Effect of seminal plasma on dendritic cell differentiation in vitro depends on the serum source in the culture medium
Introduction
Semen contains various immunomodulatory factors, such as chemokines, cytokines and prostaglandins, but also soluble HLA antigens, which can be recognized as foreign and evoke an immune response (Svanborg et al., 1982; Politch et al., 2007; Moldenhauer et al., 2009; Shima et al., 2015). The presence of seminal plasma (SP) in the female reproductive tract after coitus can lead to an influx of immune cells (Sharkey et al., 2012). Many studies have demonstrated that factors in human SP can suppress the function of several components of the immune system including T cells, B cells, natural killer (NK) cells, and the complement system (Lord et al., 1977; Allen and Roberts, 1986; Vallely et al., 1988; Lee et al., 1991; Thaler et al., 1992; Rooney et al., 1993). Furthermore, Lenicov showed that SP can redirect the differentiation of human dendritic cells (DCs) toward a regulatory phenotype (Lenicov et al., 2012). DCs are professional antigen presenting cells that have the ability to capture and present antigens to T cells, in particular for the stimulation of naïve T cells. They play a key role in inducing an active immune response as well as maintaining tolerance. DCs differentiated from human monocytes in the presence of SP expressed low levels of CD1a and high levels of CD14, which are hallmarks of semi-mature DCs (Obregon et al., 2017). While these SP-DCs showed increased expression of maturation markers HLA-DR and CD86, they were unable to develop a fully mature phenotype in response to lipopolysaccharides (LPS). Upon LPS treatment, SP-DCs produced low amounts of the inflammatory cytokines IL-12p70, IL-1β, TNF-α, and IL-6 and elevated levels of the regulatory cytokines IL-10 and TGF-β compared to control DCs.
Potent suppression of NK and T cell responses by SP components in vitro has been shown to be dependent on the addition of bovine serum factors (Vallely and Rees, 1986). NK cell mediated cytotoxicity against K562 targets was suppressed when the effectors were treated with SP in the presence of 10 % fetal calf serum (FCS), but suppression was considerably less when the effectors were treated with SP in the presence of 10 % autologous human plasma (Vallely and Rees, 1986). Furthermore, human SP has been shown to induce cytotoxic effects on lymphocytes in cultures containing FCS (Allen and Roberts, 1986). To date, the immunomodulatory effect of SP on human DCs has only been studied in in vitro cultures containing FCS. Previous studies showed that human DCs cultured in medium containing FCS are different from those cultured in medium containing human serum (HS): HS-cultured DCs are described to be more granular and have a decreased CD1a expression compared to FCS cultured DCs (Anton et al., 1998; Duperrier et al., 2000; Pietschmann et al., 2000; Tarte et al., 2000). Thus, studies showing an effect of SP on human DCs in cultures with FCS may have resulted in conclusions that are not necessarily translatable physiologically to in vivo situations. In the current study we examined whether the effect of SP on human DCs was different in cultures containing fetal bovine serum (FBS) from that in cultures containing HS. Under conditions of HS we did find a less profound effect of SP on DC phenotype and tolerogenicity toward T cells.
Section snippets
Semen samples
All semen samples were obtained from men visiting the fertility clinic at the Leiden University Medical Center (LUMC). Semen samples were produced by masturbation. The samples were produced without the use of lubricants and the complete ejaculate was collected under non-sterile conditions. Sperm quality (semen volume, sperm density, motility, morphology and viscosity) were assessed the same day. Normozoospermic samples were selected using the WHO IV guidelines (WHO, 1999). To reduce the chance
SP alters the phenotype and function of FBS-cultured DCs
In a first set of experiments, we confirmed that SP induces a change in gene expression in immature DCs cultured in FBS containing medium. In these culture conditions, CD14 mRNA levels were increased and CD1a mRNA levels were decreased in SP-DCs compared to control DCs. Also, we observed that mRNA levels of CD86 and HLA-DR were upregulated in SP-DCs compared to control DCs (Fig. 1A). Additionally, we confirmed that DCs treated with SP in FBS-containing medium for six days show an alternative
In the presence of HS, SP affects IL-10 and IL-12 production but not stimulatory capacity of activated DCs
On day 6 we added LPS to the culture for DC maturation and 48 h later we analyzed gene expression. CD14 and HLA-DR mRNA expression was still increased in SP-DCs compared to control DC, whereas CD1a mRNA expression was still decreased (data not shown). No difference was observed for mRNA expression of pro-inflammatory markers IL-12b and TNF-α (data not shown). On the other hand, mRNA expression of anti-inflammatory markers IL-10, TGF-β, COX-2, S100A8, and S100A9 was increased in mature SP-DCs
Discussion
In this study, we showed that activated SP-DCs cultured in HS containing medium resemble semi-mature DCs with regards to phenotype, gene expression patterns, and cytokine production. However, the effect of SP on DC differentiation was less pronounced under HS culture conditions than under FBS culture conditions, and it did not result in a decreased T cell stimulatory capacity.
It has been widely reported that factors in human SP are capable of affecting lymphocyte function in vitro (Lord et al.,
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank the fertility clinic of the Leiden University Medical Center for providing the SP samples and the National Reference Laboratory for Histocompatibility Testing for HLA typing. Furthermore, we thank Mieke Versluis-Lops, Merve Uyar-Mercankaya, and Els van Beelen for their assistance in the Luminex tests.
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These authors contributed equally