Differential Cytokine Pattern in the Spleens and Livers of BALB/c Mice Infected with Penicillium marneffei: Protective Role of Gamma Interferon
Institute of Microbiology, University of Milan, Milan,1 Department of Pathology, Ospedale Maggiore, Lodi, Italy,2 Department of Internal Medicine, Infectious Diseases, Algemeen Ziekenhuis Sint Jan, Bruges, Belgium3s;^u-, 百拇医药
Received 6 June 2002/ Returned for modification 12 July 2002/ Accepted 25 September 2002s;^u-, 百拇医药
ABSTRACTs;^u-, 百拇医药
Penicillium marneffei is an intracellular opportunistic fungus causing invasive mycosis in AIDS patients. T cells and macrophages are important for protection in vivo. However, the role of T-cell cytokines in the immune response against P. marneffei is still unknown. We studied by semiquantitative reverse transcription-PCR and biological assays the patterns of expression of Th1 and Th2 cytokines in the organs of wild-type (wt) and gamma interferon (IFN-) knockout (GKO) mice infected intravenously with P. marneffei conidia. At 3 x 105 conidia/mouse, a self-limiting infection developed in wt BALB/c mice, whereas all GKO mice died at day 18 postinoculation. Splenic and hepatic granulomas were present in wt mice, whereas disorganized masses of macrophages and yeast cells were detected in GKO mice. The infection resolved faster in the spleens than in the livers of wt mice and was associated with the local expression of type 1 cytokines (high levels of interleukin-12 [IL-12] and IFN-) but not type 2 cytokines (low levels of IL-4 and IL-10). Conversely, both type 1 and type 2 cytokines were detected in the livers of wt animals. Disregulation of the cytokine profile was seen in the spleens but not in the livers of GKO mice. The inducible nitric oxide synthase mRNA level was low and the TNF- level was high in both spleens and livers of GKO mice compared to wt mice. These data suggest that the polarization of a protective type 1 immune response against P. marneffei is regulated at the level of individual organs and that the absence of IFN- is crucial for the activation of fungicidal macrophages and the development of granulomas.
INTRODUCTIONn7$4j, 百拇医药
Penicillium marneffei is a dimorphic pathogen that causes disseminated mycoses in the lungs, spleens, and livers of infected subjects, especially human immunodeficiency virus (HIV)-positive patients in Southeast Asia (8, 13). In vivo (21, 29) and in vitro (6, 9), P. marneffei undergoes phase transition and multiplies as yeast cells inside macrophages. The histopathological characteristic of P. marneffei infection in humans is granuloma formation, with suppurative and anergic reactions and necrosis (9, 13, 29). In mice, disseminated granulomas are also present, with epithelioid cells containing growing yeast cells. Hepatosplenomegaly is common in both mice and humans (8).n7$4j, 百拇医药
The immune response against P. marneffei infection is mediated mainly by T cells and macrophages: in nude mice or T-cell-depleted animals, the mycosis is fatal, whereas in healthy hosts, the fungus is cleared in 2 to 3 weeks, depending on the size of the inoculum (22, 32). In vitro, both human and mouse macrophages are able to control P. marneffei growth and to kill intracellular yeast cells when activated by T-cell-derived cytokines, such as gamma interferon (IFN-) (6, 23, 30). It has been shown that fungus death is due mostly to the release of nitric oxide (NO) by activated macrophages (6, 23, 30).
Little is known about the role of inflammatory cytokines in vivo and whether resistance to P. marneffei infection is mediated by a Th1 or a Th2 immune response or both. Granuloma formation can be associated with either a predominantly Th1 response, with high levels of tumor necrosis factor alpha (TNF-), IFN-, and interleukin-12 (IL-12), as in mycobacterial infections (7, 15), or a Th2 response, with high levels of IL-10 and IL-4, as in the reaction to Schistosoma eggs (34). As reported for other fungi, such as Candida spp., Aspergillus fumigatus, or Histoplasma capsulatum (4, 20, 28, 35), the cytokine microenvironment dictates whether a protective or a deleterious immune response is generated. IFN- is the hallmark cytokine for the Th1 immune response, and its production is essential in the control of intracellular pathogens, such as Mycobacterium tuberculosis (7). In mice with a disrupted IFN- gene (IFN- knockout [GKO] mice) or in patients with defects in IFN- signaling, susceptibility to mycobacterial infections has been reported (14, 18, 19, 26).
Dissecting the mechanism(s) of P. marneffei infection in immunocompetent hosts may help to understand the pathogenicity of penicilliosis marneffei in immunocompromised patients. This goal is particularly relevant for AIDS patients living in Southeast Asia, where this infection ranks as the third most important opportunistic infection (8) and where the vast majority of HIV-infected patients cannot yet afford treatment with highly active antiretroviral therapy.d#6e8m, 百拇医药
The purpose of this study was to characterize the cytokine microenvironment leading to the formation of granulomas and to the activation of a protective immune response in healthy or GKO BALB/c mice sublethally infected with P. marneffei conidia. We also investigated the histopathological changes occurring in the organs of wild-type and GKO mice. Although the natural infection likely is acquired via the respiratory tract, in this set of experiments and in agreement with previous reports (9, 22), we preferred the intravenous (i.v.) route of infection to better control the size of the inoculum and, consequently, the extent of colonization of the different organs.
MATERIALS AND METHODS9, http://www.100md.com
Mice. Pathogen-free female BALB/c mice weighing 20 to 24 g were obtained from Charles River Italia (Calco, Como, Italy) and used at the age of 6 to 8 weeks. GKO mice (11) in a BALB/c background (BALB/c-Ifngtm1Ts) were purchased from the Jackson Laboratory (Bar Harbor, Maine). Homozygous mice were identified by PCR of tail-derived DNA with primers flanking the neo gene (sense, 5'-CAAGTGGCATAGATGTGGAAG-3'; antisense, 5'-GGCAATACTCATGAATGCATCC-3'); the wild-type gene gave an amplified fragment of 340 bp, whereas the disrupted gene gave a 2,400-bp fragment. Homozygous mice were bred and maintained at the Istituto Nazionale Tumori, Milan, Italy, and were kindly provided to us by M. P. Colombo, Istituto Nazionale Tumori. All mice were housed, fed, and handled under standard conditions according to institutional guidelines.9, http://www.100md.com
Microorganism, infections, and in vivo analysis. The strain of P. marneffei used in this study, IUM885346, was kindly provided by M. A. Viviani, Milan, Italy. It was isolated from the blood of an Italian HIV-positive i.v. drug user infected in Thailand (33). The fungus was cultured on yeast morphology agar (YMA) (Biolife, Milan, Italy) at 25°C for 10 to 14 days. P. marneffei conidia were collected by flooding the culture surface with saline, centrifuged at 1,800 x g for 20 min, and filtered to remove mixed mycelial debris. The conidia were washed twice with saline, counted with a hemocytometer, and then suspended at the desired concentration.
Mice were injected i.v. via the tail vein with a sublethal suspension of 3 x 105 conidia/mouse and sacrificed after specified time intervals. Control mice of the same sex and age were injected with sterile saline. To check the inoculum viability, the suspension was diluted and plated in triplicate on YMA, and the number of CFU was determined after 72 h at 25°C. Viability was always 60 to 80%.]i$2, 百拇医药
To evaluate in vivo the growth of the fungus, weighed organ samples were mechanically homogenized and centrifuged at 800 x g. The pellet was suspended in 0.1% Tween 20 in phosphate-buffered saline (Sigma, Milan, Italy). The suspension was plated on YMA as described above, and the results were expressed as CFU per gram of tissue. The data are the cumulative results for all available specimens from five mice per group in four experiments.]i$2, 百拇医药
For histological analysis, tissues samples were excised and immediately fixed in 10% formalin in 0.1 M phosphate buffer at pH 7.4. Serial 3-µm sections of paraffin-embedded tissues were stained with hematoxylin-eosin (HE) stain and periodic acid-Schiff (PAS) stain.
In vitro macrophage stimulation. Murine macrophage cell line J774 of BALB/c origin was maintained in minimal essential medium (Gibco BRL, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum (Euroclone, Ltd., Cellbio Sre, Milan, Italy), 1% glutamine, 2% HEPES buffer, 1% penicillin-streptomycin solution (Biological Ind., Kibbutz, Israel), and 1% nonessential amino acids (Gibco BRL) (complete medium [CM]) in 5% CO2 at 37°C in petri dishes. J774 macrophages in CM were seeded in 24-well plates (no. 3524; Costar, Milan, Italy) at 105 cells/well and incubated at 37°C in 5% CO2 for 24 h; confluent monolayers were treated with P. marneffei conidia (2 x 105/well) as described previously (30). Phagocytosis of conidia was allowed for 2 h in the absence of opsonins. J774 monolayers were then washed with warm phosphate-buffered saline to remove nonphagocytized conidia and incubated for different times in CM. Control wells were treated with CM only or activated with 10 U of recombinant murine IFN-{gamma} (Genzyme)/ml plus 1 µg of lipopolysaccharide (LPS) (Sigma)/ml. At the end of the incubation, supernatants were collected and assayed for the presence of nitrite or cytokines. For the evaluation of cytokine mRNA expression, cells were lysed with 1 ml of Trizol reagent (Invitrogen, Milan, Italy) 24 h after stimulation with recombinant murine IFN- plus LPS or 4, 24, and 48 h after treatment with P. marneffei conidia.(Francesca Sisto Annarita Miluzio Orazio Leopardi Maurizio Mirra Johan R. Boelaert and Donatella Tara)
Received 6 June 2002/ Returned for modification 12 July 2002/ Accepted 25 September 2002s;^u-, 百拇医药
ABSTRACTs;^u-, 百拇医药
Penicillium marneffei is an intracellular opportunistic fungus causing invasive mycosis in AIDS patients. T cells and macrophages are important for protection in vivo. However, the role of T-cell cytokines in the immune response against P. marneffei is still unknown. We studied by semiquantitative reverse transcription-PCR and biological assays the patterns of expression of Th1 and Th2 cytokines in the organs of wild-type (wt) and gamma interferon (IFN-) knockout (GKO) mice infected intravenously with P. marneffei conidia. At 3 x 105 conidia/mouse, a self-limiting infection developed in wt BALB/c mice, whereas all GKO mice died at day 18 postinoculation. Splenic and hepatic granulomas were present in wt mice, whereas disorganized masses of macrophages and yeast cells were detected in GKO mice. The infection resolved faster in the spleens than in the livers of wt mice and was associated with the local expression of type 1 cytokines (high levels of interleukin-12 [IL-12] and IFN-) but not type 2 cytokines (low levels of IL-4 and IL-10). Conversely, both type 1 and type 2 cytokines were detected in the livers of wt animals. Disregulation of the cytokine profile was seen in the spleens but not in the livers of GKO mice. The inducible nitric oxide synthase mRNA level was low and the TNF- level was high in both spleens and livers of GKO mice compared to wt mice. These data suggest that the polarization of a protective type 1 immune response against P. marneffei is regulated at the level of individual organs and that the absence of IFN- is crucial for the activation of fungicidal macrophages and the development of granulomas.
INTRODUCTIONn7$4j, 百拇医药
Penicillium marneffei is a dimorphic pathogen that causes disseminated mycoses in the lungs, spleens, and livers of infected subjects, especially human immunodeficiency virus (HIV)-positive patients in Southeast Asia (8, 13). In vivo (21, 29) and in vitro (6, 9), P. marneffei undergoes phase transition and multiplies as yeast cells inside macrophages. The histopathological characteristic of P. marneffei infection in humans is granuloma formation, with suppurative and anergic reactions and necrosis (9, 13, 29). In mice, disseminated granulomas are also present, with epithelioid cells containing growing yeast cells. Hepatosplenomegaly is common in both mice and humans (8).n7$4j, 百拇医药
The immune response against P. marneffei infection is mediated mainly by T cells and macrophages: in nude mice or T-cell-depleted animals, the mycosis is fatal, whereas in healthy hosts, the fungus is cleared in 2 to 3 weeks, depending on the size of the inoculum (22, 32). In vitro, both human and mouse macrophages are able to control P. marneffei growth and to kill intracellular yeast cells when activated by T-cell-derived cytokines, such as gamma interferon (IFN-) (6, 23, 30). It has been shown that fungus death is due mostly to the release of nitric oxide (NO) by activated macrophages (6, 23, 30).
Little is known about the role of inflammatory cytokines in vivo and whether resistance to P. marneffei infection is mediated by a Th1 or a Th2 immune response or both. Granuloma formation can be associated with either a predominantly Th1 response, with high levels of tumor necrosis factor alpha (TNF-), IFN-, and interleukin-12 (IL-12), as in mycobacterial infections (7, 15), or a Th2 response, with high levels of IL-10 and IL-4, as in the reaction to Schistosoma eggs (34). As reported for other fungi, such as Candida spp., Aspergillus fumigatus, or Histoplasma capsulatum (4, 20, 28, 35), the cytokine microenvironment dictates whether a protective or a deleterious immune response is generated. IFN- is the hallmark cytokine for the Th1 immune response, and its production is essential in the control of intracellular pathogens, such as Mycobacterium tuberculosis (7). In mice with a disrupted IFN- gene (IFN- knockout [GKO] mice) or in patients with defects in IFN- signaling, susceptibility to mycobacterial infections has been reported (14, 18, 19, 26).
Dissecting the mechanism(s) of P. marneffei infection in immunocompetent hosts may help to understand the pathogenicity of penicilliosis marneffei in immunocompromised patients. This goal is particularly relevant for AIDS patients living in Southeast Asia, where this infection ranks as the third most important opportunistic infection (8) and where the vast majority of HIV-infected patients cannot yet afford treatment with highly active antiretroviral therapy.d#6e8m, 百拇医药
The purpose of this study was to characterize the cytokine microenvironment leading to the formation of granulomas and to the activation of a protective immune response in healthy or GKO BALB/c mice sublethally infected with P. marneffei conidia. We also investigated the histopathological changes occurring in the organs of wild-type and GKO mice. Although the natural infection likely is acquired via the respiratory tract, in this set of experiments and in agreement with previous reports (9, 22), we preferred the intravenous (i.v.) route of infection to better control the size of the inoculum and, consequently, the extent of colonization of the different organs.
MATERIALS AND METHODS9, http://www.100md.com
Mice. Pathogen-free female BALB/c mice weighing 20 to 24 g were obtained from Charles River Italia (Calco, Como, Italy) and used at the age of 6 to 8 weeks. GKO mice (11) in a BALB/c background (BALB/c-Ifngtm1Ts) were purchased from the Jackson Laboratory (Bar Harbor, Maine). Homozygous mice were identified by PCR of tail-derived DNA with primers flanking the neo gene (sense, 5'-CAAGTGGCATAGATGTGGAAG-3'; antisense, 5'-GGCAATACTCATGAATGCATCC-3'); the wild-type gene gave an amplified fragment of 340 bp, whereas the disrupted gene gave a 2,400-bp fragment. Homozygous mice were bred and maintained at the Istituto Nazionale Tumori, Milan, Italy, and were kindly provided to us by M. P. Colombo, Istituto Nazionale Tumori. All mice were housed, fed, and handled under standard conditions according to institutional guidelines.9, http://www.100md.com
Microorganism, infections, and in vivo analysis. The strain of P. marneffei used in this study, IUM885346, was kindly provided by M. A. Viviani, Milan, Italy. It was isolated from the blood of an Italian HIV-positive i.v. drug user infected in Thailand (33). The fungus was cultured on yeast morphology agar (YMA) (Biolife, Milan, Italy) at 25°C for 10 to 14 days. P. marneffei conidia were collected by flooding the culture surface with saline, centrifuged at 1,800 x g for 20 min, and filtered to remove mixed mycelial debris. The conidia were washed twice with saline, counted with a hemocytometer, and then suspended at the desired concentration.
Mice were injected i.v. via the tail vein with a sublethal suspension of 3 x 105 conidia/mouse and sacrificed after specified time intervals. Control mice of the same sex and age were injected with sterile saline. To check the inoculum viability, the suspension was diluted and plated in triplicate on YMA, and the number of CFU was determined after 72 h at 25°C. Viability was always 60 to 80%.]i$2, 百拇医药
To evaluate in vivo the growth of the fungus, weighed organ samples were mechanically homogenized and centrifuged at 800 x g. The pellet was suspended in 0.1% Tween 20 in phosphate-buffered saline (Sigma, Milan, Italy). The suspension was plated on YMA as described above, and the results were expressed as CFU per gram of tissue. The data are the cumulative results for all available specimens from five mice per group in four experiments.]i$2, 百拇医药
For histological analysis, tissues samples were excised and immediately fixed in 10% formalin in 0.1 M phosphate buffer at pH 7.4. Serial 3-µm sections of paraffin-embedded tissues were stained with hematoxylin-eosin (HE) stain and periodic acid-Schiff (PAS) stain.
In vitro macrophage stimulation. Murine macrophage cell line J774 of BALB/c origin was maintained in minimal essential medium (Gibco BRL, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum (Euroclone, Ltd., Cellbio Sre, Milan, Italy), 1% glutamine, 2% HEPES buffer, 1% penicillin-streptomycin solution (Biological Ind., Kibbutz, Israel), and 1% nonessential amino acids (Gibco BRL) (complete medium [CM]) in 5% CO2 at 37°C in petri dishes. J774 macrophages in CM were seeded in 24-well plates (no. 3524; Costar, Milan, Italy) at 105 cells/well and incubated at 37°C in 5% CO2 for 24 h; confluent monolayers were treated with P. marneffei conidia (2 x 105/well) as described previously (30). Phagocytosis of conidia was allowed for 2 h in the absence of opsonins. J774 monolayers were then washed with warm phosphate-buffered saline to remove nonphagocytized conidia and incubated for different times in CM. Control wells were treated with CM only or activated with 10 U of recombinant murine IFN-{gamma} (Genzyme)/ml plus 1 µg of lipopolysaccharide (LPS) (Sigma)/ml. At the end of the incubation, supernatants were collected and assayed for the presence of nitrite or cytokines. For the evaluation of cytokine mRNA expression, cells were lysed with 1 ml of Trizol reagent (Invitrogen, Milan, Italy) 24 h after stimulation with recombinant murine IFN- plus LPS or 4, 24, and 48 h after treatment with P. marneffei conidia.(Francesca Sisto Annarita Miluzio Orazio Leopardi Maurizio Mirra Johan R. Boelaert and Donatella Tara)