Pathological and immunological changes in host cells in response to Leishmania mexicana infection

Alhajri, S.M., 2019. Pathological and immunological changes in host cells in response to Leishmania mexicana infection. PhD, Nottingham Trent University.

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Introduction: Leishmaniasis is a group of parasitic diseases caused by obligate intracellular protozoa of the genus Leishmania, with more than 20 pathogenic species. Leishmania infects approximately 12 million people annually in 98 countries. The deaths associated with this disease ranges between 20,000 - 30,000 per year (WHO, 2018). Therefore, the need for treatments or vaccines get more urgent. Macrophages are the ultimate host cell for the Leishmania parasite where it survives and multiplies. Though, a lot is known on how the Leishmania parasite survives and multiplies inside macrophages, there are still aspects related to pathophysiological and immunological responses to their infection that need further investigation to aid in the development of new vaccines or drugs for this disease. In this study, a virulent and avirulent L.mexicana model was developed to examine their interaction with bone marrow derived macrophages (BMDM) from susceptible (Balb/c) and resistant (C57) mice in vitro.

Methods: Virulent L.mexicana parasite MNYC/BZ/62/M379 (P1) was maintained by subcutaneous inoculation of Balb/c mice. Avirulent L.mexicana passage twenty (P20) was produced by sub culturing of L.mexicana passage one (P1) twenty times in vitro. The effects of 20 continuous passages on virulence-associated gene (LPG1, LPG2, A2, CHAT1, CPB2, CPB2.8, CPC, GP63, LACK, and MAPK9) were investigated using qPCR. The expression of LPG and PS was also investigated using flow cytometry and immunofluorescence analysis. Growth characteristics and morphology of avirulent (P20) and virulent (P1) L.mexicana parasites grown in two media (Schneider's Drosophila and RPMI1640) in vitro were investigated under different culture conditions (temperature, and oxygen) using light immunofluorescence microscopy, EM and AFM. Differentiation into amastigotes under several conditions was investigated by estimation of the number of amastigotes. The infectivity of the parasites at each passage was also assessed by hemocytometry and Alamar blue assay. Survival of parasites inside macrophages was assessed visually by labelling the parasites with CFSE stain and the ability to form PV in BMDM from C57 and Balb/c mice. qPCR was used assess the expression pro-inflammatory cytokine expression (TNF- α, IL-6, IL-1β and TGF-β) and ELISA was used for estimation of TNF-α in the culture supernatants. Annexin V stain and flow cytometry analysis was used to assess apoptosis of infected cells. qPCR was used to assess the expression of genes associated with apoptosis (Bax, BCL 2, Caspase 1, Caspase 8, Caspase 9 and PD 1). The effect of supernatants derived from cultures of infected BMDM on the P1 and P20 promastigotes growth and virulence genes regulation was also investigated by qPCR.

Results: Twenty passages of L.mexicana in vitro caused significant changes in parasite morphology, ability to differentiate into amastigotes and downregulation of all tested virulence associated genes. Expression of LPG decreased, and PS increased on the surface of L.mexicana promastigotes. P20 infected both Balb/c and C57 BMDM but failed to survive inside BMDM both mice strains. P1 survived and inhibited apoptosis accompanied by significant downregulation of Caspase 8 by qPCR. Both P1 and P20 induced the release of TNF-α as confirmed by qPCR and ELISA. P1 promastigotes incubated in conditioned media derived from Balb/c BMDM infected with P1, enhanced their growth accompanied by upregulation of LPG1, CHT1, CPB2 and CPB2.8. While, incubation in conditioned media derived from C57 BMDM infected with P1 inhibited their growth and caused downregulation of LPG1, LPG2 CPB2, CPB2.8, CHT1 and A2.

Conclusion: Culturing of L.mexicana in vitro for 20 passages has produced significant changes in their ability to differentiate from promastigotes into amastigotes, the ability to survive in macrophages and regulation of apoptosis associated genes. Supernatants produced by BMDM infected with P1 enhanced the growth rate of P1 promastigotes, when derived from Balb/c and inhibited their growth when derived from C57 cells. Understanding differences between P1 and P20 and their interaction with mammalian host may help in identifying the virulence factors of P1 L.mexicana which may aid in development of vaccines or drugs against this disease.

Item Type: Thesis
Creators: Alhajri, S.M.
Date: April 2019
Rights: This work is the intellectual property of the author, and may also be owned by the research sponsor(s) and/or Nottingham Trent University. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the first instance to the author.
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 26 Feb 2020 12:02
Last Modified: 26 Feb 2020 12:02

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