Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ T cell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Proteomic Profiling and Functional Analysis of B Cell-Derived Exosomes upon Pneumocystis Infection.

Pneumocystis is a life-threatening fungal pathogen that frequently causes fatal pneumonia (PCP) in immunocompromised individuals. Recently, B cells have been reported to play a crucial role in the pathogenesis of PCP through producing antibodies and activating CD4+ T cell response. Exosomes are nanoscale small extracellular vesicles abundant with protein cargo and can mediate immune response during infectious disease. In this study, using tandem mass tag-based quantitative proteomics coupled with bioinformatic analysis, we attempted to characterize exosomes derived from B lymphocytes in response to PCP. Several proteins were verified by parallel reaction monitoring (PRM) analysis. Also, the effects of B cell exosomes on CD4+ T cell response and phagocytic function of macrophages were clarified. Briefly, 1701 proteins were identified from B cell exosomes, and the majority of them were reported in Vesiclepedia. A total of 51 differentially expressed proteins of B cell exosomes were found in response to PCP. They were mainly associated with immune response and transcription regulation. PRM analysis confirmed the significantly changed levels of histone H1.3, vimentin, and tyrosine-protein phosphatase nonreceptor type 6 (PTPN6). Moreover, a functional study revealed the proinflammatory profile of B cell exosomes on CD4+ T cell response in PCP. Taken together, our results suggest the involvement of exosomes derived from B cells in cell-to-cell communication, providing new information on the function of B cells in response to PCP.

Myeloid C-type lectin receptors that recognize fungal mannans interact with Pneumocystis organisms and major surface glycoprotein.

Myeloid C-type lectin receptors (CLRs) are innate immune recognition molecules that bind to microorganisms via their carbohydrate recognition domains. In this study, we utilized a library of CLRs that recognize fungal mannans. We used this library to screen against Pneumocystis carinii (Pc) homogenates or purified Pc major surface glycoprotein (Msg) present on Pneumocystis. The results demonstrated that all of the mammalian CLR hFc-fusions tested displayed significant interaction/binding with Pc organisms, and furthermore to isolated Msg. Highest Pc organism and Msg binding activities were with CLR members Mincle, Dectin-2, DC-SIGN and MCL. An immunofluorescence assay with the respective CLR hFc-fusions against whole Pc life forms corroborated these findings. Although some of these CLRs have been implicated previously as important for Pneumocystis pathogenesis (Dectin-1/Dectin-2/Mincle), this is the first analysis of head-to-head comparison of known fungal mannan binding CLR-hFc fusions with Pc. Lastly, heat treatment resulted in reducted CLR binding.

IL-17 Inversely Correlated with IL-10 via the STAT3 Gene in Pneumocystis-Infected Mice.

BACKGROUND: Pneumocystis pneumonia (PCP) remains a common opportunistic infection in immunosuppressed individuals. Current studies showed that multiple immune cells and cytokines took part in the host defense against Pneumocystis (PC). However, the roles of IL-17 and IL-10 in the development of PCP have not been elucidated. METHODS: IL-10 and IL-17 levels in serum from PCP mice were detected via ELISA. The percentages of B10 cells, IL-10+ macrophages, and IL-10+ T cells in the lung from IL-17-/- PCP mice and Th17 cells and IL-17+ γδT cells in IL-10-/- PCP mice were examined via flow cytometry. Also, antibody neutralization examination was also performed to elucidate the relationship of IL-17 and IL-10 in the PCP model. RESULTS: We noted the increase of IL-17 and IL-10 levels in serum from mice infected with Pneumocystis. Furthermore, deficiency of IL-17 or IL-10 could lead to the delayed clearance of Pneumocystis and more severed lung damage. Our data also demonstrated that IL-17 deficiency enhanced the serum IL-10 level and the percentages of B10 cells, IL-10+ macrophages, and IL-10+ T cells in the lung from PCP mice. Interestingly, we also noted an increase of the IL-17 level in serum and Th17 cell and IL-17+ γδT cell percentages in the lung from IL-10-/- PCP mice. Using antibody neutralization experiments, we found that the STAT3 gene might play a critical role in the interplay of IL-17 and IL-10 in PCP. CONCLUSION: Taken together, our results demonstrated that IL-17 and IL-10 could play the protective roles in the progression of PCP and the inverse correlation of them might be mediated by STAT3.

Surfactant protein-D modulation of pulmonary macrophage phenotype is controlled by S-nitrosylation.

Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Increase in secreted airway mucins and partial Muc5b STAT6/FoxA2 regulation during Pneumocystis primary infection.

Airway mucus responses to subclinical infections may explain variations in progression of chronic lung diseases and differences in clinical expression of respiratory infections across individuals. Pneumocystis associates to more severe Chronic Obstructive Pulmonary Disease (COPD), asthma, respiratory distress of premature newborns, and is a consistent subclinical infection between 2 and 5 months of age when hospitalizations for respiratory cause and infant mortality are higher. This atypical fungus associates to increased mucin 5AC (MUC5AC), a central effector of Th2-type allergic inflammation, in infant lungs. However, mucus progression, expression of MUC5B essential for airway defense, and potential for pharmacologic modulation of mucus during Pneumocystis infection remain unknown. We measured MUC5B and Pneumocystis in infant lungs, and progression of mucin levels and effect of inhibition of the STAT6/FoxA2 mucus pathway using Kaempferol, a JAK/STAT6 inhibitor, in immunocompetent rats during Pneumocystis primary infection. Pneumocystis associated to increased MUC5B in infant lungs. Muc5b increased earlier and more abundantly than Muc5ac during experimental primary infection suggesting an acute defensive response against Pneumocystis as described against bacteria, while increased Muc5ac levels supports an ongoing allergic, Th2 lymphocyte-type response during primary Pneumocystis infection. Kaempferol partly reversed Muc5b stimulation suggesting limited potential for pharmacological modulation via the STAT6-FoxA2 pathway.

Pneumocystis infection alters the activation state of pulmonary macrophages.

Recent studies show a substantial incidence of Pneumocystis jirovecii colonization and infection in patients with chronic inflammatory lung conditions. However, little is known about the impact of Pneumocystis upon the regulation of pulmonary immunity. We demonstrate here that Pneumocystis polarizes macrophages towards an alternatively activated macrophage-like phenotype. Genetically engineered mice that lack the ability to signal through IL-4 and IL-13 were used to show that Pneumocystis alternative macrophage activation is dependent upon signaling through these cytokines. To determine whether Pneumocystis-induced macrophage polarization would impact subsequent immune responses, we infected mice with Pneumocystis and then challenged them with Pseudomonas aeruginosa 14 days later. In co-infected animals, a higher proportion of macrophages in the alveolar and interstitial spaces expressed both classical and alternatively activated markers and produced the regulatory cytokines TGFß and IL-10, as well as higher arginase levels than in mice infected with P. aeruginosa alone. Our results suggest that Pneumocystis reprograms the overall macrophage repertoire in the lung to that of a more alternatively-activated setpoint, thereby altering subsequent immune responses. These data may help to explain the association between Pneumocystis infection and decline in pulmonary function.

MyD88 signaling regulates both host defense and immunopathogenesis during pneumocystis infection.

The immune response protects against Pneumocystis infection but is also a key component of Pneumocystis pneumonia (PcP)-related immunopathogenesis. Signaling through myeloid differentiation factor 88 (MyD88) is critical for activation of immune pathways downstream of TLRs and IL-1R. To determine whether MyD88 regulates normal host defense against Pneumocystis, nonimmunosuppressed wild-type (WT) and MyD88-deficient mice were infected. MyD88(-/-) mice had higher early Pneumocystis burdens than did WT mice but mounted an effective adaptive immune response and cleared Pneumocystis similarly to WT. However, MyD88(-/-) mice displayed a more intense and prolonged pulmonary immune response than did WT mice. To determine the role of MyD88 in the development of PcP-related immunopathogenesis, WT and MyD88(-/-) mice were rendered susceptible to PcP by depletion of CD4(+) T cells. At 4 wk postinfection, CD4-depleted WT and MyD88(-/-) mice harbored similar organism burdens, but MyD88(-/-) mice were protected from the PcP-related respiratory impairment observed in WT mice. Improved pulmonary physiology in MyD88(-/-) mice correlated with lower lung CCL2 levels and reduced cell recruitment. However, by 5 wk postinfection, the overall health of MyD88(-/-) mice began to deteriorate rapidly relative to WT, with accelerated weight loss, impaired lung function, and exacerbated alveolar inflammation. This physiological decline of MyD88(-/-) mice was associated with increased TNF-α and IFN-γ in the lung, and by the inability to control Pneumocystis burden. Thus, MyD88 is not required for resistance to Pneumocystis infection, but limits the adaptive immune response in immunocompetent mice. In the setting of active PcP, MyD88 signaling contributes to both immunopathogenesis and control of fungal burden.

Pneumocystis elicits a STAT6-dependent, strain-specific innate immune response and airway hyperresponsiveness.

It is widely held that exposure to pathogens such as fungi can be an agent of comorbidity, such as exacerbation of asthma or chronic obstructive pulmonary disease. Although many studies have examined allergic responses to fungi and their effects on pulmonary function, the possible pathologic implications of the early innate responses to fungal pathogens have not been explored. We examined early responses to the atypical fungus Pneumocystis in two common strains of mice in terms of overall immunological response and related pathology, such as cell damage and airway hyperresponsiveness (AHR). We found a strong strain-specific response in BALB/c mice that included recruitment of neutrophils, NK, NKT, and CD4 T cells. This response was accompanied by elevated indicators of lung damage (bronchoalveolar lavage fluid albumin and LDH) and profound AHR. This early response was absent in C57BL/6 mice, although both strains exhibited a later response associated with the clearance of Pneumocystis. We found that this AHR could not be attributed exclusively to the presence of recruited neutrophils, NKT, NK, or CD4 cells or to the actions of IFN-γ or IL-4. However, in the absence of STAT6 signaling, AHR and inflammatory cell recruitment were virtually absent. Gene expression analysis indicated that this early response included activation of several transcription factors that could be involved in pulmonary remodeling. These results show that exposure to a fungus such as Pneumocystis can elicit pulmonary responses that may contribute to morbidity, even without prior sensitization, in the context of certain genetic backgrounds.

Microarray studies on effects of Pneumocystis carinii infection on global gene expression in alveolar macrophages.

BACKGROUND: Pneumocystis pneumonia is a common opportunistic disease in AIDS patients. The alveolar macrophage is an important effector cell in the clearance of Pneumocystis organisms by phagocytosis. However, both the number and phagocytic activity of alveolar macrophages are decreased in Pneumocystis infected hosts. To understand how Pneumocystis inactivates alveolar macrophages, Affymetrix GeneChip RG-U34A DNA microarrays were used to study the difference in global gene expression in alveolar macrophages from uninfected and Pneumocystis carinii-infected Sprague-Dawley rats. RESULTS: Analyses of genes that were affected by Pneumocystis infection showed that many functions in the cells were affected. Antigen presentation, cell-mediated immune response, humoral immune response, and inflammatory response were most severely affected, followed by cellular movement, immune cell trafficking, immunological disease, cell-to-cell signaling and interaction, cell death, organ injury and abnormality, cell signaling, infectious disease, small molecular biochemistry, antimicrobial response, and free radical scavenging. Since rats must be immunosuppressed in order to develop Pneumocystis infection, alveolar macrophages from four rats of the same sex and age that were treated with dexamethasone for the entire eight weeks of the study period were also examined. With a filter of false-discovery rate less than 0.1 and fold change greater than 1.5, 200 genes were found to be up-regulated, and 144 genes were down-regulated by dexamethasone treatment. During Pneumocystis pneumonia, 115 genes were found to be up- and 137 were down-regulated with the same filtering criteria. The top ten genes up-regulated by Pneumocystis infection were Cxcl10, Spp1, S100A9, Rsad2, S100A8, Nos2, RT1-Bb, Lcn2, RT1-Db1, and Srgn with fold changes ranging between 12.33 and 5.34; and the top ten down-regulated ones were Lgals1, Psat1, Tbc1d23, Gsta1, Car5b, Xrcc5, Pdlim1, Alcam, Cidea, and Pkib with fold changes ranging between -4.24 and -2.25. CONCLUSIONS: In order to survive in the host, Pneumocystis organisms change the expression profile of alveolar macrophages. Results of this study revealed that Pneumocystis infection affects many cellular functions leading to reduced number and activity of alveolar macrophages during Pneumocystis pneumonia.

[Enzyme change in bronchoalveolar lavage fluid of pneumocystis pneumonia rats and the effect of garlicin treatment].

OBJECTIVE: To study the change of enzymes and effect of garlicin treatment on the change in bronchoalveolar lavage fluid (BALF) of rats with Pneumocystis carinii pneumonia (PCP). METHODS: Wistar rats were injected intramuscularly continually with dexamethasone to establish the rat model of PCP. The experimental rats (group A) were injected intramuscularly with garlicin at a dose of 10 mg/(kg x d) for 5 days in the 3rd, 6th and 9th week respectively, and SMZ/TMP therapy group (B), PCP infected group (C) and normal group (D) were established as controls. Three days after the last treatment, the rats of all groups were killed and BALF was collected without contamination and enzymes AST, ALF, CHE, ALP, LDH, CK, CKMB, HBDH, AFU, 5'NT, ADA were examined. RESULTS: The ALP level in group C [(573.41 +/- 350.63)U/L] was significantly higher than that in group D [(210.56 +/- 114.41) U/L] (q = 4.682, P < 0.01), group A [(392.07 +/- 217.57) U/L] (q = 3.851, P < 0.05), and group B [(325.21 +/- 180.65) U/L] (q = 4.380, P < 0.01); the level of CK, CKMB and 5'NT in group C [948.94 +/- 403.43, 489.47 +/- 254.46 and (6.76 +/- 3.11) U/L respectively] was higher than those in group D [426.22 +/- 319.00, 213.33 +/- 144.54 and (3.22 +/- 1.20) U/L] (q = 4.696, 3.784, 3.812, P< 0.05); there was no significant difference in the level of AST, ALT, CHE, LDH, HBDH, AFU and ADA among the four groups (F = 1.852, 0.958, 2.470, 1.423, 1.178, 1.342, 0.611, P > 0.05). CONCLUSIONS: The level of ALP, CK, CKMB but the ALP level decreases distinctly after the garlicin and 5'NT increases evidently in BALF of PCP infected rats, but the ALP level decreases distinctly after the garlicin treatment.

Pneumocystis stimulates MCP-1 production by alveolar epithelial cells through a JNK-dependent mechanism.

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia (PCP) in immunocompromised individuals. Recent studies have demonstrated that the host's immune response is clearly responsible for the majority of the pathophysiological changes associated with PCP. P. carinii interacts closely with alveolar epithelial cells (AECs); however, the nature and pathological consequences of the epithelial response remain poorly defined. Monocyte chemotactic protein-1 (MCP-1) is involved in lung inflammation, immunity, and epithelial repair and is upregulated during PCP. To determine whether AECs are an important source of MCP-1 in the P. carinii-infected lung, in vivo and in vitro studies were performed. In situ hybridization showed that MCP-1 mRNA was localized to cells with morphological characteristics of AECs in the lungs of infected mice. In vitro studies demonstrated that P. carinii stimulated a time- and dose-dependent MCP-1 response in primary murine type II cells that was preceded by JNK activation. Pharmacological inhibition of JNK nearly abolished P. carinii-stimulated MCP-1 production, while ERK, p38 MAPK, and TNF receptor signaling were not required. Furthermore, delivery of a JNK inhibitory peptide specifically to pulmonary epithelial cells using a recombinant adenovirus vector blocked the early lung MCP-1 response following intratracheal instillation of infectious P. carinii. JNK inhibition did not affect P. carinii-stimulated production of macrophage inflammatory protein-2 in vitro or in vivo, indicating that multiple signaling pathways are activated in P. carinii-stimulated AECs. These data demonstrate that AECs respond to P. carinii in a proinflammatory manner that may contribute to the generation of immune-mediated lung injury.

[Effects of Fructus Psoralea and Brucea javanica on the level of IL-2 and NK cell in rats infected with Pneumocystis carinii].

SD rat model of PCP was established by subcutaneous injection with dexamethasone. The treatment groups received Fructus Psoralea (FP, 10.0 mg/kg), Brucea javanica (BJ, 1.2 mg/kg) and a mixture of the two Chinese herbs(FP 5mg/kg, BJ 0.6 mg/kg) respectively. By means of detecting the level of IL-2 in sera and NK cells in spleens, the effect of FP and BJ on the level of IL-2 and NK cells in rats with Pneumocystis carinii pneumonia (PCP) was observed, with SMZco treatment group (TMP 50 mg/kg, SMZ 250 mg/kg) and groups of infected and normal rats as controls. Compared with the infected group, the level of IL-2(526.1 +/- 5.5) pg/ml and NK cells (27.1% +/- 0.8%) significantly increased in the FP group (P < 0.01), followed by the FP/BJ combination group [(314.7 +/- 6.7) pg/ml, 22.9% +/- 0.9%) (P < 0.05)], and BJ group [(285.4 +/- 6.1) pg/ml, 20.7% +/- l.0%) (P < 0.05)]. Chinese herbs Fructus Psoralea and Brucea javanica show an immune regulatory action on the PCP rats.

Surfactant protein D enhances Pneumocystis infection in immune-suppressed mice.

To further determine the role of surfactant protein (SP)-D in the pathogenesis of Pneumocystis pneumonia, a mouse model of transgenic overexpression (OE) of SP-D was studied. These animals produce roughly 30- to 50-fold greater SP-D than their wild-type (WT) counterparts but show no other differences in lung morphology and function. Animals in both the SP-D OE and WT groups were depleted of CD4 lymphocytes with weekly injections of GK1.5 antibody, before Pneumocystis inoculation, and throughout the subsequent infection period. At various time points, mice were killed and analyzed for inflammatory parameters and organism burden. Proinflammatory cytokines in bronchoalveolar lavage fluid were elevated throughout the period of infection, with OE animals exhibiting significantly higher levels of TNF-alpha and macrophage inflammatory protein-2 compared with WT controls. The total number of cells in the lavage fluid was also increased significantly only in the OE group, whereas the cell differential composition demonstrated lymphocyte and eosinophil infiltration in both groups of animals. Significantly, the organism burden was markedly higher in the SP-D OE animals, whereas the WT mice demonstrated little alteration in organism number over the course of infection. These results further indicate that SP-D facilitates the development of Pneumocystis infection and related lung inflammation in an immunosuppressed mouse model.

Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species.

Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A(-/-)) with or without selective CD4(+)-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A(-/-) mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A(-/-) versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A(-/-) mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A(-/-) mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A(-/-) group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection.

BACKGROUND: Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level. RESULTS: The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B) to the same cells by two-color fluorescent in situ hybridization. CONCLUSIONS: The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

The impact of CMV on the respiratory burst of macrophages in response to Pneumocystis carinii.

Infection of human monocyte-derived macrophages with CMV decreased the respiratory burst when cells were stimulated with opsonized zymosan or Pneumocystis carinii (P. carinii). Such an effect, though smaller, was also seen with heat-inactivated CMV, but only when triggered by zymosan. The effect was most pronounced in cells obtained from CMV antibody-negative donors. Dexamethasone further reduced the respiratory burst, both in uninfected and CMV-infected cells. Interferon-gamma increased the response in uninfected cells and, to a lesser extend, in cells treated with heat-inactivated CMV, whereas no effect was seen with infective CMV. No overt productive infection or cytopathology could be detected, however, the monocytes incubated with infective but also heat-inactivated CMV formed clusters, a phenomenon that was equally pronounced in cultures from CMV antibody positive and negative-donors. These results might help explain the worse prognosis of P. carinii pneumonia in patients coinfected with CMV and receiving dexamethasone.

Pneumocystis carinii pneumonia in HIV-infected patients: effect of steroid therapy on surfactant level.

Previous studies have suggested alterations in pulmonary surfactant lipid in the setting of Pneumocystis carinii pneumonia in HIV-infected patients. Because pulmonary surfactant lipid is composed of a variety of lipid products and because other phospholipids might be present in bronchoalveolar lavage (BAL) lipid determinations, a single molecular species of phospholipid which comprises a substantial portion of the surfactant lipid fraction, dipalmitoyl phosphatidylcholine (DPPC), was measured by capillary column gas chromatography in BAL samples taken at the time of the diagnosis of P. carinii pneumonia, and 10 days after treatment for P. carinii pneumonia. DPPC was measured at day 0 and day 10 in seven patients who had been randomized to receive methylprednisolone adjuvant therapy for P. carinii pneumonia and in six patients who had been randomized to not receive methylprednisolone therapy. The level of DPPC in BAL from all patients at day 0 was 0.49 +/- 0.06 microgram ml-1 BAL. This level is significantly lower that the level of DPPC determined in BAL from five normal volunteers 2.48 +/- 0.40 micrograms ml-1. At day 0, the BAL level of DPPC in patients treated with methylprednisolone was not different from the BAL level of DPPC in patients not treated with methylprednisolone. By day 10 of therapy for P. carinii pneumonia, BAL levels of DPPC in all patients had increased to 1.05 +/- 0.19 micrograms ml-1 BAL. At day 10 DPPC levels in the methylprednisolone treated group were not different from the group not treated with methylprednisolone. We conclude that in HIV-infected patients, lung surfactant lipid is reduced in the setting of P. carinii pneumonia. The lipid levels return toward normal levels with treatment. Adjuvant therapy with corticosteroids does not alter the rate of recovery of surfactant lipid levels at least after 10 days of therapy.

Pneumocystis carinii glycoprotein A inhibits surfactant phospholipid secretion by rat alveolar type II cells.

Pneumocystis carinii pneumonia (PCP) remains a major cause of morbidity in AIDS. The pathogenesis of PCP is poorly understood, but evidence of surfactant abnormalities is mounting. The role of the major surface glycoprotein of P. carinii, gpA, in producing surfactant abnormalities was investigated. Rat type II pneumocytes were incubated with [3H]choline, purified gpA, and modulators. Lipid was extracted, and [3H]dipalmitoyl phosphatidylcholine (DPPC) secretion was calculated. Contaminating endotoxin had no effect on DPPC secretion. gpA inhibited basal and ATP-stimulated DPPC secretion in a dose- and time-dependent manner. An anti-gpA monoclonal antibody attenuated inhibition of DPPC secretion. Unglycosylated recombinant gpA inhibited secretion, suggesting that functional activity resides in the protein moiety of gpA. These results suggest that gpA is a specific trigger for abnormalities of surfactant lipids in PCP. This is a unique role for a microbial product in disease pathogenesis and a potentially exploitable therapeutic target.