Immune response to intravenous immunoglobulin in patients with Kawasaki disease and MIS-C

Abstract

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1β have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1β. Following IVIG treatment, activated IL-1β+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1β can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.

Keywords: Apoptosis; COVID-19; Innate immunity; Neutrophils.

Figure 1. Patient enrollment and follow-up.

Patients with KD and MIS-C were studied prior to treatment, early after IVIG administration (9 to 30 hours), or in a subacute phase 2 to 6 weeks after IVIG treatment. Blood samples were analyzed by CBC, CyTOF, flow cytometry, and live-cell imaging.

Figure 2. Flow cytometry characterization of IL-1β–positive leukocytes in patients with KD (n = 9) or MIS-C (n = 5) or in FC (n = 14) patients.

(A) Flow cytometry gating strategy used to distinguish myeloid and lymphoid cells. (B) Dot plots show the total cell numbers of each cell population per mL of whole blood from all patients prior to treatment. (C) Flow cytometry gating strategy for this study demonstrating IL-1β⁺ expression in each population. Fluorescence minus one (FMO) controls were used for each sample to assist gating. (D) Flow cytometry evaluation of absolute cell numbers of IL-1β⁺ leukocytes per mL of whole blood from all patients prior to treatment. (E) Scatter dot plots show each IL-1β⁺ cell population as a percentage of all IL-1β⁺ leukocytes from all patients prior to treatment. (F) qPCR mRNA expression of indicated genes in neutrophils isolated from 12 MIS-C patients prior to treatment. P values were determined by 1-way ANOVA and Tukey’s multiple-comparison tests with a single pooled variance. Horizontal lines on dot plots indicate median IQRs. Mat.Neuts, mature neutrophils. *P < 0.05, ***P < 0.0005.

Figure 3. CyTOF shows neutrophils are highly activated in patients with KD or MIS-C and associated with high IL-1β expression.

(A) viSNE map shows FLOWSOM automated clustering results of live CD45⁺ cells as leukocyte lineage populations from all patients prior to treatment. (B) Heatmap shows the median metal intensity of each marker in different leukocyte populations from patients with MIS-C (B, n = 9), KD (C, n = 2), and FCs (D, n = 11) obtained before treatment. (E) Total cell numbers of leukocyte populations prior to treatment from patients with KD (n = 2), MIS-C (n = 9), or from FCs (n = 11). Values determined from CyTOF data. P values were determined by 1-way ANOVA and Tukey’s multiple-comparison tests with a single pooled variance. Lines on scatter dot plots indicate median with IQR. tSNE, t distributed stochastic neighbor embedding; viSNE, visualization of t distributed stochastic neighbor embedding.

Figure 4. IVIG treatment in patients with KD or MIS-C is associated with a decrease in IL-1β–producing neutrophils and an increase in lymphocytes.

(A) Heatmap shows the median metal intensity of each marker in neutrophils from all patients before treatment. (B) Total cell number (top panels) and proportion (bottom panels) of leukocyte populations from patients with KD (n = 9) or MIS-C (n = 5) prior to IVIG treatment and 2 to 6 weeks after treatment (KD, n = 8; MIS-C, n = 9). Values determined from flow cytometry data. (C) Absolute neutrophil count (ANC) from KD patients before IVIG and 24 hours after IVIG treatment from an independent cohort (n = 95). (D) Total numbers of IL-1β⁺ leukocytes from KD patients prior to treatment (n = 9) and 2 to 6 weeks after IVIG (n = 8). (E) IL-1β expression in neutrophils from a patient with KD and a patient with MIS-C prior to treatment and 2 to 6 weeks after IVIG. Representative data shown. Fluorescence minus one of each sample was used as a staining control. (F) Total numbers of IL-1β⁺ lymphocytes, monocytes, eosinophils, and neutrophils in the peripheral blood of patients with KD (left panel) or MIS-C (right panel) determined by flow cytometry before treatment (KD, n = 9; MIS-C, n = 5) and 2 to 6 weeks after IVIG treatment (KD, n = 8; MIS-C, n = 9). P values were determined by unpaired t tests based on IVIG-alone samples. Lines on dot plots indicate median with IQR. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Figure 5. IVIG treatment in patients with KD or MIS-C is associated with a decrease of IL-1β–producing neutrophils and reduced activation.

(A) Left panel: flow cytometry gating strategy demonstrating reduced mature neutrophils after IVIG treatment in a representative KD patient (top) and a representative MIS-C patient (bottom). The percentages of neutrophils in live CD45⁺ cells are indicated on the contour plots. Right panel: absolute cell numbers of mature neutrophils per mL of whole blood. (B) Flow cytometry evaluation of NePs per mL of whole blood. (C) Serum G-CSF in patients with KD before treatment (n = 5) and 2 to 6 weeks after IVIG treatment (n = 5). (D) Flow cytometry evaluation of total numbers of IL-1β⁺ mature neutrophils (left panel) and IL-1β^– mature neutrophils (right panel) per mL of whole blood. (E) Flow cytometry evaluation of total numbers of IL-1β⁺ NePs (left panel) and IL-1β^– NePs per mL of whole blood (right panel). (F) Heatmap shows the median metal intensity of each marker in neutrophils from 5 MIS-C patients before treatment and 1 day after IVIG treatment evaluated by CyTOF. (G) Heatmap showing FLOWSOM automated clustering of neutrophil subsets from 5 MIS-C patients before treatment and 1 day after IVIG treatment. (H–J) CyTOF evaluation of total cell numbers of neutrophils (H), mature neutrophil subsets (I), and NeP subsets (J) per mL of whole blood. Samples were analyzed from 5 patients with MIS-C prior to treatment and 1 day after IVIG treatment. Differences between before and after IVIG treatment were determined via unpaired t tests (2-tailed) based on IVIG-alone samples (A, B, D, and E) or by ratio paired t tests (C, H, I, and J). Lines on dot plots indicate median with IQR. *P < 0.05; **P < 0.005; ***P < 0.0005

Figure 6. IVIG triggers rapid cell death of neutrophils.

(A) Peripheral blood neutrophils undergo unique morphological changes upon exposure to 1% IVIG. Neutrophils were labeled with 50 nM CTG and incubated in the presence of AnnV-AF647 (red) and PI (yellow) to track changes in cell viability. Representative image from KD neutrophils. Two-hour time point shown. (B) Viability of neutrophils at the 8-hour time point in the presence or absence of 2 μM PIK-75, 10 μM GDC-0941, 10 μM DPI, 10 μM QVD-OPh, 100 ng/mL FcFasL, and 1% IVIG. (C) Kinetic changes in neutrophil viability classified according to staining with CTG, AnnV, and PI. (D) Changes in cell size of neutrophils treated with 100 ng/mL FcFasL or 1% IVIG.

Figure 7. Dimension reduction analysis of neutrophil live-cell imaging reveals a unique IVIG nonapoptotic cell death signature.

For each patient, UMAP was performed on morphological and fluorescence parameters from the combined time-course data from neutrophils treated with IVIG, FasL, or saline. (A) A kernel-density estimate plot of cells across wells and over time by UMAP. Contour lines highlight the density of cells. The locations of cells in UMAP plots highlight the trajectories of cells across time and stimuli. The combined UMAP includes cells from all conditions based on equal sampling. (B) Cell features derived from live-cell imaging data overlaid on UMAP plots from cells at time points of 1 to 8 hours. The normalized intensity z score of each feature is shown for each stimuli.