Polygenic TB control and the sequence of innate/adaptive immune responses to infection: MHC-II alleles determine the size of the S100A8/9-producing neutrophil population.

Among several quantitative trait loci involved in tuberculosis (TB) control in mice, one was mapped within the chromosome 17 segment occupied by the H2 complex and another within the chromosome 3 segment comprising the S100A8/9 genes, which encode neutrophil inflammatory factor S100A8/9. Previously, we developed a panel of H2-congenic mouse strains differing by small segments of the major histocompatibility complex Class II (MHC-II) region from TB-susceptible H2j mice transferred onto the genetic background of the TB-resistant C57BL/6 (H2b) strain. Susceptible B6.I-9.3 mice differ from B6 progenitors by the alleles of their only classical MHC-II H2-Aß gene. The goals of the present study were to: (i) comprehensively characterise the differences in TB-related phenotypes between mice of the two strains and (ii) decipher interactions between the H2-Aß and S100A8/9 genes. Here, we describe the dynamics of TB-related phenotypes differentiating B6.I-9.3 and B6 mice (colony forming units counts, histopathology, lung immune cell infiltration and cytokine profiles). We show that disproportionally diminished CD4+ T-cell population, an enlarged S100A8/9-positive neutrophil population and higher S100A8/9 serum levels in B6.I-9.3 mice collectively form the 'susceptible' phenotype before infection. An increase in IL-17 and a decrease in intrferon-gamma production by CD4+ T-cells in these mice provide a mechanistic explanation of this phenotype. Using F2 segregation analysis, we show that the number of S100A8/9-producing neutrophils in lungs and spleens and the proportion of Th17 CD4+ T-cells in lungs are significantly lower in the presence of the MHC-II dominant 'resistant' b allele compared to the recessive 'susceptible' j/j genotype. This provides direct genetic evidence that MHC-II-regulated CD4+ T-cell landscapes determine neutrophil abundance before infection, an important pathogenic factor in TB immunity.

Physicochemical and Biological Properties of Vancomycin-Containing Antibacterial Polysaccharide Gels for Biocomposite Bone Implant Impregnation.

Polymeric drugs containing up to 60% by weight of the antibiotic vancomycin were synthesized based on dextran carriers activated with epichlorohydrin. Vancomycin was covalently bound, involving the primary amino group of the molecule through the hydroxypropyl radical to the C6 position of the anhydroglucose units of the dextran main chain. Covalent binding is necessary to prevent spontaneous release of the antibiotic from the gel, thereby reducing the risk of bacterial multiresistance. Antibacterial depot gels were obtained from those polymers, containing up to 17.5% by weight of polysaccharide with a cross-linking density of q = 3-5 nodes per macromolecule for the deposition of another type of drugs not covalently bound to the polymer gel. They were used to coat the surface of the internal pores of biocomposite bone implants based on bovine cancellous bone used in orthopedics. The chemical structure of the polymer was studied using 13C NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The stiffness of the gels was evaluated by the values of the accumulation modulus G' = 170-270 kPa and the loss modulus G″ = 3.7-4.2 kPa determined on a rheometer. Their values are close to those typical for materials used to replace soft tissue in plastic surgery. The minimum inhibitory concentration of the gels against Staphylococcus aureus P209 depends on the antibiotic content in the polymer. It equals 2.5 mg/L for vancomycin we used and 100 mg/L for a polymer containing 50% by weight of covalently bound antibiotic. The cytotoxic concentration measured with cell culture HEK 293T exceeds 1200 mg/L in 24 h exposure. The release dynamics of drugs not covalently bound to dextran from the depot gel were studied using fluorescein as a model. The release time is independent of the gel density and lasts up to 6 days for a 2 mm thick layer. Both the gel and the bone implants impregnated with it maintained consistently high antibacterial activity throughout the experiment, up to its completion after 168 h, with the local concentration of the released antibiotic at the site of bacterial attack exceeding the therapeutic level by 200 times.

Pleiotropic Effect of IL-6 Produced by B-Lymphocytes During Early Phases of Adaptive Immune Responses Against TB Infection.

The role of B cells migrating to the lung and forming follicles during tuberculosis (TB) inflammation is still the subject of debate. In addition to their antibody production and antigen-presenting functions, B cells secrete different cytokines and chemokines, thus participating in complex networks of innate and adaptive immunity. Importantly, lung B-cells produce high amounts of the pleiotropic gp130 cytokine IL-6. Its role during TB infection remains controversial, partly due to the fact that IL-6 is produced by different cell types. To investigate the impact of IL-6 produced by B cells on TB susceptibility and immune responses, we established a mouse strain with specific IL-6 deficiency in B cells (CD19cre-IL-6fl/fl, B-IL-6KO) on the B6 genetic background. Selective abrogation of IL-6 in B cells resulted in shortening the lifespan of TB-infected B-IL-6KO mice compare to the wild-type controls. We provide evidence that at the initial TB stages B cells serve as a critical source of IL-6. In the lung, the effect of IL-6 deficiency in B cells is associated rather with B and T cell functioning, than with macrophage polarization. TB-infected B-IL-6KO mice displayed diminished sizes of B cells themselves, CD4+IFN-γ+, Th17+, and CD4+CXCR5+ follicular T cell populations. The pleiotropic effect of B-cell-derived IL-6 on T-cells demonstrated in our study bridges two major lymphocyte populations and sheds some light on B- and T-cells interactions during the stage of anti-TB response when the host switches on a plethora of acquired immune reactions.

Prolonged infection triggered by dormant Mycobacterium tuberculosis: Immune and inflammatory responses in lungs of genetically susceptible and resistant mice.

We developed an approach for substantial attenuation of Mycobacterium tuberculosis by prolonged culturing under gradually acidifying conditions. Bacteria subjected to acidification lost the capacity to form colonies on solid media, but readily resuscitated their growth in the murine host, providing a useful model to study in vivo development of infection mimicking latent and reactivation tuberculosis (TB) in humans. Here we characterize biomarkers of lung pathology and immune responses triggered by such attenuated bacteria in genetically TB-susceptible and resistant mice. In susceptible I/St mice, CFU counts in lungs and spleens were ~1.5-log higher than in resistant B6 mice, accompanied by diffuse pneumonia and excessive lung infiltration with highly activated CD44+CD62L- T-lymphocytes resulting in death between months 7-9 post challenge. B6 mice were characterized by development of local inflammatory foci, higher production of pro-inflammatory IL-6 and IL-11 cytokines and a more balanced T-cell activation in their lungs. CFU counts remained stable in B6 mice during the whole 18-mo observation period, and all mice survived. Thus, we established a mouse model of fatal reactivation TB vs. indefinite mycobacterial possession after identical challenge and characterized the features of immune responses in the lung tissue underlining these polar phenotypes.

Reciprocal control of Mycobacterium avium and Mycobacterium tuberculosis infections by the alleles of the classic Class II H2-Aß gene in mice.

Genetic control of host susceptibility to M. avium, an important lung pathogen of immune-compromised individuals, remains incompletely defined. Apart from the slc11a1 (Nramp1) gene, which plays a pivotal role in genetic control of a few intracellular pathogens, including M. avium, in mice, we know nothing about genetic loci determining susceptibility to and/or severity of M. avium-triggered disease. Previously, our lab developed a panel of H2-congenic, recombinant mouse strains for identification of the MHC genes involved in the control of M. tuberculosis infection. In the present study, we applied a few recombinant strains from this panel to study $ possible influence of allelic variations in classical Class II genes on the development of M. avium infection. Our results demonstrate a clear difference in lung pathology, post-infection survival time, lung neutrophil influx and corresponding chemokine/cytokine responses, as well as the degree of lung T lymphocyte activation, between mouse strains differing by the alleles of a single highly polymorphic Class II H2-Aß gene. Paradoxically, mice carrying the H2-Aßb allele, which provides a notable protective effect against M. tuberculosis compared to the H2-Aßj allele, were more susceptible to M. avium infection as indicated by several parameters of the disease. We discuss possible reasons for such a reciprocal expression of phenotypes determined by a single allelic variant during two "similar" infections that may concern differences in virulence, NO-sensitivity, intracellular life style and antigenic composition between these two mycobacterial species.

B lymphocytes in anti-mycobacterial immune responses: Pathogenesis or protection?

The role of B cells and antibodies in tuberculosis (TB) immunity, protection and pathogenesis remain contradictory. The presence of organized B cell follicles close to active TB lesions in the lung tissue raises the question about the role of these cells in local host-pathogen interactions. In this short review, we summarize the state of our knowledge concerning phenotypes of B cells populating tuberculous lungs, their secretory activity, interactions with other immune cells and possible involvement in protective vs. pathogenic TB immunity.

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.

B-lymphocytes forming follicle-like structures in the lung tissue of tuberculosis-infected mice: Dynamics, phenotypes and functional activity.

During tuberculosis (TB) infection, B cells form follicles in close vicinity of lung granuloma. We assessed the dynamics of follicle formation, surface phenotypes and functional activity of lung B cells during TB course in genetically susceptible mice. The follicles appeared early post infection and peaked at weeks 7-8. Lung B cells resembled classical B2 cells (CD19+IgMloIgDhiCD1d-CD21/35intCD5-CD11b-CD43-), but differed from them by the absence of B2 marker CD23. Lung B-cells constitutively expressed MHC II molecules, presented mycobacterial antigens to immune CD4+ T-cells and produced high amounts of IL-6 and IL-11, but no classical type 1 (TNF-α, IFN-γ), or anti-inflammatory (IL-10, TGF-ß) cytokines. The total antibody response in tuberculous lung showed almost no specificity to mycobacteria. A panel of monoclonal antibodies obtained from lung B cells contained only few clones with reactivity to mycobacteria. Our results suggest that anti-TB B cell response in the lung has clear pathological and doubtful protective role.