André Kipnis

Mice Lacking Bioactive IL-12 Can Generate Protective, Antigen-Specific Cellular Responses to Mycobacterial Infection Only if the IL-12 p40 Subunit Is Present

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.

In Vivo IL-10 Production Reactivates Chronic Pulmonary Tuberculosis in C57BL/6 Mice

The production of immunosuppressive cytokines, such as IL-10 and TGF-β, has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN-γ secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in promoting reactivation tuberculosis.

Dynamics of Macrophage Cell Populations During Murine Pulmonary Tuberculosis

The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b−/CD11c+/high were defined as alveolar macrophages, CD11bhigh/CD11c+/high as dendritic cells, and CD11b+/mid/CD11c+/mid as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.

NK Cells Respond to Pulmonary Infection with Mycobacterium tuberculosis, but Play a Minimal Role in Protection

Both innate and adaptive immune systems contribute to host defense against infection with Mycobacterium tuberculosis. NK cells have been associated with early resistance against intracellular pathogens and are known to be potent producers of the cytokine IFN-γ. In C57BL/6 mice infected by aerosol exposure with M. tuberculosis, NK cells increased in the lungs over the first 21 days of infection. Expansion of the NK cell subset was associated with increased expression of activation and maturation markers. In addition, NK cells isolated from the infected lungs were capable of producing IFN-γ and became positive for perforin. In vivo depletion of NK cells using a lytic Ab had no influence on bacterial load within the lungs. These findings indicate that NK cells can become activated during the early response to pulmonary tuberculosis in the mouse model and are a source of IFN-γ, but their removal does not substantially alter the expression of host resistance.

Role of chemokine ligand 2 in the protective response to early murine pulmonary tuberculosis

Chemokines play an important role in the development of immunity to tuberculosis. Chemokine ligand 2 (CCL2, JE, monocyte chemoattractant protein‐1) is thought to be primarily responsible for recruiting monocytes, dendritic cells, natural killer cells and activated T cells, all of which play critical roles in the effective control of tuberculosis infection in mice. We show here that in mice in which the CCL2 gene was disrupted, low‐dose aerosol infection with Mycobacterium tuberculosis resulted in fewer macrophages entering the lungs, but only a minor and transient increase in bacterial load in the lungs; these mice were still able to establish a state of chronic disease. Such animals showed similar numbers of activated T cells as wild‐type mice, as determined by their expression of the CD44hi CD62lo phenotype, but a transient reduction in cells secreting interferon‐γ. These data indicate that the primary deficiency in mice unable to produce CCL2 is a transient failure to focus antigen‐specific T lymphocytes into the infected lung, whereas other elements of the acquired host response are compensated for by different ligands interacting with the chemokine receptor CCR2.

Memory T Lymphocytes Generated by Mycobacterium bovis BCG Vaccination Reside within a CD4 CD44lo CD62 Ligandhi Population

ABSTRACT In the lungs of mice vaccinated with Mycobacterium bovis BCG, there was an accumulation of CD4 cells expressing the activated effector phenotype CD44hi CD62 ligandlo (CD62Llo) which were capable of secreting gamma interferon. Upon cell transfer, however, cells expressing a resting/naïve phenotype (CD44lo CD62Lhi) were capable of protecting the recipients from a virulent challenge infection, suggesting the emergence of T-cell memory from within this subset.

The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo

The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.

Direct Effect of 10-Valent Conjugate Pneumococcal Vaccination on Pneumococcal Carriage in Children Brazil

Background 10-valent conjugate pneumococcal vaccine/PCV10 was introduced in the Brazilian National Immunization Program along the year of 2010. We assessed the direct effectiveness of PCV10 vaccination in preventing nasopharyngeal/NP pneumococcal carriage in infants. Methods A cross-sectional population-based household survey was conducted in Goiania Brazil, from December/2010-February/2011 targeting children aged 7–11 m and 15–18 m. Participants were selected using a systematic sampling. NP swabs, demographic data, and vaccination status were collected from 1,287 children during home visits. Main outcome and exposure of interest were PCV10 vaccine-type carriage and dosing schedules (3p+0, 2p+0, and one catch-up dose), respectively. Pneumococcal carriage was defined by a positive culture and serotyping was performed by Quellung reaction. Rate ratio/RR was calculated as the ratio between the prevalence of vaccine-types carriage in children exposed to different schedules and unvaccinated for PCV10. Adjusted RR was estimated using Poisson regression. PCV10 effectiveness/VE on vaccine-type carriage was calculated as 1-RR*100. Results The prevalence of pneumococcal carriage was 41.0% (95%CI: 38.4–43.7). Serotypes covered by PCV10 and PCV13 were 35.2% and 53.0%, respectively. Vaccine serotypes 6B (11.6%), 23F (7.8%), 14 (6.8%), and 19F (6.6%) were the most frequently observed. After adjusted for confounders, children who had received 2p+0 or 3p+0 dosing schedule presented a significant reduction in pneumococcal vaccine-type carriage, with PCV10 VE equal to 35.9% (95%CI: 4.2–57.1; p = 0.030) and 44.0% (95%CI: 14.–63.5; p = 0.008), respectively, when compared with unvaccinated children. For children who received one catch-up dose, no significant VE was detected (p = 0.905). Conclusion PCV10 was associated with high protection against vaccine-type carriage with 2p+0 and 3p+0 doses for children vaccinated before the second semester of life. The continuous evaluation of carriage serotypes distribution is likely to be useful for evaluating the long-term effectiveness and impact of pneumococcal vaccination on serotypes reduction.

Interleukin‐10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis

Mice on the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Brutons protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin‐10. In the current study we examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL‐10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought.

Increased neutrophil influx but no impairment of protective immunity to tuberculosis in mice lacking the CD44 molecule

Up‐regulation of expression of the cell‐surface marker CD44 is a major characteristic of T lymphocytes responding in the lungs of mice infected with Mycobacterium tuberculosis. These lymphocytes express an activated/memory phenotype as seen by their high expression of the CD44 molecule and low expression of CD62L and CD45RB cell‐surface molecules. Based on increasing evidence that the CD44 molecule participates in several aspects of the inflammatory response, we evaluated its role in the response to infection with M. tuberculosis using gene‐disrupted mice. In this report, we show that CD44 expression is not necessary for the proper trafficking of protective cells to the lungs of mice infected with M. tuberculosis or the direct expression of protective immunity leading to control and containment of the bacterial load in this organ. However, although there were no differences in the bacterial load or migration of activated T lymphocytes to the inflamed lung, the absence of the CD44 molecule resulted in a substantially increased accumulation of neutrophils in the lung. These data indicate that loss of CD44 expression does not alter expression of T helper cell type 1 immunity to tuberculosis in the lungs but has major effects on the overall cellular composition of the immunopathological response.