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Secreted fungal virulence effector triggers allergic inflammation via TLR4

Nature volume 608, pages 161–167 (2022)Cite this article

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Abstract

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3,4,5,6,7,8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9,10,11,12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

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Fig. 1: Cryptococcus promotes arginase-1 expression in macrophages via a soluble, capsule-independent mechanism.
Fig. 2: Identification of CPL1 as a fungal effector by forward genetics.
Fig. 3: CPL1 potentiates IL-4 signalling via TLR4.
Fig. 4: CPL1 promotes arginase-1 expression in pulmonary interstitial macrophages and is required for virulence.

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Data availability

The primary read files as well as expression count files for RNA-seq data in this paper are available to download from the Gene Expression Omnibus under accession number GSE203483. Source data are provided with this paper.

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Acknowledgements

We thank G. Barton for provision of Tlr2−/−, Tlr4−/− and Tlr2−/−Tlr4−/− mice, and R. Ricardo-Gonzalez for provision of Il4ra−/− and Stat6−/− mice; S. Chou for advice on protein purification; J. Cyster and E. Goldberg for critically reading the manuscript, discussions and advice; and S. Catania and M. Boucher for discussions and advice. Support was provided by the Chan–Zuckerberg Biohub, US National Institutes of Health, Jane Coffin Childs Memorial Fund for Medical Research Fellowship and Beckman Foundation.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

    Eric V. Dang, Susan Lei, Atanas Radkov & Hiten D. Madhani

  2. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

    Regan F. Volk & Balyn W. Zaro

  3. Cardiovascular Research Institute, University of California, San Francisco, CA, USA

    Regan F. Volk & Balyn W. Zaro

  4. Chan–Zuckerberg Biohub, San Francisco, CA, USA

    Hiten D. Madhani

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Contributions

E.V.D. and H.D.M. conceived and designed the project. E.V.D. and H.D.M. designed experiments and wrote the manuscript. E.V.D. performed most of the experiments (including ELISA, flow cytometry and protein purification) and analysed the generated data. S.L. performed the forward genetic arrayed screen and helped with flow cytometry experiments. A.R. helped design and perform protein purification experiments. R.F.V. and B.W.Z. provided human monocyte-derived macrophages and read and edited the manuscript.

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Correspondence to Hiten D. Madhani.

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Extended data figures and tables

Extended Data Fig. 1

(a) TNF ELISA on supernatants from BMDMs infected for 24 h with the indicated yeasts at MOI = 10. n = six biologically independent samples. Significance determined by one-way ANOVA with Bonferroni test. (b) Intracellular FACS staining for iNOS after 24 h of infection with either C. neoformans or S. cerevisiae at the indicated MOIs. n = three biologically independent samples. (c) RNA-seq heatmap depicting log2 fold changes of the indicated pro-inflammatory genes in BMDMs following 6 h of stimulation. (d) RNA-seq heatmap depicting log2 fold changes of the indicated M2/tolerized genes in BMDMs following 6 h of stimulation. (e) Representative FACS plots of ARG1 and iNOS expression in BMDMs following 24 h of stimulation with PBS, IL-4 (40 ng/ml), or LPS (100 ng/ml) and IFNγ (50 ng/ml). Data are presented as mean values +/– SD. ****p < 0.0001.

Source data

Extended Data Fig. 2

(a) Ranked Z-scores of hits from forward genetic screen for C. neoformans Arg1 induction. (b) List of validated screen hits and gene descriptions. (c) Representative FACS histograms of GXM staining on the indicated C. neoformans strains cultured overnight in 10% Sabouraud media. (d) RT-qPCR for CPL1 mRNA expression in cultures grown to OD600 = 1.0 in the indicated conditions (A.U. = arbitrary units relative to ACT1). n = three biologically independent samples. (e) Melanin production in WT or cpl1Δ strains grown at 30 °C on L-DOPA plates. (f) TNF production (measured by ELISA) to the indicated stimulations. n = six biologically independent samples. Significance determined by one-way ANOVA with Bonferroni test. Data are presented as mean values +/– SD. ****p < 0.0001.

Source data

Extended Data Fig. 3

(a) Quantification by competitive ELISA of CPL1-6xHis in supernatants from the indicated strains grown in mammalian tissue culture conditions to OD = 1.0. n = six biologically independent samples. (b) RT-qPCR for Arg1 mRNA in BMDMs stimulated with the indicated C. neoformans strains (OD = 0.1) along with IL-4 (10 ng/ml) for 24 h. Expression normalized to Actb, a.u. = arbitrary units. n = three biologically independent samples. (c) RT-qPCR for Mrc1 mRNA in PMA-differentiated THP-1 cells stimulated with the indicated C. neoformans strains (OD = 0.1) along with IL-4 (10 ng/ml) for 24 h. Expression normalized to Actb, a.u. = arbitrary units. n = six biologically independent samples (d) RT-qPCR for Mrc1 mRNA in primary human monocyte-derived macrophages stimulated for 24 h with PBS or recombinant human IL-4 (10 ng/ml) along with the indicated C. neoformans strains (MOI = 0.1). Expression normalized to Actb, a.u. = arbitrary units. n = three biologically independent samples (e) RNA-seq read counts of the indicated genes in BMDMs stimulated for 24 h with either PBS, IL-4 (10 ng/ml), rCPL1 (111 nM), or IL-4 + rCPL1. n = three biologically independent samples. (f) Transwell migration assay on splenic eosinophils towards supernatants from BMDMs stimulated as in (e). n = three biologically independent samples. Data are presented as mean values +/– SD.

Source data

Extended Data Fig. 4

(a) Representative FACS staining of surface IL-4Rα levels on BMDMs stimulated for 24 h with the indicated C. neoformans strains (MOI = 10). n = six biologically independent samples (b) Phospho-FACS for pSTAT3 (left) and pSTAT6 (right) after 30 min of stimulation with PBS, IL-4 (10 ng/ml), rCPL1 (111 nM), or IL-4 + rCPL1. (c) Western blot for pSTAT6 or total STAT6 on BMDMs stimulated for the indicated times with either IL-4 (10 ng/ml) alone, rCPL1 (111 nM) alone, or IL-4 + rCPL1. Data are representative of three independent experiments. (d) Phospho-FACS for pSTAT3 (left) or pSTAT6 (right) in BMDMs after 8 h of stimulation with PBS, IL-4 (10 ng/ml), rCPL1 (37 nM), or rCPL1+IL-4. (e) Phospho-FACS for pSTAT3 in BMDMs stimulated with 111 nM rCPL1 for the indicated time points. Data are presented as mean values +/– SD.

Source data

Extended Data Fig. 5

(a) Arginase-1 FACS in Myd88+/+ or Myd88–/– BMDMs stimulated for 24 h with the indicated concentrations of rCPL1 alone (left) or in combination with IL-4 (10 ng/ml). n = three biologically independent samples. (b) Arginase-1 FACS gated on CD45.2+ BMDMs from the indicated genotypes co-cultured with a 50:50 mix of CD45.1 BoyJ BMDMs and stimulated for 24 h with IL-4 (10 ng/ml) or IL-4 + rCPL1 (111 nM). n = three biologically independent samples. (c) Arginase-1 FACS on BMDMs stimulated for 24 h with the indicated concentrations of LPS. (d) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of LPS alone or with 10 ug/ml cholera toxin B (CTB). n = three biologically independent samples. (e) Measurement of pyroptosis by LDH release assay on BMDMs stimulated with the indicated concentrations of rCPL1 alone or with 10 ug/ml CTB. n = three biologically independent samples. (f) Arginase-1 FACS on BMDMs stimulated with rCPL1 that was either kept on ice or boiled at 100 °C for 15 min. Cells were stimulated with either rCPL1 alone (left) or in combination with IL-4 (right). n = three biologically independent samples. (g)(h) Arginase-1 FACS in in BMDMs stimulated with the indicated concentrations of rCPL1 alone (g) or in combination with IL-4 (10 ng/ml) (h) that were either treated with control or polymyxinB. n = three biologically independent samples. (i) Arginase-1 FACS on BMDMs transduced with MSCV-empty or MSCV-CPL1 retrovirus and stimulated for 24 h with the indicated concentrations of IL-4. n = three biologically independent samples. (j) Silver stain on SDS-PAGE gel of rCPL1-6xHis or rCPL1(Y160A)-6xHis purified from P. pastoris. Image is representative of three independent experiments. Data are presented as mean values +/– SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Source data

Extended Data Fig. 6

(a) Arginase-1 FACS in Tlr4+/+ or Tlr4–/– BMDMs stimulated for 24 h with the indicated concentrations of rCPL1-6xHis purified from C.n. supernatants alone or in combination with IL-4 (10 ng/ml) (b). n = three biologically independent samples. Data are presented as mean values +/– SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Source data

Extended Data Fig. 7

(a) Representative FACS gating and quantification of lung eosinophils after 10 days of intranasal infection with the indicated C.n. strains. (b) Representative FACS gating and quantification of mediastinal lymph node GC B cells. (c) Representative FACS gating and quantification of GC B cell antibody isotype. (d) Representative FACS gating and quantification of effector CD4+ T cells. (e) Representative FACS gating and quantification of cytokine production from effector CD4+ T cells after 4hrs of stimulation with PMA, Ionomycin, and GolgiSTOP. **p < 0.01 by one-way ANOVA with Bonferroni test.

Source data

Extended Data Fig. 8

(a) Quantification of YARG expression by FACS in lung interstitial macrophages in mice infected for 10 days with 5x104 CFU of the indicated strains. (b) Quantification of eosinophils in mice infected for 10 days with 5x104 CFU of the indicated strains. (c) Kaplan-Meier survival curve analysis of mice infected with WT, cpl1Δ, or cpl1Δ+CPL1 C.n. (N = 6 mice per group); ****p < 0.0001 by Mantel-Cox test. (d) Brain CFUs from mice infected with WT, cpl1Δ, or cpl1Δ+CPL1 C.n (5x104 CFU) for 14 days. (e) Lung CFUs on G418-non-resistant (left) or -resistant (right) colonies from mice infected for 10 days with a 50:50 mix of the indicated strains.

Source data

Extended Data Fig. 9

(a) Quantification of lung eosinophils in WT or Tlr4–/– mice infected for 10 days with 5x104 CFU C. neoformans. (b) Lung CFUs from Tlr4–/– or Tlr4–/– mice infected with wild type C.n. (5x104 CFU) for 10 days (c) FACS quantification of lung eosinophils in WT (N = 6 mice) or Tlr4–/– (N = 4 mice) mice sensitized intranasally with rCPL1. (d) Model of how secreted CPL1 modulates the macrophage inflammatory state (created using Biorender.com). p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Bonferroni test.

Source data

Supplementary information

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Uncropped gel images from western blots for pSTAT3 and pSTAT6.

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Dang, E.V., Lei, S., Radkov, A. et al. Secreted fungal virulence effector triggers allergic inflammation via TLR4. Nature 608, 161–167 (2022). https://doi.org/10.1038/s41586-022-05005-4

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