Immune reconstitution during Pneumocystis lung infection: disruption of surfactant component expression and function by S-nitrosylation

Abstract

Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4(+) T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4(+) cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.

FIGURE 1

CD4-depleted mouse model of PCP and IRD. A, C57BL/6 mice were immunosuppressed by selective CD4 depletion via i.p. injection twice weekly with the mAb GK1.5. One week following the initiation of CD4 depletion, mice were inoculated with P. murina cysts as described in Materials and Methods. Immunosuppression was continued for 2, 4, and 6 wk to promote P. murina infection as previously published (42). B, IRD was produced by discontinuation of immunosuppression through withdrawal of GK1.5 2 wk after inoculation. Mice were then sacrificed 2 or 4 wk (i.e. 2 wk + 2 wk or 2 wk + 4 wk groups, respectively) after removal of GK1.5.

FIGURE 2

Recovery of CD4⁺ cells after withdrawal of GK1.5 Ab results in clearance of P. murina. PCP in continuously CD4-depleted mice and IRD mice were generated as described in Materials and Methods and schematically illustrated in Fig. 1. A, For flow cytometric analysis, total spleen cells stained for CD3 and CD4 as described in Materials and Methods were subjected to FACS. The data were expressed as the percentage of CD4 expressing CD3⁺ T cells (mean ± SEM; n = 4–8 in each group). At baseline, nondepleted controls demonstrated 54% CD4⁺ T cells. *, p < 0.05 for reconstituted group vs corresponding CD4-depleted group at the same interval postinfection; #, p < 0.05 vs from corresponding treatment group; &, p < 0.05 vs nondepleted mice. B, P. murina burden after withdrawal of GK1.5. Viable P. murina were quantitated by real-time PCR measurement of rRNA copy number using a standard curve of known copy number of P. murina 18S RNA as described in Materials and Methods. Data are expressed logarithmically as copy number (mean ± SEM; n = 4–8 in each group). *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time post infection; #, p < 0.05 vs from corresponding treatment group; @, p < 0.05 vs CD4-depleted PCP group 2 wk postinoculation.

FIGURE 3

Immune reconstitution following P. murina infection induces significant worsening of lung inflammation. A, Representative morphological changes in formalin-fixed, paraffin-embedded, H&E-stained right lung sections prepared from uninfected and P. murina-infected CD4-depleted or IRD mice harvested 2, 4, and 6 wk postinoculation as labeled. Original magnification ×100. B, Histological scoring of lung inflammation. Median inflammation scores were determined by blinded evaluation of stained sections from each treatment group as described in Materials and Methods. #, p < 0.05 vs CD4-depleted 2 wk P. murina infected group.

FIGURE 4

Immune reconstitution following P. murina infection induces infiltration of lung parenchyma by CD4 and CD8 cells. Total RNA isolated from the left lungs of mice as in Fig. 2 was reverse transcribed, and CD4, CD8, and 18S RNA signals were amplified as described in Materials and Methods. Ct values obtained were normalized to 18S signals and further analyzed using the relative quantitation (ΔΔCt) method. Data are expressed as fold change (mean ± SEM; n = 5 in each group). *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinfection; &, p < 0.05 vs uninfected and nondepleted group.

FIGURE 5

P. murina in immune-reconstituted mice is associated with increased BALF cellularity. A, Total BALF cell counts and (B) differential cell counts for macrophages (MP), lymphocytes (LC), eosinophils (EO), and neutrophils (NP) were performed as described in Materials and Methods in P. murina-infected CD4-depleted or IRD mice harvested 2, 4, and 6 wk postinoculation. The data are expressed as cell numbers × 1000. Values are shown as mean ± SEM (n = 5–20 animals in each group). Multiple comparisons were made by ANOVA. *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinfection; #, p < 0.05 vs corresponding treatment group; ^, p < 0.05 vs uninfected group.

FIGURE 6

Lung injury by P. murina infection is enhanced in IRD mice. A, Total protein content of BALF fractions of uninfected and P. murina-infected CD4-depleted or IRD mice was determined as described in Materials and Methods. Data are expressed as mean ± SEM (μg) per mouse. B, Body weights and lung weights from uninfected and P. murina-infected CD4-depleted or IRD mice were recorded. Group mean data (±SEM) are expressed as a ratio of lung-to-body weight at terminal endpoints. C and D, Quantitation of total NO (C) and nitrates (D) in BALF of uninfected and P. murina-infected CD4-depleted or IRD mice. BALF samples were analyzed by chemical reduction and chemiluminesence as described in Materials and Methods. Data are expressed as mean ± SEM (in nmol); n = 4–8 in each group. For all panels, *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinfection; #, p < 0.05 vs corresponding treatment group; ^, p < 0.05 vs uninfected group.

FIGURE 7

Immunoreconstitution of C57BL/6 mice following inoculation with allogenenic lung homogenate does not produce significant lung injury. CD4-depleted C57BL/6 mice were inoculated with 100 μl of lung homogenate (LH) from uninfected nu/nu (BALB/c background) donors. A control group consisted of mice subjected to CD4 depletion only for 6 wk (CD4 Depleted). Four weeks postinoculation, GK1.5 was withheld from indicated groups (4 + 2; 4 + 4) to produce immunoreconstitution. Mice were sacrificed as indicated at 4, 6, and 8 wk after LH inoculation. A, Total BALF cell counts were determined using a Coulter counter as described in Materials and Methods. Data are expressed as cell numbers × 1000. Values are shown as mean ± SEM (n × 5–7 animals in each group). B, Total protein content of BALF fractions was determined by the method of Bradford (48). Data are expressed as mean ± SEM (μg) per mouse.

FIGURE 8

Surfactant biophysics and function are impaired in IRD mice. At the indicated time postinoculation, the biophysically active LA surfactant fraction was prepared from harvested BALF. A, Samples of LA fractions were separated by SDS-PAGE and immunoblotted with SP-B Ab as described in Materials and Methods. Band density was quantified and is expressed as percentage of uninfected level (mean ± SEM; n = 5 in each group). B, Total phospholipid in LA was estimated using a modification of the colorimetric Bartlett method as described in Materials and Methods. Data are expressed as mean ± SEM (μg) of n =10–20 samples. C, Surface activity of LA surfactant was determined by measuring capillary openness by capillary surfactometer as described in Materials and Methods. Values are obtained by averaging triplicate measurements of each sample and group mean data (mean ± SEM, expressed as percentage of capillary openness (100 being fully open); n = 4–6 samples/time point). For all panels, * p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinfection; #, p < 0.05 vs corresponding treatment group; ^, p < 0.05 vs uninfected group.

FIGURE 9

IRD alters the quaternary structure of SP-D and production of SNO-SP-D. A, Total SP-D in BALF was determined by Western blotting and densitometric scanning as described in Materials and Methods. Data were normalized to uninfected control and are reported as mean ± SEM (expressed as percentage of control; n = 4–6 samples per time point). *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinfection; #, p < 0.05 vs corresponding treatment group; ^, p < 0.05 vs uninfected group. B, BALF from P. murina-infected CD4-depleted or IRD mice 6 wk postinoculation was analyzed for SNO-SP-D content by the biotin switch assay as described in Materials and Methods. Shown are blots obtained from two separate analyses of two independent experiments. SNO-SP-D formation is consistently increased in the IRD samples. C, BALF normalized for equal calculated amounts of total SP-D (as determined by reduced SDS PAGE) in A from P. murina-infected CD4-depleted or IRD mice 6 wk postinoculation was analyzed by native PAGE and Western blotting with SP-D antiserum to determine quaternary structure of SP-D. Multimeric SP-D is incapable of entering the gel at the top, while smaller molecular mass forms are seen only in BALF from reconstituted mice. Data are representative of duplicate determinations from two independent experiments.

FIGURE 10

Immune-reconstitution induces macrophage chemotaxis through SNO-SP-D. A, BALF from uninfected or P. murina-infected CD4-depleted mice with or without IRD was harvested 2, 4, or 6 wk after inoculation and assayed for the ability to induce RAW 264.7 macrophage migration using a modified Boyden chamber. Migration, defined as the number of cells transitioning the barrier membrane after 3 h of incubation, was determined by manual counting. Data represent group mean values (±SEM) from measurements performed in triplicate from two independent experiments and analyzed by ANOVA. *, p < 0.05 for IRD mice vs corresponding CD4-depleted group at same time postinoculation; #, p < 0.05 vs corresponding treatment group; ^, p < 0.05 vs uninfected group. B, To eliminate the effect of SNO modification, BALF from P. murina-infected CD4-depleted mice or IRD mice was harvested 6 wk after inoculation and pretreated with 20 mM ascorbic acid or PBS as indicated and analyzed for the ability to induce RAW cell chemotaxis in vitro as in A. Data are normalized as percentage of P. murina-infected continuously CD4-depleted mice (6 wk). All measurements were performed in triplicate and are representative of two independent experiments analyzed by ANOVA. *, p < 0.05 for PBS treated BALF from IRD mice vs corresponding CD4-depleted group; #, p < 0.05 vs corresponding PBS-treated BAL.