Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro

Highlights

• Natural Killer cell function is impaired after simulated microgravity (SMG).

• Suppression of function without changes to phenotype.

• Decreased lysosomal degranulation and cytokine production after exposure to SMG.

• Leukemia(K562) susceptibility to killing doesn't change after SMG.

Abstract

Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

Introduction

Space travel presents a myriad of challenges to normal human physiology. Crewmembers on long duration missions, especially those that will go beyond low earth orbit, are faced with challenges presented by microgravity, radiation exposure, high gravitational forces during launch and landing, circadian rhythm disruptions, sleep disturbances and the stressors associated with prolonged periods of isolation and confinement [1]. Immune system dysregulation has been observed in response to both short and long duration spaceflight missions, which could leave crewmembers susceptible to latent viral reactivation and impaired anti-tumor surveillance [[1], [2], [3]]. Shedding of latent herpesviruses such as CMV, EBV and VZV have consistently been observed during spaceflight missions and this has been coupled with alterations in plasma cytokine profiles and impairments in T-cell function [[3], [4], [5], [6]]. NK-cells play an important role in both anti-viral and anti-tumor immune surveillance in the host. While NK cells appear to retain their ability to form complexes and kill tumor target cells while on the International Space Station ISS [23,24], we showed recently that NK cell cytotoxic activity against K562 target cells was diminished when crewmember samples collected during the flight phase of a 6-month mission to the ISS were returned to Earth for analysis [7]. It is not known, however, if these effects are due to discrete spaceflight associated factors (e.g. microgravity, stress, radiation exposure, etc.) or a synergistic effect of such factors.

Several terrestrial analogs have been used in an attempt to discern the precise aspects of the spaceflight environment that affect immunity [8]. It is possible that spaceflight alters immunity largely due to the effects of microgravity. Accepted methods for simulating microgravity (SMG) in vitro are the use of 2D rotating wall vessels (RWV), 3D rotary cell culture systems (RCCS) and random positioning machines. Such methods have shown to increase apoptosis [[9], [10], [11], [12]], reduce expression of early activation markers CD69, CD25 and CD71 in PBMCs [13,14], increase expression of cell cycle arrest protein, p21 [15], and decrease cell motility through type 1 collagen [16,17]. T-cell functionality is also suppressed as measured by reduced activation [14,18,19] and reduced proliferation in response to mitogen stimulation [20]. Only one study, to our knowledge, has examined the effects of SMG on the phenotype and function of human NK-cells [21]. Li et al. reported that ex vivo expanded NK-cells were less capable of killing K562 target cells in vitro following 48 h exposure to SMG using a 2D-RWV, and that this was accompanied by a reduced expression of both activation (NKG2D) and inhibitory (NKG2A) receptors on the cell surface. However, we contend that the use of ex vivo expanded NK-cells that require exogenous cytokines to maintain growth and viability will alter the primary expression of activating and inhibitory receptors on the surface masking the true extent and mechanisms of NK susceptibility to SMG. There is also a need to determine how SMG affects the NK-cell arsenal, specifically the expression and degranulation of the cytotoxic granules perforin and granzyme b, and the secretion of effector cytokines that facilitate NK cell killing [22].

The aim of this study was to determine the effects of SMG on the function and phenotypic characteristics of freshly isolated and non-differentiated human NK-cells. We used a 3D rotary cell culture system (RCCS) to maintain cells in a low-shear, continuous free fall and randomizing gravity vector by rotating high-aspect ratio vessels (HARV) on a horizontal axis [13,[25], [26], [27]]. Our model maintained cell viability (>90%) during 12 h of SMG without the use of cytokines or stimulants to avoid any activating or inhibitory effect on NK cells besides SMG. We report for the first time that SMG impairs the function of freshly isolated human NK-cells by ‘disarming’ them of cytotoxic granules and inhibiting their ability to degranulate and secrete effector cytokines. These findings indicate that microgravity might be partially responsible for the inhibitory effects of space travel on NK-cell function and that appropriate countermeasures might be required to protect immunity during prolonged periods of weightlessness.