Invasive fungal infections are characterized by high rates of mortality and represent an increasing problem in medicine. This is due to intensified use of immunosuppressive treatments and neutropenia but also occurs in the context of inherited diseases related to the immune system. Neutrophilic granulocytes (neutrophils) are among the first and most abundant cells at the site of an infection, to prevent invasive disease by the killing of invading pathogens. Patients with neutropenia and life-threatening infections can be treated with transfusion of donor granulocytes. Neutrophil donors are treated with G-CSF/dexamethasone (GTX) to mobilize and increase the number of circulating granulocytes for transfusion purposes. It is not known how neutrophils recognize fungal pathogens and how these cells activate signaling mechanisms for the killing of Candida and Aspergillus spp, nor whether GTX treatment of donors affects these cytotoxic mechanisms. In order to determine the neutrophil-mediated fungal killing mechanisms we studied the killing capacity of neutrophils from GTX donors and from patients with inherited immunodeficiencies and susceptibility to fungal infections. These well-defined “knockout” patients lack receptors on the neutrophil surface, signaling molecules or proteins directly involved in the cytotoxic response.

Recently, patients suffering from invasive candidiasis have been identified with an inherited deficiency of the signaling protein CARD9. We found that the neutrophils from the CARD9-deficient patients were impaired in the killing of Candida under unopsonized conditions resulting in invasive candidiasis. In contact with blood, micro-organisms are covered by blood proteins that enhance their uptake by granulocytes; this process is called “opsonization”. When the microbes enter the host directly in the tissues, this process does not take place and the microbes remain unopsonized. The neutrophils from various immunodeficient patients demonstrated the existence of distinct killing mechanisms for unopsonized Candida, which involved the integrin CR3 and CARD9, and serum-opsonized Candida¸ which depended on IgG receptors to trigger the production of toxic reactive oxygen metabolites.

The neutrophil-mediated killing of Aspergillus is differentially regulated. The CARD9-deficient neutrophils normally killed Aspergillus conidia and hyphae. We found that neutrophils recognize Aspergillus conidia by the integrin CR3 (CD11b/CD18), which triggers PI3-kinase (PI3K)-dependent signaling to a non-oxidative mechanism of killing that includes iron-sequestration. In contrast, the extracellular destruction of hyphae strictly depends on IgG opsonin recognition by neutrophils and signaling via a Syk-PI3K-PKC cascade to trigger the production of toxic reactive oxygen metabolites – independent of Neutrophil Extracellular Trap (NET) formation. Thus, neutrophils have two different mechanisms to kill Aspergillus fumigatus conidia and hyphae.

Thesis 2016: Neutrophil microbial killing mechanisms. Lessons learned from primary immunodeficiencies, Roel Gazendam