**4. Discussion**

Tuberculosis is a major health problem worldwide, causing around 2 million deaths yearly. Although more than 100 years of research have led to significant improvement in disease control (in 2009, WHO reported a 35% drop in the death rate), it has also revealed a complex landscape of intricate interactions between mycobacteria and host immune system. The relative recent appearance and description of highly virulent strains in combination with the high incidence of tuberculosis in immuno-compromised patients entitles for a deeper understanding of how the immune system reacts to mycobacteria with a broader virulence spectrum.

Since T cell expression of the PD-1 molecule has been shown associated in vivo to T cell exhaustion during chronic intracellular infections, we assessed the expression of this molecule during the infection with these three different mycobacteria. T cells showed different patterns of PD-1 expression among groups of infected mice. In M. canettii infected mice only PD-1+ CD4+ T cells are increased in lungs (figure 3, top panel-left plot), in M. tuberculosis H37Rv infection only PD-1+ CD8+ T cells are increased (figure 3, top panelright plot), and in M. Beijing infected mice both T cell subsets showed increased PD-1 expression (figure 3, top panel). Interestingly, strain virulence and PD-1 expression in both T cell subsets showed an inverse correlation in the spleen, although slight differences in CD8+ PD-1+ percentage were observed among groups (figure 3, bottom panel). Seemingly, none of the strains induced overt changes in PD-1 expression on T cells, although there is a tendency for virulent mycobacteria to increase PD-1 expression on lung CD8+ T cells while

Groups of 3-5 BALB/c mice were intra-tracheally infected with different mycobacterium strains. At 60 days after infection CD40 expression on CD103+ and CD103- DCs was determined. In all groups CD103+ DCs had the highest expression. Mtb. H37Rv (gray bars) and M. Beijing (black bars) increased CD40 expression on lung CD103+ DCs. In MedLN CD103+ DCs, M. Beijing infection reduced CD40 expression while Mtb. H37Rv increased it. In contrast, M. canettii (white bars) infection reduced the percentage of CD103+ CD40+ DC in the lungs with a slight increase in MedLN. In the spleen and compared to uninfected mice (striped bars), all infected mice had reduced expression of CD40 in both CD103+ and CD103- DCs. In infected mice, for both DC subsets in the spleen, CD40 expression followed

T cells expression of the exhaustion-associated marker PD-1 was analyzed at 60 days after infection with different mycobacteria. Overall, slight differences were observed when compared to uninfected mice (stripped bars). Mice infected with M. canettii (white bars) showed a tendency to increase PD-1 expression on lung and spleen CD4+ T cells and on spleen CD8+ T cells. Mice infected with Mtb. H37Rv (gray bars) or M. Beijing (black bars) showed no differences in PD-1 expression on both CD4+ and CD8+ T cells from the spleen or MedLN. In the lungs the two virulent mycobacteria increased PD-1 expression on CD8+

Tuberculosis is a major health problem worldwide, causing around 2 million deaths yearly. Although more than 100 years of research have led to significant improvement in disease control (in 2009, WHO reported a 35% drop in the death rate), it has also revealed a complex landscape of intricate interactions between mycobacteria and host immune system. The relative recent appearance and description of highly virulent strains in combination with the high incidence of tuberculosis in immuno-compromised patients entitles for a deeper understanding of how the immune system reacts to mycobacteria with a broader virulence

**3.3 Increased expression of PD-1 on lung CD4+ and CD8+ T cells is observed only** 

**during M. Beijing chronic infection** 

M. canettii infection affects CD4+ T cells.

T cells.

spectrum.

**4. Discussion** 

a direct correlation with virulence (Beijing>H37Rv>Canettii).

Most of the current knowledge comes from studying murine models of pulmonary infection with strains of intermediate virulence (e.g. M. tuberculosis H37Rv). In the early phase of infection, mycobacteria are recognized and internalized by resident phagocytic cells like alveolar macrophages and pulmonary dendritic cells. Within these cells, mycobacterium bacilli can escape degradation and start replication. Concomitantly, it appears that lung DC crucial role in migration and activating specific T cells in the MedLN is inhibited. During the chronic phase mycobacteria apparently avoid killing associated with apoptosis of infected cells and remain confined inside granulomas.

Infection with the highly virulent Mycobacterium tuberculosis Beijing (M. Beijing) causes a quick increase of cellular infiltrate with high numbers of colony forming units in the lungs (D. Aguilar et al., 2010; B. Marquina-Castillo et al., 2009). Conversely, infection with smoothtype M. Canettii strains rarely cause TB in humans, and in the experimental mouse model, M. canettii strains show low cellular infiltrate with limited lung bacterial burden during chronic infection (M. Fabre et al., 2010). Importantly, among these three strains, virulence showed a direct correlation with inhibition of in vivo cytotoxicity (L. Quintero-Macias et al., 2011).

In the present study we tried to further define in vivo the virulence differences by assessing the potential effects upon DCs. Regarding DC activation, we observed an apparent differential recognition of M. canettii components by the DCs. When mice were treated with M. canettii lysate, BAL and lung DCs expressed similar levels of CD86 after LPS stimulation in vivo. During infection, lung and spleen CD103+ DCs showed less CD40 expression as compared to the other mycobacterial infection and to uninfected mice. Unlike M. canetti, both Mtb H37Rv and M. Beijing components reduced DC activation in BAL, and during infection, increased CD40 expression in lung DCs. Conceivably, virulent mycobacteria might induce a strong activation of BAL DCs causing the migration towards lung parenchyma and MedLN. The differences observed between Mtb H37Rv and M. Beijing infection suggest a probable scenario where Mtb H37Rv induces DC migration to MedLN whereas M. Beijing prevents MedLN recruitment while increasing systemic distribution.

Homeostatic mechanisms during chronic inflammatory responses on mucosal surfaces tend to increase and bias T cell differentiation to anti-inflammatory and regulatory phenotypes. PD-1 expression on T cells is associated with T cell exhaustion during chronic intracellular infections. Our results showed only slight variations in PD-1 expression during chronic infection. Of note, Mtb H37Rv and M. Beijing infections induced similar increase in the percentage of CD3+CD8+PD1+ lung T cells, while M. canettii infection increased PD-1 expression on lung and spleen CD3+CD4+ T cells. Although PD-1 expression had small variations compared to uninfected mice, a tendency of virulent mycobateria to induce CD8+ PD-1+ T cells was observed and might relate to decreased in vivo cytotoxicity (L. Quintero-Macias et al., 2011).

Several mycobacterial components have been associated to immune system subversion. RD-1-encoded secreted proteins (e.g. ESAT-6) mediate macrophage inhibition by TLR2 recognition and have the potential to form pores in membranes probably facilitating bacterial escape from phagosomes. RD-1 region is associated with virulence since is absent in attenuated M. bovis BCG. However the three strains used in our experiments

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Mycobacterial cell wall also contributes to virulence. The high content of heterogeneous lipids is a hallmark of mycobacteria. Lipoarabinomanans and lipomanans are two of the major lipidic components that can reduce the activation and cytokine secretion of macrophages. Although limited evidence suggest that cell wall lipids are key in immune response subversion (L. Quintero-Macias et al., 2010; L. M. Rocha-Ramirez et al., 2008), strain-specific lipid characterization during infection represents a challenge yet to be fully engaged. Among the strains used in our experiments, clear differences on the lipid synthesis have only been described for M. Beijing (G. Huet et al., 2009; M. B. Reed et al., 2007).

On the other hand, a decade of intense research on the mucosal immune system provides an outlook of similar complexity with the interaction of an increasing number of cell types within highly specialized microenvironments. In the case of tuberculosis is necessary to consider that the interaction of at least four histological compartments (alveolar space, lung parenchyma, draining lymph nodes, blood) is affected by the balance between mycobacterial virulence and host resistance. Apparently, virulent mycobacteria would preferentially target lung CD103+ DCs and thus avoid activation of the CD103- DCs. Further research is required to determine whether this constitutes an active evasion mechanism and to clarify the role of lung CD103+ DCs in the induction of Mtb-specific T cells or regulatory T cells.
