Projecto nº:016590

Referência do Projecto:PTDC/IMI-MIC/1683/2014(POCI-01-0125-FEDER-016590)

Título:Iron hide and seek- does ferritin help the macrophage fight mycobacteria?

Montante envolvidos:

Investimento total: 199.632,00 €

IBMC-Instituto de Biologia Molecular e Celular -199.632,00€

Apoio FEDER: 169.687,20€

Apoio OE: 29.944,80€

Localização do projecto: Porto, Portugal

Sintese do projecto:

Mycobacteria are intracellular pathogens causing severe chronic infections in humans and other animals.

Mycobacterium tuberculosis is a leading cause of human death, while M. bovis, M. avium and several other nontuberculous mycobacteria act as opportunistic pathogens in immunosupressed individuals and cause important economic losses in different pecuary species. The only vaccine available to prevent human tuberculosis (BCG) has a low overall efficacy and the treatment of mycobacterial infections is especially difficult due to their intrinsic sturdiness and to the increasing spread of resistance genes among pathogenic isolates [1]. A better understanding of the interaction between mycobacteria and their hosts is thus urgently needed in order to design new and more efficient therapeutic interventions.

Our group and others have clearly established that the host?s iron status has a strong impact on resistance or

susceptibility to mycobacterial infection. Mycobacteria grow faster in the organs of an iron overloaded host while iron starvation or the administration of iron-chelating drugs can decrease bacterial growth [2-4]. During infections, the host’s innate immune response includes systemic iron re-distribution and its sequestration inside macrophages, in order to deprive pathogens from this fundamental element. However, in the case of mycobacteria, which survive and proliferate inside macrophages, it is thus far not clear what the consequences are of iron accumulation inside macrophages. Of relevance, we have recently reported that macrophages upregulate the production of the iron storage protein H-ferritin (Hft) when infected with M. avium, which could deprive the microbe of iron [5]. This increase in Hft production by macrophages during infection could also be part of a host cell protective response against oxidative stress induced by infection. We have recently shown that in a model of free heme-induced oxidative stress, host’s susceptibility to mycobacterial infection was dramatically increased, concomitantly with extensive macrophage death and a failure to form protective granulomas [6]. Other investigators have shown that Hft is a crucial host protective factor against heme-induced toxicity [7].

The general aim of this project is to elucidate the molecular mechanisms that link host iron metabolism to mycobacterial growth and pathogenesis with a special emphasis on the role played by Hft. This will include both in vitro studies, in which molecular details of Hft involvement in the macrophage-mycobacteria interaction will be addressed and in vivo assays in which the systemic role of Hft in host-mycobacteria interaction will be evaluated.

Our group has a vast experience in the study of the interaction of mycobacteria with macrophages. We have previously demonstrated that mouse primary macrophages increase their levels of Hft when infected with M. avium [5]. In this project, we will take advantage of myeloid cell-specific-Hft-knock-out (Mo-Hft-/-) mice (kindly provided by Prof. Lukas Kuhn, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) to obtain Hft-deficient macrophages.

We will evaluate the impact of Hft deficiency on the intra-macrophagic growth of M. avium and M. tuberculosis as well as on the viability of infected macrophages. The degree of autophagy and programmed cell death induced during M. avium and M. tuberculosis infections will also be evaluated, since these processes are known to be affected by iron metabolism and to have a clear impact on mycobacteria survival inside macrophages, besides being amenable to pharmacological manipulation.

Mo-Hft-/- mice will also be used to evaluate the role of macrophage Hft in host protection against M. avium and

M. tuberculosis infections in vivo. Since Mo-Hft-/- mice were not previously characterized, we will start by their

characterization in terms of hematological parameters and iron distribution in different tissues. Afterwards, we will study how the Mo-Hft-/- mice iron status is affected by infection with M. avium and M. tuberculosis. The inflammatory host response to these infections will be thoroughly characterized. Additionally, the impact of macrophage Hft deficiency on the growth of the bacteria and host pathology will be evaluated including the signs of oxidative stress and cell death in different tissues and the capacity to form granulomas.

At the end of this project we will have gathered fundamental information regarding how iron manipulation affects the outcome of infections by M. tuberculosis and other mycobacteria. This will allow an improvement of the capacity to identify host susceptibility factors and pave the way to the pharmacological manipulation of the host immune response and iron metabolism as a strategy to decrease the morbidity and mortality of these devastating diseases.

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