SARS-CoV-2 spike aggravates lupus nephritis and lung fibrosis in systemic lupus erythematosus

Abstract

Objective: COVID-19 induces the development of autoimmune diseases, including SLE, which are characterised by inflammation, autoantibodies and thrombosis. However, the effects of COVID-19 on SLE remain unclear.

Methods: We investigated the effects of COVID-19 on SLE development and progression in three animal models. Plasmids encoding SARS-CoV-2 spike protein and ACE2 receptor were injected into R848-induced BALB/C lupus mice, R848-induced IL-1 receptor antagonist knockout (KO) lupus mice and MRL/lpr mice. Serum levels of albumin and autoantibodies, lymphocyte phenotypes and tissue histology were evaluated.

Results: In R848-induced BALB/C lupus mice, the SARS-CoV-2 spike protein increased autoantibody and albumin levels compared with vehicle and mock treatments. These mice also exhibited splenomegaly, which was further exacerbated by the spike protein. Flow cytometric analysis revealed elevated T helper 1 cell counts, and histological analysis indicated increased levels of the fibrosis marker protein α-smooth muscle actin. In KO mice, the spike protein induced splenomegaly, severe kidney damage and pronounced lung fibrosis. In the MRL/lpr group, spike protein increased the serum levels of autoantibodies, albumin and the thrombosis marker chemokine (C-X-C motif) ligand 4.

Conclusion: COVID-19 accelerated the development and progression of lupus by inducing autoantibody production, fibrosis and thrombosis.

Keywords: COVID-19; lupus erythematosus, systemic; lupus nephritis; pulmonary fibrosis.

Figure 1. COVID-19 spike protein increases autoantibody levels in the R848-induced BALB/c lupus model. (A) Plasmid map of pcDNA3.1-SARS2-spike and pLEX307-ACE2-puro (Addgene). (B) BALB/c mice were treated with acetone (vehicle, n=5), and plasmids encoding blank (mock, n=5) or spike and ACE2 (spike, n=5) were injected into R848-induced lupus BALB/c mice. (C) Serum samples were harvested at the last time point and used for further analysis. Bar graph shows serum levels of antidouble stranded DNA (anti-dsDNA) total IgG, IgG1 and IgG2a in the indicated group. (D) Urine samples were harvested at the last time point and used for further analysis. Bar graph shows urine albumin levels in the indicated group. (E) Representative image showing spleen size in the indicated group. (F) Isolated peripheral blood cells were stained and subjected to flow cytometry. Bar graphs present the frequencies of interferon (IFN)-γ-positive, interleukin (IL)-4-positive and IL-17-positive cells in the blood. Data are means±SEMs (*p<0.05).

Figure 2. COVID-19 spike protein accelerates lupus nephritis and tissue fibrosis. (A) Representative images showing H&E-stained (top) and Masson’s trichome-stained (bottom) kidney sections in the indicated group. (B) Bar graph shows the glomerulonephritis score (left) and fibrosis area (right) in the indicated group. (C) Representative image showing the H&E-stained (top) and Masson’s trichome-stained (bottom) lung sections in the indicated group. (D) Bar graph showing lung Ashcroft score (left) and fibrosis area (right) in the indicated group. (E) Representative images showing α-smooth muscle actin (α-SMA)-stained lung tissues in the indicated group. (F) Bar graph showing the number of α-SMA-positive cells. Data are means±SEMs (*p<0.05, **p<0.01, ***p<0.001).

Figure 3. COVID-19 spike protein increases autoantibody production and tissue fibrosis in MRL/lpr lupus-prone mice. (A) Plasmids encoding blank (mock, n=5) or spike and ACE2 (spike, n=5) were injected in MLR/lpr mice. (B) Serum samples were harvested at the last time point and used for further analysis. Bar graph shows serum levels of total IgG, IgG2a and antidouble stranded DNA (anti-dsDNA) IgG2a in the indicated group. (C) Representative images showing H&E-stained and Masson’s trichome-stained kidney sections in the indicated group. (D) Bar graph showing glomerulonephritis, vasculitis and fibrosis in kidneys. (E) Representative images showing H&E-stained and Masson’s trichome-stained lung sections in the indicated group. (F) Bar graph showing lung Ashcroft score and lung fibrosis. (G) Serum and urine samples were harvested at the last time point and used for further analysis. Bar graphs show urine albumin levels (left) and serum chemokine (C-X-C motif) ligand 4 (CXCL4)/platelet factor 4 (PF4) levels (right) in the indicated group. Data are means±SEMs (*p<0.05, **p<0.01).

Figure 4. COVID-19 spike protein increases tissue fibrosis in IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice. (A) BALB/c mice were treated with acetone (vehicle, n=3), and plasmids encoding blank (mock, n=5) or spike and ACE2 (spike, n=3) were injected into R848-induced lupus IL-1Ra KO mice. (B) Representative images showing H&E-stained kidney images in the indicated group. (C) Bar graphs showing glomerulonephritis and vasculitis score. (D) Representative MT-stained lung images in the indicated group. (E) Bar graphs show lung Ashcroft score and lung fibrosis. Data are means±SEMs (*p<0.05, **p<0.01).