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.

Aberrant adaptive immune response underlies genetic susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a major threat worldwide, although only a fraction of infected individuals develops tuberculosis (TB). TB susceptibility is shaped by multiple genetic factors, and we performed comparative immunological analysis of two mouse strains to uncover relevant mechanisms underlying susceptibility and resistance. C57BL/6 mice are relatively TB-resistant, whereas I/St mice are prone to develop severe TB, partly due to the MHC-II allelic variant that shapes suboptimal CD4+ T cell receptor repertoire. We investigated the repertoires of lung-infiltrating helper T cells and B cells at the progressed stage in both strains. We found that lung CD4+ T cell repertoires of infected C57BL/6 but not I/St mice contained convergent TCR clusters with functionally confirmed Mtb specificity. Transcriptomic analysis revealed a more prominent Th1 signature in C57BL/6, and expression of pro-inflammatory IL-16 in I/St lung-infiltrating helper T cells. The two strains also showed distinct Th2 signatures. Furthermore, the humoral response of I/St mice was delayed, less focused, and dominated by IgG/IgM isotypes, whereas C57BL/6 mice generated more Mtb antigen-focused IgA response. We conclude that the inability of I/St mice to produce a timely and efficient anti-Mtb adaptive immune responses arises from a suboptimal helper T cell landscape that also impacts the humoral response, leading to diffuse inflammation and severe disease.

Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn-porphyrins.

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins.

During transition into a dormant state, Mycolicibacterium (Mycobacterium) smegmatis cells are able to accumulate free porphyrins that makes them sensitive to photodynamic inactivation (PDI). The formation of dormant cells in a liquid medium with an increased concentration of magnesium (up to 25 mM) and zinc (up to 62 µM) resulted in an increase in the total amount of endogenous porphyrins in dormant M. smegmatis cells and their photosensitivity, especially for bacteria phagocytosed by macrophages. To gain insight into possible targets for PDI in bacterial dormant mycobacterial cells, a proteomic profiling with SDS gel electrophoresis and mass spectrometry analysis were conducted. Illumination of dormant forms of M. smegmatis resulted in the disappearance of proteins in the separating SDS gel. Dormant cells obtained under an elevated concentration of metal ions were more sensitive to PDI. Differential analysis of proteins with their identification with MALDI-TOF revealed that 45.2% and 63.9% of individual proteins disappeared from the separating gel after illumination for 5 and 15 min, respectively. Light-sensitive proteins include enzymes belonging to the glycolytic pathway, TCA cycle, pentose phosphate pathway, oxidative phosphorylation and energy production. Several proteins involved in protecting against oxygen stress and protein aggregation were found to be sensitive to light. This makes dormant cells highly vulnerable to harmful factors during a long stay in a non-replicative state. PDI caused inhibition of the respiratory chain activity and destroyed enzymes involved in the synthesis of proteins and nucleic acids, the processes which are necessary for dormant cell reactivation and their transition to multiplying bacteria. Because of such multiple targeting, PDI action via endogenous porphyrins could be considered as an effective approach for killing dormant bacteria and a perspective to inactivate dormant mycobacteria and combat the latent form of mycobacteriosis, first of all, with surface localization.

Prolonged B-Lymphocyte-Mediated Immune and Inflammatory Responses to Tuberculosis Infection in the Lungs of TB-Resistant Mice.

During tuberculosis (TB) infection, B-lymphocytes migrate to the lungs and form B-cell follicles (BCFs) in the vicinity of TB granulomata. B-cell-lacking mice display enhanced susceptibility to TB infection, and early B-cell depletion in infected non-human primates alters T-lymphocyte cytokine responses and increases bacterial burdens in the lungs. However, the role of B cells during late TB stages remained unaddressed. Here, we demonstrate that B cells and BCFs persist up to weeks 25-45 post-challenge in the lungs of TB-resistant C57BL/6 (B6) mice. In hyper-susceptible I/St mice, B-cell content markedly drops between weeks 12-16 post-infection, paralleled by diffuse lung tissue inflammation and elevated gene expression levels for pro-inflammatory cytokines IL-1, IL-11, IL-17a, and TNF-α. To check whether B-cells/BCFs control TB infection at advanced stages, we specifically depleted B-cells from B6 mice by administrating anti-CD20 mAbs at week 16 post-infection. This resulted in more rapid cachexia, a shortened lifespan of the infected animals, an increase in (i) lung-infiltrating CD8+ T cells, (ii) IL-6 production by F4/80+ macrophages, (iii) expression levels of genes for neutrophil-attracting factors CXCL1 and IL-17, and tissue-damaging factors MMP8, MMP9, and S100A8. Taken together, our results suggest that lung B cells and BCFs are moderately protective against chronic TB infection.

An In Vivo Model of Separate M. tuberculosis Phagocytosis by Neutrophils and Macrophages: Gene Expression Profiles in the Parasite and Disease Development in the Mouse Host.

The role of neutrophils in tuberculosis infection remains less well studied compared to that of the CD4+ T-lymphocytes and macrophages. Thus, alterations in Mycobacterium tuberculosis transcription profile following phagocytosis by neutrophils and how these shifts differ from those caused by macrophage phagocytosis remain unknown. We developed a mouse model that allows obtaining large amounts of either neutrophils or macrophages infected in vivo with M. tuberculosis for mycobacteria isolation in quantities sufficient for the whole genome RNA sequencing and aerosol challenge of mice. Here, we present: (i) the differences in transcription profiles of mycobacteria isolated from liquid cultures, neutrophils and macrophages infected in vivo; (ii) phenotypes of infection and lung inflammation (life span, colony forming units (CFU) counts in organs, lung pathology, immune cells infiltration and cytokine production) in genetically TB-susceptible mice identically infected via respiratory tract with neutrophil-passaged (NP), macrophage-passaged (MP) and conventionally prepared (CP) mycobacteria. Two-hour residence within neutrophils caused transcriptome shifts consistent with mycobacterial transition to dormancy and diminished their capacity to attract immune cells to infected lung tissue. Mycobacterial multiplication in organs did not depend upon pre-phagocytosis, whilst survival time of infected mice was shorter in the group infected with NP bacilli. We also discuss possible reasons for these phenotypic divergences.

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.

Neutrophils exacerbate tuberculosis infection in genetically susceptible mice.

Mice of the I/St inbred strain genetically hyper-susceptible to TB infection and prone to form neutrophil-abundant necrotic lung lesions and relatively resistant mice of the C57BL/6 (B6) strain were infected with 100 CFU of M. tuberculosis H37Rv. To verify the role of neutrophils in TB immunity, we selectively depleted neutrophils from infected mice with highly specific 1A8 anti-Ly6G antibodies at day 2 and 6 post-challenge. Depletion of neutrophils resulted in reduced lung tissue pathology, mycobacterial CFU counts and an increase of the survival time in genetically susceptible I/St, but not in B6 mice. Furthermore, we demonstrated that in vivo neutrophil depletion at the onset of TB infection results in a significant increase in numbers of mycobacteria-specific IFN-γ-producing T-cells at the time point when the acquired immunity to mycobacteria is fully developed. These results suggest antagonistic activity of neutrophils and immune T-cells in the course of TB infection and provide further evidence of deleterious rather than protective role of the former.

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.