Targeting of activated macrophages through CD64 to elucidate their role in the pathogenesis of Multiple sclerosis

Aslanian, Eric; Fischer, Rainer (Thesis advisor); Barth, Stefan (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis

Abstract

Multiple sclerosis is a complicated autoimmune disease of the CNS that is developed and exacerbated upon inflammatory infiltrated immune cell activities and subsequent increase of demyelinated lesions. Inflammatory macrophages (M1) play a decisive role in the pathogenesis of MS through production of inflammatory mediators (TNFα, IFNγ) and stimulation of the immuno-inflammatory cascade against the myelin antigens. As there is a need for new and more effective therapeutics and considering the pivotal role of these inflammatory macrophages in MS, we investigated whether elimination of these cells could interrupt the autoimmune-reactive cascade against the neuronal myelin proteins. Characteristic for these cells is the expression of FcγRI (CD64) receptor, which was previously shown to be a suitable target to specifically eliminate M1 populations. This was done using an immunotoxin (IT) composed of an antibody fragment (H22-scFv) and the truncated pseudomonas bacterial exotoxin (ETA′). The IT induced apoptosis upon internalization. We therefore here targeted M1 macrophages with H22-ETA′ IT in the animal model for MS, called experimental autoimmune encephalomyelitis EAE.Primarily, CD64 expression was evaluated on macrophage subtypes. The peripheral monocytes were isolated from human blood’s PBMCs and polarized to M1 (by LPS) and M2 (IL-4) macrophages. Thereafter CD64 expression was confirmed to be predominantly on M1 macrophages, polarized by LPS. In addition, hCD64 transgenic mice were bred and further developed in our own animal facility center, followed by related examinations and preparation of transgenic vs. non-transgenic littermates for the experiments. Later, the proteins required for EAE induction and treatment were cloned and produced. Considering the variable factors that could influence EAE induction, different protocols were tested using various expressed MOG, purchased MOG, and EAE induction kits. Ultimately, EAE was successfully induced in human-CD64 transgenic C57BL/6 mice, using Hooke laboratory’s EAE induction kit, supplemented with complete Freund’s adjuvant and pertussis toxin. The immunotoxin was produced through bacterial fermentation and purified, while its binding and toxic abilities were further determined. Thereafter, variable doses, time points, as well as injection sites were evaluated, which enabled the establishment of a final, optimized protocol. Applying this protocol, the kinetics of different immune cells, including macrophages were studied in EAE. This was done through immunohistochemistry of brain sections at four different time points, starting from day 6 to day 12 of EAE induction. The safety of H22-ETA′ therapy was additionally verified on healthy non-EAE induced animals. For this, several immunostaining protocols were developed using a variety of combinations of antibodies and staining substrates. EAE was induced according to the developed protocol, in human-CD64 transgenic, C57BL/6, mice and treated with H22-ETA′ immunotoxin. The EAE scorings and therefore the clinical signs were recovered in the treated group, while the non-treated animals were suffering from severe clinical disabilities and progressive deterioration of the disease. The immunohistochemistry data displayed reduction of M1 cell infiltrates including CD64 and CD14 positive cells, in the treated group comparing to the non-treated and late-treated controls. Conclusively, the statistically evaluated data together with the clinical findings, confirmed the importance of CD64 targeted therapy as a candidate for further studies. Taken together, targeting macrophages could potentially be significant, as a new therapeutic strategy in MS disease. Furthermore, it has the potential to be developed as one of the disease modifying and effective therapies, for Multiple Sclerosis.

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