**3. Results**

#### **3.1 Lysates of highly virulent mycobacteria decrease activation of BAL DC in vivo**

To test whether mycobacterial components differentially affected the activation patterns of lung DCs, we intra-tracheally treated separate groups of mice with different mycobacterial

Mycobacterial Strains of Different Virulence Trigger

the MedLN.

activation of airways DCs.

increased CD86 expression in Spleen DCs.

Dissimilar Patterns of Immune System Activation *In Vivo* 171

in the lungs with moderate inhibition in the MedLN (the regional, draining lymph node) and spleen (systemic response). Mtb H37Rv induced the activation of CD103+ DC in both lungs and MedLN while inhibiting CD40 expression in the spleen; and M. canettii reduced CD40 expression in CD103+ DCs in the lungs and spleen, with a slight increase in

Fig. 1. Prior inoculation of mycobacterial lysate from virulent strains reduces subsequent

Prior inoculation of mycobacterial lysate from virulent strains reduces subsequent activation of airways DCs. Groups of 3-5 BALB/c mice received intra-tracheal (i.t.) lysates prior to intra-nasal (i.n.) LPS stimulation. After 10 hours of LPS inoculation CD86 expression on DCs was determined and compared to control groups (striped bars). Bronchio-alveolar lavage (BAL) and lung DCs from mice that received M. Beijing lysate (black bars) showed a reduction in CD86 expression (mean fluorescence intensity (MFI) and percentage of positive cells). Mtb. H37Rv lysate (gray bars) induced a reduction of CD86 only in BAL DCs while M. canettii lysate (white bars) did not induced a reduction in BAL and lung DCs and neither

lysates prepared from each mycobacterial strain. After five hours mice were challenged with intranasal LPS to determine the subsequent DC activation. CD86 expression on DCs from bronchio-alveolar lavage (BAL), lung and MedLN was assessed after 10 hours of LPS challenge.

Compared to all control groups, M. tuberculosis H37Rv and M. Beijing lysates reduced the LPS-triggered activation of BAL and lung DCs, whereas M. Canettii lysate showed no difference. Interestingly, in these two groups of mice (figure 1, top and middle panels), the reduced activation is observed regarding both the percentage of CD86+ DCs (left Y axis) and the intensity of expression of CD86 (MFI, right Y axis). In the lungs, slight differences were observed only in mice treated with lyBei. In the MedLN, all lysates increased the percentage of CD86+DCs when compared to mice that received two doses of LPS (figure 1, bottom panel, left Y axis).

Our results suggest that the one factor contributing to the different virulence observed among MTC strains lies in how DCs respond to mycobacterial components. These slight but relevant differences are clearly seen in the activation patterns of BAL and lung DCs. Although it remains uncertain whether the patterns observed with lyH37 or lyBei lysates are product of increased DC migration from the BAL to the lungs to the MedLN (which could be associated with the virulence-dependent exacerbated lung infiltrate characteristic of chronic tuberculosis) or a gradient of DC inhibition (which could be associated with the early inhibition of specific T cell responses).

#### **3.2 The CD103+ dendritic cell subset is preferentially activated during virulent Mtb infection**

To assess whether infection with different MTC strains induced divergent patterns of DC activation we evaluated the expression of CD40 on lung, MedLN and spleen DCs. CD40 in DCs is a crucial coestimulatory molecule required for naive T cell activation and –especiallyfor appropriate induction of cytotoxic T cells (P. Bjorck et al., 1997; G. Grouard et al., 1996; A. M. Moodycliffe et al., 2000), in lung, MedLN and spleen DCs. We compared CD40 expression between uninfected mice and mice infected with M. canettii, M. tuberculosis H37Rv, or M. Beijing at chronic infection (60 days after infection). Also, we analyzed two DC subsets distinguished by the expression of the CD103 molecule. In particular the lung CD103+ DC subset, as this is associated with proinflammatory responses and CD8+ T cell activation during intracellular pathogen infections (G. T. Belz et al., 2004; M. L. del Rio et al., 2010; T. S. Kim & T. J. Braciale, 2009).

We found that the infection with virulent mycobacterial strains (Mtb. H37Rv and M. Beijing) mainly activated lung CD103+ DCs (figure 2). In contrast, lung CD103+ DCs of mice infected with M. canettii had the lowest expression of CD40 (figure 2, top panel white bar). Interestingly, in M. Beijing infected mice, MedLN CD103+ DCs showed reduced expression of CD40 (figure 2, middle panel), even below the expression of the control group (uninfected mice). In the spleen, CD40 expression in both DC subsets from infected mice was below levels observed in uninfected mice (figure 2, bottom panel), but showed a direct correlation with virulence (M. Beijing infected mice had the highest expression of CD40 in spleen DCs).

These results indicate that mycobacteria of different virulence induce each a different activation pattern in the regional DCs. M. Beijing infection induced CD103+ DC activation

lysates prepared from each mycobacterial strain. After five hours mice were challenged with intranasal LPS to determine the subsequent DC activation. CD86 expression on DCs from bronchio-alveolar lavage (BAL), lung and MedLN was assessed after 10 hours of LPS

Compared to all control groups, M. tuberculosis H37Rv and M. Beijing lysates reduced the LPS-triggered activation of BAL and lung DCs, whereas M. Canettii lysate showed no difference. Interestingly, in these two groups of mice (figure 1, top and middle panels), the reduced activation is observed regarding both the percentage of CD86+ DCs (left Y axis) and the intensity of expression of CD86 (MFI, right Y axis). In the lungs, slight differences were observed only in mice treated with lyBei. In the MedLN, all lysates increased the percentage of CD86+DCs when compared to mice that received two doses of LPS (figure 1, bottom

Our results suggest that the one factor contributing to the different virulence observed among MTC strains lies in how DCs respond to mycobacterial components. These slight but relevant differences are clearly seen in the activation patterns of BAL and lung DCs. Although it remains uncertain whether the patterns observed with lyH37 or lyBei lysates are product of increased DC migration from the BAL to the lungs to the MedLN (which could be associated with the virulence-dependent exacerbated lung infiltrate characteristic of chronic tuberculosis) or a gradient of DC inhibition (which could be associated with the

**3.2 The CD103+ dendritic cell subset is preferentially activated during virulent** 

To assess whether infection with different MTC strains induced divergent patterns of DC activation we evaluated the expression of CD40 on lung, MedLN and spleen DCs. CD40 in DCs is a crucial coestimulatory molecule required for naive T cell activation and –especiallyfor appropriate induction of cytotoxic T cells (P. Bjorck et al., 1997; G. Grouard et al., 1996; A. M. Moodycliffe et al., 2000), in lung, MedLN and spleen DCs. We compared CD40 expression between uninfected mice and mice infected with M. canettii, M. tuberculosis H37Rv, or M. Beijing at chronic infection (60 days after infection). Also, we analyzed two DC subsets distinguished by the expression of the CD103 molecule. In particular the lung CD103+ DC subset, as this is associated with proinflammatory responses and CD8+ T cell activation during intracellular pathogen infections (G. T. Belz et al., 2004; M. L. del Rio et al.,

We found that the infection with virulent mycobacterial strains (Mtb. H37Rv and M. Beijing) mainly activated lung CD103+ DCs (figure 2). In contrast, lung CD103+ DCs of mice infected with M. canettii had the lowest expression of CD40 (figure 2, top panel white bar). Interestingly, in M. Beijing infected mice, MedLN CD103+ DCs showed reduced expression of CD40 (figure 2, middle panel), even below the expression of the control group (uninfected mice). In the spleen, CD40 expression in both DC subsets from infected mice was below levels observed in uninfected mice (figure 2, bottom panel), but showed a direct correlation with virulence (M. Beijing infected mice had the highest expression of CD40 in spleen DCs). These results indicate that mycobacteria of different virulence induce each a different activation pattern in the regional DCs. M. Beijing infection induced CD103+ DC activation

challenge.

panel, left Y axis).

**Mtb infection** 

early inhibition of specific T cell responses).

2010; T. S. Kim & T. J. Braciale, 2009).

in the lungs with moderate inhibition in the MedLN (the regional, draining lymph node) and spleen (systemic response). Mtb H37Rv induced the activation of CD103+ DC in both lungs and MedLN while inhibiting CD40 expression in the spleen; and M. canettii reduced CD40 expression in CD103+ DCs in the lungs and spleen, with a slight increase in the MedLN.

Fig. 1. Prior inoculation of mycobacterial lysate from virulent strains reduces subsequent activation of airways DCs.

Prior inoculation of mycobacterial lysate from virulent strains reduces subsequent activation of airways DCs. Groups of 3-5 BALB/c mice received intra-tracheal (i.t.) lysates prior to intra-nasal (i.n.) LPS stimulation. After 10 hours of LPS inoculation CD86 expression on DCs was determined and compared to control groups (striped bars). Bronchio-alveolar lavage (BAL) and lung DCs from mice that received M. Beijing lysate (black bars) showed a reduction in CD86 expression (mean fluorescence intensity (MFI) and percentage of positive cells). Mtb. H37Rv lysate (gray bars) induced a reduction of CD86 only in BAL DCs while M. canettii lysate (white bars) did not induced a reduction in BAL and lung DCs and neither increased CD86 expression in Spleen DCs.

Mycobacterial Strains of Different Virulence Trigger

Dissimilar Patterns of Immune System Activation *In Vivo* 173

Fig. 3. PD-1 expression in CD4+ and CD8+ T cells during chronic infection with different

mycobacterial strains.

Fig. 2. CD40 expression in CD103+ and CD103- DC subsets during chronic infection with different mycobacterial strains.

Fig. 2. CD40 expression in CD103+ and CD103- DC subsets during chronic infection with

different mycobacterial strains.

Fig. 3. PD-1 expression in CD4+ and CD8+ T cells during chronic infection with different mycobacterial strains.

Mycobacterial Strains of Different Virulence Trigger

cells and remain confined inside granulomas.

2011).

Macias et al., 2011).

Dissimilar Patterns of Immune System Activation *In Vivo* 175

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

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.,

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-

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

#### **3.3 Increased expression of PD-1 on lung CD4+ and CD8+ T cells is observed only during M. Beijing chronic infection**

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 M. canettii infection affects CD4+ T cells.

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

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+ T cells.
