Lerner Research Institute, Cleveland Clinic Foundation Cleveland, OH, United States
Disclosure information not submitted.
Trine Jorgensen and Lindsey Han, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH
Background/Purpose: Systemic Lupus Erythematosus (SLE) is an autoimmune disease that can cause damage to multiple organs, including the kidneys in Lupus Nephritis (LN). Current treatments for SLE are limited to conventional disease-modifying anti-rheumatic drugs, while treatments for LN are limited to corticosteroid therapy, as well as Calcineurin inhibitors or Leflunomide, both of which have been studied mainly in Asian populations and are lacking in data. A more targeted therapy is necessary to address the underlying pathogenesis of the disease. Type I interferon (IFN-I) and its receptor (IFNAR) are known to play a significant role in the progression of SLE/LN, making them a major focus of research and therapy. The exact mechanism by which IFNAR expression on myeloid cells affects SLE/LN activity remains unclear.
Methods: Female mice were used to investigate whether deficiency of IFNAR on myeloid cells will drive or reduce disease progression of SLE/LN in B6.Nba2 lupus-prone mice. Study mice were: B6.Nba2.LysMcre/cre.IFNARflx/flx (n=16) (cKO), and control mice were: B6.Nba2 (WT) lupus-prone mice (n=18) and B6 (C57Bl/6) healthy mice (n=4). The tests performed included flow cytometry of kidney cells from 9 month old mice, and ELISA of urine biomarkers S100a8, VCAM-1, and PF4, a novel methodology scarcely done in LN mouse-model studies to analyze LN-related kidney damage without invasive biopsy.
Results: Kidney 9mo flow data revealed elevated neutrophil and decreased monocytic infiltration in the WT, but normalized levels in cKO, as well as specifically reduced levels of B cells in cKO. Furthermore, we found that CD4+ T cells were reduced, CD8+ T cells were elevated, and Double positive (DP) T cells were normalized in 9mo cKO kidneys as compared to WT mice. We observed nephromegaly in cKO mice at 9 mo of age, but the urinary biomarkers S100a8 and PF4 were decreased in 9mo cKO mice compared to WT mice.
Conclusion: In summary, we observed that reduced levels of neutrophilic cells, B cells, CD4+ T cells and DP T cells were associated with reduced levels of urinary biomarkers S100a8 and PF4 in cKO mice, suggesting that neutrophils infiltrate the kidney in a type I interferon-dependent manner, which subsequently results in the secretion of urinary markers previously found to be associated with nephritis in both pediatric and adult SLE-LN patients. Thus, we suggest that in response to type I interferons neutrophilic cells are recruited to the kidney, leading to the release of chemokines and the further recruitment of inflammatory lymphocytes, ultimately resulting in renal cell damage and the development of LN.
Figure 1: Kidney infiltrating myeloid cells approach levels seen in B6 mice, while B cells are specifically reduced in cKO mice. Myeloid inflammatory cell subsets were analyzed in 9-month-old female B6.Nba2 cKO, B6.Nba2, and B6 control mice. (A) Gating strategy: All cells were gated as CD45+. (B-C) The proportion of monocytic and neutrophilic cells in myeloid-cell specific B6.Nba2 cKO mice were closer to the B6 non-autoimmune controls. (D) In B6.Nba2.IFNAR cKO, B cells were specifically and significantly reduced compared to both B6.Nba2 and B6. Each symbol represents one mouse. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001
Figure 2: Lack of IFNAR on myeloid cells affect the type of T cells infiltrating kidneys of cKO mice. T cells were detected in 9-month-old female B6.Nba2 cKO, B6.Nba2, and B6 control mice. (A) Gating strategy. All cells were gated as CD45+. (B) In CD4+ Helper T cells, WT and B6 were unaltered, while the cKO mice were specifically reduced. (C) In CD8+ T cells, WT and B6 were also unaltered, while the cKO mice were specifically increased. Further studies to determine if elevated population is representative of CD8+ Tregs. (D) In double positive T cells, cKO mice were closer to levels of B6 healthy mice compared to the WT. Each symbol represents one mouse. ** p<0.01, *** p<0.001, **** p<0.0001
Figure 3: Decreased concentrations of urinary biomarkers suggest reduced LN disease activity in cKO mice. Concentrations of urinary biomarkers were analyzed in 9-month-old female B6.Nba2 cKO, B6.Nba2, and B6 control mice. (A) Concentration of S100a8 (Saliva 100 calcium-binding protein a8) was significantly reduced in cKO mice. (B) Concentration of PF4 (Platelet Factor 4) or CXCL4 (CX Chemokine ligand 4) was significantly reduced in cKO mice. (C) Concentration of VCAM-1 (Vascular cell adhesion molecule 1) was reduced in cKO mice. Each symbol represents one mouse. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001
.gif)
