Interleukin-11 Drives Early Lung Inflammation during Mycobacterium tuberculosis Infection in Genetically Susceptible Mice

IL-11 is multifunctional cytokine whose physiological role in the lungs during pulmonary tuberculosis (TB) is poorly understood. Here, using in vivo administration of specific antibodies against IL-11, we demonstrate for the first time that blocking IL-11 diminishes histopathology and neutrophilic infiltration of the lung tissue in TB-infected genetically susceptible mice. Antibody treatment decreased the pulmonary levels of IL-11 and other key inflammatory cytokines not belonging to the Th1 axis, and down-regulated IL-11 mRNA expression. This suggests the existence of a positive feedback loop at the transcriptional level, which is further supported by up-regulation of IL-11 mRNA expression in the presence of rIL-11 in in vitro cultures of lung cells. These findings imply a pathogenic role for IL-11 during the early phase of Mycobacterium tuberculosis-triggered disease in a genetically susceptible host

In Mice, Tuberculosis Progression Is Associated with Intensive Inflammatory Response and the Accumulation of Gr-1dim Cells in the Lungs

Infection with Mycobacterium tuberculosis (Mtb) results in different clinical outcomes ranging from asymptomatic containment to rapidly progressing tuberculosis (TB). The mechanisms controlling TB progression in immunologically-competent hosts remain unclear.To address these mechanisms, we analyzed TB progression in a panel of genetically heterogeneous (A/SnxI/St) F2 mice, originating from TB-highly-susceptible I/St and more resistant A/Sn mice. In F2 mice the rates of TB progression differed. In mice that did not reach terminal stage of infection, TB progression did not correlate with lung Mtb loads. Nor was TB progression correlated with lung expression of factors involved in antibacterial immunity, such as iNOS, IFN-gamma, or IL-12p40. The major characteristics of progressing TB was high lung expression of the inflammation-related factors IL-1beta, IL-6, IL-11 (p<0.0003); CCL3, CCL4, CXCL2 (p<0.002); MMP-8 (p<0.0001). The major predictors of TB progression were high expressions of IL-1beta and IL-11. TNF-alpha had both protective and harmful effects. Factors associated with TB progression were expressed mainly by macrophages (F4-80(+) cells) and granulocytes (Gr-1(hi)/Ly-6G(hi) cells). Macrophages and granulocytes from I/St and A/Sn parental strains exhibited intrinsic differences in the expression of inflammatory factors, suggesting that genetically determined peculiarities of phagocytes transcriptional response could account for the peculiarities of gene expression in the infected lungs. Another characteristic feature of progressing TB was the accumulation in the infected lungs of Gr-1(dim) cells that could contribute to TB progression.In a population of immunocompetent hosts, the outcome of TB depends on quantitatively- and genetically-controlled differences in the intensity of inflammatory responses, rather than being a direct consequence of mycobacterial colonization. Local accumulation of Gr-1(dim) cells is a newly identified feature of progressing TB. High expression of IL-1beta and IL-11 are potential risk factors for TB progression and possible targets for TB immunomodulation

Capacity of Lung Stroma to Educate Dendritic Cells Inhibiting Mycobacteria-Specific T-Cell Response Depends upon Genetic Susceptibility to Tuberculosis

<div><p>The balance between activation and inhibition of local immune responses in affected tissues during prolonged chronic infections is important for host protection. There is ample evidence that regulatory, tolerogenic dendritic cells (DC) are developed and present in tissues and inhibit overwhelming inflammatory reactions. Also, it was firmly established that stromal microenvironment of many organs is able to induce development of immature regulatory DC (DCreg), an essential element of a general immune regulatory network. However, direct experimental data demonstrating inhibition of immune responses by stroma-instructed immature DCreg in infectious models are scarce, and virtually nothing is known about functioning of this axis of immunity during tuberculosis (TB) infection. In this study, we demonstrate that lung stromal cells are capable of supporting the development in culture of immature CD11b⁺CD11c^lowCD103^- DCreg from lineage-negative (lin^-) bone marrow precursors. DCreg developed on lung stroma isolated from mice of genetically TB-hyper-susceptible I/St and relatively resistant B6 inbred strains inhibited proliferative response of mycobacteria-specific CD4⁺ T-cell lines a dose-dependent manner. Importantly, the inhibitory activity of B6 DCreg was substantially higher than that of I/St Dcreg. Moreover, when the donors of stromal cells were chronically infected with virulent mycobacteria, the capacity to instruct inhibitory DCreg was retained in B6, but further diminished in I/St stromal cells. DCreg-provided suppression was mediated by a few soluble mediators, including PGE₂, NO and IL-10. The content of CD4⁺Foxp3⁺ Treg cells in the mediastinal, lung-draining lymph nodes at the advanced stages of chronic infection did not change in I/St, but increased 2-fold in B6 mice, and lung pathology was much more pronounced in the former mice. Taken together, these data provide genetic evidence that the capacity to maintain populations of regulatory cells during <i>M. tuberculosis</i> infection is a part of the host protective strategy.</p> </div

DCreg developed on lung stroma from naïve (N) TB-resistant (A) B6 mice are more potent inhibitors of T cell response than their counterparts from TB-susceptible (B) I/St mice (<i>P</i><0.01, ANOVA).

<p>Inhibitory activity of DCreg developed on stromal cells from infected (Inf) animals was almost fully retained in B6 (A), but significantly (<i>P</i><0.05, ANOVA) dropped in I/St mice (B). Mean triplicate CFU counts ± SD from 4 independent experiments and per cent of inhibition are displayed.</p

3 mo post aerosol infection with ~100 <i>M. tuberculosis</i> H37Rv CFU I/St mice display substantially more severe infectious course compared to B6 mice.

<p>(A) -1 log difference in lung CFU counts (N=5, P<0.001, ANOVA, 1 experiment of 3 similar); (B) – more prominent gross pathology of the lung and greater splenomegaly; (C) – granulomata with necrotizing centers (arrows) are present in the lungs of I/St but not of B6 mice (X25).</p

B6 and I/St mice differ in numbers (<i>P</i>< 0.05-0.01 at different time points, Student’s <i>t</i>-test) of regulatory (A) and activated (B) CD4⁺ T cells in mediastinal, lung-draining lymph nodes throughout the course of infection.

<p>B6 and I/St mice differ in numbers (<i>P</i>< 0.05-0.01 at different time points, Student’s <i>t</i>-test) of regulatory (A) and activated (B) CD4⁺ T cells in mediastinal, lung-draining lymph nodes throughout the course of infection.</p

Soluble mediators of DCreg suppressor activity.

<p>(A) - B6 DCreg with blocked PGE₂ production are less inhibitory compared to control cells (25-40% decrease in inhibition depending on the content of DCreg in culture, P<0.05, ANOVA), but still retain suppressor activity. (B) - Both B6 and I/St cell-free supernatants possessed inhibitory activity regarding T cell proliferation, but in I/St this activity is weaker (P<0.01, ANOVA). (C) - NO production: similar nitrite levels in B6 and I/St co-cultures with lung stroma from naïve mice and opposite shifts in the presence of infected stroma – slight decrease in I/St, but 2-fold increase in B6 cultures, resulting in 3-fold (<i>P</i><0.01, ANOVA) differences. (D) - The content of IL-10 is significantly (P=0.04, Student’s <i>t</i>-test) higher in B6 compared to I/St supernatants developed on infected stroma. Mean triplicate CFU counts ± SD from 3 independent experiments are displayed.</p

Protein levels of IL-11 affect IL-11 mRNA expression.

<p>(A) <i>In vivo</i> administration of anti-IL-11 antibodies leads to a selective down-regulation of IL-11 mRNA. The level of expression was quantified in 5 individual mice per group, using qrt-PCR and normalization against the level of GAPDH expression. Results obtained in 1 of 2 similar experiments are expressed as mean ± SEM (for IL-11 expression <i>P</i> = 0.021, for other cytokines <i>P</i>>0.05). (B) Introduction of 100 ng/ml rIL-11 in cultures of lung cells up-regulates the expression of IL-11 mRNA. Results of two similar experiments are expressed as mean of 3 wells ± SEM (<i>P</i><0.01, ANOVA, compared to negative controls and cultures stimulated with 10 ng/ml IL-11).</p

Treatment with anti-IL-11 antibodies significantly attenuates the severity of TB in I/St mice.

<p>(A) ∼3-fold decrease in lung CFU counts compared to control animals. (B and C) Lung pathology in individual animals. None of anti-IL-11-treated mice developed necrotic TB foci evident in control mice <i>a</i>, <i>d</i> and <i>g</i> (circled). (D) Statistical evaluation of the proportion of inflamed lung tissue. CFU counts and morphometry were performed in all mice included in 2 independent experiments (total N = 16 and 17 for experimental and control groups, respectively). Histology is displayed for individual mice analyzed in one experiment (N = 7 for each group).</p