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

Abstract

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.

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 between the log₁₀ FC in the expression of these genes for the wild-type mice (P<0.0001, A) but not for the CD40L-KO mice (P=0.9, B), highlighting the tight correlation between the two experiments for the responses in C57BL/6 mice, and also the strong response of C57BL/6 (A) in contrast with the absence of response of CD40L-KO mice (B). For each experiment, FC was determined by comparing expression of the gene set in exposed mice to expression in unexposed mice of the same strain.

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 mice (40L; Days 35–75). Each row represents a single gene. The brackets on the right indicate the five clusters into which the genes were grouped. Clusters A–D show the genes that were up-regulated significantly at one or more time-points, and the fifth cluster comprises significantly down-regulated genes. Colors represent the log₁₀ FC, using the scale at the top of figure (B). The graphs (A–D) show for C57BL/6 mice the log₁₀ FC over time for the individual genes in each of the four clusters, into which significantly up-regulated genes were segregated using K-means clustering.

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 mice following Pneumocystis infection included granzymes, which peaked at Day 14 and were subsequently down-regulated; IFN-γ-induced genes (including chemokine ligands and receptors), the majority of which peaked at Day 34, remained elevated but dampened at Day 41, and declined to base-line by Day 75; and Ig-related genes, the majority of which increased only at Day 41 but remained elevated through Day 75. (B) The 17 genes that were differentially expressed at Day 35 in CD40L-KO mice are shown together with results for the same genes in C57BL/6 mice at Days 7–75. The first five were also up-regulated significantly in C57BL/6 mice.

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 of the genes in these clusters. All the genes in the network are included in the two clusters. The intensity of the color is proportional to the FC seen at Day 34. The abbreviations and full gene names are included in Supplementary Tables 3 and 4.

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 by Western blot in the unexposed animals or the exposed animals at 2 weeks. GAPDH is included as a control to demonstrate that approximately equal amounts of protein were loaded into each lane.