key: cord-0297752-fmbwikx7
authors: Vangeti, S.; Falck-Jones, S.; Yu, M.; Österberg, B.; Liu, S.; Asghar, M.; Sonden, K.; Albert, J.; Johansson, N.; Färnert, A.; Smed-Sörensen, A.
title: Human influenza virus infection elicits distinct patterns of monocyte and dendritic cell mobilization in blood and the nasopharynx
date: 2022-01-21
journal: nan
DOI: 10.1101/2022.01.18.22269508
sha: 80c693beac0493564a354df0af1ed22fc63bc029
doc_id: 297752
cord_uid: fmbwikx7

During respiratory viral infections, the precise roles of monocytes and dendritic cells (DCs) in the nasopharynx in limiting infection and influencing disease severity are incompletely described. We studied circulating and nasopharyngeal monocytes and DCs in healthy individuals and in patients with mild respiratory infections (primarily influenza A virus, IAV). As compared to healthy controls (HCs), patients with acute IAV infection displayed reduced DC but increased intermediate monocytes frequencies in blood, and an accumulation of most monocyte and DC subsets in the nasopharynx. IAV patients had more mature monocytes and DCs in the nasopharynx, and higher levels of TNF, IL-6 and IFN in plasma and the nasopharynx. In blood, monocytes, the most frequent cellular source of TNF during IAV infection, remained responsive to additional stimulation with TLR7/8L. Immune responses in older patients skewed towards increased monocytes rather than DCs suggesting a contributory role for monocytes in disease severity. In patients with other respiratory virus infections, we observed changes in monocyte and DC frequencies in the nasopharynx distinct from IAV patients, while differences in blood were more similar across patient groups. Together, our findings demonstrate tissue-specific and pathogen-specific patterns of monocyte and DC function during human respiratory viral infections and highlight the importance of comparative investigations in blood and the nasopharynx.

During respiratory viral infections, the precise roles of monocytes and dendritic cells (DCs) in the nasopharynx in limiting infection and influencing disease severity are incompletely described. We studied circulating and nasopharyngeal monocytes and DCs in healthy individuals and in patients with mild respiratory infections (primarily influenza A virus, IAV). As compared to healthy controls (HCs), patients with acute IAV infection displayed reduced DC but increased intermediate monocytes frequencies in blood, and an accumulation of most monocyte and DC subsets in the nasopharynx.

IAV patients had more mature monocytes and DCs in the nasopharynx, and higher levels of TNFα, IL-6 and IFNα in plasma and the nasopharynx. In blood, monocytes, the most frequent cellular source of TNFα during IAV infection, remained responsive to additional stimulation with TLR7/8L. Immune responses in older patients skewed towards increased monocytes rather than DCs suggesting a contributory role for monocytes in disease severity. In patients with other respiratory virus infections, we observed changes in monocyte and DC frequencies in the nasopharynx distinct from IAV patients, while differences in blood were more similar across patient groups.

Together, our findings demonstrate tissue-specific and pathogen-specific patterns of monocyte and DC function during human respiratory viral infections and highlight the importance of comparative investigations in blood and the nasopharynx. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Respiratory viral infections cause significant global disease burden with influenza, or flu, being responsible for a significant portion. An estimated 1 billion cases of influenza occur annually resulting in approximately 3-5 million severe cases and 290,000-650,000 deaths (1). In addition to seasonal epidemics caused by both influenza A or B virus (IAV and IBV, respectively), IAV can also cause pandemics. The majority of infections remain asymptomatic or develop mild to moderate respiratory disease, characterized by fever, nasal congestion, cough, and muscle aches. Severe disease mainly affects infants, pregnant women, and the elderly or immunocompromised, but can also occur in otherwise healthy individuals (2, 3) . The determinants of disease severity are still incompletely understood but may include properties of the virus, environmental factors, genetics, and immune responses of the patient (4, 5) . Other agents causing an influenza-like illness during influenza season include respiratory syncytial virus (RSV) and seasonal coronaviruses (OC43, HKU1, 229E, and NL63).

Similar to IAV, coronaviruses are capable of causing pandemics, most notably the ongoing coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (6).

IAV is primarily transmitted via inhalation of virus-containing aerosols or droplets, and mainly targets respiratory epithelial cells (7, 8) , with the nasopharynx being the initial site of virus replication. IAV generally remains localized to the airways, despite signs of systemic inflammation (9) . At the site of infection, resident innate immune cells including monocytes and dendritic cells (DCs) rapidly respond to the presence of virus by secreting cytokines, interferons, and chemokines to limit viral spread and recruit immune cells (10, 11) . Monocytes and DCs shape the specificity and strength of the subsequent adaptive responses (12, 13) . In blood, three subsets of monocytes are found: the CD14+CD16-classical monocytes (CMs), the most frequent subset at steady state, and the further differentiated CD14+CD16+ intermediate monocytes (IMs) and CD14-CD16+ nonclassical monocytes (NCMs) (14) (15) (16) . Blood IMs expand rapidly in response to inflammation, infection, or vaccination (3, 12, 17) . In addition, monocytes extravasate to tissue where they play an important role in innate immune protection. Monocytes also secrete TNF, a major regulator of innate immune function that is central to the cytokine storm associated with IAV infection (18) . Of the DCs, the CD1c+ myeloid DCs (MDCs) excel at activating naïve T cells (19) ; the CD141+ MDCs can cross-present antigens via MHC-I (20) ; and the CD123+ plasmacytoid DCs (PDCs) mediate type I IFN responses (21) . Monocytes and DCs vary in distribution and function, depending on the anatomical compartment (22, 23) . Moreover, monocytes and DCs are susceptible to IAV infection in vitro, and the cytopathic nature of the virus may impair their antigen processing and presenting functions (24) (25) (26) , delaying recovery and normalization of immune cell distribution and function (27, 28) .

Studies have shown that monocytes and DCs are recruited to the nasopharynx following infection with 2009 H1N1pdm IAV strains (3) , and in individuals hospitalized with severe influenza infections (29, 30) . Disease severity in hospitalized patients has been shown to correlate with (i) monocyte recruitment and increased levels of MCP3, IFN-2 and IL-10 in the nasal compartment (3) and (ii) strong TNF-producing monocytic responses in blood (2) and inflammatory, neutrophil-dominant patterns (31) .

Despite accounting for a comparatively greater burden of disease, immune responses during mild seasonal influenza infections remain less studied. Therefore, the roles played by monocytes and DCs in contributing to or mitigating mild influenza disease . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint are largely unknown. Additionally, while the response of blood monocytes and DCs to IAV has been studied well in vitro and in animal models (24, 25, 27, (32) (33) (34) (35) , few studies compare responses between blood and the nasopharynx in human infections (31, 32, 36) . Immune cell behaviour in the nasopharynx during IBV and RSV infections has not been studied in great detail but evidence of DC mobilization to the nasal cavity has been reported (29) . Studies on immune responses to mild SARS-CoV-2 infection have also primarily focused on blood and rarely the upper airways (37) .

Here, we determined monocyte and DC subset distribution, maturation and function in both blood and, for the first time, the nasopharynx, in patients with mild to moderate seasonal influenza and influenza-like infections. The methods described in this study allowed us to investigate airway immunity in a larger cohort of patients with SARS-CoV-2 infection (38, 39) , showing that methods to study the immune responses in the nasopharynx during acute disease are essential tools as we face the possibility of future pandemics. Comparing the dynamics of systemic and nasopharyngeal immune function will add to our understanding of the roles of monocytes and DCs in shaping the nature and magnitude of inflammation and subsequently disease severity during respiratory viral infections.

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During three consecutive influenza seasons (2016-2018), 84 adults with symptoms of influenza-like illness (ILI) were included in the study. Blood, nasal swabs and nasopharyngeal aspirates were collected ( Figure 1A) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Blood from IAV patients yielded significantly fewer PBMCs/mL compared to HCs ( Figure 1C ). In contrast, 3-fold higher cell numbers were recovered from the nasopharynx of IAV patients compared to HCs (median 0.77 vs. 0.25 x 10 6 cells). In fact, 43% of IAV patients had more than 1 x 10 6 cells recovered from their NPA sample.

Viability of PBMCs and NPA cells was variable across individuals, with no statistically significant differences between patients or HCs ( Figures 1D) . We determined the immune cell distribution in blood and the nasopharynx by flow cytometry (Supplementary Figure 1) on matched PBMC and NPA samples with a minimum 10 5 cells and ≥70% viability (n=22 IAV patients and n=16 HCs) to obtain high quality data (stained with identical panels and clones of antibodies). The frequencies of live CD45+ immune cells were increased in the NPA of patients as compared to HCs but remained similar in blood between the groups (data not shown). Among the immune cells, we found significantly higher frequencies of lineage (CD3, CD19, CD20, CD56, CD66abce) negative, HLA-DR+ cells, the compartment where monocytes and myeloid DCs can be identified, in both blood (p<0.0001), and the NPA (p<0.01) of IAV patients as compared to HCs ( Figure 1E ). Therefore, our data show that acute IAV infection results in an influx of monocytes and DCs to the nasopharynx.

To identify which monocyte subsets contributed to the changes observed during IAV infection, we analyzed the distribution of the different monocyte subsets ( Figure 2A ).

As expected, CMs were the most frequent monocytes in blood in both patients and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint HCs, and remained comparable. However, in the nasopharynx of IAV patients as compared to HCs, blood CM frequencies were significantly increased ( Figure 2B ). Strikingly, IM frequencies were significantly elevated, in both blood and NPA of IAV patients ( Figure 2C ), while blood NCM appeared to be lower in patients compared to HCs ( Figure 2D ). Comparing frequencies of monocytes in blood and NPA in the same individual ( Figure 2E In future studies, longitudinal sampling of the same patient would allow for mapping kinetics of monocyte redistribution over the course of acute IAV infection and convalescence. We also compared the frequency of IMs in blood and nasopharynx with the age of IAV patients and found a negative correlation in blood (R=-0.55, p=0.0008) ( Figure 2H ) but a positive correlation in the nasopharynx (R=0.46, p=0.045) ( Figure 2I ). In HCs, age and IM frequencies were not significantly correlated in blood or NPA (data not shown). A subset of IAV patients (n=11) returned for sampling during convalescence ( 4 weeks after initial sampling). We observed that frequencies of CMs (in blood) and IMs (blood and NPA) in convalescent individuals were reduced and closer to values seen in HCs ( Figure 2J ). Collectively, we found that the increased immune cell presence in the nasopharynx during acute IAV infection could be, to a large extent, attributed to increased frequencies of IMs as well as CMs which normalized during convalescence. Acute IAV infection resulted in altered monocyte is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint distribution, in particular at the site of infection and was more pronounced in older patients.

DCs may also account for redistribution in the myeloid cell compartment. In HCs, the Figure 3H ). During convalescence, CD1c+ MDC frequencies were increased in blood and lowered in nasopharynx ( Figure 3I ). Taken together, we showed that the DC compartment also underwent substantial changes in both blood and the nasopharynx during acute IAV infection, potentially suggesting that DCs also received signals to induce migration and/or maturation. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. Figure 4A ). In contrast, in IAV patients, monocytes and DCs in the nasopharynx expressed higher levels of HLA-DR than those in blood ( Figure 4A and C). We also found that in IAV patients, nasopharyngeal CD1c+ MDCs and CD141+ MDCs expressed more CD86 than cells in blood ( Figure 4D 

Pronounced cytokinemia is a hallmark of severe influenza disease (18) . In order to characterize the degree of inflammation in IAV patients, we measured local and . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint systemic cytokine levels. In agreement with earlier reports (2, 3, 29, 30), we observed elevated levels of TNF, IL-6 and IFN in nasopharyngeal secretions (Figures 5A-C) as well as in plasma of IAV patients as compared to HCs (Figures 5D-F). We also observed elevated levels of plasma IL-10, IL-15 and IL-18 in IAV patients as compared to HCs (data not shown). We compared soluble TNF levels against frequencies of monocytes and DCs (i.e., potential cellular sources) at the respective anatomical sites;

and found a positive correlation between soluble TNF and CM frequency, both in blood ( Figure 5G ); and in the nasopharynx of IAV patients ( Figure 5H ). However, such correlation was not observed for DCs in blood or NPA (data not shown). Interestingly, we also observed positive associations between age and level of TNF in plasma and NPA in IAV patients ( Figure 5I ). Furthermore, we observed that plasma IFN levels 

The localization of mature monocytes and DCs in the nasopharynx during IAV infection, and increased cytokine levels in both compartments led us to question . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint whether the cells in circulation were directly involved in inflammation during ongoing infection, or primarily provided a cache of differentiated cells that can migrate to the site of infection. Limited by the number of viable cells obtained from the nasopharynx, we tested the functional response of circulating monocytes and DCs to in vitro stimulation with a TLR7/8 agonist which mimics ssRNA and then quantified the frequency of TNF-producing cells in each monocyte and DC subset ( Figures 6A and   B ). We observed that blood monocytes and DCs from IAV patients produced TNF spontaneously, in the absence of any external stimulus, with most of the cytokine coming from monocytes (CMs > IMs > NCMs) ( Figure 6C ). In contrast, blood monocytes and DCs from HCs only produced TNF upon stimulation with TLR7/8L ( Figure 6D ). Importantly, while many of the IAV patients had cells producing TNF spontaneously, monocytes and DCs remained responsive and had the potential for further increased frequency of TNF-producing cells in response to TLR7/8L stimulation ( Figure 6D ). In summary, our data suggest that during IAV infection, mature monocytes and DCs accumulate in the nasopharynx, and blood monocytes and DCs function as a general source of TNF, potentially contributing to the systemic inflammatory effects accompanying influenza infections.

In order to determine whether our findings were pathogen-specific to IAV or a reflection of immune responses to respiratory viral infections in general, we analyzed samples from patients with confirmed IBV, RSV or SARS-CoV-2 infections. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint IBV and RSV patients were closer to IAV patients in age (mean: 53 and 61 years, respectively) and days with symptoms (mean: 5.0 and 6.5 days respectively). In contrast, patients with mild SARS-CoV-2 were younger than IAV patients (mean: 44 years and 59 years respectively) and sought medical attention later: mean 13.2 days with symptoms (SARS-CoV-2) as compared to 3.3 days (IAV) ( Table 2 ). Similar to IAV patients, patients with SARS-CoV-2 and RSV had a higher yield of cells in the nasopharynx than HCs ( Figure 7A ). The expansion in monocyte and DC frequencies in blood we saw in IAV patients, was not observed in other infections. Interestingly, in the nasopharynx, monocytes and DCs were significantly elevated during IAV infection, but not during IBV infection ( Figure 7B ). Within the monocyte compartment, we noted differences between the different pathogens. IMs were not increased during IBV or SARS-CoV-2 infection but were increased during RSV infection in both blood and the nasopharynx ( Figure 7C ). The DC compartment too showed differences between the groups in NPA. In blood, all DC subsets were decreased in all groups ( Figure 7D ). CD1c+ MDCs were increased in the nasopharynx only during IAV and SARS-CoV-2 infections. Interestingly, CD141+ MDCs were significantly increased in the nasopharynx only during IAV infection. PDCs in NPA were increased in all groups.

In plasma, individuals with SARS-CoV-2 infection had elevated levels of TNF in comparison with HCs and IAV patients ( Figure 7E ). SARS-CoV-2 infection was associated with elevated levels of IL-6 compared to HCs, but not compared to the IAV and IBV groups. In NPA, only the IAV and IBV groups had increased levels of IL-6.

Despite having increased frequencies of PDCs in NPA, nasopharyngeal levels of IFN were not elevated in SARS-CoV-2 patients. Together, these data show different patterns of monocyte and DC engagement in the nasopharynx and in blood, and also . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint between IAV, IBV, RSV and SARS-CoV-2, suggesting a requirement for further scrutiny.

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In this study, we mapped monocyte and DC distribution and function in blood and in the nasopharynx (the initial site of infection) of patients with ongoing IAV, IBV, RSV or SARS-CoV-2 infections and healthy controls. Several studies have examined immune responses during severe flu, in particular, focusing on patients sampled during or immediately following the 2009 H1N1 pandemic (2, 3, 31, 40) , or patients hospitalized with severe respiratory symptoms (2, (29) (30) (31) 40) ; and more recently, the immune dysregulation during severe COVID-19 (41) (42) (43) (44) . Here, we elucidated the innate myeloid cell composition and responses in a cohort with relatively mild symptoms, advanced age and underlying comorbidities, typical of seasonal influenza. Finally, we showed that our methods can be adapted to study immune responses in other viral infections, including the current COVID-19 pandemic.

Flow cytometric characterization revealed an influx of monocytes and DCs to the nasopharynx during infection, in line with previous reports in more severe IAV patients (3, 29, 30, 45) . Interestingly, age appeared to differentially skew the IM response in IAV patients. Despite having higher baseline frequencies of circulating CD16expressing monocytes (46) , older patients have fewer IMs in blood during acute IAV infection and concurrently more IMs in the nasopharynx. The increase in IMs suggests a more inflammatory milieu at the site of infection in older patients, perhaps contributing to disease severity as previously suggested in pandemic influenza (2) (3) (4) (5) .

Despite the general increase in IM frequencies, CMs remained the most frequent monocyte/DC subset in either compartment implying a functional role for CMs during IAV infection. Similar to studies in paediatric patients (29, 30) , we also found that over the course of the illness, DCs appeared to migrate from blood to infiltrate the . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint nasopharynx. However, older patients displayed a weaker recruitment of CD1c+

MDCs. In vitro studies have shown that monocytes can differentiate into type I IFN producing monocyte-derived DCs in response to IAV exposure (47) . It is therefore possible that CMs recruited to the nasopharynx in older individuals preferentially differentiate into IMs rather than DCs. Moreover, CD1c+ DC function is critical for clearance of IAV infection (35) . Therefore, diminished recruitment of CD1c+ MDCs and or reduced in situ DC differentiation (and therefore delayed or attenuated CD8+ T cell responses) may also contribute more severe disease in older patients.

During acute disease, monocytes present in/recruited to the nasopharynx likely contribute to sustained DC recruitment by secreting TNF, CCL2, CCL3 and CCL7 locally (3, 29, 40) . Due to the limited availability of cells in the NPA samples, demonstrating cytokine secretion from nasopharyngeal cells was not possible.

However, we noted that in the nasopharynx, CMs, CD1c+ MDCs and CD141+ MDCs is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint apoptosis (49) . In IAV infection, monocytes and DCs are likely recruited to the nasopharynx for a shorter period of time, where they face a similar fate. Some DCs likely traffic antigens to the lymph node to support adaptive responses (21, 35) .

Nasopharyngeal CMs were also more mature in patients with higher viral RNA loads and their recruitment and/or differentiation may be aided by local cytokine production (TNF and IL-6). Key innate inflammatory cytokines, including TNF, IL-6, IFN, IL-10, IL-15, and IL-18, were all significantly elevated early during infection (days 1-5) compared to healthy controls. TNF levels strongly correlated with the presence of CMs in both blood and nasopharynx; despite the significant expansion of IMs in blood, supporting previous studies (2, 14, 31, 50) . We also demonstrated that be circulating is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. In April-May 2020, during the COVID-19 pandemic, the approach and methods described here allowed us to implement this study plan in a rapidly evolving pandemic.

Although the patient cohorts were not identical, this endeavour proved the feasibility of using nasopharyngeal aspiration to assess local immune responses during respiratory viral infections. We observed that patients with IBV, RSV or SARS-CoV-2 also . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint immune cell recruitment to the nasopharynx as compared to IAV infections. We also illustrate the value of comparative high-resolution studies of immune cells in blood and at the site of infection, in order to fully understand their individual contributions to disease and how they orchestrate inflammation synergistically. Similar studies, carried out longitudinally across tissues, will aid resolution of these findings, and allow multivariate modelling of biomarkers of disease severity. Therapeutic approaches which allow selective modulation of monocyte and DC redistribution, maturation and cytokine/chemokine function may hold the key to reducing influenza-associated disease burden and mortality in the future. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint (Table 1 ) and are extensively discussed in a previous publication (58). Total burden of comorbidities was assessed using the CCI (59).

The severity of disease was categorized using the respiratory domain of the sequential organ failure assessment score (SOFA) (60). In the absence of arterial partial pressure of oxygen (PaO2), peripheral transcutaneous haemoglobin saturation (SpO2) was used instead to calculate a modified SOFA score (mSOFA) (61). Fraction of inspired oxygen (FiO2) estimation based on O2 flow was done in accordance with the Swedish is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint Intensive Care register definition (62). Mild disease was defined as PaO2/FiO2 (PFI) >53 kPa (>400 mmHg) or SpO2/FiO2 (SFI) >400. Moderate disease was defined as PFI >27-53 kPa (>200-400 mmHg) or SFI 235-400. The disease severity score is more extensively described in a previous publication (38) .

Blood, nasal swabs and nasopharyngeal aspirates (NPA) were obtained from all patients (acute and convalescent phase samples) and healthy controls ( Figure 1A) .

Briefly, up to 30mL venous blood was collected in Vacutainer® tubes containing EDTA, for blood counts and PBMC isolation. Nasopharyngeal swabs (Sigma Virocult®) were collected for diagnostic qPCR. NPA samples were collected into a vacuum trap by inserting a thin catheter through the naris, deep into the nasopharynx and applying gentle suction for 1-3 minutes. The vacuum trap and tubing were rinsed out with 3mL sterile PBS. All samples were processed within 2 hours of sampling. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint CoV-2 infection was diagnosed similarly using the GeneXpert SARS-CoV-2 detection system (Cepheid). Supplementary data on bacterial cultures were retrieved from the microbiology lab/clinical records.

Blood samples were centrifuged at 800g/10 min/room temperature (RT) and plasma was frozen at -20°C. The blood volume was reconstituted with sterile PBS and PBMCs were obtained by density-gradient centrifugation using is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Spearman's rank correlation coefficient for nonparametric analyses. Differences between HCs or IAV patients and other patient groups were assessed using nonparametric tests after assessing normality, using the Kruskal-Wallis test with Dunn's multiple comparisons test (at 95% confidence intervals). Data were considered significant at p<0.05.

Informed consent was obtained from all patients and volunteers following verbal and written information. The study was approved by the Swedish Ethical Review Authority is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint DISCLOSURE AS-S is a consultant to Astra-Zeneca on studies not related to the present study.

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The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.18.22269508 doi: medRxiv preprint levels in the NPA (n=4) and plasma (n=8) during the acute (upward triangles) and convalescent phase (downward triangles) in IAV patients. Dashed lines depict median frequency values from HCs in blood and NPA. Differences between acute and convalescent phase values were assessed using Mann-Whitney test and considered significant at p<0.05 (*p<0.05 and **p<0.01). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. Lines indicate median concentration. Differences between IAV patients and HCs were assessed using Mann-Whitney test and considered significant at p<0.05 (*p<0.05, **p<0.01 and ***p<0.001). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 21, 2022. 

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Review of Aerosol Transmission of Influenza A Virus

Respiratory epithelial cells in innate immunity to influenza virus infection

Sensitive detection assays for influenza RNA do not reveal viremia in US blood donors

Cytokine production by primary human macrophages infected with highly pathogenic H5N1 or pandemic H1N1 2009 influenza viruses

Innate immune response of human alveolar macrophages during influenza A infection

Dengue virus infection induces expansion of a CD14(+)CD16(+) monocyte population that stimulates plasmablast differentiation

Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function

Phenotype, function, and differentiation potential of human monocyte subsets

Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes

The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

The novel subset of CD14+:CD16+ blood monocytes is expanded in sepsis patients

Into the Eye of the Cytokine Storm

Human blood mDC subsets exhibit distinct TLR repertoire and responsiveness

BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon

Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans

High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization

Influenza A virus infection of human primary dendritic cells impairs their ability to crosspresent antigen to CD8 T cells

Severe H7N9 infection is associated with decreased antigen-presenting capacity of CD14+ cells

Protection of human myeloid dendritic cell subsets against influenza A virus infection is differentially regulated upon TLR stimulation

Inflammation induced by influenza virus impairs human innate immune control of pneumococcus

Severe and persistent depletion of circulating plasmacytoid dendritic cells in patients with 2009 pandemic H1N1 infection

Differential recruitment of dendritic cells and monocytes to respiratory mucosal sites in children with influenza virus or respiratory syncytial virus infection

Mobilization of Plasmacytoid and Myeloid Dendritic Cells to Mucosal Sites in Children with Respiratory Syncytial Virus and Other Viral Respiratory Infections

Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza

Human Blood and Tonsil Plasmacytoid Dendritic Cells Display Similar Gene Expression Profiles but Exhibit Differential Type I IFN Responses to Influenza A Virus Infection

Protective influenza-specific CD8 T cell responses require interactions with dendritic cells in the lungs

Clearance of influenza virus from the lung depends on migratory langerin+CD11b-but not plasmacytoid dendritic cells

Similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ-resident dendritic cells

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses

Functional monocytic myeloid-derived suppressor cells increase in blood but not airways and predict COVID-19 severity

Airway antibodies emerge according to COVID-19 severity and wane rapidly but reappear after SARS-CoV-2 vaccination

Respiratory Mucosal Proteome Quantification in Human Influenza Infections

Increased sCD163 and sCD14 Plasmatic Levels and Depletion of Peripheral Blood Pro-Inflammatory Monocytes, Myeloid and Plasmacytoid Dendritic Cells in Patients With Severe COVID-19 Pneumonia

An inflammatory cytokine signature predicts COVID-19 severity and survival

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages

Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm

Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors

Rapid differentiation of monocytes into type I IFN-producing myeloid dendritic cells as an antiviral strategy against influenza virus infection

Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells

Kinetics of myeloid dendritic cell trafficking and activation: impact on progressive, nonprogressive and controlled SIV infections