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

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. 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. PepSpot epitope mapping identified 113 LQHLYA 118 and 302 LQVVYL 307 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 113 LQHLYA 118 and 302 LQVVYL 307 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. 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.