Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease

Abstract

While most respiratory viral infections resolve with little harm to the host, severe symptoms arise when infection triggers an aberrant inflammatory response that damages lung tissue. Host regulators of virally induced lung inflammation have not been well defined. Here, we show that enrichment for sialylated, but not asialylated immunoglobulin G (IgG), predicted mild influenza disease in humans and was broadly protective against heterologous influenza viruses in a murine challenge model. Mechanistic studies show that sialylated IgG mediated this protection by inducing the transcription factor repressor element-1 silencing transcription factor (REST), which repressed nuclear factor κB (NF-κB)-driven responses, preventing severe lung inflammation and protecting lung function during influenza infection. Therapeutic administration of a recombinant, sialylated Fc molecule in clinical development similarly activated REST and protected against severe influenza disease, demonstrating that this pathway could be clinically harnessed. Overall, induction of REST through sialylated IgG signaling is a strategy to limit inflammatory disease sequelae in infections caused by antigenically distinct influenza strains.

Keywords: CD209; IgG glycosylation; IgG sialylation; RE1-silencing transcription factor; REST; airway inflammation; alveolar macrophage; antibody signaling; influenza immunity.

Figure 1:. IgG sialylation predicts the severity of human influenza disease and protects mice against divergent influenza strains.

(A) IgG1 sialic acid distribution in hospitalized (n=10) and non-hospitalized (n=16) patients where G1 represents one and G2 represents 2 galactose residues on the biantennary N-glycan branches. Median values are depicted with a solid black line and statistical significance was two-way ANOVA with Tukey’s correction. (B) IgG1 Fc G2FS area under the receiver-operating-characteristic curve (AUC) with 95% confidence interval. (C) FcγR-humanized mice received either no treatment (Virus only), sialylated (S) human IgG (hIgG) or asialylated hIgG (S0) prior to lethal challenge with A/Netherlands/2009 (H1N1) or A/X-31 (H3N2) viruses. (D) Body weight (left) and survival (right) of mice challenged with A/Netherlands/602/2009 (H1N1). For body weight, statistical significance was determined by two-way ANOVA with Bonferroni correction for multiple comparison and body weight of sacrificed mice was defined as 85% (humane end-point) for statistical analyses. For survival analysis, statistical significance was determined by log-rank Mantel–Cox tests against the Virus only group. (*P<0.05, **P<0.01, #P<0.001). The experiment was performed twice, and representative data are shown (n=11 for the S and S0 hIgG groups, n=4 for the virus only group). (E) Body weight (left) and survival (right) for mice challenged with X31 (H3N2). For body weight, statistical significance was determined by two-way ANOVA with Bonferroni correction for multiple comparison and body weight of sacrificed mice was defined as 85% for statistical analyses. For survival analysis, statistical significance was determined by log-rank Mantel–Cox tests against the Virus only group. (*P<0.05, **P<0.01, #P< 0.001). Data from one experiment performed once (n=7 for the S and n=8 for the S0 hIgG groups, n=6 for the virus only group). (F) Lung viral titers of H1N1 infected mice on day 4 and/or day 7 post-infection. One-way ANOVA and two tailed student’s T-test analysis were used in day 4 and day 7 analysis respectively. Mice received no treatment (Virus only, Vir), sialylated (S) human IgG (hIgG) or asialylated hIgG (S0) prior to lethal challenge. Data are represented as mean ± SEM and are pooled from two independent experiments (for day 4 titers n = 12 per group, for day 7 titers, n=9 for S and n=7 for S0). (G) Lung viral titers of H3N2 infected mice on day 7 post-infection. Two tailed student’s T-test analysis was for statistical analysis. Mice received sialylated (S) human IgG (hIgG) or asialylated hIgG (S0) prior to lethal challenge. Experiment was performed once. Data are shown as mean ± SEM (n=5 per group). (H) FcγR humanized C57BL/6 mice were either pre-treated with S hIgG or received no pretreatment followed by H1N1 challenge in addition to either isotype hIgG control or CD209b blocking antibody. Experiment was performed twice, and representative data are shown (n=4 for isotype only and CD209b block, n=15-17 for S hIgG with iso or with CD209b block). Statistics by log-rank Mantel–Cox tests against the Virus only group. See also Figure S1.

Figure 2.. Sialylated hIgG reduces lung inflammation and protects lung function in influenza-infected mice.

(A) Left: UMAP plot of the myeloid lineage assessed by flow cytometry from the whole lung tissue of FcγR humanized C57BL/6 mice post H1N1 infection, with the different clusters labelled by color. Middle: pseudocolor plot of the UMAPs from the virus only group, S hIgG and S0 hIgG receiving groups, clusters that are significantly different between groups (C3- Amφ and C7-neutrophils) are outlined (red: higher density; blue: lower density). Right: The percentage of cells in each cluster among the virus only (Vir), S and S0 groups. Clusters that are significantly different between groups (C3- Amφ and C7-neutrophils) are shown in color. Statistics by two-way ANOVA with Bonferroni’s correction. Experiment was performed once (n=5 per group). (B) Neutrophils and monocyte count in BAL fluid on day 4 and day 7 post H1N1 infection. Statistics by one-way ANOVA with Tukey’s correction for day 4 analysis and two tailed student’s T test for day 7 analysis: *P<0.05, **P<0.01. Experiments were performed twice and pooled data from two independent experiments are shown (n=9-12 mice per group for day 4, n=6-8 mice per group). (C) Cytokine and chemokine concentrations in BAL fluid collected 4 days and 7 days post infection. Heatmaps represent the mean of log2 fold change for the cytokine concentrations. Statistics by one-way ANOVA with Tukey’s correction for day 4 analysis and two tailed student’s T test for day 7 analysis: *P<0.05, **P<0.01, ***P<0.001; pink = S vs Vir, grey = S0 vs Vir, black = S vs S0. Experiments was performed twice and pooled data from two independent experiments are shown for day 4 (n=8 mice per group). Experiment was performed once for day 7 (n=4-5 per group). (D) The concentration of O₂ and CO₂ in the cardiac blood collected from H1N1-infected mice on Day 5. Statistics by one-way ANOVA with Tukey’s correction: *P<0.05, **P<0.01. Experiments was performed twice and pooled data from two independent experiments are shown (n=8-9 mice per group). See also Figure S2.

Figure 3.. The presence of sialylated IgG during influenza infection induces a distinct transcriptomic signature in murine Amφ that stratifies disease severity in people with severe or mild influenza virus infections.

(A) Volcano plots showing the differentially expressed genes (DEGs) of Amφ isolated from H1N1 challenged mice pretreated with S (n=5) or S0 (n=5) hIgG, or no treatment (Vir, n=3). (B) PCA plot showing the clustering of samples based on the transcriptomic analysis of Amφ isolated from H1N1 challenged mice pretreated with S or S0 hIgG, or no treatment (Vir). (C) Top 5 most downregulated pathways via pathway enrichment analysis on common DEGs from S vs S0 and S vs Vir comparison. P values were determined by GSEA. (D) PCA depicting the stratification of influenza patients (n=328, 155 non-hospitalized, 173 hospitalized) into hospitalized (H) and non-hospitalized (NH) using the S hIgG associated gene signature. See also Figure S2.

Figure 4.. Sialylated IgG blocks NF-κB and induces REST.

(A) Expression of selected NF-κB genes by murine Amφ isolated from mice (n=4) that received sialylated (S) or asialylated (S0) immune complexes, or S IC after CD209b blockade. Statistics by Kruskal-Wallis with Dunn’s multiple comparisons test: **P<0.01, ***P<0.001, ****P<0.0001. (B) GSEA of murine Amφ transcripts (left) and human CD209+ monocyte-derived macrophage (MDM) (right) revealed enrichment of REST/NRSF target genes after sialylated (S) immune complex administration compared to asialylated (S0), n = 4 (left) and 3 (right). (C) Representative images of REST expression (red) in human MDMs stimulated with IgG with increasing abundance of Fc sialylation. 40x magnification; 10-micron scale bar; cell outline via phalloidin staining (green), nuclear outline via Hoechst staining (white). Representative pooled data from 3 independent experiments, each n=2. (D) Quantified REST expression and nuclear localization of (B): median with 95% CI. Statistics by Kruskal-Wallis with Dunn’s multiple comparisons test: *P<0.05, ****P<0.0001. Representative pooled data from 3 independent experiments, each n=2. (E) THP1 NF-κB reporter cells were differentiated and polarized toward CD209+ macrophage and treated with asialylated IgG and DMSO (control; gray), sialylated (S) IgG and DMSO (S IgG; blue), or S IgG and the REST inhibitor, X5050 (S IgG + X5050; purple) prior to stimulation with well-characterized activators of NF-κB. Statistics by two-way ANOVA with Šidák correction: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (F) Human alveolar macrophage (hAmφ) were cultured overnight with sialylated (S) or asialylated (S0) IgG prior to stimulation with an inflammatory activator, afucosylated (F0) immune complexes, or S0 IgG immune complexes. Data are from one experiment (n=4-5). (G) Expression of selected NF-κB genes in hAmφ from (E). Statistics by two-way ANOVA with post-hoc Tukey’s HSD test: *P<0.05. (H) GSEA revealed the positive enrichment of NRSF/REST target genes in hAmφ from (E) that were treated with sialylated IgG (S) compared to asialylated (S0) IgG. (I) Cytokine production by human Amφ from (E). Statistics by two-way ANOVA with post-hoc Tukey’s HSD test: *P<0.05, **P<0.01, ***P<0.001. See also Figures S3-S6.

Figure 5.. REST depletion ablates sialylated IgG-mediated protection against influenza disease.

(A) Neutrophils and cytokines within BALF of FcγR humanized C57BL/6 mice pretreated with either X5050 or DMSO followed by administration of sialylated IgG or PBS. Statistical significance for neutrophils was determined by one-way ANOVA with Tukey’s correction and for cytokines by two-way ANOVA with Tukey’s correction. Experiment was performed once (n=2-5 mice per group). (B) Neutrophils within BALF of FcγR humanized C57BL/6 mice pretreated with either X5050 or DMSO followed by administration of sialylated (S) IC or asialylated (S0) IC. Statistical significance for determined by one-way ANOVA with Tukey’s correction. Experiment was performed once (n=5 mice per group). (C) FcγR humanized C57BL/6 mice received sialylated hIgG or no IgG prior to H1N1 infection, along with the REST inhibitor, X5050 or sham (DMSO alone). (D) Weight (top) and survival (bottom) of mice in (A) were monitored daily. Error bars depict SEM. Statistical significance for weight lost (top) was determined by two-way ANOVA with Bonferroni’s correction. Body weight of sacrificed mice was defined as 85% for statistical analyses. Statistical significance for survival (bottom) was determined by log-rank Mantel–Cox tests against Vir group. For body weight, data are represented as mean ± SEM. Experiment was performed once (n=4 for X5050, n=8 for DMSO, n=10 for S hIgG + DMSO and n=9 for S hIgG + X5050).

Figure 6.. Recombinant, sialylated Fc induces REST and protects against influenza disease.

(A) Representative images showing REST induction in CD209+ human monocyte-derived macrophages stimulated with neutrally glycosylated and either S Fc or S0 Fc. 40x magnification; 10-micron scale bar; REST (red), cell outline via phalloidin staining (green), nuclear outline via Hoechst staining (white). Representative pooled data of N = 3 from 2 independent experiments. (B) Quantified REST expression and nuclear localization of (A): median with 95% CI. Statistics by Mann-Whitney test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Representative pooled data from 2 independent experiments (n=3). (C) FcγR humanized C57BL/6 mice received no treatment (Vir), S0 Fc, or S Fc on days 2, 4, and 6 post-lethal H1N1 challenge. (D) Weight (left) and survival (right) of mice in (B) were monitored daily. Statistical significance for weight loss (left) was determined by two-way ANOVA with Bonferroni’s correction. The body weight of sacrificed mice was defined as 85% for statistical analyses. Statistical significance for survival (right) was determined by log-rank Mantel–Cox tests against Vir group. The experiment was performed twice, and representative data are shown (n=5-10 mice per group).