Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung

Abstract

Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.

Figure 1

Effect of cryptococcal urease on fungal clearance. Mice were intratracheally infected with 10⁴ C. neoformans and lungs were harvested at weeks 1, 2, and 3 post–infection for analysis of fungal burden. Data, pooled from separate matched experiments, are expressed as the mean CFU per lung ± SE from mice infected with the following strains: wt (strain H99, urease-producing; gray bars, n = 14 week1, n = 33 week 2, n = 12 week 3), ure1 (knockout strain, urease-deficient; white speckled bars, n = 13 week 1, n = 13 week 2, n = 5 week 3), and ure1::URE1 (revertant strain, urease-producing; black bars, n = 7 week 1, n = 17 week 2, n = 7 week 3). *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice.

Figure 2

Effect of cryptococcal urease on pulmonary leukocyte recruitment. Mice were intratracheally infected with 10⁴ C. neoformans. Lungs were harvested at weeks 1, 2, and 3 post–infection and total lung leukocytes were isolated from infected lungs following enzymatic digestion and assessed by visual identification (see Materials and Methods). Data were pooled from separate matched experiments and expressed as mean absolute numbers of recruited leukocytes per lung ± SE for mice infected with the following strains of C. neoformans: wt (strain H99, urease-producing; gray bars), ure1 (knockout strain, urease-deficient; white speckled bars), and ure1::URE1 (revertant strain, urease-producing; black bars). *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice (n = 5 to 33 per group of infected mice as per Figure 1).

Figure 3

Effect of cryptococcal urease on recruitment of leukocyte subsets. Mice were intratracheally infected with 10⁴ C. neoformans. Lungs harvested at weeks 2 (A) and 3 (B) post–infection were enzymatically digested and lung leukocyte subsets were assessed by visual identification (see Materials and Methods). Data were pooled from separate matched experiments and expressed as mean absolute numbers of recruited leukocytes per lung ± SE for mice infected with the following strains of C. neoformans: wt (strain H99, urease-producing; gray bars), ure1 (knockout strain, urease-deficient; white speckled bars), and ure1::URE1 (revertant strain, urease-producing; black bars). *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice (n = 5 to 33 per group of infected mice as per Figure 1).

Figure 4

Effect of cryptococcal urease on the production of serum IgE. Total serum IgE concentrations were measured by ELISA at 3 weeks post–infection C. neoformans. Bars represent mean serum IgE concentration ± SE from mice infected with the following strains of C. neoformans: wt (strain H99, urease-producing; gray bars), ure1 (knockout strain, urease-deficient; white speckled bars), and ure1::URE1 (revertant strain, urease-producing; black bars). *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice (n = 6 to 7 per group of infected mice).

Figure 5

Effect of cryptococcal urease on the morphological pattern of pulmonary inflammation. Mice were either uninfected (A) or intratracheally infected (B–D) with 10⁴ C. neoformans. Representative photomicrographs (H&E-stained; ×100 magnification) taken of lung sections harvested (3 weeks post–infection) from mice which were (A) uninfected or infected with one of the following strains of C. neoformans: B: wt (H99; urease-producing), ure1 (C urease-deficient knockout), or ure1: URE1 (D urease-producing revertant). Note the diffuse infiltrates in mice infected with urease-producing strains (wt, panel B and ure1::URE1, panel D), which consisted of loose conglomerates of visible C. neoformans, eosinophils and macrophages. In contrast, infiltrates in mice infected with the urease-deficient strain (ure1, panel C) contained tightly packed granulomas and bronchovascular infiltrates in which extracellular C. neoformans and eosinophils were notably diminished.

Figure 6

Effect of cryptococcal urease on pulmonary YM1 crystal deposition. Mice were intratracheally infected with 10⁴ C. neoformans. Representative photomicrographs of lung sections (3 weeks post–infection) stained with H&E followed by counterstaining with mucicarmine (to identify C. neoformans) from mice that were infected with wt (urease-producing H99, A–D, I), ure1::URE1 (urease-producing revertant, E–H, J), or ure1 (urease-deficient knockout, K). At high power magnification (A, C, E, G ×400 magnification; B, D, F, H ×1000 magnification), note the presence of both intracellular and extracellular YM1 crystals (arrows) in the lungs of mice infected with wt or ure1::URE1 (urease-producing). At low power magnification (I–K, ×200 magnification), note the absence of crystals in the lungs of mice infected with ure1 (urease-deficient).

Figure 7

Effect of cryptococcal urease on arginase expression by pulmonary macrophages. Mice were either uninfected (A) or intratracheally infected with 10⁴ C. neoformans (B–E). A–C: Representative high power (×1000 magnification) photomicrographs taken of lung sections stained with anti-Arginase1 antibody (peroxidase detection, brown substrate) from (A) uninfected mice or mice infected 2 weeks with (B) wt (urease-producing H99) or (C) ure1 (urease-deficient knockout) strains of C. neoformans. Note the absence of staining in resting alveolar macrophages (RAM; panel A), whereas strong staining indicative of alternatively-activated macrophages (AAM) was evident in mice infected with the urease-producing strain (wt; panel B). In contrast, weak staining suggestive of classically activated macrophages (CAM) was noted in mice infected with the urease-deficient strain (ure1; panel C). D, E: Representative low power (×200 magnification) photomicrographs demonstrate that AAM (strong arginase expression) are more abundant in the lungs of mice infected with wt (D) compared with ure1 (E).

Figure 8

Effect of cryptococcal urease on pulmonary cytokine production. Total lung leukocytes were isolated 2 weeks post–infection with C. neoformans and cultured for 24 hours at 5 × 10⁶ cells/ml in the absence of any stimulation. Supernatants were harvested, and cytokine levels were detected by ELISA. Bars represent mean cytokine (A, IL-4; B, IL-13; C, IFN-γ) concentration ± SE (pg/ml) or (D), the IL-4/IFN-γ ratio, for paired leukocyte cultures obtained from the lungs of individual mice infected with the following strains of C. neoformans: wt (strain H99, urease-producing; gray bars), ure1 (knockout strain, urease-deficient; white speckled bars), and ure1::URE1 (revertant strain, urease-producing; black bars). *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice (n = 6 to 7 per group of infected mice).

Figure 9

Effect of cryptococcal urease on the number and phenotype of conventional dendritic cells. Flow cytometric analysis was performed to identify conventional dendritic cells (cDC) among leukocytes obtained from the lung (A, B) and lung associated lymph nodes (LALN; C–F) 2 weeks following infection with C. neoformans. A, C: Representative histogram demonstrating the gating strategy used to identify cDC (oval gates) among lung (A), and LALN (C) leukocytes as cells expressing modest to high amounts of both MHC Class II (I-A^b; FL-1 channel, fluorescein isothiocyanate) and CD11c (FL-2 channel, phycoerythrin). B, D: Total numbers of cDC within leukocyte populations were determined by multiplying the frequency of cDC by the total number of leukocytes present in each tissue. Bars represent the mean number of DC in lung (B), and LALN (D) ± SE from mice infected with the following strains of C. neoformans: wt (strain H99, urease-producing; gray bars), ure1 (knockout strain, urease-deficient; white speckled bars), and ure1::URE1 (revertant strain, urease-producing; black bars). E, F: Three color flow cytometric analysis was performed on cDC identified within LALN at 2 weeks postinfection and assessed for the expression of the costimulatory molecule, CD80 (FL-3, PerCp). Results are expressed as the percentage (E) and total number (F) of CD80_low cDC identified within LALN leukocytes (same key as above). Bars represent mean ± SE, n = at least three animals per group, *P < 0.05, in comparison with wt, **P < 0.05, in comparison with ure1::URE1 infected mice.