Co-infection of macrophages modulates interferon gamma and tumor necrosis factor-induced activation against intracellular pathogens.

Co-infection of macrophages (M phi) with Toxoplasma gondii and Mycobacterium avium-intracellulare complex (MAC) has been observed in patients with acquired immunodeficiency syndrome (AIDS). In this study we have demonstrated that co-infected murine M phi respond differently to cytokine stimulation than M phi infected with either of the microorganisms alone. Whereas treatment with interferon gamma (IFN- gamma) activated both single and co-infected groups of M phi to kill T. gondii, treatment with TNF did not influence the rate of MAC growth in co-infected M phi, in contrast with the inhibition of growth observed in MAC-infected M phi. These results suggest that in AIDS patients suffering infection with multiple intracellular pathogens, the ability of cytokines to stimulate microbicidal or static activity in mononuclear phagocytes can be impaired by the presence of more than one of the intracellular organisms.

T oxoplasma gondii and organisms belonging to the Mycobacterium avium complex (MAC) are two of the most common pathogens in patients with AIDS (1-2) . Both T. gondii and MAC block phagosome-lysosome fusion (3,4) and replicate inside of human monocyte-derived macrophages and murine macrophages (MO) (5,6). While the IFN-. y-activated Mo has been shown to be the key effector cell in host defense mechanisms against T. gondii (7), IFN -,y paradoxically induces enhanced intracellular survival of MAC within MO (5,8). TNF elicits no apparent effect on intracellular replication of T. gondii within MO (9) . In contrast, TNF-treated Mcb exhibit static or cidal activity against intracellular MAC (5) . Since in vivo production of these cytokines is probably triggered sequentially by infection, the outcome of TNF-and IFN-. y-induced activation of Mo during co-infection with these two intracellular pathogens becomes critical in severely immunocompromised patients. Stimulated by the clinical observation ofmononuclear phagocytes harboring both T. gondii and MAC in the brain of a patient with AIDS, toxoplasmic encephalitis, and mycobacterial meningoencephalitis, we investigated the response of co-infected Mo to cytokine activation, and assessed whether successful fusion of lysosomes with phagosomes occurred in co-infected activated Mo.

Materials and Methods
Strains and Cytokines. T. gondii strain RH and M. avium complex strain 101(serotype 1) were harvested and processed bymethods described previously (5,6) . Recombinant murine IFN-y (2.3 x 10' U/mg protein ; <25 pg endotoxin/mg protein) and recombinant murine TNF-tx (2.8 x 10' Wing protein ; 12 pg endotoxin/mg protein) were supplied by Genentech, Inc. (South San Francisco, CA) . IFN-y was stored before use at 4*C in a solution of 1 x 10' U/ml PBS containing 1 mg/ml BSA. TNF was stored undiluted at 4°C and never frozen . Treatment ofMacrophages. Unelicited murine peritoneal MO were prepared as described (6) and incubated with 230 U/ml IFN-y, 1,000 U/ml TNF, or medium alone (IMDM [Gibco Laboratories, Grand Island, NY] containing 10% FCS and 40 Ftg gentamicin/ml) as a control for 24 h at 37°C . The monolayers were then challenged with T. gondii alone (2 tachyzoites per MO) ; MAC alone (10 bacilli per MO); or with both microorganisms simultaneously at the same multiplicities of infection . After 2 h, all monolayers were thoroughly washed with PBS to remove extracellular microorganisms. At this time, to correspond to the 0 h time point, some ofthe monolayers on slides were fixed and stained with Dif-Quik stain (American Scientific Products, McGaw Park, IL) with the third step of the procedure omitted. The Kinyoun stain was then performed on the stained monolayers, omitting the counterstain step. This combined staining procedure allowed easy visualization and distinction between intracellular T. gondii and MAC organisms. The remaining monolayers were incubated an additional 20 h before staining.
Phagosome-Lysosome Fusion. Cytokine-treated and control macrophage monolayers were incubated for 20 min with acridine orange (5 Fcg/ml) in HBSS (Gibco Laboratories) at 37°C in a humidified atmosphere containing 5% C0 2 and infected as described above. This method by which phagosome-lysosome fusion was quantitated within infected MO has been described and verified by electron microscopy (10, 11) . After quantitation of fused phagolysosomes, the coverslips were removed and the slides were soaked briefly in xylene. After staining with modified DiffQuik as described above, numbers of intracellular microorganisms per 100 MO and percentages of infected cells were determined .

Results
Co-infection in Normal Mo. The percentage of infected Mo was 56 .5 ± 10 .2% for monolayers infected with T. gondii only, and 70 .8 ± 12 .7% for monolayers infected only with MAC . The percentages of infected Mo in co-infected monolayers were: 6 .0 ± 0.9% contained intracellular T. gondii only, 21 .2 ± 3 .7% contained intracellular MAC only, and 50 .8 ± 9 .9% Mo contained both organisms. These percentages were unaffected by treatment of the Mo with cytokines . After 20 h of infection, numbers of intracellular T. gondii per 100 Mo increased nearly fourfold in Mo infected with T, gondii alone ( Fig. 1 A) . Numbers of intracellular MAC remained approximately the same after 20 h of infection in untreated Mo infected with MAC alone (Fig . 1 B) . In Mo co-infected 978 Co-infection Alters Activation of Macrophages  with both pathogens, numbers of intracellular T. gondii increased similarly as in Mo infected with T. gondii alone ( Fig.  1 A ; the apparently higher mean value for co-infected Mo was not significant), and numbers of intracellular MAC increased 'x+21% ( Fig. 1 B) . The results of experiments for which colony-forming units of MAC were counted in parallel by lysing infected and co-infected MO and plating on agar showed percentage increases with time of infection similar to those measured by microscope assay (not shown) . Co-infection in IFN-y-and TNF-treated Mo. In Mo activated in vitro with IFN-y and infected with T gondii alone or co-infected, numbers of intracellular T gondii per 100 cells decreased ten-fold by 20 h post infection (Fig . 1 A) . In marked contrast, numbers of intracellular MAC within IFN-yactivated Mo increased 70% in singly infected cells and 215% in co-infected cells (Fig. 1 B) . The intracellular growth of T. gondii within Mo treated with TNF was not significantly different from that within untreated control Mo regardless of co-infection (Fig. 2 A) . However, smaller increases in numbers of intracellular T. gondii occurred consistently in coinfected Mrs treated with TNF as opposed to Mo treated with TNF and infected with T. gondii alone (Fig . 2 A) . Whereas the numbers of intracellular MAC increased 182% in singly infected, untreated control MO and 165% in coinfected control Mo, this value increased only by 23% in TNF-treated Mo that were infected with MAC alone (Fig.  2 B) . In contrast to this result, TNF-treated Mo that were co-infected with MAC and T. gondii showed a 144 % increase in intracellular mycobacteria per 100 Mo. Since the 20-h in-   Table 1) . In MO activated with IFN-y, however, the percentage of cells exhibiting fusion of parasitophorous phagosomes with lysosomes increased to over 25% in MO infected with T. gondii alone and 14% in co-infected MO (Table 1) . In contrast, IFN-y-activated MO that were infected with MAC showed no significant increases in fusion of phagosomes with lysosomes. A reverse trend was observed in MO treated with TNF : fusion of phagosomes 979 Black et al. Brief Definitive Report phagosomes within coinfected cells that contained both infecting agents .
with lysosomes was significantly increased in MO that were infected singly or co-infected and contained only MAC within the phagolysosome, whereas, in contrast, little fusion was observed within MO infected with T. gondii alone and within phagosomes of co-infected MO that contained only T. gondii. In the case of phagosomes of co-infected MO that contained both T. gondii and MAC, fusion was inhibited regardless of whether MO were treated with cytokines (Table 1) . These co-infected phagosomes were only observed occasionally, however. Most phagosomes observed contained only one species of infecting agent (not shown) .
The results presented in this report (summarized in Fig .  3) demonstrate that when MO are co-infected with both T. gondii and MAC, the inhibition of intracellular growth of MAC that is usually observed within TNF-treated MO is abrogated by the presence of the co-infecting T. gondii, in spite of successful fusion of phagosomes containing MAC with lysosomes . This result suggests that the clinical outcome of co-infections may be complicated due to exacerbation of one infection by the other, and that resolution of the infections may be prolonged regardless of therapeutic intervention .
For most intracellular pathogens, the ability to inhibit phagosome-lysosome fusion correlates with intracellular replication . This was true for T. gondii in all cases, and was true for MAC in normal and IFN-y-treated MO, in which the mycobacteria inhibited phagosome-lysosome fusion and replicated intracellularly. However, agents such as Mycobacterium Both11 0 .0 lepraemurium (12), Nocardia asteroides (10), and Leishmania donovani (13) are capable of multiplying within the fused phagolysosome. Our finding that MAC was capable of intracellular replication in co-infected cells in the face of fusion of phagosomes with lysosomes in TNF-treated Mo is therefore not wholly unexpected. It is surprising, however, that coinfection of TNF-treated Mo with both organisms did not alter the successful fusion of lysosomes with phagosomes containing MAC, but did alter the ability of the Mo to inhibit replication of MAC. This strongly suggests that, within TNFtreated Mo infected only with MAC, some antimycobacterial mechanism other than phagosome-lysosome fusion plays a critical role in the inhibition of intracellular growth . It is