Cell-Specific Type I IFN Signatures in Autoimmunity and Viral Infection: What Makes the Difference?

<div><p>Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4⁺) and monocyte subsets (CD16⁻ inflammatory and CD16⁺ resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4⁺ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.</p></div

For the validation of the 125 monocyte- (a) and the 68 T helper lymphocyte-specific (b) common-IFN signature genes their expression were followed in PBMC's of an independent cohort of juvenile SLE patients.

<p>In addition, we used these samples to validate the 132 monocyte- (c) and the 72 T helper cell-specific (d) gene signatures, which have been identified as autoimmune-specific type I IFN response signatures in these cell types. The common IFN signatures of both cell types allowed a correct classification in 8 out of 10 samples. Only the autoimmune-specific IFN signature of monocytes allowed a correct grouping of all SLE samples.</p

Number of probe-sets that are differentially expressed in various cell types from SLE patients and immunised healthy donors.

<p>Healthy donors (ND) immunised with the yellow fever vaccine are designated as “Viral infection”. (A) Total number of significantly differentially expressed probe-sets. (B) Number of significantly differentially expressed probe-sets from a reference list of 2442 IFN-related genes. (C) Number of significantly differentially expressed probe-sets from (B) with the additional cutoff of fold change (FC; ≥2 or ≤−2).</p

Distribution of “common” and “autoimmune-specific” IFN signature probe-sets in SLE patients and immunised healthy donors (ND).

<p>Red circles (described as “Autoimmunity”) indicate the number of IFN signature gene probes observed in the SLE patient samples. Blue circles (described as “Viral infection”) indicate the number of IFN signature gene probes observed in ND immunised with the yellow fever vaccine. The overlaps of the red and blue circles indicate “common” IFN signatures that were detected in both SLE and viral infection. When genes had several probe-sets that categorised them into multiple groups, they were excluded from the “autoimmune-/immunisation-specific” groups and only included in the “common” group. There were 11/1 (SLE/immunised ND) of these probe sets in the CD4⁺ T cells, 13/5 in the CD16⁻ monocytes and 19/1 in the CD16⁺ monocytes. Two different numbers in the area of overlaps, for example 94/79 in CD4⁺ T cells, are shown because only probe-sets that meet the cutoff of fold-change values > = 2 or < = −2 were counted in this figure.</p

Cluster diagrams of SLE patients and healthy donors before and after immunisation with the yellow fever vaccine.

<p>Healthy donors (ND) immunised with the yellow fever vaccine are designated as “ND_YF_day7”, and ND before immunisation are designated as “ND_YF_day0”. (A) IFN signature in CD4⁺ T cells. The 94 probe-sets of “common” IFN signatures observed both in the SLE patients and immunised ND and the 86 probe-sets of “autoimmune-specific” IFN signatures observed only in the SLE patients distinguish the SLE patients from the immunised ND. (B) IFN signature in CD16⁻ monocytes. The 165 probe-sets of “common” IFN signatures, 164 probe-sets of “autoimmune-specific” IFN signatures and 8 probe-sets of “immunisation-specific” IFN signatures distinguish the SLE patients from the immunised ND. (C) IFN signature in CD16⁺ monocytes. The 173 probe-sets of “common” IFN signatures, 120 probe-sets of “autoimmune-specific” IFN signatures and 5 probe-sets of “immunisation-specific” IFN signatures distinguish the SLE patients from the immunised ND.</p

For the validation of 165 monocyte- (a, b and c) and 94 T helper lymphocyte-specific (d, e and f) common-IFN signature genes, their expression were followed in PBMC's of an independent cohort of yellow-fever vaccinated individuals.

<p>Expression profiles were generated from PBMC's at day 3 (a and d), 7 (b and e) and 21 (c and f) after vaccination and compared to baseline levels at d0. Both, the common type I interferon signatures of monocytes and CD4 lymphocytes allowed the monitoring of the induction of interferon responses in PBMCs at d3, peaking at d7 and almost declining at d21.</p

Number of probe-sets differentially expressed in SLE patients and immunized healthy donors with yellow fever vaccine.

<p>This table summarizes differentially expressed probe sets as obtained by comparing arrays of the experimental group versus baseline group. Indicated are the total number of differentially expressed probe sets, the number of overlapping IFN-associated genes (absolute number and percentage of total number of differentially expressed probe sets) and the number of IFN-associated genes with fold changes ≥2 or ≤−2.</p><p>^a 2.442 interferon (IFN) signature genes were extracted from previous publications by Romos PS et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083776#pone.0083776-Ramos1" target="_blank">[17]</a> and Smiljanovic B et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083776#pone.0083776-Smiljanovic1" target="_blank">[11]</a>.</p><p>^b FC: fold change.</p><p>^c Expression data of healthy donors (ND) before immunization with yellow fever vaccine (YFV) was used as baseline for all comparisons.</p><p>^d ND 7 days after immunization with YFV.</p

Comparison of the enrichments of genes with selected biological functions in SLE patients and immunised healthy donors.

<p>CD4⁺ T cells, CD16⁻ monocytes and CD16⁺ monocytes from patients with SLE and healthy donors immunised with the yellow fever vaccine (designated as “Viral infection”) were analysed using the Ingenuity Pathway Analysis (IPA) tool.</p

Comparison of the enrichments of genes in the top-ranked canonical pathways in SLE patients and immunised healthy donors.

<p>CD4⁺ T cells, CD16⁻ monocytes and CD16⁺ monocytes from patients with SLE and healthy donors immunised with the yellow fever vaccine (designated as “Viral infection”) were analysed using the Ingenuity Pathway Analysis (IPA) tool. (A) Commonly observed pathways in all compared groups. (B) Dominantly observed pathways in the CD4⁺ T cells from the SLE patients. (C) Dominantly observed pathways in the CD16⁻ monocytes from the SLE patients. (D) Dominantly observed pathways in the CD16⁺ monocytes from the SLE patients.</p

Expression of immunoproteasomal subunits in immune cells:

<p>Gene expression of immunoproteasomal subunits (PSMB8–10) in CD4+, CD8+, CD19+, CD14+ and DCs of patients with myopathies (PM, DM, OM, NIM) and controls (HD). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**). Significantly higher expression of PSMB8 was observed in CD14+ cells and DC of PM patients compared to HD or DM, NIM and HDs, respectively. PSMB9 was increased in CD8+ and CD14+ of DM and in DCs of PM and DM patients compared to NIM. PSMB10 was found increased in PM patients compared to DM, NIM and HD in CD8+, compared to HD in CD19+ and CD14+ cells and compared to OM, NIM, and HD in DCs.</p