Topical CCL3 Is Well-Tolerated and Improves Liver Function in Diabetic Mice: Evidence from a 14-Day Toxicity Study

1. Introduction

Diabetic foot ulcers (DFUs) are one of the most prevalent and debilitating consequences of diabetes, which account for 60-75.5% of all lower extremity amputations (LEA) and cost ~$9-12 billion in the annual care in the United States alone [1]. The staggering number of ulcers that progress to LEA underscores the inadequacy of current treatments and the urgent need for innovative approaches. Infection is a major co-morbidity and a driver of chronic state and LEA in DFUs [1]. Unlike chronic ulcers that are locked in a state of persistent and non-resolving inflammation [2,3], inflammatory responses are significantly diminished during the acute phase of healing early after injury, making diabetic wounds susceptible to infection with pathogenic bacteria such as Pseudomonas aeruginosa which further exacerbate tissue damage and impair tissue repair through the production of their virulence factors that can further dampen inflammatory responses and adversely affect a multitude of cellular processes underlying tissue repair [4,5,6,7,8,9,10,11].

Neutrophils are the first line of defense against invading pathogens, immediately migrating to the site of injury to facilitate bacterial clearance by phagocytosis, the generation of reactive oxygen and nitrogen species, antimicrobial peptides, and the development of neutrophil extracellular traps [12,13]. However, antimicrobial and bactericidal functions are impaired in neutrophils under hyperglycemia and diabetic conditions, rendering them ineffective to control infection [14]. In addition, neutrophils isolated from the blood of diabetic patients exhibit impaired chemotactic responses, leading to reduced neutrophil trafficking into the wound and toward infection during the early stages of diabetic wound healing. This neutrophil dysfunction is attributed to impaired signaling through the formyl peptide receptor (FPR), which normally mediates the initial wave of neutrophil trafficking to injury and infection [4,15]. Importantly, some auxiliary chemokine receptors, such as CCR1, remain functional under diabetic conditions; however, they are not activated in diabetic wounds in the early stages due to low expression of their endogenous ligands [4,6].

There are currently no approved biologic therapies that target infection in diabetic wounds. The only approved biologic therapy for DFUs is Becaplermin gel (0.01% rhPDGF-BB). However, Becaplermin has limited efficacy in stimulating healing and is not effective against infection [16,17]. Antibiotics are routinely included in the management of diabetic ulcers [18,19], but they are linked to emergence of antibiotic resistance, toxicity, and interference with tissue repair [20,21,22]. These reports underscore the urgent need for novel therapeutics (preferably antibiotic-free) to address infection in diabetic wounds. We have previously demonstrated that topical treatment with CCL3/MIP-1α immunomodulator reduced infection by >99% and improved healing in diabetic mice by bypassing impaired FPR signaling and restoring timely neutrophil trafficking, highlighting its potential in diabetic wound care [4]. However, regulatory approval of novel biologics requires comprehensive evaluation of their efficacy and toxicity in relevant animal models [23,24].

The present study investigated the toxicity profile of topical CCL3 wound therapy in diabetic mice, including physiological monitoring, biochemical and hematological analyses, and histopathological assessment of major organs. Our data demonstrate that topical CCL3 treatment was well tolerated and did not result in any cytotoxic effects. Intriguingly, it resulted in modest yet significant systemic benefits, including hepatoprotection and reduced inflammation. These results provide essential insights into the immunological safety and systemic tolerability of CCL3 therapy in diabetic wound models, supporting its advancement toward clinical application.

2.Material and Methods

2.1. Chemicals

Phosphate buffer saline (PBS) pH 7.4 (Gibco™), recombinant mouse CCL3/mip-1 alpha protein (Biotechne/R&D systems), ketamine, hydrochloride, xylazine, buprenorphine, sterile alcohol prep pads (Fisherbrand) biopsy punch (Acuderm, inc), 10% neutral buffered formalin (Epredia), xylene (Merck), hematoxylin (Epredia), eosin (Epredia™), Invitrogen mouse CRP ELISA kit, Invitrogen mouse IgG1 uncoated ELISA kit, Invitrogen mouse IgE ELISA kit.

2.2. Animals and Grouping

We used 8-week-old B6.BKS(D)-Lepr^db/J (db/db) obese diabetic mice (strain #00697; The Jackson Laboratory) as animal model in this study [25]. Heterozygous (db/+) mice served as normoglycemic controls. All animals were obtained from The Jackson Laboratory and housed in the University of California Davis Health Animal Vivarium. Mice were maintained under controlled environmental conditions (temperature: 22 ± 2 °C; relative humidity: 50 ± 10%) with a 12 h light/dark cycle and ad libitum access to standard chow and water. All experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC, Protocol #24063).

Mice were surgically wounded by 5-mm sterile biopsy punches as described [5,26]. They were then randomly assigned to four experimental groups (6 mice/group): 1) control (db/+ receiving PBS); 2) diabetic (db/db receiving PBS); 3) diabetic treated with CCL3 at low dose (1 µg per wound); And 4) diabetic treated with CCL3 at high dose (10 µg per wound). The schematic diagram of the research strategy is depicted in Figure 1. All wounds in the same mouse received the same treatment to prevent immune confusion that has been reported [27].

2.3. Wounding and Treatment

One day prior to the wounding (day -1), the dorsal hair was removed with an electric trimmer to expose the skin surface. On day 0, mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), followed by analgesia with buprenorphine (0.01 mg/kg) for pain management. The dorsal skin was sanitized by wiping with a sterile alcohol pre pad, and full-thickness excisional wounds (5-mm diameter) were made using a sterile biopsy punch. Following wounding, PBS or CCL3 (1 µg or 10 µg per wound) was applied topically based on group allocation. Mice were divided into control and treatment groups, wounded, and then treated with PBS or CCL3 topically (Figure 1). They were also observed every day for changes in behavior, body weight, and food consumption. To ascertain the systemic safety profile of CCL3 treatment, main organs were histologically evaluated, and blood was studied for hematological and biochemical markers at the end of the trial.

2.4. Monitoring and Clinical Observations

Following treatment, mice were observed for survival, movement, posture, and any signs of discomfort. Body weight and food intake were measured at the start of the trial and at regular intervals during the 14-day period. Daily observations were also made to note any behavioral changes as described [28] that could suggest systemic toxicity associated with CCL3 treatment.

2.5. Immunological, Hematological and Biochemical Analysis

To assess the systemic safety profile of CCL3 treatment in diabetic mice, immunological, hematological, and biochemical parameters were evaluated using established procedures [29]. On the 14 day post-injury and treatment, the mice were anesthetized, and blood was collected via cardiac puncture. For plasma collection, blood was drawn into BD Vacutainer™ collection tubes containing EDTA as an anticoagulant and centrifuged at 1000× g for 10 min at 8 °C, after which the supernatant plasma was collected. For serum separation, blood was collected in BD Microtainer™ serum separator tubes with gel, allowed to clot at room temperature for 30 min, and centrifuged at 4000× g for 10 min at room temperature to obtain serum [29]. Plasma and serum samples were prepared and sent to the Comparative Pathology Laboratory at the University of California Davis, School of Veterinary Medicine for extensive hematological and biochemical testing. Red and white blood cell counts (RBC & WBC), hemoglobin (Hb), platelet counts, and differential leukocyte distribution (monocyte, eosinophil, and basophil) were all analyzed using an automated hematology analyzer (Hemavet 950FS). The serum biochemistry was analyzed by Roche Cobas Integra 400 Plus, for indicators of liver and kidney functions including, Alanine Transaminase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), total bilirubin, Blood Urea Nitrogen (BUN), creatinine, protein levels (total protein and albumin), electrolytes, and glucose as described [30].

Furthermore, serum samples were analyzed for immunological parameters, including inflammatory and allergy markers such as, C-reactive protein (CRP), Immunoglobulin G1 (IgG1), and Immunoglobulin E (IgE), using enzyme-linked immunosorbent assay (ELISA) methodology.

2.6. Histopathological Analysis

Histopathological examination was used as a gold-standard method to visualize and confirm whether CCL3 treatment altered tissue architecture or caused signs of organ toxicity, providing direct insight beyond hematological and biochemical measurements. Following blood collection, the mice were euthanized, and key organs such as the liver, kidney, spleen, heart, and pancreas were taken and fixed in 10% neutral buffered formalin. The organs were processed, paraffin-embedded, sectioned at 4-5 µm, and stained with Hematoxylin & Eosin (H&E). Histological evaluation was performed under light microscopy, and semi-quantitative scoring was utilized to quantify necrosis, inflammation, and tissue structure [31]. Semi-quantitative scoring for necrosis and inflammation was performed according to previously published criteria [32]. Necrosis was graded on a scale of 0–5: 0 = no necrosis, 1 = minimal (<5% tissue), 2 = mild (5–20%), 3 = moderate (21–40%), 4 = severe (41–60%), and 5 = very severe (>60%). Inflammation was similarly graded on a scale of 0–5: 0 = no infiltrates, 1 = minimal infiltration (few scattered cells), 2 = mild (<10% tissue), 3 = moderate (10–30%), 4 = severe (31–50%), and 5 = very severe (>50%). Necrosis was graded on a scale of 0–5: 0 = no necrosis, 1 = minimal (<5% tissue), 2 = mild (5–20%), 3 = moderate (21–40%), 4 = severe (41–60%), and 5 = very severe (>60%). Inflammation was similarly graded on a scale of 0–5: 0 = no infiltrates, 1 = minimal infiltration (few scattered cells), 2 = mild (<10% tissue), 3 = moderate (10–30%), 4 = severe (31–50%), and 5 = very severe (>50%).

2.7. Statistical Analysis

One-way ANOVA was used to compare groups, followed by Tukey’s multiple comparison post hoc test. Statistical significance was determined as p < 0.05. Statistical differences in the figures are given as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****) and p ≥ 0.05 (not significant). All analyses were carried out with GraphPad Prism software (version 10.4.2). All results were reported as mean ± SEM. Of note, we evaluated normality and variance homogeneity for all datasets included in parametric analyses before proceeding to student’s t test and one way ANOVA by using GraphPad Prism (version 10.4.2). Normality was assessed using the Shapiro–Wilk test (n < 30), and homogeneity of variance was evaluated using the Brown-Forsythe test (n > 2). In all cases, the data met these assumptions.

3. Results

The experimental design is illustrated in Figure 1. Briefly, mice were surgically wounded using 5-mm sterile biopsy punches as previously described [5,26,33]. They were then randomly assigned to four experimental groups (6 mice per group): (1) control (db/+ receiving PBS); (2) diabetic (db/db receiving PBS); (3) diabetic treated with low-dose CCL3 (1 µg per wound); and (4) diabetic treated with high-dose CCL3 (10 µg per wound). Food intake, body weight, and any signs of mortality or behavioral changes were monitored over a 14-day period. On day 14, mice were euthanized, and their blood and major organs were collected for biochemical, immunological, and histological analyses to evaluate the systemic effects of CCL3 treatment on overall health.

3.1. Physiological and Behavioral Monitoring Following CCL3 Treatment

Following wound surgery on day 0, mice in all groups showed reduced mobility and decreased grooming behavior, which is consistent with acute post-surgical discomfort [34,35]. However, by day 2, behavioral patterns had recovered, and the animals resumed normal movement and feeding, with no further abnormalities noted. Nondiabetic control mice (db/+) consumed about 3-4 g of food per day, whereas diabetic mice (db/db) consumed significantly higher amounts (6.5-8 g) per day, which is consistent with increased appetite in db/db obese diabetic mice [36]. Of note, food intake was temporarily reduced in all groups following surgery on days 0 and 1, but this reduced appetite for food was temporary, and by day 2 the animals had resumed their normal eating behavior (Figure 2A). Compared to db/+ nondiabetic control mice, db/db diabetic mice continued to consume more food throughout the duration of this study regardless of their treatments. PBS-treated and CCL3-treated db/db mice did not differ significantly at either dose, indicating that topical CCL3 at 1 µg and 10 µg had no effect on feeding behavior. Throughout the study, db/db mice in all groups weighed significantly more (40-50 g) than nondiabetic db/+ mice (20-30 g) controls at baseline (day 0) (p < 0.0001). Despite loss of appetite on day 1, mice did not experience any significant weight loss following wounding procedure and their weights remained constant throughout this 14-day period (Figure 2B). Notably, the db/db groups treated with PBS and CCL3 did not differ significantly in their eating behavior or weight, indicating that CCL3 treatments were well tolerated and did not change metabolic status in mice (Figure 2B).

3.2. CCL3 Therapy Reduces Serum IgG1 in Diabetic Mice

We assessed serum biochemistry, hematology, immunological parameters, and circulating biomarkers in day 14 blood to determine whether CCL3 treatment adversely altered immune responses or caused systemic toxicity. In terms of immunological parameters associated with systemic inflammation, compared to nondiabetic control group, diabetic mice treated with PBS (mock) exhibited approximately 48.5% higher serum IgG1 levels (285.75 ± 86.27 µg/mL vs. 192.45 ± 13.51 µg/mL, p < 0.01); 125% higher IgE (207.35 ± 57.64 vs. 92.29 ± 22.03, p <0.01); and 335% higher C-reactive protein (CRP) levels (8.14 ± 1.86 vs. 1.83 ± 0.33, p <0.001) (Table 1). These findings are consistent with clinical reports showing that diabetic patients exhibit elevated serum levels of IgG1, IgE, and CRP, associated with increased risks of kidney nephropathy, allergic reactions, systemic inflammation, and diabetic retinopathy [37,38,39,40,41,42,43].

Interestingly, CCL3 treatment lowered serum IgG1 in diabetic mice to levels similar to those of nondiabetic controls (Figure 3A), suggesting a durable therapeutic effect of topical CCL3 treatment on systemic IgG1-associated inflammation. Of note, IgE and CRP levels in the diabetic groups treated with CCL3 were not significantly different compared to PBS-treated diabetic group (Table 1). These results indicate that CCL3 therapies do not adversely affect these immune biomarkers. Rather, they potentially have long-term beneficial impact on serum IgG.

3.3. CCL3 Treatment Confers Modest but Significant Hepatoprotective Benefits in Diabetic Mice

Liver enzymes—including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)—are frequently elevated in pre-diabetic and diabetic individuals and have been associated with obesity, visceral adiposity, dyslipidemia, increased diabetes risk, and metabolic dysfunction–associated steatosis liver disease (MASLD) [44,45,46,47,48,49]. In line with these observations, by day 14 post-treatment the PBS-treated diabetic (db/db) control mice exhibited markedly elevated serum ALT (275.65 ± 22.94 U/L), AST (247.58 ± 11.97 U/L), and ALP (217.00 ± 6.32 U/L) levels compared with the nondiabetic control group (ALT: 23.27 ± 1.33 U/L; AST: 83.39 ± 6.18 U/L; ALP: 97.93 ± 16.37 U/L) (Table 2 and Figure 4A). Interestingly, topical CCL3 treatment significantly reduced ALT levels in diabetic mice at both 1 µg (210.35 ± 11.43 U/L) and 10 µg (211.18 ± 22.02 U/L) compared with PBS-treated diabetic controls (p < 0.05; Figure 4A, and Table 2). While serum AST and ALP levels were significantly elevated in diabetic mice compared to nondiabetic mice, treatment with CCL3 did not alter these levels any further.

To further assess the impact of CCL3 on liver injury, we performed histological analysis of hepatic tissue (Materials & Methods). Compared with nondiabetic mice, diabetic control mice (db/db + PBS) exhibited marked steatosis, hepatocyte ballooning, disruption of normal lobular architecture, and elevated necrosis and inflammation scores (Figure 4A–D). These histopathological features are consistent with nonalcoholic fatty liver disease (NAFLD) and liver injury reported in patients with type 2 diabetes [50,51,52]. In contrast, mice treated with CCL3 at either dose showed reduced steatosis, attenuated hepatocyte ballooning, and preservation of hepatic architecture (Figure 4A–C). Collectively, these findings indicate that CCL3 treatment does not exacerbate liver injury and suggest a potential hepatoprotective effect of CCL3 in diabetic mice.

3.4. CCL3 Does not Cause Organ Toxicity: Histopathological Evidence

Kidney, spleen, pancreas, and heart are among other important organs that are adversely affected by diabetic condition [53,54,55,56,57,58]. We performed histopathological evaluation of these organs to assess potential systemic toxicity following topical CCL3 treatment in diabetic db/db mice. Representative hematoxylin and eosin–stained sections from liver, kidney, spleen, pancreas, and heart are shown, together with semi-quantitative scoring of necrosis and inflammation (Materials & Methods).

Renal histology in db/db + PBS mice revealed characteristic diabetic nephropathy–like changes [53], including tubular vacuolation and glomerular hypertrophy (Figure 5A). In contrast, kidneys from CCL3-treated mice displayed comparable histological features without additional structural damage. Quantitative scoring confirmed that necrosis and inflammation levels remained similar across all treatment groups, with no evidence of worsening renal pathology following topical CCL3 application (Figure 5B-C).

Diabetes causes significant histological changes in the spleen, leading to structural alterations like white pulp atrophy, reduced lymphoid nodules/germinal centers, decreased lymphocyte density, and altered immune cell distribution, often linked to increased oxidative stress and inflammation, resulting in impaired immune function [54]. The spleen of db/+ control mice showed normal histology, with clearly demarcated white pulp (lymphocyte-rich areas) and red pulp (erythrocyte-rich areas). In contrast, diabetic PBS treated control mice showed disrupted architecture with a lower white pulp percentage compared to non-diabetic control group (53.82 ± 2.94% vs. 69.11 ± 4.13%, p < 0.0001), indicating impaired immune organization (Figure 5D). CCL3-treated diabetic mice regardless of dose, showed a similar reduction in white pulp (57.33 ± 2.75% and 57.64 ± 4.9% respectively), with no statistical differences from db/db PBS control group, indicating that treatment did not impact splenic changes (Figure 5D-G).

Diabetic patients display significant histological changes in the pancreas, especially in the islets of Langerhans, including beta-cell loss, insulitis (inflammation in Type 1), and islet amyloidosis (protein deposits in Type 2), alongside potential exocrine tissue damage like fat accumulation and fibrosis, affecting both insulin production and overall pancreatic structure and function [55,56]. As expected, nondiabetic db/+ control mice had normal islet and acinar tissue, whereas db/db control mice showed islet distortion and vascular congestion (Figure 5G). Consistent with these data, diabetic mice showed significantly higher necrosis and inflammation scores than controls (Figure 5H-I, p < 0.0001). Semi-quantitative scoring showed no evidence of additional CCL3-associated pancreatic toxicity (Figure 5H-I).

Cardiomyopathy, and cardiovascular problems, including focal myofiber alterations have been reported in diabetic patients [57,58]. Consistent with these reports, cardiac sections from db/db + PBS mice exhibited myocardial disruption consistent with diabetic cardiomyopathy, including focal myofiber alterations and increased necrosis scores (Figure 5J-K). Importantly, topical CCL3 treatment did not increase myocardial rupture, necrosis, or inflammatory changes at the examined doses.

Collectively, histopathological analysis across multiple organs demonstrates that topical CCL3 treatment, even at 10-fold higher dose, does not induce systemic organ toxicity or exacerbate diabetes-associated tissue pathology, supporting its favorable safety profile in this model.

3.5. Assessment of Hematological and Serum Biochemical Parameters

To further evaluate the systemic safety of CCL3 therapy, serum biochemical and hematological parameters were assessed on day 14. As anticipated, glucose levels were significantly higher in db/db mice (402.48 ± 34.02 mg/dL) compared with db/+ controls (231.68 ± 24.41 mg/dL); however, CCL3 treatment did not further affect serum glucose levels (Table 2). Serum electrolytes (sodium, potassium, chloride, calcium, and phosphorus), as well as total protein and bilirubin concentrations, were similar between PBS- and CCL3-treated diabetic mice, indicating that CCL3 administration did not alter these parameters (Table 2).

White blood cell (WBC) counts were significantly elevated in db/db mice compared with their db/+ control counterparts (Table 3), a finding that is consistent with previously reported hematological alterations [59]. Importantly, administration of CCL3 at either the 1 µg or 10 µg dose did not further alter circulating WBC levels, indicating that CCL3 treatment does not exacerbate diabetes-associated leukocytosis. In addition, no significant differences were detected in hemoglobin concentrations or in the relative proportions of major leukocyte subsets, including neutrophils, lymphocytes, monocytes, eosinophils, and basophils, when comparing diabetic and nondiabetic mice or PBS-treated and CCL3-treated diabetic groups (Table 3). Consistent with these findings, platelet counts and mean corpuscular volume (MCV) remained comparable across all experimental conditions, and mean platelet volume (MPV) showed no detectable variation among groups (Table 3). Collectively, these data demonstrate that CCL3 administration at the doses tested does not induce measurable hematological abnormalities, supporting the conclusion that CCL3 therapy is not associated with hematological toxicity under these experimental conditions.

5. Conclusions

This study shows that topical administration of CCL3 in diabetic mice is safe and well-tolerated, with no evidence of systemic organ toxicity. CCL3 reduced hepatocellular injury markers, preserved hematological and biochemical homeostasis, and did not cause abnormal immune responses. CCL3 accelerates wound closure in db/db mice, demonstrating a dual role in restoring impaired immune function and promoting tissue repair. These new safety studies highlight CCL3 as a promising biologic for diabetic wound therapy, combining immunomodulatory efficacy with a favorable systemic safety profile, and represent an important step toward clinical translation.