Pathological and Immunological Changes in Host Cells in Response to Leishmania mexicana Infection

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2019-04

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Nottingham Trent University, UK

Abstract

Abstract 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. v 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.

Description

Continuous passaging of L.mexicana for twenty passages in vitro caused major morphological and functional changes. Cells became rounded and elongated, increased in body size, and their growth was inhibited in RPMI1640 medium. This was accompanied by a loss of metacyclic stage promastigotes, the ability to differentiate into amastigotes and virulence and the downregulation of all ten virulence-associated genes tested (LPG1, LPG2, A2, CHAT1, CPB2, CPB2.8, CPC, GP63, LACK, and MAPK9). Expression of lipophosphoglycan (LPG) and phosphatidylserine (PS) on the surfaces L.mexicana promastigotes was affected by 20 passages, the expression of LPG was significantly decreased, whereas the expression of PS significantly increased in P20. Therefore, this is a point to consider when preparing a vaccine from old culture parasites. Virulent (P1) L.mexicana can infect and survive in both Balb/c and C57 bone marrow derived macrophages in vitro. Although avirulent (P20) L.mexicana was engulfed by both Balb/c and C57 bone marrow derived macrophages in vitro, it failed to differentiate into amastigotes and failed in the formation PV, subsequently it did not survive inside the macrophage. Therefore 20 passages did not affect the infectivity of L.mexicana but affects the survival of parasites which was noticed in both resistant and susceptible mice strains. The strategy to survive in the macrophages may be a result of parasite virulence factors such as LPG, GP63. Virulent L.mexicana was able to modulate gene regulation in Balb/c more than that in C57 BMDM. P1 was able to modulate pro-inflammatory cytokine gene regulation in Balb/c, which was also confirmed by detection of TNF-α by ELISA. The generation and secretion of TNF-α cytokines (that activate Th1) was decreased by cytokines in Balb/c and increased in C57 after 24 hours infection. Virulent L.mexicana can inhibit apoptosis in both infected Balb/c and C57 Balb/c BMDM combined with downregulation of caspase 8 and upregulation of BCL 2 by qPCR. P1 promastigote, treated with supernatants derived from Balb/c BMDM infected with P1 for 24 hours enhanced their growth and upregulated LPG1, CHT1, CPB2 and CPB2.8, while when treated with supernatant derived from C57 infected with P1 inhibited their growth and caused downregulation of LPG1, LPG2, CPB2, CPB2.8, CHT1 and A2.

Keywords

Keywords: L.mexicana, Leishmania mexicana, twenty passages, Passage 20, lipophosphoglycan, LPG, promastigotes, Virulent, Balb/c and C57 bone marrow derived macrophages in vitro, PCR, ELISA. Mice, LPG1, LPG2, A2, CHAT1, CPB2, CPB2.8, CPC, GP63, LACK, and MAPK9, pro-inflammatory cytokine, TNF- α, IL-6, IL-1β and TGF-β, Avirulent, Leishmania Parasite, supernatant, treatments or vaccines, bone marrow derived macrophages, Differentiation into amastigotes, Alamar blue, CFSE stain, qPCR, Apoptosis

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Alhajri,2019

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