Mycobacterium avium: pathogenicity in HIV1 infection

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1 lth F O R U M I N M I C R O B I O L O G Y

Mycobacterium avium: pathogenicity in HIV1 infection H. Shiratsuehi, J.L. Johnson and J,J. Ellner Case Western Reserve University, University Hospitals and Cleveland Veterans Administration Medical Center, Cleveland, O H 44106 (USA)

Introduction Mycobaeterinm avium is considered a rare human pathogen in healthy individuals with normal innnune responses. Disseminated M. avium infection is, however, the most frequent infectious complication in patients with AIDS (Green et al., 1982; Hawkins et aL, 1986) and occurs late in the course of HIV infection. AIDS patients with disseminated M. avium infection are severely immunocompromised, with peripheral CD4 counts of < 100/p.1 (Nightingale et al., 1992; Horsburgh, 1991). About 47-56 % of AiDS patients undergoing autopsy have evidence of disseminated M. avium infection (Fauci, 1984; Wallace and Hannah, 1988). Disseminated M. avium infection is increasing in frequency, probably due to longer survival resulting from antiretroviral chemotherapy and prevention of otiler opportunistic infections. A prospective study of patterns with AIDS with CD4 counts of less than 200/id indicated that 17 % of AIDS patients will develop disseminated infection wi~h M. aviurn by 1 year and 32 Vo by 2 years (Nightingale et al., 1992). Development of disseminated M. avium infection is associated with shortened smvival (median survival of 4 months compared with l I months for AIDS patients without disseminated M. avium infection) (Horsburgh et at., 1991). Disseminated M. avium infection clearly contributes to morbidity and mortality in patients with AIDS.

Pathogenicity of 1t4. avium Virulence factors of M. avium are largely unknown. Plasrrtid content and colonial morphotype (smooth, flat and transparent (SmT), as compared with smooth, domed and opaque (StuD) appearing colonies), are associated with increased intracellular M. avium growth (Crawford et al., 1986; Gangadharam et aL, t988). Fresh clinical isolates from AIDS patients usually exhibit SmT colony morpho-

type (although our recent studies raise questions concerning this issue Oaeobs et aL, 199])) and are highly virulent for intraccllular growth in human monoeytederived macrophages (Meylan et al., 1990). After serial passage in in vitro culture, the SmT colonial morphotype is transformed into the SmD morphotype and loses the capacity to replicate intraeeltularly in mice, chickens and humans (Dunbar et aL, 1968; Schaefer et aL, 1970; Shiratsuchi et al., 1990). M. avium of SmD morphotypes are phagoeytosed more efficiently, but replicate more slowly within human monocytes than isogenic SmT morphotypes of the same M. avium strains (Shiratsuchi et al., 1990). Infection of human monocytes with SmT morphotype induced less ILl and TNF= release into culture supernatants than did the StuD morphotype (Michelini-Norris et aL, 1992; Shiratsuchi et al., 1993); however, the expression of cytokine-speeific mRNA was comparable, indicating differences in translational efficiency (Shiratsuchi et aL, 1993). After phagocytosis, M. avium replicates inside macrophages in non-acidic vesicles (Crowle et al., 1991). On electron microscopy, intracelluiar organisms are enclosed in membrane-bound phagosomes and are surrounded by a multilamellar electron-translucent zone (ETZ) which resembles ;~ycopeptidolipid (GPL), an important cell wall component of myeobaeteria structurally (Frehel et al., 71986; Ruiong et ale 1991). The ETZ may represent a barrier to prevent intracellular killing of the organisms. GPL expression may be responsible for differences in colonial morphology between SmT and SmD organisms. GPL isolated from StuD morphotypes induced more ILi[~ and TNFa than ¢3PL from SmT morphotypes (unpublished data), observations paralleling those from experiments with intact bacteria, M. avium GPL also inhibited the killing of Candida atbicans by bovine macrophages and mitogen-induced lymphocyte proliferation (Hines II etaL, 1993 ; Tassell et at., 1992; Brownbach and Barrow, 1988) and therefore may be considered a mycobacterial virulence factor.

,*) Correspondingauthor: HiroeShiratsuchi, Ph.D., Case Western ReserveUniversity.BiomedicalResearch Building, 10th Floor. West Administration. 10900 Euclid Avenue,CleveIan+".OH 44106-49S4. USA.

L A B O R A TOR Y A N D C L I N I C A L A S P E C T S O F M, AVIUM E P I D E M I C H I V infection and M. avium

The late stages of HIV infection are characterized by repeated, often incurable opportunistic infections in the setting of progressive immunosuppressionwith decliningceU-mediated host immumty and increased HIV replication. Considerable controversy exists regarding whether HiV or specific viral constituents or products directly impair host immune defences against opportunistic pathogens or whether the increased occurrence of these infections is due to indirect effects of HIV mediated by progressive depletion of the CD4 helper T-cell population. M. avium is an intraceilular pathogen; its major site of growth in human tissues is within tissue macrophages. Disseminated M. avium infection in AIDS patients is characterized by a continuous high grade bacteraemia and enormous tissue burdens of the organism. Tissue macrophages in AIDS patients with disseminated M. avium infection are filled with innumerable mycobacteria (109-101° AFB per gram tissue) OVong et al,, 1985), Earlier studies produced conflicting data regarding whether mononuclear phagocytes of AIDS patients were intrinsicallymore permissive to intraeellular M. avium growth. Effector functions of monocytes from patients with AIDS were intact when tested against certain intracellutar pathogens (Washburn et aL, 1985), although cytokine production and Fe- and C3-receptor-mediated phagocytosis were defective (Murray et el., 1984; Bender et el., 1988). We and others demonstrated preserved effector cell function of monocytes obtained from AIDS patients against in vitro M. aviura infection (Johnson et aL, 1991 ; Sebnittman eta!., 1988). In contrast, Crowle et aL reported that uio,,~ytc-u~,vgu macrophageg of AIDS patients exhibited enhanced intracellular M . avium growth (Crowle at al., 1992). Some of the disparities in these studies may be due to differences in conditions and . . . . .

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length of culture and in the virulenceof M. avium strains used in the studies. The direct effects of HIV infection on human monoeyte effeetor functions against M. avium have been studied by in vitro infection of monocytes from healthy subjects with HIV. Dual infection of human monocyte-derived macrophages with HIV and M. avium did not alter intracellular myeobacterial growth in shert-term culture (Meylan el aL, 1992), but increased pe~'missivenessof HIVl-infected macrophages for intracellular M. avium growth was present after 14 days of culture (Kfillenius et at., 1992). Rapid imracellular M. avium replication has been reported at early time points wi~en haman monocyte-deri~d-macrophages were chronically infected with HIVI before infection with M . avium (Newman et aL, 1993). Dual infection with HIVI and M. avium also decreased cell viability (Newman et ai., 1993). In vitro infection ef monocytes with HIV may not, however, accurately reflect conditions in

231

the tissues of patients with AIDS. HIV infection of monocytes and tissue macrophages in AIDS patients is quantitatively lower than that of T cells (Sehnittman et aL, 1989; Spear et al., 1990), Therefore, the effects of HIVI infe~ion on mononuclearphagocyte effector functions against M. avium are likely to be indirect. Constituents and byproducts of HIVI might enhance the pathogenicity of AIDS-associated opportunistic infectious agents by interfering with host defence mechanisms. Effects of HIV1 proteins on M. avlura pathogenesis 1. Phagocytosis and intracellular growth

Using an in vitro human monocyte infection model, we examined the effect of HIVI proteins on monocle phagocytosis and imracellular growth inhibition of M. avium. Recombinant envelope protein gpl20, transmembrane protein p121 and core proteins were studied. Monocytes obtained from HIV-negative healthy subjects were pretreated with or without HIV1 proteins for 2 days and then infected with M. avium. We found that pretreatment with gpl20 for 2 days prior to infection inhibited monocyte phagocytosis of M. avium (table I), We also found that, despite inhibitionof phagocytosis, HIV envelope protein gp 120 enhanced intmee!lular growth of 6 strains of M. avium in human monocytes from healthy sul3jects (fig. 1) (Shiratsuctfi el aL, 1994 in press). The other HIVI proteins tested, core and transmembrane proteins, did not inhibit M. avium phagocytosis nor increase intracellulargrowth of M. a',ium when monocytes were pretreated with these HIV proteins before M. avium infection (data not shown). The effects of gp120 appear to be mediated through specific interactions of HIV proteins with monocytes, as shown in figure 2, l-IlVl gpl20 specifically hinds to CD4 receptors (Ktatzmann et at., 1984; Melendez-Guerrero el ul., 1990} and may activate monocytes via ~his interactions (Merrill et aL, 1989). Although monocytcs/macrophages express much lower numbers of CIM receptors than peripheral blood T 13nnphocytes,binding of gpl20 to human monocytes has been demonstrated (Finbloom et al., 1991). Soluble CD4 (fig. 2) and OKT4A, but not OKT4, zbroge.ted the effects of gpl20, indicating that the effects ofgpl20 on phagocytosis and intracellular growth of M. avium are specifically mediated, at least in part, via CD4 receptors on the monoeyte surface (Shiratsuehi et al.. 1994). In a preliminary report, Wagner et aL demonstrated that HIV1 gpi20 reduced the phagocytosis and enhanced the intracellutar growth of Cryptococcus neoforroans, another common opportunistic pathogen in AIDS patients, by alveotm macrophages (Wagner et aL. 1990). It is not yet clear whether" the effects of gp120 on effe:tor function against M. ,.:,i.~,.rnand C. neoformans are mediated similarly.

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llth FORUM IN MICROBIOLOGY

Table I, Effect of gpl20 on M. avium phagocytosis by h u m a n monocytes. gpl20 {ttg/mi)

no.

% Infected monoeytes

AFB/100 monocytes

0 0.95 0.2

4 4 4

43.04-3.7 39.7 ± 5.2 35,3±4.1 (')

165 ± 19 162 4- 28 131 --+7.8 ~')

(*1 p < 0.01 compared to conuol (without gpl20). Monocytesobtained from healthysubjectswereprccultured with or without recombinantH]VI gpl20 (rangingfrom 0 to 0.2 pg/ml) for 2 days before infection. Data wereexpressedas the mean:l:SEof the ~'0 monacytesinfected with t> 1 AFB or the number of AFB per tO0 monocytes.

2. Modulation o f cytokines that affect M. avium growth 100

!

J

M, avlum Strain

O: LR11~mT • : 2151SmO ZL: SV4(4/44) &: LR147 [:: 10-153 l : 86m2096

! 1

(-)

gpl 2o

o~. ~ m ; Fig. !. EIfect of gpl20 pretreatment on intracellular growth of various M. avium strains in human monocytes. Monocyzes obtained from healthy HIV-negative donors were preeultured with or without gpl20 0.2 Fg/ml for 2 days and then infected with 2 avirulem (2151Stud and SV4(4/44)) and 4 virulent M. avium Strains (LRll4SmT, LRI4?, 10-153 and 86m2096). After infection, monoeytes were cultured with medium alone for 7 days. Samples were hatwe~ted at day 0 (immediately after infection) and "/days after infection. The number of 114.avium in each sample was assessed by CFU assay. Data were expressed as mean -4- SE of a growth index (GI) calculated as: CFU in day 7 monocyte lysat¢ divided by CFU in day 0 monocyte lysate. Prctteatment whh gp 120 enhanced intr~cellular growth of 34. aviptm strzdns LRI14SmT and 10-153 (p < 0.05 campared to control culture) {paired t-tes0.

Several investigators have shown that HIVI infection and HIV envelope protein gp120 activate human monocytes to express cytokines (Clouse et al., 1991 ; Merrill et al., 1989; Nakajima et aL, 1989). M. avium infection itself also induces cytokine production by mononuclear phagocytes such as 1L 1, IL6, ILt0, TNFa and TGF~ (Blanchard et at., 1991a; Shiratsuchi et al., 1993 ; Bermudez, 1993; Bermudez and Champsi, 1993). Several cytokines possess macrophage-activating factor (MAF) activity and augment mononuclear phagocyte effe~tor c¢il functions. IFNy, a prototypie agent with MAF activity, inhibits the replication of Leishmania species (Douvas et al., 1985; Passwell et aL, 1986) and other pathogens (Rothermel et aL, 1983; Bhardwaj et al., 1986). IFNy enhances intracellular growth inhibition of M. tuberculosis by murine macrophages (FIesch and Kaufmann, 1987). Previously, we demonstrated that after M. avium infection, cocultuting with IFN't' decreased inlracellular growth in human monocytes; further enhancement of intracellular growth inhibition was observed when indomethacin was added to these cultures (Shiratsuchi et al., 1990, 1991 ; Johnson el al., 1991). TGF[~ and ILl0 inhibited the effects of TNF~t or GM-CSF to augment intracellular M . avium killing by macrophages (Bermudez~ 1993 ; lqermudez and Ehampsi, 1993). GM-CSF and TNFa also have demonstrated M A F activity to decrease intracellular growth of M. avium (Bermudez and Young, 1988, 1990; Blanchard et aL, 1991b; Denis, 1991). Beside blocking the effects of MAF, cytokines also may directly promote intraceilular growth of M. avium (Denis and Oregg, 1990; Shiratsuchi et al., 1991). Cytokines including IL3, M-CSF and IL6 increased the intracellular growth of one of two M. avium strains tested, and I L i a enhanced the growth of both strains. Interestingly, I L i a and IL6 promoted myeobacterial replication in cell-free medium. Direct binding of IL6 to M. avium and downregula-

L A B O R A T O R Y A N D C L I N I C A L A S P E C T S OF M. A VIUM E P I D E M I C I ~ b ' T R A I . ~ T I ~ I OF gpI20 EFFEGT ON F t 4 . ~ a ' ~ O 6 1 BY ~[:D4

:f

10

o

ll'ti o'

gpt20

-

÷

SGD4

-

-

-

+

glsl20

+

+

sC04

-

-~

-

+

+

+

Fig. 2. Neutralization of the effects of gpl20 on monocyte phagocytosis of M. avium by sCD4 or monoclonal OKT4A antibody. Phagocytosis of M. avium wa~ assayed by couming AFB. gpl20 (0.2 pg/ml) was preincubatcd with recombinant sCD4 (0.2 pg/ml) at room temperature for 30 rain before addition to monocyte cultures. Monocytes from 3 healthy subjects were cultured with medium alone, gpl20 0.2 ~g/ml, sCD4 0.2 pg/ml or gpl20 preincubated with sCD4 for 2 days and *.hen infected with M. avium strain LRI I4SmT. Data are shown as the mean +_ SE of monocytes ingesting ~ l AFB or the number of AFB per tOO monocytes in 3 independent experiments using monocytes from healthy subjects, gpl20 decreased M. avium phagocytosis (p < 0.02 compared to control culture, or sCD4 + gpl20, or sCD4 atone; paired l-test).

tion of TNF receptor expression by IL6 on human monocyte-derived-macrophages have been demonstrated (Bermudez et al., 1992). Clearly, direct effects of cytokines on bacterial replication may alter intraeellular killing of M. avium by mononuclear phagocytes. Sera from AIDS patients contain high levels of IL6 (Breen et at., 1990). Spontaneous production of high levels of TNF~t and IL6 by peripheral monocytes (Wright et al., 1988; Breen, 1990) and IL6 and GM-CSF by alveolar macrophages has been demonstrated in AIDS patients (Agostini et al., 1992). Increased TGF[~ p r o d u c t i o n by P B M C from HIVl-infected subjects also has been reported after stimulation with antigens such as tuberculin-purified protein and tetanus toxoid (Kekow et aL, 1990). TNF~ production by M. avium-infeeted monocytes from AIDS patients and healthy individuals was comparable; however, monocytes from AIDS patients released lower concentrations of ILl into culture su-

233

pernatants after infection with M. avium (Johnson et al., 1993). Newman et aL reported that chronically HIV-infected human monocyte-derived macrophages ,.'xhibited prolonged cytokine m R N A expression and produced more TNF,,, ILl and IL6 after LPS stimulation in vitro ffgewman et aL, 1993). In o,~r recent study, pretreatment of human monocymes with gpl20 synergistically augmented production of I L l , "INF~ and [L6 when monocytes were infected with M. avium {fig. 3) (Shiratsuchi et aL, 1994). Induction of these and other cytokines by HIVt proteins might medalate the ability of mononuclear phagocytes to limit intrace|lular mycobacterial growth. ILl0 and TGF[3 are known as immunosuppressive cytokines that inhibit lymphocyte proliferation and deactivate mononuclear phago~ cyte functions {Bogdan et al., 1991 ; Fiorentino et ul., 1991). Bermudez et al. have shown that TGF[3 and I L l 0 enhanced intraeellular M. avium growth, and induction of TGF[I production was associated with the capacity of M. avium to replicate intracellularly (Bermudez, 1993; Bermudez and Champsi, 1993), Alterations in the dynamic local balance of these mycobacterial growth inhibitory and growth enhancing factors, therefore, may lead to increased mtraceilular replication of this pathogen~ Clearly, HIV infection may aker host responses to M. avium infection. It also is conceivable that M.

Effect of Reco,~,ltat~ gp120 of 1-~ I:n Ck;ok,Me b .,*Monoc/les Stlmu~ed wllh LRll 4F Sham of M. m4um

Fig. 3. Effect of gpl20 on cytokine induc~.ion by human monocytes. Monocytes from healthy ~ubjects were precultured with or without gpl20 (1 ~g/mi) for 2 days and then stimulated with M. a~.,ium strain LRlI4SmT for another 24 h. Cytokine activities in culture supernatants were assayed by ELISA.

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i lth FORUM IN MICROBIOLOGY

avium infection may alter the course of HIV infection. Lymphocytes and other cells capable of harbouring HIV are exposed to cytokines in the circulation and at the tissue level. HIV infection of quiescent T cells results in incomplete HIV replication; cellular activation is required for complete transcription of retroviral RNA (Zack et eL, 1990). TNFa, ILl[~ and IL6 have been shown to augmen,~ HIV replication in vitro in HIV-infected cells (Osborn et el., 1989; Mellors et el., 1991 ; Poll et eL, 1990; Clous¢ et el., I989), leading to further depression of host immune defences in a potential vicious cycle. Cytokine induction due to M. avium infection could therefore theoretically accelerate the course of HIV infection. hi addition to alterations in cytokine expression, HIV1 products may iml~air other aspects of mononuclear vhago~vl¢ defences against intracellulet pathogens. Ma:rophage phagosome-lysosome fusion is another important intermediate step in the killing of intracellular pathogens such as mycobacteria. Inhibition of phagosomalolyso~omal fusion occurs after phagocytosis of virulent strains of M. avium by murine bone marrow-derived macrophages and correlates with a capacity for rapid intracellular mycobacterial replication (Frehei et el., 1986). Estevez et aL have shown that gp 120 inhibits macrophage phagosom¢.4ysosome fusion after phagocytosis of opsonized zymosan particles (Estcvez et el., 1992), providing another potent.ial basis for its action en intracellular M. evium growth. In AIDS patiexlts with disseminated M. avium infection, therefore, abnormalhies in effector ftmetions of mononuclear phagocytes induced by HIVI products may contribute to uncontrolled intracellular bacterial growth. Further understanding of the underlying mechanisms of intraccllular parasitism by M. avium may provide new avenues for therapeutic intervention.

Re[erences

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