Effects of burosumab on macrophage polarization and TRPV1 expression in children with XLH

Abstract

Purpose

X-linked hypophosphatemia (XLH) is a rare genetic disorder caused by PHEX mutations, leading to hypophosphatemia and impaired bone mineralization. Burosumab, a monoclonal antibody targeting FGF23, improves phosphate levels and bone health. Although burosumab improves musculoskeletal pain, it remains unclear whether this benefit results solely from restoration of phosphate homeostasis or also involves immunomodulatory mechanisms. Given the role of FGF23 in modulating macrophage function and the involvement of TRPV1 – expressed by macrophages – in pain transmission, we investigated the effects of burosumab on macrophage polarization and TRPV1 expression in children with XLH.

Methods

Macrophages were isolated from untreated XLH patients (n = 4), burosumab-treated XLH patients (n = 8), and healthy donors (n = 5). Western blot was used to assess M1 (CCR7, CD86, iNOS) and M2 (CD206, p-STAT6) markers, as well as TRPV1 expression. To evaluate burosumab’s direct effects, macrophages from healthy donors were stimulated with LPS and treated with 1.5 or 3 μg/mL burosumab, followed by the same analyses.

Results

Untreated XLH macrophages predominantly exhibited an M1 phenotype with higher TRPV1 protein levels compared with healthy controls. In contrast, macrophages from burosumab-treated patients showed a tendency to acquire an M2-like phenotype and reduced TRPV1 expression. Similar effects were observed in vitro, with LPS-stimulated healthy macrophages shifting toward the M2 phenotype and showing decreased TRPV1 after burosumab exposure.

Conclusion

In untreated XLH patients, macrophages exhibit a pro-inflammatory M1 phenotype with upregulated TRPV1 expression. Burosumab reverses this profile, driving M2 polarization and reducing TRPV1 levels, thereby suggesting that it exerts therapeutic effects beyond phosphate homeostasis through immunomodulation.

Plain summary

X-linked hypophosphatemia (XLH) causes rickets and chronic pain. We found that immune cells from untreated patients show a pro-inflammatory profile. Treatment with burosumab shifted these immune cells toward an anti-inflammatory state and reduced the expression of TRPV1, a molecule involved in neuroimmune signaling, suggesting that burosumab may exert biological effects beyond bone health.

Introduction

X-linked hypophosphatemia (XLH) is a rare genetic disorder primarily caused by inactivating mutations in the PHEX gene, which encodes a phosphate-regulating endopeptidase. These mutations result in elevated circulating levels of fibroblast growth factor 23 (FGF23), an osteocyte-derived hormone that plays a central role in phosphate and vitamin D metabolism. Abnormal FGF23 levels impair renal phosphate reabsorption and suppress 1α-hydroxylase activity, leading to chronic hypophosphatemia.

Short stature, bone malformations such as lower limb varus deformity, growth retardation, rickets, and dental abnormalities are the main clinical signs of XLH. Among the recognized features of XLH, particularly in adolescents and adults, there are musculoskeletal pain and muscle weakness (1, 2). These symptoms are likely related to chronic phosphate loss and altered energy metabolism in muscle tissue and can significantly impair mobility, functional capacity, and quality of life (3). In recent years, additional extraskeletal complications have emerged, including a predisposition to overweight/obesity and a potentially increased cardiovascular risk, as suggested by higher BMI and blood pressure values in XLH patients compared with the general population (4, 5). Conventional treatment for XLH has traditionally relied on oral phosphate supplementation and active vitamin D analogs. Although considered the standard of care since the 1980s, it is limited by poor adherence, incomplete correction of skeletal abnormalities, and adverse effects such as hypercalcemia, nephrocalcinosis, and hyperparathyroidism (6). More recently, burosumab – a fully human IgG1 monoclonal antibody targeting FGF23 – has emerged as a targeted therapeutic option, representing a major advancement in the management of XLH. Clinical trials have demonstrated that burosumab significantly improves biochemical and clinical outcomes, including increased serum phosphate levels, improved radiographic scores, greater height gain, and reduced pain (6, 7, 8, 9). Emerging evidence further suggests that the clinical benefits of burosumab extend beyond skeletal health. Improvements in motor performance and reductions in musculoskeletal pain and fatigue may not be solely attributable to its effects on bone metabolism. These findings have prompted interest in the broader biological actions of FGF23 and its contribution to systemic inflammation and neuromuscular dysfunction in XLH.

In particular, FGF23 has been implicated in several non-classical functions, including bone remodeling (10, 11), cardiovascular regulation (12, 13), and immune responses (14), although its precise contribution to inflammatory pathways has yet to be fully elucidated. Accordingly, sustained FGF23 elevation in XLH may contribute not only to the classical skeletal features but also to extraskeletal complications, including cardiovascular, muscular, and immunometabolic alterations.

Among the mechanisms potentially underlying these immunometabolic effects, experimental studies have suggested an interaction between FGF23 signaling and macrophage biology. In particular, macrophages have been identified not only as target cells responsive to FGF23 signaling through FGFR-dependent pathways but also as a potential source of FGF23 under inflammatory conditions, supporting a bidirectional interaction between FGF23 and innate immune responses (14, 15, 16, 17).

As key regulators of innate immunity, macrophages display remarkable functional plasticity, acquiring phenotypes ranging from the classically activated, pro-inflammatory M1 state to the alternatively activated, anti-inflammatory, and tissue repair-associated M2 state, depending on the local microenvironment.

In animal studies, FGF-23 is released by pro-inflammatory M1 macrophages and has been proposed to modulate macrophage inflammatory responses through paracrine mechanisms and, possibly, to act on other cells in inflamed and injured tissues to promote fibrosis (15). These findings suggest that macrophages may contribute to local FGF23 production, supporting its potential paracrine role in the regulation of inflammatory responses (14, 15, 16, 17).

In addition, in peritoneal macrophages isolated from Hyp mice, a murine model of XLH, FGF23 expression has been shown to be increased compared with wild-type controls; moreover, peritoneal macrophages from Hyp mice presented a greater response to LPS + IFN-γ-induced TNF-α expression compared with peritoneal macrophages from wild-type mice (15). Although the biological relevance of these observations in XLH patients remains to be clarified, they raise the possibility that macrophage phenotype may contribute to the inflammatory milieu associated with the disease.

Another potentially relevant player in this context is transient receptor potential vanilloid 1 (TRPV1). Beyond its well-established role in nociception, TRPV1 is expressed in macrophages, where it participates in their activation and polarization while contributing to neuroimmune interactions involved in chronic pain (18, 19). Interestingly, TRPV1 activation may promote polarization toward an M2 phenotype, suggesting that this channel may modulate both inflammatory responses and pain perception (20). Therefore, TRPV1 represents a potential molecular link between macrophage function and neuroimmune pathways in XLH.

Based on these findings, we investigated, for the first time, the macrophage profiles in pediatric patients with XLH, either untreated or receiving burosumab therapy, compared with those of healthy controls. In particular, we aimed to dissect whether burosumab could influence macrophage polarization, with particular emphasis on the balance between the pro-inflammatory M1 and anti-inflammatory M2 phenotypes, as well as modulate TRPV1 expression. To address this question, we performed both ex vivo experiments using macrophages isolated from patients with XLH and healthy donors and in vitro experiments using LPS-stimulated macrophages derived from healthy donors and treated with different concentrations of burosumab.

Material and methods

Patients

We isolated macrophages from the peripheral blood of 4 treatment-naive XLH patients (XLH-naive), 8 XLH patients receiving burosumab treatment (XLH treatment), and 5 healthy donors (CRT NT). Samples were collected during routine clinical visits performed according to standard clinical practice.

At the time of evaluation, the XLH-naive group comprised 4 females, with a median age of 1.84 years (range: 0.19–3.4 years), and all were prepubertal. The XLH treatment group comprised 8 patients (3 males), with a median age of 5.35 years (range: 1.98–14.5 years) and a median pubertal stage of 1 (range: 1–4). The CTR NT group comprised 5 patients (3 males), with a median age of 6.2 years (range: 3.42–13.75 years) and a median pubertal stage of 1 (range: 1–3).

XLH patients in treatment were receiving burosumab for a median time of 31.5 months (range: 6–67 months), with an average per kg dose of 1.62 mg (1–1.79), while XLH-naive patients and CRT NT were not receiving any medication at the time of the analysis.

Macrophage cultures

Peripheral blood mononuclear cells (PBMCs) were isolated from XLH-naive patients, the XLH treatment group, and healthy controls by density gradient centrifugation using Ficoll-Paque and subsequently differentiated into macrophages under standard culture conditions (1.077 g/mL; Lympholyte, Cedarlane Laboratories Ltd, The Netherlands). PBMCs were resuspended at a concentration of 1 × 10⁶ cells/mL in α-Minimal Essential Medium (α-MEM; Lonza, Belgium), enriched with 10% fetal bovine serum (FBS; Euroclone, Italy), 100 IU/mL penicillin, 100 μg/mL streptomycin, and L-glutamine (Gibco Limited, UK), and then seeded into 24-well cell culture plates.

To promote full differentiation into human macrophages, the cells were maintained in culture for 15 days in the presence of 25 ng/mL of recombinant human macrophage colony-stimulating factor (rh-MCSF; Peprotech, UK). The culture medium was refreshed twice weekly. All cells were incubated at 37°C in a humidified environment containing 5% CO₂. Following the 15-day differentiation period, cells from both XLH patients and CTR were collected for protein extraction.

In vitro treatments with burosumab

To model an inflammatory environment, PBMCs derived from the CTR group were isolated and differentiated into macrophages as described in the section titled Macrophage cultures and stimulated with lipopolysaccharide (LPS, 500 ng/mL). Lipopolysaccharide (LPS) (Sigma-Aldrich, USA) was dissolved in phosphate-buffered saline (PBS) containing dimethyl sulfoxide (DMSO) and used to stimulate macrophages derived from CTR at a final concentration of 500 ng/mL. The final concentration of DMSO in cell cultures was 0.01%. Untreated control cells were maintained in the incubation medium for the equivalent exposure time in the presence or absence of the vehicle (DMSO 0.01%).

After 24 h of LPS exposure, burosumab was added for 24 h at escalating concentrations (0.03; 0.3; 1.5; 3 μg/mL). Dose selection was guided by preliminary dose–response experiments and by the need to balance efficacy testing with safety (see the section titled MTT cell proliferation and cytotoxicity assay).

Burosumab (Kyowa Kirin) was prepared by diluting in physiological saline to a final concentration of 1 mg/mL. CTR cells not subjected to treatment were kept in culture medium for the same duration as the treated samples, with or without the corresponding vehicle.

MTT cell proliferation and cytotoxicity assay

Cell viability and proliferation of XLH-derived macrophages after 24 h of in vitro treatment with burosumab were evaluated using the colorimetric MTT assay, following the manufacturer’s instructions (Elabscience, USA). The assay is based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into formazan crystals by mitochondrial dehydrogenase enzymes present in metabolically active cells.

Formazan, a purple insoluble product, was subsequently solubilized in DMSO, and its absorbance was measured at approximately 570 nm. The intensity of the color correlates directly with the number of viable and proliferating cells: a darker color indicates higher cell viability and proliferation, while a lighter color reflects reduced metabolic activity, typically due to cytotoxic effects. The colorimetric response is linearly proportional to the cell number. CTR macrophages were stimulated with LPS for 24 h and then exposed for an additional 24 h to escalating concentrations of burosumab (0.03, 0.3, 1.5, and 3 μg/mL). These doses were selected to span a broad experimental range, from very low (sub-pharmacological) concentrations to higher levels.

Protein isolation and western blot

Proteins were isolated from both XLH and CTR macrophage cultures using Radio-Immunoprecipitation Assay (RIPA) Lysis Buffer (Millipore, USA), following the instructions provided by the manufacturer. Total protein extracts from the cell cultures were subjected to western blot analysis to assess the expression of CCR7, CD86, iNOS, CD206, pSTAT6, FGF23, TRPV1, FPN-1, TfR1, and DMT1.

For immunodetection, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CCR7 (1:500, Rabbit, Elabscience, USA), anti-CD86 (1:500, Rabbit, Elabscience, USA), anti-CD206 (1:200, Mouse, Santa Cruz Biotechnology, USA), anti-pSTAT6 (1:500, Rabbit, Elabscience, USA), anti-FGF23 (1:2,000, Goat, Invitrogen by Thermo Fisher, USA), anti-TRPV1 (1:1,000, Rabbit, Novus Biologicals, Italy), anti-FPN-1 (1:1,000, Rabbit, Novus Biologicals, Italy), anti-TfR1 (1:1,000, Rabbit, Abcam, UK), and anti-DMT1 (1:100, Mouse, Santa Cruz Biotechnology, USA).

Detection of protein bands was performed using enhanced chemiluminescence (Clarity Max Western ECL Substrate, Bio-Rad, USA) and visualized with the Bio-Rad CHEMIDOC system (Bio-Rad, Hercules, USA). A mouse monoclonal anti-β-actin antibody (1:500, Mouse, Santa Cruz Biotechnology, USA) and anti-GAPDH (1:2,000, Mouse, Elabscience, USA) were used as internal loading controls. Image acquisition, processing, and densitometric analysis were conducted using the Image Lab.Ink 6.1 software.

Statistical analysis

Statistical analyses were carried out on data obtained from western blot. To compare XLH-naive samples with CTR samples, an unpaired t-test was applied.

To compare XLH treatment samples with XLH-naive samples, we first assessed the distribution of the data using the Shapiro–Wilk normality test. If the data followed a normal distribution, we applied a one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis. For datasets that did not meet normality criteria, we used the Kruskal–Wallis test, followed by Dunn’s test as a post hoc comparison. To compare CTR samples treated with burosumab at various concentrations, we assessed them against untreated CTR samples as well as CTR samples treated with LPS. Normality was first evaluated using the Shapiro–Wilk test. For datasets that were normally distributed, a one-way ANOVA was performed, followed by Tukey’s HSD test for post hoc comparisons. For datasets that did not meet normality criteria, the Friedman test was applied, followed by Dunn’s test for post hoc analysis. All statistical analyses were performed using GraphPad Prism software (version 8.4.2).

Each experiment was conducted on samples derived from three independent donors, and results are expressed as mean ± standard deviation (SD). Differences were considered statistically significant at P ≤ 0.05.

Results

Clinical and biochemical characteristics of patients

A comprehensive assessment of bone metabolism was performed in XLH patients: as expected, XLH-naive patients showed lower phosphate levels compared with the XLH treatment group (P: 3 mg/dL, range: 2.6–3.8, vs 3.6 mg/dL, range: 2.6–4.4, respectively), higher ALP levels (624 U/L, range: 441–810, vs 338 U/L, range: 126–418), and reduced phosphate tubular reabsorption (median TmPO4/GFR: 2.37, range: 2.23–3.36, vs 2.99, range: 2.25–4.11). Serum phosphate levels in treatment-naive patients were below the age-specific reference range, whereas burosumab-treated patients showed phosphate concentrations within the expected range for age. XLH-naive patients presented a higher rickets severity score than the XLH treatment group (10 vs 6, respectively). In CRT NT patients, basal bone metabolism (calcium, phosphate, ALP, 25-hydroxy vitamin D (25OHD), and PTH) on blood withdrawal was performed in order to rule out any electrolyte imbalance or 25OHD deficiency. All blood biochemical parameters measured in the healthy control group fell within the corresponding age-adjusted normal ranges (see Supplementary file 1 (see section on Supplementary materials given at the end of the article) for further details).

In two adolescent patients with XLH on burosumab treatment, the Brief Pain Inventory (BPI) questionnaire and the WOMAC questionnaire were administered and resulted in scores suggesting no pain or fatigue (0 for both BPI and WOMAC in both patients). However, no information on scores before treatment was available, as they were too young when they started treatment (21, 22). The other younger patients on burosumab treatment did not report pain on clinical interview, but they were too young for a structured pain evaluation.

Effect of burosumab on polarization of macrophages isolated from XLH patients

To investigate the expression levels of macrophage polarization markers, we performed western blot analysis targeting CCR7, CD86, and iNOS as M1 markers and CD206 and pSTAT6 as M2 markers. We first compared protein levels in macrophages derived from XLH-naive patients with those from CTR NT. In XLH-naive patients, the biochemical analysis showed a statistically significant upregulation of CCR7 and CD86 (Fig. 1A and B) and a trend toward an increase in iNOS (Fig. 1C). We also revealed a trend toward a decrease in pSTAT6 in XLH-naive macrophages compared with CTR NT (Fig. 2B), suggesting a predominant M1 pro-inflammatory phenotype in cells derived from untreated XLH patients. Subsequently, we analyzed the phenotype of macrophages isolated from XLH patients who had previously received burosumab treatment. Compared with XLH-naive samples, macrophages from burosumab-treated patients (XLH treatment) displayed a statistically significant reduction of CCR7 and CD86 protein expression levels (Fig. 1A and B), a downward trend in iNOS levels (Fig. 1C), and a concomitant increase, albeit not statistically significant, in CD206 and pSTAT6 protein expression levels (Fig. 2A and B) compared with XLH-naive patients. These findings suggest that burosumab treatment may promote a shift toward an anti-inflammatory M2 macrophage phenotype.

Figure 1.

Effect of burosumab on M1 macrophages isolated from XLH patients. CCR7 (A), CD86 (B), and iNOS (C) protein expression levels in macrophages derived from healthy donors (CTR), XLH-naive patients (XLH-naive (N)), and burosumab-treated patients (XLH treatment (T)), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. If the data followed a normal distribution, one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used. For datasets that did not meet normality criteria, the Kruskal–Wallis test, followed by Dunn’s test for a post hoc comparison, was used. *P ≤ 0.05 compared with CTR; ^P ≤ 0.05 compared with XLH-naive patients.

Figure 2.

Effect of burosumab on M2 macrophages isolated from XLH patients. CD206 (A) and pSTAT6 (B) protein expression levels in macrophages derived from healthy donors (CTR), XLH-naive patients (XLH-naive (N)), and burosumab-treated patients (XLH treatment (T)), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. If the data followed a normal distribution, one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used. For datasets that did not meet normality criteria, the Kruskal–Wallis test, followed by Dunn’s test for a post hoc comparison, was used. *P ≤ 0.05 compared with CTR; ^P ≤ 0.05 compared with XLH-naive patients.

Effect of burosumab on macrophage viability

To determine a non-cytotoxic working range for subsequent mechanistic assays, LPS-stimulated CTR macrophages were exposed for 24 h to increasing concentrations of burosumab (0.03, 0.3, 1.5, and 3.0 μg/mL). Cell viability remained >80% at all tested doses, with values of 93.0% at 0.03 μg/mL, 88.7% at 0.3 μg/mL, 85.5% at 1.5 μg/mL, and 81.1% at 3.0 μg/mL. Although none of these concentrations exerted overt cytotoxicity, only the two highest doses (1.5 and 3.0 μg/mL) were selected for subsequent experiments. The intermediate dose (1.5 μg/mL) was considered optimal to detect maximal biological effects without approaching toxicity, whereas the highest dose (3.0 μg/mL) was included to assess whether macrophage responses plateau or decline at increased antibody exposure. Lower concentrations (0.03 and 0.3 μg/mL), while non-toxic, did not elicit measurable changes in the endpoints of interest and were therefore not pursued further (Table 1).

Table 1.

Effects of lipopolysaccharide (LPS) and burosumab (Bur) on CTR macrophages viability. Cell survival rate in CTR macrophages after 24 h of treatment with LPS (500 ng/mL) and Bur at different concentrations (0.03, 0.3, 1.5, and 3 μg/mL). The results are presented as mean percentage ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. One-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used for statistical analysis.

Molecule concentration	Cell survival rate (%)
CTR (untreated)	100 ± 1.7
LPS	91.3 ± 4.2
Bur 0.03	93.1 ± 2.5
Bur 0.3	85.6 ± 0.6
Bur 1.5	85.5 ± 2.6
Bur 3	81.1 ± 1.5

Effect of burosumab on polarization of LPS-inflamed macrophages isolated from CTR subjects

To investigate the impact of burosumab on macrophage polarization, we performed western blot analysis assessing the expression of M1 markers (CCR7, CD86, and iNOS) and M2 markers (CD206 and pSTAT6). We used macrophages derived from CTR, which were stimulated with LPS to induce an inflammatory state that mimics the condition previously demonstrated in XLH patients, where we observed a predominance of the M1 pro-inflammatory phenotype.

As expected, LPS stimulation alone increased the expression of M1 markers (CCR7, CD86, and iNOS) (Fig. 3A, B, C) and concomitantly reduced the levels of M2 markers, specifically CD206 and pSTAT6 (Fig. 4A and B).

Figure 3.

Effect of burosumab on the M1 phenotype of LPS-inflamed macrophages isolated from CTR subjects. CCR7 (A), CD86 (B), and iNOS (C) protein expression levels in macrophages derived from healthy donors (CTR NT), stimulated with LPS (CTR LPS), and treated with burosumab at different concentrations (1.5 and 3 μg/mL) (CTR LPS BUR 1.5 and CTR LPS BUR 3), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. Normality was first assessed using the Shapiro–Wilk test. For datasets that were normally distributed, one-way ANOVA was performed, followed by Tukey’s HSD test for post hoc comparisons. For datasets that did not meet normality criteria, the Friedman test was applied, followed by Dunn’s test for post hoc analysis. *P ≤ 0.05 compared with CTR NT; ^P ≤ 0.05 compared with CTR LPS.

Figure 4.

Effect of burosumab on the M2 phenotype of LPS-inflamed macrophages isolated from CTR subjects. CD206 (A) and pSTAT6 (B) protein expression levels in macrophages derived from healthy donors (CTR NT), stimulated with LPS (CTR LPS), and treated with burosumab at different concentrations (1.5 and 3 μg/mL) (CTR LPS BUR 1.5 and CTR LPS BUR 3), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. Normality was first assessed using the Shapiro–Wilk test. For datasets that were normally distributed, one-way ANOVA was performed, followed by Tukey’s HSD test for post hoc comparisons. For datasets that did not meet normality criteria, the Friedman test was applied, followed by Dunn’s test for post hoc analysis. *P ≤ 0.05 compared with CTR NT; ^P ≤ 0.05 compared with CTR LPS.

Following this, CTR macrophages were treated in vitro with burosumab at concentrations of 1.5 and 3 μg/mL after LPS stimulation. The biochemical analysis showed that burosumab treatment led to a marked reduction in M1 marker expression (CCR7, CD86, and iNOS) (Fig. 3A, B, C) compared with LPS-induced inflammation and a corresponding increase in M2 markers, CD206 and pSTAT6 (Fig. 4A and B). However, although the results suggested slightly greater effects at 1.5 μg/mL than at 3 μg/mL, no significant differences were detected, indicating variability without a true dose-dependent effect.

These in vitro results confirm what we previously observed in XLH patient-derived macrophages, reinforcing the conclusion that burosumab may modulate macrophage polarization, promoting the shift from the M1 pro-inflammatory phenotype to an anti-inflammatory M2 profile under inflammatory conditions that mimic the pathological context of XLH.

Effect of burosumab on TRPV1 expression

To investigate the potential modulatory role of burosumab on inflammatory signaling pathways, we analyzed the expression levels of transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel known to be involved in inflammatory responses, nociception, and macrophage activation. Western blot analysis showed that macrophages derived from XLH-naive patients exhibited a marked upregulation of TRPV1 compared with macrophages from healthy donors (CTR), consistent with the presence of a pro-inflammatory macrophage phenotype in XLH.

Conversely, in macrophages isolated from burosumab-treated XLH patients (XLH treatment), TRPV1 expression was slightly reduced (Fig. 5A). A similar result was observed in LPS-stimulated macrophages from healthy donors (Fig. 5B), where burosumab treatment (1.5 and 3 μg/mL) led to a significant dose-dependent decrease in TRPV1 expression compared with LPS alone (Fig. 5B). However, although the results suggested slightly greater effects at 1.5 than at 3 μg/mL, no significant differences were detected, indicating variability without a true dose-dependent effect.

Figure 5.

Effect of burosumab on TRPV1 expression. (A) TRPV1 protein expression levels in macrophages derived from healthy donors (CTR), XLH-naive patients (XLH-naive (N)), and burosumab-treated patients (XLH treatment (T)), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. Since the data were normally distributed, one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used. *P ≤ 0.05 compared with CTR. (B) TRPV1 protein expression levels in macrophages derived from healthy donors (CTR NT), stimulated with LPS (CTR LPS), and treated with burosumab at different concentrations (1.5 and 3 μg/mL) (CTR LPS BUR 1.5 and CTR LPS BUR 3), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. Since the datasets were normally distributed, one-way ANOVA was performed, followed by Tukey’s HSD test for post hoc comparisons. *P ≤ 0.05 compared with CTR NT; ^P ≤ 0.05 compared with CTR LPS.

Effect of burosumab on FGF23 levels

To investigate the effects of burosumab on fibroblast growth factor 23 (FGF23) levels, we assessed intracellular protein content by western blot (WB) and quantified secreted levels in cell culture supernatants using ELISA. In macrophages from treatment-naive XLH patients, ELISA revealed an increased extracellular secreted fraction of FGF23 compared with healthy controls (Fig. 6A), while intracellular levels were significantly lower (P = 0.051) (Fig. 6B), likely reflecting an active secretion process in which FGF23 is rapidly exported once synthesized. This secretion pattern suggests that macrophages in the XLH setting may contribute to the elevated circulating FGF23 levels typically observed in patients. In XLH treatment group subjects, both intracellular and extracellular FGF23 levels were reduced compared with untreated patients, with intracellular FGF23 levels notably lower even than those of healthy controls.

Figure 6.

Effect of burosumab on FGF23 in macrophages isolated from XLH patients. (A) FGF23 release from macrophages derived from healthy donors (CTR), XLH-naive patients (XLH-naive (N)), and burosumab-treated patients (XLH treatment (T)), measured using an enzyme-linked immunosorbent assay (ELISA). The graphs show extracellular FGF23 levels (pg/mL) as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. Since the data followed a normal distribution, one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used. (B) Intracellular FGF23 protein expression levels in macrophages derived from healthy donors (CTR), XLH-naive patients (XLH-naive (N)), and burosumab-treated patients (XLH treatment (T)), evaluated by western blotting, starting from 15 μg of total lysates. The most representative images are shown. The protein bands were detected using Image Lab Ink 6.1 software (Bio-Rad). The relative quantification for these proteins, normalized to the housekeeping protein β-actin, is presented in the histograms as mean ± SD. The distribution of the data was assessed using the Shapiro–Wilk normality test. Since the data followed a normal distribution, one-way ANOVA, followed by Tukey’s HSD test for post hoc analysis, was used. *P ≤ 0.05 compared with CTR.

Discussion

X-linked hypophosphatemia (XLH) is a rare disease most often caused by inactivating mutations in the PHEX gene, which causes an increased production of fibroblast growth factor 23 (FGF23). This hormone is normally produced by osteocytes, and it is implicated in phosphate reabsorption and calcitriol production reduction in the kidneys. Considering these aspects, XLH could be considered a disorder of mineral metabolism and skeletal development. However, with advancing age, muscle weakness and pain often become more prominent and debilitating than the classic skeletal manifestations, contributing to fatigue and reduced physical performance (23, 24).

The therapeutic landscape of XLH has been substantially transformed by the introduction of burosumab, a fully human IgG1 monoclonal antibody that selectively neutralizes circulating FGF23 and directly targets the underlying pathophysiological mechanism of the disease. Beyond restoring phosphate homeostasis and improving skeletal mineralization, burosumab has consistently been associated with clinically meaningful improvements in musculoskeletal pain, physical function, fatigue, and other extraskeletal manifestations (8). These clinical observations are consistent with the hypothesis that the biological consequences of FGF23 excess extend beyond phosphate metabolism. Consistent with this view, accumulating evidence has identified FGF23 as a modulator of innate immunity, capable of directly interacting with macrophages through its binding to FGFR1 or FGFR4 and the activation of intracellular signaling pathways – including MAPK, ERK1/2, STAT1, and NF-κB – that promote the expression of pro-inflammatory genes (15).

Accordingly, the elevated FGF23 levels characteristic of XLH may contribute to a chronic low-grade inflammatory state, potentially underlying several extraskeletal manifestations. Supporting this hypothesis, Meux et al. recently observed a strong increase in pro-inflammatory cytokine levels in PBMCs from XLH patients compared with healthy controls. Notably, this inflammatory profile was observed in both burosumab-treated and conventionally treated patients, suggesting that systemic inflammation may persist despite current therapeutic approaches (25).

Despite these observations, the mechanisms linking FGF23 signaling to macrophage dysfunction and the potential impact of FGF23 inhibition on macrophage phenotype remain largely unexplored.

In this context, the first aim of our study was to characterize the macrophage phenotype associated with XLH by comparing treatment-naive patients with healthy controls, including the evaluation of macrophage polarization, TRPV1 expression, and macrophage-derived FGF23. We found that macrophages isolated from untreated XLH patients displayed a predominant pro-inflammatory M1 phenotype, characterized by increased expression of CCR7, CD86, and iNOS together with reduced expression of the M2-associated markers CD206 and phosphorylated STAT6. The predominance of M1 macrophages may contribute to the chronic inflammatory milieu associated with XLH and could partly account for the musculoskeletal manifestations commonly observed in these patients (26).

This interpretation is supported by previous experimental evidence showing that M1 macrophage polarization contributes to persistent inflammation and tissue dysfunction in several chronic inflammatory conditions, including inflammatory myopathies (27, 28). Moreover, FGF23 has been reported to enhance M1 macrophage polarization by promoting the production of pro-inflammatory cytokines such as TNF-α and IL-6 through FGFR4, independently of Klotho (17). Han et al. further demonstrated that FGF23 activates NF-κB signaling in macrophages, supporting the concept that FGF23 signaling may participate in macrophage inflammatory responses under specific experimental conditions (15). Although the relevance of these mechanisms in human XLH remains to be fully established, our findings are consistent with the presence of a pro-inflammatory macrophage phenotype in untreated patients.

Interestingly, untreated XLH macrophages showed reduced intracellular FGF23 levels together with increased extracellular FGF23 concentrations, suggesting enhanced FGF23 secretion under inflammatory conditions. This finding is consistent with previous experimental evidence showing increased FGF23 expression in activated macrophages and in peritoneal macrophages isolated from Hyp mice, an established murine model of XLH, suggesting that macrophages may represent a local source of FGF23 through a potential paracrine mechanism (15, 16, 17).

The second aim of the study was to investigate whether treatment with burosumab is associated with changes in macrophage phenotype. We have, in fact, reported that treatment with burosumab appears to be associated with a marked reversal of the pro-inflammatory profile observed in XLH. Both macrophages isolated from burosumab-treated patients and lipopolysaccharide (LPS)-stimulated macrophages from healthy donors exposed to burosumab in vitro exhibited a shift toward an anti-inflammatory M2 phenotype, characterized by reduced expression of M1-associated markers together with increased levels of CD206 and phosphorylated STAT6 (pSTAT6) compared with untreated XLH children. Consistent with its biological function, burosumab treatment appeared to reduce intracellular and extracellular FGF23 levels, although this effect was observed as a trend. Although these findings suggest that the biological effects of burosumab may extend beyond restoration of phosphate homeostasis, including modulation of macrophage phenotype, the underlying mechanisms remain to be elucidated.

Low-grade chronic inflammation could lead to metabolic and cardiovascular consequences in XLH, such as an increased BMI and high blood pressure, particularly during adulthood. Future studies focusing on the effects of burosumab on inflammation and macrophage polarization in adult XLH patients could be useful to better understand this aspect.

In addition, given the well-documented presence of pain, including neuropathic pain, in patients with X-linked hypophosphatemia (XLH), we sought to further investigate the role of transient receptor potential vanilloid 1 (TRPV1), a key mediator of both inflammatory and neuropathic pain pathways. TRPV1 is a non-selective cation channel expressed in sensory neurons and in immune cells, such as macrophages (18), where it plays a critical role in pain transmission and peripheral sensitization (19, 29, 30). During inflammation, TRPV1 expression is upregulated, determining calcium influx and subsequent neuronal activation, thus enhancing pain perception. In macrophages, TRPV1 activation has been shown to amplify inflammation and neuroinflammation, supporting its role in immune–nociceptive crosstalk (29). Elevated TRPV1 expression has been associated with chronic pain and inflammatory hypersensitivity (31), conditions that are frequently reported by XLH patients in the form of persistent musculoskeletal pain. TRPV1 expression paralleled macrophage phenotype, being increased in untreated XLH patients and reduced following burosumab treatment both ex vivo, with a slight effect, and in vitro, where it is more pronounced. Although these findings suggest that TRPV1 may participate in macrophage activation pathways, the functional significance in pain modulation in XLH patients remains to be established in larger studies including pain quantification.

A limitation of our study is the relatively small sample size, reflecting the rarity of XLH and the consequent challenges in recruiting larger patient cohorts. Although differences in age were present among the study groups, age-related immune maturation is unlikely to be the sole determinant of macrophage polarization, as in vitro M1/M2 phenotypic responses are strongly influenced by disease-related factors and by differentiation and culture conditions (32). Furthermore, although the in vitro findings mirrored those observed ex vivo, the influence of systemic factors cannot be excluded. Larger, multicenter longitudinal studies will therefore be needed to validate these findings and better characterize the immunoregulatory effects of burosumab. Finally, our study was not designed to directly determine whether FGF23 is responsible for the differences observed between untreated and burosumab-treated patients. However, this aspect represents an interesting area for future research, given that, to date, the available evidence is limited and derives exclusively from studies conducted in mice.

In conclusion, our findings suggest that burosumab not only is able to modulate bone metabolism, but also exerts significant immunomodulatory effects. In particular, it prompts a macrophage polarization toward the anti-inflammatory M2 phenotype and a reduction in TRPV1 protein expression levels and FGF23 synthesis. Future studies will be required to determine the molecular mechanisms underlying these effects and to establish their relationship with clinical outcomes.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.

Funding

This work was supported by the ‘RachiExtra’ project (ex. L.183/1987) funded by the Campania Region.

Author contribution statement

ADP contributed to conceptualization, formal analysis, data curation, methodology, writing of the original draft, and review and editing. SP contributed to conceptualization, methodology, formal analysis, data curation, writing of the original draft, and review and editing. LA, GDF, and ODD contributed to formal analysis and methodology. FA performed investigation and contributed resources. GC, GP, MMM, and EMDG contributed to validation and writing, review, and editing. AG and FR contributed to conceptualization, project administration, and supervision. All authors read and approved the final manuscript and take full responsibility for its content.

Ethics approval and consent to participate

The study involving human participants was approved by the Ethics Committee of the University of Campania ‘Luigi Vanvitelli’ and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from the parents or legal guardians, and assent was provided by the participating children prior to the initiation of any study-related procedures.