The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1^LysM Mice

Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation.

Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1^LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1^LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions.

Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients' blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA　treated animals (2.1 x 10⁵ CFU/lung +/- 0.5 (CsA) vs. 1.7 x 10⁵ +/- 0.2, p<0.005). In our murine knockout model, NFATc1^LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1^LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1^LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 10⁵ cells +/- 0.2 (NFATc1^LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence.

Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined.