Targeting biglycan-TLR2/TLR4 interaction with synthetic peptides mitigates inflammation and ameliorates renal function in ischemia-reperfusion injury.

Abstract

Background: Renal ischemia-reperfusion injury (IRI), characterized by enhanced inflammation, is a leading cause of acute kidney injury (AKI) with limited treatments beyond supportive dialysis. Biglycan through toll-like receptors (TLR)2/TLR4 signaling aggravates tissue damage in acute renal IRI. This study aimed to identify the TLR2/TLR4 binding sites of biglycan and test the efficacy of biglycan-derived peptides in vitro and in vivo.

Methods: Epitope mapping was conducted using PepSpot peptide arrays with overlapping 15-mer biglycan peptides. Binding of wildtype (WT) and mutant biglycan proteins and synthesized peptides to TLR2 and TLR4 was analyzed by microscale thermophoresis. Nuclear Factor (NF)-κB activation was assessed in HEK-Blue-TLR2 and HEK-Blue-TLR4 reporter cells following stimulation with biglycan variants and peptides. Primary murine and human monocyte-derived macrophages were used to evaluate inflammatory responses in vitro. Renal IRI was induced in C57BL/6 mice with intravenous peptide treatment administered prior to surgery. Renal function, immune cell infiltration, and cytokine expression were assessed using serum creatinine measurements, immunohistochemistry, and molecular analyses.

Results: PepSpot epitope mapping identified ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ as the TLR2/TLR4 binding sequences in leucine-rich repeat (LRR) 2 and LRR10, respectively. Sequence-swapped (swLRR2 and swLRR10) and point mutant (LRR2ΔF and LRR10ΔF) biglycans exhibited decreased binding to TLR2 and TLR4 than WT-biglycan. Compared with WT-biglycan, these mutants elicited reduced NF-κB activation in HEK-Blue-TLR2 and HEK-Blue-TLR4 cells, consistent with decreased TNFα, CCL2, and CXCL1 expression in primary murine macrophages. Peptides 1 and 2, containing the ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences of LRR2 and LRR10, respectively, exhibited significant binding affinity to TLR2 and TLR4 and inhibited biglycan-induced NF-κB activation, ERK phosphorylation, and TNFα, CCL2, and CXCL1 expression. These findings were corroborated in human monocyte-derived macrophages. In the renal IRI mouse model, peptide treatment mitigated immune cell infiltration, decreased Cxcl1 ,Ccl2, and Tnfα expression, and improved renal function, evidenced by lower serum creatinine.

Conclusions: The findings delineate conserved biglycan-TLR2/TLR4 recognition sequences and design of inhibitory peptides with therapeutic efficacy in vitro and in vivo. Peptides that inhibit biglycan-TLR2/TLR4 binding offer a targeted therapeutic approach for mitigating biglycan-TLR-associated sterile and pathogen-induced inflammatory diseases, with significant translational relevance for IRI.