Unexpected involvement of IL‐13 signalling via a STAT6 independent mechanism during murine IgG2a development following viral vaccination

Abstract

In this study, recombinant pox viral vaccination was shown to induce highly elevated IgG2a and low IgG1 antibody expression in mice lacking IL-4 or STAT6, whilst IL-13 mice exhibited elevated IgG1, but very low IgG2a. These findings revealed that IL-13 and IL-4 differentially regulated antibody development. To understand this further, when STAT6 mice were given a vaccine co-expressing IL-13Rα2 that temporarily sequestered IL-13, significantly reduced IgG2a expression, was detected. These findings for the first time demonstrated that IL-13 regulated IgG2a differentiation utilising an alternative IL-13R signalling pathway independent of STAT6 (IL-13Rα2 pathway). This was further corroborated by the (i) elevated IL-13Rα2 expression detected on STAT6 lung MHCII CD11c cells 24 h post IL-13 inhibitor vaccination and ii) significant up-regulation of IL-13Rα2 expression on spleen and lung derived MHCII CD11c following inhibition of STAT6 signalling in vitro, or vaccination with IL-4R/STAT6 antagonist in vivo. When T follicular helper (Tfh) cells which regulate antibody differentiation were assessed post vaccination, although no difference in IL-4 expression was observed, greatly reduced IFN-γ expression was detected in IL-13 and STAT6 mice compared to wild-type. These findings support the notion that the balance of IL-13 level at the vaccination site can differentially regulate T and B-cell immune outcomes.

Unique IL-13Rα2/STAT3 mediated IL-13 regulation detected in lung conventional dendritic cells, 24 h post viral vector vaccination

“…rMVA or rVV boost vaccination strategy) can induce high avidity/poly-functional mucosal and systemic T cells with better protective efficacy 15,17 , which was associated with elevated cDC recruitment 17,19 . These studies also showed that IL-13 was necessary for effective antibody differentiation 15 , which was regulated via a STAT6 independent pathway 28 . When trying to unravel how IL-13 modulated these different vaccine-specific outcomes current study revealed that, i) under low IL-13 conditions/rFPV vaccination (which induced low IL-13 at the lung mucosa), IL-13Rα2 expression was up-regulated on DC; ii) under low IL-13/STAT3 inhibition IL-13Rα2 expression was up-regulated whilst TGF-β1 was down-regulated on lung DCs, as opposed to STAT6 inhibition; iii) Moreover, up-regulation of phosphorylated STAT3 and TGF-β1 was detected on STAT6 −/− cDCs post rFPV vaccination.…”

“…Studies have shown that STAT6 and STAT3 can be differentially regulated, according to the state of viral infection/vaccination. Specifically, in the context of viral vector-based vaccination whilst IL-13/STAT6 signalling has been shown to dampen effective antiviral immunity 13,28 , however in acute and primary viral infections, it has shown to improve antiviral immunity 50,51 . This study showed that viral vector induced IL-13 "level" in the cell milieu significantly altered the STAT3/STAT6 equilibrium.…”

“…Knowing that IFN-γ can regulate IL-13 responses 30 , these observations indicated that following viral vector-based vaccination, at the cDC level the differential environmental immune responses to IL-13 are not only determined/regulated by STAT3/STAT6, but also by TGF-β1 and IFN-γ either collaboratively or independently, which was consistent with cancer/inflammation studies [53][54][55][56] . Interestingly, rapid STAT3 activation has shown to control some viral infections 51,57,58 whilst, STAT6 independent mechanisms have also been associated with effective antibody differentiation 28 . Moreover, IL-13 mediated enhanced IFN-γ signalling has been shown to exacerbate respiratory viral infections 31,59 .…”

“…Unimmunised BALB/c lung suspensions were treated with either 100 nM of small molecule STAT6 inhibitor (Axon Medchem) or 20 μM Stattic (small molecule STAT3 inhibitor) in PBS overnight followed by low (100 pg/ml) or high (10,000 pg/ml) IL-13 stimulation for 3 h or 0.5 h before evaluation of IL-4 and IL-13 receptor expression on lung DCs using flow cytometry as described above. Biologically relevant inhibitor concentrations were used in this study as reported previously 28,60 .…”

“…Furthermore IL-13Rα2 has also shown to induce transforming growth factor beta 1 (TGF-β1) under certain chronic infections and autoimmune disease conditions 2 . Recently, we have also shown that in the context of viral vector-based vaccination, the STAT6 independent pathway (likely associated with IL-13Rα2) was involved in antibody differentiation 28 . Therefore, knowing that both STAT3 and STAT6 are involved in IL-13 regulation and that IFN-γ can also modulate IL-13 activity [29][30][31] , this study focused on deciphering the IL-13 signalling mechanisms lung cDCs employ under different IL-13 conditions (different viral vector-based vaccination conditions), to induce vastly different adaptive immune outcomes.…”