The trophic life cycle stage of Pneumocystis species induces protective adaptive responses without inflammation-mediated progression to pneumonia

The trophic life cycle stage of Pneumocystis species induces protective adaptive responses... Abstract Pneumocystis species are fungal pathogens that cause pneumonia in immunocompromised hosts. Lung damage during Pneumocystis pneumonia is predominately due to the inflammatory immune response. Pneumocystis species have a biphasic life cycle. Optimal innate immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, the trophic life cycle stage broadly suppresses proinflammatory responses to multiple pathogen-associated molecular patterns (PAMPs), including β-1,3-glucan. Little is known about the contribution of these life cycle stages to the development of protective adaptive responses to Pneumocystis infection. Here we report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B-cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised recombination-activating gene 2 knockout (RAG2−/−) mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia. Pneumocystis, echinocandin, pneumonia, lung, AIDS Introduction Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Patients at risk for developing Pneumocystis pneumonia (PcP) include those whose immune systems have become compromised due to acquired immunodeficiency syndrome (AIDS) or immunosuppressive treatments such as chemotherapy, steroid treatment, and anti-tumor necrosis factor alpha (TNFα) biologic therapy.1–6 The clearance of Pneumocystis organisms from the host lung is dependent on a broad range of effector responses, including CD4+ T-cell, B-cell, and macrophage activity.7–10 In the absence of an efficient adaptive immune response, immunocompromised patients may progress to PcP, which is characterized by inflammation-mediated alveolar damage.11 Basic research into the interactions between this fungal pathogen and the host immune response has the potential to inform novel approaches to reduce morbidity and mortality due to Pneumocystis pneumonia. Pneumocystis species have a biphasic life cycle consisting of trophic forms and cysts. Trophic forms are single-nucleated organisms typically found in clusters surrounded by a biofilm-like substance consisting of a conglomeration of DNA, β-glucan, and other sugars.12 Cysts are ascus-like structures that consist of multiple nuclei surrounded by a fungal cell wall consisting of β-1,3 glucan and β-1,6 glucan.13–15 Trophic forms do not express β-glucan and do not form a cell wall.14 We have previously reported that the life cycle stages of Pneumocystis murina have opposing effects on the immune response. The immune response to primary infection with P. murina trophic forms alone was less robust than the response to infection with a physiologically normal mixture of cysts and trophic forms.16 Infection with trophic forms alone resulted in reduced numbers of CD11c+ innate immune cells in the lungs, as well as reduced recruitment of B cells and T cells, compared to infection with a normal mixture of trophic forms and cysts.16In vitro, trophic forms suppressed production of the proinflammatory cytokines interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and TNFα by bone marrow-derived dendritic cells (BMDCs) stimulated with β-glucan, zymosan, depleted zymosan, lipoteichoic acid (LTA), or lipopolysaccharides (LPS).16 In addition, trophic form-stimulated BMDCs failed to stimulate production of the T helper 1 (Th1)-type cytokine interferon gamma (IFN-γ) by CD4+ T cells.16 Despite these delays, the mice inoculated with trophic forms were able to clear the infection within a similar period of time as the mice inoculated with mixed organisms.16 However, the rapid turnover of trophic forms into cysts in these previous experiments precludes the formation of conclusions regarding which life cycle stage(s) initiate the immune responses that lead to clearance.16 Here we demonstrate that the trophic stage of P. murina is sufficient to provoke CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to PcP. The adoptive transfer of CD4+ T cells generated in the presence of trophic forms was sufficient to mediate the clearance of both trophic forms and cysts from the lungs of RAG2−/− mice. In addition, primary infection with trophic forms was sufficient for the development of antibody-mediated secondary responses leading to clearance of both trophic forms and cysts following reinfection. Recent studies demonstrate that protective immunization against Pneumocystis infection is a feasible goal, even in the context of simian immunodeficiency virus (SIV)-induced immunosuppression or CD4+ T-cell depletion.17,18 Our data suggest that trophic form-mediated immune suppression of innate responses does not impede the development of protective secondary adaptive responses. Conversely, our data suggest that the depletion of the cyst life cycle stage may be sufficient for protection against the inflammation-mediated pathology associated with PcP. Methods Mice Mice were maintained at the University of Kentucky Department of Laboratory Animal Resources (DLAR) under specific-pathogen-free conditions. BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our animal facilities. C.129S6(B6)-Rag2tm1Fwa N12 (Rag2−/−) mice, originally from Taconic (Germantown, NY) were bred at DLAR in sterile microisolator cages with sterilized food and water. The University of Kentucky Institutional Animal Care and Use Committee approved all protocols regarding animal use. All protocols conformed to the Guide for the Care and Use of Laboratory Animals (2011 edition) of the Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. P. murina isolation and infection Lungs were excised from P. murina-infected Rag2−/− mice and pushed through stainless steel mesh in Hank's balanced salt solution (HBSS) containing 0.5% glutathione at pH 7.3. Cell debris was broken up by aspirating through 20 and 26-gauge needles, then removed by centrifugation at 100 × g for 3 min. Trophic forms were isolated by removing the supernatant following centrifugation at 400 × g for 7 min. This preparation results in greater than 100 trophic forms per cyst.16 The pellet from the 400 × g spin contained a mixed population of cysts and trophic forms, at a typical ratio of 10 : 1 trophic forms to cysts. Erythrocytes in the pellet were lysed with water and organisms suspended in an equal volume of 2× phosphate-buffered saline (PBS). Organisms were incubated with 200 U DNase (Sigma-Aldrich, St. Louis MO) at 37°C for 30 min. Clumps were broken up by aspirating through a 26-gauge needle. The remaining cell debris was removed by centrifugation at 100 × g for 3 min, followed by passage over a 70 μm filter. Pneumocystis life forms were pelleted by centrifugation at 1300 × g for 15 min, then resuspended in 100 μl HBSS containing 10 units/ml penicillin, 10 μg/ml streptomycin, and 1 μg/ml gentamicin. Aliquots of mixed P. murina organisms or enriched trophic forms were diluted, and 100 μl aliquots were spun onto a 28.3 mm2 area of glass slides. Slides were fixed in methanol and stained with DiffQuik (Siemens Healthcare Diagnostics, Inc., Deerfield, IL, USA). The numbers of trophic forms, cysts, and total fungal nuclei were determined microscopically using the 100× oil immersion objective of a Nikon microscope. Cysts may contain up to eight nuclear contents, which may excyst and live as trophic forms within the alveolar spaces. Therefore, the trophic and mixed P. murina inoculation doses were normalized based on the nuclei count. The growth kinetics during primary infection are similar following inoculation with equal nuclei burdens of trophic forms or mixed P. murina in the absence of anidulafungin.16 Adoptive transfer of CD4+ T cells Adult (8 weeks old) BALB/cJ mice were treated with saline or 1 mg/kg anidulafungin (Pfizer, New York, NY, USA) by the intraperitoneal route 1 day prior to infection and three times per week thereafter. Mice were anesthetized lightly with isoflurane anesthesia to suppress the diver's reflex and inoculated intratracheally with a dose of enriched trophic forms or mixed P. murina equivalent to 106 total nuclei in 100 μl HBSS containing 10 units/ml penicillin, 10 μg/ml streptomycin, and 1 μg/ml gentamicin. The mice were placed on an upright support rack, and the inoculations were performed using a blunted intratracheal needle designed with a curve to accommodate the trachea. Infected mice were euthanized at 14 days post-infection. Tracheobronchial lymph nodes (TBLN) were collected from the infected mice followed by passage over a 70 μm filter. Erythrocytes were removed using hypotonic ammonium-chloride-potassium (ACK) lysing buffer.19 CD4+ T cells were isolated with mouse CD4+ T cell enrichment columns (R&D Systems, Minneapolis, MN, USA). CD4+ T cells were washed, resuspended, and split for phenotyping or adoptive transfer. Greater than 90% of recovered cells were CD4+ T cells. In sum, 105 CD4+ T cells were adoptively transferred by the retro-orbital route to RAG2−/− mice (“recipients”). Three days after adoptive transfer, the recipient mice were treated with either 1 mg/kg anidulafungin or saline by the intraperitoneal route. This treatment continued three times per week throughout the course of the experiment. One day after the initiation of drug or saline treatment, the recipient mice were infected with a dose of enriched trophic forms or mixed P. murina organisms equivalent to 5 × 105 total nuclei. Animals were euthanized at 15 and 30 days post-infection. Rechallenge with P. murina in CD4+ T cell-depleted mice Adult BALB/cJ mice were treated with 1 mg/kg anidulafungin by the intraperitoneal route one day prior to intratracheal infection with 3 × 106 enriched trophic forms. Animals were treated with anidulafungin three times per week throughout the primary infection. Following clearance of the primary infection, anidulafungin treatment was stopped at day 34 post-infection to permit clearance of the drug prior to reinfection. At day 40 post-infection, 0.15 mg anti-CD4 antibody (clone GK1.5, Bio X Cell, Lebanon, NH, USA) in 200 μl sterile saline was administered to deplete CD4+ T cells. The anti-CD4 antibody treatment was administered every 4 days for the remainder of the study. One day after CD4+ T cell depletion, mice were treated with either anidulafungin or saline followed by reinfection with a dose of enriched trophic forms or mixed P. murina organisms equivalent to 5 × 106 total nuclei. The anidulafungin or saline treatment was administered 3 days per week for the remainder of the study. Animals were euthanized at day 15 post-infection. Isolation of cells from alveolar spaces, lungs, and lymph nodes Mice were exsanguinated under deep isoflurane anesthesia, and lungs were lavaged with five washes of HBSS containing 3 mM ethylenediaminetetraacetic acid (EDTA). Bronchial alveolar lavage fluid (BALF) was centrifuged to obtain cells, and cell-free supernatant from the first wash was frozen for subsequent cytokine assays using TNFα, IFNγ, IL-13, and IL-17A ELISA kits (eBioscience, San Diego, CA, USA). Right lung lobes were excised, minced, and digested in Roswell Park Memorial Institute medium (RPMI) medium supplemented with 3% heat-inactivated fetal calf serum, 1 mg/ml collagenase A (Sigma-Aldrich, St. Louis, MO, USA), and 50 U/ml DNase for 1 h at 37°C. Digested lungs were pushed through 70 μm nylon mesh screens to obtain single-cell suspensions, and aliquots were taken for enumeration of P. murina. Tracheobronchial lymph nodes (TBLN) were also excised and pushed through 70 μm nylon mesh screens in HBSS. Erythrocytes were removed using hypotonic ACK lysing buffer. Cells were washed and counted by hemocytometer. Flow cytometric analysis BALF, lung digest, and TBLN cells were washed with PBS containing 0.1% bovine serum albumin and 0.02% NaN3 and stained with appropriate concentrations of fluorochrome-conjugated antibodies specific for murine surface proteins (anti-CD4 clone GK1.5, anti-CD8a clone 53–6.7, anti-CD19 clone 1D3, anti-CD44 clone MEM-85, anti-CD62L clone MEL-14, anti-CD11c clone N418, anti-CD11b clone M1/70, and anti-F4/80 clone BM8). Antibodies were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). Expression of these molecules on the surface of the cells was determined by multiparameter flow cytometry using a LSRII flow cytometer (BD Biosciences). 50,000 events were routinely acquired and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Analysis P. murina-specific IgG in serum Blood was collected from rechallenged mice following euthanasia at day 15 post-rechallenge (n = 3 mice per group). Blood samples were centrifuged to pellet cells, and serum was collected and frozen at −80°C for later use. P. murina-specific immunoglobulin G (IgG) was measured by enzyme-linked immunosorbent assay (ELISA). A sonicate of P. murina trophic forms or mixed organisms (10 μg protein/ml) was coated onto 96-well plates, and wells were blocked with 5% dry milk in HBSS containing 0.05% Tween-20. Sera were diluted and incubated on plates overnight. Serum collected from an uninfected mouse was used as a negative control. Plates were extensively washed, and bound IgG was detected using alkaline phosphatase-conjugated anti-mouse IgG (Sigma). Plates were washed and secondary antibodies detected using p-nitrophenylphosphate at 1 mg/ml in diethanolamine buffer. Optical density was read at 405 nm using a plate reader equipped with KC Junior software (Bio-Tek Instruments, Inc., Winnoski, VT, USA). An OD of 0.1 was considered a cutoff value based on previous results from sera collected from uninfected mice. The OD of the negative control serum employed in these experiments remained below the 0.1 cutoff value. Enumeration of Pneumocystis in the lungs of mice Aliquots of lung homogenates were spun onto glass slides and stained as described above. The number of P. murina trophic forms or cysts in 50 microscopic oil immersion fields was used to calculate fungal burden. Lung burden is expressed as the number of P. murina organisms per right lung lobe, and the limit of detection was Log10 3.42 organisms per right lung lobe. Statistical analysis Data were analyzed utilizing the SigmaStat statistical software package (SPSS Inc., Chicago, IL, USA). Student's t-test, one-way or two-way analysis of variance (ANOVA) was used to determine differences between groups, with Student-Newman-Keuls multiple comparisons post hoc tests. Kruskal-Wallis one-way ANOVA on ranks was used to analyze differences between groups when the data were nonparametric. Data were determined to be significantly different when the P-value was < .05. Results Trophic forms are sufficient to provoke CD4+ T cell-mediated clearance of infection. To evaluate the role of trophic forms in the generation of protective CD4+ T-cell responses, we treated immunocompetent, wild-type mice (“donors”) with the β-1,3-glucan synthesis inhibitor anidulafungin to prevent encystment. Anidulafungin-treated mice were infected with enriched trophic forms. A control group of mice was treated with saline and infected with a mixture of trophic forms and cysts. At 14 days post-infection, CD4+ T cells were isolated from the TBLN and adoptively transferred to RAG2−/− mice (“recipients”). Recipient mice were treated with either anidulafungin or saline followed by infection with trophic forms or mixed organisms, respectively. As expected, cysts were not detected below the limit of detection in recipient mice treated with anidulafungin (Fig. 1A). Mice that received trophic form-stimulated CD4+ T cells followed by infection with trophic forms had more trophic forms in the lungs at day 15 post-infection compared to all other groups (Fig. 1A). However, all of the recipient mice infected with trophic forms cleared their trophic burden by day 30 post-infection, while only one of four mice infected with mixed organisms cleared its trophic and cystic burdens by day 30 post-infection. A higher number of activated CD4+ T cells were also observed at day 15 post-infection in the lung parenchyma of the group that received trophic form-stimulated CD4+ T cells followed by infection with trophic forms compared to all other groups (Fig. 1B). These differences were resolved by day 30 post-infection, and no differences in the numbers of activated CD4+ T cells in the alveolar spaces (BALF) or TBLN were observed among the groups. Figure 1. View largeDownload slide Infection with trophic forms is sufficient to provoke clearance of P. murina trophic forms following adoptive transfer of CD4+ T cells. Fungal lung burdens (A) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD44high CD62Llow CD4+ T cells (B) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare differences among the groups at individual timepoints when the data were nonparametric (A). Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cell numbers where the data were parametric (B), *P ≤ .05. Figure 1. View largeDownload slide Infection with trophic forms is sufficient to provoke clearance of P. murina trophic forms following adoptive transfer of CD4+ T cells. Fungal lung burdens (A) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD44high CD62Llow CD4+ T cells (B) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare differences among the groups at individual timepoints when the data were nonparametric (A). Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cell numbers where the data were parametric (B), *P ≤ .05. Trophic forms promote early CD4+ T cell-mediated IFNγ production in response to infection with trophic forms in the absence of cysts. Adoptive transfer of CD4+ T cells from trophic form-infected donors lead to increased expression of IFNγ at day 15 post-infection in the alveolar spaces of recipient mice infected with trophic forms compared to adoptive transfer of CD4+ T cells from mixed P. murina-infected donors into recipient mice infected with mixed P. murina organisms (Fig. 2). Statistically significant differences were not observed amongst the groups in regards to TNFα, IL-13, and IL-17A production in the alveolar spaces (Fig. 2). Figure 2. View largeDownload slide Transfer of CD4+ T cells from trophic form-infected mice promotes increased production of IFNγ in recipient mice challenged with trophic forms, compared to challenge with mixed Pneumocystis organisms. TNFα, IFNγ, IL-13, and IL-17A concentrations in the supernatant of the first BALF wash were quantified by ELISA. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cytokine expression, *P ≤ .05. Figure 2. View largeDownload slide Transfer of CD4+ T cells from trophic form-infected mice promotes increased production of IFNγ in recipient mice challenged with trophic forms, compared to challenge with mixed Pneumocystis organisms. TNFα, IFNγ, IL-13, and IL-17A concentrations in the supernatant of the first BALF wash were quantified by ELISA. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cytokine expression, *P ≤ .05. Trophic forms are sufficient to induce B cell-mediated clearance of P. murina infection. To evaluate the role of trophic forms in the generation of protective B cell responses, we treated immunocompetent, wild-type mice with anidulafungin, followed by infection with enriched trophic forms. Following clearance of the primary infection, anti-CD4 antibody was administered to deplete CD4+ T cells. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after the initial dose (Fig. 3A) and at the end of the study (data not shown). Mice were then treated with either anidulafungin or saline and reinfected with enriched trophic forms or mixed P. murina organisms, respectively. Figure 3. View largeDownload slide Infection with trophic forms is sufficient to provoke antibody-mediated clearance of P. murina trophic forms and cysts. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after treatment with the first dose of anti-CD4 monoclonal antibody (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD19+ B cells (C) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean fungal burden or total cell number between the groups, *P ≤ .05. Figure 3. View largeDownload slide Infection with trophic forms is sufficient to provoke antibody-mediated clearance of P. murina trophic forms and cysts. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after treatment with the first dose of anti-CD4 monoclonal antibody (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD19+ B cells (C) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean fungal burden or total cell number between the groups, *P ≤ .05. The memory B-cell response to trophic forms was sufficient to mediate clearance of P. murina trophic forms and cysts by day 15 post-infection (Fig. 3B). No differences in the numbers of CD19+ B cells in the alveolar spaces, lung parenchyma, and TBLN were observed between the mice rechallenged with trophic forms or mixed organisms (Fig. 3C). Likewise, no differences were observed in the expression of the costimulatory molecules CD80 and CD86 on the surface of B cells (data not shown). No differences in the numbers of activated CD8+ T cells in the alveolar spaces, lung parenchyma, and TBLN were observed between the groups (data not shown). Serum antibody from trophic form- or mixed P. murina-infected mice binds mixed P. murina antigen at higher titer than enriched trophic forms. To test the specificity of the antibody produced in our B cell rechallenge model, serum was collected at day 15 post-rechallenge. The titer of serum IgG against sonicated mixed P. murina organisms was 1:5000, compared to a titer of 1:1000 against an equal protein concentration of sonicated trophic forms (Fig. 4). A similar trend was observed when serum IgG was probed against an equal organism concentration of sonicated trophic forms versus mixed P. murina organisms (data not shown). The source of the serum (mice rechallenged with trophic forms versus mixed organisms), did not have an impact on the titer against trophic forms nor mixed organisms (Fig. 4). These data are comparable to the anti-trophic form and anti-mixed organism titers of serum collected from immunocompetent mice following primary infection with mixed organisms (data not shown). Figure 4. View largeDownload slide Serum antibody collected from trophic form or mixed P. murina-infected mice binds trophic forms at lower titer than mixed P. murina. Serum antibody was collected 15 days after rechallenge. ELISAs were performed on serum to determine the level of trophic form- or mixed Pneumocystis-specific IgG. Serum was probed against equal protein concentrations of sonicated trophic or mixed P. murina antigen. Data are expressed as the optical density at 405 nm. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare mean optical density at 405 nm between trophic antigen and mixed antigen groups at individual dilutions, *P ≤ .05. Figure 4. View largeDownload slide Serum antibody collected from trophic form or mixed P. murina-infected mice binds trophic forms at lower titer than mixed P. murina. Serum antibody was collected 15 days after rechallenge. ELISAs were performed on serum to determine the level of trophic form- or mixed Pneumocystis-specific IgG. Serum was probed against equal protein concentrations of sonicated trophic or mixed P. murina antigen. Data are expressed as the optical density at 405 nm. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare mean optical density at 405 nm between trophic antigen and mixed antigen groups at individual dilutions, *P ≤ .05. Trophic forms promote early antibody-mediated innate responses to infection with trophic forms in the absence of cysts. B cells may enhance innate immunity to Pneumocystis organisms via antibody-mediated opsonization of the fungal organisms and by the production of cytokines and other signals to maximize antifungal responses.9,20–22 Here, innate immune cells were phenotyped and quantified at day 15 post-infection in the lungs of CD4-depleted mice rechallenged with trophic forms or mixed P. murina (Fig. 5). A twofold increase in activated macrophages and lung dendritic cells (CD11c+ CD11b+ innate immune cells) was observed at day 15 post-infection in the alveolar spaces of mice rechallenged with trophic forms (Fig. 5). Differences in the numbers of immature alveolar macrophages and lung dendritic cells (CD11c+ CD11b−), and non-resident innate immune cells (CD11c− CD11b+) were not observed between the groups (Fig. 5). Figure 5. View largeDownload slide The antibody-mediated secondary response to trophic forms promotes an increase in CD11c+ CD11b+ innate immune cells in the alveolar spaces. Flow cytometry was used to phenotype CD11c+ CD11b−, CD11c+ CD11b+, and CD11c− CD11b+ nonlymphocytes with high granularity and size from the BALF and lung digest at day 15 post-rechallenge. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean total cell number between the groups, **P ≤ .01. Figure 5. View largeDownload slide The antibody-mediated secondary response to trophic forms promotes an increase in CD11c+ CD11b+ innate immune cells in the alveolar spaces. Flow cytometry was used to phenotype CD11c+ CD11b−, CD11c+ CD11b+, and CD11c− CD11b+ nonlymphocytes with high granularity and size from the BALF and lung digest at day 15 post-rechallenge. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean total cell number between the groups, **P ≤ .01. Infection with trophic forms in the absence of cysts is not sufficient for progression to PcP in immunocompromised mice. Pneumocystis pneumonia is characterized by inflammation-mediated alveolar damage. Here, we evaluate the role of the trophic forms in the development of Pneumocystis pneumonia (PcP) using mice deficient in T and B cells. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with enriched trophic forms (Fig. 6A–B). Saline-treated control mice were euthanized at day 72 post-infection due to symptoms of advanced PcP, including severe weight loss (Fig. 6A). In contrast, mice treated with anidulafungin had no overt symptoms of PcP, and continued to gain weight through the end of the study at day 120 post-infection (Fig. 6A). The lungs of saline-treated control mice contained 8 × 107 trophic forms and 5 × 106 cysts at day 72 post-infection (Fig. 6B). No cysts were detected in the lungs of the anidulafungin-treated mice at day 120 post-infection (Fig. 6B). A small population of 4 × 105 trophic organisms remained in the lungs of the anidulafungin-treated mice (Fig. 6B). Figure 6. View largeDownload slide Cysts are required for progression to pneumonia in immunocompromised mice infected with P. murina. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with trophic forms. The change in average body weight was recorded (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Data represent the mean ± SD of three to four mice per group and are representative of two separate experiments. T tests were used to compare body weight between the groups at individual timepoints, *P ≤ .05. Figure 6. View largeDownload slide Cysts are required for progression to pneumonia in immunocompromised mice infected with P. murina. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with trophic forms. The change in average body weight was recorded (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Data represent the mean ± SD of three to four mice per group and are representative of two separate experiments. T tests were used to compare body weight between the groups at individual timepoints, *P ≤ .05. In the absence of adaptive immunity, infection with mixed P. murina organisms induces non-protective inflammatory responses, including recruitment of innate immune cells.23,24 We evaluated the innate immune responses in the alveolar spaces (BALF) and lung parenchyma of the saline- and anidulafungin-treated mice following euthanasia at day 72 and day 120 post-infection, respectively (Fig. 6). A direct comparison between the groups is not advisable due to the difference in study endpoints. Rather, the following data are presented as a snapshot of the lungs at the time of euthanasia. An average of 119 pg/ml TNFα was detected in the BALF of the saline-treated mice at day 72 post-infection, while the lavage of anidulafungin-treated mice contained an average of 17 pg/ml TNFα at day 120 post-infection. The majority of the cells in the alveolar spaces and lung parenchyma of saline-treated mice were CD11c− CD11b+ non-resident innate immune cells. The bulk of cells within this population were F4/80low neutrophils. In contrast, most of the cells in the alveolar spaces of the anidulafungin-treated groups were CD11c+ CD11b− immature macrophages or dendritic cells, and the majority of the CD11c− CD11b+ non-resident cells were F4/80high, indicating recruitment of monocytes or macrophages rather than neutrophils. These data indicate that cysts are required for the development of inflammatory responses associated with Pneumocystis pneumonia. Additionally, trophic forms are able to survive in immunocompromised lungs for extended periods of time in the absence of cysts and without being able to complete their life cycle. Discussion The data reported here confirm that the trophic stage of P. murina is sufficient to induce CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to Pneumocystis pneumonia. We have previously reported that cysts are required for robust early innate and adaptive immune responses, including recruitment of CD4+ T cells and B cells into the alveolar spaces.16 While the magnitude of the early response was reduced in the animals inoculated with trophic forms alone, both the mice inoculated with trophic forms and the mice inoculated with mixed P. murina cleared the infection by 30 days post-inoculation. Previously we found that mice infected with trophic forms developed detectable cysts by day 7 in adult mice and day 14 post-infection in neonates.16 Because of this, we were unable to discern whether trophic forms failed to induce protective immune responses, or if the response to trophic forms was merely delayed.16 Here, we used the β-1,3-glucan synthase inhibitor anidulafungin to evaluate the adaptive and innate immune responses stimulated by trophic forms in the absence of cysts. Both CD4+ T cells and B cells are required for clearance of P. murina during primary infection, but either CD4+ T cells or antibody are sufficient for clearance during secondary infection.7,9,10,25–27 We report here that adoptive transfer of CD4+ T cells from trophic form- or mixed P. murina-infected animals was sufficient to mediate clearance of trophic forms in recipient mice. However, adoptive transfer of CD4+ T cells from trophic form- or mixed P. murina-infected animals was sufficient to mediate clearance of mixed P. murina organisms in only one mouse among the group of four animals by day 30 post-infection. Previous studies from our laboratory and others demonstrate that transfer of CD4+ T cells from mixed P. murina-infected animals is sufficient to mediate clearance of mixed P. murina organisms in SCID mice.7,26,28,29 Therefore, we predict that mixed P. murina-stimulated CD4+ T cells would eventually mediate clearance of the infection, given sufficient time. While macrophages are required for clearance of Pneumocystis organisms, these cells are not competent to resolve the infection in the absence of adaptive immunity.7–10 Here, a control population of RAG2−/− mice was infected with mixed Pneumocystis organisms and treated with saline, but were not given CD4+ T cells. As expected, this group contained a relatively high average fungal burden of 9.89 × 106 trophic forms and 8.69 × 105 cysts at day 30 post-infection, with an average of 11.4 trophic forms to cysts (data not shown). This population reconfirms that the adoptive transfer of CD4+ T cells was required for the control of fungal growth. In addition, this control population demonstrates that while the majority of the CD4+ T cell recipient mice failed to clear the mixed P. murina organisms by day 30 post-infection, the adoptive transfer of CD4+ T cells was sufficient to limit fungal growth. This observation is consistent with our prediction that the transfer of P. murina-stimulated CD4+ T cells would eventually mediate clearance of the mixed P. murina infection, given sufficient time. Intriguingly, our data suggest that the CD4+ T cell-mediated clearance of trophic forms during secondary infection occurs more rapidly in the absence of cysts. This phenomenon occurs independently of the composition of the priming infection. Our previous evidence indicates that cysts enhance, rather than inhibit the inflammatory response.16 Therefore, we suggest that the more efficient clearance of trophic forms observed in the absence of cysts may be due to slower growth kinetics of trophic forms in the absence of cysts. Linke et al. demonstrate that anidulafungin does not limit the expansion of trophic forms during the first 2 weeks of treatment.30 However, our long-term anidulafungin model (Fig. 6) demonstrates poor expansion of the trophic population due to the prolonged absence of the cyst stage. In the absence of anidulafungin, a single mature cyst produces eight progeny which develop into trophic forms. Clearance of the trophic population by immune cells may be accelerated in the absence of this source of nascent trophic forms. Previously reported data demonstrate that cysts are required for robust numbers of activated CD4+ T cells within the alveolar spaces and lung parenchyma of infected animals.16 However, we report here that the adoptive transfer of trophic form-stimulated CD4+ T cells leads to increased numbers of activated CD4+ T cells in the lung parenchyma of recipient mice at day 15 post-infection compared to transfer of CD4+ T cells from mixed infection. It is noteworthy that fewer CD4+ T cells were harvested from the draining lymph nodes of the wild-type donor mice infected with trophic forms compared to mixed P. murina infection (data not shown). However, the adoptive transfer of equal numbers of CD4+ T cells reveals that these CD4+ T cells are fully capable of mediating clearance. Our previous data indicated that inoculation with trophic forms leads to fewer IFN-γ-producing CD4+ T cells and suboptimal IFN-γ concentrations in the alveolar spaces compared to inoculation with mixed P. murina organisms.16 Furthermore, trophic form-stimulated BDMCs failed to produce TNFα, IL-1β, and IL-6 in response to various other stimuli, and failed to induce IFNγ production during co-culture with CD4+ T cells.16 Conversely, the data reported here show that trophic forms are sufficient to drive CD4+ T cell-mediated TNFα, IFN-γ, and IL-13 production in vivo. These data suggest that stimulation with trophic forms does not skew the immune response away from Th1 or Th2-type responses. While Th17 responses are critical for control of many fungal infections, including candidiasis, a mixed T helper response is observed in response to Pneumocystis infection. This mixed response appears to be largely redundant, as Th1-, Th2-, and Th17-type responses have all been associated with clearance of P. murina organisms.30–35 Cumulatively, our data suggest that trophic forms suppress the development of innate immunity, and thus delay the initiation of adaptive responses16, but trophic forms do not have a direct suppressive effect on CD4+ T cells in vivo. We have reported that cysts are required for an early increase in B cells in the lungs of infected mice.16 However, here we report that primary infection with trophic forms is sufficient to induce antibody-mediated clearance of P. murina trophic forms and cysts during secondary infection. No differences in B cell count were observed in the animals rechallenged with trophic forms or mixed P. murina organisms. Rechallenge of CD4+ T cell-depleted mice with trophic forms promoted increased numbers of activated macrophages and dendritic cells (CD11c+ CD11b+) in the alveolar spaces compared to mice rechallenged with mixed P. murina. However, these data were generated by a single timepoint following clearance (day 15), and differences between the groups may be due to the kinetics of clearance, rather than differences in the magnitude of the response. Intriguingly, serum antibody bound sonicated mixed P. murina antigen at higher titer than trophic antigen. This phenomenon was observed even in serum from anidulafungin-treated animals that had never developed cysts. Previous studies suggest that there is significant overlap in the glycoproteins, including glycoprotein A (gpA, alternatively, major surface glycoprotein, MSG), on the surface of trophic forms and cysts.36,37 Our data suggest that serum antibody raised against trophic forms binds conserved material on the cyst life cycle stage. The data may also indicate that there is more of this material on the cysts than on the trophic forms. Regardless, our data indicate that while serum antibody binds mixed P. murina antigen at higher titer than trophic forms, antibody-mediated responses are sufficient for clearance of both trophic forms and cysts. While the initiation of innate and adaptive responses is required for clearance of Pneumocystis organisms in immunocompetent hosts, the nonspecific provocation of inflammation leads to immune-mediated damage in immunocompromised hosts that progress to Pneumocystis pneumonia.23,24 It has been previously reported that treatment of immune-reconstituted mice with the β-1,3-glucan synthase inhibitor anidulafungin results in depletion of cysts and a reduced inflammatory response.30 Here we report that long-term anidulafungin treatment of trophic form-infected RAG2−/− mice results in the carriage of a relatively small population of trophic forms without progression to pneumonia. Control RAG2−/− mice developed cysts and progressed to pneumonia characterized by weight loss, and recruitment of neutrophils into the lungs. In contrast, the majority of innate immune cells within the alveolar spaces of anidulafungin-treated mice were immature alveolar macrophages and lung dendritic cells. Linke et al. propose that depletion of cysts would reduce inflammation-induced lung damage in patients, and our data further corroborates this suggestion.30 However, our data highlight the need for a greater understanding of the impact of trophic forms on inflammatory responses and host health. While no overt symptoms of pneumonia were detected in our anidulafungin-treated mice, it has been reported that trophic forms induce direct damage to alveolar epithelial cells.38 In our model, the fungal lung burden of RAG2−/− mice with severe pneumonia may consist of as many as 1 × 108 trophic forms. It is unclear why the expansion of trophic forms was limited in RAG2−/- mice during long-term treatment with anidulafungin. Linke and Cushion et al. demonstrate that a short-term course of β-1,3-glucan synthase inhibitor therapy does not hamper expansion of trophic forms.30,39 However, the extended time course of our study suggests that cysts enhance the growth of the trophic forms by an unknown mechanism. It is thought that the formation of the cyst requires sexual reproduction, and it is possible that the growth of the fungus is severely limited when the trophic forms are restricted to asexual reproduction.40 Meiosis followed by multiple rounds of mitosis within the formed cyst results in a quadrupling of the number of organisms which may be important for more rapid expansion of organisms. In the absence of this rapid expansion of organisms via sexual reproduction, it is possible that innate immune cells may kill enough of the trophic forms to limit the growth of the population, but not enough to clear the organisms from the lungs. Alternatively, a signal or product from the cyst or cyst-stimulated host cells could be required for expansion of trophic forms. Trophic forms are found clustered within a biofilm-like substance that includes material from previously ruptured cysts.12 Depletion of cysts may deprive the trophic forms of this protective shelter, and may permit killing of trophic forms by innate immune cells. Whatever the mechanism, it is interesting that in an immunosuppressed environment the trophic population does not collapse in the absence of cysts. This would certainly limit the use of glucan synthase inhibitors as routine monotherapy for PCP. We have previously reported that infection with trophic forms in the absence of cysts leads to the delayed initiation of innate and adaptive responses against Pneumocystis infection.16 Despite these delays, we found here that the trophic stage of P. murina is sufficient to induce CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to Pneumocystis pneumonia, as defined by severe weight loss and infiltration of innate immune cells into the lungs. These data suggest that immune evasion by the trophic forms may hinge on the suppression of the initiation of the innate immune response. The development of adaptive immunity may represent a “point of no return” at which the trophic forms are no longer able to escape clearance. Manipulation of the immune response to trophic forms and cysts may provide new options for the treatment and prevention of Pneumocystis infection, while a failure to consider these differential responses may hamper future efforts. Here, our data indicate that trophic forms elicit adaptive responses, but do not provoke the non-protective inflammation characteristic of Pneumocystis pneumonia. Further evaluation of the antigenic determinants on trophic forms and cysts may elicit a vaccine that provides protection while limiting immune-mediated damage, including immune reconstitution syndrome. Acknowledgements We thank Melissa Hollifield for technical assistance. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Funding This work was supported by a Public Health Services grant from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [R21 AI118818]. 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Nollstadt KH , Powles MA , Fujioka H , Aikawa M , Schmatz DM . Use of beta-1,3-glucan-specific antibody to study the cyst wall of Pneumocystis carinii and effects of pneumocandin B0 analog L-733,560 . Antimicrob Agents Chemother . 1994 ; 38 : 2258 – 2265 . Google Scholar CrossRef Search ADS PubMed 15. Kottom TJ , Hebrink DM , Jenson PE , Gudmundsson G , Limper AH . Evidence for proinflammatory beta-1,6 glucans in the Pneumocystis carinii cell wall . Infect Immun . 2015 ; 83 : 2816 – 2826 . Google Scholar CrossRef Search ADS PubMed 16. Evans HM , Bryant GL 3rd , Garvy BA . The life cycle stages of Pneumocystis murina have opposing effects on the immune response to this opportunistic, fungal pathogen . Infect Immun . 2016 ; pii: IAI.00519-16 . 17. Kling HM , Norris KA . Vaccine-induced immunogenicity and protection against Pneumocystis pneumonia in a nonhuman primate model of HIV and pneumocystis coinfection . J Infect Dis . 2016 ; 213 : 1586 – 1595 . 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APMIS . 2003 ; 111 : 405 – 415 . Google Scholar CrossRef Search ADS PubMed 22. Empey KM , Hollifield M , Schuer K , Gigliotti F , Garvy BA . Passive immunization of neonatal mice against Pneumocystis carinii f. sp. muris enhances control of infection without stimulating inflammation . Infect Immun . 2004 ; 72 : 6211 – 6220 . Google Scholar CrossRef Search ADS PubMed 23. Gigliotti F , Wright TW . Immunopathogenesis of Pneumocystis carinii pneumonia . Expert Rev Mol Med . 2005 ; 7 : 1 – 16 . Google Scholar CrossRef Search ADS PubMed 24. Wright TW , Gigliotti F , Finkelstein JN , McBride JT , An CL , Harmsen AG . Immune-mediated inflammation directly impairs pulmonary function, contributing to the pathogenesis of Pneumocystis carinii pneumonia . J Clin Invest . 1999 ; 104 : 1307 – 1317 . Google Scholar CrossRef Search ADS PubMed 25. Harmsen AG , Chen W , Gigliotti F . Active immunity to Pneumocystis carinii reinfection in T-cell-depleted mice . Infect Immun . 1995 ; 63 : 2391 – 2395 . 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J Infect Dis . 2000 ; 181 : 2011 – 2017 . Google Scholar CrossRef Search ADS PubMed 34. Rudner XL , Happel KI , Young EA , Shellito JE . Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection . Infect Immun . 2007 ; 75 : 3055 – 3061 . Google Scholar CrossRef Search ADS PubMed 35. Myers RC , Dunaway CW , Nelson MP , Trevor JL , Morris A , Steele C . STAT4-dependent and -independent Th2 responses correlate with protective immunity against lung infection with Pneumocystis murina . J Immunol . 2013 ; 190 : 6287 – 6294 . Google Scholar CrossRef Search ADS PubMed 36. Angus CW , Tu A , Vogel P , Qin M , Kovacs JA . Expression of variants of the major surface glycoprotein of Pneumocystis carinii . J Exp Med . 1996 ; 183 : 1229 – 1234 . Google Scholar CrossRef Search ADS PubMed 37. De Stefano JA Myers JD , Du Pont D , Foy JM , Theus SA , Walzer PD . 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The trophic life cycle stage of Pneumocystis species induces protective adaptive responses without inflammation-mediated progression to pneumonia

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Oxford University Press
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10.1093/mmy/myx145
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Abstract

Abstract Pneumocystis species are fungal pathogens that cause pneumonia in immunocompromised hosts. Lung damage during Pneumocystis pneumonia is predominately due to the inflammatory immune response. Pneumocystis species have a biphasic life cycle. Optimal innate immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, the trophic life cycle stage broadly suppresses proinflammatory responses to multiple pathogen-associated molecular patterns (PAMPs), including β-1,3-glucan. Little is known about the contribution of these life cycle stages to the development of protective adaptive responses to Pneumocystis infection. Here we report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B-cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised recombination-activating gene 2 knockout (RAG2−/−) mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia. Pneumocystis, echinocandin, pneumonia, lung, AIDS Introduction Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Patients at risk for developing Pneumocystis pneumonia (PcP) include those whose immune systems have become compromised due to acquired immunodeficiency syndrome (AIDS) or immunosuppressive treatments such as chemotherapy, steroid treatment, and anti-tumor necrosis factor alpha (TNFα) biologic therapy.1–6 The clearance of Pneumocystis organisms from the host lung is dependent on a broad range of effector responses, including CD4+ T-cell, B-cell, and macrophage activity.7–10 In the absence of an efficient adaptive immune response, immunocompromised patients may progress to PcP, which is characterized by inflammation-mediated alveolar damage.11 Basic research into the interactions between this fungal pathogen and the host immune response has the potential to inform novel approaches to reduce morbidity and mortality due to Pneumocystis pneumonia. Pneumocystis species have a biphasic life cycle consisting of trophic forms and cysts. Trophic forms are single-nucleated organisms typically found in clusters surrounded by a biofilm-like substance consisting of a conglomeration of DNA, β-glucan, and other sugars.12 Cysts are ascus-like structures that consist of multiple nuclei surrounded by a fungal cell wall consisting of β-1,3 glucan and β-1,6 glucan.13–15 Trophic forms do not express β-glucan and do not form a cell wall.14 We have previously reported that the life cycle stages of Pneumocystis murina have opposing effects on the immune response. The immune response to primary infection with P. murina trophic forms alone was less robust than the response to infection with a physiologically normal mixture of cysts and trophic forms.16 Infection with trophic forms alone resulted in reduced numbers of CD11c+ innate immune cells in the lungs, as well as reduced recruitment of B cells and T cells, compared to infection with a normal mixture of trophic forms and cysts.16In vitro, trophic forms suppressed production of the proinflammatory cytokines interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and TNFα by bone marrow-derived dendritic cells (BMDCs) stimulated with β-glucan, zymosan, depleted zymosan, lipoteichoic acid (LTA), or lipopolysaccharides (LPS).16 In addition, trophic form-stimulated BMDCs failed to stimulate production of the T helper 1 (Th1)-type cytokine interferon gamma (IFN-γ) by CD4+ T cells.16 Despite these delays, the mice inoculated with trophic forms were able to clear the infection within a similar period of time as the mice inoculated with mixed organisms.16 However, the rapid turnover of trophic forms into cysts in these previous experiments precludes the formation of conclusions regarding which life cycle stage(s) initiate the immune responses that lead to clearance.16 Here we demonstrate that the trophic stage of P. murina is sufficient to provoke CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to PcP. The adoptive transfer of CD4+ T cells generated in the presence of trophic forms was sufficient to mediate the clearance of both trophic forms and cysts from the lungs of RAG2−/− mice. In addition, primary infection with trophic forms was sufficient for the development of antibody-mediated secondary responses leading to clearance of both trophic forms and cysts following reinfection. Recent studies demonstrate that protective immunization against Pneumocystis infection is a feasible goal, even in the context of simian immunodeficiency virus (SIV)-induced immunosuppression or CD4+ T-cell depletion.17,18 Our data suggest that trophic form-mediated immune suppression of innate responses does not impede the development of protective secondary adaptive responses. Conversely, our data suggest that the depletion of the cyst life cycle stage may be sufficient for protection against the inflammation-mediated pathology associated with PcP. Methods Mice Mice were maintained at the University of Kentucky Department of Laboratory Animal Resources (DLAR) under specific-pathogen-free conditions. BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our animal facilities. C.129S6(B6)-Rag2tm1Fwa N12 (Rag2−/−) mice, originally from Taconic (Germantown, NY) were bred at DLAR in sterile microisolator cages with sterilized food and water. The University of Kentucky Institutional Animal Care and Use Committee approved all protocols regarding animal use. All protocols conformed to the Guide for the Care and Use of Laboratory Animals (2011 edition) of the Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. P. murina isolation and infection Lungs were excised from P. murina-infected Rag2−/− mice and pushed through stainless steel mesh in Hank's balanced salt solution (HBSS) containing 0.5% glutathione at pH 7.3. Cell debris was broken up by aspirating through 20 and 26-gauge needles, then removed by centrifugation at 100 × g for 3 min. Trophic forms were isolated by removing the supernatant following centrifugation at 400 × g for 7 min. This preparation results in greater than 100 trophic forms per cyst.16 The pellet from the 400 × g spin contained a mixed population of cysts and trophic forms, at a typical ratio of 10 : 1 trophic forms to cysts. Erythrocytes in the pellet were lysed with water and organisms suspended in an equal volume of 2× phosphate-buffered saline (PBS). Organisms were incubated with 200 U DNase (Sigma-Aldrich, St. Louis MO) at 37°C for 30 min. Clumps were broken up by aspirating through a 26-gauge needle. The remaining cell debris was removed by centrifugation at 100 × g for 3 min, followed by passage over a 70 μm filter. Pneumocystis life forms were pelleted by centrifugation at 1300 × g for 15 min, then resuspended in 100 μl HBSS containing 10 units/ml penicillin, 10 μg/ml streptomycin, and 1 μg/ml gentamicin. Aliquots of mixed P. murina organisms or enriched trophic forms were diluted, and 100 μl aliquots were spun onto a 28.3 mm2 area of glass slides. Slides were fixed in methanol and stained with DiffQuik (Siemens Healthcare Diagnostics, Inc., Deerfield, IL, USA). The numbers of trophic forms, cysts, and total fungal nuclei were determined microscopically using the 100× oil immersion objective of a Nikon microscope. Cysts may contain up to eight nuclear contents, which may excyst and live as trophic forms within the alveolar spaces. Therefore, the trophic and mixed P. murina inoculation doses were normalized based on the nuclei count. The growth kinetics during primary infection are similar following inoculation with equal nuclei burdens of trophic forms or mixed P. murina in the absence of anidulafungin.16 Adoptive transfer of CD4+ T cells Adult (8 weeks old) BALB/cJ mice were treated with saline or 1 mg/kg anidulafungin (Pfizer, New York, NY, USA) by the intraperitoneal route 1 day prior to infection and three times per week thereafter. Mice were anesthetized lightly with isoflurane anesthesia to suppress the diver's reflex and inoculated intratracheally with a dose of enriched trophic forms or mixed P. murina equivalent to 106 total nuclei in 100 μl HBSS containing 10 units/ml penicillin, 10 μg/ml streptomycin, and 1 μg/ml gentamicin. The mice were placed on an upright support rack, and the inoculations were performed using a blunted intratracheal needle designed with a curve to accommodate the trachea. Infected mice were euthanized at 14 days post-infection. Tracheobronchial lymph nodes (TBLN) were collected from the infected mice followed by passage over a 70 μm filter. Erythrocytes were removed using hypotonic ammonium-chloride-potassium (ACK) lysing buffer.19 CD4+ T cells were isolated with mouse CD4+ T cell enrichment columns (R&D Systems, Minneapolis, MN, USA). CD4+ T cells were washed, resuspended, and split for phenotyping or adoptive transfer. Greater than 90% of recovered cells were CD4+ T cells. In sum, 105 CD4+ T cells were adoptively transferred by the retro-orbital route to RAG2−/− mice (“recipients”). Three days after adoptive transfer, the recipient mice were treated with either 1 mg/kg anidulafungin or saline by the intraperitoneal route. This treatment continued three times per week throughout the course of the experiment. One day after the initiation of drug or saline treatment, the recipient mice were infected with a dose of enriched trophic forms or mixed P. murina organisms equivalent to 5 × 105 total nuclei. Animals were euthanized at 15 and 30 days post-infection. Rechallenge with P. murina in CD4+ T cell-depleted mice Adult BALB/cJ mice were treated with 1 mg/kg anidulafungin by the intraperitoneal route one day prior to intratracheal infection with 3 × 106 enriched trophic forms. Animals were treated with anidulafungin three times per week throughout the primary infection. Following clearance of the primary infection, anidulafungin treatment was stopped at day 34 post-infection to permit clearance of the drug prior to reinfection. At day 40 post-infection, 0.15 mg anti-CD4 antibody (clone GK1.5, Bio X Cell, Lebanon, NH, USA) in 200 μl sterile saline was administered to deplete CD4+ T cells. The anti-CD4 antibody treatment was administered every 4 days for the remainder of the study. One day after CD4+ T cell depletion, mice were treated with either anidulafungin or saline followed by reinfection with a dose of enriched trophic forms or mixed P. murina organisms equivalent to 5 × 106 total nuclei. The anidulafungin or saline treatment was administered 3 days per week for the remainder of the study. Animals were euthanized at day 15 post-infection. Isolation of cells from alveolar spaces, lungs, and lymph nodes Mice were exsanguinated under deep isoflurane anesthesia, and lungs were lavaged with five washes of HBSS containing 3 mM ethylenediaminetetraacetic acid (EDTA). Bronchial alveolar lavage fluid (BALF) was centrifuged to obtain cells, and cell-free supernatant from the first wash was frozen for subsequent cytokine assays using TNFα, IFNγ, IL-13, and IL-17A ELISA kits (eBioscience, San Diego, CA, USA). Right lung lobes were excised, minced, and digested in Roswell Park Memorial Institute medium (RPMI) medium supplemented with 3% heat-inactivated fetal calf serum, 1 mg/ml collagenase A (Sigma-Aldrich, St. Louis, MO, USA), and 50 U/ml DNase for 1 h at 37°C. Digested lungs were pushed through 70 μm nylon mesh screens to obtain single-cell suspensions, and aliquots were taken for enumeration of P. murina. Tracheobronchial lymph nodes (TBLN) were also excised and pushed through 70 μm nylon mesh screens in HBSS. Erythrocytes were removed using hypotonic ACK lysing buffer. Cells were washed and counted by hemocytometer. Flow cytometric analysis BALF, lung digest, and TBLN cells were washed with PBS containing 0.1% bovine serum albumin and 0.02% NaN3 and stained with appropriate concentrations of fluorochrome-conjugated antibodies specific for murine surface proteins (anti-CD4 clone GK1.5, anti-CD8a clone 53–6.7, anti-CD19 clone 1D3, anti-CD44 clone MEM-85, anti-CD62L clone MEL-14, anti-CD11c clone N418, anti-CD11b clone M1/70, and anti-F4/80 clone BM8). Antibodies were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). Expression of these molecules on the surface of the cells was determined by multiparameter flow cytometry using a LSRII flow cytometer (BD Biosciences). 50,000 events were routinely acquired and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Analysis P. murina-specific IgG in serum Blood was collected from rechallenged mice following euthanasia at day 15 post-rechallenge (n = 3 mice per group). Blood samples were centrifuged to pellet cells, and serum was collected and frozen at −80°C for later use. P. murina-specific immunoglobulin G (IgG) was measured by enzyme-linked immunosorbent assay (ELISA). A sonicate of P. murina trophic forms or mixed organisms (10 μg protein/ml) was coated onto 96-well plates, and wells were blocked with 5% dry milk in HBSS containing 0.05% Tween-20. Sera were diluted and incubated on plates overnight. Serum collected from an uninfected mouse was used as a negative control. Plates were extensively washed, and bound IgG was detected using alkaline phosphatase-conjugated anti-mouse IgG (Sigma). Plates were washed and secondary antibodies detected using p-nitrophenylphosphate at 1 mg/ml in diethanolamine buffer. Optical density was read at 405 nm using a plate reader equipped with KC Junior software (Bio-Tek Instruments, Inc., Winnoski, VT, USA). An OD of 0.1 was considered a cutoff value based on previous results from sera collected from uninfected mice. The OD of the negative control serum employed in these experiments remained below the 0.1 cutoff value. Enumeration of Pneumocystis in the lungs of mice Aliquots of lung homogenates were spun onto glass slides and stained as described above. The number of P. murina trophic forms or cysts in 50 microscopic oil immersion fields was used to calculate fungal burden. Lung burden is expressed as the number of P. murina organisms per right lung lobe, and the limit of detection was Log10 3.42 organisms per right lung lobe. Statistical analysis Data were analyzed utilizing the SigmaStat statistical software package (SPSS Inc., Chicago, IL, USA). Student's t-test, one-way or two-way analysis of variance (ANOVA) was used to determine differences between groups, with Student-Newman-Keuls multiple comparisons post hoc tests. Kruskal-Wallis one-way ANOVA on ranks was used to analyze differences between groups when the data were nonparametric. Data were determined to be significantly different when the P-value was < .05. Results Trophic forms are sufficient to provoke CD4+ T cell-mediated clearance of infection. To evaluate the role of trophic forms in the generation of protective CD4+ T-cell responses, we treated immunocompetent, wild-type mice (“donors”) with the β-1,3-glucan synthesis inhibitor anidulafungin to prevent encystment. Anidulafungin-treated mice were infected with enriched trophic forms. A control group of mice was treated with saline and infected with a mixture of trophic forms and cysts. At 14 days post-infection, CD4+ T cells were isolated from the TBLN and adoptively transferred to RAG2−/− mice (“recipients”). Recipient mice were treated with either anidulafungin or saline followed by infection with trophic forms or mixed organisms, respectively. As expected, cysts were not detected below the limit of detection in recipient mice treated with anidulafungin (Fig. 1A). Mice that received trophic form-stimulated CD4+ T cells followed by infection with trophic forms had more trophic forms in the lungs at day 15 post-infection compared to all other groups (Fig. 1A). However, all of the recipient mice infected with trophic forms cleared their trophic burden by day 30 post-infection, while only one of four mice infected with mixed organisms cleared its trophic and cystic burdens by day 30 post-infection. A higher number of activated CD4+ T cells were also observed at day 15 post-infection in the lung parenchyma of the group that received trophic form-stimulated CD4+ T cells followed by infection with trophic forms compared to all other groups (Fig. 1B). These differences were resolved by day 30 post-infection, and no differences in the numbers of activated CD4+ T cells in the alveolar spaces (BALF) or TBLN were observed among the groups. Figure 1. View largeDownload slide Infection with trophic forms is sufficient to provoke clearance of P. murina trophic forms following adoptive transfer of CD4+ T cells. Fungal lung burdens (A) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD44high CD62Llow CD4+ T cells (B) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare differences among the groups at individual timepoints when the data were nonparametric (A). Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cell numbers where the data were parametric (B), *P ≤ .05. Figure 1. View largeDownload slide Infection with trophic forms is sufficient to provoke clearance of P. murina trophic forms following adoptive transfer of CD4+ T cells. Fungal lung burdens (A) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD44high CD62Llow CD4+ T cells (B) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare differences among the groups at individual timepoints when the data were nonparametric (A). Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cell numbers where the data were parametric (B), *P ≤ .05. Trophic forms promote early CD4+ T cell-mediated IFNγ production in response to infection with trophic forms in the absence of cysts. Adoptive transfer of CD4+ T cells from trophic form-infected donors lead to increased expression of IFNγ at day 15 post-infection in the alveolar spaces of recipient mice infected with trophic forms compared to adoptive transfer of CD4+ T cells from mixed P. murina-infected donors into recipient mice infected with mixed P. murina organisms (Fig. 2). Statistically significant differences were not observed amongst the groups in regards to TNFα, IL-13, and IL-17A production in the alveolar spaces (Fig. 2). Figure 2. View largeDownload slide Transfer of CD4+ T cells from trophic form-infected mice promotes increased production of IFNγ in recipient mice challenged with trophic forms, compared to challenge with mixed Pneumocystis organisms. TNFα, IFNγ, IL-13, and IL-17A concentrations in the supernatant of the first BALF wash were quantified by ELISA. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cytokine expression, *P ≤ .05. Figure 2. View largeDownload slide Transfer of CD4+ T cells from trophic form-infected mice promotes increased production of IFNγ in recipient mice challenged with trophic forms, compared to challenge with mixed Pneumocystis organisms. TNFα, IFNγ, IL-13, and IL-17A concentrations in the supernatant of the first BALF wash were quantified by ELISA. Data represent the mean ± SD of four mice per group and are representative of two separate experiments. Two-way ANOVA with Student-Newman-Keuls post hoc test was used to compare cytokine expression, *P ≤ .05. Trophic forms are sufficient to induce B cell-mediated clearance of P. murina infection. To evaluate the role of trophic forms in the generation of protective B cell responses, we treated immunocompetent, wild-type mice with anidulafungin, followed by infection with enriched trophic forms. Following clearance of the primary infection, anti-CD4 antibody was administered to deplete CD4+ T cells. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after the initial dose (Fig. 3A) and at the end of the study (data not shown). Mice were then treated with either anidulafungin or saline and reinfected with enriched trophic forms or mixed P. murina organisms, respectively. Figure 3. View largeDownload slide Infection with trophic forms is sufficient to provoke antibody-mediated clearance of P. murina trophic forms and cysts. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after treatment with the first dose of anti-CD4 monoclonal antibody (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD19+ B cells (C) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean fungal burden or total cell number between the groups, *P ≤ .05. Figure 3. View largeDownload slide Infection with trophic forms is sufficient to provoke antibody-mediated clearance of P. murina trophic forms and cysts. The depletion of CD4+ T cells was confirmed by flow cytometry 3 days after treatment with the first dose of anti-CD4 monoclonal antibody (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Flow cytometry was used to phenotype activated CD19+ B cells (C) in the BALF, lung digest, and TBLN. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean fungal burden or total cell number between the groups, *P ≤ .05. The memory B-cell response to trophic forms was sufficient to mediate clearance of P. murina trophic forms and cysts by day 15 post-infection (Fig. 3B). No differences in the numbers of CD19+ B cells in the alveolar spaces, lung parenchyma, and TBLN were observed between the mice rechallenged with trophic forms or mixed organisms (Fig. 3C). Likewise, no differences were observed in the expression of the costimulatory molecules CD80 and CD86 on the surface of B cells (data not shown). No differences in the numbers of activated CD8+ T cells in the alveolar spaces, lung parenchyma, and TBLN were observed between the groups (data not shown). Serum antibody from trophic form- or mixed P. murina-infected mice binds mixed P. murina antigen at higher titer than enriched trophic forms. To test the specificity of the antibody produced in our B cell rechallenge model, serum was collected at day 15 post-rechallenge. The titer of serum IgG against sonicated mixed P. murina organisms was 1:5000, compared to a titer of 1:1000 against an equal protein concentration of sonicated trophic forms (Fig. 4). A similar trend was observed when serum IgG was probed against an equal organism concentration of sonicated trophic forms versus mixed P. murina organisms (data not shown). The source of the serum (mice rechallenged with trophic forms versus mixed organisms), did not have an impact on the titer against trophic forms nor mixed organisms (Fig. 4). These data are comparable to the anti-trophic form and anti-mixed organism titers of serum collected from immunocompetent mice following primary infection with mixed organisms (data not shown). Figure 4. View largeDownload slide Serum antibody collected from trophic form or mixed P. murina-infected mice binds trophic forms at lower titer than mixed P. murina. Serum antibody was collected 15 days after rechallenge. ELISAs were performed on serum to determine the level of trophic form- or mixed Pneumocystis-specific IgG. Serum was probed against equal protein concentrations of sonicated trophic or mixed P. murina antigen. Data are expressed as the optical density at 405 nm. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare mean optical density at 405 nm between trophic antigen and mixed antigen groups at individual dilutions, *P ≤ .05. Figure 4. View largeDownload slide Serum antibody collected from trophic form or mixed P. murina-infected mice binds trophic forms at lower titer than mixed P. murina. Serum antibody was collected 15 days after rechallenge. ELISAs were performed on serum to determine the level of trophic form- or mixed Pneumocystis-specific IgG. Serum was probed against equal protein concentrations of sonicated trophic or mixed P. murina antigen. Data are expressed as the optical density at 405 nm. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. Kruskal-Wallis one-way ANOVA on ranks with Student-Newman-Keuls post hoc test was used to compare mean optical density at 405 nm between trophic antigen and mixed antigen groups at individual dilutions, *P ≤ .05. Trophic forms promote early antibody-mediated innate responses to infection with trophic forms in the absence of cysts. B cells may enhance innate immunity to Pneumocystis organisms via antibody-mediated opsonization of the fungal organisms and by the production of cytokines and other signals to maximize antifungal responses.9,20–22 Here, innate immune cells were phenotyped and quantified at day 15 post-infection in the lungs of CD4-depleted mice rechallenged with trophic forms or mixed P. murina (Fig. 5). A twofold increase in activated macrophages and lung dendritic cells (CD11c+ CD11b+ innate immune cells) was observed at day 15 post-infection in the alveolar spaces of mice rechallenged with trophic forms (Fig. 5). Differences in the numbers of immature alveolar macrophages and lung dendritic cells (CD11c+ CD11b−), and non-resident innate immune cells (CD11c− CD11b+) were not observed between the groups (Fig. 5). Figure 5. View largeDownload slide The antibody-mediated secondary response to trophic forms promotes an increase in CD11c+ CD11b+ innate immune cells in the alveolar spaces. Flow cytometry was used to phenotype CD11c+ CD11b−, CD11c+ CD11b+, and CD11c− CD11b+ nonlymphocytes with high granularity and size from the BALF and lung digest at day 15 post-rechallenge. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean total cell number between the groups, **P ≤ .01. Figure 5. View largeDownload slide The antibody-mediated secondary response to trophic forms promotes an increase in CD11c+ CD11b+ innate immune cells in the alveolar spaces. Flow cytometry was used to phenotype CD11c+ CD11b−, CD11c+ CD11b+, and CD11c− CD11b+ nonlymphocytes with high granularity and size from the BALF and lung digest at day 15 post-rechallenge. Data represent the mean ± SD of three mice per group and are representative of two separate experiments. T tests were used to compare mean total cell number between the groups, **P ≤ .01. Infection with trophic forms in the absence of cysts is not sufficient for progression to PcP in immunocompromised mice. Pneumocystis pneumonia is characterized by inflammation-mediated alveolar damage. Here, we evaluate the role of the trophic forms in the development of Pneumocystis pneumonia (PcP) using mice deficient in T and B cells. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with enriched trophic forms (Fig. 6A–B). Saline-treated control mice were euthanized at day 72 post-infection due to symptoms of advanced PcP, including severe weight loss (Fig. 6A). In contrast, mice treated with anidulafungin had no overt symptoms of PcP, and continued to gain weight through the end of the study at day 120 post-infection (Fig. 6A). The lungs of saline-treated control mice contained 8 × 107 trophic forms and 5 × 106 cysts at day 72 post-infection (Fig. 6B). No cysts were detected in the lungs of the anidulafungin-treated mice at day 120 post-infection (Fig. 6B). A small population of 4 × 105 trophic organisms remained in the lungs of the anidulafungin-treated mice (Fig. 6B). Figure 6. View largeDownload slide Cysts are required for progression to pneumonia in immunocompromised mice infected with P. murina. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with trophic forms. The change in average body weight was recorded (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Data represent the mean ± SD of three to four mice per group and are representative of two separate experiments. T tests were used to compare body weight between the groups at individual timepoints, *P ≤ .05. Figure 6. View largeDownload slide Cysts are required for progression to pneumonia in immunocompromised mice infected with P. murina. RAG2−/− mice were treated with anidulafungin or saline, followed by infection with trophic forms. The change in average body weight was recorded (A). Fungal lung burdens (B) in the right lung lobes were determined by enumeration of organisms on DiffQuik stained slides under a microscope. Data represent the mean ± SD of three to four mice per group and are representative of two separate experiments. T tests were used to compare body weight between the groups at individual timepoints, *P ≤ .05. In the absence of adaptive immunity, infection with mixed P. murina organisms induces non-protective inflammatory responses, including recruitment of innate immune cells.23,24 We evaluated the innate immune responses in the alveolar spaces (BALF) and lung parenchyma of the saline- and anidulafungin-treated mice following euthanasia at day 72 and day 120 post-infection, respectively (Fig. 6). A direct comparison between the groups is not advisable due to the difference in study endpoints. Rather, the following data are presented as a snapshot of the lungs at the time of euthanasia. An average of 119 pg/ml TNFα was detected in the BALF of the saline-treated mice at day 72 post-infection, while the lavage of anidulafungin-treated mice contained an average of 17 pg/ml TNFα at day 120 post-infection. The majority of the cells in the alveolar spaces and lung parenchyma of saline-treated mice were CD11c− CD11b+ non-resident innate immune cells. The bulk of cells within this population were F4/80low neutrophils. In contrast, most of the cells in the alveolar spaces of the anidulafungin-treated groups were CD11c+ CD11b− immature macrophages or dendritic cells, and the majority of the CD11c− CD11b+ non-resident cells were F4/80high, indicating recruitment of monocytes or macrophages rather than neutrophils. These data indicate that cysts are required for the development of inflammatory responses associated with Pneumocystis pneumonia. Additionally, trophic forms are able to survive in immunocompromised lungs for extended periods of time in the absence of cysts and without being able to complete their life cycle. Discussion The data reported here confirm that the trophic stage of P. murina is sufficient to induce CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to Pneumocystis pneumonia. We have previously reported that cysts are required for robust early innate and adaptive immune responses, including recruitment of CD4+ T cells and B cells into the alveolar spaces.16 While the magnitude of the early response was reduced in the animals inoculated with trophic forms alone, both the mice inoculated with trophic forms and the mice inoculated with mixed P. murina cleared the infection by 30 days post-inoculation. Previously we found that mice infected with trophic forms developed detectable cysts by day 7 in adult mice and day 14 post-infection in neonates.16 Because of this, we were unable to discern whether trophic forms failed to induce protective immune responses, or if the response to trophic forms was merely delayed.16 Here, we used the β-1,3-glucan synthase inhibitor anidulafungin to evaluate the adaptive and innate immune responses stimulated by trophic forms in the absence of cysts. Both CD4+ T cells and B cells are required for clearance of P. murina during primary infection, but either CD4+ T cells or antibody are sufficient for clearance during secondary infection.7,9,10,25–27 We report here that adoptive transfer of CD4+ T cells from trophic form- or mixed P. murina-infected animals was sufficient to mediate clearance of trophic forms in recipient mice. However, adoptive transfer of CD4+ T cells from trophic form- or mixed P. murina-infected animals was sufficient to mediate clearance of mixed P. murina organisms in only one mouse among the group of four animals by day 30 post-infection. Previous studies from our laboratory and others demonstrate that transfer of CD4+ T cells from mixed P. murina-infected animals is sufficient to mediate clearance of mixed P. murina organisms in SCID mice.7,26,28,29 Therefore, we predict that mixed P. murina-stimulated CD4+ T cells would eventually mediate clearance of the infection, given sufficient time. While macrophages are required for clearance of Pneumocystis organisms, these cells are not competent to resolve the infection in the absence of adaptive immunity.7–10 Here, a control population of RAG2−/− mice was infected with mixed Pneumocystis organisms and treated with saline, but were not given CD4+ T cells. As expected, this group contained a relatively high average fungal burden of 9.89 × 106 trophic forms and 8.69 × 105 cysts at day 30 post-infection, with an average of 11.4 trophic forms to cysts (data not shown). This population reconfirms that the adoptive transfer of CD4+ T cells was required for the control of fungal growth. In addition, this control population demonstrates that while the majority of the CD4+ T cell recipient mice failed to clear the mixed P. murina organisms by day 30 post-infection, the adoptive transfer of CD4+ T cells was sufficient to limit fungal growth. This observation is consistent with our prediction that the transfer of P. murina-stimulated CD4+ T cells would eventually mediate clearance of the mixed P. murina infection, given sufficient time. Intriguingly, our data suggest that the CD4+ T cell-mediated clearance of trophic forms during secondary infection occurs more rapidly in the absence of cysts. This phenomenon occurs independently of the composition of the priming infection. Our previous evidence indicates that cysts enhance, rather than inhibit the inflammatory response.16 Therefore, we suggest that the more efficient clearance of trophic forms observed in the absence of cysts may be due to slower growth kinetics of trophic forms in the absence of cysts. Linke et al. demonstrate that anidulafungin does not limit the expansion of trophic forms during the first 2 weeks of treatment.30 However, our long-term anidulafungin model (Fig. 6) demonstrates poor expansion of the trophic population due to the prolonged absence of the cyst stage. In the absence of anidulafungin, a single mature cyst produces eight progeny which develop into trophic forms. Clearance of the trophic population by immune cells may be accelerated in the absence of this source of nascent trophic forms. Previously reported data demonstrate that cysts are required for robust numbers of activated CD4+ T cells within the alveolar spaces and lung parenchyma of infected animals.16 However, we report here that the adoptive transfer of trophic form-stimulated CD4+ T cells leads to increased numbers of activated CD4+ T cells in the lung parenchyma of recipient mice at day 15 post-infection compared to transfer of CD4+ T cells from mixed infection. It is noteworthy that fewer CD4+ T cells were harvested from the draining lymph nodes of the wild-type donor mice infected with trophic forms compared to mixed P. murina infection (data not shown). However, the adoptive transfer of equal numbers of CD4+ T cells reveals that these CD4+ T cells are fully capable of mediating clearance. Our previous data indicated that inoculation with trophic forms leads to fewer IFN-γ-producing CD4+ T cells and suboptimal IFN-γ concentrations in the alveolar spaces compared to inoculation with mixed P. murina organisms.16 Furthermore, trophic form-stimulated BDMCs failed to produce TNFα, IL-1β, and IL-6 in response to various other stimuli, and failed to induce IFNγ production during co-culture with CD4+ T cells.16 Conversely, the data reported here show that trophic forms are sufficient to drive CD4+ T cell-mediated TNFα, IFN-γ, and IL-13 production in vivo. These data suggest that stimulation with trophic forms does not skew the immune response away from Th1 or Th2-type responses. While Th17 responses are critical for control of many fungal infections, including candidiasis, a mixed T helper response is observed in response to Pneumocystis infection. This mixed response appears to be largely redundant, as Th1-, Th2-, and Th17-type responses have all been associated with clearance of P. murina organisms.30–35 Cumulatively, our data suggest that trophic forms suppress the development of innate immunity, and thus delay the initiation of adaptive responses16, but trophic forms do not have a direct suppressive effect on CD4+ T cells in vivo. We have reported that cysts are required for an early increase in B cells in the lungs of infected mice.16 However, here we report that primary infection with trophic forms is sufficient to induce antibody-mediated clearance of P. murina trophic forms and cysts during secondary infection. No differences in B cell count were observed in the animals rechallenged with trophic forms or mixed P. murina organisms. Rechallenge of CD4+ T cell-depleted mice with trophic forms promoted increased numbers of activated macrophages and dendritic cells (CD11c+ CD11b+) in the alveolar spaces compared to mice rechallenged with mixed P. murina. However, these data were generated by a single timepoint following clearance (day 15), and differences between the groups may be due to the kinetics of clearance, rather than differences in the magnitude of the response. Intriguingly, serum antibody bound sonicated mixed P. murina antigen at higher titer than trophic antigen. This phenomenon was observed even in serum from anidulafungin-treated animals that had never developed cysts. Previous studies suggest that there is significant overlap in the glycoproteins, including glycoprotein A (gpA, alternatively, major surface glycoprotein, MSG), on the surface of trophic forms and cysts.36,37 Our data suggest that serum antibody raised against trophic forms binds conserved material on the cyst life cycle stage. The data may also indicate that there is more of this material on the cysts than on the trophic forms. Regardless, our data indicate that while serum antibody binds mixed P. murina antigen at higher titer than trophic forms, antibody-mediated responses are sufficient for clearance of both trophic forms and cysts. While the initiation of innate and adaptive responses is required for clearance of Pneumocystis organisms in immunocompetent hosts, the nonspecific provocation of inflammation leads to immune-mediated damage in immunocompromised hosts that progress to Pneumocystis pneumonia.23,24 It has been previously reported that treatment of immune-reconstituted mice with the β-1,3-glucan synthase inhibitor anidulafungin results in depletion of cysts and a reduced inflammatory response.30 Here we report that long-term anidulafungin treatment of trophic form-infected RAG2−/− mice results in the carriage of a relatively small population of trophic forms without progression to pneumonia. Control RAG2−/− mice developed cysts and progressed to pneumonia characterized by weight loss, and recruitment of neutrophils into the lungs. In contrast, the majority of innate immune cells within the alveolar spaces of anidulafungin-treated mice were immature alveolar macrophages and lung dendritic cells. Linke et al. propose that depletion of cysts would reduce inflammation-induced lung damage in patients, and our data further corroborates this suggestion.30 However, our data highlight the need for a greater understanding of the impact of trophic forms on inflammatory responses and host health. While no overt symptoms of pneumonia were detected in our anidulafungin-treated mice, it has been reported that trophic forms induce direct damage to alveolar epithelial cells.38 In our model, the fungal lung burden of RAG2−/− mice with severe pneumonia may consist of as many as 1 × 108 trophic forms. It is unclear why the expansion of trophic forms was limited in RAG2−/- mice during long-term treatment with anidulafungin. Linke and Cushion et al. demonstrate that a short-term course of β-1,3-glucan synthase inhibitor therapy does not hamper expansion of trophic forms.30,39 However, the extended time course of our study suggests that cysts enhance the growth of the trophic forms by an unknown mechanism. It is thought that the formation of the cyst requires sexual reproduction, and it is possible that the growth of the fungus is severely limited when the trophic forms are restricted to asexual reproduction.40 Meiosis followed by multiple rounds of mitosis within the formed cyst results in a quadrupling of the number of organisms which may be important for more rapid expansion of organisms. In the absence of this rapid expansion of organisms via sexual reproduction, it is possible that innate immune cells may kill enough of the trophic forms to limit the growth of the population, but not enough to clear the organisms from the lungs. Alternatively, a signal or product from the cyst or cyst-stimulated host cells could be required for expansion of trophic forms. Trophic forms are found clustered within a biofilm-like substance that includes material from previously ruptured cysts.12 Depletion of cysts may deprive the trophic forms of this protective shelter, and may permit killing of trophic forms by innate immune cells. Whatever the mechanism, it is interesting that in an immunosuppressed environment the trophic population does not collapse in the absence of cysts. This would certainly limit the use of glucan synthase inhibitors as routine monotherapy for PCP. We have previously reported that infection with trophic forms in the absence of cysts leads to the delayed initiation of innate and adaptive responses against Pneumocystis infection.16 Despite these delays, we found here that the trophic stage of P. murina is sufficient to induce CD4+ T cell- and antibody-mediated responses leading to clearance of infection but not progression to Pneumocystis pneumonia, as defined by severe weight loss and infiltration of innate immune cells into the lungs. These data suggest that immune evasion by the trophic forms may hinge on the suppression of the initiation of the innate immune response. The development of adaptive immunity may represent a “point of no return” at which the trophic forms are no longer able to escape clearance. Manipulation of the immune response to trophic forms and cysts may provide new options for the treatment and prevention of Pneumocystis infection, while a failure to consider these differential responses may hamper future efforts. Here, our data indicate that trophic forms elicit adaptive responses, but do not provoke the non-protective inflammation characteristic of Pneumocystis pneumonia. Further evaluation of the antigenic determinants on trophic forms and cysts may elicit a vaccine that provides protection while limiting immune-mediated damage, including immune reconstitution syndrome. Acknowledgements We thank Melissa Hollifield for technical assistance. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Funding This work was supported by a Public Health Services grant from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health [R21 AI118818]. 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Medical MycologyOxford University Press

Published: Dec 18, 2017

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