Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4

Abstract

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-β signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the <i>IgH</i> α locus <i>Iα</i> promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets <i>Smad2, Smad3</i> and <i>Smad4</i> mRNA 3'UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-β, B cells downregulated miR-146a, thereby reversing the silencing of <i>Smad2, Smad3</i> and <i>Smad4</i>, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline <i>Iα-Cα</i> transcription. Deletion of miR-146a in <i>miR-146a</i>^-/- mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In <i>miR-146a</i>^-/- mice, the elevated systemic IgA levels were associated with increased IgA⁺ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline <i>Iα-Cα</i> transcription and CSR to IgA in <i>miR-146a</i>^-/- B cells <i>in vitro</i> proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed <i>in vivo</i> by construction and OVA immunization of mixed bone marrow <i>μMT/miR-146a</i>^-/- chimeric mice. Thus, by inhibiting <i>Smad2</i>, <i>Smad3</i> and <i>Smad4</i> expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.