Classically (M1) and alternatively (M2) activated macrophages infected with generalists and specialists Mycobacterium tuberculosis complex isolates display differential phagosomal acidification patterns
M. Chiacchiaretta1, N. Bresciani1, A. Agresti2, S. Zambrano2,3, D. Mazza3,4, F. Cugnata3, C. Tassan Din5, D.M. Cirillo1, P. Miotto1

1 Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS Ospedale San Raffaele, Milano, Italy; 2 Chromatin Dynamics Unit, San Raffaele University and Scientific Institute, Milano, Italy; 3 Vita-Salute San Raffaele University, Milano, Italy; 4 Cancer Imaging Unit, Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milano, Italy; 5 Infectious Diseases Clinical Department, Division of Immunology, Transplantation and Infectious Diseases, IRCCS Ospedale San Raffaele, Milano, Italy

The contribution of host/pathogen heterogeneity to the pathogenesis of tuberculosis (TB) remains poorly characterized. Our study aims at understanding the contribution of the bacterial genotype (lineage) and of the macrophage (mφ) phenotype (M1/M2) in determining the fate of host-pathogen interaction during M. tuberculosis (Mtb) infection.
dsRED fluorescent Mtb strains (EAI –L1, Beijing –L2, Haarlem, H37Rv, H37Ra, CDC1551 –L4, Africanum –L6) were used for THP-1 derived classically (M1) and alternatively activated (M2) mφ-like cells infection. Strains were grouped as generalist/modern/more virulent or specialist/ancient/less virulent isolates. Live single-cell confocal microscopy was used to analyze (i) lysosomal acidification (LysoSensor Green) at 6 h and 24 h post-infection (p.i.), (ii) autophagy (CYTO-ID), and (iii) apoptosis and necrosis (cytocalcein violet, apopxin, and 7-AAD) at 24 h p.i. Linear mixed-effects (LME) models were employed to evaluate differences among groups.
A total of 7384 single cells were considered for the analysis of phagosomal acidification. In M1 mφs acidification is delayed by generalist/modern/more virulent strains, whereas at earlier time points specialist/ancient/less virulent strains are not blocking acidification. At 24 h p.i. none of the strains is blocking phagosomal acidification, but specialist/ancient/less virulent strains displayed lower levels of acidification compared to generalist/modern/more virulent strains. Generalist/modern/more virulent isolates displayed higher colocalization with regions at lower pH. The number of responsive mφs remained relatively low (15-25%). In M2 mφs acidification is observed at early time points with clinical isolates, but not with laboratory strains. At 24 h p.i. generalist/modern/more virulent strains blocked acidification, whereas specialist/ancient/less virulent strains were less efficient in blocking the acidification of phagosomes. No differences in terms of colocalization of mycobacteria within acidified compartments were observed in M2 mφs. The number of responsive mφs was found to vary depending upon the features of the infecting strains, with the generalist/modern/more virulent strains inducing the lowest percentages of acidifying cells (<10%).
To evaluate the autophagic flux, a total of 1960 single cells were considered. None of the strains induced autophagy in M1 nor M2 mφs.
A total of 2470 single cells were considered for the analysis of apoptosis and necrosis. In M1 mφs, bystander non-infected cells of generalist/modern/more virulent strains showed increased apoptosis (40%). In contrast, none of the category induced apoptosis in M2 mφs at the time point considered. In our testing conditions (24 h p.i.) necrosis was not observed.
The use of a single-cell analytical approach highlighted the heterogeneity of the macrophagic response to Mtb infection, emphasizing the role played in the infection by (i) the mφ’s activation status and (ii) the isolate’s features.

Paolo Miotto PhD

IRCCS Ospedale San Raffaele

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