Alterations in Innate Immune Cells During the Progression of Systemic Autoimmune Rheumatic Diseases

Objectives Systemic autoimmune rheumatic diseases (SARD) exhibit a prolonged pre-clinical phase during which they have anti-nuclear antibodies (ANAs).[1] However, ANAs cannot be reliably used to predict impending disease because a subset of healthy women are ANA+ (~20%) and the majority of these individuals will not progress to SARD.[2] Why some individuals progress while others remain asymptomatic is unknown. Our objective is to evaluate functional alterations in innate immune populations during SARD development.

Methods CITE-Seq was conducted on innate immune cells from 24 patients including healthy controls (HC, n=5), non-progressor (NP, n=6) IFN high or low, progressors (P, n=5) or SARD (n=8) patients. Differential gene expression analysis was performed to identify genes of interest. Spectral flow cytometry and plasma ELISAs were conducted in an expanded group of patients to validate differences in CITE-Seq genes of interest. A monocyte cell line (THP-1) was used to investigate the kinetics of 1 gene identified by CITE-Seq to gain a better understanding of potential functional implications.

Results Non-progressors (NP) exhibited increased gene expression of heat shock proteins (HSPs) like HSP70 and CD52 compared to P (Figure 1A). Both proteins are proposed to promote immune regulation through tolerogenic effects on innate immune cells. Conversely, P exhibited increased gene expression of MHC class II alleles, which are associated with immune activation. Using flow cytometry, we confirmed the differences between groups of surface expression of CD52 and MHC class II on innate immune cells (Figure 1B). Little is known about how HSPs are regulated and expressed. To better understand the kinetics of HSP70, THP-1 cells were heat shocked. Gene expression showed rapid and transient upregulation which was attenuated by 18h, while soluble HSP70 increased steadily following activation of the heat shock response (Figure 1C). Protein HSP70 was decreased at the 12h timepoint using immunofluorescence (Figure 1D), following heat shock, suggesting the release of HSP70. We therefore measured plasma HSP70 in our patient cohort and found that soluble HSP70 was increased in NP compared to P (Figure 1E), in support of our CITE-Seq results. Ongoing experiments are being conducted to evaluate the release of HSP70 by purified innate immune cells from our patient cohort.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Innate immune cells during the progression of SARD. A) Feature plots from CITE-Seq of monocytes and DCs shotting HSPA1A (HSP70), CD52 (CD52) and HLA-DRB5 (MHC Class II). B) Spectral flow cytometry showing representative results from classical monocytes tor HLA-DR and CD52 protein expression. C) Gene expression of HSPA1A (HSP70) by RT-qPCR and soluble HSP70 (sHSP70) concentration by supernatant ELISAs measured in a THP-1 monocyte cell line. Cells were heat shocked (43°C for 1h) and subsequently rested for 6h, 12h, 18h, 24h before collection. E) Heat shocked THP-1 cells were rested for 12h and used for cytospin with subsequent immunofluorescence staining with DAPI for cell nuclei and HSP70 (scale bar = 50 μm). E) Plasma ELISAs for soluble HSP70 in patient cohort. Data was analyzed using a Kruskal-Wallis test with Dunn post hoe and Bonferroni correction (* = p-value < 0.05).

Conclusion Our data shows that NPs exhibit mechanisms of immune suppression that are decreased in P. Importantly, P exhibits immune dysregulation prior to clinical progression. These results will allow us to further investigate the immunological differences in innate cells that may drive or inhibit progression in SARD.

References [1.] Goldblatt F. Lancet 2013;382:797-808. [2.] Wither J. Arthritis Res Ther 2017;19:41.