Bernadette M. Saunders

Life and death in the granuloma: immunopathology of tuberculosis

During tuberculosis (TB) infection, the granuloma provides the microenvironment in which antigen‐specific T cells colocate with and activate infected macrophages to inhibit the growth of Mycobacterium tuberculosis. Although the granuloma is the site for mycobacterial killing, virulent mycobacteria have developed a variety of mechanisms to resist this macrophage‐mediated killing. These surviving mycobacteria become dormant, however, if host cellular immunity or the signals maintaining granuloma structure wane, or if mycobacteria resume replication, leading to reactivation of TB. This balance of life and death applies not only to the mycobacterium but also to the host macrophages that may undergo apoptosis or necrosis, leading to the characteristic caseous necrosis within the granuloma, and the potential spread of TB infection. The immunological factors controlling the development and maintenance of the granuloma will be reviewed.

Restraining mycobacteria: Role of granulomas in mycobacterial infections

The generation of prolonged immunity to Mycobacterium tuberculosis requires not only an antigen‐specific IFN‐γ‐producing T cell response, including both CD4 and CD8 T cells, but also the generation of protective granulomatous lesions, whereby the close apposition of activated T cells and macrophages acts to contain bacterial growth. The importance of the granulomatous lesion in controlling this immune response and in limiting both tissue damage and bacterial dissemination has been considered a secondary event but, as the present review illustrates, is no less important in surviving mycobacterial infection than an antigen‐specific T‐cell response. The formation of a protective granuloma involves the orchestrated production of a host of chemokines and cytokines, the upregulation of their receptors along with upregulation of addressins, selectins and integrins to coordinate the recruitment, migration and retention of cells to and within the granuloma. In the present review, the principal components of the protective response are outlined and the role of granuloma formation and maintenance in mediating prolonged containment of mycobacteria within the lung is addressed.

Neutrophils Play a Protective Nonphagocytic Role in Systemic Mycobacterium tuberculosis Infection of Mice

ABSTRACT Evidence showing that neutrophils play a protective role in the host response to infection by different intracellular parasites has been published in the past few years. We assessed this issue with regard to the infection of mice with Mycobacterium tuberculosis. We found a chronic recruitment of neutrophils to the infection foci, namely, to the peritoneal cavity after intraperitoneal infection and to the spleen and liver after intravenous inoculation of the mycobacteria. However, bacilli were never found associated with the recruited neutrophils but rather were found inside macrophages. The intravenous administration of the antineutrophil monoclonal antibody RB6-8C5 during the first week of infection led to selective and severe neutropenia associated with an enhancement of bacillary growth in the target organs of the mice infected by the intravenous route. The neutropenia-associated exacerbation of infection was most important in the liver, where a bacterial load 10-fold higher than that in nonneutropenic mice was found; the exacerbation in the liver occurred both during and after the neutropenic period. Early in infection by M. tuberculosis, neutropenic mice expressed lower levels of mRNAs for gamma interferon and inducible nitric oxide synthase in the liver compared to nondepleted mice. These results point to a protective role of neutrophils in the host defense mechanisms against M. tuberculosis, which occurs early in the infection and is not associated with the phagocytic activity of neutrophils but may be of an immunomodulatory nature.

Interleukin-6 induces early gamma interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection.

ABSTRACT Immunity to Mycobacterium tuberculosis is dependent upon the generation of a protective gamma interferon (IFN-γ)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-γ. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity to M. tuberculosis, IL-6 and IL-4 knockout mice were infected with M. tuberculosis via aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-γ. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-γ production, it is not essential for the development of protective immunity against M. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-γ production, this had no significant effect upon bacterial growth.

CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis

To confirm the primary role of CD4 T cells in pulmonary tuberculosis, mice with a disruption of their CD4 gene (CD4 KO) were exposed to an aerosol of Mycobacterium tuberculosis and survival, cellular responses in the lung and granuloma development followed. CD8 and NK cells from the lungs of infected CD4 KO mice expressed IFN-gamma and were recruited in numbers similar to those seen in the C57BL/6 mice; recruitment correlated with initial control of bacteria. The major defect in mice lacking CD4 was the significant reduction in total cellular recruitment into the lungs. CD4 KO mice did not generate the typical mononuclear granulomatous lesions, instead the cellular influx was macrophage in character and was localized as perivascular cuffing. Early control of M. tuberculosis growth is therefore independent of CD4+ cells but such cells are required to ensure recruitment of mononuclear cells to the lung and thus ensure long-term survival.

Transmembrane TNF Is Sufficient to Initiate Cell Migration and Granuloma Formation and Provide Acute, but Not Long-Term, Control of Mycobacterium tuberculosis Infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF−/−, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF−/− mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF−/− mice, into either RAG−/− or TNF−/− mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.

A thr357 to ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs atp-induced mycobacterial killing by macrophages

The P2X7 receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X7 receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X7 polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr357 to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X7 function was measured by ATP-induced ethidium+ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10–65% in heterozygotes, 1–18% in homozygotes, and 0–10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X7 in either HEK-293 cells or Xenopus oocytes gave P2X7 function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X7, restored P2X7 function to near normal in cells heterozygous for T357S and to a value 50–65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X7 function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X7 receptor function.

Chemokine Gene Expression during Fatal Murine Cerebral Malaria and Protection Due to CXCR3 Deficiency

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. Using murine models of malaria, we found much greater up-regulation of a number of chemokine mRNAs, including those for CXCR3 and its ligands, in the brain during fatal murine CM (FMCM) than in a model of non-CM. Expression of CXCL9 and CXCL10 RNA was localized predominantly to the cerebral microvessels and in adjacent glial cells, while expression of CCL5 was restricted mainly to infiltrating lymphocytes. The majority of mice deficient in CXCR3 were found to be protected from FMCM, and this protection was associated with a reduction in the number of CD8+ T cells in brain vessels as well as reduced expression of perforin and FasL mRNA. Adoptive transfer of CD8+ cells from C57BL/6 mice with FMCM abrogated this protection in CXCR3−/− mice. Moreover, there were decreased mRNA levels for the proinflammatory cytokines IFN-γ and lymphotoxin-α in the brains of mice protected from FMCM. These data suggest a role for CXCR3 in the pathogenesis of FMCM through the recruitment and activation of pathogenic CD8+ T cells.

A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria

Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X7 pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-primed, mycobacteria-infected macrophages from wild-type individuals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from individuals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X7-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.

Mycobacterium bovis BCG-Specific Th17 Cells Confer Partial Protection against Mycobacterium tuberculosis Infection in the Absence of Gamma Interferon

ABSTRACT Protective immunity against tuberculosis (TB) requires the integrated response of a network of lymphocytes. Both gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-secreting CD4+ T cells have been identified in subjects with latent TB infection and during experimental Mycobacterium tuberculosis infection, but the contribution of Th17 cells to protective immunity is unclear. To examine their protective effects in vivo, we transferred mycobacterium-specific IL-17- and IFN-γ-secreting CD4+ T cells isolated from M. tuberculosis BCG-immunized IL-12p40−/− and IFN-γ−/− or wild-type mice, respectively, into M. tuberculosis-infected IL-12p40−/− or RAG−/− mice. In the absence of IL-12 and IL-23, neither IL-17-secreting (Th17) nor IFN-γ-secreting (Th1) BCG-specific T cells expanded or provided protection against M. tuberculosis. In RAG−/− recipients with an intact IL-12/IL-23 axis, both Th17 and Th1 cells were activated and induced significant protection against M. tuberculosis. The reduction in the bacterial load following transfer of IFN-γ−/− Th17 cells was associated with significant prolongation of survival compared to recipients of naïve IFN-γ−/− T cells. This effect was at the cost of an increased inflammatory infiltrate characterized by an excess of neutrophils. Therefore, Th17 cells can provide IFN-γ-independent protection against M. tuberculosis, and this effect may contribute to the early control of M. tuberculosis infection.