A population of atypical CD56⁻CD16⁺ natural killer cells is expanded in PTSD and is associated with symptom severity

Highlights

• PTSD subjects show higher frequencies of dysfunctional CD56⁻CD16⁺ natural killer (NK) cells.

• PTSD subjects show lower frequencies of functional CD56^brightCD16⁻ NK cells.

• The frequency of atypical CD56⁻CD16⁺ NK cells is near significantly correlated with lifetime PTSD severity.

Abstract

Introduction

Post-traumatic stress disorder (PTSD) has been associated with immune disturbances, including a higher incidence of infections and autoimmune diseases as well as a net pro-inflammatory state. Natural killer (NK) cells, a key component of the innate immune system, have been less well-studied in PTSD despite their importance in immunity.

Methods

We studied two independent samples of combat-exposed male war veterans with or without PTSD, the first (“Discovery Sample”) to generate hypotheses, and the second (“Validation Sample”) to replicate the findings. The Discovery Sample was comprised of 42 PTSD subjects and 42 controls. The Validation Sample was comprised of 25 PTSD subjects and 30 controls. Participants had fasting, morning blood samples collected for examination of the frequency of NK cell subsets, determined by flow cytometry. The current and lifetime Clinician Administered PTSD Scale (CAPS) was used to assess symptom severity. Statistical analyses were adjusted for age and BMI.

Results

PTSD subjects compared to controls had (i) a significantly higher relative frequency of atypical CD56⁻CD16⁺ NK cells in the Discovery Sample (p = 0.027), which was replicated in the Validation Sample (p = 0.004) and the combined sample (p < 0.001), and (ii) a non-significantly lower relative frequency of CD56^brightCD16⁻ NK cells in the two samples (p = 0.082; p = 0.118), which became statistically significant in the combined sample (p = 0.020). Further, within subjects with PTSD of both samples, the relative frequency of atypical CD56⁻CD16⁺ NK cells was near significantly positively correlated with lifetime PTSD severity (p = 0.074).

Discussion

This study is the first to characterize NK cell subsets in individuals with PTSD. The results suggest that combat-exposed men with PTSD exhibit an aberrant profile of NK cells with significantly higher frequencies of an atypical population of CD56⁻CD16⁺ cells and possibly lower frequencies of the functional CD56^brightCD16⁻ NK cell subsets. Higher proportions of dysfunctional CD56⁻CD16⁺ cells have been reported in certain chronic viral infections and in senescent individuals. It is possible that this could contribute to immune dysfunctions and prematurely senescent phenotypes seen in PTSD.

Introduction

Post-traumatic stress disorder (PTSD) is a debilitating mental illness characterized by re-experiencing distressing memories of an initial traumatic event, avoidance, negative cognitions and mood, and hyperarousal (American Psychiatric Association, 2013). In addition to the traditional psychiatric symptoms, individuals with PTSD have a substantially higher incidence of various disorders including those reflecting immune senescence or dysfunction, such as autoimmune diseases and infections (Levine et al., 2014). Several studies, in fact, show that PTSD individuals have increased innate immune responses and a low-grade systemic pro-inflammatory state with increased circulating pro-inflammatory markers that are positively related to psychopathological severity (Levine et al., 2014, Bauer et al., 2010, Lindqvist et al., 2014).

Natural killer (NK) cells, a key component of the first-line anti-viral and anti-tumor defence, have been suggested to play a relevant role in the possible PTSD-associated immune impairments (Gotovac et al., 2010, Kawamura et al., 2001, Laudenslager et al., 1998, Mosnaim et al., 1993, Segerstrom and Miller, 2004). NK cells are an important population of cytotoxic cells linking innate and cellular immunities (Campbell and Hasegawa, 2013, Farag et al., 2002, Vivier et al., 2011). They originate from common lymphoid progenitors along with B and T cells, and mature in lymphoid tissues (spleen, bone marrow, tonsil) to express a diverse array of activating and inhibitory receptors (Campbell and Hasegawa, 2013, Farag et al., 2002, Vivier et al., 2011). NK cells can react very quickly upon stimulation, faster than T cells, as one of their primary functions is to kill tumor and virally infected target cells that lack Major Histocompatibility Complex I (MHC I) expression without any need for previous sensitization, antibody binding, or peptide presentation (Campbell and Hasegawa, 2013, Farag et al., 2002, Vivier et al., 2011).

NK cells are traditionally identified as CD3⁻, CD14⁻, CD19⁻ lymphocytes expressing CD56 (neural cell adhesion molecule) and are typically characterized into two main subsets. Approximately 90% of circulating NK cells are defined as CD56^dimCD16⁺ NK cells and are considered mature while approximately 10% are defined as CD56^brightCD16⁻ NK cells and represent immature NK cells (Caligiuri, 2008, Lanier et al., 1986). CD56^dimCD16⁺ NK cells predominantly contribute to innate immunity through direct cytotoxicity, although they also influence innate and adaptive immunity through cytokine production (Campbell and Hasegawa, 2013, Camous et al., 2012). CD56^brightCD16⁻ NK cells predominantly secrete cytokines and are more resistant to oxidative stress and apoptosis (Campbell and Hasegawa, 2013, Camous et al., 2012). A third subset of NK cells, defined as CD56⁻CD16⁺, was originally described as an expanded NK cell population in persons with Human Immunodeficiency Virus type 1 (HIV-1) and other viral infections (Tarazona et al., 2002, Bjorkstrom et al., 2010, Gonzalez et al., 2009, Hu et al., 1995). Recent data also suggest an expansion of CD56⁻CD16⁺ NK cells during the process of normal senescence (Camous et al., 2012). While a recent study suggests CD56⁻CD16⁺ NK cells may represent activated, mature NK cells that have recently encountered target cells (Milush et al., 2013), CD56⁻CD16⁺ NK cells are generally considered to represent an unusual, highly dysfunctional NK cell subset with poor proliferative and cytotoxic capacity, which secrete lesser amounts of cytokines and higher amounts of chemokines (Camous et al., 2012, Bjorkstrom et al., 2010, Hu et al., 1995). Elevated frequencies of CD56⁻CD16⁺ NK cells are postulated to account for impaired function of the total NK cell population in certain conditions, such as HIV (Mavilio et al., 2005).

In comparing patients with or without PTSD, studies of NK cell cytotoxicity have yielded inconsistent results, with increases (Laudenslager et al., 1998), decreases (Gotovac et al., 2010, Kawamura et al., 2001) and non-significant differences in cytotoxicity reported (Mosnaim et al., 1993). However, no studies so far have explored the relative preponderance of the different NK cells subsets in PTSD individuals compared to controls. Determining differences in NK cell subset distribution could reveal a new aspect of immune dysregulation that contributes to immune dysfunction and immunosenescence in this disorder.

It is possible that the PTSD-associated immune disturbances (Levine et al., 2014) might be explained by an expansion of the dysfunctional (i.e. CD56⁻CD16⁺), rather than the functional (CD56^brightCD16⁻ and CD56^dimCD16⁻), NK cell subsets. In this study, we assessed the frequencies of CD56^brightCD16⁻, CD56^dimCD16⁻ and CD56⁻CD16⁺ NK cells in a sample of combat-exposed male war veterans with PTSD in comparison with combat-exposed male war veterans without PTSD (i.e. controls). We hypothesized that veterans with PTSD would show a more dysfunctional profile of NK cells, with higher frequencies of CD56⁻CD16⁺ cells and lower frequencies of CD56^brightCD16⁻ and CD56^dimCD16⁻ cells.