Inbred SJL mice recapitulate human resistance to Cryptococcus infection due to differential immune activation

Abstract

Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.

Keywords: Cryptococcus neoformans; QTL analysis; Th1/Th2; atopic immunity; complement.

Fig 1

SJL/J strain mice are uniquely resistant to cryptococcal infection using highly virulent strains. Various strains of laboratory mice were infected with 5,000 yeast cells of indicated Cryptococcus strains. C57BL/6J (black line), SJL/J (blue), MHC class II KO (dotted dark red), TCR KO (dashed red), and Rag1 KO (solid red) mice were infected with (A) high virulence Cryptococcus neoformans strain H99 and (B) moderate virulence C. neoformans strain D52. For (A) and (B), statistical comparisons indicated strain compared to C57BL/6J mice. N = 8 mice per condition for C57BL/6J and SJL/J and 12–15 mice per condition for the knockout strains divided between two independent experiments. (C) Swiss-Webster (purple), SJL/J (blue), C57BL/6J (black), FVB/J (red), NOD/SHiLtJ (red dashed), and SWR/J (red dotted) mice were infected with high virulence C. neoformans strain H99. (D) SJL/J (blue), C57BL/6J (black), Balb/C (black dashed), A/J (gray), DBA2 (orange dashed), DBA1 (orange), and 129S1/SvImJ (129; green) mice were infected with high virulence C. neoformans strain H99. (E) AKR (green), C3H/HeH (green dashed), CBA (green dotted), and CD-1 mice (purple dashed) mice were infected with high virulence C. neoformans strain H99. For (D) and (E), N = 5 mice per group except C57BL/6J (20) and SJL/J (15). Several similar experiments were combined for these plots. (F) Swiss-Webster (purple), SJL/J (blue), C57BL/6J (black), and FVB/J (red) mice were infected with high virulence C. gattii strain R265. For (C) and (F), N = 10–13 mice per group divided into two independent experiments, except N = 5 for NOD/SHiLtJ. For (C)–(F), statistical comparisons are indicated strain compared to SJL/J mice. For these survival experiments, the mice in (A), (B), (C), and (F) are combined data from females and males with no notable differences observed between females and males, whereas (D) and (E) were performed with female mice. SJL/J (blue), C57BL/6J (black), FVB/J (red), and SJL/J mice dosed with anti-IFN-γ neutralizing monoclonal antibody (blue dashed) were infected with C. neoformans strain H99. At the indicated intervals following infection, lungs (G) and brains (H) were collected and fungal loads were measured. Statistical comparisons are the indicated (color matched) condition compared to SJL/J mice at the same timepoint. For (G) and (H), N = 16 total mice per group per timepoint with four total experimental repeats except for N = 8 with two experimental repeats for 17 days p.i.

Fig 2

Histological analysis shows that cryptococcal infection in SJL/J mice is similar to susceptible strains early but diverges late. Lung sections from C57BL/6J, FVB/J, and SJL/J mice infected with C. neoformans strain H99 at indicated times post-infection. Sections stained with alcian blue, which stains Cryptococcus yeast blue, and PAS, which stains host mucin and microbial carbohydrate bright pink. Note that structures that stain with both alcian blue and PAS appear dark blue or purple. DPI indicates days post-infection. The scale bar indicates 200 µm.

Fig 3

SJL/J mice infected with cryptococci induce a robust cytokine response not featuring Th2-associated cytokines. Cytokine and chemokine levels in lung homogenates from SJL/J (blue), C57BL/6J (black), FVB/J (red), and SJL/J mice dosed with anti-IFN-γ neutralizing monoclonal antibody (blue dashed) mice infected with C. neoformans strain H99 at the indicated timepoints. Statistical comparisons are the indicated (color matched) condition compared to SJL/J mice at the same timepoint. N = 8 total mice over two independent experiments.

Fig 4

Immunophenotyping of lung leukocytes from cryptococcus-infected SJL/J, FVB/J, and C57BL/6J mice. (A–F) Indicated populations measured from lung leukocyte preparations acquired 14 days post-infection in C56BL/6J (black/gray), FVB/J (red), and SJL/J (blue) mice infected with C. neoformans H99. (G–J) Percentage of CD4+ T cells (G and H) and CD8+T cells (I and J) isolated from lungs or spleens expressing IFN-γ (G and I) or IL-17a (H and J) following in vitro stimulation with heat-killed C. neoformans H99. Statistical comparisons are the indicated (color matched) condition compared to SJL/J mice. N = 6 mice per group combined from two independent experiments.

Fig 5

CD4+ T cells, CD8+ T cells, IFN-γ, and TNF-α are all critical for the effective SJL/J response to cryptococcal infection. Day 21 lung fungal loads (A) and brain fungal loads (B) from SJL/J mice dosed with the indicated monoclonal antibodies as well as control FVB/J mice infected with C. neoformans H99. Statistical comparisons are the indicated condition compared to SJL/J mice not dosed with antibodies (blue circles). Data are combined from several similar experiments with at least N = 8 over two experiments for each antibody depletion condition except n = 4 with one experiment for anti-Ly6C. Lung histological sections from similarly treated mice at (C) 17 and (D) 21 days post-infection. For each timepoint, the top rows are alcian blue stained sections and the bottom row sections are stained with H&E. For all images, the scale bar indicates 200 µm.

Fig 6

Recovered SJL/J mice are immune to fatal secondary infection. (A) Survival plot of SJL/J mice infected with C. neoformans H99 (blue lines) or left uninfected (“No 1⁰”; green lines) then allowed to clear infection for 60 days then reinfected with 5,000 (solid lines) or 50,000 (dashed lines) C.g. VGI as indicated. N = 15–19 mice per group combined from three independent experiments. (B) SJL/J mice were dosed with live, formalin-killed (“fK”), or heat-killed (“HK”) C. neoformans H99 or left uninfected and then reinfected with 50,000 C.g. VGI. For B, N = 5 mice per group. Statistical comparisons are the indicated condition compared to SJL/J mice infected with live C. neoformans H99 and then reinfected.

Fig 7

SJL/J resistance to cryptococcal infection is a heritable quantitative trait linked to regions on chromosomes 2 and 11. (A) Cartoon depicting breeding strategies and summarizing outcomes. Initially, SJL/J mice were crossed with FVB strain mice generating the SJL × FVB F1 mice (top row) used in (B). Some of these SJL × FVB F1 mice were backcrossed with FVB/J mice generating SJL × FVB N2 mice used in C and D. (B) Survival plots of two experimental repeats of SJL × FVB F1 (purple), FVB/J (red), SJL/J (blue), and C57BL/6J mice (black) infected with C. neoformans H99. For experiment 1, F1 mice were bred in-house, N = 5 for FVB, SJL, and C57BL/6J and 51 for SJLxFVB F1. For experiment 2, F1 mice were bred under contract by Taconic, N = 5 for FVB, SJL, and C57BL/6J and 20 for SJL × FVB F1. (C) Survival plot of SJL × FVB N2 (purple), FVB/J (red), SJL/J (blue), and C57BL/6J mice (black) infected with C. neoformans H99. N = 10 for FVB, SJL, and C57BL/6J and 208 for SJL × FVB N2. (D) Logarithm of odds plot of linkage analysis from SNP genotyping of the SJL × FVB N2 mice depicted in C. (E) Replot of survival of the SJL × FVB N2 data in (C). Mice were grouped by genotype of the SNPs corresponding to the LOD peaks on chromosomes 2 and 11 depicted in (D). Statistical comparisons in (E) are the indicated condition compared to mice expressing the SJL/J variants at both loci (Blue). These experiments used mixed female and male mice.