Background Systemic Lupus Erythematosus (SLE) is an interferon-related autoimmune disease characterized by immune cell dysfunction and is common among women and minorities. A loss of tolerance to self-antigens leads to increased levels of autoantibodies against nuclear components (ANAs) prior to clinical disease onset. However, only about 4-8% develop an autoimmune disease. Patients with incomplete lupus erythematosus (ILE) exhibit some SLE clinical symptoms with most never progressing to SLE. Exact mechanisms involved in myeloid cell dysregulation and the progression of autoimmune disease remains unclear.

Objectives Determine whether alterations in myeloid cell population frequencies or activation of particular cellular processes is dysregulated in subjects with pre-clinical disease.

Methods PBMCs from 32 subjects (ANA-, ANA+, ILE and SLE) were sorted with Nanocellect Wolf microfluidic flow cytometer to remove dead and dying cells. Viable isolated single cells were used to do a multiomics single-cell analysis using 10x Genomics 5’ scRNA-Seq/137-plex Total-Seq multiomics kit, that also enable BCR and TCR repertoire analysis. Single-cell transcript and proteogenomics library preparation was done on a 10x Chromium X targeting 20,000 cells per channel, each sample encapsulated in a single channel. Normalized, pooled UDI labeled libraries were sequenced on Illumina Nova-Seq S4 flowcell (PE100, depth of 50,000 reads/cell). Data were analyzed for cluster identification, differential gene signatures in Python. Pathway analysis was performed in Ingenuity Pathway Analysis (IPA).

Results Across all subjects, 9 distinct myeloid clusters (Classical, Non-classical, Intermediate, CCR4+ monocytes) were identified with a community detection algorithm and visualized with Uniform Manifold Approximation Projection (UMAP). The proportion of cells in those clusters varied by disease group. Fractions of non-classical monocytes appear to be higher in ILE, SLE patients than in ANA- and ANA+ subjects. Classical monocytes have increasing cell fractions with disease progression, except ILE subjects that appear to have lower fractions than the other groups. Intermediate monocytes fractions are higher in ANA- controls and lower in the other groups. Pathway analysis revealed further differences within ethnicities (African, European Americans). Upregulation of autophagy related pathways was observed in AA ANA+ compared to ANA- and ILE, however it is downregulated in EA. Oxidative phosphorylation is upregulated in AA SLE compared to ILE and upregulated in AA ANA+ compared to ANA-. On the contrary, that pathway is upregulated in EA ANA+ compared to ANA- and ILE, as well as SLE compared to ILE. Further differences between subpopulations of major monocyte clusters were also observed.

Conclusion Dysregulation of signaling in monocyte activation appears to be manifesting in either increased oxidative phosphorylation or alteration in cellular apoptotic or autophagy pathway regulation. Alterations in these processes may vary by ancestral background reflected in the heterogeneity one sees in the presentation of lupus or trajectory of disease.

References [1]Dorner, T. and R. Furie, Novel paradigms in systemic lupus erythematosus. Lancet, 2019

[2]Slight-Webb, S., et al., Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype. J Allergy Clin Immunol, 2020

[3]Slight-Webb, S., et al., Autoantibody-Positive Healthy Individuals Display Unique Immune Profiles That May Regulate Autoimmunity. Arthritis Rheumatol, 2016

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Figure 1.

A. UMAP projection of 9 distinct myeloid clusters. B. Myeloid cell density for total population, African Americans and European Americans across 4 disease groups. C. Cell fractions for total population and each distinct cluster by disease group. D. Expression of genes involved in autophagy, death receptor signaling and oxidative phosphorylation pathways by race and disease group.

Acknowledgements: NIL.

Disclosure of Interests None Declared.