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.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s11658-026-01019-2.

Background

Renal ischemia–reperfusion injury (IRI) results from transient restriction of blood flow, leading to renal tissue hypoperfusion, followed by reperfusion and reoxygenation, which impairs renal function. Renal IRI is characterized by inflammation, immune cell infiltration, cytokine and chemokine secretion, apoptosis, and necroptosis, culminating in tubular damage, decreased glomerular filtration rate (GFR), and elevated serum creatinine levels [1]. Renal IRI poses a significant challenge to graft survival in renal transplantation [2] and is a major cause of acute kidney injury (AKI), frequently progressing to chronic kidney disease (CKD), which remains a leading cause of mortality worldwide [3, 4]. The current management of AKI and CKD is limited to dialysis or renal transplantation, with no effective curative therapies, underscoring the need for novel therapeutic strategies.

The pathophysiology of renal IRI is critically influenced by innate immune pattern recognition receptors (PRRs), particularly toll-like receptors (TLR) 2 and TLR4, which recognize endogenous damage-associated molecular patterns (DAMPs) released from ischemic renal tissues during reperfusion. Activation of TLR2/TLR4 signaling triggers sterile inflammation, exacerbating tissue injury in acute renal IRI [5]. The expression of TLR2 and TLR4 is significantly upregulated in renal tubular epithelial cells (TECs) following IRI [6]. Experimental models, including TLR2 and TLR4 knockout mice and donor kidneys from humans with TLR4 mutations, demonstrated attenuated acute inflammatory responses and tubular damage in renal IRI [6, 7]. Therapeutic approaches targeting TLRs, such as small-molecule inhibitors [8], TLR-specific antibodies [9], and cell-penetrating peptides [10], aim to mitigate TEC injury and restore renal function in renal IRI models. Thus, inhibiting TLR2/TLR4-induced sterile inflammation is a promising strategy for attenuating acute renal inflammation and tubular damage.

Biglycan, a ubiquitous class I small leucine-rich proteoglycan (SLRP) in the extracellular matrix (ECM), comprises a 42-kDa core protein with 12 tandem leucine-rich repeats (LRRs) flanked by cysteine clusters and two covalently linked glycosaminoglycan (GAG) chains [11]. Biglycan modulates diverse biological functions by interacting with various molecules and cell surface receptors, including the regulation of cell–matrix interactions, innate immunity, and inflammatory responses [12–18]. Upon proteolytic cleavage from the ECM, soluble biglycan functions as a DAMP and ligand for TLR2 and TLR4 [19], activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducing the expression of proinflammatory cytokines and chemokines, including tumor necrosis factor α (TNFα) and chemokines including C-X-C motif chemokine (CXCL) 1 across diverse renal pathologies [5, 20–25]. Elevated serum biglycan levels correlate with the etiology and progression of acute renal IRI and CKD [5, 21], establishing soluble biglycan as a biomarker for inflammatory renal diseases [26]. Biglycan–TLR2/TLR4 signaling exacerbates pathological outcomes in diverse renal disorders, including nephritis, [21] diabetic nephropathy, [27] ischemic AKI, [5] and chronic graft dysfunction post-renal transplantation [28]. The identification of the TLR2/TLR4-binding sites of biglycan and the subsequent development of inhibitory peptides hold therapeutic potential for acute sterile inflammatory renal diseases.

Building on previous findings demonstrating the adverse effects of biglycan–TLR2/TLR4 signaling in a murine model of ischemic AKI [5], the present study aimed to identify the TLR2/TLR4-binding sequence of biglycan, design and validate novel inhibitory peptides in vitro using primary murine and human monocyte-derived macrophages, and evaluate the therapeutic efficacy of these peptides in attenuating biglycan–TLR2/TLR4 signaling and ameliorating renal IRI pathology in vivo.

Methods

PepSpot peptide array

Epitope mapping was performed using a PepSpot peptide array with SPOT-synthesized overlapping linear 15-meric biglycan peptides, following the manufacturer’s instructions (JPT Peptide Technologies GmbH, Berlin, Germany). Peptides were covalently bound by their C-termini to Whatman 50 cellulose membranes and acetylated at the N-terminus to enhance stability. Membranes were blocked with 5% weight/volume (w/v) bovine serum albumin for 2 h at 37 °C, incubated with human TLR2 and TLR4 recombinant proteins (R and D Systems, Wiesbaden, Germany), and probed with the following primary and secondary antibodies: anti-TLR2 (NB100-56058, Novus Biologicals, Colorado, USA), anti-TLR4 (NB100-56579, Novus Biologicals), sheep anti-mouse-HRP (Abcam, Cambridge, UK), and donkey anti-rabbit-HRP (GE Healthcare, Munich, Germany).

Modelling and sequence analysis

Biglycan peptide sequences binding to TLR2 and TLR4, corresponding to LRR2 and LRR10, along with class I SLRPs, decorin, asporin, and extracellular matrix protein 2, and class II SLRPs lumican and fibromodulin, were subjected to sequence analysis with Clustal Omega [29]. The accession numbers for the SLRPs used in sequence analysis were P21810 (biglycan), P07585 (decorin), P51884 (lumican), Q06828 (fibromodulin), Q9BXN1 (asporin), and O94769 (extracellular matrix protein 2). The three-dimensional (3D) structure of biglycan (Protein Data Bank entry: 2FT3) was visualized using PyMOL (version 2.5.4).

Human biglycan mutants and peptides

HEK 293 cells (American Type Culture Collection, Manassas, VA, USA; cat. no. CRL-1573) were cultured in minimum essential medium (Life Technologies, Darmstadt, Germany) supplemented with 1 g/L glucose, 1% (w/v) penicillin and streptomycin, and 10% volume/volume (v/v) fetal bovine serum (Biochrom, Berlin, Germany) at 37 °C in 5% (v/v) CO₂. Human biglycan mutants swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF were generated using pcDNA3.1hBGN (corresponding to 1–368 aa) as a template with the QuickChange II site-directed mutagenesis kit (Agilent Genomics, California, USA) according to the manufacturer’s instructions. Cells were transfected with each mutant using lipofectamine LTX and PLUS reagent (Thermo Fisher Scientific, Dreieich, Germany). Stable overexpression was selected with 0.75 mg/mL geneticin disulphate solution (Carl Roth, Karlsruhe, Germany). Primers designed for site-directed mutagenesis (Sigma-Aldrich, Munich, Germany) are listed in Supplementary Table S1. Wildtype (WT) and mutant biglycan proteins with GAG chains were purified from conditioned media of transfected cells using DEAE-Tris acryl-M packed columns (Tosoh Bioscience, Tokyo, Japan) and separated by a discontinuous sodium chloride gradient using high-performance liquid chromatography (Shimadzu Europa GmbH, Duisburg, Germany). Purified proteoglycans were treated with chondroitinase ABC lyase (Seikagaku Corporation, Tokyo, Japan) and identified by silver staining. Peptide 1, ₁₀₉DFKGLQHLYALVLVNNKIS₁₂₇ and peptide 2, ₂₉₃PSGLPDLKLLQVVYLHSNNITKV₃₁₅, containing the human biglycan–TLR2/TLR4 binding sequence ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ of LRR2 and LRR10, respectively, and control peptide ₁₆₅RIHDNRIRKVPKGVFSGLR₁₈₃, representing LRR5, which was not identified by peptide mapping as a TLR2/TLR4-interacting motif, were synthesized and supplied by Genosphere Biotechnologies, Clamart, France. To determine whether biglycan, mutants, and synthetic peptides exhibited cytotoxic effects under the experimental conditions, cell viability was evaluated using the CCK-8 assay. Treatment with WT-biglycan (20 nM), swLRR2-, swLRR10-, LRR2ΔF-, and LRR10ΔF-biglycan mutants (20 nM), as well as peptide 1 (18 µM), peptide 2 (16 µM), and the control peptide (18 µM), did not significantly affect cell viability compared with untreated control cells (ordinary one-way analysis of variance [ANOVA] followed by Dunnett’s multiple comparisons test; all P > 0.05). In contrast, treatment with 50 nM staurosporine almost completely abolished cell viability, confirming the responsiveness of the assay (Supplementary Fig. S1).

Microscale thermophoresis (MST) binding assay

MST enables the detection of quantitative binding affinities in solutions without the immobilization of either binding partner, preserving native conformations, and minimizing potential artifacts associated with surface-coupled methods. The direct binding of WT-biglycan, biglycan mutants (swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF), and peptides 1 and 2 to TLR2 and TLR4 was assessed using MST. Recombinant human TLR2 or TLR4 (R and D Systems) was labelled with the Monolith protein labeling kit RED-NHS second generation (NanoTemper Technologies, Munich, Germany) according to the manufacturer’s instructions. The concentration of labelled TLR2 or TLR4 was maintained at 100 nM. Serial 1:1 dilutions of biglycan (WT or mutants), peptide 1, peptide 2, or albumin (negative control), starting from 2 µM, were prepared in phosphate-buffered saline containing 0.05% (v/v) Tween 20 (Sigma-Aldrich). The reaction mixtures were incubated in the dark for 30 min and centrifuged at 18,000×g for 5 min. Samples were then loaded into glass capillaries. The binding affinity was measured using Monolith NT.115 and analyzed with the NT Analysis software (MOAffinity analysis, version 3.0; NanoTemper Technologies). Dissociation constants (K_d) were calculated from three independent thermophoresis measurements.

Macrophage isolation and stimulation

Primary murine macrophages were isolated from C57BL/6 J mice as described previously [30]. Human monocyte-derived macrophages were obtained from peripheral blood mononuclear cells isolated by density gradient centrifugation from the whole blood of four healthy donors, as previously described [25]. The cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum. The cells were stimulated with 20 nM WT-biglycan or biglycan mutants (swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF) in serum-free medium for specified durations. For the inhibition assays, cells were preincubated with peptides 1 (18 µM) and 2 (16 µM), and control peptide (18 µM) for 1 h prior to biglycan stimulation.

Immunoblotting

Total protein was extracted from primary murine macrophages 30 min post-stimulation, as described previously [30]. The primary antibodies used were anti-phospho-ERK (Cat. no. 9101) and anti-ERK (Cat. no. 9102) (both 1:1000; cell signaling technology, Frankfurt, Germany). The secondary antibody used was goat anti-rabbit immunoglobulin G (IgG) (H + L)-HRP conjugate (Cat. no. 1706515, 1:10,000; Bio-Rad Laboratories GmbH, Feldkirchen, Germany).

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from mouse kidneys (20 h post-IRI) or macrophages (3 h post-stimulation) was isolated using TRI Reagent (Sigma-Aldrich). Complementary DNA (cDNA) synthesis was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Darmstadt, Germany) following the manufacturer’s instructions. The resulting cDNA, together with TaqMan fast advanced master mix (Applied Biosystems) and custom FAM-MGB-labeled TaqMan gene expression assay probes, was subjected to qRT-PCR using the AbiPrism 7500 sequence detection system (Applied Biosystems). Assay probes included mouse Tnfα (Mm_00443260_g1), mouse chemokine (C–C motif) ligand 2 (Ccl2, Mm00441242_m1), mouse chemokine (C-X-C motif) ligand 1, (Cxcl1, Mm04207460_m1), mouse glyceraldehyde−3-phosphate dehydrogenase (Gapdh, Mm_ 03302249_g1), human TNFα (Hs01113624_g1), human CCL2 (Hs00234140_m1), human CXCL1 (Hs00236937_m1), and human 18S (Hs03003631_g1) (Life Technologies). Relative gene expression was quantified using the 2^−ΔΔCt method, normalized to Gapdh or 18S, and compared with that of the controls.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to quantify cytokines and chemokines in culture supernatants, 6 h post-stimulation. The following ELISA kits were used in accordance with the manufacturer’s protocol: mouse TNF-α (DY410), mouse CXCL1/KC (DY453), mouse CCL2/JE/MCP-1 (DY479), human TNF-α (DY210), human CXCL1/GRO alpha (DY275), and human CCL2/MCP-1 (DY279) (all from R and D Systems).

Renal ischemia–reperfusion injury (IRI)

All animal experiments were performed in accordance with the German Animal Protection Law and were approved by The Ethics Review Committee for Laboratory Animals of the District Government of Darmstadt, Germany (approval no: FU/1063, 16.07.2015–15.07.2020). Male C57BL/6 J mice, aged 8–12 weeks, were purchased from Charles River Laboratories (Sulzfeld, Germany). Male mice were used to minimize sex-dependent differences in tissue susceptibility, hormone-driven confounding variables, and baseline experimental standardization. A total of 18 mice were divided into four experimental groups: sham-operated control group (n = 3), sham-operated peptide group (n = 3), IRI group (n = 6), and peptides-treated IRI group (n = 6). For peptide administration, the mice were anesthetized with 2% isoflurane (Abbott, Germany) and 1 L/min oxygen. Peptides 1 (4.0 nmol) and 2 (3.0 nmol) (8 µg each/mouse) were administered intravenously via the tail vein in a total volume of 100 µL 0.9% saline (Braun, Melsungen, Germany) 30 min prior to IRI induction. The sham-operated control group received 0.9% saline. Renal IRI was induced, as previously described [30]. Briefly, a midline incision was made, followed by ligation of the left renal pedicle with sutures, cut distal to the suture, and the kidney was removed. The renal artery of the right kidney was clamped for 25 min with atraumatic microaneurysm clamps (Braun). Body temperature was maintained by placing the mice on a 37 °C heating pad, and the kidney was kept moist with sterile 0.9% saline. Following the removal of the clamp, the kidney was inspected for 1 min to ensure restoration of blood flow and return to its original color, before closing the incision. Sham-operated mice underwent identical surgical procedures, except for nephrectomy and microaneurysm clamp applications. The painkiller buprenorphine (100 μg/kg; RB Pharmaceuticals Limited, Berkshire, UK) was administered subcutaneously every 4 h. The mice were euthanized 20 h post-reperfusion. Blood was drawn for serum creatinine analyses. The kidney was excised and cut into two parts, one of which was snap-frozen for expression analysis of inflammatory markers, while the other was fixed with phosphate-buffered saline containing 4% (v/v) paraformaldehyde and 0.1% Tween 20 (Sigma-Aldrich) for immunohistochemical analysis.

Determination of renal function and histopathology

Serum creatinine levels were determined using a colorimetric microplate assay (Cayman Chemical, Michigan, USA). Tissue sections were prepared as previously described [30]. The primary antibodies used for staining were anti-F4/80 (1:250; MCA497, Bio-Rad AbD Serotec GmbH, Neuried, Germany) and anti-7/4 (1:250; ab53457, Abcam). Neutrophils were quantified by calculating the reciprocal of the staining intensity, expressed in arbitrary units (a.u.). The number of macrophages was estimated per high-power field (HPF; 200 ×, with a minimum of three fields counted; Soft Imaging System, Olympus, Germany). Histological examinations were performed by two observers who were blinded to the conditions.

Statistical analysis

Data are expressed as the mean ± standard deviation. An unpaired, 2-tailed, Student’s t test was used to evaluate the differences between groups. Comparisons among multiple groups were performed using ordinary one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test, with the untreated control or WT-biglycan groups designated as the reference group. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results

Biglycan LRR2 and LRR10 harbor TLR2/TLR4 binding sites

Having deciphered the biglycan–TLR2/TLR4 signaling pathway and its associated immunological significance [19], this study aimed to identify the binding sites in biglycan that mediate the interactions with TLR2 and TLR4. Structurally, the biglycan core protein consists of a signal peptide (aa 1–16) and propeptide (aa 17–37), followed by 12 tandem LRRs (aa 82–368) that are flanked by cystine clusters (Fig. 1a). For epitope mapping, human biglycan sequences were divided into partially overlapping peptides representing the signal peptide, pro-peptide, cys-rich residues, and LRR 1–12 (Supplementary Fig. S2). Epitope mapping revealed that peptide sequences corresponding to biglycan LRR2 and LRR10 bound to TLR2 (Fig. 1b and Supplementary Fig. S2), whereas TLR4 showed strong signals with LRR2, LRR3, LRR4, and LRR10 and modest signals with LRR6, LRR8, LRR11, and LRR12 (Fig. 1c and Supplementary Fig. S2). LRR2 and LRR10 were identified as common regions with strong binding affinities for both TLR2 and TLR4. Sequence analysis of the binding peptides from LRR2 and LRR10 (Fig. 1d) showed ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇, (located near the N-terminus in LRR2 and C-terminus in LRR10, respectively) as the sequences likely responsible for the interactions, i.e., given their similarity, which can be described by the consensus: L-Q-X₁-V/L-Y-X_2, where X₁ and X₂ represent variable residues. Structural visualization revealed that these peptides lie mostly on one face of the biglycan dimer, consistent with their potential role in mediating receptor interactions (Fig. 1e). These putative binding sequences informed site-directed mutagenesis of biglycan and the synthesis of peptides 1 and 2 used in subsequent analyses.

Fig. 1.

Binding sequence of biglycan to TLR2 and TLR4 located in LRR2 and LRR10. (a) Basic structure of biglycan characterized by two glycosaminoglycan (GAG) chains and a protein core with 12 leucine-rich repeats (LRRs) (upper left panel). Protein sequence of biglycan depicting the signal peptide, propeptide, and LRR1–LRR12 (upper right panel). LRR2 and LRR10 (upper left panel) and sequences with biglycan–TLR2/TLR4 binding sites (upper right panel) are highlighted in blue and green, respectively. (b–c) Epitope mapping of biglycan using PepSpot peptide array screening for (b) TLR2 and (c) TLR4. (d) Clustal alignment of the identified 15-mer biglycan peptides, demonstrating their binding affinity to TLR2 and TLR4. Similar amino acid residues are denoted by an asterisk, whereas those identical in all but one are indicated by a colon. (e) The position of the putative binding sequences in LRR2 and LRR10 are shown as spheres on a cartoon representation of dimeric bovine biglycan (Protein Data Bank entry: 2ft3)

Mutations at LRR2/LRR10 inhibit biglycan–TLR2/TLR4 interactions

To further investigate ligand–receptor binding specificity, the identified sequences at LRR2 and LRR10 were swapped with those of adjacent LRRs. Accordingly, ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ were mutated to ₁₃₇LRKLQK₁₄₂ of LRR3 (swLRR2) and ₂₇₉LRELHL₂₈₄ of LRR9 (swLRR10), respectively (Fig. 2a). Their binding affinities to fluorescence-conjugated TLR2 and TLR4 were determined by MST. WT-biglycan and mutants swLRR2 and swLRR10 bound to TLR2 with K_d values of 39.2 ± 7.6, 453.4 ± 29.6, and 287.3 ± 17.4 nM, respectively (Fig. 2b), and to TLR4 with K_d values of 23.5 ± 4.2, 1022.1 ± 103.4, and 302.2 ± 26.8 nM, respectively (Fig. 2c).

Fig. 2.

Swapping and point mutation of biglycan sequences decreases binding efficacy to TLR2 and TLR4. (a) Sequences ₁₁₃LQHLYA₁₁₈ in leucine-rich repeats (LRR)2 and ₃₀₂LQVVYL₃₀₇ in LRR10 in wildtype (WT)-biglycan were swapped with ₁₃₇LRKLQK₁₄₂ of LRR3 (swLRR2-biglycan) and ₂₇₉LRELHL₂₈₄ of LRR9 (swLRR10-biglycan), respectively. (b) Microscale thermophoresis binding analysis of fluorescence-labeled TLR2 with WT-biglycan, swLRR2-biglycan, and swLRR10-biglycan. (c) Microscale thermophoresis binding analysis of fluorescence-labeled TLR4 with WT-biglycan, swLRR2-biglycan, and swLRR10-biglycan. (d) The polar amino acids glutamine (Q) and tyrosine (Y) in sequences ₁₁₃LQHLYA₁₁₈ in LRR2 and ₃₀₂LQVVYL₃₀₇ in LRR10 in WT-biglycan were substituted with nonpolar isoleucine (I) and phenylalanine (F) residues to generate ₁₁₃LIHLFA₁₁₈ (LRR2ΔF-biglycan) and ₃₀₂LIVVFL₃₀₇ (LRR10ΔF-biglycan), respectively. (e) Microscale thermophoresis binding analysis of fluorescence-labeled TLR2 with WT-biglycan, LRR2ΔF-biglycan, and LRR10ΔF-biglycan. (f) Microscale thermophoresis binding analysis of fluorescence-labeled TLR4 with WT-biglycan, LRR2ΔF, and LRR10ΔF. The dissociation constant (K)_(d)) was determined from three independent thermophoresis measurements

The polar, noncharged nature of glutamine and tyrosine, along with the surface accessibility of QH-Y in LRR2 and Q in LRR10, suggests that these residues may facilitate interactions with TLR2/TLR4. Accordingly, the exposed polar amino acids in ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ were substituted with nonpolar isoleucine and phenylalanine residues to generate ₁₁₃LIHLFA₁₁₈ (LRR2ΔF) and ₃₀₂LIVVFL₃₀₇ (LRR10ΔF), respectively (Fig. 2d). WT-biglycan and the mutants LRR2ΔF and LRR10ΔF bound to TLR2 with K_d values of 36.5 ± 5.3, 174.4 ± 19.5, and 235.1 ± 27.6 nM, respectively (Fig. 2e), and to TLR4 with affinities of 25.7 ± 3.9, 187.5 ± 22.4, and 721.4 ± 108.2 nM, respectively (Fig. 2f).

Thus, altering the LRR2 and LRR10 sequences or specifically mutating glutamine and tyrosine residues significantly decreased the binding affinity of biglycan to TLR2 and, to a greater extent, to TLR4, supporting the relevance of these sequences in mediating biglycan–TLR2/TLR4 interactions.

Biglycan LRR2 and LRR10 are critical for TLR2/TLR4-mediated proinflammatory signaling

Biglycan, a ligand of TLR2 and TLR4, triggers proinflammatory signaling [19]. The effect of altering the ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences on biglycan–TLR2/TLR4-mediated NF-κB activation was analyzed using the SEAP assay. Biglycan mutants swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF induced lower SEAP activity than that induced by WT-biglycan, both in HEK-Blue-TLR2 cells (Supplementary Fig. S3A, C), and HEK-Blue-TLR4 cells (Supplementary Fig. S3B, D).

The specificity of the ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences in biglycan–induced proinflammatory signaling was further analyzed in primary murine macrophages. The phosphorylation of ERK was significantly lower post-stimulation with biglycan mutants swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF compared with that of WT-biglycan (Fig. 3a, b). NF-κB and Erk activation in innate immune cells, such as macrophages, promotes the expression of proinflammatory cytokines and chemokines. The mRNA and protein expression levels of the chemokine CXCL1(Fig. 3c, d) and CCL2 (Fig. 3e, f), and the cytokine TNFα(Fig. 3g, h) were substantially lower post-stimulation with biglycan mutants swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF, compared with that of WT-biglycan. Thus, altering the ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences of biglycan significantly inhibited its ability to activate TLR2 and TLR4, resulting in mitigated proinflammatory effects. These findings indicate the potential for developing inhibitory peptides that target biglycan–TLR2/TLR4 interactions.

Fig. 3.

Biglycan LRR2 and LRR10 sequences are crucial for TLR2/TLR4-mediated proinflammatory signaling—evidence based on biglycan–swapped and point mutants. (a, b) Phosphorylation of (a) ERK (b) quantification, induced by wildtype (WT)-biglycan and biglycan mutants swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF. (c–h) Expression of CXCL1 (c) mRNA and (d) protein, CCL2 (e) mRNA and (f) protein, and TNFα (g) mRNA and (h) protein induced by WT-biglycan and the biglycan mutants. Primary C57BL/6 macrophages were stimulated with 20 nM WT-biglycan, swLRR2, swLRR10, LRR2ΔF, and LRR10ΔF, 30 min for immunoblotting, 3 h for quantitative polymerase chain reaction (qPCR), and 6 h for ELISA. mRNA levels in cells were quantified using qPCR and protein levels in the culture supernatant were measured using enzyme-linked immunoassay (ELISA). qPCR data are presented as fold induction normalized to Gapdh. Data represent mean ± standard deviation (SD) from a minimum of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Similarity of biglycan binding sequence and peptide design for mimicking biglycan–TLR2/TLR4 interactions

The similarity and distribution of the biglycan ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences and flanking regions were analyzed in structurally similar SLRPs to inform the development of small peptides capable of inhibiting the biglycan–TLR2/4 interaction. Sequence analysis of biglycan, class I SLRPs (decorin, asporin, and extracellular matrix protein 2), and two principal class II SLRPs (lumican and fibromodulin) revealed that the ₁₁₃LQHLYA₁₁₈ sequence in biglycan LRR2 was not very similar to the other SLRPs analyzed (Fig. 4a). However, the ₃₀₂LQVVYL₃₀₇ sequence in LRR10 displayed greater similarity to sequences in decorin, asporin, lumican, and fibromodulin (Fig. 4b). Accordingly, two longer (19–22-mer) peptides were designed harboring the binding sequences to target the biglycan–TLR2/TLR4 interaction. Peptide 1, ₁₀₉DFKGLQHLYALVLVNNKIS₁₂₇, and peptide 2, ₂₉₃PSGLPDLKLLQVVYLHSNNITKV₃₁₅, corresponding to the sequences in biglycan, contained LQHLYA of LRR2, and LQVVYL of LRR10 (Fig. 4c). The binding affinities of these peptides for TLR2 and TLR4 were analyzed using MST. Peptide 1 bound to TLR2 and TLR4 with K_d values of 37.8 ± 23.4 and 28.1 ± 26.6 nM, respectively (Fig. 4d, e), and peptide 2 with K_d values of 42.0 ± 24.2 and 23.4 ± 13.5 nM, respectively (Fig. 4d, e). Albumin (10 nM) served as a control and showed no association with TLR2 or TLR4 (Fig. 4f, g). Thus, the peptides demonstrated significant binding affinities to TLR2 and TLR4 in the nanomolar range, albeit with somewhat lower affinities than those of WT-biglycan. AlphaFold-Multimer analyses were performed to address the possibility of peptide self-association. The predicted interface confidence scores (ipTM) were 0.38 for peptide 1 homodimers (Supplementary Fig. S4A), 0.43 for peptide 2 homodimers (Supplementary Fig. S4B), and 0.55 for the peptide 1–peptide 2 heterodimer (Supplementary Fig. S4C). The results do not indicate the formation of stable homo- or heterodimeric peptide complexes (Supplementary Table S2). In silico prediction using the PlifePred platform estimated blood half-lives of approximately 6.0 min for peptide 1 and 11.5 min for peptide 2 (Supplementary Table S3).

Fig. 4.

Peptides containing biglycan LRR2 and LRR10 sequences exhibit binding affinity for TLR2 and TLR4. (a, b) Clustal alignment illustrating sequence similarity among class I small leucine-rich proteoglycans (SLRPs) biglycan, decorin, asporin, and extracellular matrix protein 2, and class II SLRPs lumican and fibromodulin at (a) leucine-rich repeat (LRR2), and (b) LRR10. Identical amino acid residues are denoted with an asterisk, while those found in all but one are indicated by a colon, and similar residues are designated by a period. (c) Schematic representation of peptides 1 and 2 containing the sequences LQHLYA of LRR2 and LQVVYL of LRR10, respectively. (d–e) Microscale thermophoresis binding analysis of biglycan peptide 1 and 2 with fluorescence-labeled (d) TLR2 and (e) TLR4. The dissociation constant (K_d) was calculated from three independent thermophoresis measurements. (f, g) Microscale thermophoresis binding analysis of albumin (10 nM) with fluorescence-labeled (f) TLR2 and (g) TLR4, which served as a negative control. Fnorm [‰] indicates normalized fluorescence per mL

Peptides mitigate biglycan–TLR2/TLR4-mediated proinflammatory signaling in vitro

Having established the binding affinity of peptides 1 and 2 to TLR2 and TLR4, their efficacy in inhibiting biglycan–induced proinflammatory signaling was further evaluated in vitro. Preincubation with peptides 1 and 2 significantly inhibited WT-biglycan–mediated NF-κB activation-induced SEAP activity in HEK-Blue-TLR2 cells (Supplementary Fig. S5A) and HEK-Blue-TLR4 cells (Supplementary Fig. S5B). In primary murine macrophages, preincubation with peptides 1 and 2 significantly inhibited biglycan–induced ERK phosphorylation (Fig. 5a, b), and corresponding mRNA and protein expression of CXCL1 (Fig. 5c, d), CCL2 (Fig. 5e, f), and TNFα(Fig. 5g, h). Corresponding results with the control peptide are shown in Supplementary Fig. S6. These findings indicate the potential of peptides 1 and 2 to inhibit biglycan–TLR2/TLR4-mediated proinflammatory signaling, with therapeutic implications for sterile inflammation-associated pathologies.

Fig. 5.

Peptides containing biglycan LRR2 and LRR10 sequences inhibit biglycan–TLR2/4-induced proinflammatory signaling in macrophages. (a–b) Phosphorylation of (a) ERK (b) quantification, following incubation with peptides 1 and 2 and stimulation with biglycan. (c–h) Expression of CXCL1 (c) mRNA and (d) protein, CCL2 (e) mRNA and (f) protein, and TNFα (g) mRNA and (h) protein following incubation with peptides 1 and 2 and stimulation with biglycan. mRNA levels in cells were quantified using quantitative polymerase chain reaction (qPCR), and protein levels in the culture supernatants were measured by enzyme-linked immunoassay (ELISA). qPCR data are presented as fold induction normalized to Gapdh. Primary C57BL/6 macrophages were incubated with peptides 1 (18 µM) and 2 (16 µM) for 1 h prior to biglycan stimulation (20 nM), 30 min for immunoblotting, 3 h for qPCR, and 6 h for ELISA. Data are presented as the mean ± standard deviation (SD) from a minimum of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Peptide-treatment attenuates inflammation and improves renal function in IRI in vivo

The efficacy of peptide treatment in inhibiting TLR2/TLR4 signaling and renal damage was analyzed in vivo, given that soluble biglycan aggravates disease outcomes in a model of renal IRI [5]. The peptides exhibited nonimmunogenicity in vivo, as indicated by the lack of induction of inflammatory markers in the sham-operated peptide group compared with the sham-operated control group (Fig. 6). Immunostaining for neutrophils (7/4, Fig. 6a, b) and macrophages (F4/80, Fig. 6c, d) revealed significant inhibition of these immune cell infiltration in renal sections of peptides-treated IRI group compared with that of IRI group, which showed enhanced infiltration compared with sham-operated controls 20 h-post surgery. Correspondingly, the expression of Cxcl1 (Fig. 6e), and Ccl2 (Fig. 6f) was substantially inhibited in the renal tissue of peptide-treated IRI group compared with that of the IRI group, with enhanced expression compared with sham-operated controls. Accordingly, the expression of Tnfα (Fig. 6g), an early response proinflammatory cytokine, and serum creatinine levels (Fig. 6h), an indicator of renal function, were significantly decreased in the peptide-treated IRI group compared with that in the IRI group, with enhanced expression compared with sham-operated controls, indicating that peptides may have potential therapeutic applications in mitigating renal damage and rescuing renal function in IRI.

Fig. 6.

Peptides harboring LRR2 and LRR10 sequences attenuate inflammation improving renal function in murine ischemia–reperfusion injury (IRI). a–d Immunohistochemical staining for (a) neutrophils (7/4+, brown) (b) quantification, and (c) macrophages (F4/80+, brown) (dE) quantification in renal sections of IRI and sham-operated groups, with and without treatment with peptides 1 and 2. Scale bar = 50 µm. e–g The mRNA expression of (e) Cxcl1, (f) Ccl2, and (g) Tnfα in renal tissue of IRI and sham-operated groups, with or without treatment with peptides 1 and 2. Results are expressed as fold induction normalized to Gapdh, as determined by quantitative polymerase chain reaction (qPCR). (h) Serum creatinine levels in IRI and sham-operated groups, with and without treatment with peptides 1 and 2, measured by colorimetric microplate assay. The peptide-treated groups were intravenously administered peptides 1 (4.0 nmol) and 2 (3.0 nmol) (8 µg each) 30 min prior to surgery. Samples were collected 20 h pos-tsurgery. Data are presented as mean ± standard deviation (SD). n = 6 for the IRI mouse models and n = 3 for sham-operated mice with and without peptide treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

The results suggest the translational potential of peptides 1 and 2 in inhibiting the proinflammatory effects of biglycan–TLR2/TLR4 signaling in renal IRI, resulting in improved renal function and disease phenotype.

Translational potential of peptides in inhibiting biglycan–induced inflammation in human monocyte-derived macrophages

Having established the efficacy of peptides 1 and 2 in inhibiting biglycan–TLR2/TLR4 signaling both in vitro and in vivo, their translational potential in suppressing biglycan–TLR2/TLR4-induced inflammation was evaluated in human monocyte-derived macrophages. Preincubation with peptides 1 and 2 significantly inhibited biglycan–induced mRNA and protein expression of CXCL1 (Fig. 7a, b), CCL2 (Fig. 7c, d), and TNFα (Fig. 7e, f) in human monocyte-derived macrophages.

Fig. 7.

Peptides harboring LRR2 and LRR10 sequences inhibit biglycan–TLR2/4-induced proinflammatory signaling in human monocyte-derived macrophages. (a–f) Expression of CXCL1 (a) mRNA and (b) protein, CCL2 (c) mRNA and (d) protein, and TNFα (e) mRNA and (f) protein following incubation with peptides 1 and 2 and stimulation with biglycan. The mRNA levels in cells were quantified using quantitative polymerase chain reaction (qPCR) and protein levels in culture supernatants were measured by enzyme-linked immunoassay (ELISA). qPCR data are presented as fold induction normalized to18S. Primary human monocyte-derived macrophages were incubated with peptides 1 (18 µM) and 2 (16 µM) for 1 h prior to biglycan stimulation (20 nM), 3 h for qPCR and 6 h for ELISA. Data are presented as mean ± standard deviation (SD) from n = 4 samples. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

The consistent inhibition of biglycan–TLR signaling by inhibitory peptides in both mouse models and human macrophages suggests a conserved mechanism of action and broad applicability, reinforcing their potential for clinical translation in mitigating biglycan–induced inflammation in various diseases.

Discussion

Having deciphered biglycan as a DAMP that acts via TLR2/TLR4 signaling with pathological implications in sterile renal inflammation [5], this study identified the TLR2/TLR4 binding sequences of biglycan as ₁₁₃LQHLYA₁₁₈ in LRR2 and ₃₀₂LQVVYL₃₀₇ in LRR10. Modifying these sequences either through sequence swapping or polar residue substitution significantly inhibited biglycan–TLR2/TLR4 interactions and subsequent proinflammatory signaling in vitro. Peptides 1 and 2, containing the “related” ₁₁₃LQHLYA₁₁₈ and ₃₀₂LQVVYL₃₀₇ sequences from LRR2 and LRR10, respectively, effectively inhibited biglycan–TLR2/TLR4 binding and proinflammatory signaling in murine and human monocyte-derived macrophages. Additionally, administration of these peptides mitigated infiltration and inflammation, leading to improved renal function in a murine model of renal IRI (Fig. 8).

Fig. 8.

Inhibitory peptides mitigate biglycan–induced TLR2/TLR4 signaling and acute inflammation, both in vitro and in vivo. Peptides containing LRR2 and LRR10 sequences inhibit biglycan–TLR2/TLR4-induced inflammation, as evidenced by reduced NF-κB and ERK activation and decreased CXCL1, CCL2, and TNFα expression in murine primary C57BL/6 and human monocyte-derived macrophages in vitro. In a renal IRI model, administration of peptides 1 and 2 mitigated infiltration, including neutrophils and macrophages, in the ischemic renal tissue and improved renal function with lower serum creatinine levels. These findings suggest potential therapeutic applications of inhibitory peptides in alleviating the pathology of biglycan–induced inflammatory diseases. The scheme was assembled in Adobe Illustrator using graphical elements adapted from BioRender

ECM-derived soluble biglycan has been identified as a biomarker for inflammatory renal diseases, highlighting its function as a DAMP released during tissue injury in renal pathologies [26, 27, 31]. Our previous research elucidated ECM-derived soluble biglycan as a signaling molecule and proinflammatory endogenous ligand of TLR2/TLR4/CD14, activating the ERK/NF-κB pathways, inducing TNFα and CXCL13 expression, thereby mediating sterile inflammation in diseases such as renal IRI and nephritis [5, 19, 21, 24]. The de novo expression of circulating biglycan augments its accumulation in the kidneys, triggering an inflammatory response through the MyD88/TRIF pathway, resulting in leukocyte infiltration, and elevated levels of chemoattractants CXCL1 , CXCL2, CCL2, and CCL5 [20]. The biglycan/TLR/TRIF/MyD88-signaling pathway promotes the recruitment of Th1 and Th17 cells and contributes to renal fibrosis [23]. As a mineralocorticoid receptor target, the biglycan/TLR4/NF-κB/CCL3 cascade is implicated in aldosterone/salt-induced glomerular injury and macrophage infiltration [32]. The interaction of biglycan with TLR2/TLR4 and purinergic P2X(4)/P2X(7) receptors enhances receptor cooperativity, activating NLR family pyrin domain-containing 3/apoptosis-associated speck-like protein containing caspase activation and recruitment domain (NLRP3/ASC) inflammasome and caspase−1, leading to the release of mature IL-1β [33]. These intricate biglycan–induced proinflammatory signaling cascades implicate biglycan in the pathogenesis of renal disease. The identification of biglycan as an endogenous ligand of TLR2 and TLR4 is a common feature of all biglycan–mediated signaling pathways. Given that TLRs and PRRs often recognize DAMPs through evolutionarily conserved sequences, this study aimed to identify TLR2/TLR4 binding sites in biglycan, revealing ₁₁₃LQHLYA₁₁₈ in LRR2 and ₃₀₂LQVVYL₃₀₇ in LRR10 as TLR2/TLR4 binding sites. Ligand-receptor binding specificity was confirmed using biglycan swap and amino acid substitution mutants, which significantly reduced the binding affinity for TLR2/TLR4 and proinflammatory signaling. These findings suggest that targeting biglycan–TLR interactions may serve as a potential therapeutic strategy for mitigating biglycan–TLR signaling in sterile inflammation-associated diseases.

TLRs are potential biomarkers for renal diseases [34]. Increased expression and activation of TLRs are associated with the etiology and progression of various renal pathologies, including ischemic kidney damage, AKI, end-stage renal disease, nephritis, renal transplant rejection, and CKD [35, 36]. In renal IRI, TLR2 and TLR4 are significantly upregulated in TECs and play critical roles in pathogenesis, with the single deletion of either TLR2 or TLR4 conferring protection comparable to that of double genetic deletion [6]. TLRs are integral to the development and progression of renal diseases, making them potential therapeutic targets [36, 37]. TJ-M2010-2, a small-molecule inhibitor of TLR/MyD88 signaling, ameliorates renal IRI-induced tubular interstitial fibrosis by suppressing transforming growth factor-beta 1-induced epithelial–mesenchymal transition in renal TECs [8]. OPN301, a mouse anti-human TLR2 antibody, has proven effective against IRI in a murine kidney transplantation model [9]. Peptides designed to inhibit TLR signaling pathways have been developed as therapeutic agents for inflammatory diseases [38, 39]. TIP1, a novel cell-penetrating peptide derived from the TIR domain-containing adapter protein, effectively inhibits multiple TLR signaling pathways and has shown promising results in rodent models of arthritis and sepsis [40]. The decoy peptide 2R9 disrupts TIRAP recruitment to TLRs, thereby providing protection against influenza in murine models [41]. TLR inhibitory peptide 1, a multiple-TLR-blocking peptide, efficiently attenuates elevated plasma creatinine levels, tubular injury, inflammatory cytokines, apoptosis, and oxidative stress in postischemic kidneys [10]. Despite these advances, the broad inhibition of TLR signaling limits translational clinical outcomes because of its role in innate immunity, tissue repair, and regeneration. An alternative approach involves the use of peptides to inhibit TLR binding to endogenous DAMPs, offering a promising strategy for mitigating sterile inflammation and the acute proinflammatory phase of inflammatory diseases. In this study, peptides were synthesized following the identification of the TLR2/TLR4 binding domain of biglycan. These peptides demonstrated high binding affinity for TLR2 and TLR4 and significantly inhibited the biglycan–induced proinflammatory response in primary murine and human monocyte-derived macrophages. The consistent inhibition of the biglycan–TLR2/TLR4-induced acute sterile proinflammatory response by peptide treatment across both mouse and human macrophages underscores the targeting of a conserved mechanism and suggests the potential for broad translational applications.

Renal IRI is a major cause of AKI in renal pathologies, with effective treatment limited to supportive dialysis. Hence, IRI substantially contributes to increased patient morbidity and mortality, lower quality of life, and reduced life expectancy [42, 43]. The involvement of TLR and DAMP signaling is critical to the pathophysiology, progression, and outcome of renal IRI, particularly during the acute proinflammatory phase [35]. Following renal tissue injury, the recognition of soluble DAMPs, including biglycan, by TLR2 and TLR4 triggers a signaling cascade that exacerbates the adverse outcomes associated with renal IRI [5]. Several therapeutic targets, including cell-penetrating peptides, inhibit TLR2 and TLR4 signaling, resulting in favorable outcomes in renal IRI [8–10]. Although TLR2 and TLR4 signaling contribute to adverse pathologies during the acute phase of IRI, these pathways are essential for recovery, disease resolution, and tubular repair [44]. Pharmacological inhibition of extracellular endogenous ligands that induce acute exaggerated sterile inflammation is considered safer and more effective for mitigating tissue damage in IRI than directly targeting TLRs. Therapeutic interventions targeting TLR2/TLR4 ligands, such as anti-HMGB1 antibodies [45] and anti-histone IgG [46], indicate promising results in ameliorating renal tissue damage in IRI. We previously demonstrated that biglycan–triggered TLR2/TLR4 signaling exacerbates ischemic AKI pathophysiology, characterized by increased infiltration and secretion of proinflammatory cytokines and chemokines, resulting in compromised renal function [5]. The proinflammatory effects of biglycan in acute renal IRI were significantly attenuated in Tlr2^−/− and Tlr4^−/− mice [5], suggesting that targeting soluble biglycan may represent a promising therapeutic strategy to alleviate renal damage. Accordingly, the present study evaluated the efficacy of biglycan–inhibitory peptides in an experimental IRI model, a well-established and effective preclinical mouse model that closely simulates the pathophysiology of IRI in vivo [47]. This study highlights the significant efficacy of inhibitory peptides in inhibiting biglycan–induced infiltration and the expression of chemokines Cxcl1 and Ccl2 in renal tissue. The intervention resulted in mitigated Tnfα expression and improved renal function, as evidenced by lower serum creatinine levels 20 h post-IRI, an acute phase characterized by maximal immune cell infiltration. The lack of effective treatments for renal IRI highlights the critical need for novel therapeutic interventions. Additionally, IRI extends beyond renal complications, manifesting as a frequent and inevitable consequence of sepsis, cardiac surgery, and trauma [48]. Considering the significant involvement of biglycan–TLR2/TLR4 signaling in the pathophysiology of sepsis and other inflammatory diseases, including renal IRI, the development of biglycan–inhibitory peptides represents a promising therapeutic strategy. These peptides can effectively mitigate excessive acute proinflammatory responses and tissue damage while maintaining the necessary long-term TLR2/TLR4 signaling for disease resolution and tissue repair.

In addition to its role as an acute DAMP, biglycan is a key component of the ECM that contributes to collagen fibril organization and structural integrity in connective tissues, including bone and tendons [49, 50]. Systemic blockade of biglycan signaling affects ECM maintenance and skeletal homeostasis, potentially influencing bone density and tissue mechanical properties, warranting pharmacological consideration. Importantly, the peptide-therapeutic intervention described here is intended to selectively block the acute proinflammatory soluble biglycan DAMP–TLR2/4 pathway during tissue injury rather than abolish all structural or receptor-mediated functions of biglycan, including off-target skeletal toxicities seen in conventional knockouts arising from chronic, lifetime depletion. In this study, therapeutic intervention relied on an acute, transient pretreatment strategy (administered as a single injection 30 min prior to IRI), with peptides featuring rapid single-digit minute clearance kinetics, which selectively neutralize the acute, pathologically shed soluble biglycan DAMP-mediated TLR2/4 signaling in the injured kidney parenchyma, without depleting or interfering with the deeply sequestered structural matrix of bones and tendons. Nonetheless, further in vivo studies are required to evaluate the chronic safety, tissue-specific effects, and long-term consequences of skeletal density parameters to fully confirm the systemic structural safety of this approach. Although the present findings highlight the therapeutic potential of disrupting the biglycan–TLR2/4 interaction, they are restricted to the proof-of-concept of the short-term acute effects of biglycan–inhibitory peptides. Their long-term effects on receptor dynamics, tissue regeneration, and repair in renal IRI and diverse clinically relevant animal renal disease models, including renal transplantation, are limited by their in vivo stability. Future research should focus on chemical modifications and specialized delivery systems to improve bioavailability and tissue targeting in vivo. Additionally, while the specificity of LQHLYA at LRR2 presents the potential for targeting the biglycan–TLR2/TLR4 interaction, the more highly conserved sequence at LRR10 may necessitate further modifications to enhance selectivity. Comparative studies of peptides 1 and 2 with existing antiinflammatory drugs could provide valuable insights into their potential benefits or synergistic effects. The translation of short peptides targeting the biglycan–TLR2/4 interaction into clinical applications presents several challenges, including potential limitations related to peptide stability in vivo, susceptibility to proteolytic degradation, bioavailability, tissue-specific delivery, pharmacokinetics, potential off-target effects, immunogenicity, and differences between experimental animal models and human diseases that affect therapeutic efficacy and safety. Thus, while the current findings support the therapeutic potential of targeting the biglycan–TLR2/4 axis, additional studies are required to optimize peptide design and evaluate their long-term pharmacological and toxicological profiles in relevant preclinical models. Long-term investigations evaluating the safety and efficacy of these peptides in human cell cultures could aid in predicting their applicability to chronic inflammatory conditions.

Conclusions

This study identified the binding sequence of biglycan with TLR2/TLR4 and demonstrated the efficacy of the peptides in inhibiting biglycan–induced TLR2/TLR4 signaling and acute inflammation, both in vitro and in vivo (Fig. 8). By designing peptides that specifically target endogenous DAMPS, such as the biglycan–TLR2/TLR4 interaction, rather than broadly inhibiting TLR2/TLR4 signaling, it is possible to preserve the beneficial renoprotective effects of TLR-mediated tissue repair. This method offers a promising strategy for alleviating tissue damage caused by acute inflammation in various clinical pathologies associated with sterile inflammation. Peptides that inhibit biglycan hold potential for translational applications in clinical contexts where IRI is anticipated, such as during surgical procedures, including renal transplantation.

Abbreviations

AKI

Acute kidney injury

CCL2

Chemokine (C–C motif) ligand 2

CKD

Chronic kidney disease

CXCL1

C-X-C motif chemokine 1

DAMP

Damage-associated molecular pattern

ECM

Extracellular matrix

ELISA

Enzyme-linked immunosorbent assay

GAG

Glycosaminoglycan

GAPDH

Glyceraldehyde−3-phosphate dehydrogenase

IRI

Renal ischemia–reperfusion injury

K_d

Dissociation constant

LRR

Leucine-rich repeat

MST

Microscale thermophoresis

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

PRR

Pattern recognition receptor

qRT

Quantitative real-time

SEAP

Secreted embryonic alkaline phosphatase

SLRP

Small leucine-rich proteoglycan

TEC

Tubular epithelial cell

TLR

Toll-like receptor

TNF

Tumor necrosis factor

WT

Wild-type

Author contributions

All authors conceptualized the study. R.M. and L.T.H. performed in vitro and in vivo experiments, analyzed the data, and interpreted the results. L.T.H. and J.Z.-B. performed microscale thermophoresis analysis. R.M., L.T.H., D.C.B., and A.J.D. performed sequence analysis and designed biglycan mutants. L.S., M.W., and R.B. secured the funding and resources. R.M., L.T.H., and L.S. drafted and revised the manuscript. S.N. and R.V. performed the histochemical analysis. R.M. and S.N. prepared the figures. R.M., L.T.H., J.Z.-B., R.V., D.C.B., A.J.D., M.W., S.N., R.B., and L.S. read, revised, and approved the final manuscript.

Funding

This project is supported by grants from the German Research Council SFB 1177, 259130777, Project F11 (to L.S.); the Cardio-Pulmonary Institute (CPI), EXC 2026, Project ID: 390649896 (to L.S. and M.W.), the German Center for Lung Research 82 DZL 005A1 (to M.W.), and the Polish National Science Center, Project ID: UMO-2022/47/B/NZ7/00051 (to R.B.).

Data availability

This study did not involve large datasets or custom-designed algorithms. All data generated or analyzed in this study are included in this published article. Detailed information on any animal models or analytical techniques is available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were performed in accordance with the German Animal Protection Law and were approved by The Ethics Review Committee for Laboratory Animals of the District Government of Darmstadt, Germany (Approval No: FU/1063, 16.07.2015–15.07.2020), the local ethics committees act on International Council for Laboratory Animal Science (ICLAS).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.