A Short Allelic Epitope Coded by HLA-DRB1*03:01 Activates a Systemic Lupus Erythematosus Cell Aberration Cascade

Bruna Miglioranza Scavuzzi¹, Bhavneet Kaur¹, Vincent Van Drongelen¹, Raquel Mesquita-Ferrari², Amr Sawalha³, Frederick Miller⁴ and Joseph Holoshitz¹, ¹University of Michigan, Ann Arbor, MI, ²Universidade Nove de Julho (UNINOVE), São Paulo, Brazil, ³University of Pittsburgh, Pittsburgh, PA, ⁴Environmental Autoimmunity Group, Clinical Research Branch, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health, Bethesda, MD

Meeting: ACR Convergence 2022

Keywords: genetics, macrophages, Systemic lupus erythematosus (SLE)

Session Information

Date: Sunday, November 13, 2022

Title: SLE – Etiology and Pathogenesis Poster

Session Type: Poster Session B

Session Time: 9:00AM-10:30AM

Background/Purpose: Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from an interaction of genetic, epigenetic, hormonal, environmental and immunoregulatory influences. It has been well established that the HLA-DRB1*03:01 allele is a major risk factor in SLE; however, the mechanistic basis of the association is unclear. Our group has long proposed that in addition to presenting antigens, rheumatoid arthritis (RA)-associated HLA-coded molecules interact with a non-MHC receptor and trigger innate signaling events and lead to the manifestation of the disease. Here we report that, reminiscent of RA-associated HLA-DRB1 alleles, a lupus associated HLA-DRB1*03:01 epitope triggers cellular aberrations akin to known cellular events in SLE.

Methods: PMA-differentiated THP-1 cells and RAW 264.7 cells were treated with synthetic peptides corresponding to the third allelic hypervariable region (residues 65-79) of the DRb chain coded by three DRB1 alleles: *03:01, *04:01 and *04:02 (100 µg/mL) with or without recombinant mouse IFN-g (5 ng/mL). Cytotoxicity was determined by Lactate dehydrogenase (LDH) Kit (Roche). Intracellular ATP levels were measured with the ATPlite Luminescence Assay (PerkinElmer). Mitochondrial membrane potential and ROS were determined using the tetramethylrhodamine (TMRM) dye (Invitrogen), and MitoSOX Red (Molecular Probes), respectively, following the supplier’s protocol. Protein expression was determined by Western blotting. Griess Reagent assay (Promega) was used to estimate nitrite concentration. Cells were incubated for 2 hours with the following concentrations of ER-stress inhibitors: 5 mM 4-Phenylbutyric acid, (4-PBA); 10 μM 4μ8C; 25 μM AMG PERK 44; 10 μM Ceapin-A7.

Results: In the presence of IFN-g, human THP-1 and mouse RAW 264.7 macrophage cell lines stimulated with 15-amino acid peptide sequence coded by HLA-DRB1*03:01 induced allele-specific signature SLE transcriptome, with activation of genes related to ER-stress and proteasomal degradation and triggered a cascade of cellular aberrations associated to SLE, including endoplasmic reticulum stress, mitochondrial dysfunction, DNA damage, cell death by necroptosis and release of proinflammatory cytokines. Cell death (p < 0.0001) and mitochondrial function assessed by intracellular ATP levels (p=0.0029), mitochondrial membraned potential (p < 0.0001) and mitochondrial ROS (p < 0.0001) were completely restored upon inhibition with chemical chaperone, 4-PBA. When investigating the possible ER-pathways involved, restoration of intracellular ATP levels pointed to the ATF6 pathway (p=0.008).

Conclusion: This study suggests a noncanonical, antigen presentation-independent mechanism of HLA-disease association in SLE and could lay new foundations for our understanding of key molecular mechanisms that trigger and propagate this devastating autoimmune disease. Translation of the findings to primary human macrophages derived from healthy controls and SLE patients could open the door to better understanding of the etiology and pathogenesis of SLE and in the long run, could potentially introduce new preventive, diagnostic and therapeutic solutions for the disease.

Fig. 1: The LE activates a SLE transcriptome and activates ER stress, UPR, mitochondrial dysfunction, cell death, pro-inflammatory cytokine production and DNA damage, which can be mitigated by chemical chaperone 4-PBA in human and mouse macrophages.
a, Notable KEGG pathway enrichment among 65-79*LE-upregulated DEGs, versus 65-79*SE.
Data are based on DEGs with a fold change >1.5 and Padj < 0.05. All data are based on 6 biological replicates in 2 independent experiments.
b, Representative immunoblots of ER stress markers (pIRE1-α, GRP78, CHOP) and proteasomal degradation marker (p62) in THP_1 macrophages in the presence of 65-79*LE, 65-79*SE, or 65-79*PE.
c, Representative immunocytochemistry images of poly-ubiquitinated protein accumulation (red) in RAW 264.7 macrophages. Scale bar = 2 μm.
d, Intracellular ATP levels reduction in allelic epitope-treated human THP_1 macrophages (n=3).
e, Mitochondrial membrane potential in allelic epitope-treated THP_1 macrophages, as determined by tetramethylrhodamine ethyl ester (TMRE), red (n=4).
f, Mitochondrial ROS in RAW 264.7 macrophages exposed to allelic epitopes in the presence of IFN-γ and measured by MitoSOX.
g, Cell death and h, DNA damage, quantified by the comet assay, in THP_1 cells exposed to different allelic epitopes (n=3).
i-k, Supernatant levels of the pro-inflammatory cytokines i, TNF-alpha j, IL-6 and k, IL_1β in THP_1 macrophages exposed to different allelic epitopes (n=3).
l, Immunoblots of Receptor-Interacting Protein 1 (RIP) and pMLKL in RAW 264.7 macrophages.
m, Cell death of RAW 264.7 macrophages in the presence (dotted bars) or absence (solid bars) of the RIP1 inhibitor Necrostatin_1 (50 μM) (n=3).
n, Cell death of RAW 264.7 macrophages in the presence (dotted bars) or absence (solid bars) of the MLKL inhibitor Necrosulfonamide (10 μM) (n=3).
o, Cell death in allelic epitope-treated RAW 264.7 macrophages in the presence (dotted bars) or absence (solid bars) of 4-PBA (n=3).
p, Intracellular ATP levels q, mitochondrial membrane potential and r, mitochondrial ROS levels in RAW 264.7 macrophages in the presence (dotted bars) or absence (solid bars) of 4-PBA (n=3).
s, Immunoblots of RIP1 and pMLKL in allelic epitope-treated RAW 264.7 macrophages in the presence (dotted bars) or absence (solid bars) of 4-PBA.
Blots are representative of 3 independent experiments. Data represent mean ± SEM. One-way (e,f,h,k) and two-way (d,g,I,j,m-r) ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001.

Figure 2. A proposed model of cell aberration cascade activated by allelic epitope coded by HLA-DRB1*03:01. In the presence of IFN-γ (1), the LE – possibly through interaction with a yet unknown cell surface receptor – triggers ER stress (2), which leads to mitochondrial dysfunction (3), DNA damage (4), release of pro-inflammatory cytokines
and necroptosis (5).

Disclosures: B. Miglioranza Scavuzzi, None; B. Kaur, None; V. Van Drongelen, None; R. Mesquita-Ferrari, None; A. Sawalha, None; F. Miller, None; J. Holoshitz, Zydus-Cadila.

To cite this abstract in AMA style:

Miglioranza Scavuzzi B, Kaur B, Van Drongelen V, Mesquita-Ferrari R, Sawalha A, Miller F, Holoshitz J. A Short Allelic Epitope Coded by HLA-DRB1*03:01 Activates a Systemic Lupus Erythematosus Cell Aberration Cascade [abstract]. Arthritis Rheumatol. 2022; 74 (suppl 9). https://acrabstracts.org/abstract/a-short-allelic-epitope-coded-by-hla-drb10301-activates-a-systemic-lupus-erythematosus-cell-aberration-cascade/. Accessed .

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