A new model for chronic and reactivation tuberculosis: Infection with genetically attenuated Mycobacterium tuberculosis in mice with polar susceptibility.

TB infection in mice develops relatively rapidly which interferes with experimental dissection of immune responses and lung pathology features that differ between genetically susceptible and resistant hosts. Earlier we have shown that the M. tuberculosis strain lacking four of five Rpf genes (ΔACDE) is seriously attenuated for growth in vivo. Using this strain, we assessed key parameters of lung pathology, immune and inflammatory responses in chronic and reactivation TB infections in highly susceptible I/St and more resistant B6 mice. ΔACDE mycobacteria progressively multiplied only in I/St lungs, whilst in B6 lung CFU counts decreased with time. Condensed TB foci apeared in B6 lungs at week 4 of infection, whilst in I/St their formation was delayed. At the late phase of infection, in I/St lungs TB foci fused resulting in extensive pneumonia, whereas in B6 lungs pathology was limited to condensed foci. Macrophage and neutrophil populations characteristically differed between I/St and B6 mice at early and late stages of infection: more neutrophils accumulated in I/St and more macrophages in B6 lungs. The expression level of chemokine genes involved in neutrophil influx was higher in I/St compared to B6 lungs. B6 lung cells produced more IFN-γ, IL-6 and IL-11 at the early and late phases of infection. Overall, using a new mouse model of slow TB progression, we demonstrate two important features of ineffective infection control underlined by shifts in lung inflammation: delay in early granuloma formation and fusion of granulomas resulting in consolidated pneumonia late in the infectious course.

Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.

Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.

B cells delay neutrophil migration toward the site of stimulus: tardiness critical for effective bacillus Calmette-Guérin vaccination against tuberculosis infection in mice.

Mutations in the btk gene encoding Bruton's tyrosine kinase cause X-linked immune deficiency, with impaired B lymphocyte function as the major phenotype. Earlier, we demonstrated that CBA/N-xid mice, unlike the wild-type CBA mice, were not protected by bacillus Calmette-Guérin (BCG) vaccination against tuberculosis infection. Because IFN-gamma-producing T cells and activated macrophages are key elements of antituberculosis protection, it remained unclear how the mutation predominantly affecting B cell functions interferes with responses along the T cell-macrophage axis. In this study, we show that B cell deficiency leads to an abnormally rapid neutrophil migration toward the site of external stimulus. Using adoptive cell transfers and B cell genetic knockout, we demonstrate a previously unappreciated capacity of B cells to downregulate neutrophil motility. In our system, an advanced capture of BCG by neutrophils instead of macrophages leads to a significant decrease in numbers of IFN-gamma-producing T cells and impairs BCG performance in X-linked immune-deficient mice. The defect is readily compensated for by the in vivo neutrophil depletion.

Analysis of cellular phenotypes that mediate genetic resistance to tuberculosis using a radiation bone marrow chimera approach.

Adoptive transfer of bone marrow cells from tuberculosis-resistant (I/St x A/Sn)F(1) donor mice into lethally irradiated susceptible I/St recipients changed their phenotype following infection with virulent Mycobacterium tuberculosis. Compared to I/St-->I/St control animals, F(1)-->I/St chimeras demonstrated (i) prolonged survival time, (ii) increased antimycobacterial function of lung macrophages, (iii) elevated gamma interferon production by lung cells, and (iv) decreased infiltration of the lungs with CD4(+) and CD8(+) T cells and Ly-6G(+) neutrophils.

Different innate ability of I/St and A/Sn mice to combat virulent Mycobacterium tuberculosis: phenotypes expressed in lung and extrapulmonary macrophages.

Mice of the I/St and A/Sn inbred strains display a severe and moderate course, respectively, of disease caused by Mycobacterium tuberculosis. Earlier, we showed that the response to mycobacterial antigens in I/St mice compared to that in A/Sn mice is shifted toward Th2-like reactivity and a higher proliferative activity and turnover of T cells. However, the physiologic basis for different expressions of tuberculosis severity in these mice remains largely unknown. Here, we extend our previous observations with evidence that I/St interstitial lung macrophages are defective in the ability to inhibit mycobacterial growth and to survive following in vitro infection with M. tuberculosis H37Rv. A unique feature of this phenotype is its exclusive expression in freshly isolated lung macrophages. The defect is not displayed in ex vivo macrophages obtained from the peritoneal cavity nor in macrophages developed in vitro from progenitors extracted from various organs, including the lung itself. In addition, we show that, in sharp contrast to peritoneal macrophages, the mycobactericidal capacity of lung macrophages is not elevated in the presence of exogenous gamma interferon. Our data suggest that the in vivo differentiation in a particular anatomical microenvironment determines the pattern of macrophage-mycobacterium interaction. Thus, caution should be exercised when conclusions based upon the results obtained in a particular in vitro system are generalized to the functions of all phagocytes during M. tuberculosis infection.