Immune response to atypical mycobacteria
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Immune response to atypical mycobacteria R. Appelberg University
of Porio, Centro de Citotogia
Interest in the study of atypical mycobacteria, often called non-tuberculous mycobacteria, was dramatically stimulated by the finding of a very high incidence of infection by a subgroup of these mycobacteria, the Mycobacterium avium complex (MAC), in patients infected with HIV and at terminal stages of AIDS. M. avium and M. intracellulare, the most important species in the MAC, are widely disseminated in the environment and seldom infect immunocompetent human beings. Infection is mostly found among immunodepressed individuals, and AIDS patients are at a particularly high risk of infection in some countries. Infection is also found in patients suffering from chronic obstructive pulmonary disease, where the distinction between active infection and colonization is often difficult to make, and in children where these mycobacteria cause lymphadenitis. Virulence of MAC and the pathogenesis of the infection clearly differ from those of M. tuberculosis. The clinical aspects of MAC infection as well as much microbiological information have been reviewed recently in considerable detail (Inderlied et al., 1993). Here we will concentrate on the discussion of data obtained in experimental infections by M. avium using mouse models. Microbiological
M. avium is a facultative intracellular pathogen that survives and proliferates inside the macrophages of host organisms (Frehel et al., 1991). It can be isolated from numerous sources in the environment and during culture on solid media exhibits a polymorphic colonial aspect, giving rise to smooth transparent (SmTr), smooth opaque (SmOp) or rough (Rg) colonial variants. Although in many instances we can find a high rate of variation between morphotypes upon subculturing, inocula of particular morphotypic appearance may be prepared. The use of the mouse model to study M. avium infections has revealed an interesting system where the interplay of the virulence mechanisms of the bacterium and the defence mechanisms of the host
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show a wide spectrum of outcomes. Indeed, unlike M. tuberculosis, M. avium exhibits an extraordinary variation in virulence which is strain-associated as well as dependent on the morphotypes under study. Therefore, there is the possibility of studying virulence factors in mycobacteria as well as the strategies adopted to escape from the host defence mechanisms. Virulence of a particular strain of M. avium varies according to the morphotype used to infect the animal. Thus, SmTr variants are the most consistently virulent and SmOp are usually non-virulent. Rg variants have variable virulence that may reflect some molecular heterogeneity among those morphotypes not easily detectable by the crude visual inspection of the colonies growing on agar plates. The molecular and genetic bases of morphotypeassociated virulence have been analysed and shown to be related to the synthesis of glycopeptidolipds (Belisle and Brennan, 1994). When we studied a panel of 41 strains of M. avium from various sources by infecting BALB/c mice with inocula prepared from SmTr variants, we found that these isolates showed an extremely wide spectrum of virulence (Pedrosa et al., 1994). The most virulent strains were isolated from naturally infected animals and from some HIV-negative human patients. The isolates from AIDS patients varied from non-virulent to low or intermediate virulence for mice. According to what was described above, it is clear that there are virulence factors in M. avium that are related to the colony morphology as well as other virulence factors that are morphotype-independent. As we will discuss below, some of the mechanisms of virulence relate to the capacity of certain strains of M. avium to avoid the triggering of the antimycobacterial mechanisms of the macrophage.
The dissection of the mechanisms M. &urn in mice
In our studies, we have concentrated on the analysis of a few strains of M. avium, paying particular
attention to their relative virulence in vivo and correlating it with the pattern of the immune response observed in the infected animals. The dissection of protective mechanisms has mostly used a low virulence strain with which we could easily detect some protective mechanisms at work. Innate immunity
to hf. avium
In most instances, the first cell the bacilli encounter is the macrophage. Several groups have shown that different morphotypes of hf. avium elicit distinct secretory responses on macrophages: SmOp mycobacterial inocula trigger higher oxidative burst responses (Gangadharam and Edwards, 1984) and higher monokine release (Fattorini ef ul., 1994 ; Michelini-Norris et al., 1992 ; Shiratsuhi et al., 1993) than SmTr variants. Isolates of M. avium with a lower ability to proliferate in in vitro cultured macrophages were shown to trigger higher amounts of TNFa secretion than isolates with good capacity to proliferate inside those cells (Furney et al., 1992). We showed that the mycobacterial multiplication of a “poor grower” could be enhanced both in vitro and in viva by the treatment of either cultures of macrophages or BALB/c mice, respectively, with neutralizing anti-TNFa monoclonal antibodies, suggesting that the triggering of TNFo! secretion by such nonvirulent strains of M. avium induced, in an autocrine or paracrine way, the restriction of growth of that mycobacterial strain (Sarmento and Appelberg, 1995). Recently, we found that such bacteriostasis was at least partially dependent on the reactive oxygen intermediates whose production was primed by TNFa (Sarmento and Appelberg, 1996). Other strains or variants of M. avium, however, remain avirulent despite their inability to trigger TNFa secretion (Appelberg et al., 1995b). These latter strains are probably susceptible to macrophage functions expressed constitutively or induced by monokines other than TNFa. The involvement of macrophage functions in early innate resistance to h4. avium was also evidenced by the clear participation of the Beg gene in the genetically-determined resistance of different strains of mice to this mycobacterial species (Appelberg and Sarmento, 1990). The exact mechanism by which the Beg gene product (the Nramp protein) exerts its antimycobacterial function is still not clear, although it could influence both the macrophage antimicrobial functions and the accessory cell function of the macrophage during an immune response to M. avium. It has been shown that the degree of phagosome-lysosome fusion in in vitro infected macrophages is influenced by the Beg gene (de Chastellier et al., 1993), with the Beg’ cells inducing a higher degree of fusion between the lysosomal compartment and the h4. &urn-containing
phagosomes than the BcgS cells. Whether such phagosome-lysosome fusion influences M. uvium growth or not is still not yet established with certainty. The fact that only virulent mycobacteria will induce an inhibition of such fusion is suggestive of a pathogenetic role for this phenomenon. Another cell type believed to participate in innate mechanisms of resistance to M. avium is the NK cell. Purified NK cells were shown to be able to activate the bacteriostasis or killing of hf. avium in in vitro cultured macrophages (Bermudez and Young, 1991). It has also been shown that the in vivo depletion of NK cells by anti-NK cell antibodies led to an exacerbation of the infection by M. avium in mice (Harshan and Gangadharam, 1991). However, we have been unable to reproduce these latter results using either anti-NKl. 1 monoclonal antibodies or anti-asialo GM1 polyclonal sera (Fl6rido et al., submitted). There is, however, compelling evidence for a role of NK cells in the induction of some resistance to infection by M. avium. SCID mice are known to have a hypertrophic NK cell compartment as compared to immunocompetent animals and have been used as a model for the study of NK cell function during infection. We have shown that neutralization of either IFNy or IL12 caused an exacerbation of the growth of a low virulence strain of M. avium in SCID mice, suggesting the involvement of NK cells in innate immunity to that mycobacterium, through the lL12-dependent secretion of IFNy (Appelberg et al., 1994b, Castro et al., 1995a). Recently, we found that anti-asialo-GM1 antibodies would deplete SCID mice of their cytolytic NK cells, but not of a cell population which produced IFNY in response to M. burn. This latter population was, on the other hand, sensitive to anti-Thy1.2 antibodies (Fl6rido and Appelberg, in preparation). It is likely that a cell type related to the cytotoxic NK cells is the major source of IFNy during 44. avium infection of SCID mice. The identity of this CD3-, Thyl.2+, anti-asialo GMl-resistant cell is not clear at the moment. Neutrophils have never been convincingly shown to kill mycobacteria. However, we have shown that the depletion of neutrophils in C57BIf6 mice using a granulocyte-specific monoclonal antibody induced an exacerbation of the growth of M. avium in the liver of the infected animals (Appelberg et al., 1995a). Interestingly, the defect of C57BL/6bg& (beige) mice in the ability to control the proliferation of M. avium was reversed in the liver by the infusion of neutrophils from wild-type C57BIJ6 mice. These results clearly point to a modest albeit significant participation of the neutrophil in the early phases of the infection by some strains of M. avium with low to intermediate virulence for mice. We have been unable (for technical reasons) to maintain selective neutropenias for longer than a week and therefore, it
is still plausible that a more important role for the neutrophil in resistance to M. avium has been underestimated. Since neutrophils do not ingest M. avium which is found inside vacuoles of infected macrophages, we postulated that their protective activity is dependent on the transfer of neutrophilic molecules to the macrophage, therefore arming this latter cell and increasing its antimycobacterial activity (Silva ef al., 1989). In this respect, it is noteworthy to mention that Kupffer cells are particularly deficient in antimicrobial mechanismsand that it is in the liver that we have found a major influence of the protective ability of the neutrophil. Finally, mycobacterial infections including infections by M. avium induce the chronic recruitment of neutrophils into the infectious lesions (Appelberg and Silva, 1989 ; Appelberg, 1992a, b), suggesting a role for these cells in the defence against the mycobacterial challenge. Acquired immunity to M. avium The exquisite sensitivity of AIDS patients with very low CD4+ T-cell counts to M. avium disseminated infections has illustrated the relevance of this T-cell subpopulation in acquired resistance mechanisms againt M. avium infection. Mouse models have confirmed the requirement of CD4+ T cells for immunity to strains of M. avium with low to intermediate virulence for mice (Appelberg et al., 1994b; Orme et al., 1992; Saunders and Cheers, 1995). In contrast, CD8+ T cells do not appear to play any influential role in the overall resistance to infection. Highly virulent strains of M. avium, however, seem to avoid or resist the CD4+ T-cell-mediated mechanisms of resistance (Appelberg and Pedrosa, 1992). Finally, avimlent M. avium may be eliminated from immunodeficient murine hosts as efficiently as from immunocompetent hosts (Collins and Stokes, 1987). The induction of a protective T-cell response is dependent on at least two major cytokines: IL6 and IL12. Neutralization of IL6 with specific monoclonal antibodies induced the inability to control the infection by M. avium (Appelberg et al., 1994a). Further studies showed that such exacerbation of the infection was due to the inhibition of the induction of protective T cells as assessedby an adoptive transfer assay (Appelberg, unpublished results). No effect was seen on the early phase of the infection, in contrast to the effects obtained with the neutralization of IL12, showing that, unlike IL12, IL6 is not involved in innate immunity. The neutralization of IL12 caused a major increase in mycobacterial proliferation dependent, in a first phase, on an inhibition of the innate immunity mechanismsand, in a second phase, on the inhibition of the induction of protective CD4+ T cells (Castro ef al., 1995b). The neutralization of IL12 caused a partial inhibition of the IFNy expression induced by the M. avium infec-
tion with a concomitant increase in the expression of IL4 and ILlO. It was also shown that the requirement for IL12 as regards protection was only seenin the first weeks of the infection, since the delay in administration of the neutralizing antibodies until the third week of the infection significantly reduced the effects of such treatment on mycobacterial growth. The expression of CD4’ T-cell-mediated protection involves IFNy and TNFa. Both cytokines are able to induce bacteriostasisof M. avium in in vitro cultured macrophages(Appelberg and Orme, 1993). Furthermore, IFNy is involved in the priming of the macrophages for the enhanced secretion of TNFa during M. avium infections (Appelberg et al., 1994b). These studies therefore illustrate, in M. avium, infections the cytokine cascades shown in other models, whereby IL12 will prime T cells (and NK-related cells) to secrete IFNy which will act on the macrophage,priming it for TNFcx secretion. Both effector cytokines IFNy and TNFcc cooperate in the induction of antimycobacterial activity in the mononuclear phagocyte. They are also involved in the regulation of the inflammatory response, namely affecting the organization of granulomas (Hansch et al., 1996). The susceptibility of mice to highly virulent strains of M. avium was not associated with the development of Th2 cells. There was no significant expression of IL4 during such infections, and the neutralization of either IL4 or IL10 led to no alteration in the pattern of growth of the virulent M. avium isolate (Castro et al., 1995a; Appelberg, 1995). Also, the addition of IL4 to IFNy-treated macrophage cultures was able to inhibit the generation of reactive oxygen intermediates but not the induction of bacteriostasis of M. avium (Appelberg et al., 1992). Likewise, IL10 treatment of macrophages inhibited the oxidative burst but only marginally the induction of bacteriostasis by IFNy. On its own, IL10 was able to induce some protection on infected macrophages (Appelberg, 1995). Finally, T cells from M. avium-infected, anti-IL12-treated donors did not exacerbate the infection by M. avium upon adoptive transfer to naive recipients despite the fact that IL4 and IL10 expression was increased as compared to control immune donors (Castro et al., 1995b). All these results show that Th2 cells are not induced during M. avium infection of normal mice, and even when a Thl to Th2 switch is promoted by antibody treatment this is not associated with a diseasepromoting effect. Mice infected with highly virulent strains of M. avium have highly activated macrophagesas assessed by the secretion of oxygen radicals and reactive nitrogen intermediates triggered by PMA or LPS, respectively, and by the expression of considerable amounts of cytoplasmic immunoreactive inducible nitric oxide
synthase (Florid0 and Appelberg, unpublished results). These data suggest that the highly virulent strains of hf. avium resist the antimicrobial machinery of the macrophage or, alternatively, that the appropriate bacteriostatic mechanisms are not coinduced with the oxidative metabolism.
Being the host cells, macrophages have been considered the main effector cells in immunity to mycobacteria. Virulent mycobacteria are resistant to killing by neutrophils and are detected inside macrophages throughout the infection (with the exception of the tubercle bacilli found in caseous necrosis foci). M. avium proliferates inside phagocytic vacuoles that do not interact with the endocytic compartments in a normal way (Frehel et al., 1986, 1991). M. avium-containing phagosomes lose the competence to fuse with lysosomes and vesicles from the endocytic pathway (Frehel et al., 1986) ; they fail to acidify (Crowle et al., l991), possibly due to their lack of interaction with the acidic lysosomal compartment and also to the lack of activity of the membrane-associatedproton pumps that seem not to assemblein those vacuoles (Sturgill-Koszycki ef al., 1994). These alterations in vacuolar trafficking may confer some protective advantage to M. avium.
The activation of the infected macrophage with different cytokines may lead to bacteriostasis and, according to some reports, to killing of M. avium. The efficacy among different cytokines varies according to the different reports. This may be due to the use of different macrophage populations, the use of mouse versus human cells, and the assay used (viable counts versus isotope incorporations assays). We also found that the ability of a given cytokine to induce bacteriostasis varies with the isolate of M. avium studied (Appelberg and Orme, 1993). The members of MAC are resistant to the bactericidal effects of NO (Appelberg and Orme, 1993; Bermudez, 1993). We recently found that we could induce bacteriostasis of M. avium equally well in normal mice and in iNOS gene-disrupted animals (Gomes and Appelberg, unpublished). Most strains (except for a small percentage of avirulent strains) are resistant to the oxygen-dependent mechanisms of the macrophages.Also, the bacteriostasis induced by IFNy is independent of these latter mechanisms (Appelberg and Orme, 1993). What are the effector molecules responsible for the bacteriostasis ? We postulated that the acidification of the phagosomes might play a role (Appelberg and Orme, 1993). However, the degree of phagosome-lysosomefusion and the phagosomalpH in M. avium-infected macrophages did not change after in vitro activation by
IFNy (Games, Appelberg and Silva, unpublished). The sequestration of iron may play a role in the induction of some degree of bacteriostasis (Douvas et al., 1993). It is, however, not known whether this latter mechanism is relevant in vivo. In summary, much has been learned regarding the cell biology of the infection by M. avium and the cellular and molecular mediators of immunity. However, we still do not understand the basis of virulence in these mycobacteria and we do not know the mechanisms of bacteriostasis and killing mediated by the macrophage. Hopefully, the knowledge gathered using experimental models may clarify these aspectsand one day lead to the design of new therapeutic approaches to infection by members of the MAC.
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