Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

As suggested by this reviewer, Neuropilin1 was blocked by the addition of specific monoclonal Ab to isolated pDC before stimulation with SARS-CoV-2. A dose dependent inhibition of type I IFN release was observed when the anti-Neuropilin1 Ab was used, while no difference was found in presence of anti-human IgG , thus indicating a role of this molecule in facilitating entry of virus particles in pDC. These results are inserted in new Figure 4C.
Reviewer #2: 1. The introduction should be more comprehensive about published work on pDC in . This is the main topic of the study. Several references are cited by the authors, but the associated work is not presented in the introduction: in particular Refs 39,45,[47][48][49]. Authors should also introduce pDC response to other coronaviruses, which forms important background information: refs 42 and 44. On the contrary, other parts of the introduction appear less relevant to the present study and could be moved to discussion.
Both introduction and discussion sessions were changed according to the reviewer's suggestions. Furthermore, new references on the manuscript topic were added and can be found highlighted in yellow in the tracked version of the revised manuscript. The requested control experiments were performed on both PBMC and isolated pDC and results are now inserted in the new Supplementary Figure 2 (see also description on pages 5 and 6, lanes 133-139 and on page 8, lanes 186-187 of revised manuscript). An oligonucleotide with an unrelated sequence was also used as internal control. In addition, to verify the specificity of TLR7/8 inhibitors Flu virus, a well-known TLR7 agonist, was also used to stimulate PBMC culture and then the effects of the inhibitor were analyzed as reduction of IFN-a release.
Mock description was present in the Methods section ("Cell stimulation and supernatant collection"). Nonetheless, we further detailed its meaning in Methods (see on page 21, lanes 488-490 of revised manuscript) and Results section (see on page 5, lanes 127-128). We also changed throughout all the figure legends from "mock medium only" to "mock-treated cells".

Results presented in figure 5 are completely expected given the antiviral functions of type I interferons. It could be moved to supplementary.
We agree with the reviewer on the fact that some of the data shown in old Figure 5 are somehow expected. However, collectively these results provide a proof of evidence on the presence of biologically active type I IFN released in the supernatants of PBMC stimulated with SARS-CoV-2, that is able to promote in CALU-3 cells both the expression of MxA as well as a reduction of virus replication. Based on this we would prefer to leave this figure in the main body of the manuscript as new Figure 3. We hope that the reviewer agrees.

In figure 6D, it is very surprising that the inhibitor induces TNF and IL-6 production by pDC. Could authors coment on that ? Is the figure just mislabled ?
The effects induced by TLR7/8 inhibitor reported in old Figure 6C (new Figure 5C) reflect the wellknown cross-regulation between type I IFN release and the production of maturation-related cytokines TNF-a and IL-6 previously described by Palucka and collaborators (ref. 59 of the revised manuscript). The description of this effect was already present in the discussion section of the original manuscript (now on page 15, lanes 364-369). Interestingly, in presence of TLR7/8 inhibitor, a profound change in maturation marker profile occurs in isolated pDCs. In particular, mirroring the switch from type I IFN-a-to TNF-a-and IL-6-producing cells, in the presence of the TLR-7/8 inhibitor the P1 phenotype induced by SARS-CoV-2 stimulation in pDC reverted into the more mature/adaptive P2 and P3 populations, exemplified by the induction of the co-stimulatory marker CD86 (new Figure 5B). Similar data were recently obtained by our group in pDC stimulated with another single-stranded RNA virus, Tick-borne encephalitis virus, where the treatment with a TLR7/8 inhibitor blocking type I IFN production enhances TNF-a and IL-6 production (also this aspect is now discussed on page 15, lanes 366-369 and new Ref. 60). Figure 8 : authors should depict individual patient/donnor results in each graph (as in 8A), instead of histograms.

In
Old Figure 8 (now Figure 7) was revised according to the reviewer's suggestion. Figure 8, IL-10 levels are actually very low in all conditions. Are these levels and differences biologically meaningful? What is the sensitivity limit of the assay? (should be shown on the graph)

In
We agree with the reviewer on the IL-10 levels found in COVID-19 patient sera are low (now in new Figure 7C). However, these data are in line with values previously published by several groups showing a direct correlation between IL-10 increase and COVID-19 severity (just a couple of papers on the topic: doi: 10.1172/JCI137244; doi: 10.1080/22221751.2020.1770129). Agreeing with the reviewer's concern on the limit of assay sensitivity, we verified this parameter, which resulted to be 3.3 pg/ml according to the datasheet of the product, thus being below the values found in the analyzed sera. Moreover, while performing the CBA assay we always consider only values within the standard curves, never those falling below.