PMC8636939_01
Clinical and Representative Histologic Findings. Infant at approximately 1 week of age. (A) Skin eruption on the ventral torso. (B) 4th proximal interphalangeal joint arthritis. (C, D) Hematoxylin-eosin stains showing mixed dermal inflammation with numerous eosinophils. Skin, left leg biopsy. Step level sections with unremarkable dermis. Papillary dermal edema and a dense perivascular, periadnexal and interstitial infiltrate of lymphocytes and numerous eosinophils. No vasculitis.
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On the face, neck, trunk, and upper extremities were confluent, erythematous, blanchable macules and polycyclic patches ( Figure 1A ). After several days, arthritis developed in the right 4th and left 2nd proximal interphalangeal joints ( Figure 1B ). During the initial admission, the pathologic evaluation included hematoxylin-eosin stain of a punch biopsy of the left leg demonstrated papillary dermal edema, dense perivascular, periadnexal, and interstitial infiltrate ( Figure 1C ). The perieccrine infiltrate included lymphocytes, numerous eosinophils, and scattered neutrophils ( Figure 1D ).
Timeline of clinical events and treatment strategies. mo, month.
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The heterozygous de novo NLRP3 mutation M408T causes a gain of function for inflammasome activation. (A) Genomic DNA from a healthy control, the NOMID patient, and each of his parents was collected. PCR was used to amplify a region of NLRP3 surrounding the patient's mutation, and these PCR amplicons were subjected to Sanger sequencing using a second set of specific primers. A T>C single nucleotide change, causing the missense mutation M408T in NLRP3 protein, was confirmed to be heterozygous in the NOMID patient and absent from each parent, indicating this is a de novo mutation. (B) Plasmids were constructed, encoding an empty vector (EV), wild-type NLRP3 (WT), or the NOMID patient's NLRP3 mutation (M408T). HEK293T cells were either left non-transfected (NT) or were transfected with these plasmids, and whole cell lysates were used for western blotting. Using an antibody specific for NLRP3, expression of both WT and M408T NLRP3 protein was observed at the expected size (118kDa) with no notable difference in expression resulting from the mutation. Alpha-tubulin is used as a protein loading control. (C) HEK293ASC-YFP cells were plated on coverslips, then were either left non-transfected (NT), or transfected with an empty vector (EV) or plasmids encoding WT or M408T NLRP3. Cells were stained with DAPI, mounted and visualized by fluorescence microscopy. Images are representative of 8 or more fields per condition. (D) Summary of ASC specks per nuclei for the experiment presented in (C), and replicate experiments (n=4). P value is from a Mann-Whitney t-test. (E) HEK293T cells were either left non-transfected (NT, lane 1) or were transfected with the indicated combinations of plasmids encoding pro-IL-1beta-V5, Caspase-1, ASC, and WT or M408T versions of NLRP3. After 8 hours, whole cell lysate was prepared and used for western blotting. Pro-IL1beta is expected to migrate at 35.8kDa, while the mature cleaved form of IL-1beta is 22.4kDa.
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Sanger sequencing of genomic DNA from the patients' parents demonstrated the M408T mutation was de novo in this patient ( Figure 3A ). No difference in the WT and M408T protein amounts were observed ( Figure 3B ). These plasmids were expressed in a HEK293ASC-YFP cell line and formation of ASC specks, a marker of inflammasome activation, were visualized by fluorescence microscopy ( Figure 3C ). Cells transfected with M408T showed significantly increased ASC specks per nuclei relative to WT ( Figure 3D ). Eight hours post-transfection, western blot with anti-V5 antibody demonstrated increased cleavage of Pro-IL1beta-V5 (35.8kDa) to the mature IL1beta-V5 (22.4kDa) by inflammasomes containing M408T NLRP3, compared to WT NLRP3 ( Figure 3E ).
NOMID infant response to anakinra. The x-axis is a time course in months. Vertical red lines designate initiation and then dose increases of anakinra. Gray bands indicate normal values. (A) Weight-based anakinra dosing over time. (B) Markers of inflammation, CRP and ESR. CRP reference range is 0.3-6.1 mg/L. (C) Complete blood count values. (D) White blood cell differential absolute values.
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The time course in months showing initiation of IL-1beta pathway blockade with anakinra along with clinical laboratory results are shown ( Figure 4 ). Anakinra, which competitively binds the IL-1 receptor to inhibit IL-1beta signaling, is prescribed by weight-based dosing that is crucial for a growing infant ( Figure 4A ). CRP and ESR, remained elevated after initiation of anakinra and improved upon dose increase ( Figure 4B ). Clinical CBC values, including white blood cells, hemoglobin, and platelets, returned to normal upon dose increase. The white blood cell differential highlights that while neutrophils, lymphocytes and monocyte levels normalize with increasing IL-1beta blockade, eosinophils remain persistently elevated ( Figures 4C, D ).