ATF3 is a member of the CREB/ATF family of BZip TFs that participate in a wide variety of biological phenomena including development, memory, and apoptosis (17
). We recently demonstrated that ATF3 is a negative regulator of TLR4-induced inflammation (12
), and we show in this paper that it significantly dampens the development of allergic airway inflammation. Our observations provide a clear link between severe asthmatic inflammation and airway disease with the defect in transcriptional control mediated by the TF ATF3.
ATF3 is activated in response to allergen challenge in the lungs of WT mice and contributes to the regulation of the Th2 cytokines IL4, IL5 and IL13, as well as many CCL chemokine genes that contribute significantly to immune cell influx into the lung (15
). These increased cytokine levels can account for some of the key effects on the asthma phenotype seen in these mice. IL13 induces differentiation of goblet cells and secretion of mucin in the airways of mice after allergen challenge (18
). IL4 induces typical Th2 responses, including airway goblet cell metaplasia in a mouse asthma model (19
). IL5 promotes growth and maturation of eosinophil precursors and stimulates chemotaxis of mature eosinophils, prolonging their survival in allergic inflammatory tissue sites by inhibition of apoptosis (20
Similar levels of OVA-specific IgE and IgG1 were induced in ATF3-null mice, indicating that they are competent in their ability to generate OVA-specific T cell responses and undergo Ig isotype switching, although this is not enhanced despite increased Th2 cytokine production. It has been previously shown that IL4, through the induction of the TF STAT6, directs the transactivation of the Cε and Cγ1 promoters (21
). Thus, ATF3 is not absolutely required for IgE and IgG1 expression, nor does it appear to play a role in dampening IgE production. The diminished OVA-specific IgG2a in ATF3-null mice are interesting, as IFN-γ regulation of the transcription factor TBX21 (T-bet) in B-lymphocytes controls IgG class switching to IgG2a (22
). As IFN-γ levels were undetectable in the BAL of ATF3-null mice (unpublished data), it is possible that the severe Th2 skewing in the ATF3-null animals accounts for this stark phenomenon. However, direct induction of T-bet transcription by ATF3 cannot be ruled out and will require further investigation.
We previously identified ATF3 as a component of NF-κB–containing transcriptional complexes in LPS-triggered macrophages. In this study, we show that ATF3 functions in AP-1–containing complexes in Th2-skewed CD4 lymphocytes and that ATF3 binds to the Th2 cytokine promoters in close proximity to the AP-1 family TF, Junb. Junb has previously been shown to be necessary for the induction of IL4, IL5, and IL13 (23
). Previous work indicates that ATF3 can physically interact with many AP-1 family members including Junb (25
). Thus, in Th2-skewed CD4 lymphocytes, ATF3 and Junb function in a feed-forward loop.
The selective allergen-induced production of Th2 cytokines and chemokines in association with diminished IFN-γ production is a hallmark of the asthmatic response (26
). Th2 skewing has been mechanistically linked to changes in chromatin structure (6
). Our previous observations demonstrating changes in chromatin structure after regulated binding of ATF3 and HDAC1 (12
) suggest a possible mechanism for Th2 cytokine regulation by this TF.
The promoters of IL4 and IL13 share similar cis-regulatory elements within 500 bp of the transcription start site (TSS) that bind multiple TFs, particularly NFAT and AP-1 (Junb), which strongly synergize to induce transcription (27
). These NFAT/AP-1 regions are called P sites and act via cooperative binding to form a higher order transcriptional complex. We identified ATF3 recruitment to the essential P2 region of the IL4 promoter (28
). Importantly, Type I HDAC (HDAC1, 2, or 3) activity has been identified in this region of the promoter (29
). Therefore, ATF3-mediated HDAC1 recruitment and resulting effects on histone acetylation levels offer a mechanistic explanation for this observation. Significantly less is known about regulatory regions of the IL13 promoter, although the ATF3 binding site is also, amid P sites, closely opposed to the TSS (30
). These similarities in promoter binding site architecture and histone regulation may lead to the shared expression kinetics of IL4 and IL13.
Transcriptional control of the IL5 promoter is less completely described. There are four cis elements necessary for full transcriptional activation (31
). The first of these, termed IL5A, is found −940 bp from the TSS and is within ~40 bp of the ATF3 binding site. This is the upstream enhancer region of IL5, and deletion of these sites inhibits transcription by ~50% (31
). As ATF3 can recruit HDAC activity to these sites, resulting in condensed chromatin and inhibited gene transcription, this may be a necessary regulatory switch in Th2 cytokine production.
A significant hallmark of asthma is accumulation of eosinophils, neutrophils, lymphocytes, and macrophages in the lung, a process which is highly dependent on chemokines. This cellular influx is directly proportional to disease severity. Many cell types contribute to chemokine mRNA profiles seen in asthmatic lung tissue (32
). IL13 is known to induce chemokine expression, a mechanism that was only partly dependent on STAT6 expression (33
). Macrophages are significant producers of lung chemokines and can respond to IL13 through PPAR receptor signaling pathway, which may explain this phenomenon (35
). Conversely, ATF3 may play a direct regulatory role in controlling chemokine expression, as has been previously shown for RANTES (36
) and CCL4 (37
). Like the Th2 cytokine locus, CCL and CXCL chemokines are also clustered on mouse chromosomes 11 and 5, respectively (38
). Our data showing the enhanced up-regulation of these clustered CCL chemokines define a potential mechanism to explain the pronounced inflammatory cell infiltrate in ATF3-null mice leading to asthmatic exacerbation. Given the complex interplay between diverse cell types, signaling pathways, and cis-regulatory regions contributing to the exacerbated chemokine profiles seen in the lungs of ATF3-null mice, elucidation of this mechanism will require more in-depth and cell-specific examination.
In conclusion, ATF3-null mice generate a complete asthma-like phenotype, including airway inflammation, remodeling, and resulting hyperresponsiveness, and a lack of responsiveness from the ATF3 pathway confers susceptibility to severe allergic disease.