The TGF-β pathway is involved in the control of the primary and secondary immune response
Acvr1b (ALK4, activin receptor type 1B) was identified to be a hub protein in both primary and secondary infection (Tables 1 and 2). Activins are members of the TGF superfamily that act as local regulators of biological processes and are associated with cell growth and differentiation [19]. Correspondingly, activin is crucial to the control of innate and adaptive immune responses [10].
To regulate the immune response, TGF-β mediates its effects via SMAD proteins [9]. To clarify the role of TGF-β in the innate and adaptive immune response, we focused on the protein interactions of SMAD proteins and Acvr1b in primary and secondary infection, and compared the differences between initial and recurring infections. In primary infection, the SMAD protein Smad2 was found to interact with Acvr1b; whereas in secondary infection, Smad2, Smad3, and Smad7 were found to interacted with Acvr1b. The SMAD proteins can be divided into three major groups according to their function: receptor-regulated SMADs (R-SMAD), the common mediator SMAD (Co-SMAD), and inhibitory SMADs (I-SMAD) [9]. The R-SMADs Smad2 and Smad3, which, upon phosphorylation, interact with Co-SMAD and translocate to the nucleus [11], were identified as key proteins in primary and secondary infection. However, Smad7, an I-SMAD, was found to interact with Acvr1b during secondary, but not primary, infection (Figures 3 and 4).
Smad7 has been reported to play an essential role in the negative regulation of TGF-β signaling by interfering with the binding of TGF-β to type I receptors [25]. Furthermore, we compared the expression profile of Smad7 over time during primary and secondary infection to see if there was a significant change in between infections (Figure 5).
The time course of Smad7 expression showed a significant increasing trend of inhibitory Smad7 expression during secondary infection, suggesting that TGF-β signaling is suppressed in secondary infection relative to primary infection. This is in agreement with previous findings suggesting that Smad7 is involved in the reciprocal inhibition of TGF-β and IFN-γ [26].
In the reciprocal inhibition of TGF-β and IFN-γ, Smad7 is the key component responsible for polarizing responses toward either immunity or tolerance to infection. More specifically, although Smad7 can suppress the TGF-β signaling pathway to initiate infection tolerance, it can also promote immunity triggered through the IFN-γ signaling pathway. This is of interest, as the dual role of Smad7 in determining whether immunity or pathogen tolerance occurs is suggestive of a key mechanism that controls the immune response.
The gene expression time course in primary and secondary infection showed that Smad7 expression, which is at basal level in primary infection, increased rapidly during secondary infection (Figure 5). The difference in Smad7 expression between the primary and secondary infections may indicate that after initial low-dose infection, the zebrafish immune system was able to tolerate the invading pathogen, thereby shifting the immune response toward infection tolerance. However, in secondary infection with a lethal pathogen dose, increased Smad7 expression suggests that the immune response is triggered to defend against the invading pathogen; thus, the pathway responsible for infection tolerance is inhibited in this phase (Figure 6).
In addition, TGF-β has also been suggested to inhibit the function of inflammatory cells and immune responses [8],[27]. The regulation of TGF-β and its relationships with various immune cells are depicted in Figure 6. Under normal conditions, there is a feedback system that characterizes the relationship between the TGF-β pathway, the innate immune response, and the adaptive immune response (Figure 6). Even though immune cells can secrete cytokines that promote TGF-β signaling, TGF-β signaling can inhibit activation of these immune cells, thus acting as a feedback system. Smad7 during secondary infection was found to suppress TGF-β signaling, leading to attenuated inhibition of immune cells (Figure 6). Consequently, the increased proliferation of immune cells such as T and B cells in the adaptive immune response promotes defense against the invading pathogen.
In summary, the identification of Acvr1b in primary and secondary infection suggests that TGF-β signaling is indeed involved in the control of innate and adaptive immune responses. Furthermore, the discovery that Smad7 interacted with Acvr1b only during secondary infection suggests that TGF-β controls immune responses via a SMAD-dependent pathway. Therefore, the control mechanism can be described as a feedback system involving TGF-β signaling and the adaptive immune response (Figure 6).
The proteasome plays a role in controlling the adaptive immune response
Psmd1 and Psmd13, 26S proteasome regulatory subunits, were identified to be significant primarily during secondary infection. Proteasomal activity has been shown to be related to inflammatory and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis because of its role in activating an anti-apoptotic and pro-inflammatory regulator of cytokine expression [28]. Therefore, the identification of Psmd1 and Psmd13 in our constructed intracellular PPI networks for secondary infection indicates that the proteasome system plays a pivotal role in the zebrafish immune response. Furthermore, the numbers of linkages from primary to secondary infection for both Psmd1 and Psmd13 increased significantly, suggesting that the proteasome is more active during secondary infection, and is therefore more important in the adaptive immune response of zebrafish.
Regulation of apoptosis in primary and secondary infection
Many of the ten most significant hub proteins discussed above are related to the apoptotic process, as shown in Table 1. Further investigation revealed that apoptosis was activated during primary infection but was inhibited during secondary infection. In our constructed zebrafish intracellular PPI networks, we identified three proteins (Casp2, Acvr1b, Hsp90a.1) that were involved in apoptosis out of the ten hub proteins (Table 4).
For Casp2, the number of linkages increased significantly during secondary infection relative to the number during primary infection. The caspase family of proteins has a dominant role in activating apoptosis [29]. Analysis of Casp2 protein interactions revealed that it interacted with Bcl2 during secondary but not primary infection (Figures 7 and 8). Bcl2 is a member of the Bcl2 family that regulates cell death by inhibiting the apoptotic process [30]. Thus, the finding that Casp2 and Bcl2 interacted during secondary infection suggests that apoptosis is suppressed during secondary infection, in contrast to the induction of apoptosis during primary infection.
Acvr1b, a type 1B activin receptor, has been shown to be related to the apoptotic process in both primary and secondary infection. Activins are members of the TGF-β superfamily and are local regulators of biological processes that are associated with cell growth and differentiation [19]. The TGF-β pathway is also involved in inducing apoptosis and the SMAD family of molecules act as key signal transducers during this apoptotic process [31]. Smad7 protein was found to interact with Acvr1b during secondary but not primary infection (Figures 3 and 4). Smad7 is an inhibitory protein that interferes with the phosphorylation of pathway-restricted SMAD proteins such as Smad2 and SMAD3 by binding to type I receptors [11],[32]. Therefore, the interaction between Acvr1b and Smad7 supports our observation that apoptosis is inhibited during secondary C. albicans infection.
Hsp90a.1, a heat shock protein, was identified to be a key hub protein in the zebrafish intracellular PPI networks. The number of its linkages increased significantly from primary to secondary infection. Hsp70 and Hsp90 directly interact with proteins regulating the programmed cell death machinery and thus block the apoptotic process [23]. The identification of Hsp90a.1 as an important protein mainly during secondary infection in our constructed network again suggests that apoptosis is inhibited during secondary infection. Furthermore, Hsp90 stabilizes the 26S proteasome (Psmd1 and Psmd13, 26S proteasome proteins that are a part of the ten hub proteins, as listed in Table 4), and thereby enables the cell to remove unwanted or harmful proteins.
In summary, the finding that apoptotic proteins such as Casp2, Acrv1b, and Hsp90a.1 are more prominent during secondary rather than primary infection is intriguing. Increasing evidence supports that apoptosis has a crucial role in innate and adaptive immunity during infection [33]-[36]. Our results indicate that apoptosis was inhibited in secondary but not primary infection, suggesting that during infection, apoptosis can be adopted as an offensive or defensive strategy by the pathogen or zebrafish, respectively.
The identification of Ncstn implies a relationship between bacteria- and fungus-induced immune responses
Ncstn, a part of the γ-secretase protein complex, was found to play a significant role during both primary and secondary infection in our constructed PPI networks. Ncstn can generate a peptide epitope that facilitates immune recognition of intracellular mycobacteria with related components of γ-secretase through MHC II-dependent priming of T cells [18]. Such pathogen recognition mechanisms are crucial to adaptive immunity in the host. The identification of Ncstn during C. albicans infection of zebrafish suggests that Ncstn responds not only to bacterial infection but also to fungal infection.
Taken together, initial investigation of our constructed PPI networks for primary and secondary infection revealed that the immune responses activated after secondary infection are generally stronger. As shown in the in vivo experiment, zebrafish that had been infected with 1 × 105 CFU C. albicans have a higher survival rate and survive longer after secondary infection with a more lethal C. albicans dose (1 × 107 CFU) compared with zebrafish without prior infection (Figure 9). Identification of the aforementioned hub proteins in our constructed zebrafish intracellular PPI networks encouraged us to explore how the zebrafish immune system responds to infection and whether the response differs in primary and secondary infection.
Note that our dynamic modeling approach is not free from errors. False-positive and false-negative interactions in the initial putative PPI network can affect the accuracy of our constructed network. In order to minimize the effect of false-positive interactions, we applied AIC in the last step of network construction to eliminate the false-positive interactions based on model order selection. False-negative interactions are harder to avoid since if a PPI link is missing in the initial putative network, there is no effective method to recover the link. Therefore, we used BioGRID and InParanoid7 database, the most comprehensive PPI database available, to build our initial candidate network. We understand that PPI links may still be missing in BioGRID and InParanoid7. Such error can be improved when more PPI databases are available and can be integrated to form a comprehensive initial putative network.