Optimal AID expression and efficient immunoglobulin class switch recombination are dependent on the hypoxia‐inducible factor

During immune responses, B cells engaging a cognate antigen are recruited to GCs in secondary lymphoid organs where they will diversify their BCR to generate highly specific and adapted humoral responses. They do so, by inducing the expression of activation‐induced cytidine deaminase (AID), which initiates somatic hypermutation (SHM) and class switch recombination (CSR). AID deaminates cytosines in ss DNA, generating U:G mismatches that are processed to induce ds DNA break intermediates during CSR that result in the expression of a different antibody isotype. Interestingly, hypoxia regions have been reported in GCs and suggesting that hypoxia could modulate the humoral response. Furthermore, hypoxia inducible transcription factor (HIF) can bind to the AID promoter and induce AID expression in a non‐B‐cell setting, suggesting that it might be involved in the transcriptional induction of AID in B cells, hence, regulating SHM and CSR. We, thus, hypothesized that HIF could regulate the efficiency of CSR. Here, we show that the inactivation of both the HIF‐1α and HIF‐1β subunits of the HIF transcription factor in murine CH12 B cells results in defective CSR and that this is due to the suboptimal induction of AID expression.


Introduction
During immune responses, the BCR repertoire is diversified in GC through somatic hypermutation (SHM) and class switch recombination (CSR) [1]. While SHM diversifies the variable region of Ig genes by introducing mutations and modifying antibody affinity for its cognate antigen, CSR provides novel antibody effector functions through a DNA recombination reaction (occurring at the Ig heavy chain locus) that replaces the isotype expressed (from IgM to IgG, IgE, or IgA). Both of these processes are initiated in GC B cells by the inducible expression of activation-induced cytidine deaminase (AID). AID triggers these molecular events by deaminating cytosines to uracils in DNA, which are then processed by uracil DNA glycosylase and mismatch repair proteins to induce mutations or double stranded DNA breaks [1]. While AID-induced DNA lesions are essential to establish highly specific and adapted humoral responses, AID carries a significant oncogenic potential, which has been implicated at the origin of GC-derived B-cell lymphomas [2].
Given the strong oncogenic potential of AID, its expression and activity need to be tightly regulated. At the transcriptional level, AID expression is only induced in activated B cells and results from a tight balance between the activity of transcription factors (Batf, Hoxc4, etc.) and transcriptional repressors [3]. Interestingly, it has been previously shown that during immune responses, regions of hypoxia are observed within GCs and that the response to hypoxia modulates the humoral response [4,5]. Nevertheless, little is known about the physiological mechanisms for regulating these processes within the GC microenvironment, and it is possible that oxygen tension and the physiological response to hypoxia may play a key role in regulating the humoral response within GCs [5][6][7].
The cellular response to hypoxia is mediated by the hypoxia inducible factor complex (HIF), formed by the HIF-1α and HIF-1β subunits. In normoxia, the HIF-1α protein is unstable, it fixes the tumor suppressor factor von Hippel-Lindau (pVHL), an E3 ubiquitin ligase that ubiquitinates it and triggers its degradation through the proteasome. Hypoxia then triggers the stabilization of HIF-1α, its association with HIF-1β, and the nuclear translocation of the HIF complex, where it associates with other co-factors, such as p300/CBP, to bind to hypoxia response elements (HREs; RCGTG) and induce the transactivation of hypoxia-sensitive genes [5]. In addition, the HIF signaling pathway can also be activated in an oxygen-independent manner through the activation of the BCR, TLRs, or cytokine receptors, leading to the stabilization of the HIF-1α protein in B cells [5].
It has been shown that HIF-1α is capable of binding to the HREs in the Aicda promoter (located at -16 and -1620) inducing AID's mRNA production in a non-B-cell setting [8]. We, therefore, hypothesized that the HIF complex could be implicated in regulating the transcriptional activation of the Aicda gene in B cells. Here, we explore the role of HIF-1α and HIF-1β in the process of CSR in CH12 B cells.

Hif1a is required for optimal CSR in CH12 cells
To determine whether HIF-1α plays a role in CSR, we inactivated the Hif1a gene in CH12 cells (Supporting Information Fig. S1A), a murine B-cell line, that when cultured in vitro in the presence of TGF-β, IL4, and CD40 ligand is capable of undergoing CSR from IgM to IgA very efficiently [9]. The absence of HIF-1α was verified by Western blot (Fig. 1A). To determine whether CSR is affected by inactivation of Hif1a, WT and Hif1a −/− cells were induced to undergo CSR. After 72 h, the efficiency of CSR was evaluated by flow cytometry (Fig. 1B, C and Supporting Information Fig. S1D). While the parental cell line displayed 60% of IgA-expressing cells, this percentage was reduced by threefold in Hif1a −/− CH12 cells (Fig. 1B,C). As CSR is dependent on proliferation, we induced CSR in CFSE-labeled cells and found that all genotypes diluted the CFSE dye at the same extent (Supporting Information Fig.  S1C). To confirm that the defective CSR phenotype observed is due to the lack of Hif1a, we transduced Hif1a −/− cells with a retrovirus expressing HIF-1α. We find that re-expression of HIF-1α is able to rescue significantly the percentage of IgA-expressing cells (Fig. 1B,C). We conclude that Hif1a is required for optimal CSR in CH12 cells, independently of proliferation defects.

Induction of the AID mRNA is compromised in Hif1a −/− cells
To determine whether AID expression is defective in the absence of Hif1a, we quantified the level of AID protein in mRNA by RT-qPCR ( Fig. 1D) and Western blot (Fig. 1E). We found that induction of the AID mRNA is compromised in Hif1a −/− cells (Fig. 1D) and that this results in delayed AID expression at the protein level (Fig. 1E) and a concomitant defect in CSR (Fig. 1B,C). Interestingly, we can observe that the re-expression of HIF-1α can rescue the levels of AID mRNA (Fig. 1D) and protein (Fig. 1E).

Hif1b is required for optimal AID expression and CSR
The nuclear translocation of HIF-1α is dependent on HIF-1β. Thus, in the absence of HIF-1β, we should observe a similar phenotype. To test this hypothesis, we inactivated the Hif1b gene in CH12 cells (Supporting Information Fig. S1B). Absence of HIF-1β was verified by Western blot (Fig. 2A). To determine whether CSR is affected by inactivation of Hif1b, WT and Hif1b −/− cells were induced to undergo CSR (  Fig. S1C). To confirm that the defective CSR phenotype observed is due to the lack of HIF-1β, we transduced Hif1b −/− cells with a retrovirus expressing HIF-1β. We find that re-expression of HIF-1β is able to rescue the CSR defect (Fig. 2B, C). Phenocopying Hif1adeficiency, Hif1b −/− cells displayed delayed AID mRNA expression and protein levels (Fig. 2D, E). We conclude that Hif1b is required for optimal AID expression and CSR in CH12 cells, independently of proliferation defects.

The CSR defect in Hif1a-and Hif1b-deficient B cells is due to defective AID expression
If HIF-1α and HIF-1β are involved in mediating optimal levels of AID mRNA production, then the CSR defect observed in Hif1α −/− and Hif1β −/− cells should be rescued by overexpressing AID. To test this hypothesis, we transduced Hif1α −/− and Hif1β −/− cells with a retrovirus expressing AID (Fig. 3A,C) and cultured them for 3 days in the presence of TGF-β, IL4, and CD40 ligand to induce CSR. We find that overexpressing AID in Hif1α −/− or Hif1β −/− cells is able to rescue CSR (Fig. 3B,D). We conclude that the CSR defect observed in Hif1α −/− and Hif1β −/− cells is related to suboptimal expression of AID.
We have shown that CSR is defective in Hif1a-and Hif1bdeficient B cells. We observe that in these cells there is a delayed expression of the AID mRNA and protein that is visible after 16 h (Fig. 1D,E and 2D,E) and that lasts through the peak of AID expression (24 h). This, per se, explains the CSR defect observed (Fig. 1B,C and 2B,C) and allows us to conclude that deficiency in the HIF transcription factor results in suboptimal levels of AID expression and, hence, defective CSR. This conclusion is supported by the fact that overexpression of AID bypasses the CSR defect observed in a Hif1a-or Hif1b-deficient background (Fig. 3B,D).
While it is clearly established that hypoxia and the HIF1 transcription factor play a fundamental role in B-cell biology [5][6][7], it is difficult to pinpoint their precise role in shaping the humoral response at the GC reaction in vivo. This might be due, in part, to the pleiotropic effect of HIF1 in controlling intrinsic B-cell responses to hypoxia such as metabolism, cell survival, signaling, or AID expression [5,7,10]. In addition, hypoxia can also influence the function of T-follicular helper cells and/or follicular DCs, thus, having a potential impact on B-cell selection and affinity maturation. Furthermore, there are some apparent contradictions concerning hypoxia in GCs and antibody diversification through CSR and SHM. For example, it has been shown that CSR occurs at the pre-GC stage outside of GCs [11] and that the region in which hypoxia is more pronounced is in the light zone [6] and not in the dark zone, where AID expression is predominant [12]. It is possible that hypoxia triggers the transactivation of the Aicda mRNA in the light zone and that its expression, at the protein level, is maximal once B cells reach the dark zone. Furthermore, as the secondary lymphoid organs are in general in a hypoxic environment relative to other tissues [7], this per se may poise follicular and/or GC B cells to enforce optimal AID expression.
Supporting the role of the HIF transcription factor in controlling AID expression, it has been shown that the inactivation of pVHL leads to a strong accumulation of the HIF-1α protein, and consequently to increased protein levels of the members of the APOBEC3 cytidine deaminase (A3B) family, which includes AID [13]. Thus, we would have expected that inactivation of pVHL would lead to enhanced HIF-dependent transcriptional activity [13], resulting in increased production of the AID mRNA and protein and more efficient CSR and SHM in a hypoxic environment in the GC or in response to cytokine signaling. Nevertheless, a B-cell-specific KO of pVHL, which resulted in constitutive HIF-1α stabilization [6,13], surprisingly led to impaired affinity maturation and defective CSR [6]. These results highlight the underlying difficulty of exploring the role of hypoxia and HIF in antibody diversification in vivo.
As the HIF signaling pathway can be activated (independently of oxygen) through cytokine signaling [5,10], notably through TGF-β, IL4, and CD40, our results highlight the possibility of an oxygen-independent role of the HIF transcription factor in AID's expression and, hence, modulating the efficiency of the humoral response. Nevertheless, the role of HIF in SHM, which requires the generation of a KO mouse model, remains to be determined. In addition, as the efficiency of CSR in the absence of Hif1a, is not completely abolished, it is possible that there is a functional compensation by Hif2a, whose role in B cells is unclear [5].
While it has been shown that hypoxia leads to contrasting effects on CSR [4,6,14], here we demonstrate that inactivation of the HIF transcription factor in murine CH12 B cells (independently of pleiotropic effects on metabolism, cell survival, or oxygen dependence) results in defective CSR and that this is due to the suboptimal induction of AID expression. This has implications for the effectiveness of the humoral response within the GCs, as regions of hypoxia and cytokine stimulation might potentiate AID expression to enforce the diversification of the BCR and to accomplish the establishment of highly specific and adaptive immunity.