The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1^LysM Mice

Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models.

Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1^LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1^LysM mice using flow cytometry and simultaneously counting PMN in PB.

Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 10⁵ CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 10⁵ +/- 0.2 (control), p<0.005). Along with that, NFATc1^LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1^LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 10³ PMN/µl +/- 0.2 (NFATc1^LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1^LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 10⁵ cells +/- 0.2 (NFATc1^LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged.

Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1^LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1^LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings.