Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

English version

Autoři: Patrícia Alves de Castro ^aff001; Ana Cristina Colabardini ^aff001; Adriana Oliveira Manfiolli ^aff001; Jéssica Chiaratto ^aff001; Lilian Pereira Silva ^aff001; Eliciane Cevolani Mattos ^aff001; Giuseppe Palmisano ^aff002; Fausto Almeida ^aff003; Gabriela Felix Persinoti ^aff004; Laure Nicolas Annick Ries ^aff003; Laura Mellado ^aff001; Marina Campos Rocha ^aff005; Michael Bromley ^aff006; Roberto Nascimento Silva ^aff003; Gabriel Scalini de Souza ^aff007; Flávio Vieira Loures ^aff007; Iran Malavazi ^aff005; Neil Andrew Brown ^aff008; Gustavo H. Goldman ^aff001
Působiště autorů: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil ^aff001; Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil ^aff002; Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil ^aff003; Laboratório Nacional de Biorrenováveis (LNBR), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, São Paulo, Brasil ^aff004; Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil ^aff005; Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom ^aff006; Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo, São José dos Campos, Brazil ^aff007; Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom ^aff008
Vyšlo v časopise: Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance. PLoS Genet 15(12): e1008551. doi:10.1371/journal.pgen.1008551
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008551

Souhrn

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Klíčová slova:

Aspergillus fumigatus – Calcium signaling – Cell walls – Gene expression – Mouse models – Osmotic shock – Phosphatases – Transcription factors

Zdroje

1. van de Veerdonk F.L., Gresnigt M.S., Romani L., Netea M.G., & Latgé J.P. Aspergillus fumigatus morphology and dynamic host interactions. Nat. Rev. Microbiol. 15, 661–674 (2017). doi: 10.1038/nrmicro.2017.90 28919635

2. Brakhage A.A. Systemic fungal infections caused by Aspergillus species: Epidemiology, infection process and virulence determinants. The J. Current Drug Targets 6, 875–886 (2005).

3. Steinbach W.J., Lamoth F., & Juvvadi P.R. Potential Microbiological Effects of Higher Dosing of Echinocandins. Clin. Infect. Dis. 61 (Suppl 6), S669–S677 (2015).

4. Brown G.D., Denning D.W., & Levitz S.M. Tackling human fungal infections. Science 336, 647 (2012). doi: 10.1126/science.1222236 22582229

5. Brown G.D., et al. Hidden killers: human fungal infections. Sci Transl Med 4, 165rv13 (2012).

6. Lackner M., & Lass-Flörl C. Up-date on diagnostic strategies of invasive aspergillosis. Curr. Pharm. Des. 19, 3595–3614 (2013). doi: 10.2174/13816128113199990323 23278540

7. Walsh T.J., et al. Infectious Diseases Society of America. 2008. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin. Infect. Dis. 46, 327–360 (2008). doi: 10.1086/525258 18177225

8. Fisher M.C., Hawkins N.J., Sanglard D., & Gurr S.J. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 360, 739–742 (2018). doi: 10.1126/science.aap7999 29773744

9. Brown N.A., & Goldman G.H. The contribution of Aspergillus fumigatus stress responses to virulence and antifungal resistance. J. Microbiol. 54, 243–253 (2016). doi: 10.1007/s12275-016-5510-4 26920884

10. Fox D.S., & Heitman J. Good fungi gone bad: the corruption of calcineurin. Bioessays 24, 894–903 (2002). doi: 10.1002/bies.10157 12325122

11. Cyert M.S. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochem. Biophys. Res. Commun. 311, 1143–1150 (2003). doi: 10.1016/s0006-291x(03)01552-3 14623300

12. Juvvadi P.R., Lee S.C., Heitman J., & Steinbach W.J. Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach. Virulence 8, 186–197 (2017). doi: 10.1080/21505594.2016.1201250 27325145

13. Crabtree G.R., & Graef I.A. Bursting into the nucleus. Sci. Signal. 1, pe54 (2008).

14. Thewes S. Calcineurin-Crz1 signaling in lower eukaryotes. Eukaryot. Cell 13, 694–705 (2014). doi: 10.1128/EC.00038-14 24681686

15. Espeso E. A. The CRaZy Calcium Cycle. Adv. Exp. Med. Biol. 892, 169–186 (2016). doi: 10.1007/978-3-319-25304-6_7 26721274

16. Stathopoulos A.M., & Cyert M.S. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev. 11, 3432–3444 (1997). doi: 10.1101/gad.11.24.3432 9407035

17. Silva Ferreira M.E., et al. Functional characterization of the Aspergillus fumigatus calcineurin. Fungal Genet. Biol. 44: 219–230 (2007). doi: 10.1016/j.fgb.2006.08.004 16990036

18. Steinbach W.J., et al. Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5, 1091–1103 (2006). doi: 10.1128/EC.00139-06 16835453

19. Liu S., et al. Synergistic effect of fluconazole and calcium channel blockers against resistant Candida albicans. PLoS One 11, e0150859 (2016). doi: 10.1371/journal.pone.0150859 26986478

20. Zhang J., et al. Calcineurin is required for pseudohyphal growth, virulence, and drug resistance in Candida lusitaniae. PLoS One 7, e44192 (2012). doi: 10.1371/journal.pone.0044192 22952924

21. Chen Y-L., et al. Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis. Eukaryotic Cell 10, 803–819, (2011). doi: 10.1128/EC.00310-10 21531874

22. Kojima K., Bahn Y-S., & Heitman J. Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans. Microbiology 152, 591–604 (2006). doi: 10.1099/mic.0.28571-0 16514140

23. Lamoth F., Alexander B.D., Juvvadi P.R., & Steinbach W.J. Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species. J. Antimicrob. Chemother. 70, 1408–1411 (2015). doi: 10.1093/jac/dku549 25558076

24. Steinbach W.J., et al. In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus. Antimicrob. Agents Chemother. 48,1664–1669 (2004). doi: 10.1128/AAC.48.5.1664-1669.2004 15105118

25. Narreddy S., Manavathu E., Chandrasekar P.H., Alangaden G.J. & Revankar S.G. In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. J. Antimicrob. Chemother. 65, 701–703 (2010). doi: 10.1093/jac/dkq020 20130026

26. Cramer R.A. Jr. et al. Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot. Cell 7, 1085–1097 (2008). doi: 10.1128/EC.00086-08 18456861

27. Soriani F.M., et al. Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA. BMC Microbiol. 10, 12 (2010). doi: 10.1186/1471-2180-10-12 20078882

28. Soriani F.M., et al. Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA. Mol Microbiol. 67, 1274–1291 (2008). doi: 10.1111/j.1365-2958.2008.06122.x 18298443

29. Dinamarco T.M. et al. Molecular characterization of the putative transcription factor SebA involved in virulence in Aspergillus fumigatus. Eukaryot. Cell 11, 518–531 (2012). doi: 10.1128/EC.00016-12 22345349

30. de Castro P.A., et al. ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway. Mol. Microbiol. 94, 655–674 (2014). doi: 10.1111/mmi.12785 25196896

31. de Castro P.A., et al. The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence. Virulence 8, 797–809 (2017). doi: 10.1080/21505594.2016.1239010 27652896

32. Ries L.N.A., et al. The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect. MBio 8, pii: e00705–17 (2017). doi: 10.1128/mBio.00705-17 28611248

33. Denning D.W. A new class of antifungal. J. Antimicrob. Chemother. 49, 889–891 (2002). doi: 10.1093/jac/dkf045 12039879

34. Valiante V., Macheleidt J., Föge M., & Brakhage A.A.. The Aspergillus fumigatus cell wall integrity signaling pathway: drug target, compensatory pathways, and virulence. Front. Microbiol. 6, 325 (2015). doi: 10.3389/fmicb.2015.00325 25932027

35. Fortwendel J.R. et al., Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin. Antimicrob. Agents Chemother. 54:1555–1563 (2010). doi: 10.1128/AAC.00854-09 20124000

36. Juvvadi P.R., et al. Calcium-mediated induction of paradoxical growth following caspofungin treatment is associated with calcineurin activation and phosphorylation in Aspergillus fumigatus. Antimicrob. Agents Chemother. 59, 4946–4955 (2015). doi: 10.1128/AAC.00263-15 26055379

37. Walker L.A., Lee K.K., Munro C.A., & Gow N.A. Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies. Antimicrob. Agents Chemother. 59, 5932–5941 (2015). doi: 10.1128/AAC.00862-15 26169407

38. Pott G.B., Miller T.K., Bartlett J.A., Palas J.S. & Selitrennikoff C.P. The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus. Fungal Genet. Biol. 31, 55–67 (2000). doi: 10.1006/fgbi.2000.1225 11118135

39. Hagiwara D. et al. The SskA and SrrA response regulators are implicated in oxidative stress responses of hyphae and asexual spores in the phosphorelay signaling network of Aspergillus nidulans. Biosci. Biotechnol. Biochem. 71, 1003–1014 (2007). doi: 10.1271/bbb.60665 17420584

40. Chapeland-Leclerc F. et al. Systematic gene deletion and functional characterization of histidine kinase phosphorelay receptors (HKRs) in the human pathogenic fungus Aspergillus fumigatus. Fungal Genet. Biol. 84, 1–11 (2015). doi: 10.1016/j.fgb.2015.09.005 26365385

41. Silva L.P., et al. Genome-wide transcriptome analysis of Aspergillus fumigatus exposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakA(HOG1) and MpkC dependent. Cell Microbiol. 19 (2017).

42. Beauvais A. et al. Glucan synthase complex of Aspergillus fumigatus. J. Bacteriol. 183, 2273–2279 (2001) doi: 10.1128/JB.183.7.2273-2279.2001 11244067

43. Lee M.J. et al. Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides. J. Biol. Chem. 289, 1243–1256 (2014). doi: 10.1074/jbc.M113.522516 24257745

44. Mouyna I., et al. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J. Biol. Chem. 275, 14882–14889 (2000). doi: 10.1074/jbc.275.20.14882 10809732

45. de Groot P.W. et al., Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans. Fungal Genet. Biol. 46, Suppl 1, S72–81 (2009).

46. Winkelströter L.K., et al. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus. G3 (Bethesda), 5, 1525–1539 (2015).

47. Angeles de la Torre-Ruiz M., et al. Sit4 is required for proper modulation of the biological functions mediated by Pkc1 and the cell integrity pathway in Saccharomyces cerevisiae. J. Biol. Chem. 277, 33468–33476 (2002). doi: 10.1074/jbc.M203515200 12080055

48. Vaughan C.K., et al. Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol. Cell. 31, 886–895 (2008). doi: 10.1016/j.molcel.2008.07.021 18922470

49. Tal R., et al. Aup1p, a yeast mitochondrial protein phosphatase homolog, is required for efficient stationary phase mitophagy and cell survival. J. Biol Chem. 282, 5617–5624 (2007). doi: 10.1074/jbc.M605940200 17166847

50. Bom V.L., et al. The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence. Eukaryot. Cell. 14, 728–744 (2015). doi: 10.1128/EC.00008-15 25911225

51. Balloy V., & Chignard M. The innate immune response to Aspergillus fumigatus. Microbes and Infection 11, 919–927 (2009). doi: 10.1016/j.micinf.2009.07.002 19615460

52. Brown A.J.P., Cowen L.E., di Pietro A., & Quinn J. Stress Adaptation. Microbiol. Spectr. 5(4) (2017).

53. Kim H.R., Chae K.S., Han K.H., & Han D.M. The nsdC gene encoding a putative C2H2-type transcription factor is a key activator of sexual development in Aspergillus nidulans. Genetics. 182, 771–783 (2009). doi: 10.1534/genetics.109.101667 19416940

54. Lamarre C., Ibrahim-Granet O., Du C., Calderone R., & Latgé J.P. Characterization of the SKN7 ortholog of Aspergillus fumigatus. Fungal Genet. Biol.44: 44, 682–690.

55. Spielvogel A., et al. Two zinc finger transcription factors, CrzA and SltA, are involved in cation homoeostasis and detoxification in Aspergillus nidulans. Biochem. J. 414, 419–429 (2008). doi: 10.1042/BJ20080344 18471095

56. Blankenship J.R., & Heitman J. Calcineurin is required for Candida albicans to survive calcium stress in serum. Infect. Immun. 73, 5767–5774 (2005). doi: 10.1128/IAI.73.9.5767-5774.2005 16113294

57. Cunningham K.W., & Fink G.R. Ca2+ transport in Saccharomyces cerevisiae. J. Exp. Biol. 196,157–166 (1994). 7823019

58. Bruder Nascimento A.C., et al. Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence. Mol. Microbiol. 100, 841–859 (2016). doi: 10.1111/mmi.13354 26878695

59. Hagiwara D., Suzuki S., Kamei K., Gonoi T., & Kawamoto S. The role of AtfA and HOG MAPK pathway in stress tolerance in conidia of Aspergillus fumigatus. Fungal Genet. Biol. 73,138–149 (2014). doi: 10.1016/j.fgb.2014.10.011 25459537

60. Lindén M., Laan G., & Anderson P. Neutrophils, interleukin-17A and lung disease. Europ. Respir. J. 25, 159–172 (2005).

61. Loures F.V., Pina A., Felonato M., & Calich V.L. TLR2 is a negative regulator of Th17 cells and tissue pathology in a pulmonary model of fungal infection. J. Immunol. 183, 1279–1290 (2009). doi: 10.4049/jimmunol.0801599 19553529

62. Zelante T., et al. Th17 cells in the setting of Aspergillus infection and pathology. Med. Mycol. 47(Suppl 1), S162–S169 (2009).

63. Becker K.L., et al. Aspergillus cell wall chitin induces anti- and proinflammatory cytokines in Human PBMCs via the Fc-y recepctor/Syk/PI3K pathway. MBio 7, e01823–15 (2016). doi: 10.1128/mBio.01823-15 27247234

64. Rocha M.C., et al. Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence. G3 (Bethesda) 6, 2983–3002 (2016).

65. Ost K.S., et al. The Cryptococcus neoformans alkaline response pathway: identification of a novel rim pathway activator. PLoS Genet. 11, e1005159 (2015). doi: 10.1371/journal.pgen.1005159 25859664

66. Ost K.S., et al. Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation. MBio 8, pii: e02290–16 (2017). doi: 10.1128/mBio.02290-16 28143983

67. Käfer E. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv. Genet. 19, 33–131 (1977). doi: 10.1016/s0065-2660(08)60245-x 327767

68. Furukawa, T. The Negative Cofactor 2 complex is a master regulator of drug resistance in Aspergillus fumigatus (submitted to Nature Communications).

69. Manfiolli A.O. et al. Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence. Cell Microbiol. 19(12) (2017).

70. Bayram O., Bayram O. S., Valerius O., Jöhnk B., & Braus G. H. Identification of protein complexes from filamentous fungi with tandem affinity purification. Methods in Molecular Biology 944, 191–205 (2012). doi: 10.1007/978-1-62703-122-6_14 23065618

71. Park H.S., et al. Calcineurin Targets Involved in Stress Survival and Fungal Virulence. PLoS Pathog. 12, e1005873 (2016). doi: 10.1371/journal.ppat.1005873 27611567

72. Kussmann M., Lässing U., Stürmer C.A., Przybylski M., & Roepstorff P. Matrix-assisted laser desorption/ionization mass spectrometric peptide mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation. J. Mass Spectrom. 32, 483–493 (1997). doi: 10.1002/(SICI)1096-9888(199705)32:5<483::AID-JMS502>3.0.CO;2-J 9180051

73. Cox J. & Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008). doi: 10.1038/nbt.1511 19029910

74. Graham L.M., et al. Soluble dectin-1 as a tool to detect beta-glucans. J. Immunol. Methods 314, 164–169 (2006). doi: 10.1016/j.jim.2006.05.013 16844139

75. Winkelströter L.K. et al., High osmolarity glycerol response PtcB phosphatase is importante for Aspergillus fumigatus virulence. Mol. Microbiol. 96, 42–54 (2015). doi: 10.1111/mmi.12919 25597841

76. François J.M. A simple method for quantitative determination of polysaccharides in fungal cell walls. Nat. Protoc. 1, 2995–3000 (2006). doi: 10.1038/nprot.2006.457 17406560

77. Scheneider C.A., Rasband W.S. & Eliceiri K.W. NIH image to imageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). doi: 10.1038/nmeth.2089 22930834

78. Semighini C.P., Marins M., Goldman M.H., & Goldman G.H. Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl. Environ. Microbiol. 68, 1351–1357 (2002). doi: 10.1128/AEM.68.3.1351-1357.2002 11872487

79. Yim A.K., et al. Using RNA-Seq Data to Evaluate reference genes suitable for gene expression studies in soybean. PLoS One 10, e0136343 (2015). doi: 10.1371/journal.pone.0136343 26348924

80. Bolger A.M., Lohse M., & Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). doi: 10.1093/bioinformatics/btu170 24695404

81. Kopylova E., Noé L., & Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012). doi: 10.1093/bioinformatics/bts611 23071270

82. Nierman W.C., et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151–1156 (2005). doi: 10.1038/nature04332 16372009

83. Kim D., et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013). doi: 10.1186/gb-2013-14-4-r36 23618408

84. Wang L., Wang S., & Li W. RSeQC: quality control of RNA-seq experiments. Bioinformatics 28, 2184–2185 (2012). doi: 10.1093/bioinformatics/bts356 22743226

85. Liao Y., Smyth G.K., & Shi W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013). doi: 10.1093/nar/gkt214 23558742

86. Love M.I., Huber W., & Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 550 (2014). doi: 10.1186/s13059-014-0550-8 25516281

87. Benjamini Y., Drai D., Elmer G., Kafkafi N., & Golani I. Controlling the false discovery rate in behavior genetics research. Behav. Brain Res. 125, 279–284 (2001). doi: 10.1016/s0166-4328(01)00297-2 11682119

88. Cano L.E., Singer-Vermes L.M., Vaz C.A.C., Russo M., & Calich V.L.G. Pulmonary paracoccidioidomycosis in resistant and susceptible mice: relationship among progression of infection, bronchoalveolar cell activation, cellular immune response, and specific isotype patterns. Infect. Immun. 63, 1777–1783 (1995). 7729885

89. Francke A., Herold J., Weinert S., Strasser R.H., & Braun-Dullaeus R.C. Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties. J. Histochem. Cytochem. 59, 813–825 (2011). doi: 10.1369/0022155411416007 21705645