Introduction

Several infectious agents, including viruses, parasites, bacteria, and fungi, tend to attack the human body and modulate the host for their own advantage. When this happens, the human immune system works to protect the body against foreign organisms through a network of cells, proteins, tissues, and organs1. The natural protective mechanism is also responsible for the maintenance of homeostasis through the removal of damaged cells. In some cases, dying cells may also be eliminated to prevent the issue of autoimmune diseases.

The human immune system is primarily composed of the innate and adaptive immune systems. The former is responsible for protecting the body against conserved pathogenic sequences and similar threats. The latter helps to identify specific infections and provides memory recall about them. In this way, the system can initiate or recall the previously used immune response to deal with the infectious agents2. Based on this knowledge, scientists are continuously working to develop prophylactic measures to fight against several infectious agents. These foreign bodies have been reported to cause significant health challenges and even death.

Several studies have looked into the possibility of immunotherapy for disease treatment by modulating or inhibiting the immune system3,4. This therapeutic approach identifies certain immunomodulators, which are usually chemical compounds. They could be designed, synthesised, or identified as natural compounds from different sources5,6. More so, immunotherapeutic approaches have been found to be effective in treating different health conditions. This is especially true for cancer, colitis, allergies, infectious diseases, and arthritis3,7. Immunomodulators help to support the immunological performance by boosting or suppressing the immune system, as required. In this way, it provides an alternative solution to conventional antibiotic solutions5. This works primarily because immune-modulating molecules are able to activate the humoral antibody responses. When this happens, they alter the cell-mediated immune responses and trigger other nonspecific mechanisms in the system8.

Naturally existing immunomodulators are more promising due to their effectiveness and reduced side effects compared with synthetic alternatives. In fact, natural small molecules from plants and animal origins are the most common sources of medications used to treat malaria, cancer, and others. Hence, these natural sources are considered excellent candidates for identifying immunomodulators9. Additionally, studies have shown that many plants contain bioactives with anti-infective effects. This is possible through their direct engagement with pathogens and modulation of the immune responses to strengthen the host immunity against known diseases6. Furthermore, these bioactive compounds can stimulate the macrophages and lymphocytes to change the cytokine levels, leading to enhanced alertness to foreign agents and subsequent chemosuppression10.

One of the previously reported plants with pharmacological values is Vernonia amygdalina, commonly referred to as bitter leaf. The small perennial shrub grows with rough bark and dark green leaves, especially in tropical Africa11. In Nigeria, V. amygdalina is known by different names across various regions. These include Ewuro, Edidot, Olubu, Oriwo, Shuwaka, and Onigbu12. In folkloric medicine, the popular local plant has been reported to be used to treat malaria infection, which was also validated in a study conducted by Afolayan and colleagues13. Furthermore, multiple studies have claimed that V. amygdalina has been used to treat gastrointestinal disorders, bacterial infections, inflammatory diseases, and parasitic infections11,12. These vast health-benefit attributes could be due to the abundance of bioactive compounds present in the herbal plant.

A computational study conducted by Afolayan and Abdulkareem11 showed that multiple chemical agents from the herbal plant have strong antimalarial and anti-inflammatory qualities. These agents range from PA, 9,12,15-Octadecatrienoic acid (Z, Z, Z), isophytol to 9,12,15-Octadecatrien-1-ol, (Z, Z, Z). Guthrod and colleagues14 described palmitic acid as a branched-chain unsaturated alcohol present in chlorophyll. Multiple studies have associated PA with different pharmacological benefits, including the treatment of tumours, diabetes, epilepsy, spasms, depression, and inflammation-induced health conditions15,16.

Palmitic acid is widely available in animal-based foods. These include meat, plants, dairy products, and micro-organisms17, which means isolating the compound for drug development should pose little to no challenge. A study conducted by Abdel-Naime and colleagues18 showed that the PA is capable of suppressing soil-borne pathogens in plants. At the same time, it can facilitate seedling growth. Another study has shown that PA is toxic to cancerous cells, reduces the abundance of fungal infection, and can enhance the growth of bone marrow mesenchymal stem cells19. A combination of these benefits, along with PA’s role as a signalling molecule in disease development and progression20, has led to increasing interest in understanding how PA modulates the immune system in healthy and diseased states.

Based on previous studies, PA is capable of regulating immune cell activity and can impact the outcome of infectious diseases and other immune-related health conditions11. Nevertheless, there is still a paucity of information about whether PA can modulate key cytokines associated with immune responses. Additionally, since dosage is crucial in keeping a balance between pro-inflammatory and anti-inflammatory cytokines, it becomes essential to evaluate safe doses to ascertain the potential of PA as an immunomodulator.

Thus, this study assessed the potential of palmitic acid to modulate the immune system through a combination of bioinformatic approaches and in vivo techniques.

Materials and methods

Computational assessment

Retrieval of compounds

Palmitic acid structural data file (compound ID: 985) was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in SDF format.

Target protein retrieval and preparation

Proteins involved in immune regulation and inflammatory responses during tissue injury were retrieved from the Protein Data Bank (PDB) (https://www.rcsb.org/). Six proteins were retrieved: tumour necrosis factor-alpha (TNF-α, PDB ID: 2AZ5), interferon-gamma (IFN-γ, PDB ID: 1FG9), interleukin-12 (IL-12, PDB ID: 1F45), IL-6 (PDB ID: 1ALU), transforming growth factor-beta (TGF-β, PDB ID: 5USQ), and IL-10 (PDB ID: 1LQS). The proteins were prepared using Biovia Discovery Studio Visualizer Software (https://discover.3ds.com/discovery-studio-visualizer-download). Non-essential components, including heteroatoms, bound ligands, and irrelevant chains, were removed. The proteins were then imported into USCF Chimera (https://www.cgl.ucsf.edu/chimera/download.html) for final preparation, where hydrogen atoms and Gasteiger charges were added.

Active site identification

The active sites of the target proteins were identified using the Computed Atlas for Surface Topography of Proteins (CASTp) (http://sts.bioe.uic.edu/castp/index.html?2pk9). All the proteins were available in the database, and the identified active sites were cross-validated with existing literature.

Molecular docking assessment

Molecular docking analysis was performed using the AutoDock Vina tool integrated into PyRx. Target proteins were loaded as molecules and converted to macromolecules. The ligand was imported as a chemical table file, energy-minimized, and converted to PDBQT format.

Visualisation of the ligand-receptor complex

Post-docking, PA was complexed with each protein, and their 2D and 3D interactions were visualised using Biovia Discovery Studio Visualizer21.

In vivo assessment

Drugs/chemicals

Palmitic Acid was obtained from Chemscene LLC (Princeton, USA), while cyclophosphamide, which was used as a positive immunosuppressant22 and levamisole, as a positive immunostimulant23, were sourced from pharmaceutical tablet formulations. All additional chemicals were of analytical grade.

Animal housing and ethics

Male Swiss mice (Mus musculus) weighing 22 ± 2 g were obtained from the Department of Biochemistry, University of Ibadan, Nigeria. The animals were housed in transparent rubber cages covered with wire mesh and maintained at the animal facility of the Department of Zoology, University of Ibadan. Throughout the experiment, the mice had unrestricted access to commercial rodent feed (Vital Feeds, Ibadan) and dechlorinated water. Ethical approval for this study was granted by the University of Ibadan Animal Care, Use, and Research Ethical Committee (UI-ACUREC/001-0123/11) while the procedures are in compliance with the ARRIVE guidelines 2.0 (https://arriveguidelines.org/arrive-guidelines). All methods were performed in accordance with the relevant guidelines and regulations.

Experimental grouping and drug administration

A total of 84 albino mice (6–8 weeks old, 22 ± 2 g) were used to evaluate the immunomodulatory effects of PA using various bioassays. The mice were acclimatized to laboratory conditions for 14 days before random assignment into 3 groups: A, B, and C. For the haemagglutination assay, 36 mice from group A (Haemagglutination Assay) were further subdivided into 6 subgroups of 6 mice each and treated as follows: Group 1—PA (2 mg/kg); Group 2—PA (5 mg/kg); Group 3—PA (10 mg/kg); Group 4—Cyclophosphamide (50 mg/kg); Group 5—Levamisole (LEV, 50 mg/kg); and Group 6—Vehicle.

For the remaining bioassays, groups B; 24 mice (Delayed Type Hypersensitivity Assay) and C, 24 mice (Phagocytic Index Assay) were each divided into six subgroups comprising four mice per group and received the same treatment as previously described. The different doses of palmitic acid (PA) were properly constituted using the vehicle (Ethanol-Phosphate Buffer Saline [2:1]), which served as solvent to dissolve the compound and all treatments were orally administered.

Antigen preparation (sheep red blood cell suspension [SRBC])

Fresh blood was obtained from healthy sheep slaughtered at the Akinyele International Sheep and Goat Market abattoir, Akinyele, Ibadan, and collected into tubes containing Alsever’s solution in a 1:1 ratio. The samples were gently mixed by inversion and transported on ice to the laboratory. Sheep red blood cells were isolated by centrifugation at 3000 rpm for 10 min using a CR3 Joan cold centrifuge. The supernatant was discarded, and the pellet was washed three times in a large volume of pyrogen-free 0.9% normal saline. The resulting SRBCs were resuspended in saline and stored at 4 °C until further use.

Evaluation of cell-mediated immune response

The delayed-type hypersensitivity (DTH) response to PA treatment was assessed following the methods described by Afolayan and colleagues3. On day 0, each mouse received an intraperitoneal injection of 0.2 mL of 5% SRBC, after which treatments (as described in Section “Experimental grouping and drug administration”) were administered for 7 days. On day 8, baseline paw thickness was measured using a digital vernier calliper. Subsequently, the mice were challenged by injecting 0.04 mL of 5% SRBC into the left footpad and 0.04 mL of normal saline into the right footpad. Paw thickness was measured 4 h post-challenge and at 24- and 48-h intervals. The difference in paw thickness served as an indicator of cell-mediated immune response in the treated mice.

Evaluation of phagocytic activity and index

Macrophage phagocytic activity was determined after 7 days of treatment, following the method of Zhou et al.24, with slight modifications. Briefly, 24 h after the last gavage, mice were intraperitoneally injected with 0.2 mL of 5% SRBC suspension. After 1 h, the mice were euthanized by cervical dislocation, and 2 mL of physiological saline was injected into the peritoneal cavity. The peritoneal fluid was collected, smeared on glass slides, and incubated at 23 °C for 24 h. Slides were rinsed with physiological saline, fixed in a 1:1 acetone-methanol solution, and stained with 4% (v/v) Giemsa for 15 min. Once dried, macrophages were observed and counted under a microscope. The phagocytic rate and index were calculated using the formula:

$$Phagocytic \; rate\left(\%\right)= \frac{Number \; of \; macrophages \; that \; engulfed \; SRBCs}{Total \; number \; of \; macrophages}\times 100 \%$$
$$Phagocytic \; index= \frac{Number \; of \; macrophages \; that \; engulfed \; SRBCs}{Total \; number \; of \; macrophages}$$

Preparation of peritoneal exudates

Peritoneal exudates were prepared as described by Zhou et al.24. Briefly, small incisions were made on the skin of the euthanized mice, and the abdominal skin was pulled back to expose the peritoneal wall. A 2 mL volume of cold physiological saline was injected into the peritoneal cavity. The cavity was gently massaged to dislodge the cells, and the resulting suspension was aspirated and transferred into a centrifuge tube kept on ice. The suspension was then centrifuged at 2000 rpm for 8 min. The supernatant was discarded, and the cell pellets were resuspended in physiological saline for further analysis.

Assessment of humoral immune response

Haemagglutination assay was performed according to the method described by Afolayan et al.3. On day 1, each mouse received an intraperitoneal injection of 0.2 mL of 5% SRBC. The animals were then grouped and treated as outlined in section “Experimental grouping and drug administration” for 7 days. Blood samples were collected from each mouse via retro-orbital bleeding into clean Eppendorf tubes 24 h after the final treatment for primary antibody titre assessment. Subsequently, the mice were given another intraperitoneal injection of 0.1 mL of 5% SRBC, followed by another 7-day treatment period. Blood was then collected for secondary antibody titre assessment.

The blood samples were kept at 23 °C for 30 min and then centrifuged at 3000 rpm for 20 min to separate the serum. The sera were titrated against 1% SRBC using a 96-well microplate. For each sample, 50 µL of serum was serially diluted in 25 µL of normal saline, followed by the addition of 25 µL of 1% SRBC into each well. The microplate was gently agitated by handshaking and incubated at 23 °C for 2 h. The plates were then examined visually against a white background to assess haemagglutination and left overnight for further observation. The antibody titre was determined as the highest serum dilution displaying at least 50% visible haemagglutination. The antibody titre value was expressed as:

$$Antibody \; titre \; value= \frac{1}{dilution \; factor}$$

Haematological analysis and organ coefficient

Animals used for the DTH assessment were treated for an additional 7 days, after which whole blood was collected from each mouse for haematological analysis. Red blood cell (RBC), platelet, and total white blood cell (WBC) counts were determined using a Neubauer haemocytometer25. Packed cell volume (PCV) was measured using a microhematocrit capillary tube, and haemoglobin (Hb) concentration was determined using the cyanomethaemoglobin method. Differential leukocyte counts, including lymphocytes, monocytes, eosinophils, neutrophils, and basophils, were evaluated following standard procedures. After blood collection, the mice were euthanized, and the liver and spleen were excised and immediately weighed using a sensitive digital balance. Relative organ weights were computed using the formula:

$$Organ \; coefficient \left(\%\right)= \frac{absolute \; organ \; weight \left(g\right)}{animal \; body \; weight \left(g\right)} \times 100$$

Statistical data analysis

All experimental data are presented as mean ± standard error of the mean (mean ± SEM). Statistical comparisons of group means were performed using a one-way analysis of variance and Dunnett’s multiple comparison post-test. Differences were considered statistically significant at p < 0.05. Data analysis was conducted using Microsoft Office Excel 7 for Windows and GraphPad Prism (version 5.0.1).

Results

Molecular docking assessment

The binding affinities of PA with the various cytokines are presented in Table 1. Among the interactions, TGF-β, IFN-γ, and IL-12 exhibited the least binding affinities, with values of − 6.1, − 4.9, and − 4.9 kcal/mol, respectively. A lower binding affinity score with negative values between a protein and ligand indicates stronger and more favourable binding.

Table 1 Binding affinity scores of the palmitic acid-cytokine complexes.
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Ligand-receptor complex analysis

Figures 1, 2, 3, 4, 5 and 6 present the 2D and 3D visualisations of the interactions between the ligand (PA) and the amino acid residues of the various cytokines. PA was effectively embedded within the binding pockets of each cytokine. In Fig. 1, PA formed a stable complex with TNF-α via one hydrogen bond and multiple hydrophobic interactions. This suggests that the interaction is stable to a certain extent. Figure 2 shows that IFN-γ interacted with PA primarily via hydrophobic bonds, lacking hydrogen bonds, suggesting reduced stability compared with the PA-TNF-α complex.

Fig. 1
figure 1

3D (Left) and 2D (Right) visuals of the Molecular interaction between Palmitic acid and Tumour necrosis factor-alpha (TNF-α).

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Fig. 2
figure 2

3D (Left) and 2D (Right) visuals of the molecular interaction between Palmitic acid and Interferon-gamma (IFN-γ).

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In Fig. 3, PA established a more compact interaction with TGF-β, involving one conventional hydrogen bond, van der Waals forces, and an alkyl bond. This suggests that the complex is very stable. Figure 4 illustrates PA’s interaction with IL-6. The interaction is less compact, involving two hydrogen bonds and multiple hydrophobic interactions. Figure 5 reveals that IL-10 formed three hydrogen bonds with PA, the highest among all complexes, alongside hydrophobic interactions, indicating a highly stable complex. Figure 6 shows that PA exhibited good interaction in the binding pocket of IL-10, forming one conventional hydrogen bond, pi-alkyl bond, alkyl bonds, and van der Waals forces.

Fig. 3
figure 3

3D (Left) and 2D (Right) visuals of the molecular interaction between Palmitic acid and Transforming growth factor-beta (TGF-β).

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Fig. 4
figure 4

3D (Left) and 2D (Right) visuals of the molecular interaction between Palmitic acid and Interlekin-6 (IL-6).

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Fig. 5
figure 5

3D (Left) and 2D (Right) visuals of the molecular interaction between Palmitic acid and Interleukin-10 (IL-10).

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Fig. 6
figure 6

3D (Left) and 2D (Right) visuals of the molecular interaction between Palmitic acid and Interleukin-12 (IL-12).

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Overall, the high binding affinities and interactions with multiple amino acid residues suggest that PA may modulate the signalling pathways associated with these cytokines.

Effect of palmitic acid on delayed-type hypersensitivity response

Table 2 presents the effect of PA on cell-mediated immune responses, evaluated via DTH-induced footpad oedema. A significant increase in paw thickness was observed in the experimental groups 4 h after SRBC challenge. Treatment with PA at 2 mg/kg significantly increased paw thickness by 69% at 4 h, with a slight increase at 24 h, while no increase was observed at 48 h. PA treatment at 5 mg/kg caused a significant increase in paw thickness at 4 and 24 h (p < 0.05), while no effect was observed at 48 h. Conversely, PA treatment at 10 mg/kg yielded no significant increase in paw thickness at any of the evaluated time points.

Table 2 Delayed type hypersensitivity response in animals and corresponding percentage increase in paw volume to treatments after 4, 24, and 48 h.
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Effects of PA on macrophage phagocytic index

PA induced a significant, dose-dependent increase in the macrophage phagocytic index and overall phagocytic activity in Mus musculus (Fig. 7). Compared with the control group, treatment with 50 mg/kg cyclophosphamide reduced macrophage activity.

Fig. 7
figure 7

Macrophage phagocytic index across treatment groups CPE, Cyclophosphamide; PA, Palmitic acid; LEV, Levamisole. *Indicates a significant difference compared with the control group (*p < 0.05). Data are expressed as Mean ± SEM.

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Effects of palmitic acid on antibody haemagglutinating titre value

The humoral immune response of treated mice to the SRBC challenge was evaluated via haemagglutinating antibody titre assessment. Figure 8 shows the antibody titre obtained after treating mice with PA and other standard immunomodulatory agents. Assessment of agglutination titre against SRBC antigen at various serum dilutions revealed significant differences in primary and secondary haemagglutinating antibody titres in the cyclophosphamide-treated group compared with the control. Notably, the 10 mg/kg dose of PA elicited the strongest primary humoral response, and the secondary humoral response showed no significant difference from that of the primary.

Fig. 8
figure 8

Effects of palmitic acid on humoral antibody response. CPE, Cyclophosphamide; PA, palmitic acid; LEV, Levamisole; *Indicates a significant difference compared with the control group (*p < 0.05). Data are expressed as Mean ± SEM.

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Effects on blood parameters and organ coefficients

The haematological parameter analysis presented in Table 3 shows the effects of PA on blood parameters in Mus musculus. Six different groups of mice were used: a control group, a cyclophosphamide group (used as a positive control for immunosuppression), an LEV group (used as a positive control for immunostimulation), and three groups treated with different doses of PA (2, 5, and 10 mg/kg). We discovered that treatment with 50 mg/kg of cyclophosphamide resulted in a significant decrease in neutrophils compared with the control group (p < 0.05). However, although the treatment with 2, 5, or 10 mg/kg of PA exhibited a dose-dependent decrease in the mean of PCV, Hb, and platelet count, none of the changes were statistically significant compared with the control group.

Table 3 Effects of palmitic acid on blood parameters in Mus musculus.
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Table 4 shows some morphometric measures of mice treated with PA, as well as those treated with standard immunosuppressants and immunostimulators (Cyclophosphamide and LEV). There was a dose dependent increase in the liver coefficient of PA-treated mice, which was lower compared with that in the LEV-treated mice. The spleen coefficient in the cyclophosphamide group was significantly lower than in all other groups.

Table 4 Organ coefficient and weight of mice treated with palmitic acid.
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Discussion

Immune responses need modulation to maintain body homeostasis and initiate the mechanisms associated with the host defence system. In a diseased state, modulatory small molecules are essential to adjust the immune system to trigger immunity against foreign agents, as previously shown with compounds from Tinospora cordifolia and Curcuma longa26. For this reason, uncovering potential immunodulators is crucial in immunotherapy research. One of the most reliable means of identifying promising modulatory compounds is through natural sources, especially herbal plants27.

Several previous studies suggest that palmitic acid (PA) can stimulate lymphocyte proliferation and cytokine production28,29. This is believed to have potential implications for immune-related disorders and can be harnessed as an adjuvant to chemotherapy. Fortunately, in silico approaches has increasingly become efficient and affordable means of identifying promising bioactives from various sources, which can then be validated using in vivo techniques to identify promising drug leads13,30. Thus, this study used a combination of in silico (computational) approaches and in vivo experiments to assess the immunomodulatory activity of PA in Mus musculus.

Using the molecular docking technique, the interactions between PA and six regulatory cytokines were investigated. These included Tumour Necrosis Factor-alpha (TNF-α), Interferon-gamma (IFN-γ), Interleukin-6 (IL-6), Transforming Growth Factor-beta (TGF-β), and Interleukin-10 (IL-10). Additionally, in vivo experiments on cell-mediated immunity, blood parameters, antibody humoral responses, and mouse organ morphometric measures were performed to investigate the capability of PA in modulating immune response.

According to Parameswaran and Patial31, TNF-α is a pro-inflammatory cytokine, responsible for the regulation of macrophage function. Post-infection, TNF-α is induced and plays a part in several inflammatory molecules, including cytokines and chemokines32. Therefore, the molecule helps regulate responses to inflammation. Inhibiting TNF-α blocks the downstream inflammatory signalling pathways. This has therapeutic implications, especially in the treatment of inflammatory conditions. Meanwhile, studies have shown that IFN-γ raises the level of immune system performance. This is particularly important in mounting anti-microbial functions. However, excessive expression of IFN-γ can lead to tissue damage, emergence of autoimmune diseases, and hyper-inflammatory-related health conditions33. Another crucial inflammatory cytokine is IL-12, which is involved in promoting the immune system response, according to Ayuthaya and colleagues34. This cytokine plays a key role in T-natural killer cell activation, which is important for mounting defence against intracellular pathogens. When the level of IL-12 is raised, it can result in inflammatory-related diseases and autoimmune conditions. Studies have associated IL-6 with B-cell activation and differentiation. Chronic inflammatory conditions can materialize when there is an overexpression of IL-635. Nevertheless, another interleukin (IL-10) helps in regulating the immune responses. The mechanism involves the inhibition of pro-inflammatory cytokine synthesis, such as IL-6 and TNF-α36. This particular cytokine can promote immune responses and help moderate excessive inflammation.

The outcome of molecular docking and visualisation shows favourable binding between PA and the active sites of each cytokine. The binding score ranging from − 3.5 to − 6.1 kcal/mol suggests favourable interactions of the compounds with the cytokines. Meanwhile, the visualisation of the interactions showed beneficial interactions, including conventional hydrogen bonds, van der Waals forces, and hydrophobic interactions, such as Akyl, Pi-Akyl, and Pi-Sigma between the amino acid residues of the cytokines and PA. With the exception of IFN-γ/PA complex showing unfavourable donor-donor interactions, every other interaction indicates potential stability between the ligand (PA) and the receptors (cytokines). The potentially stable ligand-receptor complexes suggest that the bioactive can modulate the activities of the cytokines and can be harnessed for therapeutic uses.

When memory T cells try to recognize and respond to an antigen upon secondary exposure, there is a mediation of Delayed-Type Hypersensitivity (DTH) immune response. In this case, immune cells are recruited to mediate inflammation in the affected tissues. Concerned cells often include macrophages and T-helper cells (CD4+ and CD8+). Also, there is production of cytokines, including IFN-γ3,37. When safeguarding the body against intracellular pathogens, DTH reactions play key roles3. In this study, treatment with PA showed inducement of DTH response, while the extent and duration of the response varied depending on the doses. For instance, the strongest initial response was seen in the 2 mg/kg PA treatment. However, the 50 mg/kg LEV treatment produced a sustained response even after 24 and 48 h. Meanwhile, the cyclophosphamide treatment (50 mg/kg) showed the weakest effect, especially after 2 days. These outcomes align with the previous results that sought to demonstrate the effect of LEV when it comes to the inducement of DTH responses38.

In a study conducted by Zhang and colleagues39, 3, 5, 7, 3′, 4′-pentamethoxyflavone was assessed in terms of its anti-inflammatory effects on DTH-induced inflammation using the mice model. The scientists showed that the compound reduced paw-thickness, though its effect varied depending on doses. In fact, the highest reduction was observed at 24 h post-treatment. Likewise, Huang et al.40 observed that the aqueous bark extracts of Ximena americana treatment reduced the paw volume in DTH-induced inflammation using mice. Some studies on PA have also shown that the bioactive is effective in reducing inflammation and immune responses41,42, with a need to determine the optimal dose and timing for its administration to reduce DTH responses.

Among the crucial indicators of immune function is macrophage phagocytosis. Macrophages are effective in engulfing particles, and this has been demonstrated in vitro and in vivo43. Research has shown that macrophage phagocytosis can be measured by assessing the percentage of cells that have undergone phagocytosis, following a specific incubation period, especially particulate materials such as SRBCs44. Due to the multiple protein complexes available on the surface of SRBCs, the cells are widely used to initiate macrophages in experiments to stimulate phagocytosis45. A good case study is that of Kemaladewi and colleagues46, who showed that injecting SBRCs into the peritoneal cavity of mice not only activated the macrophages but also induced phagocytosis. In this study, the dose-dependent nature of PA in potentiating macrophage phagocytosis of SRBC was observed.

At 2 and 10 mg/kg PA treatment, the primary antibody production increased in comparison to the control molecules. This suggests the immunostimulatory potential of palmitic acid. However, a sustained production was not observed, as there was no significant difference when compared with the controls. At the same time, it showed that PA is a potential adjuvant to prime the immune system. Furthermore, a reduction in neutrophils and lymphocytes was observed. These could have significant implications for the immune systems of the mice. Neutrophils play a critical role during the early stages of the initial immune response. Meanwhile, lymphocytes play key roles in adaptive immune response, as reported by Janeway Jr. et al.47. When lymphocytes or neutrophils decrease, there could be an impairment of the immune response. In this study, the cyclophosphamide-treated group was observed to show a significant reduction in the neutrophil percentage in comparison to the control group, suggesting immunosuppression. Meanwhile, the LEV group was observed to experience a significant rise in neutrophil percentage. This suggests a possible case of immunostimulation. In short, the outcomes indicate an appropriate experimental design, with the cyclophosphamide and LEV groups acting as immunosuppressants and immunostimulants, respectively.

The three PA-treated groups (at varying doses) showed no significant differences in platelet, RBC, PCV, and Hb counts compared with the control groups. Consequently, PA exerted no significant influence on the parameters, indicating a minimal stimulatory effect on the adaptive immune response. Nevertheless, the PA-treated groups of 2 mg/kg doses showed significantly higher impacts on the WBC count compared with the control group—the impacts were not significant in the case of 5 and 10 mg/kg PA-treated groups. In translation, PA can affect the WBC count at certain dose levels.

Regarding the immune cell percentages, PA-treated groups of 5 and 10 mg/kg doses showed no statistically significant difference from the control groups. However, an increase in eosinophil percentage was observed in the 10 mg/kg group. This suggests that PA may have limited effects in potentiating prolonged activation of the adaptive immune response. These findings differ from those of Montes-Nieto and colleagues48, which showed that PA treatment increased the expression of neutrophils and reduced the expression of lymphocytes in Sprague Dawley rats models. Some factors such as animal models, treatment duration, and doses, could have played a factor in the different outcomes.

According to Zhou et al.24 the weight of the immune organs indicates the performance of the innate immune system. This is especially true for the spleen, which is a location for the maturation and differentiation of immune cells. Hence, any changes in the spleen coefficient indicate the efficiency of the innate immune response. In our study, PA-treated groups of varying doses showed no significant differences compared with the controls in terms of spleen coefficient. However, the same cannot be said for the average spleen coefficients of the various PA-treated groups. At higher doses, the average coefficient was slightly higher than that of the control groups, indicating a potential effect on the innate immune response.

Clearly, a combination of molecular docking, delayed-type hypersensitivity (DTH) response, phagocytic activity, and haemagglutination assay strongly indicates that PA modulates cytokines and cell-mediated immune responses. By assessing the interaction of PA with cytokines such as IL-12, it is apparent that PA can potentiate the activation and differentiation of CD4+ and CD8+ during inflammation. Meanwhile, its ability to engage with anti-inflammatory cytokines such as IL-10 and TGF-beta also shows that it can regulate immune responses and consequently, help maintain immune homeostasis.

Based on the findings in our study, palmitic acid, as a bioactive compound with immunomodulatory activity, can serve as a therapeutic agent. For instance, it could be harnessed to boost host defence in conditions such as viral infections, immunodeficiencies, and early-stage cancers. Its ability to stimulate the TLR4 pathways, as previously reported by Nicholas and colleagues49 and cytokine release with limited influence on antibody-mediated responses, as shown herein, means PA can serve as a candidate for adjuvant development in immunotherapy or vaccine formulations. Hence, palmitic acid is recommended for further in vitro and biochemical studies to assess its efficacy, especially in specific immunodeficiencies.

Conclusions

In this study, the immunostimulatory effect of palmitic acid was investigated using in silico techniques and in vivo experiments. Computational molecular docking revealed that PA can engage the active sites within the binding pocket of key cytokines involved in the immune system. This gave insights into the potential immunomodulatory effect of the bioactive. Wet-lab experiments through DTH and macrophage phagocytosis studies revealed that PA modulated innate and cell-mediated immunity significantly and in a dose-dependent manner. Meanwhile, the bioactive had no effect on the antibody-mediated immune response. Additionally, the naturally occurring compound had an effect on the WBC count at lower doses. Overall, it is recommended that PA be further explored to understand the molecular mechanisms underlying its immunostimulatory effect on the immune system.