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. 2023 Jan 10;24(2):1339.
doi: 10.3390/ijms24021339.

Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti-Myeloperoxidase Autoimmunity and Glomerulonephritis

Sharon L Ford  1 , Kim M O'Sullivan  1 , A Richard Kitching  1   2   3 , Stephen R Holdsworth  1   2 , Poh Yi Gan  1 , Shaun A Summers  1   2
Affiliations

Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti-Myeloperoxidase Autoimmunity and Glomerulonephritis

Sharon L Ford et al. Int J Mol Sci. .

Abstract

ANCA-associated vasculitis (AAV) is intricately linked with infections. Toll-like receptors (TLR) provide a potential link between infection and anti-myeloperoxidase (MPO) autoimmunity. TLR9 ligation has been shown to promote anti-MPO autoimmunity and glomerular vasculitis in murine MPO-AAV. This study investigates dendritic cell TLR9 ligation in murine experimental anti-MPO glomerulonephritis. We analyzed autoimmune responses to MPO following transfer of TLR9 stimulated, MPO pulsed dendritic cells and kidney injury following a sub-nephritogenic dose of sheep anti-mouse glomerular basement membrane globulin. TLR9 ligation enhanced dendritic cell activation upregulating CD40 and CD80 expression, promoting systemic anti-MPO autoimmunity and T cell recall responses and exacerbating kidney injury. CD40 upregulation by TLR9 was critical for the induction of nephritogenic autoimmunity. The presence of DEC205, which transports the TLR9 ligand to TLR9 located in the endosome, also promoted kidney injury. This confirms TLR9 mediated dendritic cell activation as a mechanism of anti-MPO autoimmunity in AAV and further defines the link between infection and the generation of MPO specific autoimmune inflammation.

Keywords: ANCA associated vasculitis; TLR9; autoimmunity; dendritic cell; glomerulonephritis; kidney injury; myeloperoxidase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TLR9 ligation enhances MPO pulsed dendritic cell activation upregulating dendritic cell CD40 and CD80 expression. For fluorescence-activated cell sorting analysis, gates were set based on the mean fluorescence intensity for single color negative controls. (A,B): DCs assessed following rmMPO and TLR9 ligand, CpG incubation; Viability (A) and Cell Specificity (B). (C,D): DCs assessed following recombinant mouse MPO (rmMPO) and Control, GpC (C) and TLR9 ligand, CpG (D); Assessment of cell surface activation markers MHCII and CD40. CD40 expression was higher in rmMPO/CpG incubated DCs (91% CD40hi) compared to rmMPO/Control incubated DCs (44% CD40hi). MHCII expression was unchanged between DC groups ((D):72% vs. (C):79%). (E,F): DCs assessed following rmMPO and Control, GpC (E) and TLR9 ligand, CpG (F). Assessment of cell surface activation markers CD86 and CD80. CD80 expression was higher in rmMPO/CpG incubated DCs (91% CD80hi) compared to rmMPO/Control incubated DCs (39% CD80hi). CD86 expression was unchanged between DC groups ((F): 74% vs. (E): 75%). DC = Dendritic cell, PI = Propidium iodine.
Figure 2
Figure 2
TLR9 ligation of MPO pulsed dendritic cells promotes early MPO autoimmunity. Early Immune Responses. MPO-stimulated mouse splenocyte cytokine production (day 10). (A): Increased production of interleukin 17A (IL-17A) in mice receiving TLR9 stimulated MPO pulsed DCs compared to Control stimulated MPO pulsed DCs. (B): Increased production of interferon gamma (IFN-γ) in mice receiving TLR9 stimulated MPO pulsed DCs. (C): Increased MPO specific dermal DTH response in mice receiving TLR9 stimulated MPO pulsed DCs. (D): No difference in serum MPO-ANCA was observed between groups. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. Bars show the mean +/− SEM. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001. DC = Dendritic cell. Mouse numbers for experiment: Control: n = 10, TLR9 Ligand: n = 10.
Figure 3
Figure 3
TLR9 ligation of MPO pulsed dendritic cells promotes late MPO autoimmunity, t cell recall responses and subsequent kidney injury. Immune responses (day 18): (A): Increased production of IL-17A in mice receiving TLR9 stimulated MPO pulsed DCs compared to control stimulated MPO pulsed DCs. (B): No significant increase in production of IFN-γ in mice receiving TLR9 stimulated MPO pulsed DCs at Day 18. (C): No difference in serum MPO-ANCA IgG in mice receiving TLR9 stimulated and control stimulated MPO pulsed DCs compared to OVA injected mice. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. Glomerular Injury Measurements: (D,E): Kidney injury as assessed by percentage of abnormal glomeruli on formalin fixed paraffin embedded periodic acid Schiff sections taken at original magnification ×400. Increased kidney injury in mice receiving TLR9 stimulated MPO pulsed DCs. (F): Serum urea measurement in µmol/L. Increased in mice receiving TLR9 stimulated MPO pulsed DCs. (G): Increase in glomerular macrophage recruitment, measured as average FA11+ cells per glomerular cross section (cells/glom) in mice receiving TLR9 stimulated MPO pulsed DCs. (H): Increase in glomerular neutrophil recruitment, measured as average Gr1+ cells per glomerular cross section (cells/glom) in mice receiving TLR9 stimulated MPO pulsed DCs. (I): Increase in glomerular CD4+ T Cell recruitment, measured as average GK1.5+ cells per glomerular cross section (cells/glom) in mice receiving TLR9 stimulated MPO pulsed DCs. Bars show the mean +/− SEM. * = p <0.05, ** = p < 0.01, **** = p < 0.0001. DC = Dendritic cell. Mouse numbers for experiment: Control: n = 6, TLR9 Ligand: n = 6.
Figure 4
Figure 4
Enhanced TH17 Additionally, TH1 MPO autoimmunity induced by TLR9 stimulated MPO pulsed dendritic cells is dependent on TLR9 ligation. (A): Decreased production of IL-17A in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs compared to WT DCs. (B): Decreased (ns) production of IFN-γ in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs. (C): Decreased production of tissue necrosis factor alpha (TNFα) in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs. (D): Increased production of interleukin 4 (IL-4) in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs. (E): Decreased serum MPO-ANCA IgG in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs compared to WT DCs. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. Kidney injury was assessed at Day 18. (F): Decreased kidney injury in mice receiving TLR9 stimulated MPO pulsed Tlr9−/− DCs. (G) Periodic-acid Schiff-stained micrographs of glomeruli were taken at original magnification ×400. Bars show the mean +/− SEM. * = p <0.05, ** = p < 0.01, *** = p < 0.001. DC = Dendritic cell. Mouse numbers for experiment: WT: n = 8, Tlr9−/−: n = 8.
Figure 5
Figure 5
CD40 upregulation on TLR9 stimulated MPO pulsed dendritic cells is critical for the enhancement of MPO autoimmunity. Early Immune Responses Model (Day 10): (A): Reduced production of IFN-γ in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs compared to WT DCs. (B): Reduced production of interleukin 2 (IL-2) in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (C): Reduced production of TNFα in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (D): Reduced production of interleukin 10 (IL-10) in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (E): Reduced production of IL-17A in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (F): Reduced production of interleukin 6 (IL-6) in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (G): Decreased serum MPO-ANCA IgG in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs compared to WT DCs. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. Bars show the mean +/− SEM. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. DC = Dendritic cell. Mouse numbers for experiment: WT: n = 8, CD40−/−: n = 8.
Figure 6
Figure 6
CD40 upregulation on TLR9 simulated MPO pulsed dendritic cells is critical for the development of kidney injury following the transfer of TLR9 stimulated MPO pulsed dendritic cells and the triggering of kidney injury. (A,B): Abrogated kidney injury in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. Periodic-acid Schiff-stained micrographs of glomeruli were taken at original magnification ×400. (C): Serum urea measurement in µmol/L. Reduced in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs. (D): Decreased serum MPO-ANCA IgG in mice receiving TLR9 stimulated MPO pulsed CD40−/− DCs compared to WT DCs. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. Bars show the mean +/− SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. DC = Dendritic cell. Mouse numbers for experiment: WT: n = 7, CD40−/−: n = 8.
Figure 7
Figure 7
DEC205 is required for the development of Th17 MPO autoimmunity on transfer of TLR9 stimulated MPO pulsed dendritic cells. dendritic cell DEC205 deficiency protects from subsequent kidney injury. (A) Reduced production of IL-17A in mice receiving TLR9 stimulated MPO pulsed DEC205−/− DCs compared to WT DCs. (B): Increased production of TNF-α in mice receiving TLR9 stimulated MPO pulsed DEC205−/− DCs. (C): No difference in production of IFN-γ in mice receiving TLR9 stimulated MPO pulsed DEC205−/− DCs. (D): No difference in serum MPO-ANCA IgG in mice receiving both TLR9 stimulated MPO pulsed DEC205−/− and WT DCs compared to OVA injected mice. Dotted line represents the OD450 of sera from control, OVA immunized mice receiving anti-GBM globulin. (E,F): Decreased kidney injury in mice receiving TLR9 stimulated MPO pulsed DEC205−/− DCs. Periodic-acid Schiff-stained micrographs of glomeruli were taken at original magnification ×400. (G): Serum urea measurement in µmol/L. No difference in mice DEC205−/− TLR9 stimulated MPO pulsed DEC205−/− DCs. (H): Proteinuria measured in mg/day. Reduced in mice receiving TLR9 stimulated MPO pulsed DEC205−/− DCs. Bars show the mean +/− SEM. * = p <0.05, ** = p < 0.01. DC = Dendritic cell. Mouse numbers for experiment: WT: n = 6, DEC205−/−: n = 5.
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