**1. Introduction**

166 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Velayati, A.A., Farnia, P., Merza. M.A., Zhavnerko, G.K, Tabarsi, P., Titov, L.P, . Ghanavei,

Velayati, A.A., Farnia, P., Masjedi, M.R., Zhavnerko, G.K., Merza, M.A., Ghanavei, J.,

Vera, H.D., and L.F. Rettger.1940. Morphological variations of the tubercle bacillus and

Vicente, M., and A.I.Rico. 2006. The order of the ring: assembly of *Escherichia coli* cell

Vollmer, W., and J.V. Holtje .2004.The architecture of the murein (peptidoglycan) in gram – negative bacteria :vertical scaffold or horizontal layer(s). J.Bacteriol.186:5978-5987. Wayne, L.G.1994. Dormancy of Mycobacterium tuberculosis and latency of disease.

Wayne, L.G., and L.G.Hayes.1996. An in vitro model for sequential study of shiftdown of

Wayne, L.G., andH.A.Sramek.1994.Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis. Antimicrob .Agents.Chemother.38:2054-2058. Wietzerbin, J., Das, B.C., Petit, J.F., Lederer, E., Leyh-Bouille, M., and J.M.Ghuysen.1974.

Weidel, W., Frank, H., and H.H.Martin. 1960.The rigid layer of the cell wall of *Escherichia coli*

Weidel, W., and H, Pelzer.1964.Bagshaped macromolecules – a new outlook on bacterial cell

Werner, G.H. 1951. La cytologie des bacilles tuberculex etudièe en relation avec leurs

Young, M., Mukamolova, G.V., and A.S. Kaprelyants. 2005. Mycobacterial dormancy and its

Young, K.D. 2006. The selective value of bacterial shape. Microbiol . Mol. Biol.Rev. 70 :660-

Young, K.D. 2006.The selective value of bacterial shape. Microbiol.Mol.Biol.Rev.70:660-703 Yuan, Y., Barrett, D., Zhang, Y., Kahne, D., Silz, P., and S, Walker. 2007.Crystal structure Of

relation to persistence .In :parish T, editor .Mycobacterial molecular biology.

a peptidoglycan glycosyltransferase suggests a model for processive glycan chain

atomic force microscopy. Eur .Respir. J. 36: 1490-3.

(AFM). Int J Clin Exp Med 2011;4(in press).

division components .Mol.Microbiol.61:5-

Eur.J.Clin.Microbiol. Infect. Dis. 13:908-914.

J.Bacteriol.39:659-687.

Infect. Immun. 64:2062-2069.

mycobacteria. Biochem.13:3471-3476.

strain. J .Gen. Microbiol. 22:158-166.

caractèe de virulence 59:1043

703.

walls. Adv.Enzmol.Relat.Areas Mol.Biol. 26:193-232.

Young, K.D. 2003. Bacterial shape. Mol. Microbiol. 49:571-580

Norwich: Horizon Scientific Press: 265-320

synthesis.Proc.Natl.Acad.Sci.USA.104;5348-5353.

Ziehl, F. 1882. Zur Farbung des Tuberkelbacillus. 8:451.

55:303-7

Mycobacterium tuberculosis: using transmission electron microscopy. Chemo.

J., Farnia, P., Setare, M., Poleschuyk, N.N., Owlia, P., Sheikolslami, M., Ranjbar, R., Masjedi, M.R. 2010. New insight into extremely drug-resistant tuberculosis: using

Tabarsi, P., Farnia, P., Poleschuyk, N.N., and G.Ignatyev.2011. Sequential adaptation in latent tuberculosis bacilli: observation by atomic force microscopy

certain recently isolated soil acid fasts with emphasis on filterability.

Mycobacterium tuberculosis through two stages of nonreplicating persistence .

Occurrence of D-alanyl(D)-meso-diaminopimelic acid and meso-diaminopimelymeso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Tuberculosis (TB), one of the major world health problems, is a chronic infection caused by members of the Mycobacterium tuberculosis complex (MTC). In 2009, tuberculosis (TB) caused 1.7 million deaths and 9.4 million new cases. Although recent efforts to improve TB prevention, diagnosis and treatment have contributed to a 35% decrease in the death rate, the emergence of mycobacterial strains with highly virulent phenotypes combined with pandemic HIV infections has added new challenges to control TB.

Host-pathogen interactions during experimental pulmonary tuberculosis have been studied using laboratory mycobacterial strains of well defined, relatively homogeneous virulence. These studies have contributed to uncover immune evasion mechanisms evolved by mycobacteria, and their role to establishing chronic infections. Despite the successful models of experimental tuberculosis and the high homology among MTC strains, the immune mechanisms and the mycobacterial characteristics that cause the remarkable varying degrees of clinical virulence remain barely studied. Although previous reports partially described differences in immunopathogenesis and bacterial growth (R. Chacon-Salinas et al., 2005; J. Dormans et al., 2004; B. Lopez et al., 2003), the effects of different MTC strains both on airways DC and on T cell activation have not been assessed, especially in vivo.

Broadly, mycobacterium of intermediate virulence (e.g. M. tuberculosis H37Rv) seems to reduce DC migration to the mediastinal lymph nodes (A. J. Wolf et al., 2007) which could be associated with a delayed onset of specific effector T cell responses (G. S. Garcia-Romo et al., 2004; R. J. North & Y. J. Jung, 2004; A. J. Wolf et al., 2008), thus allowing early (during the first 4 weeks of infection) exponential Mtb replication. Around 30 days post-infection,

<sup>\*</sup> Selene Meza-Pérez1, Fernando Muñoz-Teneria1,

Dulce Mata3, Juana Calderon-Amador1, Sergio Estrada-Parra2,

Rogelio Hernández-Pando3, Iris Estrada-García2 and Leopoldo Flores-Romo1

*<sup>1</sup>Department of Cell Biology Cinvestav-IPN,Mexico City, Mexico*

*<sup>2</sup>Department of Immunology ENCB-IPN, Mexico City, Mexico 3Department of Pathology INNSZ, Mexico City, Mexico*

Mycobacterial Strains of Different Virulence Trigger

for the various analysis.

**3. Results** 

Dissimilar Patterns of Immune System Activation *In Vivo* 169

Then, 10 hours after LPS challenge, a time which is around the peak of lung DC activation induced by LPS alone, we obtained cell suspensions from lung, BAL (Bronchio-Alveolar Lavage) and MedLN to assess CD86 expression in (Gr1-, MHC-II hi, CD11c+) DCs.

*M. tuberculosis* strains were grown in Middlebrook 7H9 medium (Difco Laboratories) supplemented with OADC (Difco Laboratories). After 1 month of culture, mycobacteria were harvested, adjusted to 2.5x105 bacteria in 100µl sterile endotoxin-free saline solution, aliquoted, and maintained at -70ºC until used. Before use, bacteria were stained with fluorescein diacetate (InvitroGen, F1303) and viable bacteria (Kvach, J. T. and Veras, J. R. 1982) (green fluorescence) were counted with an epifluorescence microscope and adjusted to the infective dose. We used the murine model of intra-tracheal infection as described

Briefly, 3-5 male BALB/c mice from 6-8 weeks of age were anaesthetized with sevoflurane, and 100 μl isotonic sterile endotoxin-free saline solution with 2.5 x 105 viable bacilli were inoculated intra-tracheally. Control animals were inoculated only with isotonic, sterile endotoxin-free saline solution without bacilli. Animals were then maintained in cages fitted with microisolators in a P-3 biosecurity level facility. The protocol was institutionally approved according to ethical norms for use of animals in experimentation. Following infection, at least three to five mice per group were euthanized at every time point selected

Monoclonal antibodies used for phenotypic analysis of DC and T cells were anti-CD3-FITC (BD Pharmingen 553062), anti-CD4-PerCP (BD Pharmingen 553052), anti-CD8a-APC (BD Pharmingen 553035), PD-1-PE (BD Pharmingen 551892), anti-CD11c-APC (BD Pharmingen 550261), anti-CD40-PE (BD Pharmingen 553791), anti-MHCII-FITC (BD Pharmingen 553623), anti-Ly6c-A700 (e-biosciences 56-5981-32), and Streptavidin-conjugated with PerCP fluorochrome (SAV-PerCP, BD Pharmingen 554064), biotinylated anti-CD103 (R&D Systems BAF1990). Cell suspensions were prepared by disgregating the organs using a 70m cell strainer (BD Falcon 352350) and the piston of a 3mL Syringe (BD 309585). Spleen, Lungs, BAL and Mediastinal lymph nodes cell suspensions were washed, incubated 10 min at 4°C with Power Block reagent (Biogenex, HK085-5K) to block Fc receptors, washed, and stained with fluorochrome-coupled mAbs for 15 min at 4°C. Cells were centrifuged and resuspended in FACS buffer. 106 and 105 live MHC-II high or CD3+ cell cells were acquired respectively. Data was acquired in a Dako Cyan Flow Cytometer and analyzed with FlowJo

**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 lysates were prepared by one of us (I. Estrada-Garcia, ENCB-IPN).

**2.2 Experimental model of airways-induced pulmonary tuberculosis in mice** 

previously (Hernandez-Pando, R. 1996), with some modifications.

**2.3 Staining of cell suspensions for flow cytometry analysis** 

Software 7.2 (Tree Star, Inc., San Carlos, CA).

mycobacterial replication rate is diminished (B. J. Rogerson et al., 2006) while diverse immune evasion mechanisms avoid bacterial killing by T cell-activated macrophages (J. A. Armstrong & P. D. Hart, 1971) and cytotoxic CD8+ T cells (E. M. Weerdenburg et al., 2010). The analysis of these evasion mechanisms used by MTC strains, however, have barely been comparatively assessed (L. Quintero-Macias et al., 2011).

To evaluate the in vivo differences in host-pathogen interaction across the wide range of virulence among MTC strains we used three mycobacterial strains as representative of low (*Mycobacterium canettii*), intermediate (*Mycobacterium tuberculosis* H37Rv), and high (*Mycobacterium beijing*) virulence degrees. The recently defined *Mycobacterium tuberculosis* Beijing (M. Beijing) strains are associated with high virulence and multidrug resistance (I. Parwati et al., 2010), and cause in mice a quick increase of cellular infiltrate with high numbers of colony forming units in the lungs (J. Dormans et al., 2004). Conversely, smoothtype *Mycobacterium tuberculosis* Canettii (M. canettii) strains rarely cause TB in humans and in the experimental mouse model show low cellular infiltrate with limited chronic infection (M. Fabre et al., 2010). Interestingly, our previous results assessing the mechanisms causing the difference in virulence showed an inverse correlation between strain virulence and in vivo cytotoxic responses, as well as higher bacterial burden in the lungs of M. Beijing infected mice (L. Quintero-Macias et al., 2011).

We decided to assess a profile of dendritic cell maturation and T cell exhaustion in vivo during pulmonary infection with these three mycobacterial strains. Since MTC strains are intracellular pathogens, T cells have an important role in mediating cytolysis of infected cells and to induce activation of other immune cells (Y. He et al., 2001; S. Inoue et al., 2005; E. A. Murphy et al., 2001; S. C. Oliveira et al., 2002). For intracellular pathogens like MTb, one way to subvert T cell responses could well be by altering activation/maturation of DCs. Importantly, DCs play an important role both in inducing effector cytotoxic T cells in vivo as well as in Ag-surveillance of mucosal surfaces and in the uptake and transport of mycobacterial bacilli to the lung draining lymphoid tissue, the mediastinal lymph nodes (MedLN)(G. S. Garcia-Romo et al., 2004; A. Pedroza-Gonzalez et al., 2004; A. J. Wolf et al., 2007).

We aimed to describe our recent findings regarding the differential stimulation of DCs and T cells by MTC strains with different virulence. We consider that increasing the research on the differences among MTC strains pathogen-host interactions in vivo might help to better understand, among other things, the underlying limitations of anti-TB vaccines.
