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J Leukoc Biol. Aug 2008; 84(2): 420–430.
Published online May 8, 2008. doi:  10.1189/jlb.1207816
PMCID: PMC2493075

Immune responses to Pneumocystis murina are robust in healthy mice but largely absent in CD40 ligand-deficient mice


Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. Microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knockout (CD40L-KO) mice over time following exposure to Pneumocystis murina. Immunocompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the up-regulation of 349 primarily immune response-associated genes. Temporal changes in the expression of these genes identified an early (Week 2), primarily innate response, which waned before the infection was controlled; this was followed by primarily adaptive immune responses that peaked at Week 5, which coincided with clearance of the infection. In conjunction with the latter, there was an increased expression of B cell-associated (Ig) genes at Week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no up-regulation of immune response-associated genes at Days 35–75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust, biphasic response to infection by Pneumocystis; CD40L is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia.

Keywords: immunodeficiency diseases, rodent, lung


Pneumocystis jirovecii is a ubiquitous fungus that causes life-threatening pneumonia in immunodeficient patients, especially those with HIV infection [1]. Although clinically significant pneumonia does not occur in immunocompetent individuals, serological studies have demonstrated that most humans have been infected by the age of 2, presumably as a result of a clinically inapparent infection [2, 3]. Studies of immune responses to Pneumocystis have largely used immunodeficient animal models or cell-depletion studies to identify important immunoregulatory mechanisms. Such studies have demonstrated a critical role for CD4 cells in immunity against Pneumocystis infection. However, only a limited number of studies have examined immune responses in unmanipulated, immunocompetent hosts [4].

Pneumocystis can infect a variety of mammalian species. Each species is infected by a genetically distinct member of the genus: P. jirovecii infects humans, Pneumocystis carinii and Pneumocystis wakefieldiae infect rats, and Pneumocystis murina infects mice. Key features of P. murina infection have rendered it experimentally useful for studying immunity to Pneumocystis, in part as a result of the availability of mice with defined immune defects. Following exposure to the organism, immunocompromised murine hosts, such as scid mice, or those deficient in CD40 ligand (CD40L), develop a progressive pulmonary disease, which histologically and clinically resembles human Pneumocystis pneumonia (PcP) [5,6,7]. In contrast, as we and others have shown recently [8, 9], healthy mice become infected following exposure but have a peak organism load ~5 weeks after exposure that is two to three logs below the peak seen in immunodeficient hosts and can clear the infection in a matter of weeks with little residual compromise. Little is known about early events, including innate immune responses, in such healthy hosts.

CD40L is a costimulatory molecule expressed on activated T cells that plays a critical role in T cell- and B cell-mediated responses [10, 11]. CD40L is also expressed on a number of additional immune-related cells, including γδ T, NK, NKT, and dendritic cells (DC) and macrophages, as well as nonimmune cells. CD40L knockout (KO) mice have a specific immune defect that makes them highly susceptible to PcP, with levels of infection similar to that seen in scid or Rag-1-deficient mice; humans with a homologous abnormality, resulting in the hyper-IgM syndrome, are similarly highly susceptible to Pneumocystis infection [12, 13]. However, the interaction of CD40L with other aspects of the immune response to Pneumocystis has not been defined. In the current study, we sought to investigate the responses to Pneumocystis infection that occur in healthy hosts and to determine which of these responses were deficient in CD40L-KO hosts. We used microarray techniques to examine changes in expression of over 12,000 genes in the lungs of wild-type and CD40L-KO mice infected with P. murina. Such a genome-wide approach provides a means to simultaneously examine a broad range of immune responses and thereby yield biologically meaningful results, not only about individual genes but also about gene networks and multi-network genetic programs of host defense.



Healthy, ~8-week-old, female C57BL/6 mice were obtained from the National Cancer Institute (NCI; Frederick, MD, USA) or from The Jackson Laboratory (Bar Harbor, ME, USA). CD40L-KO (B6;129S2-Tnfsf5tm1Imx/J) and scid (CBySmn.CB17-Prkdcscid/J) mice were bred in-house, initially using breeders purchased from The Jackson Laboratory. All studies were carried out under protocols approved by the National Institutes of Health (NIH) Clinical Center Animal Care and Use Committee (Bethesda, MD, USA).

Study design

As a primary goal was to characterize immune responses to naturally acquired infection (similar to what occurs in humans), infection was transmitted via the respiratory route by co-housing study animals with immunosuppressed seeder animals (scid or CD40L-KO) that had active PcP, which was subsequently verified by a quantitative real-time PCR (Q-PCR) assay [8]. This has been previously shown to result in infection of co-housed animals with predictable kinetics and infection of healthy animals peaking at 35–42 days [8]. We used this approach rather than intratracheal inoculation of organisms, as the latter, although commonly used in studies of Pneumocystis, will deliver a substantially larger organism load as a bolus and may induce immune responses that differ qualitatively or kinetically from those seen during naturally acquired infection [14]. In four separate experiments, C57BL/6 and/or CD40L-KO mice were co-housed with a P. murina-infected seeder or remained unexposed. Mice (three to 10 per time-point per group) were killed at varying time-points ranging from 7 to 75 days.

Lungs and blood were obtained from each animal when they were killed. Lungs were split in two and stored at –20°C; one portion was placed in RNAlater™ (Qiagen, Valencia, CA, USA) for microarray analysis and the other in PBS for quantitation of organism load by Q-PCR. Blood was obtained by cardiac puncture, and serum was stored at –20°C for subsequent detection of anti-P. murina antibodies by ELISA [8]. P. murina Q-PCR and ELISA results for animals from the first experiment have been reported previously in a study describing the Q-PCR assay [8]. For immunohistochemistry studies, mice were exposed to P. murina, animals were killed at 14, 35, or 42 days after exposure, and portions of the lung were stored at –20°C for Q-PCR or Western blot analysis or frozen with O.C.T. compound (Sakura Finetek USA, Torrance, CA, USA) in cryomolds on a slurry of dry ice with isopentane and stored at –20°C for subsequent immunohistochemical staining.


DNA extraction from lung homogenates and subsequent quantitation of P. murina dihydrofolate reductase (DHFR) gene copies per mg lung by means of a real-time PCR were performed as described previously [1, 8]. As DHFR is a single-copy gene, the number of copies reflects the number of nuclei. The presence of anti-P. murina antibodies was detected using an ELISA technique as described previously [8].

Microarray hybridization and analysis

Total RNA was extracted from lung tissue using an RNeasy mini kit including Qiashredder columns and DNase treatment (Qiagen), following the manufacturer’s instructions.RNA (4–10 μg) was reverse-transcribed using a SuperScrip™ ds cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, CA, USA) and the GeneChip® T7-Oligo(dT) promoter primer kit (Affymetrix, Santa Clara, CA, USA). The cDNA was purified following the procedures described by Affymetrix and was transcribed in vitro and biotin-labeled using the BioArray High Yield RNA transcript labeling kit (ENZO Life Science Inc., Farmingdale, NY, USA). The resulting labeled cRNA was purified, and 20 μg was fragmented using 5× fragmentation buffer and heated to 95°C for 35 min.

Hybridization cocktail (Affymetrix) containing 15 μg fragmented cRNA was added to an MG-U74Av2 array (Affymetrix) and hybridized for 16 h at 45°C in a Hybridization Oven 640 (Affymetrix). The arrays were stained and washed using the GeneChip® Fluidics Station 400 using R-PE streptavidin (Molecular Probes, Eugene, OR, USA) and a biotinylated goat anti-streptavidin antibody (Vector Laboratories, Burlingame, CA, USA). Experiments 1 and 2 were scanned using the GeneArray® 2500 scanner. The GeneChip® Scanner 3000 was used to scan Experiments 3 and 4. The signal intensity was quantitated using Microarray Suite Software, Version 4.0 (MAS 4.0), or GeneChip® Operating Software v1.2 (GCOS 1.2; both from Affymetrix). The data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI; U.S. National Library of Medicine, Bethesda, MD, USA) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession Number GSE11005.

RT-PCR analysis of gene expression

To validate the microarray gene expression results, expression of a panel of differentially expressed genes, including genes that were up-regulated, unchanged, and down-regulated, was examined using a real-time, two-step, semi-QRT-PCR assay, based on TaqMan® technology (Applied Biosystems, Foster City, CA, USA). The oligonucleotide sequences for individual genes are provided as supplemental data (Supplemental Table 1). For CCR2, CCL2, and CXCL10, reagents were purchased as Taqman® predeveloped assay reagents for gene expression. The cDNA was amplified in triplicate in an ABI PRISM™ 7900 HT sequence detection system instrument. Relative standard curves were used to calculate the fold-change (FC) of the gene of interest after normalizing the values to those of GAPDH (rodent GAPDH control reagents), which were run in parallel. We used the same RT-PCR assay for determining the change in expression of TLR-2 and TLR-4 in the early exposure to P. murina, as they are important, innate immunity effectors but are not included in the MG-U74Av2 array. The oligonuclotide sequences are provided as supplemental data (Supplemental Table 1).

Statistical analysis

Affymetrix MAS 5 signal and present call values were stored in the NIHLIMS, a local database for storage and retrieval of Affymetrix GeneChip data, which were retrieved and analyzed using the Mathematical and Statistical Computing Lab Analyst’s Toolbox (P. J. Munson, J. Barb, 2004; http://abs.cit.nih.gov/MSCLtoolbox/) and the JMP statistical software package (SAS Inc., Cary, NC, USA). Data were first normalized to the median value of each chip and then logarithmically transformed after the addition of a small value (1% of the median) to dampen the influence of extremely small data values. A one-way ANOVA was performed to determine the significance of changes within each experiment. Affymetrix genes were selected if the P value was less than 0.001, and the FC between the exposed compared with the unexposed animals of the same strain was at least 1.5-fold or at most 1/1.5-fold. K-means and hierarchical clustering were performed using JMP. Heat maps were generated using Genesis [15].

Gene list analysis

NCBI databases, primarily LocusLink, its substitute Entrez Gene, and Pubmed, as well as Mouse Genome Informatics 3.2 database (The Jackson Laboratory) were used to further explore the gene lists provided by the statistical treatment of the data. Gene list subsets assigned to specific gene ontology categories were annotated by means of the National Institute of Allergy and Infectious Diseases-developed tool Database for Annotation, Visualization and Integral Discovery (DAVID; http://david.abcc.ncifcrf.gov/home.jsp) and Expression Analysis Systematic Explorer (EASE; http://david.abcc.ncifcrf.gov/ease/ease.jsp) [16, 17]. To explore the relations of genes within the relevant gene lists and the possible existence of networks comprising them, we used EASE as well as Ingenuity Pathways analysis (Ingenuity® Systems, Redwood City, CA, USA). The latter was also used for additional data annotation and mining. Genes annotated by means of the Affymetrix website reflect the available data as of March 2005.


To verify the results of the microarray analysis that suggested the presence of different cell populations during the course of Pneumocystis infection, in situ immunohistochemistry was performed using frozen lung sections from healthy C57BL/6 mice obtained at Weeks 2, 5, and 6 following exposure and from CD40L-KO mice obtained at Weeks 2 and 5 following exposure. Unexposed mice were used as controls. The following primary antibodies were used: anti-Ly49s, a cocktail of three mAb (4D11, 4E5, and 1F8, kindly provided by Drs. John Ortaldo and Robin Winkler-Pickett, NCI-Frederick) to detect NK cells, anti-CD4 (clone H129.19), and anti-CD8 (clone 53-6.7) T cell markers (BD Biosciences PharMingen, San Diego, CA, USA); anti-CD19 (clone 1D3) B cell marker (BD Biosciences PharMingen); and anti-CD68 (clone FA-11) macrophage marker (AbD Serotec, Raleigh, NC, USA). The secondary antibody was an Alexa Fluor 488-conjugated donkey anti-rat IgG (H&L; 20 μg/ml, Molecular Probes). Staining was performed by Histoserv, Inc. (Germantown, MD, USA). Images were collected on a Leica TCS-NT/SP1 confocal microscope (Leica Microsystems, Exton, PA, USA) using a 40× oil-immersion objective numerical aperture (NA) 1.32. Images of CD19 staining were collected on a Nikon Eclipse E800 using a 40× plan-fluor oil-immersion objective NA 1.30.

All regions of the specimen were examined to ensure that observed changes were consistent throughout the sample. To quantitate cell populations, positive cells were visually counted in 10 random fields per section using an epifluorescence microscope and a 40× or 60× objective; the same objective was used for all samples of a given mouse strain and cell type. The average number of positive cells per field was compared. To quantitate reactivity with anti-mouse CD68, the mean fluorescence intensity (MFI) was calculated for 10 fields per lung-tissue section using Leica TCS-SP software. MFIs were compared using Student’s t-test.

Western blot

Lung tissue was homogenized at 100 mg tissue per ml homogenization buffer [0.05 M Tris, pH 8.0, 0.12 M NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 10 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 1× protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, USA)], separated on 4–20% Tris-glycine SDS-PAGE gels (Invitrogen Life Technologies), and transferred to nitrocellulose membranes (Invitrogen Life Technologies). After blocking, the membrane was incubated with anti-ClCa3 antibody (kindly provided by Dr. Hiroki Iwashita, Takeda Pharmaceutical Co. Ltd., Osaka, Japan), followed by peroxidase-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Anti-mouse GAPDH mAb (Calbiochem, La Jolla, CA, USA) reactivity was analyzed simultaneously to demonstrate that equal amounts of protein were loaded on the gel. Reactivity was detected using Amersham ECL™ advance Western blotting detection kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).


Microarray techniques identify substantial immune responses to Pneumocystis

We initially sought to determine if microarray techniques could detect changes in gene expression in the lungs of animals following infection with P. murina. To address this, the lungs of C57BL/6 (n=3) and CD40L-KO (n=3) animals were examined 32 days after exposure, which approximately corresponds to the peak of infection in the wild-type animals. At this time, the mean P. murina load (log organisms per mg lung tissue) was similar for the two groups of animals: 2.39 log copies/mg (~245 copies/mg) for the wild-type C57BL/6 mice and 3.05 log copies/mg (~1100 copies/mg) for the CD40L-KO mice. Results from co-housing experiments previously reported by our group showed that from this time-point on, the organism load increases in the CD40L-KO mice, reaching 5.5 log copies/mg (~300,000 copies/mg) at Day 75 of exposure, and the infection is cleared by 7–8 weeks in wild-type C57BL/6 mice [8]. Unexposed, control cages were negative for P. murina by Q-PCR. No anti-P. murina antibodies were detected in any cage at this time-point [8].

Microarray results for Experiment 1 were analyzed initially by ANOVA, identifying 418 probe sets that were differentially expressed (P<0.003) in exposed compared with unexposed animals of the same strain. EASE analysis revealed that this list was highly enriched in immune response-related gene categories, as all eight categories selected using a Bonferroni-corrected P value <0.05 were immune response-related. EASE analysis, restricted to up-regulated genes in the healthy, exposed group (n=152), found 18 over-represented categories, of which 17 (94.4%) were immune response-related (Table 1), including genes induced by IFN-γ, macrophage, and T cell-related genes and genes for chemokine receptors and ligands. Up-regulated genes in the CD40L-KO-exposed group (n=70) and down-regulated genes in either group (n=132) were not over-represented significantly in any category. RT-PCR for six genes encompassing a broad range of levels of expression showed FC similar in magnitude to the microarray results for this experiment (Table 2).

EASE Analysis of Genes Significantly Up-Regulated in Healthy Mice at Day 32 of Exposure
Validation of FC in Gene Expression by Real-Time RT-PCR

Biphasic immune response to Pneumocystis in immunocompetent animals

This initial experiment demonstrated that microarray analysis of lung homogenates was able to identify robust immune responses in wild-type but not in CD40L-KO mice. We undertook additional experiments to better characterize the kinetics of immune responses to P. murina infection in wild-type mice. In all studies, exposed animals showed the expected increase in organism load and anti-P. murina antibodies with time, and unexposed animals were negative for both (data not shown).

ANOVA analysis was performed for each experiment separately to identify those genes that were differentially regulated in exposed compared with unexposed animals, using a P value ≤0.001 and a FC ≥1.5 or ≤1/1.5 at any time-point as criteria for selection, following which the selected genes from all experiments were combined into a single list for further analysis. A total of 448 genes fulfilled the selection criteria, of which 349 were significantly up-regulated (FC≥1.5) in at least one experiment. To validate the reliability of our model, we compared the log10 FC in all 448 genes in wild-type mice at Day 34, Experiment 2, to the log10 FC for the same genes at Day 32, Experiment 1. We found a highly significant correlation (R=0.85; P<0.0001) between the values for the two samples (Fig. 1).

Fig. 1.
Correlation between Experiments 1 (32 days of exposure to P. murina, C57BL/6, and CD40L-KO mice) and 2 (34 days of exposure to P. murina, C57BL/6 mice) for the 448 genes that were differentially expressed in the study. There was a highly significant correlation ...

K-means clustering was used to categorize the up-regulated genes into four clusters (Fig. 2A), which segregated by time following exposure. Complete lists of the genes that were included in each cluster are included as supplemental material (Supplemental Tables 2–5). Cluster A represents 52 genes that share a similar expression profile, showing an early increase with peak expression at Day 14, followed by a return to baseline levels after Day 21. Cluster B represents 218 genes that exhibit a mild peak at Day 34, whereas cluster C represents a smaller group of 32 genes that exhibits a more dramatic peak at the same time-point. Finally, cluster D represents a group of 47 genes that rises dramatically after Day 34 (Fig. 2B).

Fig. 2.
(A) Heat map showing the log10 FC over time for the 448 genes identified in the study. Days following exposure to Pneumocystis are shown at the top. The values on the left (Days 7–75) are for C57BL/6 mice (57B); those on the right are for CD40L-KO ...

Each of these gene clusters was analyzed individually by EASE as well as by a literature search for individual genes to characterize biological responses represented by that cluster. Cluster A has hallmarks of a primarily innate, NK cell-associated, immune response to Pneumocystis in wild-type mice, characterized by a marked increase in expression of granzymes B–G (Fig. 3A) [18,19,20,21], killer cell lectin-like receptors (Klrd1 and Klra3) [22,23,24,25], and cathepsin W [26]. Two genes involved in the signaling transduction of NK receptors are also expressed {TYRO protein tyrosine kinase-binding protein (Tyrobp) and phospholipase Cγ2 [27,28,29,30,31]}. At this stage of P. murina infection, there was significant overexpression of three IFN-type I (α, β)-related genes: IFN regulatory factor 7 (Irf7), which appears to be a critical regulatory factor for production of Type 1 IFNs [32,33,34]; IFN-α-inducible protein (G1P2; clone IFI15; and a similar expressed sequence tag), which among other properties, plays a role in NK differentiation and proliferation and is involved in stimulating IFN-γ production by T cells [35,36,37,38,39,40]; and IFN-stimulated exonuclease gene 20 kDa (Isg20), which is induced by type-1 IFNs.

Fig. 3.
Heat map for select subgroups of genes. Colors represent the log10 FC, using the scale at the top of the figure. Each row represents a unique gene and is labeled with the Affymetrix probe set ID and gene title. (A) Genes that were up-regulated in C57BL/6 ...

Up-regulation of cathepsin E [41], CCR2, Irf7, Isg20, and Tyrobp suggests activation of DC, and increased expression of the γ-chain of the TCR likely reflects the influx of γδ T cells, a subset usually present in the airway mucosa with limited antigen recognition that contributes to host defense early in infection [42]. Additional up-regulated genes include those encoding acute-phase proteins such as calgranulin A (S100a8) and calgranulin B (S100a9), which form a heterodimer and are secreted by macrophages or neutrophils [43,44,45], and a subset of chemokine-related genes, including CCR2, CCR5, and CCL5 (RANTES).

As the genes in cluster A waned, those in clusters B and C, which represent aspects of an adaptive immune response, increased. Both clusters represent common immunological processes taking place at Day 34 of exposure; the clusters differ, mainly in the intensity of the up-regulation. Genes in clusters B and C are expressed mainly in DC, macrophages/monocytes, and T cells. There are also some genes expressed in B cells and lung cells (alveolar macrophages and epithelial and endothelial cells). We found a subset of genes coding for cell surface molecules corresponding to antigen receptors, mainly the TCR, and chemokine receptor CXCR3 (genes associated with signaling pathways of both receptors were also up-regulated at this time-point), costimulatory molecules, FcRs, and CD and histocompatibility antigens. The functional analysis of clusters B and C also identified a substantial number of IFN-γ-induced genes [46] (Fig. 3A), including members of the CC and CXC chemokine families, complement components, histocompatibility class II antigens, FcRs, GTPases, immunoproteasome-active sites, and signal transducer activators and inhibitors. Two other gene subsets related to antigen presentation and involved in the immunological synapse were also identified. Signatures of adaptive immune response such as genes related to phagocyte function and T cell activation, differentiation, and regulation were also categorized, together with genes involved in B cell function regulation. A network created by means of Ingenuity Pathways highlights the widely interconnected relations among some of the up-regulated key genes included in clusters B and C (Fig. 4).

Fig. 4.
Network created by means of Ingenuity Pathways using genes up-regulated in clusters B and C in C57BL/6 mice. The network was one of many generated following input of the genes in clusters B and C and was selected to illustrate the complex interactions ...

Taken together, the pattern of up-regulated genes following 34 days of exposure of healthy mice to P. murina suggests a Th1 response. In addition to evidence of IFN-γ responses, there is up-regulation of three chemokine receptors expressed by Th1 cells: CCR2, CCR5, and CXCR3. CCR2 and CCR5 peak at Day 14 and therefore, are included in cluster A but remain up-regulated at Day 34. Among the chemokines expressed by Th1 cells, CCL2 (MCP-1), CCL4 (MIP-1β), CCL5 (RANTES at Day 14), CXCL9 (monokine induced by IFN-γ), and CXCL10 (IFN-inducible protein 10) are all up-regulated. These in turn attract cells bearing the aforementioned chemokine receptors, colocalizing Th1 cells, NK cells, and macrophages [47]. Also, four MCPs, CCL2, CCL7, CCL8, and CCL12, are up-regulated at this time-point.

Two of the most highly up-regulated genes at Day 34 of exposure are ClCa3 (also know as Gob-5; FC=47.9) and Saa3 (FC=17.9). ClCa3 is a member of the calcium-activated chloride channel family and is selectively expressed in the airway goblet cells [48,49,50]. Western blot analysis confirmed the high ClCa3 expression found by microarray and RT-PCR (Fig. 5). Although its function is not elucidated completely, it has been shown to increase mucin (muc5ac) expression and mucus production and to have a role in diseases with secretory dysfunctions and airway hyper-responsiveness (asthma and cystic fibrosis) [48,49,50]. Recently, its role as a chloride channel has been questioned, as no transmembrane region seems to be present [51, 52]. Also, its role in mucus overproduction has been found not to be essential in a ClCa3 KO mouse [53], but a role in regulation of tissue inflammation in the innate immune response has been suggested recently [49]. Saa3 is a major acute-phase protein that is expressed in macrophages, and although its role in immune defense is yet to be determined, it has been described as a T cell chemoattractant [54, 55].

Fig. 5.
Western blot showing the differential expression of ClCa3 in C57BL/6 mice unexposed and exposed to P. murina for 2, 5, and 6 weeks. Increased levels of protein can be seen in four of the six exposed animals at Weeks 5 and 6. No expression is detected ...

The fourth cluster (D) represents a group of genes that is up-regulated primarily at Day 41. This cluster is composed almost exclusively of Ig genes (Fig. 3A) and signals B cell infiltration and/or activation. It is noteworthy that up-regulation of these genes is maintained through Day 75 following exposure.

Microarray gene expression results for a subset of genes were confirmed by RT-PCR. Table 2 shows the FC in gene expression, as determined by microarray and RT-PCR, for selected genes when compared with unexposed animals. Although the absolute FC varied between the two assays, the rank order and relative magnitude of change were in general similar. By RT-PCR, TLR-2 was up-regulated significantly at Day 14 of exposure (P=0.005), and TLR-4 was not up-regulated significantly at any tested time-point.

Lack of immune response in CD40L-KO mice

In contrast to the robust immune response detected in C57BL/6 mice, CD40L-KO mice exposed to Pneumocystis were lacking such immune responses at the later stages of infection that were examined by microarray analysis. At 35 days post-exposure, only 17 genes met the selection criteria for differential expression (P value ≤0.001 and a FC ≥1.5 or ≤1/1.5). Nine were up-regulated, five of which were also up-regulated in C57BL/6 mice (Fig. 3B), and eight were down-regulated. At 39 days post-exposure, no gene with a false discovery rate of <20% met the selection criteria. At 75 days post-exposure, at a time when the Pneumocystis organism load was ~300,000 copies/mg lung tissue, 96 genes were differentially expressed, of which 56 were up-regulated and 40 down-regulated. Only 11 of these genes were also included in the 448 genes identified in immunocompetent C57BL/6 mice, and none was immune-function genes. EASE analysis of these 96 genes did not identify enrichment of any immune response-related gene categories; identified categories were primarily related to intracellular organelles and metabolic processes (Supplementary Table 4).

Immunohistochemical staining identifies immune cell influx into the lungs of immunocompetent but not CD40L-KO mice

Frozen lung tissue sections were stained to examine the frequency of various cell populations at different time-points (Supplemental Fig. 1). In healthy C57BL/6 animals, an increase in CD4+, CD19+, and CD68+ cells was seen when compared with unexposed controls, most prominently at Week 6, at which point, the changes were significant for all three populations (Table 3). No changes were seen at Week 2 in any of these cell populations, and no increase in CD8+ or Ly49+ cells was seen at any time. Among the CD40L-KO animals, no changes were seen at Weeks 2 or 5 compared with unexposed animals.

Quantitation of Different Cell Populations Over Time Following Exposure to P. murina


Our studies have shown the evolution of the immune response to naturally acquired Pneumocystis in the healthy host, which progresses in a bimodal manner. In contrast to this, CD40L-KO animals, which are highly susceptible to Pneumocystis infection, showed little evidence by microarray or immunohistochemical analysis of an immune response to infection at the later time-points that appear critical to clearance in the healthy host.

Our animal model mimics infection of humans by Pneumocystis, as mice get the infection through inhalation of presumably a small number of organisms rather than by direct inoculation into the respiratory tract. Inoculation of a bolus (106–108) of Pneumocystis organisms in healthy animals results in more rapid clearance of the organisms in a response that is characterized by a profound eosinophilic pulmonary infiltration, which is different from what was observed in the current study [14].

We used microarray analysis to obtain a broad survey of host responses following Pneumocystis infection. The advantage of this approach is that it permits the simultaneous evaluation of a large number of genes; thus, the role of different cell types and pathways can be explored at the same time. The reproducibility of this approach was validated by the high level of correlation between the results of the first two experiments (R=0.845; P<0.0001).

Initially there is an up-regulation of genes in the healthy host that reflects an innate immune response (cluster A), which peaks at Day 14 and wanes through Day 21. This initial response is ineffective in clearing Pneumocystis, as the organism load continues to rise until it peaks at ~5 weeks. The main effector cells of this first phase are likely NK or NKT cells and DC, as reflected by the gene networks and families that were up-regulated, such as granzymes. Although the immunohistochemical staining did not identify an increase in NK cells, this may reflect activation in situ without recruitment of such cells or recruitment of a small subset that was not apparent by the staining used. The influx may also be short-lived and not detected by the single, early time-point that was stained.

Also, during this first phase, type I IFN activity was increased, which in other models, down-regulates Th2 responses in Pneumocystis-infected mice, lowering pulmonary complications associated with infection [14]. There is also evidence of an influx of γδ T cells, which have previously been reported to be associated with slower clearance in intratracheally inoculated models of Pneumocystis infection [4, 56]. Although TLRs are not interrogated by the array, we studied the gene expression of TLR-2 and -4 and found that only TLR-2 was up-regulated significantly at Day 14 of infection. In accordance with this, a recent study [57] has shown that normal C57BL/6 mouse macrophages respond to P. murina through TLR-2, with subsequent production of TNF-α and CXCL1, and TLR-4 transcription was not increased. Interestingly, CXCL1 is one of the chemokines highly up-regulated in our study at Day 34.

As the innate response declines, robust, adaptive immune responses are apparent at Days 34 and 41. Coincident with this, there is a stabilization and subsequent decline in organism load, with clearance of Pneumocystis by Day 75 or earlier. At Day 34, genes included in clusters B and C are up-regulated; these genes largely reflect IFN-γ induction and influx or activation of T cells and macrophages. There is evidence of a Th1 response, characterized by the up-regulation of a large set of IFN-γ-induced genes and chemokine signaling genes (Th1-related receptors and ligands). Although previous studies have shown that IFN-γ is not essential for clearance of Pneumocystis, the current study demonstrates a prominent role for IFN-γ-induced genes in the immunocompetent host response [58, 59]. Also, CXCR3 and its ligands CXCL9 and CXCL10 were greatly up-regulated. CXCR3 is expressed by CD4 (preferentially Th1), CD8, NK cells, and B cells. A recent study by McAllister et al. [60] found that clearance of Pneumocystis was delayed, but not impaired, in otherwise immunocompetent, CXCR3-deficient mice, demonstrating that CXCR3 plays an important but not essential role in clearance of Pneumocystis. Our data suggest that the increased expression of a broad range of chemokines and chemokine receptors provides a redundancy to the clearance mechanisms.

Our studies are consistent with the previously established, critical role of CD4 cells and macrophages in clearance of Pneumocystis [61,62,63]. Both cell populations increased at Weeks 5 and 6, and multiple genes related to their function are up-regulated. There was no increase in CD8 cell number, suggesting that this population does not play an important role in clearance of Pneumocystis in healthy animals. Although most studies in immunodeficient models also support this conclusion, CD8 cells can be manipulated to facilitate clearance of the organism in CD4-depleted models [64, 65].

A surprising and previously unreported finding was the prominent influx of B cells at Day 41, which was initially suggested by up-regulation of Ig-related genes in cluster D and confirmed by immunohistochemistry. The role of these B cells in anti-Pneumocystis immunity is unclear. It is noteworthy that genes in this cluster remained up-regulated at Day 75, suggesting that B cells may play a role in long-term immunity to Pneumocystis. Consistent with our data, two studies have recently highlighted an important role for B cells in clearance of Pneumocystis that is independent of antibody production and may be related to the antigen-presenting function of B cells [66, 67].

One of the most striking findings of this study was the marked absence of late immune responses in CD40L-KO mice. Among the immune-related genes up-regulated in C57BL/6 mice, only CCL9, CXCR1, and integrin-β2 were up-regulated in these animals. In two separate experiments looking at an earlier time-point (Day 14) in CD40L-KO animals, inconsistent results were obtained, and no significant up-regulation of innate responses was noted for the group as a whole (data not shown). However, in individual animals, there was up-regulation of granzymes as well as calgranulin A and B, suggesting that innate responses do occur in these animals (Supplementary Fig. 2). Thus, CD40L is a critical molecule for induction of adaptive immune responses to Pneumocystis and is upstream of essentially all of these adaptive responses. This is consistent with reports that CD40L is essential in certain models to the link between innate and adaptive immune responses [68]. Additional studies are needed to better understand the mechanisms by which CD40L orchestrates anti-Pneumocystis responses.

Our study highlights the intricate mechanisms involving multiple cell types that lead to the eradication of Pneumocystis in the healthy host and the critical role that CD40L plays in the generation of these responses. These studies have identified a number of potentially important cellular responses that can now be explored in greater detail to determine the role that they play in Pneumocystis infection in healthy and immunocompromised hosts.

Supplementary Material

[Supplemental Figures and Tables]


This research was supported by the Intramural Research Program of the NIH Clinical Center. The authors have no conflicting financial interests. This paper was presented in part at VIII International Workshops on Opportunistic Protists (IWOP-8) and International Conference on Anaerobic Protists (ICAP; Hilo, HI, USA), July 25–29, 2003, Abstract B29; 42nd Annual Meeting of the Infectious Diseases Society of America (IDSA; Boston, MA, USA), September 30–October 3, 2004, Abstract 1078; and 44th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC; Washington, DC, USA), October 30–November 2, 2004, Abstract B-144. We thank Drs. Meggan Czapiga and Owen Schwartz for performing the confocal imaging and quantitation of fluorescence; Drs. John Orlando and Robin Winkler-Pickett for providing anti-Ly49s antibodies; Dr. Hiroki Iwashita for providing anti-ClCa3 antibody; Drs. James Shelhamer and Anthony Suffredini for providing support and advice; and Rene Costello and Howard Mostowski for providing animal care.


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