Effect of Moringa oleifera and ivermectin nanoparticles on the immunopathological response during experimental trichinosis in mice

The immune response is composed of an orchestra of fine-tuned interlacing pathways that exert varying defense strategies against various microbial threats. Macrophage polarization represents a vivid example of the delicate balance of immunological mechanisms. Macrophages are generally categorized into classically activated macrophages (M1) and alternatively activated macrophages (M2). These 2 spectra of macrophage phenotype represent the traditional poles of the immune response, the killing response against various threats versus the healing and regenerative potential after the resolution of combat [1]. M1 macrophages are stimulated by several pro-inflammatory mediators and augment the T helper (Th)1 immune response by producing pro-inflammatory chemokines and cytokines such as TNF-α and interleukins 1,6,12,18, and 23. In parallel, M2 macrophages enhance the Th2 immunity arm and are particularly important in parasitic infections. IL-10 serves both as an inducer and effector of alternatively activated macrophages [2].

A key marker of M2 macrophages, arginase-1, converts L-arginine into L-ornithine and urea. Induction or activation of this enzyme depletes L-arginine, limiting its use by inducible nitric oxide synthase (iNOS), which in turn is a marker for M1 macrophages. Inducible NOS converts L-arginine into nitric oxide (NO) and L-citrulline. NO is an important effector molecule in the process of phagocytosis and antimicrobial immune response [3]. Macrophage polarization is a dynamic and flexible process that adapts to the status of the immune-pathological response. An important regulator of macrophage phenotype switching is the nuclear factor kappa beta (NF-κB) pathway. During the early inflammatory phase of tumor formation, macrophages exhibit a high activity of NF-κB and M1 activity profile. Later in disease progression, an immunosuppressive state is established, which is characterized by an M2 phenotype and low NF-κB [4].

Trichinella spiralis infection has been employed as a rich and comprehensive model to study both local tissue-specific and systemic immune responses. Several signaling pathways synergize and interlace to shape the host reaction against this unique nematode. M1 polarization is an important feature of intestinal trichinellosis, where M1 macrophages play an important role in adult worm expulsion [5]. The intestinal phase of T. spiralis infection starts after the release of L1 larvae from the digested collagen capsules upon ingestion of infected meat. The freed larvae invade the small intestinal epithelium to mature into adult worms, which copulate at around day 7 post-infection (PI) to generate a new progeny of larvae, that will then migrate to finally encyst in skeletal muscles [6].

Herbal agents are successful synergistic and sometimes alternative remedies to conventional anti-parasitic agents [7].

Moringa oleifera, a member of the Moringaceae family, has been traditionally used as a medicinal plant due to its multiple health benefits, which include antioxidant, anti-hyperglycemic, anti-hyperlipidemic, and antihypertensive activities [8].

In addition to the rediscovery of herbal agents as potent anti-parasitic effectors, scientists sought to enhance the effect of conventional anti-parasitic drugs through the addition of nanoparticles. The integration of nanomedicine and anti-parasitic agents aims to overcome drug limitations such as low solubility and bioavailability, as well as high dosing and elimination [9]. Ivermectin (IVM), also termed the ‘wonder drug’, was shown to be effective against a variety of parasitic agents [10]. Nano-preparations of IVM include the employment of liposomes, solid lipid nanoparticles and nano-suspensions as drug delivery systems [11‐13].

Parasitic infections modulate the host immunity by various mechanisms, including the regulation of macrophage polarization and phenotype switching [2]. Owing to the diversity of tissue involvement in T.spiralis infection, both M1 and M2 phenotypes contribute to the anti-Trichinella immune response as demonstrated by the study conducted by Sun et al. [5]. They conducted a time-dependent study involving different tissues to assess the activity of macrophages using flow cytometry. The authors found that early during infection (between day 1–5 post-infection), the M1 macrophage response was dominating in the small intestine of the study animals. After 15 days of infection, the small intestine showed a mixed M1 and M2 response which was replaced by a predominantly M2 type response during late stages of infection (after 30 days). In our study, protein expression of iNOS, a type 1 macrophage marker, and relative gene expression of arginase-1, a type 2 macrophage marker, were both elevated after 7 days of T. spiralis infection, indicating the establishment of a mixed macrophage response during the intestinal phase of the disease. Kang et al. [28] studied the effect of T. spiralis on macrophage function and the potential anti-inflammatory effect of parasite-derive excretory-secretory (ES) products on experimentally induced colitis. They found that the peritoneal transfer of macrophages from T. spiralis infected animals led to an increased secretion of IL-10 and a suppression of pro-inflammatory cytokine production. In addition, treatment of lipopolysaccharide- induced macrophages with T. spiralis-ES products led to the suppression of iNOS production and a shift towards a M2 phenotype.

The role of T. spiralis derived proteins in macrophage polarization was also investigated by Liu et al. [29], who investigated the effect of co-incubation of T.spiralis cathepsin L with murine macrophages. The authors observed an increased release of iNOS, IL-6, IL-1β and TNF-α, indicating a type 1 macrophage response. Furthermore, the study revealed upregulated expression of phosphorylated NF-κB and its inhibitor IκBα, along with enhanced nuclear translocation of the NF-κB p65 subunit, suggesting activation of the NF-κB signaling pathway. The addition of a NF-κB inhibitor interfered with the macrophage potential to polarize towards a type 1 response.

Management of T. spiralis is challenged by the low bioavailability of anti-parasitic drugs, where intestinal inflammation per se decreases the absorption of anti-parasitic agents [30]. Nanotechnology has emerged as a potential solution for improving drug delivery, bioavailability and decreasing drug toxicity [31]. The current study investigated the effect of IVM-NP and MOL-NP on macrophage-related immune activity during intestinal trichinosis. Infection with T. spiralis led to a significant increase in arginase-1 and TNF-α gene expression, whereas the relative expression of IL-10 significantly decreased. Both IVM-NP and MOL-NP reversed these effects, and the combination of both therapeutic agents was most effective in decreasing arginase-1 and TNF-α relative gene expression and decreasing the expression of IL-10. The combined treatment was also the most effective in decreasing intestinal worm count. In contrast, dual administration of IVM-NP and MOL-NP were less efficient than the monotherapy of both drugs in attenuating the increased protein expression of NF-κB and iNOS observed after infection with T. spiralis.

Nanotechnology has provided a breakthrough in the improvement of anti-parasitic drug delivery. Gamboa et al. [11] demonstrated that IVM lipid loaded nanoparticles achieve a higher peak plasma concentration and longer elimination half-life in the liver, small intestine and lungs when compared to commercial IVM preparations.

Elmehy and his colleagues [32] investigated the effect of 2 different IVM nano-formulations on different stages of T. spiralis infection. They employed a niosomal IVM preparation, which is lipid based, and a nano-crystalline IVM formulation. Niosomal IVM was superior in alleviating intestinal inflammation and improving oxidative status during the intestinal phase infection. In addition, it was more efficient than the nano-crystalline preparation in reducing inflammation and angiogenesis, where niosomal IVM achieved a higher reduction in the expression vascular endothelial growth factor (VEGF), matrix-metalloproteinase-9 (MMP-9) and regulated on activation, normal T cell expressed and secreted (RANTES). VEGF is an angiogenesis marker, MMP-9 is a marker of inflammation and tissue remodeling, while RANTES is an inflammatory chemokine that attracts immune cells [32].

The synergistic effect of IVM nanoparticles to conventional antiparasitic therapy was investigated by Moawad et al. [33] by adding IVM chitosan loaded nanoparticles to albendazole in the treatment of T. spiralis-infected mice. They observed that the combination of both agents was most efficient in decreasing the intestinal worm count and the expression of IFN γ, TNF-α and iNOS genes. Similar to our study, an increase in IL-10 expression was also observed.

Faheem et al. [34] investigated the effect of MOL and MOL loaded on chitosan NP in comparison to metronidazole therapy on parasite burden and intestinal pathology in experimental giardiasis. Although MOL significantly decreased the G. lamblia cyst count (76.89% reduction), it was not as effective as metronidazole therapy (79.76% reduction). MOL NPs, however, were superior to both drug regimen in reducing parasite count (86.01%). Nevertheless, MOL both alone and as NP preparation was more efficient in improving intestinal pathology than metronidazole.

Abdel-Latif et al. [35] demonstrated that Moringa oleifera extracts (MOE) had an immunomodulatory and protective effect against intestinal Hymenolepis nana infection by exerting an anti-oxidant response and inducing T helper 2 cytokine expression. Mice immunized with MOE prior to infection showed attenuated intestinal pathology, increased goblet cells, mast cells and secretory IgA, in addition to decreased parasite shedding.

The biological actions of Moringa oleifera are attributed to the wide array of its biochemical constituents, such as flavonoids, phenols, polysaccharides, amino acids, minerals and phytohormones. Flavonoids were found to exert an immune-modulatory effect during experimental T. spiralis infection by decreasing the intestinal expression of NOD-like receptor-pyrin domain containing 3 (NLRP3). This intracellular protein is critical for the inflammasome formation as part of the innate immune response and is dependent on NF-κB for its upregulation [36]. Flavonoids were also investigated as potential anti-cancer agents, due to their effects on angiogenesis, cell signaling and cell death [37].

Polyphenols present in Moringa oleifera contribute to the antioxidant potential of the plant. In addition to flavonoids and phenols, anti-inflammatory and anti-bacterial effects are also exerted by tannins, saponins and vitamins, particularly vitamin C [38]. MOL polysaccharides were found to have a regenerative effect on intestinal immunopathological changes as represented in an experimental colitis model studied by Hussein et al. [39]. MOL polysaccharides significantly improved the pathological changes resulting from dextran sulfate induced colitis in mice. In addition, induction of the mRNA expression of the anti-inflammatory cytokine IL-10 and inhibition of the mRNA expression of pro-inflammatory cytokines such as TNFα and IL-1β was observed. Moreover, the authors demonstrated that the anti-inflammatory effect of MOL was also achieved by inhibiting the TLR4/MyD88/NF-κB signaling pathways.

The incorporation of herbal medicines in the management of parasitic infections has re-surfaced to overcome limitations of conventional anti-parasitic agents, such as low bioavailability, toxicity and development of resistance [40]. Literature on the combination of IVM and Moringa oleifera is relatively scarce, especially regarding their nanoparticle preparations. Uko et al. reported that MOL extracts and vitamin C improved the reproductive toxicity induced by IVM in rabbits through their antioxidant properties [41]. Similarly, Boudjema et al. stated that MOL extracts improve the oxidative damage produced by abamectin on the brains and erythrocytes of rats [42].

In conclusion, this study illustrated a pivotal role of IVM-NP and MOL-NP in immune regulation by significantly downregulating pro-inflammatory markers (iNOS and TNF-α) while upregulating the anti-inflammatory cytokine IL-10 in T.spiralis infection. Furthermore, combination therapy synergistically decreased parasite burden and intestinal pathology more effectively than monotherapies. While this study demonstrates promising anti-parasitic effects of both nanoparticle preparations and their combination, future studies targeting the effect of batch variability and nanoparticle properties on the desired drug effects are recommended. The notable effects of IVM-NP on the adult worm burden and inflammatory response during the intestinal phase, strongly encourages the design of additional studies on the effect of this agent on the muscle phase of the disease, posing the question on whether the employment of nanotechnology can aid to overcome the relatively limited efficacy of IVM on encysted T. spiralis larvae.