Date of Award

January 2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Bibhuti B. Mishra

Abstract

Neurocysticercosis (NCC) is the most common parasitic infection of the central nervous system (CNS) caused by the helminth parasite Taenia solium (T. solium) and affects 50-100 million people worldwide. Its clinical symptoms include headache, seizures, hydrocephalus, epilepsy and stroke. These clinical symptoms can have life-long detrimental effects. As many as half of the total adult-onset seizures and 10% of the stroke cases in the endemic areas are attributed to NCC, whereas 20% to 30% of symptomatic patients display hydrocephalus. Treatment of NCC remains a major challenge as the severity of the symptoms is thought to be elicited by the degenerating larvae resulting from therapeutic treatment or normal attrition. Specifically, the inflammatory response in the CNS detected during symptomatic phase is thought to be responsible for the neuropathology and underlining cause of clinical symptoms. Interestingly, NCC has a long asymptomatic phase, typically lasting 3-5 years before the onset of the symptomatic phase. This asymptomatic phase is characterized by little or no signs of inflammation detected around the live cysts. Thus it is commonly believed that viable cysticerci induce immune suppressive effects, and loss of these effects when the parasite dies likely leads to activation of uncontrolled inflammatory response and neuropathology. Therefore, treatments with antiparasitic drugs combined immunosuppressive/ anti-inflammatory factors such as corticosteroids are predominantly used to control the host immune inflammatory response. However, their longtime use can cause other problematic side effect. The characterization of the immunoregulatory mechanisms in the CNS during helminth parasitic infection is critical to develop novel therapeutic targets for NCC, which can aid in finding treatment of a plethora of chronic neuro-inflammatory diseases as inflammation plays central role in development of majority of the chronic CNS inflammatory/autoimmune diseases.

The severity of the symptoms is associated with the intensity and chronicity of the local immune response in the CNS, use of a mouse model is essential to perform kinetic of analysis of the entire infection process. Because T. solium is not infectious to mice, use of Mesocestoides corti (M. corti), a relative cestode, has allowed for a more controlled study of the entire infection process, and to better characterize the immune response in the CNS microenvironment. Importantly, analysis of the infected CNS in the murine NCC model paralleled results obtained with brain specimens from NCC patients showing release of glycan antigens that are uptaken by host immune cells in the parasite infected CNS of humans and mice. In this regard, multiples researchers had indicated that glycans from pathogens, including helminths, act as pathogen-associated molecular pattern molecules (PAMPs), which are knowns to be recognized by C-type lectin receptors (CLRs) through their carbohydrate recognition domains (CRDs) and function as important pattern recognition receptors (PRRs). We, therefore, hypothesized that M. corti larvae in the CNS release their antigens glycoconjugates molecules which induce the expression of specific lectin receptors (LRs) such as galectins. The LRs may play important role in regulating the immune response and pathology during NCC.

Parasites, particularly helminths, tend to shift the balance of immunity to a Th2 type of response, and it is likely that Th2 immunity evolved in response to infections with these parasites. The Th2 response activates or expands alternatively activated macrophages (M2 macrophages) which are associated with suppressive activity. The development of M2 macrophages is thought to be crucial in establishment of chronic parasitic infections as they express various negative signaling accessory molecules to downregulate the proliferation of activated T cells, and promote healing and tissue remodeling (1-3). In this regard, previous studies involving the mouse model of NCC showed the presence of macrophages with M2 phenotype in the CNS microenvironment. Moreover, a reduction in CNS levels of M2 macrophages in STAT6-/- mice undergoing NCC is correlated with widespread tissue damage and greater mortality (4). This is exciting as M2 macrophage mediated anti-inflammatory/ neuro protective functions could be important in nervous tissue repair as well as containment of neuropathology in myriad neuro-inflammatory diseases. However, the specific role of these M2 cells during NCC infection still remind to be determined.

The tegument in helminths such as T. solium and M. corti is dynamically responsive to changing host environments or immune attack in the CNS. During CNS infection of both these parasites, glycan antigens from the rapidly released in the CNS. Interestingly the glycan antigens containing terminal galactose and galactosamine (detected by the lectin IB4) is rapidly released (1-2d p.i.), and it is almost absent after 1 wk of infection. This was correlated with differential upregulation of the Galectins (Galectin-3, -7, and -9), a family of the galactose specific LRs, in the CNS during murine NCC. The aim of the studies is to study the specific roles of galectin 3, galectin 7, and galectin 9, in regulation of immune functions in the CNS microenvironment during murine CNS infection.

Here we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin 3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin 3 in this process. Indeed, Galectin 3 deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in-vitro. Furthermore, adoptive transfer of M2 macrophages from Galectin 3 sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin 3-/- mice. Together, these results demonstrate for the first time a novel role of galectin 3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.

Analysis of helminth-infected mice revealed an abundant expression of Galectin 7 (Gal 7) that recognize galactose moieties in brain endothelial cells. The Galectin 7-/- NCC mice displayed a reduced accumulation of M2 macrophages in the parasite-infected brain. This reduction in M2 macrophage numbers in the CNS was correlated with an increased neuropathology and shortened survival. The use of adoptive transfer of fluorescently labeled cells demonstrated a decreased influx of M2 macophage into the CNS of helminth-infected NCC brains. The use of in-vitro transmigration assay conclusively demonstrated a key function of Galectin 7 expressed on endothelial cells in centrally regulating M2 macrophage entry into the CNS. The results are highly significant as the BBB restricts the flow of both cells into the brain, and the fact that galectin-7 regulates M2 macrophage extravasation into the CNS could be of great importance how to contain neuropathology in CNS infection.

Galectin 9 was the first to be upregulated as well as at a relatively high level in M2 macrophage. Parasite-infected Galectin 9-/- mice displayed significantly increased susceptibility to parasite infection, despite a similar parasite burden in the CNS compared to infected WT mice. The accumulation of macrophages, γδ T cells, αβ T cells, or B cells in the brains of parasite-infected Galectin 9-/- mice was similar to the WT mice. Moreover, AAMs which play a profound protective role in murine NCC were observed an increased level in infected WT and Galectin 9-/- mice. Instead, the parasite-infected Galectin 9-/- mice exhibited a massive neutrophilia in the CNS and an increase in neutrophil-associated neuroinflammation. These results suggest that galectin 9 may be involved in controlling and resolving neutrophilia and inflammation, however, not by efferocytosis. For the first, we showed in the possible correlation between the presence of Galectin 9 and neutrophilia in NCC infection. Further experiments need to be done to determine the specific mechanism by which Galectin 9 is involved in this process.

In toto, our studies make an important contribution in establishing the novel protective mechanisms of M2 macrophages functions in the CNS pathological conditions. Our studies provide a better understanding of the immunopathogenesis of NCC which should lead to new therapeutic strategies in other CNS inflammatory disorders as well.

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