Regulation of gap junction protein, Connexin 43 in macrophages upon infection with Leishmania donovani

Das, Damayantee (2015) Regulation of gap junction protein, Connexin 43 in macrophages upon infection with Leishmania donovani. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Damayantee Das (13IP15)) Damayantee_Das_13IP15.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Connexins or gap junction intracellular channel (GJIC) proteins (ranging in molecular weight from 25 to 60 kDa) assemble into gap junction channels where it oligomerizes into hexameric hemichannels to form connexons. Connexons from two adjacent cells form a gap junction intercellular channel (GJIC). Molecules and ions of less than 1 kDa can be transported across the GJIC and helps in maintaining the homeostasis of the cells. Recent studies demonstrated several role of connexins or GJIC in immune cell activation and inflammation. Several different alpha and beta connexins are known to be expressed in different immune cells. Macrophages/monocytes are the major immune cells in our body and are known to play a major role in inflammation and infection. It is known that Cx43 is primarily expressed in macrophages and may also be present in its phagosome. Question remains whether Cx43 or Cx43 mediated GJIC have any role in macrophage activation and phagosome maturation. We have chosen Leishmania donovani infection in mouse macrophages as a model to study the alteration of Cx43 as Leishmania donovani is known to profusely infect macrophages. Macrophage cell lines J774A.1 cells and RAW 264.7 cells are infected with Leishmania donovani. Infection is characterized by DAPI staining.Cell lysate are prepared from infected cells as well as from uninfected cells. Total protein is isolated from the cell lysate and subjected for Cx43 immunoblotting. Preliminary immunoblotting data demonstrated that there could be an upregulation of Cx43 upon infection which needs to be confirmed further. As Leishmania donovani shows very limited infection rate in transformed macrophage cell lines, so experiments are in progress to test whether infection rate can be higher in peritoneal macrophages that are isolated from adult C57BL/6 mouse. Our preliminary data shows that the infection rate is considerably high in peritoneal macrophages compared to macrophage cell lines. It will be interesting if we can confirm that there is an upregulation of Cx43 expression in macrophages due to Leishmania donovani infection. Leishmania donovani infection in macrophages will provide a potential model to understand the role of Cx43 in the phagocytosis of macrophages and immune activation.

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