Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells

Abstract Type I and type III interferons (IFNs) are fundamental for antiviral immunity, but prolonged expression is also detrimental to the host. Therefore, upon viral infection high levels of type I and III IFNs are followed by a strong and rapid decline. However, the mechanisms responsible for this suppression are still largely unknown. Here, we show that IgG opsonization of model viruses influenza and respiratory syncytial virus (RSV) strongly and selectively suppressed type I and III IFN production by various human antigen‐presenting cells. This suppression was induced by selective inhibition of TLR, RIG‐I‐like receptor, and STING‐dependent type I and III IFN gene transcription. Surprisingly, type I and III IFN suppression was mediated by Syk and PI3K independent inhibitory signaling via FcγRIIa, thereby identifying a novel non‐canonical FcγRIIa pathway in myeloid cells. Together, these results indicate that IgG opsonization of viruses functions as a novel negative feedback mechanism in humans, which may play a role in the selective suppression of type I and III IFN responses during the late‐phase of viral infections. In addition, activation of this pathway may be used as a tool to limit type I IFN‐associated pathology.

A revised version of your manuscript that takes into account the comments of the referees will be reconsidered for publication. Should you disagree with any of the referees' concerns, you should address this in your point-by-point response and provide solid scientific reasons for why you will not make the requested changes.
You should also pay close attention to the editorial comments included below. **In particular, please edit your figure legends to follow Journal standards as outlined in the editorial comments. For all data, including the new data generated for the revision of the manuscript, please report in the Figure legends the number of independent experiments and number of samples per experiment (or experimental replicates). For flow cytometry data please show the full gating strategy, including the percentage of cells in the region or gate or event count. In the histograms/dot plots shown please report which fluorochromes were used and the scaling in the axis (log/lin). Failure to do this will result in delays in the re-review process.** Please note that submitting a revision of your manuscript does not guarantee eventual acceptance, and that your revision will be re-reviewed by the referees before a decision is rendered.
If the revision of the paper is expected to take more than three months, please inform the editorial office.
Revisions taking longer than six months may be assessed by new referees to ensure the relevance and timeliness of the data.
Once again, thank you for submitting your manuscript to European Journal of Immunology and we look forward to receiving your revision. Newling et al., "IgG opsonization of viruses suppresses type I and III interferon production by human myeloid antigen-presenting cells via inhibitory signaling by Fc gamma receptor IIa" The authors demonstrate that immune complexes, including opsonized influenza and respiratory syncytial viruses, attenuate type I and III interferon (IFN) production by human monocyte-derived myeloid DCs. This affects also IFN stimulated gene (ISG) expression, but not production of IL-12 and IL-27 or co-stimulatory molecule up-regulation like CD70. The authors identify FcgammaRIIa as the responsible receptor, but the activating signaling by this receptor does not seem to be involved in this type I and III IFN production inhibition. From these data the authors speculate that immune complexes terminate anti-viral IFN production after viral infection via novel FcgammaRIIa signaling.
The reported findings could be potentially interesting, if the authors can clarify that viral infection is unaltered under their experimental conditions, how their monocyte-derived DCs detect CpG and give some indication on the FcgammaRIIa signaling that is involved in the inhibition of IFN production.
Major comments:

1.
In order to exclude that opsonization blocks infection and thereby prevents all cytosolic RNA detection in response to RNA virus infection, the authors have to demonstrate that infection is similar between virus exposure with and without antibody addition, for example for the reported conditions after adding antibodies 30 minutes after infection.

2.
TLR9 is thought to be primarily expressed by plasmacytoid DCs. How do the authors envision that CpG induces type I and III interferon in myeloid DCs, which they then report to be attenuated by immune complexes? Do they authors detect expression of TLR9 by human monocyte-derived DCs?
3. How does FcgammaRIIa inhibit type I and III interferon production? Some information how an activating Fcgamma receptor can achieve this, should be provided. To make this statement the authors should generate DCs under the same conditions. Moreover, DCs exist in many subsets, which may or may not react in the way as shown for blood derived DCs. This should be discussed in much greater detail.

First Revision -authors' response -17-Jul-2018
We thank the reviewers for their comments and suggestions and are glad to hear that they consider this work of potential interest for publication in the European Journal of Immunology. Below we have made a point-by-point response to the reviewer's comments.

Reviewer 1
The The reviewer is right that it should be verified that the infection rate is similar between virus exposure with and without antibodies. We now show this in Figure 1B (previously, this was shown as supplementary data, but we realize that this important control should be included as a main figure). Since the issue of TLR9 expression by moDCs is still unclear, and also since we have merely used TLR9 as one of many examples of IFN-inducing receptors that are inhibited by FcγRIIa stimulation, we have decided to remove the CpG data from the manuscript. Please note that the data in Figure 2 still clearly demonstrates that IgG immune complexes inhibit type I IFN production by various receptors, including TLRs, RIG-I/MDA5, and cytosolic DNA sensors.

How does FcgammaRIIa inhibit type I and III interferon production? Some information how an activating
Fcgamma receptor can achieve this, should be provided.
As requested by the reviewer, we have further looked into the mechanism of FcγRIIa-mediated type I IFN suppression. Since we identified that FcγRIIa inhibits type I IFN production at the level of gene transcription, we have assessed the expression of the main transcription factors of type I IFN, i.e. IRF1, IRF3, and IRF7. Importantly, while we did not observe any difference in IRF3 and IRF7 expression, we identified that FcγRIIa co-stimulation down-regulates the expression of IRF1 ( Figure 6D). This finding nicely correlates with very recent transcriptomics data that also identified IRF1 as one of the main As requested, we have now added statistical evaluation for several key experiments. These are depicted in Figures 1D, 1F, 2B, and Supplementary Figure 1A.

While the authors show that adding IgG 0.5 hrs after virus does not alter virus mRNA in the cell they do not show proof that a virus IC is generated under these conditions. The could elute virus IgG complexes from the cell surface to proof this.
The reviewer is right that it is important to verify that the antibodies indeed recognize and bind to the viruses. Therefore, we have assessed binding of antibodies to both influenza and RSV using an ELISA setup ( Figure 1A), which confirmed IgG binding to the viruses (previously, this was shown as supplementary data, but we agree that this control should be included as a main figure).
Most forward, I have a major concern with the results in Figures 2-6  Another good point by the reviewer. To really make sure that soluble immune complexes induce the same responses as plate-bound IgG we have tested this not in one, but in two different ways. First we have co-stimulated our cells with the soluble fraction of heat-aggregated immunoglobulins (also known as "haggs"). Second, we have co-stimulated our cells with IgG-coated beads, which we have compared with control (BSA-coated) beads. Importantly, both soluble IgG immune complexes yielded the exact same results as plate-bound IgG and IgG opsonized viruses ( Figure 2D and 2E Taken together, we believe that the additional experimental data and textual adjustments have greatly strengthened the manuscript. We thank the reviewers for their comments and suggestions and look forward to their reply.
Kind regards, Jeroen den Dunnen (also on behalf of all co-authors)

Second Editorial Decision -20-Aug-2018
Dear Mrs. Newling, It is a pleasure to provisionally accept your manuscript entitled "Inhibitory signaling by Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells" for publication in the European Journal of Immunology. For final acceptance, please follow the instructions below and return the requested items as soon as possible as we cannot process your manuscript further until all items listed below are dealt with.
Please note that EJI articles are now published online a few days after final acceptance (see Accepted Articles: https://onlinelibrary.wiley.com/toc/15214141/0/ja). The files used for the Accepted Articles are the final files and information supplied by you in Manuscript Central. You should therefore check that all the information (including author names) is correct as changes will NOT be permitted until the proofs stage.
We look forward to hearing from you and thank you for submitting your manuscript to the European Journal of Immunology.