Animals in nature need to balance resource allocation on reproduction and self-maintenance whose major component is immunity [12]. The reproduction and preservation of giant pandas concern the world [11, 13]. However, the immune performance of male giant pandas during reproduction has been rather little studied. Here we investigated the immune changes in 8 male giant pandas over the breeding season compared with 5 males in non-breeding season. We monitored the expression of immune-related genes based on peripheral blood transcriptome and identified 45 immune-related genes with altered expression, mostly up-regulated, in the breeding season compared to non-breeding season.
The GO term enrichment of “translation”, “peptide biosynthetic process” and “structural constituent of ribosome” and KEGG pathway enrichment of “ribosome” were observed in up-regulated genes. This suggests an increased requirement for protein synthesis in male giant pandas in reproductive phases. The amplification of protein synthesis was also reported in male freshwater spotted snakehead during reproductive phases [14]. The enrichment of ribosome pathway was in agreement with the study in sheep testes, which indicates that the normal function of ribosome plays essential roles in spermatogenesis [15]. The dramatic up-regulated genes were enriched in spliceosome who removes noncoding introns from transcribed mRNA precursors, suggesting spliceosome is very important in producing necessary gene products related to male sexual development [16]. Oxidative phosphorylation was another enriched pathway in our study. This pathway as an important ATP-related metabolic pathway provides energy for male breeding [16]. Moreover, two hub genes HSPA4 and SOD1 during the breeding season were about 3.36 and 3.25 folder higher than during the non-breeding season respectively. The expression of HSPA4 is higher in germ cells of prenatal gonads [17] and SOD1 activity is higher in stallion during the breeding season [18]. This suggests HSPA4 and SOD1 are involved in spermatogenesis and antioxidant protection of sperm for male giant pandas [17, 18]. The up-regulated genes and enriched pathways may indicate that male giant pandas are prepared for the breeding in terms of protein synthesis, energy generating and spermatogenesis.
Innate Immune Changes
The innate immune subsystem typically includes pattern recognition receptors, autophagy, antimicrobial peptides and many cell types (e.g. dendritic cells, macrophages and natural killer cells), establishing the first line of defence against a wide range of invading pathogens [19, 20]. Moreover, innate immune subsystem is responsible for the activation of adaptive immune subsystem [19]. During the breeding season, males tree lizards reduced innate immunity in the laboratory [3], while the innate immunity showed no change in male Eurasian tree sparrows and temperate bat [6, 21]. Arabian and Thoroughbred horses presented a increased innate immunity [22]. Considering methodological difference, we explored the alteration of innate immunity from several aspects and found an enhanced innate immunity in male giant pandas.
We found several key genes referred to pattern recognition receptors (PRRs) were upregulated, including CLEC4E (also known as Mincle), SUGT1 (SGT1 homolog), HSP90AA1, IL1B and GABARAPL1 (LC3 paralog). Pattern recognition receptors mainly include Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and NOD-like receptors (NLRs) [23]. CLRs were found to recognize microorganisms such as viruses, bacteria and fungi, and then regulate the production of proinflammatory cytokines [23]. CLEC4E encodes macrophage-inducible C-type lectin (Mincle) who is a member of the CLRs family [24]. Mincle has been known to recognize dead cells and bacteria [24]. In late spring, some skin mites were commonly found in captive giant pandas [25]. Evidence showed that Mincle was strongly up-regulated after skin injury and irritation, and mediated a severe inflammatory response [26]. The up-regulation of Mincle in our study may protect giant pandas from infectious diseases.
NOD1 as a member of NLRs family recognizes invasive bacteria by specific peptidoglycans [23]. The SGT1 was reported to positively regulate NOD1 activation and depletion of SGT1 block multiple cellular responses caused by NOD1 activation [27]. Besides, HSP90 protects NOD1 from degradation and functions as a stabilizer which is an evolutionarily conserved molecular chaperone [27, 28]. NLRs can interact indirectly with LC3 through a signaling cascade to regulate autophagy [28, 29]. Autophagy-associated genes were also up-regulated in our analysis. Marcin et al. demonstrated that NLRs were down-regulated in the pregnant pigs to maintain the earliest stages of pregnancy [30]. The up-regulation of positive regulator who contributes to the activation of NLRs may imply the potential function of NLRs in breeding giant pandas.
Autophagy is a fundamental intracellular bulk degradation process with multiple roles in innate immune responses and cellular stress [31, 32]. Beclin1 and LC3 encoded by BECN1, GABARAPL1, respectively, were both up-regulated. Mammalian core autophagy-related proteins mainly involves several functional units, including the PI3K complex which is composed of Beclin1, the LC3 conjugation system and so on [33]. LC3 conjugation system regulates the elongation of the phagophore and promotes the completion of autophagosome formation [29]. During the breeding stage of testicular recovery, the expression of BECN1 and LC3 began to increase in South American plains vizcacha [34]. Anna et al. observed the increase in beclin1 and LC3 synthesis and confirmed the function of autophagy in adult reproductive male European bison [32]. Up-regulated expressions of BECN1 and LC3 suggests the increased demand for maintaining homeostasis in male giant pandas during the period of reproductive activity.
Peroxisomes are crucial metabolic organelles which play central roles in lipid metabolism and ROS turnover [35]. Accumulating evidence suggest a new function for peroxisomes in microbial infection resolution and antiviral response [35, 36]. What’s more, peroxisomes have be pointed to an important role for cell type-specific metabolic function in the testis and spermiogenesis [37]. Two antioxidant genes SOD1 and PRDX5 were up-regulated in present study. ROS which has recently emerged as a signal factor in innate immune responses, is influenced by the disruption of redox balance in enzymes and subcellular compartments [36, 38]. Kwang et al. demonstrated that SOD1 tightly regulate the generation of ROS during virus infection [38]. Bernard et al. reported that human PRDX5 interacted with or binded to PRRs to activate a proinflammatory response [39]. PRDX5 can trigger the expression and release of IL1B [39]. The expression of PRDX5 and IL1B were both up-regulated in giant pandas, confirming an association between PRDX5 and IL1B. Collectively, peroxisomes are essential for the activation of the innate immune system and the normal function of testis in giant pandas.
The proteasome is responsible for the poly-ubiquitinated substrates recognizing and intracellular proteins degradation [40]. The proteasome system and autophagy are closely interconnected [41]. Proteasome is a multi-subunit protein complex, consisting of a 20S core particle and 19S regulatory particles [41]. Standard 20S proteasomes can be replaced by immunoproteasomes which are activated by PA28 complex in conditions of infection, inflammation and an intensified immune response [41]. PSME1 encoding PA28 alpha, one of PA28 complex, was up-regulated about 2.8 fold in giant pandas during breeding season. Furthermore, the proteasomes generate spliced peptides from major histocompatibility complex type I (MHC class I) molecules and PA28 enhances the presentation of several viral epitopes [42]. Proteasome subunits was reported to increase the immune tolerance of the rhesus monkey during early pregnancy [43]. The upregulation of PA28 may indicate the enhancement of the immunity in giant pandas.
NK cells comprise 5–10% of lymphocytes in peripheral blood and varies with age [44]. Natural killer (NK) cells play an immensely significant role in innate immunity by defending against virus infections [19]. CD94, encoded by KLRD1, was down-regulated. CD94-NKG2A receptor complex which recognizes MHC class I, is an inhibitor of the cytotoxic activity of NK cells [45]. PLCG2 (PLC-gamma2) which encodes phospholipase C-gamma2 belongs to PLC-gamma proteins family and was up-regulated. PLC-gamma proteins, serving as cytoplasmic enzymes, involve in NK cell activation [46]. VAV1, which was also up-regulated in this study, is indispensable for polarization of lytic granules secreted by NK cells toward target cells [46]. The balance of activator and inhibitor signals regulate whether the NK cells become activated or not [44]. Integration of down-regulated inhibitor and up-regulated activator may imply the partial activation states of NK cells. The increased expression level of NK cells was also reported in breeding horses, suggesting a slightly increased innate immunity in the breeding season [22].
Considered together, these results suggest an enhanced innate immunity in male giant pandas during breeding season, which is consistent with some previous findings. The energy investment in reproduction does not lead to a corresponding decrease innate immune investment. One possible explanation is that the captive pandas is not in a resource-limited environment [3].
Adaptive Immune Changes
The two typical cellular subsets T and B cells comprise the adaptive immune system [47]. In terms of cellular immunity, male ruffs showed a decreased immunity while tree frogs showed a increased immunity during the breeding season via phytohaemagglutinin challenge test [8, 48]. When it comes to humoral immunity, many studies have documented breed-associated alterations in the immune system. Male bank voles and Eurasian tree sparrows had lower humoral immunocompetence [7, 21], While the immunoglobulin concentration of the Great Tit increased during breeding in accordance with previous studies on birds [49]. In this study, we observed some breed-associated alterations in cellular immunity and humoral immunity of male giant pandas.
Several key genes involved in antigen presentation and processing were up-regulated. Antigen processing pathway is required for proteasomes which produce peptide fragments of MHC class I ligands [42]. The activation of the proteasome relies on PA28 who enhances the liberation of immunopeptidome [42]. Not only PA28 but also HSP70 and HSP90 were up-regulated in our study. HSP70 stimulates antigen cross-presentation of dendritic cells and immune response of activated NK cells [50]. HSP90 contributes to the translocation of extracellular antigen and associates with peptides implicated as precursors of MHC class I ligands [51]. Our data indicate that male giant pandas may have a great capability of antigen presentation and processing compared to non-breeding males.
T cell receptors consists of an antigen-binding subunit (TCRαβ) and three dimers of protein CD3 signaling subunit assemble in a coordinated way [52]. CD3D and CD3G coding genes showed elevated transcript levels in the current study, which are involved in TCR activation [53]. Moreover, Aykut et al. also documented that male horses had higher CD3 expression level during breeding season [22]. The upregulation of IL1R2 was found in T-cell activation [53], and IL1R2 was up-regulated in male giant pandas. However, we found the down-regulation of IL15. IL15 is an important cytokine in lymphocyte survival [53] as well as T cell proliferation and differentiation [54]. Moreover, some co-receptors are also indispensable for the activation of T cells [53]. Therefore, T cells did not show proliferative and differentiation potential in male giant pandas during breeding season.
B cells can differentiate into plasma cells and secrete immunoglobulins against the pathogen [54]. For male temperate bats, reproductive states did not influence the concentration of immunoglobulin G (IgG) [6]. However, for male Eurasian tree sparrows, birds during the breeding stage had lower IgA levels than those from the wintering stage [21]. In our study, we found the down-regulation of IGIP. IGIP has the capability of inducing IgA production by B cells [55]. IGIP produced primarily by Dendritic cells, acts as a switch or differentiation factor to regulate IgA [55, 56]. The down-regulation of IGIP may indicate the low concentration of IgA and a reduced humoral immunity in giant pandas.