Monocytes migrate to the site of infection from blood, in response to inflammatory signals (cytokines and chemokines) that are often produced by macrophages as well.12
Only a fraction of macrophages in the granuloma are actually infected with Mtb, although this proportion is likely to be higher in a granuloma that is poorly functioning and not controlling the infection. It is difficult to estimate the fraction of cells infected in human granulomas, or the number of bacilli per cell because acid-fast staining for the microbe is notoriously inefficient in human and non-human primate tissues. Nonetheless, we consider the macrophage populations to consist of both infected and uninfected cells, both of which can be influenced by other factors (cells and soluble factors) in the granuloma. Mtb can have effects on macrophages from within by interacting with host receptors, such as Toll-like receptors in the phagosome.35–42
It has been suggested that under some circumstances Mtb can escape the vacuole to reside in the cytoplasm,43,44
and that mycobacterial molecules can exit the phagosome and interact with cytoplasmic receptors to induce responses.45–48
Mycobacteria and microbial factors can interact with cells from the outside as well, again through Toll-like receptors or other receptors. The variety of cytokines induced by interaction of Mtb with macrophages can then act on other cells within the granuloma, including macrophages, to induce various functional and phenotypic changes in cells that then modulate the environment of the granuloma.
In recent years, the complexity of macrophage populations has gained appreciation.49,50
For ease of discussion, macrophages differentiated in response to cytokine signals have been termed either classically activated macrophages (CAMs) or alternatively activated macrophages (AAMs).49–52
CAMs arise in response to TH1 T-cell signals (interferon-γ and TNF). These macrophages produce proinflammatory cytokines (TNF and IL-12) and chemokines and are capable of killing bacilli; in mice, a marker for CAM is inducible nitric oxide synthase (iNOS). iNOS uses arginine as a substrate for production of nitric oxide that can kill Mtb. In mice, iNOS is essential for control of Mtb infection.53–55
iNOS expression in human tuberculous lung macrophages has been reported,56,57
and we have detected iNOS expression in macrophages in macaque granulomas (J.T. Mattila and J.L. Flynn, personal communication).
AAMs are anti-inflammatory in nature, and were initially described as arising in response to TH2 cytokines IL-13 and IL-4.50,51
These macrophages can produce IL-10, TGF-β, and IL-6. In a TH2 environment, induction of the AAM phenotype is STAT-6 (signal transducer and activation of transcription 6) dependent.51
There are a set of genes and proteins used to characterize AAMs, with the primary marker being arginase.51
Arginase also uses arginine as a substrate and directly competes with iNOS for arginine, making the relative expression of these genes in a macrophage an important balancing feature for whether the macrophage will be pro- or anti-inflammatory, and directly affects the ability of a macrophage to kill Mtb ().
There are several recent studies of AAMs in tuberculosis. Unlike parasitic and worm diseases, or asthma, where AAMs have been predominantly studied, tuberculosis induces a TH1-mediated immune response. It is difficult to measure IL-4 or IL-13 in tuberculous granulomas, and interruption of these genes in mice has little effect on Mtb infections, although there are a few reports that these cytokines can interfere with adequate control of tuberculosis.58–60
The generation of AAMs in tuberculous (TH1) granulomas appears to have a different mechanism. Mtb induces arginase (Arg1
) gene expression in a MyD88-dependent but STAT-6-independent fashion in murine macrophages.36
induction by mycobacteria was mediated by Toll-like receptor-induced IL-6, IL-10, and granulocyte colony-stimulating factor production by macrophages.61
However, only a subset of AAM markers were induced by these cytokines, suggesting perhaps an intermediate AAM phenotype compared with a TH2 environment. Other studies using a mouse that overexpressed IL-10 from macrophages supported that IL-10 can strongly induce arginase expression in the lungs of Mtb-infected mice.61
The latter study also suggested that IL-10 enhanced the sensitivity of macrophages to IL-4, exacerbating induction of AAMs even in the presence of very low IL-4 levels, as might occur in a granuloma. These mice had higher numbers of Mtb in the lungs, which was correlated with increased AAMs. Mice lacking arginase specifically in macrophages controlled Mtb infection better than wild-type mice, and Arg1
−/− macrophages had enhanced iNOS expression and increased killing of Mtb in vitro
. Thus, although Mtb appears to have a different mechanism for induction of arginase than a TH2-mediated disease, arginase and AAM appear to inhibit control of Mtb infection.
Some studies have suggested that alveolar macrophages are inherently alternatively activated, and may allow Mtb bacilli to gain a foothold immediately upon entering the airway, as they are impaired in their ability to kill bacilli. 15
Gordon and Martinez51
have suggested that true AAMs require a signal of “activation” to attain the qualities of AAMs, and hence although alveolar macrophages before infection are not classically activated, they may not be true AAMs, either. There is clearly a spectrum of activation for macrophages, and incompletely activated cells may possess some but not all qualities of AAMs or CAMs, depending on the local cytokine environment.