Pneumocystis infection in an immunocompetent host can promote collateral sensitization to respiratory antigens

Abstract

Infection with the opportunistic fungal pathogen Pneumocystis is assumed to pass without persistent pathology in immunocompetent hosts. However, when immunocompetent BALB/c mice were inoculated with Pneumocystis, a vigorous Th2-like pulmonary inflammation ensued and peaked at 14 days postinfection. This coincided with a 10-fold increase in the number of antigen-presenting cells (APCs) in the lung, and these cells were capable of presenting antigen in vitro, as well as greater uptake of antigen in vivo. When mice were presented with exogenous antigen at the 14-day time point of the infection, they developed respiratory sensitization to that antigen, in the form of increased airway hyperresponsiveness upon a later challenge, whereas mice not infected but presented with antigen did not. Like other forms of collateral sensitization, this response was dependent on interleukin-4 receptor signaling. This ability to facilitate sensitization to exogenous antigen has been previously reported for other infectious disease agents; however, Pneumocystis appears to be uniquely capable in this respect, as a single intranasal dose without added adjuvant, when it was administered at the appropriate time, was sufficient to initiate sensitization. Pneumocystis infection probably occurs in most humans during the first few years of life, and in the vast majority of cases, it fails to cause any overt direct pathology. However, as we show here, Pneumocystis can be an agent of comorbidity at this time by facilitating respiratory sensitization that may relate to the later development or exacerbation of obstructive airway disease.

Fig. 1.

Kinetics of the immune response to Pneumocystis in BALF of immunocompetent BALB/c mice. NK cells and neutrophils are the first immune cells to increase in large numbers in the BALF, followed closely by CD4 and CD8 T cells and then B cells and eosinophils, together with a gradual increase in alveolar macrophages. Anti-Pneumocystis antibody titers increase in BALF between 14 and 21 days, and this coincides with the clearance of Pneumocystis (arrow, assay detection limit). Values are means ± standard errors of the means (n = 5), representative of three independent experiments. O.D., optical density. *, P = 0.05 to 0.01; ***, P < 0.001.

Fig. 2.

The Th2 cytokine IL-4 is moderately elevated in BALF during the peak of inflammation (A), while the Th2 cytokine IL-5 is elevated only early after Pneumocystis inoculation (B). Values are means ± standard errors of the means (n = 4), representative of two independent experiments. *, P = 0.05 to 0.01; **, P = 0.01 to 0.001.

Fig. 3.

Vigorous pulmonary inflammation in wild-type immunocompetent mice 14 days after inoculation with 10⁷ Pneumocystis organisms (A and B) compared to normal lung histology (C). In addition to influx of inflammatory cells, there is hypertrophy of airway epithelial cells (arrows) and formation of BALT near airways and blood vessels (arrowheads). A Th2 environment is suggested by the presence of multinucleate giant cells (open arrowhead). Staining is standard H&E. Magnification, ×200.

Fig. 4.

Antigen-presenting cells in the lung during Pneumocystis infection. (A) Levels of putative APCs (MHC-II^hi CD11c⁺) peak in the lung at 14 days after Pneumocystis inoculation. Cells are from collagenase digestion of the lung and are measured as CD45⁺ MHC-II^hi CD11c⁺ using flow cytometry. Values are means ± standard errors of the means (n = 5), representative of three independent experiments. (B) In vitro proliferation of ova-specific (D011.10) CD4 lymphocytes when they were cocultured with CD11c⁺ cells obtained from collagenase digestion of cells from mice obtained 14 days after Pneumocystis inoculation or noninfected control mice incubated with increasing concentrations of ovalbumin. Data are representative of those from two independent experiments, with each data point measured in triplicate. (C) Relative expression of cell surface markers on APCs obtained by collagenase digestion and analyzed by flow cytometry. Dark histogram, expression on cells obtained 14 days after inoculation with Pneumocystis; light gray histogram, expression of the same molecule on cells from uninfected mice. Histograms are representative of staining seen in two independent experiments with 4 to 5 mice per group. ***, P < 0.001.

Fig. 5.

Percentages of CD11c⁺ cells that are positive for fluorescent label 24 h after i.n. inoculation with 50 μg of Alexa Fluor 633-tagged ovalbumin (OVA-633). Cells are either BALF cells (BAL) or cells from a collagenase digestion of the lung after alveolar lavage (Lung). Mice are either uninfected controls or mice tested 14 days after inoculation with Pneumocystis (PC 14d). Values are means ± standard errors of the means (n = 4), representative of two independent experiments. Bars indicate the groups being compared by Tukey's test. ***, P < 0.001.

Fig. 6.

AHR (Penh) is elevated in BALB/c mice in response to antigen challenge after intranasal exposure to that antigen at 14 days after Pneumocystis inoculation. Methacholine response curves after challenge with ova are presented as Penh values. Three groups were inoculated with 10⁷ Pneumocystis organisms, and then the first group was given 50 μg ova i.n. at 14 days postinoculation (OVA 14d), the second group was given 50 μg ova i.n. at 21 days postinoculation (OVA 21d), and the third group was not exposed to ova until the final challenge (PC only). The other three groups were not inoculated, and then one group was sensitized to ova with an i.p. injection of ova-alum 21 days before challenge [positive control, (+) CON], no ova was given to the second uninoculated group (No PC-OVA), and one group was left untreated until the final challenge [negative control, (−) CON]. All groups received antigen challenge (3 successive days of 1 dose 20 μg ova i.n and then the methacholine challenge and Penh measurements on the subsequent day). Final challenges occurred 30 to 35 days after previous ova exposure. Values are means ± standard errors of the means (n = 4), representative of three independent experiments. P values (**, P = 0.01 to 0.001) are from comparison of the results for the group treated with ova at 14 days postinoculation to those for both the uninoculated group not receiving ova and the group inoculated with Pneumocystis only. Other significant differences are not shown here for purposes of clarity.

Fig. 7.

BALF eosinophil numbers and serum ova-specific IgE of mice that were exposed to ova at different times after Pneumocystis inoculation. Groups are the same as those described in the Fig. 6 legend. Values are means ± standard errors of the means (n = 4), representative of three independent experiments. *, P = 0.05 to 0.01. OD, optical density.

Fig. 8.

IL-4 signaling is necessary for antigen sensitization to occur during Pneumocystis infection. (A) AHR (Penh) of IL-4r KO mice and BALB/c mice in response to antigen challenge. PC, mice inoculated with 10⁷ Pneumocystis organisms. All groups except the positive and negative controls were given 50 μg ova i.n. 14 days postinoculation. One group was sensitized i.p. with ova-alum. The negative control group [(−)CON] was untreated until the final challenges. All groups received antigen and methacholine (Mch) challenges as described in the text. Values are means ± standard errors of the means (n = 4), representative of three independent experiments. **, P = 0.01 to 0.001. (B) Lung inflammation in IL-4r KO mice 14 days after Pneumocystis infection is absent the epithelial hypertrophy, multinucleate giant cells, and organized clusters of inflammatory cells seen in wild-type mice (Fig. 3).