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. 2014 Dec 1;21(16):2231-45.
doi: 10.1089/ars.2013.5828. Epub 2014 Jul 29.

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature

Tiina Kelkka  1 , Deborah Kienhöfer, Markus Hoffmann, Marjo Linja, Kajsa Wing, Outi Sareila, Malin Hultqvist, Essi Laajala, Zhi Chen, Júlia Vasconcelos, Esmeralda Neves, Margarida Guedes, Laura Marques, Gerhard Krönke, Merja Helminen, Leena Kainulainen, Peter Olofsson, Sirpa Jalkanen, Riitta Lahesmaa, M Margarida Souto-Carneiro, Rikard Holmdahl
Affiliations

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature

Tiina Kelkka et al. Antioxid Redox Signal. .

Abstract

Aims: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. VSports手机版.

Results: A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli V体育安卓版. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. .

Innovation: Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans V体育ios版. .

Conclusion: We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans. VSports最新版本.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Chronic granulomatous disease patients display a type I interferon signature. (A) All significantly differentially regulated transcripts (adjusted p-value<0.05 and FC>1.5) that were upregulated in chronic granulomatous disease (CGD) patients were curated into interferon-regulated (IRG), lymphocyte-related (LC), and inflammatory (INFL) transcripts. The numbers and percentages of genes in each category are presented. (B) Gene expression analysis results describing the ten significantly differentially regulated genes (corrected p-value<0.05) with the largest fold change between CGD patients and healthy controls are presented. CGD patients n=7, x-linked carriers n=4, healthy controls n=13. Benjamini–Hochberg corrected p-values for the correlation between the gene expression and phenotype are given. Boxplots present the minimum, maximum, median, and the 25% and 75% quartiles for each gene; the angular dashed line illustrates the slope of the correlation.
<b>FIG. 2.</b>
FIG. 2.
Chronic granulomatous disease patients have alterations in their B-lymphocyte populations. (A) Total cell counts per ml of blood of the main leukocyte populations. Total CD4+ T cells and total CD8+ T cells were calculated within total CD3+ cells. CD11b++SSChiFCShi are total phagocytes. (B) The frequency of total CD19+ B cells was calculated within total lymphocytes. The frequencies of the different B-cell subsets were calculated within total CD19+ B cells. (C) Frequency of total CD11b+ monocytes and CD11b++SSChi granulocytes were calculated within total leukocytes. Frequencies of monocyte subsets were calculated within CD11b+ cells. (D) The frequency of CD3+ T cells was calculated within total lymphocytes. The frequency of the CD4+, CD8+, and CD4+CD8+ cell subsets was calculated within total CD3+ T cells. The frequency of the CD4+ subsets was calculated within total CD4+ T cells, and that of the CD8+ subsets was calculated within the total CD8+ T cells. Bars represent average frequencies,±SEM, Kruskall–Wallis test with Bonferroni–Dunn post hoc analysis. *Represents p<0.05, **represents p<0.01, and ***represents p<0.001 between the indicated groups.
<b>FIG. 3.</b>
FIG. 3.
ROS-deficient Ncf1m1J mutated mice share the type I IFN response. (A) All significantly differentially regulated transcripts (adjusted p-value<0.05 and FC>1.5) that were upregulated in ROS-deficient BQ.Ncf1m1J mice were curated into interferon-regulated (IRG), lymphocyte-related (LC), and inflammatory (INFL) transcripts. The percentages and the numbers of genes in each category are presented. BQ.Ncf1m1J n=12 and in BQ n=12. (B) STAT1 expression in wild-type (BQ) and Ncf1 mutated (BQ.Ncf1m1J) mice was analyzed in two independent pooled experiments by flow cytometry. Heparinized blood was subjected to red blood cell lysis and stained for STAT1 expression. The normalized GeoMean values (BQ signal set as 100) of the STAT1-specific signal are reported. Mean±SEM, n=11–12, two-tailed unpaired Student's t-test. (C) Heparinized blood was suspended into IMDM (Gibco) supplemented with 10% FCS and heparin. The cells were stimulated with recombinant mouse IFN-β (1000 U/ml; GenWay Biotech) at +37°C for 10 min and stained with pSTAT1-PE (pY701) antibody to assess the levels of activated STAT1. Results were calculated as the GeoMean for PE fluorescence, mean±SEM, n=6, two-tailed Mann–Whitney U test. The relative frequencies of CD3+, CD3+CD8+, CD3+CD4+ T lymphocytes, and B220+ B lymphocytes were analyzed in the (D) blood and (E) spleen samples from BQ.Ncf1m1J and BQ mice. Phagocytes were divided into CD11b+Gr-1HI granulocytes and CD11b+Gr-1− monocytes (and CD11c+ dendritic cells in spleen samples). Mean±SEM, n=16, two-tailed unpaired Student's t-test. ***p<0.001, **p<0.01, *p<0.05, ns, not significant.
<b>FIG. 4.</b>
FIG. 4.
ROS deficient Ncf1m1J mutated mice have a reduced total memory B-cell compartment. Flow cytometry analysis of heparinized peripheral blood from the Ncf1m1J mutated and BQ wild-type mice was performed to determine B-cell subset frequencies. The frequency of total B cells (CD19+) was calculated within the total lymphocyte gate based on FSCxSSC. Total memory B cells (CD19+CD23+CD21+IgM+/−IgD−) and naïve B cells (CD19+IgM+IgD++) were calculated within total B cells. B cells expressing activation marker CD69 (CD19+CD69+), homing receptor (CD19+CXCR4+), or the apoptosis-associated Fas receptor (CD19CD95+) were calculated within total B cells. Mean±SEM, n=6/group, two-way Mann–Whitney test.
<b>FIG. 5.</b>
FIG. 5.
Anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-cyclic citrullinated protein antibodies in chronic granulomatous disease patients. (A) IgG and (B) IgA ASCA were analyzed from serum samples from all subjects enrolled in the study. (C) Similarly, the titers of anti-cyclic citrullinated protein (CCP) antibodies were measured. Bar indicates the mean of each group, Kruskall–Wallis test followed by Bonferroni post hoc test. CGD patients n=7, x-linked carriers n=4, and healthy controls n=13. *Represents p<0.05, **represents p<0.01, and ***represents p<0.001 between the indicated groups.
<b>FIG. 6.</b>
FIG. 6.
ROS-deficient naïve Balb/c.Ncf1m1J mutated mice spontaneously produce lupus-associated autoantibodies and develop inflammatory kidney inflammation. (A) Anti-dsDNA, (B) anti-Sm/RNP, and (C) anti-histone autoantibodies were measured in sera of wild-type and Ncf1m1J mutated Balb/c mice using ELISA. Horizontal lines represent the mean medians. Two-tailed Mann–Whitney U test, *p<0.05, and **p<0.01, ns, not significant, n=47 in Balb/c.Ncf1m1J and n=21 in Balb/c mice. (D) Representative immunofluorescence images of glomeruli from wild-type and Ncf1m1J mutated mice stained with FITC-conjugated antibodies to mouse IgG and complement factor C3. Glomeruli of Ncf1m1J mutated mice revealed moderate granular IgG deposits, mostly in mesangial areas, and a marked mesangial pattern of complement factor C3-deposition. In contrast, glomeruli from wild-type mice showed only minimal depositions of IgG or C3 in the glomeruli. Microscope; Nikon Eclipse 80i, Nikon Plan Apo 20×0.75 objective, Camera: Nikon DS-QiMc, Original magnification×200, DAKO Fluorescent Mounting Medium, scale bar 50 μm. (E) Glomerular deposition of IgG and C3 was analyzed using a semiquantitative scoring system. 0=no deposits, 1=mild mesangial deposition, 2=marked mesangial deposition, 3=mesangial and slight capillary deposition, 4=intense mesangial and mesangocapillary deposition. Horizontal lines represent the medians. n=5 in Balb/c.Ncf1m1J and n=6 in Balb/c mice. Horizontal lines represent the medians, two-tailed Mann–Whitney U test, *p<0.05, ns, not significant.
<b>FIG. 7.</b>
FIG. 7.
The type I IFN signature was confirmed in naïve germ-free BQ.Ncf1m1J mutated mice. (A) Anti-mouse norovirus antibodies in mouse sera were analyzed by fluorometric immunoassay. Results are presented as arbitrary units (A.U.). Mean±SEM, in BQ.Ncf1m1J n=8 and in BQ n=9, two-tailed unpaired Student's t-test, mean values±SEM are presented, **p<0.01. Spleen and blood samples from germ-free mice were subjected to genome-wide gene expression analysis. (B) All significantly differentially regulated transcripts (p-value<0.05 and FC>1.3) that were upregulated in the spleen samples collected Ncf1m1J mutated were curated into interferon-regulated (IRG), lymphocyte-related (LC), and inflammatory (INFL) transcripts. A similar analysis was performed for the transcripts yielding the largest possible difference between the genotypes but the smallest possible difference between the Ncf1m1J mutated replicates. The numbers and percentages of genes in each category are presented. In spleen samples, n=6 in both groups, in blood samples BQ.Ncf1m1J n=2, and in BQ n=1. (C) Ten significantly differentially regulated transcripts with the largest difference (FC) between the genotypes in spleen samples and (D) ten transcripts with the largest difference between the genotypes but the smallest difference between the Ncf1m1J mutated replicates are presented with functional curation into the groups mentioned earlier (IRG, LC, and INFL).
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