Caracterización funcional de la proteína scavenger AIM en la respuesta anti-infecciosa del macrófago

Abstract

[eng] AIM (Apoptosis Inhibitor of Macrophages) a soluble protein belonging to the SRCR superfamily, is expressed mostly by macrophages in both lymphoid and inflamed tissues. Research undertaken over the last decade has uncovered critical roles of CD5L as a pattern recognition receptor of bacterial and fungal components and in the control of key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In the context of bacterial infection, AIM influences the monocyte inflammatory response to PAMPs by reducing TNF-α secretion. However, no AIM-dependent regulation of other inflammatory mediators nor the mechanisms involved in its anti-inflammatory function had been described at the beginning of our work. Considering this, the main objective of the present work was to analyze the immunomodulatory role of human AIM protein (hAIM) in macrophage response to bacterial aggression. To study hAIM function, we used two cellular models, namely THP1 cell line and peripheral blood monocytes obtained from healthy donors. As AIM expression disappears in cultured cells, we generated a THP1 cell line that stably expresses hAIM and produced a new recombinant form of this protein with improved yield to supplement monocyte cultures. The first specific objective of the present work was to deepen the analysis of AIM anti-inflammatory properties in the context of macrophage responses to PAMPs. Our goal was to try to decipher the macrophage signalling pathways that could be affected by AIM resulting in its inhibition of TNF-α. Our results indicated that modulation of cytokine responses by hAIM was not restricted to TNF-α. Lowered IL-1β and enhanced IL-10 protein secretion suggest a broader anti-inflammatory role of hAIM on macrophages. We also proved that the anti-inflammatory pattern induced by AIM is mediated through enhanced autophagy, a highly conserved cellular degradation process. Examination of autophagy markers in both THP1 macrophages and PB monocytes reinforced this notion. Furthermore, electron microscopy analysis showed increased presence of cytoplasmic autophagosomes in THP1 macrophages overexpressing hAIM. Silencing experiments indicated that the receptor CD36 was required for hAIM-induced autophagy, thereby revealing a novel function for the CD36-hAIM axis in the induction of macrophage autophagy and in the control of cellular homeostasis. These observations suggest that the modulation of hAIM-CD36 activity may offer therapeutic options for severe inflammatory conditions associated with deregulated autophagy. Phagocytosis, like autophagy, is an ancient and highly conserved cellular function. However, the nature of the interactions between these two critical processes remains unclear. The second goal of the present work was to analyze hAIM role in macrophage phagocytosis. In order to achieve this goal, a set of in vitro experiments were performed comparing the phagocytic abilities of murine and human AIM proteins. Our data suggested that hAIM does not share the pro-phagocytic function of its murine homolog. A lack in direct evidences of the role of AIM in macrophage bactericidal mechanisms brought us to our third objective: to analyze the involvement of AIM in the response of macrophages to infection by bacteria. An in vitro model of Mycobacterium tuberculosis infection of THP1-MΦ was established in the laboratory. This helped us to prove that AIM potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy. In summary, our results provide new findings on hAIM that are highly relevant for our knowledge of macrophage in homeostasis as well as in response to inflammation and infection. They further provide a new function for the CD36 receptor as an inducer of MΦ autophagy through hAIM, and therefore they open a new perspective of the role of the hAIM-CD36 axis participating in the cross-talk between the immune (pathogen-sensing) and metabolic systems that is emerging as a crucial homeostatic mechanism.