A Role for NFAT in Innate Immunity: Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation Under Cyclosporine Α Treatment
Authors: Plein Katharina , Teschner Daniel , Michel Christian , Wagner Eva-Maria , Stein Pamela , Theobald Matthias , Schild Hansjoerg , Radsak Markus 
Published: October 2019

Abstract
Background and Aims: Patients after allogeneic hematopoietic stem cell transplantation (HSCT) suffer from immunodeficiency, in part due to long-term immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA). Additionally, these patients have an increased risk for opportunistic fungal infections like invasive aspergillosis (IA). The nuclear factor of activated T cells (NFAT) is known as an important transcription factor in signaling-pathways downstream of calcineurin in the adaptive immune systems, e.g. in T cells, but also plays an important role in innate immune response as indicated by recent data in rodent models. These studies showed a relevant impact of NFAT-/calcineurin inhibition on the innate immune response by polymorphonuclear neutrophils (PMN) in a candida sepsis model. To clarify whether this is also relevant in humans, we investigated the role of NFAT signaling pathways in PMN activation and effector functions in healthy volunteer donors and patients under immunosuppressive treatment with CsA after HSCT.
Methods: Firstly, we performed in vitro experiments using PMN from healthy donors analyzing their effector functions in absence or presence of CsA in titrated doses according to therapeutic levels. In detail, we examined phagocytosis, activation, generation of reactive oxygen species (ROS) and release of inflammatory mediators like IL-8 and TNF-α. After activation with lipopolysaccharide (LPS) and zymosan, phagocytosis and activation-induced shedding of CD62L were measured by flow cytometry using polychromatic microspheres and matching surface markers (CD11b, CD62L, CD66b). In addition, generation of ROS was analyzed by dichlorofluorescein assay (DCF), whereas activation-induced release of inflammatory mediators was measured by enzyme-linked immunosorbent assay (ELISA) and intracellular flow cytometry. In addition, blood samples of patients after HSCT under continuous CsA medication (n=6) and healthy volunteer donors (n=6) were analyzed ex vivo at two different time points after allogeneic HSCT (day 25-35 and 125-135) concerning their PMN effector functions in the same manner as described above.
Results: Analysis of healthy donors PMN in vitro showed that CsA had no significant influence on expression of activation markers and shedding of CD62L. Moreover, no substantial influence of CsA on generation of ROS was detected compared to untreated controls (5245 RFU +/- 354 (CsA) vs. 5763 +/- 520 (control) after stimulation with LPS, mean +/- SEM). Furthermore, activation-induced synthesis of IL-8 was not reduced in presence of CsA (519pg/ml +/- 81 vs. 463 +/- 131 (control) after stimulation with LPS). In contrast, CsA rather enhanced phagocytosis after stimulation with LPS (83.5% +/- 1.7 vs. 71.0 +/- 1.5 (control)). Regarding the ex vivo analysis of HSCT patient and healthy donor blood samples, production of ROS was not affected under CsA therapy (35.1% +/- 9.4 vs. 31.1 +/- 6.6 (control) after stimulation with zymosan). Furthermore, CsA medication showed a stimulating effect on PMN phagocytosis which is in line with our in vitro data (58.4% +/- 7.1 vs. 44.3 +/- 2.3 (control) after stimulation with LPS). Interestingly, in several patients an increased production of IL-8 and TNF-α was detectable after stimulation with zymosan (IL-8: 9.1% +/- 3.6 vs. 1.5 +/- 0.3 (control); TNF-α: 16.6% +/- 6.2 vs. 1.0 +/- 0.4 (control)).
Conclusions: In contrast to previous results by others in murine model systems, we found an increased phagocytic activity in vitro and ex vivo in human PMN upon NFAT/calcineurin inhibition, whereas other effector mechanisms were unaffected. In addition, HSCT patients under CsA treatment displayed enhanced inflammatory mediators production in PMN. It is currently unclear whether these findings are clinically relevant for the innate immune response after HSCT, e. g. in terms of anti-fungal immunity. Further studies are needed to address whether these enhanced PMN functions in the presence of CsA contribute to a dysregulated innate immune response in humans. Alternatively, our discrepant results may be due to differences in PMN functionality and CsA responsiveness in mice and man. However, while CsA strongly suppresses adaptive immune responses, i.e. T-cell responses in graft-versus-host disease (GvHD), our results so far suggest that CsA does not affect innate immune effector functions by humans PMN in a comparable way.

Disclosures
Radsak: Celgene: Research Funding.