Research Interests

 

Research Achievements of the Lab

 

pic1Fungal infections are under recognized causes of human disease with huge economical and health impact [1, 2].  In particular, Aspergillus fumigatus, an airborne ubiquitous saprophytic fungus (mold), is a major cause of asthma, allergy and chronic inflammation of the lung in immunocompetent adults [1-4]. In immunocompromised patients, A. fumigatus is the most important cause of invasive fungal pneumonia worldwide [1, 2] Opportunistic molds are also implicated in recent fatal outbreaks of fungal meningitis associated with the use of contaminated steroid injections [5]. Collectively, invasive mold infections (IMI) are major causes of death in an expanding population of immunocompromised patients with mortality rates that exceed 90% upon disease dissemination [1, 2]. Better understanding of immunopathogenesis of IMI is required to improve patient’s outcome.

Professional phagocytes, mainly alveolar macrophages (AMs) and neutrophils, phagocytose hundreds of inhaled conidia on a daily basis and prevent fungal germination, inflammation and invasive growth [2]. Physiologically, conidia are efficiently eliminated inside acidified phagolysosomes of AMs via incompletely understood mechanisms.

Our research in centered on the discovery of novel molecular pathways regulating biogenesis of fungal phagosomes. We have recently identified the important role of a new specialized autophagy pathway termed LC3 associated phagocytosis (LAP) [6], in immunity against Aspergillus fumigatus and identified the signaling requirements for activation of LAP [7]. Specifically, we found that LAP is selectively activated during intracellular swelling of A. fumigatus conidia (spores) via a Dectin-1/Syk Kinase/NADPH oxidase signaling pathway. The physiological role of LAP in Aspergillus immunity was also revealed by the discovery that LAP is compromised in patients with genetic defects in NADPH oxidase activity and following administration of corticosteroids, two uniquely predisposed patient populations for development of invasive aspergillosis.

img2In a follow up study we discovered that cell wall melanin selectively targets LAP to promote fungal pathogenicity via excluding the p22phox NADPH oxidase subunit from the phagosome membrane. Of interest, melanin-induced LAP blockade was a principal virulence mechanism because the avirulent melanin deficient A. fumigatus mutant regained full virulence in transgenic mice with conditional inactivation of Atg5 in myeloid cells [8].

Collectively, our findings led to a new model of Aspergillus pathogenicity by demonstrating that two fundamental events for elimination of fungal conidia that occur concomitantly during intracellular swelling (i) exposure of PAMPs and (ii) removal of melanin are required for phagosome maturation and fungal killing. Importantly, LAP blockade is a general property of melanin pigments, a finding with broad implication in pathogenesis of human diseases beyond fungal infections.

 

Selected References

  1. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. Hidden killers: human fungal infections. Sci Transl Med. 2012 Dec 19;4(165):165rv13.
  2. Romani L. Immunity to fungal infections. Nat Rev Immunol. 2011 Apr;11(4):275-88.
  3. Knutsen AP, Bush RK, Demain JG, Denning DW, Dixit A, Fairs A, Greenberger PA, Kariuki B, Kita H, Kurup VP, Moss RB, Niven RM, Pashley CH, Slavin RG, Vijay HM, Wardlaw AJ. Fungi and allergic lower respiratory tract diseases. J Allergy Clin Immunol. 2012 Feb;129(2):280-91; quiz 292-3. doi: 10.1016/j.jaci.2011.12.970.
  4. Denning DW, Pleuvry A, Cole DC. Global burden of allergic bronchopulmonary aspergillosis with asthma and its complication chronic pulmonary aspergillosis in adults. Med Mycol. 2013 May;51(4):361-70
  5. Smith RM, Schaefer MK, Kainer MA, Wise M, Finks J, Duwve J, Fontaine E, Chu A,Carothers B, Reilly A, Fiedler J, Wiese AD, Feaster C, Gibson L, Griese S,Purfield A, Cleveland AA, Benedict K, Harris JR, Brandt ME, Blau D, Jernigan J,Weber JT, Park BJ; Multistate Fungal Infection Outbreak Response Team. Fungal infections associated with contaminated methylprednisolone injections. N Engl JMed. 2013 Oct 24;369(17):1598-609.
  6. Sanjuan MA, Dillon CP, Tait SW, Moshiach S, Dorsey F, Connell S, Komatsu M, Tanaka K, Cleveland JL, Withoff S, Green DR. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007 Dec 20;450(7173):1253-7
  7. Kyrmizi I, Gresnigt MS, Akoumianaki T, Samonis G, Sidiropoulos P, Boumpas D, Netea MG, van de Veerdonk FL, Kontoyiannis DP, Chamilos G. Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling. J Immunol. 2013 Aug 1;191(3):1287-99.
  8. Akoumianaki T, Kyrmizi I, Valsecchi I, Gresnigt MS, Samonis G, Drakos E, Boumpas D, Muszkieta L, Prevost MC, Kontoyiannis DP, Chavakis T, Netea MG, van de Veerdonk FL, Brakhage AA, El-Benna J, Beauvais A, Latge JP, Chamilos G. Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity. Cell Host Microbe. 2016 Jan 13;19(1):79-90.

Research Interests

  • Identifying Novel signaling pathways regulating phagosome biogenesis
  • Delineating the molecular mechanisms of phagosome maturation arrest induced by airborne fungi
  • Exploring the mechanisms of intracellular persistence of certain fungal pathogens
  • Exploring the role of NETosis and other specialized innate effector mechanisms in antifungal host defense
  • Dissect molecular mechanisms of sepsis induced immunosuppression
  • Define common defects in signaling pathways regulating phagosome biogenesis in acquired human immunodeficiencies
  • Explore mechanisms of nutritional immunity in antifungal host defense
  • Develop immunomodulatory strategies harnessing phagosome biogenesis to improve the outcome of infectious and inflammatory diseases

 

 

 

Future Research

Developing A Systems Medicine approach to identify novel pathways regulating phagosome biogenesis with physiological role in fungal disease pathogenesis

img3Patients developing IMIs typically possess an array of complex, epigenetically-mediated immune defects related to the receipt of immunosuppressive therapies and other medical interventions, co-morbidities, underlying disease, and co-infections, coupled with incompletely characterized genetic predisposition to the disease. At the cellular level, all these acquired immune defects in diverse molecular pathways lead to impaired phagocyte effector function and the development of IMI. Because phagosome is the central regulator of phagocyte effector function, there is an unmet need of dissecting the molecular pathways of fungal phagosome biogenesis in order to gain insight in critical aspects of disease pathogenesis. As a proof of this concept, we have recently identified common defects in Aspergillus phagosome biogenesis in monocytes of patients with primary (chronic granulomatous disease) and secondary immunodeficiencies (receipt of high doses of corticosteroids, sepsis), uniquely predisposing for development of invasive aspergillosis.

In view of the complexity and diversity of immunosuppressive conditions leading to development of IMI a Systems Medicine approach is required to dissect the master regulatory pathways regulating fungal phagosome biogenesis that determine phagocyte dysfunction and development of disease. High throughput genomic studies in patients with acquired immunodeficiencies will lead in the identification of novel molecular targets associated with phagosome biogenesis defects.

Establishment of a high throughput assay in Drosophila melanogaster larvae for functional validation of novel candidate genes regulating phagosome biogenesis. In order to explore and validate the role of identified novel genes/targets identified in the proteomics approach we will perform an in vivo forward genetic screen in Drosophila. Phagocytosis and phagosome biogenesis is an evolutionarily conserved mechanism for pathogen elimination that relies on circulating phagocytic blood cells. Extensive functional characterization of the role of novel candidate genes in phagosome biogenesis will be performed ex vivo with the use of Drosophila S2 phagocytic cell line and in vivo

Validation of master regulatory pathways in human and murine models of invasive fungal infection. Importantly, the complexity of mammalian immune system relies on cross-talk and functional specialization of different immune cell subsets. In particular, among professional phagocytic cells, including monocytes/macrophages, dendritic cells and neutrophils, there are major differences in immune responses and signaling pathways following interaction of a particular PRR (e.g., Dectin-1) with its ligand (e.g. β-glucan). Therefore, priority will be given on in vivo validation of the relevant gene-targets in murine models of IMI. Our lab has acquired significant experience over the past 10 years on physiologically relevant models of fungal infections and conditional inactivation of genes in myeloid cells with the use of Cre LoxP system. Methods for assessment of primary murine phagocyte effector function ex vivo, including different source of macrophages and neutrophils, and in vivo following Broncho alveolar lavage (BAL) have been widely utilized in our lab.