The location of the V341A substitution is displayed (b) Mapping of Val-341 onto the crystal structure of murine NLRC4 in the ADP-bound state (PDB accession code 4KXF) 12,25 . The ribbon diagram excludes the N-terminal CARD domain which was not included in its crystal structure. ADP is drawn as sticks, and the position of Val-341 is indicated with red spheres. The zoomed-in region (structure rotated 90° toward the reader) shows the position of Val-341 on α-helix 12. Neighboring hydrophobic residues within the HD1 (black outlines) and adjacent α-helices are numbered. NLRC4 V341A promotes spontaneous cleavage of pro-caspase-1 and ASC multimerization in HEK293 cells. (a) Increased cleavage of FLAG-procaspase-1 in HEK293 cells expressing NLRC4 V341A versus NLRC4 WT . Western blot at top of figure shows results of blotting for NLRC4, pro-caspase-1 (p45) and its p35 and p10 cleavage products as well as actin controls in cells expressing constructs shown below, as described in Methods. Levels of p35 and p10 are normalized in each case to the level of pro-caspase-1. Alternate analysis normalizing p35 and p10 to levels of actin and NLRC4 also yielded statistically significant differences between lines transfected with NLRC4 WT and NLRC4 V341A . Mean and standard deviation of four independent transfections is shown. A two-sided Student's t-test was used to determine statistical significance. (b) Spontaneous ASC multimerization (white arrows) in HEK293 cells expressing GFP-ASC and either NLRC4 WT (left panel) or NLRC4 V341A (right panels) using epifluorescent microscopy. A total of 1422 cells transfected with wildtype NLRC4 and 1155 cells transfected with mutant NLRC4 were scored. * the frequency of ASC puncta + cells in lines transfected with NLRC4 WT is significantly different (P<0.0001) from lines transfected with NLRC4 V341A (chi-square testing). Scale bars, 20 μm. Increased production of IL-1β,IL-18 and increased cell death in macrophages harboring NLRC4 V341A . Monocyte-derived macrophages from SCAN4 patients (II.3 and III.2) and WT controls (one related and four unrelated) were cultured for 18 hours in media containing low-dose LPS (1 ng/ml) followed by measurement of (a) IL-1-β(b) IL-18, and (c) LDH as described in Methods. LDH release is reported relative to result following total lysis by TritonX-100 (0.1%). Cytokine secretion and cell death results were similar in macrophages from patients (II.3 and III.2). Bar graphs show mean ± S.E.M from three separate experiments. Significance by unpaired Student's t-test is indicated.