Defective calcineurin/NFAT signaling in myeloid cells and susceptibility to aspergillosis in post-transplant patients

KEYWORDS:

• calcineurin inhibitors
• myeloid cells
• solid organ transplantations
• hematopoietic stem cell transplantations
• aspergillosis

This article refers to:

Treatment of Cyclosporin A retains host defense against invasive pulmonary aspergillosis in a non-immunosuppressive murine model by preserving the myeloid cell population

Solid organ and hematopoietic stem cell transplantations are life-saving therapeutic interventions considered for an increasing variety of severe and irreversible end-stage diseases. Immunosuppressive agents are often used to prevent rejection of the transplanted organ,¹ and Graft-versus-host disease.² Because immunosuppression depresses both detrimental and anti-microbial immunity,³ these agents render the recipient more susceptible toward viral, bacterial or fungal infections.⁴

Calcineurin inhibitors (CNIs), such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and treatment of a variety of inflammatory and immune-mediated diseases.⁵ These immunosuppressant drugs inhibit T-cell activation by binding to intracellular immunophilins; CsA binds to cyclophilin, whereas FK506 binds to FK-binding protein-12, in either case leading to calcineurin inhibition. Calcineurin dephosphorylates inactive nuclear factor of T cells (NFAT) thereby activating NFAT family proteins. NFATs transcription factors regulate T cell development and activation, especially the activation of IL-2, IL-4, and CD40 ligand by NFAT 1, NFAT 2, and NFAT 4. By inhibiting calcineurin, and, in turn, NFAT and T cells, CNIs interfere directly or indirectly with the activation or expression of IκB, Na-K-ATPase, IL-3, TNF-alpha, IFN-γ, granulocyte/macrophage colony-stimulating factor, and nitric oxide synthase.⁶

Because CNIs are relatively T-cell selective, use of these agents allows for better preservation of myeloid cell functions,⁷ allowing clinicians to reduce corticosteroid doses. Early studies have shown that this CNIs treatment regimen has reduced the overall incidence of infection,⁸ however treatment of post-transplant patients with CNIs is associated with a higher incidence of opportunistic fungal infections mainly aspergillosis, associated with considerable morbidity and mortality.^9,10 Analysis of opportunistic infections after transplantation in 1476 kidney transplant recipients showed a total of 110 episodes of systemic mycoses occurred in 98 transplant recipients, predominantly Aspergillus infections. Importantly, the use of CsA was associated with nearly a 4-fold risk of invasive aspergillosis (IA) within the first 6 months from the time of transplantation when compared with combination of prednisolone and azathioprine therapy.¹¹ Aspergillus species are saprophytic filamentous fungi that are commonly found in soil worldwide, and are the etiologic agents for a diverse set of human diseases collectively named aspergillosis, particularly in patients with immune deficiency.¹² Both experimental and clinical studies indicated that myeloid leukocytes including macrophages and neutrophils are the primary cellular mediates of host protection against invasive Aspergllus infections.¹³ As the most common immune population in a healthy lung, alveolar macrophages (AM) are thought to be the first cells to respond to the initial inhalation of Aspergillus conidia. AM have been shown to phagocytose and kill conidia through reactive oxygen species (ROS) generation and acidification of the phagolysosome.¹⁴ Additionally, AM play an important role in the inflammatory response through sensing of fungal infection via the ligation of pattern recognition receptors (PRRs) resulting in cytokine and chemokine production, including neutrophil attractants such as macrophage inflammatory protein-2 (MIP-2/CXCL2) and CXCL1.¹⁵ A recent study has shown that AMs control the majority of fungal germination.¹⁶ However, some aspergillus conidia escape killing by AM, these microbes germinate and grow into vegetative hyphae, which are resistant to macrophage killing.^17,18 In individuals with normal immune function neutrophils are fungicidal to hyphal aspergillus, by the generation of ROS via NADPH oxidase, nonoxidative lactoferrin-mediated iron sequestration, the formation of neutrophil extracellular traps, and through the release of antimicrobial proteases.^19,20 Neutrophils also express multiple pattern recognition receptors (PRRs) involved in A. fumigatus recognition, including the β-glucan receptors Dectin-1 and Mac-1 (CR3). Neutrophils have been shown to be the essential defense against A. fumigatus, and neutropenia is the primary risk factor for developing IA.²¹

While T-cells were the initial focus of CNIs, a number of recent studies demonstrated that inhibition of calcineurin/NFAT signaling plays a key role in the control of the innate immune response by myeloid cells,²² and significantly affects the outcome of IA.²³ Defective Calcineurin–NFAT signaling in murine myeloid cells impaired phagocytic fungal killing in alveolar macrophages, leading to sustained inflammatory responses in the lung, enhanced tissue destruction and increased mortality rate in A. fumigatus infection.²⁴ The calcineurin inhibitor FK506 results in uncontrolled fungal germination in macrophages.²⁵ Similarly, administration of calcineurin inhibitors was associated with defects in neutrophil control of A. fumigatus hyphal growth and increased susceptibility to invasive aspergillosis.²⁶ Impaired calcineurin-NFAT signaling in CD11c-expressing cells including macrophages and granulocytes also was correlated with downregulation of the antifungal protein pentraxin-3 expression in a murine model of intravenous A. fumigatus infection.²⁷ Pentraxin-3 secretion is one of the major strategies used by myeloid cells to eliminate fungal infections.²⁸ Furthermore, calcineurin signaling in CD11c-expressing cells from donor bone marrow was a critical component promoting IA survival in irradiated recipients.²⁷ The essential role for calcineurin–NFAT signaling pathway in macrophages and neutrophils in protection against Candida infections has also been described previously.²³

The finding that inhibition of calcineurin/NFAT signaling in myeloid cells is a risk factor for opportunistic fungal infections such as IA among organ-transplant recipients provides a novel insight into understanding how the CNIs treatment may increase susceptibility to aspergillosis. In this issue of Virulence, Wong et al.,²⁹ investigated the direct growth inhibitory activity of CsA against A. fumigatus in vitro and in vivo, and whether CsA renders mice susceptible to IA. After 10-h incubation with 6.25 to 400 mg/mL CsA, the in vitro growth of A. fumigatus resting conidia was inhibited by 18% to 31% as compared with the untreated control. CsA also exerted a profound effect on A. fumigatus hyphal elongation by inhibiting 48% to 66% of the mean hyphal length in the same range of concentration.²⁹ One possible explanation for the observed activity of CsA on A. fumigatus is that the calcineurin signaling pathway is relatively conserved between fungus and mammals.³⁰ CsA has been shown to orchestrate morphogenesis, growth, virulence and drug resistance in a variety of fungal pathogens such as A. fumigatus, Cryptococcus neoformans and Candida albicans.³¹ The synergistic activity of calcineurin inhibitors against C. albicans and A. fumigatus has also been shown with echinocandin and azoles³² In the study of Wong et al.,²⁹ daily injections of CsA did not produce an antifungal effect sufficient to rescue cyclophosphamide immunosuppressed mice from lethal infection. However, non-immunosuppressed CsA treated mice were completely protected from Aspergillus infection, resulting in a 100% survival rate. CsA treatment significantly decreased the number of T- and B-lymphocytes as well as natural killer cells in peripheral blood samples, but did not reduce the myeloid cell populations. Therefore, the authors concluded that CsA does not impair the host defense against IA since the myeloid cells were preserved. Of note, a major limitation in the study of Wong et al.²⁹ is that the function of these preserved myeloid cells was not investigated. Since previous studies showed that functional calcineurin/NFAT signaling in myeloid cells is important for protective immunity against A. fumigatus, it could be therefore concluded that preserving the numbers of myeloid cells may not compensate their loss in function during immune responses against IA.

While CNIs have observed activity in T cells, innate immune cells and Aspergillus fumigatus, the preponderance of IA cases in CNI-receiving patients suggests that ultimately the balance of the in vivo effect of current CNI formulations is toward patient susceptibility to IA, at least in certain vulnerable groups. Clearly, the incidence of opportunistic infections after transplantation depends on a number of factors.³³ The type of organ transplanted, the degree of immunosuppression, the need for additional immunosuppressive therapy, and the occurrence of technical or surgical complications all affect the incidence of infections.³³

Overall, invasive fungal infections such as IA are a major determinant of the outcome of transplantation. The number of studies addressing the interaction between immunosuppressive drugs and myeloid cell function in order to unravel critical importance of calcineurin signaling in the control of fungal infections are increasing. Apart from causing T cell dysfunction, calcineurin inhibitors were found to directly affect important immunoprotective functions of myeloid cells, which still warrants further investigation. That said, the finding that CsA directly inhibits A. fumigatus, suggests that development of fungal-specific CNIs may be a worthy area of future study.

Disclosure of potential conflicts of interest

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Funding

This work was supported by a research fund from the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.