In modern “24-h society,” flexible working schedules are becoming increasingly common, with the healthcare industry having one of the highest percentages of night shift (NS) workers to guarantee continuous assistance to patients (1, 2). However, irregular sleep/wake patterns and chronic sleep deprivation could result in disruption of circadian rhythms, i.e., biochemical and behavioral fluctuations synchronized with external 24 h light/dark cycles, with detrimental consequences on both physical and mental health; indeed, circadian rhythms control a wide array of cellular processes, including metabolic homeostasis and innate immunity (3). Therefore, it is not surprising that NS work has been associated with an increased risk of several pathological conditions characterized by altered immune and inflammatory responses, such as infectious diseases, cancer, obesity, and cardiometabolic diseases (4, 5). In this scenario, the identification of molecular modifications mirroring such a complex pattern of biological processes affected by NS work could be of great interest. Besides, this could also help monitor the efficacy of health promotion strategies aimed at reducing NS detrimental effects. Epigenetic modifications such as DNA methylation (i.e., the covalent addition of a methyl group to the C5 position of cytosines, generally occurring at CpG sites) may be suitable candidates since they are known to modulate gene expression in response to a wide array of environmental and lifestyle stressors (6–8). A growing body of literature has reported that NS workers retain an altered methylation profile of clock genes, i.e., the set of genes controlling circadian rhythmicity (9–12). Also, genes involved in the inflammatory process (13, 14) and cancer development (3, 14, 15) are differentially methylated in subjects working at night. Such methylation modifications could also be exacerbated by additional stressful conditions associated with systemic low-grade inflammation, such as being overweight (16).

In this framework, human endogenous retroviruses (HERVs) are one of the most abundant classes of repetitive elements (REs), accounting for 8% of the human genome (17). These sequences originated from ancestral retroviruses that infected the primates' germ line and integrated into the host genome, thus being vertically transmitted across generations; however, the progressive accumulation of sequence changes within HERVs has re-shaped them into novel elements at the service of the host cell innate immune system, modulating the inflammatory response after different environmental triggers (18, 19).

Based on this evidence, HERV sequences may likely respond to NS work by changing their methylation status, with potential implications in the innate immune/inflammatory processes. Being NS work a potential risk factor for infectious and cancer diseases, the immune escape mechanisms might also be involved. Indeed, environmental stressors can affect the capacity of pathogen and tumor cells to evade the immunogenic response by altering the host defense mechanisms (20, 21).

To test this hypothesis, we designed a cross-sectional study to investigate the effects of NS work on inflammation and the methylation of HERV elements and a subset of immune escape-related genes. The analyses were conducted on blood samples of healthy nurses with different NS working schedules, who have been recruited in the context of previous research (15, 22). First, we evaluated whether NS work is associated with the plasmatic concentration of the NLR family pyrin domain containing 3 (NLRP3) and the tumor necrosis factor-alpha (TNF-alpha) inflammatory markers. Second, we tested the association between NS work and the methylation of a panel of 10 HERV elements and 10 genes involved in immune evasion. Finally, we explored the possible role of the body mass index (BMI) as an additional susceptibility factor, potentially influencing the association of NS work with gene methylation.

In the present study conducted on 94 female workers with different working schedules, we first evaluated the association of NS work with plasmatic concentrations of NLRP3 and TNF-alpha.

NLRP3 and TNF-alpha were chosen as paradigmatic indicators of innate immune and inflammatory alterations (25). We found that plasmatic levels of NLRP3 were increased in NS nurses compared to colleagues with daily schedules. NLRP3 is a crucial player in host immune defenses by mediating the activation of caspase-1 and the secretion of proinflammatory cytokines IL-1β and IL-18 (26). NLRP3 expression is controlled by many endogenous and exogenous factors, including circadian rhythms (27). A recent study reported that the core clock component NR1D1 negatively regulates NLRP3 expression and activation, as well as the downstream release of IL-1β and IL-18 (28). Of note, circadian rhythm disruption results in impaired NLRP3 levels, as supported by a study reporting NLRP3 overexpression in mice subjected to sleep deprivation (29). On the contrary, we found no difference in plasma TNF-alpha concentrations between the two study groups. TNF-alpha alterations could be expected among NS workers, as the secretion of this proinflammatory cytokine is under circadian control (30). However, data available in the literature are inconsistent, with studies reporting either positive (31) or negative (30) associations between NS and plasmatic TNF-alpha levels, while others observed non-significant differences (32–34). The recent finding that TNF-alpha knockout and wild-type mice do not respond differently to sleep deprivation (35) led to speculation that other pathways (such as the NLRP3 one) might have a greater impact on the response to circadian disruption (25, 35).

Being DNA methylation affected by environmental triggers, including NS (22, 36), we also examined the methylation levels of HERV elements, which are implied in immune and inflammatory responses (18).

Three HERV genes were differentially methylated between NS and DS workers. In particular, ERVFRD-1 methylation was lower, while ERVL and HERV-P methylation levels were higher in nurses working at night. HERVs derive from ancestral retroviruses that infected the primates' germ line and subsequently integrated into the human genome, thus becoming endogenous elements stably inherited across generations (18). Indeed, the structural organization of HERVs resembles that of retroviral DNA, with the gag, pol, and env genes sided by two long terminal repeats (LTRs) with regulatory functions. However, the progressive accumulation of sequence changes within ancestral retroviral elements has led to the “domestication” of HERVs, leading in most cases to the loss of protein-coding capacity and the acquisition of novel functions at the interface between the “self” and “non-self.” HERVs are emerging as important players in the host's innate immune system by modulating the inflammatory response to pathogens and other external triggers (18). Being non-unique sequences, HERVs are generally subjected to transcriptional silencing to prevent genomic instability (37). Nevertheless, changes in HERV methylation levels have been reported in response to many proinflammatory stressors (e.g., environmental toxicants and lifestyle stressors) with possible pathological outcomes (19). Given their numerous and largely unexplored physiological functions, their alterations have been implicated in cancer, as well as in several autoimmune and inflammatory diseases (38, 39). HERVs can act as pathogen-associated molecular patterns, triggering both innate and adaptive immune responses (18, 40–42) by providing antigenic epitopes recognized by lymphocytes and stimulating the onset of specific T- and B- cells (43, 44). These mechanisms might be crucial for the involvement of HERVs in autoimmune diseases, such as multiple sclerosis (ERVW-1), systemic lupus erythematosus (ERVE and HRES), rheumatoid arthritis (ERVK), and amyotrophic lateral sclerosis (ERVK) (45, 46).

On the other hand, HERVs have also been involved in immune response downregulation, starting from their crucial role in maternal immune tolerance to their suggested protective action against undue immune activation (41). The immune response downregulation underlies HERV involvement in tumorigenesis. Interestingly, aberrant methylation and expression of HERV elements have been reported in many cancer types, including breast, colorectal, and prostate cancer (39, 46), for which an increased risk has been reported also in association with NS work (47, 48).

We observed that ERVE, ERVW-1, and ERVL were differentially methylated depending on the BMI. In particular, ERVE and ERVW-1 were differentially methylated between NS and DS at high BMI values, while ERVL methylation was different for lower-mid BMI values. These results suggest that the overweight condition might be an additional susceptibility factor interacting with NS in fostering an altered immune/inflammatory response modulation. We recently reported an increase in age acceleration per year in NS work in subjects with overweight/obesity (22). Thus, HERV sequences might be considered sensitive indicators of immune alterations caused by multiple environmental stress factors.

We further investigated the association between NS work and the methylation of a subset of immune-related genes. We found that BIRC2 and SIRT1 methylation was higher in NS nurses. BIRC2 (Baculoviral IAP Repeat Containing 2) encodes a member of the inhibitors of apoptosis (IAP) family of proteins. BIRC2 expression is downregulated by promoter hypermethylation (49, 50), which was also associated with increased IL-1β production (49, 51). Similarly, hypermethylation of the SIRT1 gene (encoding the Sirtuin 1, a deacetylase whose downregulation has been found in many disease conditions) correlates with lower transcript levels and with an increased risk of inflammatory and metabolic diseases (52, 53). SIRT1 affects multiple biological processes by de-acetylating a variety of proteins including histones and non-histone proteins. Alterations to SIRT1 methylation, expression, and activity were linked to inflammatory diseases (52). It is therefore possible that NS-induced hypermethylation of such genes might determine an increased inflammation and susceptibility to related pathological conditions.

On the contrary, FLRT3 and MIG6 methylation levels were reduced in NS workers. FLRT3 (Fibronectin leucine-rich transmembrane protein 3) is a protein involved in cell adhesion and receptor signaling. Although to our knowledge methylation levels of the FLRT3 gene have never been investigated before, FLRT3 overexpression was found to be protective toward tumorigenesis as it promotes apoptosis and suppresses epithelial-mesenchymal transition, as well as colorectal cancer cell proliferation, migration, and invasion (54). Besides, FLRT3 is overexpressed in breast cancer cells and participates in immune escape processes by regulating the immune receptor Tim-3 pathway (55). Also, MIG6 (Mitogen-inducible gene-6, also known as ERRFI1) has been principally studied in the context of cancer biology, where it poses as a tumor suppressor that inhibits EGFR signaling. MIG6 hypermethylation results in transcriptional downregulation (56), which has been observed in many cancer types; however, the impact of DNA methylation on MIG6 expression could depend on cancer type, as treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine did not always affect MIG6 expression (57).

In the present study, we selectively included workers without major pathological conditions at the time of enrollment to specifically focus on the effects of NS work on DNA methylation and exclude possible confounding due to ongoing disease states. The modifications observed might mirror alterations in biological processes affected by NS work. Future functional studies are needed to evaluate the effects of DNA methylation differences reported in the present associative study. Our findings suggest that DNA methylation of HERV elements and immune-related genes could be a promising marker to highlight the complex pattern of inflammatory alterations found in NS workers (supported by plasmatic NLRP3 increase). However, prospective studies are needed to explore the causal relationship between NS exposure and the observed biological modifications, and eventually evaluate their potential as biomarkers of NS-related diseases.

We acknowledge some limitations of the present study. First, the study population is quite small, especially when it comes to considering BMI classes (the number of study participants with BMI>29.1 kg/m² is very small). Second, we selected the genes of interest based on current evidence in the literature, and not based on unbiased genome-wide methylation analysis; therefore, we could have missed additional methylation alterations occurring in uninvestigated regions. Third, the observed methylation differences between night and day shifters are slight. These results are expected upon exposure of a healthy population to environmental/occupational factors and have emerged thanks to the high precision technology employed and the analytical method applied. However, it is possible that the reduced variations detected might have been underestimated due to the analysis of bulk samples (i.e., buffy coat cell populations). Future studies should be conducted to clarify this aspect.

In the present study, although explorative and based on a small sample, we observed that exposure to NS work is associated with altered modulation of immune and inflammatory-related factors. Moreover, our findings indicate DNA and in particular HERV methylation as a potential biomolecular sensor to monitor the cumulative effect of multiple stressors (NS and metabolic alterations).

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board Comitato Etico Milano Area 2 of the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico (approval number 702_2015). The patients/participants provided their written informed consent to participate in this study.

LF, ACP, and VB contributed to the conception and design of the study. LF, PM, and LT performed experimental analyses. CF and MC organized the database and performed the statistical analysis. CM and MB enrolled the subjects. LF and PM wrote the first draft of the manuscript. LF and VB supervised the whole study. All authors contributed to the manuscript revision, read, and approved the submitted version.