Andrea J. Wolf

Mycobacterium tuberculosis Infects Dendritic Cells with High Frequency and Impairs Their Function In Vivo

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4+ T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4+ T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.

Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs

The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4+ T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4+ T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4+ T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.

CCR2-Dependent Trafficking of F4/80dim Macrophages and CD11cdim/intermediate Dendritic Cells Is Crucial for T Cell Recruitment to Lungs Infected with Mycobacterium tuberculosis

We previously reported that CCR2−/− mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2+/+ or CCR2−/− while maintaining the genotype of the myeloid cells as CCR2+/+. After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80dim macrophages and CD11cdim/intermediate DCs to the infected lung.

Suboptimal Activation of Antigen-Specific CD4+ Effector Cells Enables Persistence of M. tuberculosis In Vivo

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

Dynamic Roles of Type I and Type II IFNs in Early Infection with Mycobacterium tuberculosis

Although the protective role of type II IFN, or IFN-γ, against Mycobacterium tuberculosis has been established, the effects of type I IFNs are still unclear. One potential confounding factor is the overlap of function between the two signaling pathways. We used mice carrying null mutations in the type I IFNR, type II IFNR, or both and compared their immune responses to those of wild-type mice following aerosol infection with M. tuberculosis. We discovered that, in the absence of a response to IFN-γ, type I IFNs play a nonredundant protective role against tuberculosis. Mice unable to respond to both types of IFNs had more severe lung histopathology for similar bacterial loads and died significantly earlier than did mice with impaired IFN-γ signaling alone. We excluded a role for type I IFN in T cell recruitment, which was IFN-γ dependent, whereas both types of IFNs were required for optimal NK cell recruitment to the lungs. Type I IFN had a time-dependent influence on the composition of lung myeloid cell populations, in particular by limiting the abundance of M. tuberculosis-infected recruited macrophages after the onset of adaptive immunity. We confirmed that response to IFN-γ was essential to control intracellular mycobacterial growth, without any additional effect of type I IFN. Together, our results imply a model in which type I IFN limit the number of target cells that M. tuberculosis can infect in the lungs, whereas IFN-γ enhances their ability to restrict bacterial growth.

Codominance of TLR2-Dependent and TLR2-Independent Modulation of MHC Class II in Mycobacterium tuberculosis Infection In Vivo

Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-γ. Although IFN-γ is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-γ. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-γ induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-γ induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-γ is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR+/+ and TLR2−/− cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2+/+ and TLR2−/− macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-γ are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity.

Peptidoglycan recognition by the innate immune system

The innate immune system recognizes microbial products using germline-encoded receptors that initiate inflammatory responses to infection. The bacterial cell wall component peptidoglycan is a prime example of a conserved pathogen-associated molecular pattern (PAMP) for which the innate immune system has evolved sensing mechanisms. Peptidoglycan is a direct target for innate immune receptors and also regulates the accessibility of other PAMPs to additional innate immune receptors. Subtle structural modifications to peptidoglycan can influence the ability of the innate immune system to detect bacteria and can allow bacteria to evade or alter host defences. This Review focuses on the mechanisms of peptidoglycan recognition that are used by mammalian cells and discusses new insights into the role of peptidoglycan recognition in inflammation, metabolism, immune homeostasis and disease.