Background/Purpose: Infiltrating macrophages are one of the hallmarks of renal inflammation and kidney damage in lupus nephritis (LN). Surprisingly overexpression of TLR8, but not other endosomal TLRs, was observed in renal macrophages of nephritic NZB/W mice and in LN kidneys. Myeloid cells and neutrophils primarily express TLR8 but as its function differs from mouse to man, its role in autoimmunity remains poorly understood. Recently, TLR8 activation was shown to drive differentiation of T-follicular helper (T_FH) cells which are crucial regulators of the germinal-center response and humoral immunity. Herein, we aimed to evaluate the functional consequence of one or two copies of huTLR8 on autoimmunity and renal inflammation in murine SLE.

Methods: NZW/B6, NZW/B6.Yaa and Sle1.Yaa mice expressing 1 (NZW/B6 strains) or 2 (Sle1.Yaa strain) copies of huTLR8 as a BAC transgene (huTR8tg) were generated and followed clinically. HuTLR8 DNA copy number and mRNA expression was confirmed by qDigital and qRT-PCR respectively. 24-week-old huTLR8tg NZW/B6.Yaa mice were administered TL-506 (TLR8-agonist) subcutaneously for 4 weeks and spleens and kidneys were harvested for analysis at 8 weeks. Serum autoantibodies were assessed over time. Spleen weights were assessed and splenocytes, kidney cells and bone marrow cells were characterized by flow cytometric analysis.

Results: A single copy of huTLR8 did not exacerbate clinical disease, however, subcutaneous TLR8-agonist administration appeared to enhance germinal center formation and plasma cell generation in male NZW/B6.Yaa huTLR8tg mice. Strikingly, huTLR8.tg SLE1.Yaa males expressing 2 copies of huTLR8 showed accelerated renal disease and mortality by 4 months of age compared with >9 months in Sle1.Yaa controls. Anti-RNP and anti-cardiolipin antibodies developed by 7-10 weeks of age in both male and female huTLR8.tg mice but maximal titers were the same as in their SLE1.Yaa counterparts. Splenomegaly was observed in both male Sle1.Yaa- (p<0.001) and female Sle1-huTLR8.tg mice (p=0.0013). Flow cytometric analysis of splenocytes from heterozygous huTLR8.tg SLE1.Yaa males showed an acceleration of memory T cell expansion but similar numbers of germinal center (GC) B cells and T_FH cells as their wild type Sle1.Yaa counterparts. By contrast there was a 10 fold increase in splenic myeloid cells compared to their SLE1.Yaa counterparts including macrophages, dendritic cells and neutrophils. Interestingly, aged huTLR8tg SLE1 females often showed distended caeca, intestinal obstruction, cataract, periocular alopecia and blepharitis, indicating a disrupted epithelial and mucosal homeostasis in these mice.

Conclusion: One copy of huTLR8 does not exacerbate lupus in NZW/B6.Yaa mice. One copy of huTLR8 induces splenomegaly with myeloid cell expansion and memory T cell activation in Sle1.Yaa mice, but does not cause B cell expansion or an increase in germinal center cells; two copies of huTLR8 in this strain causes early mortality. Further elucidating how dysregulated TLR8 signaling accelerates disease and disrupts homeostasis is crucial to better understand the role of this TLR in autoimmunity and will facilitate more precise therapeutic targeting.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/human-tlr8-at-the-interface-between-innate-and-adaptive-immunity-in-systemic-lupus-erythematosus/