Therapeutic blockade of CCL17 in obesity-exacerbated osteoarthritic pain and disease

Kevin Ming-Chin Lee; Tanya Lupancu; Stacey N Keenan; Georgina Bing; Adrian A Achuthan; Mark Biondo; Kim Gia Lieu; Matthew J Watt; Eugene Maraskovsky; Bronwyn A Kingwell; John A Hamilton

doi:10.1371/journal.pone.0317399

. 2025 Jan 16;20(1):e0317399. doi: 10.1371/journal.pone.0317399

Therapeutic blockade of CCL17 in obesity-exacerbated osteoarthritic pain and disease

Kevin Ming-Chin Lee ^1,^*, Tanya Lupancu ¹, Stacey N Keenan ², Georgina Bing ³, Adrian A Achuthan ¹, Mark Biondo ³, Kim Gia Lieu ³, Matthew J Watt ², Eugene Maraskovsky ³, Bronwyn A Kingwell ³, John A Hamilton ¹

Editor: Xindie Zhou⁴

¹Faculty of Medicine Dentistry and Health Sciences, Department of Medicine, Royal Melbourne Hospital, Melbourne Medical School, University of Melbourne, Parkville, Victoria, Australia

²Faculty of Medicine Dentistry and Health Sciences, Department of Anatomy and Physiology, School of Biomedical Sciences, University of Melbourne, Parkville, Victoria, Australia

³Bio21 Institute, CSL Innovation Pty Ltd, Parkville, Victoria, Australia

⁴The Affiliated Changzhou No 2 People’s Hospital of Nanjing Medical University, CHINA

Competing Interests: KGL, MB, GB, BAK and EM are employees and shareholders of CSL. The authors have declared that no conflicts of interest exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

^✉

* E-mail: mingchinl@unimelb.edu.au

Roles

Kevin Ming-Chin Lee: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing

Tanya Lupancu: Data curation, Formal analysis, Investigation, Methodology

Stacey N Keenan: Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Georgina Bing: Data curation, Formal analysis, Investigation, Writing – review & editing

Adrian A Achuthan: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing

Mark Biondo: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing

Kim Gia Lieu: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – review & editing

Matthew J Watt: Data curation, Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review & editing

Eugene Maraskovsky: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – review & editing

Bronwyn A Kingwell: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – review & editing

John A Hamilton: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing

Xindie Zhou: Editor

PMCID: PMC11737751 PMID: 39820104

Abstract

Objectives

We previously reported that CCL17 gene-deficient mice are protected from developing pain-like behaviour and exhibit less disease in destabilization of medial meniscus (DMM)-induced OA, as well as in high-fat diet (HFD)-exacerbated DMM-induced OA. Here, we explored if therapeutic neutralization of CCL17, using increasing doses of a neutralizing monoclonal antibody (mAb), would lead to a dose-dependent benefit in these two models.

Design

DMM-induced OA was initiated in male mice either fed with a control diet (7% fat) or 8 weeks of a 60% HFD, followed by therapeutic intraperitoneal administration (i.e. when pain is evident) of an anti-CCL17 mAb (B293, 25mg/kg, 5mg/kg or 1mg/kg) or isotype control (BM4; 25mg/kg). Pain-like behaviour and arthritis were assessed by relative static weight distribution and histology, respectively. The effects of B293 (25mg/kg) on HFD-induced metabolic changes, namely oral glucose tolerance test, insulin tolerance test and liver triglyceride levels, were examined.

Results

Therapeutic administration of B293 results in a dramatic amelioration of DMM-induced OA pain-like behaviour and the inhibition of disease progression, compared to BM4 (isotype control) treatment. A similar therapeutic effect was observed in HFD-exacerbated OA pain-like behaviour and disease. B293 treatment did not alter the measured HFD-induced metabolic changes.

Conclusions

Based on the data presented, CCL17 could be a therapeutic target in OA patients with joint injury alone or with obesity.

Introduction

Osteoarthritis (OA) is by far the most common musculoskeletal disease with chronic pain being the major concern for patients [1]. Treatment options for OA patients are limited and are often centered on pain relief. OA is characterized by cartilage damage, synovial inflammation and bone remodeling. There are a number of risk factors that can dramatically increase OA prevalence and severity, with obesity being prominent among these [2–4]. The OA enhancement by obesity is due not only to “so-called” mechanoinflammation (i.e. inflammation due to increased weight bearing) [5], but also to systemic low-grade inflammation (i.e. metainflammation)–several studies have implicated cytokines and adipokines in the pathogenesis of obesity-associated OA [6,7].

We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/CC motif chemokine ligand 17 (CCL17) pathway in which the pro-inflammatory cytokine, GM-CSF, upregulates dramatically the production of CCL17 in monocytes/macrophages [8]. This pathway was shown to drive pain-like behaviour and disease in a number of monoarticular inflammatory mouse arthritis models [8–10]. Recently, evidence was provided, using gene-deficient mice, for the involvement of the GM-CSF/CCL17 pathway in the development of pain-like behaviour and maximal disease in three experimental OA models, as well as for the exacerbated OA development due to obesity [11]. In light of these findings, we explored the efficacy of anti-CCL17 mAb therapy for the treatment of OA in lean and obese mice.

The mouse destabilization of medial meniscus (DMM)-induced OA model is the most widely used post-traumatic OA model. Given that Ccl17-gene deficient mice fed either a control diet or a HFD are protected from developing DMM-induced OA pain-like behaviour and exhibit less severe arthritis [11], we reasoned it to be important to determine whether an anti-CCL17 mAb could ameliorate therapeutically DMM-induced OA pain-like behaviour and disease in a dose-dependent manner under both dietary conditions.

Materials and methods

A detailed Materials and Methods section is available in the online S1 File.

Anti-CCL17 (B293) monoclonal antibody and isotype control purification

The sequence for the anti-CCL17 (huB293-mG1K-aCCL17) monoclonal antibody (mAb) was derived from International PCT Publication No. WO 2015/069865 A1. Both the anti-CCL17 (B293) mAb and the isotype control (muBM4-muG1K) antibody were purified from the culture supernatant from ExpiCHO cells, which were transiently transfected with either the sequence of the anti-CCL17 mAb or the isotype control antibody. The antibodies were then sterile filtered and stored at -80°C.

Tango™ β-Arrestin recruitment reporter assay

Tango™ CCR4-bla U2OS cells (Invitrogen) were resuspended in assay medium (Thermo Fisher Scientific, FreeStyle™ 293 Expression Medium supplemented with Glutamax™) and incubated with murine CCL17 (10 nM) and increasing concentrations of B293. The fluorescence intensities at 450 nm (blue) and 520 nm (green) were used to determine the response ratio (blue:green signal) as per the manufacturer’s instructions. B293 pre-complexed with murine CCL17 resulted in a dose-dependent inhibition of β-lactamase production when compared to a BM4 isotype control, indicating that B293 is able to inhibit murine CCL17-dependent signaling in vitro.

Mice

Male C57BL/6 wild-type (WT) and GM-CSF^-/- mice (6–10 weeks) [8,11] were used. A total of 108 mice was used in this study. All mice (n = 2–5 mice/cage) were kept under a 12-hour light and dark cycle at 22°C and allowed free access to food and water ad libitum. All animal experiments were approved by The University of Melbourne Animal Ethics committee (#21555).

Destabilization of medial meniscus (DMM)-induced OA model

The mouse destabilization of medial meniscus (DMM)-induced OA model is the most widely used post-traumatic OA model. Mice were anesthetized with 4% isoflurane mixed with oxygen at a flow rate of 0.5 L/min, followed by 2% isoflurane for maintenance. DMM-induced OA was then initiated by cutting the ligament that attaches the medial meniscus to the tibia [11] in the left knee. Buprenorphine (0.1 mg/kg) was administered subcutaneously before the surgery and eight hours post-surgery to alleviate surgery-associated discomfort. Mice were killed by CO₂ asphyxiation for histologic analysis 12 weeks post DMM induction. For experiments examining the role of obesity, mice were fed with 60% high-fat diet (HFD) (SF02-006, Specialty Feeds) for 8 weeks prior to the initiation of the OA model [11]. All mice fed with control diet showed a slow steady increase in their body weight (approximately 15% weight increase by the end of the experiment), while all mice fed with 60% high fat diet had significant weight increase prior to the induction of the DMM OA model. For mAb treatments, mice received intraperitoneal (i.p.) injection twice weekly of 25 mg/kg, 5 mg/kg or 1 mg/kg of anti-CCL17 (B293) mAb or 25 mg/kg of isotype control antibody (BM4). These treatments commenced at week 9 for experiments with a control diet and week 7 for experiments with a HFD.

Behavioural pain assessment

As an indicator of pain-like behaviour (referred to as pain throughout), a ratio between two knees (left vs. right) was used as a measure of static weight-bearing joint pain using an incapacitance meter (IITC Life Science Inc, USA) and expressed as percentage weight on the contralateral hindlimb. Values between 90 and 100 for the percentage (%) weight on the contralateral hindlimb are within a normal range of variation (i.e. no pain); a value below 90 indicates pain [8–13]. Mice were acclimatized to the incapacitance meter on at least three occasions prior to the commencement of the experiment. Three measurements were taken for each time point and averaged. All equipment was pre-calibrated.

Histopathologic assessment of arthritis

For Safranin-O and Fast green stained knee sections, articular cartilage damage, osteophyte maturity and osteophyte size were scored using The Osteoarthritis Research Society International (OARSI) scoring system [11,14]. For H&E-stained knee sections, the degree of synovitis was assessed (Supplemental Materials and Methods in S1 File). All scores were acquired in a blinded manner by two independent investigators. All images are representative images of the means and taken from the mid-joint region.

CCL17 ELISA

Sera were collected. Mouse CCL17 (R&D Systems) was measured by ELISA as per manufacturer’s instructions.

Oral glucose tolerance test and insulin tolerance test

Following 4 weeks of HFD, WT mice received intraperitoneal (i.p.). injection twice weekly of either B293 (25 mg/kg) or BM4 (25 mg/kg). WT and GM-CSF^-/- mice received an oral gavage of D-glucose (2 g/kg body mass) and i.p. injection of insulin (1 U/kg body mass, Actrapid) for the oral glucose test (oGTT) and the insulin tolerance test (ITT), respectively. Blood obtained from a tail nick was assessed for glucose (Accu-Chek, Victoria, Australia) before and throughout the tests as indicated. Additional blood was obtained before and at 15 and 30 min after glucose administration. The blood was spun (2,500 g, 5 min, 4°C) and the plasma used for later analysis of plasma insulin by ELISA (#90080, Crystal Chem, Elk Grove Village, IL).

Quantification of liver triglyceride levels

Liver triglycerides were extracted and were determined by colorimetric assay (Triglycerides GPO-PAP; Roche Diagnostics) as previously described [15].

Statistical analysis

For longitudinal incapacitance meter measurements, linear mixed effects models were used for repeated measures over time and a Dunnett post-hoc test was used when comparing between treatments (i.e. B293 vs. BM4) or between genotypes (WT vs. GM-CSF^-/- mice). For histology measurements, the Shapiro-Wilk and Levene’s test was used for assessing normality of data and homogeneity of variance. Due to the violation of normality assumption, which did not improve after the logarithmic or square root transformation, a non-parametric Kruskal-Wallis test, following Benjamini and Hochberg adjustment for p-values in multiple comparison, was performed to examine differences in mean histopathologic arthritis assessments, osteophyte scores and synovitis. Statistical analysis was performed using GraphPad Prism Software (10.1.2). and based on a 0.05 significance level. Plots were generated using GraphPad Prism Software (10.1.2). Data were plotted as means with corresponding 95% confidence interval (CI).

Results

Therapeutic efficacy of CCL17 neutralization in experimental OA

DMM-induced OA

We explored the therapeutic effect of increasing doses of the anti-CCL17 (B293) mAb on the development of established DMM-induced OA pain-like behaviour and disease. The neutralizing potency of purified B293 was first assessed in vitro (see Materials and Methods and Supplemental Materials and Methods in S1 File). Following the initiation of DMM-induced OA [11], all WT mice developed significant pain-like behaviour by week 9 (p = 0.004); following sham surgery WT mice showed no pain nor disease development [11]. Following the administration of BM4 (isotype control, 25 mg/kg) (week 9), WT mice continued to exhibit pain-like behaviour until the cessation of the experiments at week 12 (Fig 1A). On the other hand, WT mice treated with B293 (25 mg/kg or 5 mg/kg), but not with B293 (1 mg/kg), effectively ameliorated DMM-induced OA pain-like behaviour (Fig 1A and S1 Table). In comparison to WT mice treated with BM4, mice treated with B293 (25 mg/kg or 5 mg/kg) also showed at termination reduced cartilage damage (Fig 1B and 1C) with the highest B293 dose also resulting in reduced osteophyte maturity and size (Fig 1D and 1E); mice treated with B293 (1 mg/kg) had comparable histologic scores to mice treated with BM4 (Fig 1B–1E). At termination, no differences were seen for the synovitis score between treatment groups (Fig 1F). Serum CCL17 concentrations were significantly elevated in mice treated with B293 (25mg/kg) (Fig 1G), possibly due to the reported accumulation of cytokine/anti-cytokine complexes [16,17].

Fig 1 — The DMM-induced OA model was initiated in WT male mice. The administration of B293 (anti-CCL17 mAb; 25mg/kg, 5mg/kg or 1mg/kg) or BM4 (isotype control; 25mg/kg) began at week 9 twice weekly. (A) Change in weight distribution (pain-like behaviour) over time. Numerical summaries are provided in detail in S1 Table. (B) Representative histologic pictures of knee joints (Safranin O/fast green stain, original magnification x4) for cartilage damage (indicated by black arrows) and osteophyte formation (indicated by orange arrows) (week 12). (C) DMM-induced OA cartilage damage, (D and E) osteophyte maturity and size, and (F) synovitis were quantified. (G) Sera were collected at week 12 for CCL17 quantification via ELISA. Results are expressed as the mean ± 95% CI; n = 10/treatment group; F, femur; T, tibia. Scale bar indicates 100μm. *represents p-values between BM4 vs. B293 (25mg/kg) or B293 (5mg/kg).

Thus, therapeutically administered B293 dramatically suppressed DMM OA pain-like behaviour and reduced the disease severity.

HFD-exacerbated DMM-induced OA

In the DMM-induced OA model, HFD-fed mice demonstrated earlier pain and more severe disease [11,18–20], which we have termed HFD-exacerbated OA [11]. We have recently provided some clinical and preclinical evidence in Ccl17 gene deficient mice for CCL17 involvement in HFD-exacerbated OA [11]. Given the data above and obesity as a comorbidity for OA [21], we therefore tested whether therapeutic administration of B293 would be as effective in ameliorating DMM-induced OA in HFD-fed mice. Consistent with the literature [11,18–20], following the initiation of DMM-induced OA, HFD-fed WT mice developed significant pain-like behaviour by week 7 (p = 0.0043) (Fig 2A), which is earlier than for the experiment whose data are shown in Fig 1, and developed more severe OA disease at termination, as judged by summed histologic score (Fig 2B and 2C); following sham surgery HFD-fed WT mice again showed no pain nor disease development [11]. HFD-fed mice treated with B293 (25 mg/kg or 5 mg/kg) showed reversal of pain-like behaviour compared with mice treated with BM4 or B293 (1 mg/kg) (Fig 2A and S2 Table). HFD-fed WT mice treated with the two highest doses of B293 also showed at termination reduced cartilage damage (Fig 2B and 2C) and, consistent with the results in control diet-fed mice, only the highest dose of B293 reduced osteophyte maturity and size compared with the other treatment groups (Fig 2D and 2E). At termination, no differences were seen for the synovitis score between treatment groups (Fig 2F). There was again an increase in serum CCL17 levels in response to B293 administration (Fig 2G), which is consistent with the data shown in the experiment above (Fig 1G).

Fig 2 — WT male mice were fed with 60% high-fat diet (HFD) for 8 weeks, after which DMM-induced OA was initiated. The administration of B293 (anti-CCL17 mAb; 25mg/kg, 5mg/kg or 1mg/kg) or BM4 (isotype control; 25mg/kg) began at week 7 twice weekly. (A) Change in weight distribution (pain-like behaviour) over time. Numerical summaries are provided in detail in S2 Table. (B) Representative histologic pictures of knee joints (Safranin O/fast green stain, original magnification x4) for cartilage damage (indicated by black arrows) and osteophyte formation (indicated by orange arrows) (week 12). (C) DMM-induced OA cartilage damage, (D and E) osteophyte maturity and size, and (F) synovitis were quantified. (G) Sera were collected at week 12 for CCL17 quantification via ELISA. Results are expressed as the mean ± 95% CI; n = 10/treatment group; F, femur; T, tibia. Scale bar indicates 100μm. *represents p-values between BM4 vs. B293 (25mg/kg) or B293 (5mg/kg).

Together, these data demonstrate that therapeutically administered B293 also dramatically suppressed the HFD-exacerbated DMM-induced early OA pain-like behaviour and reduced the disease severity.

CCL17 and HFD-induced metabolic changes

CCL17 expression has been reported to be dependent on GM-CSF in a number of clinical [22–24] and preclinical studies [8–12,25,26]. More specifically, we have prior evidence, using gene deficient mice, suggesting that GM-CSF and CCL17 are both involved in obesity-associated spontaneous joint damage and the elevation of inflammatory mediators in the synovial tissue [11], which could help explain their involvement in obesity-exacerbated experimental OA. Consistent with other metabolic studies using GM-CSF^-/- mice [27–30], we also observed that HFD-fed GM-CSF^-/- male mice were found to be heavier than their WT counterparts (Fig 3A) and they showed improved glucose tolerance and had reduced plasma insulin levels during fasting and the oGTT (Fig 3B); insulin sensitivity, however, was similar between the genotypes as indicated by the ITT (Fig 3C). Additionally, liver triglyceride levels were found to be similar between the genotypes (Fig 3D). Based on these data, we explored the effects of anti-CCL17 mAb therapy on these obesity comorbidities.

Fig 3 — WT and *GM-CSF*^-/- male mice were fed with 60% high-fat diet (HFD) for 8 weeks. (A) Absolute body weight (g). (B) Plasma glucose and insulin levels during oGTT. (C) Plasma glucose levels during ITT. (D) Triglyceride levels in liver. Results are expressed as the mean ± 95% CI; n = 9/group. *p<0.05, **p<0.01, WT vs. *GM-CSF*^-/- mice.

Mice were fed with a HFD for 4 weeks to induce mild obesity and glycemic dysregulation [31,32]. They were then treated with B293 (25 mg/kg) or BM4 (25 mg/kg) for a further 4 weeks, while being maintained on the HFD. Serum CCL17 levels were again increased in mice treated with B293, indicating the effectiveness of the neutralization (Fig 4A). Both treated groups showed a similar steady increase in body weight (Fig 4B), comparable responses during the oGTT and ITT (Fig 4C and 4D) and similar liver triglyceride content (Fig 4E). Together, these data suggest that CCL17 does not regulate body mass gain or obesity-associated metabolic changes in mice fed a HFD.

Fig 4 — WT male mice were fed with 60% high-fat diet (HFD) for 8 weeks with B293 (anti-CCL17 mAb; 25mg/kg) or BM4 (isotype control; 25mg/kg) being administered at week 4 twice weekly. (A) Sera were collected at week 8 for CCL17 quantification (ELISA). (B) Absolute body weight (g). (C) Plasma glucose and insulin levels during oGTT. (D) Plasma glucose levels during ITT. (E) Triglyceride levels in liver. (E) Sera were collected for CCL17 quantification (ELISA). Results are expressed as the mean ± 95% CI; n = 5/treatment group.

Discussion

We have reported using gene-deficient male mice that CCL17 is involved in pain-like behaviour and disease in a number of OA mouse models in lean mice [11,12,25] and that this involvement is conserved in obesity-exacerbated OA models [11]. In this study, we extended these observations by exploring the therapeutic effects of targeting CCL17 using different doses of a neutralizing mAb in both DMM-induced OA and HFD-exacerbated DMM-induced OA.

The DMM surgical model in mice is a frequently used chronic OA model, with clinical relevance particularly for post-traumatic OA [33]. Following the surgical induction, mice develop moderate histologic features of OA (i.e. cartilage damage, osteophyte formation and synovitis). In the current study, we found that increasing doses of an anti-CCL17 mAb inhibit DMM-induced pain-like behaviour and reduce disease severity in male mice. Such benefit is consistent with our previous study using a single dose of a commercially available neutralizing anti-CCL17 mAb (MAB529) in a collagenase-induced OA model [12,25]. We also observed a similar therapeutic effect for HFD-exacerbated DMM-induced OA pain-like behaviour and disease in male mice. Together, these independent studies using different antibodies targeting different CCL17 epitopes and models of OA highlight the therapeutic potential of anti-CCL17 mAbs for the treatment of OA. Although the mode of action of CCL17 was not explored in the current study, CCL17-driven pain-like behaviour was previously shown to be modulated by the inhibition of neuropeptides/neurotrophins and eicosanoids [10] and Ccl17 gene deficiency in OA synovial tissues also led to reduced gene expression of cyclooxygenase-2, an enzyme responsible for eicosanoid production [11]. One limitation of this study is that static weight bearing measurement as a pain readout does not fully recapitulate the pain phenotype observed in clinical trials. Another limitation is that this study only included male mice. It has been reported that DMM-induced OA progresses less in female mice [34]. Future studies using anti-CCL17 mAb therapy should examine other measurements of pain-like behaviour as well as examining the efficacy of anti-CCL17 mAb therapy in female mice. Interestingly, accumulation of circulating CCL17 in response to the administration of B293 was observed in both OA models tested above, consistent with other reports of increased circulating cytokine/mAb complexes following anti-cytokine mAb therapies (e.g. anti-IL-6 and anti-GM-CSF mAbs [16,17]).

We have previously shown that mice fed with HFD have (i) elevated CCL17 levels in both sera and synovial tissue, (ii) spontaneous joint pathology and (iii) elevated cyclooxygenase-2 levels in synovial tissue, with the last two readouts being dependent on CCL17 [11]. We have also reported that obese knee OA patients have elevated circulating CCL17 levels and that synovial CCL17 gene expression correlated with body mass index [11], the circulating CCL17 levels confirming what has been reported recently in morbidly obese patients [35]. Obesity leads to the metabolic syndrome and is known to contribute to OA severity [36]. In an attempt to gain insights as to whether CCL17 could be involved in obesity-induced metabolic changes, we used the highest dose of B293 tested in this study in a model of HFD-induced obesity. While we observed some improvements in glucose tolerance in GM-CSF^-/- mice (Fig 3), consistent with the literature [27–30], we did not see an improved glucose response in obese mice treated with B293, as judged by the oGTT and ITT (Fig 4)–these data are interesting given the reported dependence on CCL17 on GM-CSF actions in a number of studies [8–12,22–26]. This lack of effect of B293 treatment on obesity-induced metabolic changes could possibly be due to a sub-optimal dosing protocol for these readouts.

A recent Phase I clinical trial indicates that an anti-CCL17 mAb has shown efficacy as an analgesic in patients with knee OA pain, demonstrating favorable safety and tolerability profiles [37]. While long-term use of analgesics can be associated with radiographic progression of knee OA and an increased risk of future knee replacement [38], it is likely that anti-CCL17 mAb therapy may not have these adverse effects since our preclinical data indicate that CCL17 inhibition can provide both analgesic and cartilage protective benefits (see also references [12,39]). We suggest from our data above that CCL17 could be a therapeutic target in OA patients following joint injury alone or who are also obese for both pain and disease progression. It could also be a target in other diseases where CCL17 is expressed and where obesity is a risk factor, for example, asthma [40], and where serum levels of CCL17 are significantly elevated in the obese group [11].

Supporting information

S1 File. Supplemental materials and methods.

(DOCX)

pone.0317399.s001.docx^{(23.3KB, docx)}

S1 Table. Between-group mean differences in incapacitance meter analyses.

(DOCX)

pone.0317399.s002.docx^{(15KB, docx)}

S2 Table. Between-group mean differences in incapacitance meter analyses.

(DOCX)

pone.0317399.s003.docx^{(15.6KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

KMCL, TL, AAA and JAH were supported in part by funding from the University of Melbourne and CSL (Grant no. Lex34425). SNK and MJW were supported by the University of Melbourne.

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PLoS One. doi: 10.1371/journal.pone.0317399.r001

Decision Letter 0

Xindie Zhou

11 Nov 2024

PONE-D-24-36038Therapeutic blockade of CCL17 in obesity-exacerbated osteoarthritic pain and diseasePLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments :

Reviewer's comments form Reviewer 3#:

The current study aims to determine the therapeutic effects of B293 in the DMM-induced OA model. The authors have provided data on IP administration of B293 in mice and have looked at OA severity at 12-week post DMM under different conditions; however, there are many controls missing from this study. The baseline data for 9 week or 7 week for both the studies are not provided. Overall the study is premature and lacks convincing data to support the conclusion. Below are some specific comments.

The manuscript is well written but can be improved by correcting for English, for example, in lines 69-70; “mice was”; line 110 “a Dunnett post-hoc test used when”

Abstract and introduction; CCL17 gene KO mice do not develop OA or develop less severe OA?

In the materials and methods, please provide the exact time of treatment instead of “when pain like behavior was evident”

I suggest to change from “pain-like” to “pain-related” behavior.

For statistical analyses, did the author test data distribution before applying non-parametric test?

The authors have not included females in this study, which is a major limitation. There are reports which prove that female do not develop or do develop OA in DMM model.

Results:

Line 118-124: This whole paragraph is based on data not shown. the CCL13 mAb mediated inhibition is important for this study. If the authors believe that this is important to be discussed in a separate paragraph under a subheading, the authors should include the data in the manuscript instead of saying data not shown.

Line 126, authors are proposing to study the effect of B293 on the development of OA in DMM model, but administer B293 after 9 week of DMM. Multiple studies have shown that mice already develop moderate to severe OA at 8-week post DMM.

Authors have not included any data on the severity of OA at 8-week post DMM to set a baseline for the study. This time point is important to understand if B293 is inhibiting further degradation of the cartilage or the protection is through some other mechanism.

There is no sham control included in this study.

There is no no-treatment control included in this study.

The whole joint images make it difficult to see if there is any damage to the cartilage in their DMM model. The difference in the safranin o staining intensity appears be due to overstaining of safranin o as can be seen by the strong staining of growth plate which cant be seen in BM4 group image. Authors should provide better images to show cartilage damage, osteophytes.

This reviewer does not understand the rationale of combining DMM with HFD-induced OA. HFD-induced OA fits well with aging-related spontaneous OA.

Same comments for images in figure 2.

This reviewer does not understand why the results for “CCL17 and HFD-induced metabolic changes” were put in the supplementary figure.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: This is an interesting report on CCL-17 blockade in an experimental model of OA in mice (DMM°. Previous results in mice KO for CCL-17 and in a collagenase induced OA model have shown promising results.

Several points need to be clarified.

1. How many mice per group have been tested?

2. It is not made clear in DMM induced OA what was the evolution of BMI in the different groups. This is an important bias.

3. Only male mice have been selected meaning that this strategy does not work in female mice. Please comment (at least) in the discussion and explain why.

4. It does not seem that the level of pain behaviour in the first weeks is different between non fed and fed mice, while it is assume that pain may be increased in HFD-exacerbated DMM- OA ? Please comment.

5. In the histopathologic assessment where the cartilage lesions predominated?

6. Please provide information on the synovial membrane inflammation.

7. Do the authors look to anti-anti CCL-17 Ab?

General remarks

- Please comment on the risk of a long-term dramatic analgesia (according to what we have learned from the anti-NGF model) on the cartilage potential degradation.

- Please comment of the potential risk (infections) of CCL-17 inhibition.

Reviewer #2: I enjoyed reading this manuscript. Congratulations for this admirable work. These findings are very interesting indeed for relieving the OA pain in the future. I would prefer a more thorough explanation as a reader to comprehend the increased levels of CCL17 in the sera of patients with osteoarthritis despite the use of antibodies against CCl17. What is the reason that immune complexes may lead to these high levels in the sera.

**********

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Reviewer #1: No

Reviewer #2: Yes: Konstantina Bounia

**********

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PLoS One. 2025 Jan 16;20(1):e0317399. doi: 10.1371/journal.pone.0317399.r002

Author response to Decision Letter 0

17 Dec 2024

Response to Reviewer #1: Many thanks for your thorough review and for the opportunities for clarification. We believe we have responded appropriately to your constructive comments leading to an improved manuscript.

1. How many mice per group have been tested?

Response to Reviewer: We already had indicated in the Figure legends that there are 10 mice per treatment group (see Figure Legends, lines 395, 407, 412 and 419).

2. It is not made clear in DMM induced OA what was the evolution of BMI in the different groups. This is an important bias.

Response to Reviewer: All mice fed with control diet showed a slow steady increase in their body weight (approximately 15% weight increase by the end of the experiment), while all mice fed with 60% high fat diet had significant weight increase prior to the induction of the DMM OA model (Figs 3 and 4, and Materials and Methods); mice continued to increase their body weight until the end of the experiment.

3. Only male mice have been selected meaning that this strategy does not work in female mice. Please comment (at least) in the discussion and explain why.

Response to Reviewer: We have added a comment that only male mice were used in this study with a suggestion that future studies should include female mice to assess the efficacy of anti-CCL17 mAb therapy (see lines 239-243).

Response to Reviewer: We have previously reported that, in comparison to control diet-fed DMM OA mice, HFD-fed DMM OA mice develop earlier onset of pain-like behaviour (around week 9 vs. week 7, respectively) and had more severe disease (PMID 37225052). This background data provided the rationale for the current study (see lines 175-178). The pain-like behaviour between control diet-fed (Fig. 1) and high-fat diet-fed mice (Fig. 2) was similar in the first few weeks post DMM OA induction, which is consistent with our previous study (PMID 37225052).

5. In the histopathologic assessment where the cartilage lesions predominated?

Response to Reviewer: In this study, the scoring criteria for cartilage lesions in regions of lateral tibia, latera femur, medial tibia and medial femur are in accordance with the recommendations from OARSI, which is often used in the OA DMM literature (see, for example, PMID 33609695 and PMID 29434267).

6. Please provide information on the synovial membrane inflammation.

Response to Reviewer: We have previously reported that DMM OA wild-type mice develop some synovial membrane inflammation (i.e. synovitis) and that CCL17 KO mice have no effect on this readout (PMID 37225052). In response, we have now provided synovitis scores in the updated Figures (see new Figs. 1F and 2F). Consistently, mice treated with B293 showed no effects on synovitis compared to mice treated with BM4 (see lines 168-169 and 190-191). Relevant scoring methods are now also included in the Materials and Methods (lines 121-122) and in the Supplemental Materials and Methods (lines 51-55).

7. Do the authors look to anti-anti CCL-17 Ab?

Response to Reviewer: It would be very interesting to know if auto-antibodies against anti-CCL17 Ab were generated. However, we did not have an assay to explore the levels of anti-anti-CCL17 Ab in the sera.

General remarks

- Please comment on the risk of a long-term dramatic analgesia (according to what we have learned from the anti-NGF model) on the cartilage potential degradation.

Response to Reviewer: We have added comments on the risk of long-term analgesia on the potential cartilage degradation (see lines 263-267).

- Please comment of the potential risk (infections) of CCL-17 inhibition.

Response to Reviewer: We have added comments about safety profile of CCL17 inhibition (see lines 262-263).

Response to Reviewer #2: Many thanks for your positive comments. With regards to the elevated circulating levels of CCL17 in response to anti-CCL17 therapy, we presume that the CCL17 epitope recognized by the detection antibody from the mouse CCL17 ELISA kit (R&D Systems) is different to the binding epitope of B293; a similar approach is well described in the literature (see, for example, references 16 and 17) as a tool to monitor the effectiveness of a neutralizing anti-cytokine monoclonal antibody as a therapy.

Reviewer's comments from Reviewer 3#:

Response to Reviewer: Many thanks for your thorough review and for the opportunities for clarification. We believe we have responded appropriately to your constructive comments leading to an improved manuscript.

The manuscript is well written but can be improved by correcting for English, for example, in lines 69-70; “mice was”; line 110 “a Dunnett post-hoc test used when”

Response to Reviewer: Many thanks for pointing it out and we have rectified these mistakes.

Abstract and introduction; CCL17 gene KO mice do not develop OA or develop less severe OA?

Response to Reviewer: We have shown that CCL17 gene KO mice were protected from pain-like behaviour and developed less OA disease (PMID 37225052). This information has now been included (lines 25-26 and 64-66).

In the materials and methods, please provide the exact time of treatment instead of “when pain like behavior was evident”

Response to Reviewer: The exact time of treatment has now been included (lines 107-108).

I suggest to change from “pain-like” to “pain-related” behavior.

Response to Reviewer: Thank you for this reasonable suggestion. However, we have used “pain-like” behaviour in our prior publications (see, for example, PMID 32028021 and PMID 37225052) and, for reasons of consistency, we prefer to continue using this term. Other research groups also use our terminology (see, for example, PMID 23169679).

For statistical analyses, did the author test data distribution before applying non-parametric test?

Response to Reviewer: Yes, the test of data distribution was performed using a Shapiro-Wilk and Levene’s test. This information is now available in Material and Methods (lines 143-145).

The authors have not included females in this study, which is a major limitation. There are reports which prove that female do not develop or do develop OA in DMM model.

Response to Reviewer: We agree that the exclusion of female mice is a major limitation – this exclusion is now mentioned in the Discussion (lines 239-243).

Results:

Line 118-124: This whole paragraph is based on data not shown. the CCL17 mAb mediated inhibition is important for this study. If the authors believe that this is important to be discussed in a separate paragraph under a subheading, the authors should include the data in the manuscript instead of saying data not shown.

Response to Reviewer: Many thanks for this comment. In response, we have now moved this paragraph into Materials and Methods as information about the purification and the inhibitory effects of B293 (see lines 72-87).

Response to Reviewer: We agree that moderate to severe OA can be observed at 8 weeks post DMM. However, one of the aims of our study was to determine if CCL17 neutralization would affect both pain and disease in OA mice; therefore the chosen time point for anti-CCL17 mAb administration was based on when pain is robustly evident in our particular mice (i.e. 9 weeks post DMM surgery) to ensure that the therapeutic inhibitory effect on OA pain is clearly captured.

Response to Reviewer: We agree that having data on the severity of OA at 8-week post DMM to set a baseline would be ideal to indicate a reduction in the further degradation of cartilage. However, our data showed that B293 treatment commencing at week 9 led to reduced cartilage degradation at termination, which we have now clarified (see lines 165 and 187).

There is no sham control included in this study.

Response to Reviewer: We have shown previously that, in comparison to mice induced with DMM OA, mice with sham surgery showed no disease nor pain-like behaviour development (PMID 37225052). Given that the aim of this study was to focus on the treatment efficacy towards OA pain-like behaviour and disease, a sham control group was therefore not included again. However, we agree that ideally a sham control group should be included as a comparison. We have now included this information (see lines 159-160 and 184-185).

There is no no-treatment control included in this study.

Response to Reviewer: In this study, we used BM4 (25mg/kg) as a treatment control to assess the efficacy of different doses of B293 (see Figures 1 and 2). In previous publications (PMID 29622035 and PMID 32028021), we have found no difference in pain-like behaviour nor disease severity between PBS-treated and isotype control-treated groups; importantly, we observed differences between BM4-treated and B923-treated mice, indicating a role for CCL17.

The whole joint images make it difficult to see if there is any damage to the cartilage in their DMM model. The difference in the safranin o staining intensity appears be due to overstaining of safranin o as can be seen by the strong staining of growth plate which can’t be seen in BM4 group image. Authors should provide better images to show cartilage damage, osteophytes.

Response to Reviewer: As requested, better histologic images have now been included in Figure 1B. We have also indicated cartilage damage and osteophytes with black and orange arrows, respectively (see Figure 1B). As indicated in the Supplemental Materials and Methods, the OARSI scoring system is mainly based on the intactness of cartilage surface with a minor component being the safranin O staining intensity.

This reviewer does not understand the rationale of combining DMM with HFD-induced OA. HFD-induced OA fits well with aging-related spontaneous OA.

Response to Reviewer: Our rationale for this study is to determine the efficacy of anti-CCL17 mAb therapy in an OA mouse model, namely the DMM-induced OA (see Figure 1). Given that obesity is known as a comorbidity for OA (PMID 34405518) and, based on our prior evidence implicating CCL17 in obesity-exacerbated DMM-induced OA (PMID, we therefore sought to assess also the efficacy of anti-CCL17 mAb therapy in this context with this information being now included (see lines 175-180)

Same comments for images in figure 2.

Response to Reviewer: As requested, better histologic images have now been included in Figure 2. We have also indicated cartilage damage and osteophytes with black and orange arrows, respectively (see Figure 2B).

This reviewer does not understand why the results for “CCL17 and HFD-induced metabolic changes” were put in the supplementary figure.

Response to Reviewer: As recommended, Figures associated with “CCL17 and HFD-induced metabolic changes” have now become main Figures (see new Figures 3 and 4).

Attachment

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PLoS One. doi: 10.1371/journal.pone.0317399.r003

Decision Letter 1

Xindie Zhou

29 Dec 2024

Therapeutic blockade of CCL17 in obesity-exacerbated osteoarthritic pain and disease

PONE-D-24-36038R1

Dear Dr. Lee,

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Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0317399.r004

Acceptance letter

Xindie Zhou

7 Jan 2025

PONE-D-24-36038R1

PLOS ONE

Dear Dr. Lee,

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Associated Data

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Supplementary Materials

S1 File. Supplemental materials and methods.

(DOCX)

pone.0317399.s001.docx^{(23.3KB, docx)}

S1 Table. Between-group mean differences in incapacitance meter analyses.

(DOCX)

pone.0317399.s002.docx^{(15KB, docx)}

S2 Table. Between-group mean differences in incapacitance meter analyses.

(DOCX)

pone.0317399.s003.docx^{(15.6KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0317399.s004.docx^{(27.2KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Therapeutic blockade of CCL17 in obesity-exacerbated osteoarthritic pain and disease

Kevin Ming-Chin Lee

Tanya Lupancu

Stacey N Keenan

Georgina Bing

Adrian A Achuthan

Mark Biondo

Kim Gia Lieu

Matthew J Watt

Eugene Maraskovsky

Bronwyn A Kingwell

John A Hamilton

Roles

Abstract

Objectives

Design

Results

Conclusions

Introduction

Materials and methods

Anti-CCL17 (B293) monoclonal antibody and isotype control purification

Tango™ β-Arrestin recruitment reporter assay

Mice

Destabilization of medial meniscus (DMM)-induced OA model

Behavioural pain assessment

Histopathologic assessment of arthritis

CCL17 ELISA

Oral glucose tolerance test and insulin tolerance test

Quantification of liver triglyceride levels

Statistical analysis

Results

Therapeutic efficacy of CCL17 neutralization in experimental OA

DMM-induced OA

Fig 1. Therapeutic blockade of CCL17 ameliorates DMM-induced OA pain and disease.

HFD-exacerbated DMM-induced OA

Fig 2. Therapeutic blockade of CCL17 ameliorates HFD-exacerbated DMM-induced OA pain and disease.

CCL17 and HFD-induced metabolic changes

Fig 3. The effects of GM-CSF gene deficiency on obesity-associated metabolic changes.

Fig 4. The effects of CCL17 neutralization on obesity-associated metabolic changes.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Xindie Zhou

Roles

Author response to Decision Letter 0

Decision Letter 1

Xindie Zhou

Roles

Acceptance letter

Xindie Zhou

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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