The immunopathogenesis of sarcoidosis.

Sarcoidosis is a granulomatous multiorgan disease, thought to result from exposure to yet unidentified antigens in genetically susceptible individuals. The exaggerated inflammatory response that leads to granuloma formation is highly complex and involves the innate and adaptive immune system. Consecutive immunological studies using advanced technology have increased our understanding of aberrantly activated immune cells, mediators and pathways that influence the formation, maintenance and resolution of granulomas. Over the years, it has become increasingly clear that disease immunopathogenesis can only be understood if the clinical heterogeneity of sarcoidosis is taken into consideration, along with the distribution of immune cells in peripheral blood and involved organs. Most studies offer an immunological snapshot during disease course, while the cellular composition of both the circulation and tissue microenvironment may change over time. Despite these challenges, novel insights on the role of the immune system are continuously published, thus bringing the field forward. This review highlights current knowledge on the innate and adaptive immune responses involved in sarcoidosis pathogenesis, as well as the pathways involved in non-resolving disease and fibrosis development. Additionally, we describe proposed immunological mechanisms responsible for drug-induced sarcoid like reactions. Although many aspects of disease immunopathogenesis remain to be unraveled, the identification of crucial immune reactions in sarcoidosis may help identify new treatment targets. We therefore also discuss potential therapies and future strategies based on the latest immunological findings.

Comparison of SARS-CoV-2 spike-specific IgA and IgG in nasal secretions, saliva and serum.

Introduction: Several novel vaccine platforms aim at mucosal immunity in the respiratory tract to block SARS-CoV-2 transmission. Standardized methods for mucosal sample collection and quantification of mucosal antibodies are therefore urgently needed for harmonized comparisons and interpretations across mucosal vaccine trials and real-world data. Methods: Using commercial electrochemiluminescence antibody panels, we compared SARS-CoV-2 spike-specific IgA and IgG in paired saliva, nasal secretions, and serum from 1048 healthcare workers with and without prior infection. Results: Spike-specific IgA correlated well in nasal secretions and saliva (r>0.65, p<0.0001), but the levels were more than three-fold higher in nasal secretions as compared to in saliva (p<0.01). Correlations between the total population of spike-specific IgA and spike-specific secretory IgA (SIgA) were significantly stronger (p<0.0001) in nasal secretions (r=0.96, p<0.0001) as opposed to in saliva (r=0.77, p<0.0001), and spike-specific IgA correlated stronger (p<0.0001) between serum and saliva (r=0.73, p<0.001) as opposed to between serum and nasal secretions (r=0.54, p<0.001), suggesting transudation of monomeric spike specific IgA from the circulation to saliva. Notably, spike-specific SIgA had a markedly higher SARS-CoV-2 variant cross-binding capacity as compared to the total population of spike specific IgA and IgG in both nasal secretions, saliva and serum, (all p<0.0001), which emphasizes the importance of taking potential serum derived monomeric IgA into consideration when investigating mucosal immune responses. Discussion: Taken together, although spike-specific IgA can be reliably measured in both nasal secretions and saliva, our findings imply an advantage of higher levels and likely also a larger proportion of SIgA in nasal secretions as compared to in saliva. We further corroborate the superior variant cross-binding capacity of SIgA in mucosal secretions, highlighting the potential protective benefits of a vaccine targeting the upper respiratory tract.

Development of a proteomic signature associated with severe disease for patients with COVID-19 using data from 5 multicenter, randomized, controlled, and prospective studies.

Significant progress has been made in preventing severe COVID-19 disease through the development of vaccines. However, we still lack a validated baseline predictive biologic signature for the development of more severe disease in both outpatients and inpatients infected with SARS-CoV-2. The objective of this study was to develop and externally validate, via 5 international outpatient and inpatient trials and/or prospective cohort studies, a novel baseline proteomic signature, which predicts the development of moderate or severe (vs mild) disease in patients with COVID-19 from a proteomic analysis of 7000 + proteins. The secondary objective was exploratory, to identify (1) individual baseline protein levels and/or (2) protein level changes within the first 2 weeks of acute infection that are associated with the development of moderate/severe (vs mild) disease. For model development, samples collected from 2 randomized controlled trials were used. Plasma was isolated and the SomaLogic SomaScan platform was used to characterize protein levels for 7301 proteins of interest for all studies. We dichotomized 113 patients as having mild or moderate/severe COVID-19 disease. An elastic net approach was used to develop a predictive proteomic signature. For validation, we applied our signature to data from three independent prospective biomarker studies. We found 4110 proteins measured at baseline that significantly differed between patients with mild COVID-19 and those with moderate/severe COVID-19 after adjusting for multiple hypothesis testing. Baseline protein expression was associated with predicted disease severity with an error rate of 4.7% (AUC = 0.964). We also found that five proteins (Afamin, I-309, NKG2A, PRS57, LIPK) and patient age serve as a signature that separates patients with mild COVID-19 and patients with moderate/severe COVID-19 with an error rate of 1.77% (AUC = 0.9804). This panel was validated using data from 3 external studies with AUCs of 0.764 (Harvard University), 0.696 (University of Colorado), and 0.893 (Karolinska Institutet). In this study we developed and externally validated a baseline COVID-19 proteomic signature associated with disease severity for potential use in both outpatients and inpatients with COVID-19.

Super-resolution proximity labeling reveals anti-viral protein network and its structural changes against SARS-CoV-2 viral proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates in human cells by interacting with host factors following infection. To understand the virus and host interactome proximity, we introduce a super-resolution proximity labeling (SR-PL) method with a "plug-and-playable" PL enzyme, TurboID-GBP (GFP-binding nanobody protein), and we apply it for interactome mapping of SARS-CoV-2 ORF3a and membrane protein (M), which generates highly perturbed endoplasmic reticulum (ER) structures. Through SR-PL analysis of the biotinylated interactome, 224 and 272 peptides are robustly identified as ORF3a and M interactomes, respectively. Within the ORF3a interactome, RNF5 co-localizes with ORF3a and generates ubiquitin modifications of ORF3a that can be involved in protein degradation. We also observe that the SARS-CoV-2 infection rate is efficiently reduced by the overexpression of RNF5 in host cells. The interactome data obtained using the SR-PL method are presented at https://sarscov2.spatiomics.org. We hope that our method will contribute to revealing virus-host interactions of other viruses in an efficient manner.

Delayed generation of functional virus-specific circulating T follicular helper cells correlates with severe COVID-19.

Effective humoral immune responses require well-orchestrated B and T follicular helper (Tfh) cell interactions. Whether these interactions are impaired and associated with COVID-19 disease severity is unclear. Here, longitudinal blood samples across COVID-19 disease severity are analysed. We find that during acute infection SARS-CoV-2-specific circulating Tfh (cTfh) cells expand with disease severity. SARS-CoV-2-specific cTfh cell frequencies correlate with plasmablast frequencies and SARS-CoV-2 antibody titers, avidity and neutralization. Furthermore, cTfh cells but not other memory CD4 T cells, from severe patients better induce plasmablast differentiation and antibody production compared to cTfh cells from mild patients. However, virus-specific cTfh cell development is delayed in patients that display or later develop severe disease compared to those with mild disease, which correlates with delayed induction of high-avidity neutralizing antibodies. Our study suggests that impaired generation of functional virus-specific cTfh cells delays high-quality antibody production at an early stage, potentially enabling progression to severe disease.

Correlates of protection and viral load trajectories in omicron breakthrough infections in triple vaccinated healthcare workers.

Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.

Human influenza virus infection elicits distinct patterns of monocyte and dendritic cell mobilization 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 controls (HCs) and in patients with mild to moderate infections (primarily influenza A virus [IAV]). As compared to 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 than HCs. In blood, monocytes were the most frequent cellular source of TNFα during IAV infection and remained responsive to additional stimulation with TLR7/8L. Immune responses in older patients skewed towards increased monocyte frequencies 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 infection groups. Using SomaScan, a high-throughput aptamer-based assay to study proteomic changes between patients and HCs, we found differential expression of innate immunity-related proteins in plasma and nasopharyngeal secretions of IAV and SARS-CoV-2 patients. 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.

Respiratory and systemic monocytes, dendritic cells, and myeloid-derived suppressor cells in COVID-19: Implications for disease severity.

Since the beginning of the SARS-CoV-2 pandemic in 2020, researchers worldwide have made efforts to understand the mechanisms behind the varying range of COVID-19 disease severity. Since the respiratory tract is the site of infection, and immune cells differ depending on their anatomical location, studying blood is not sufficient to understand the full immunopathogenesis in patients with COVID-19. It is becoming increasingly clear that monocytes, dendritic cells (DCs), and monocytic myeloid-derived suppressor cells (M-MDSCs) are involved in the immunopathology of COVID-19 and may play important roles in determining disease severity. Patients with mild COVID-19 display an early antiviral (interferon) response in the nasopharynx, expansion of activated intermediate monocytes, and low levels of M-MDSCs in blood. In contrast, patients with severe COVID-19 seem to lack an early efficient induction of interferons, and skew towards a more suppressive response in blood. This is characterized by downregulation of activation markers and decreased functional capacity of blood monocytes and DCs, reduced circulating DCs, and increased levels of HLA-DRlo CD14+ M-MDSCs. These suppressive characteristics could potentially contribute to delayed T-cell responses in severe COVID-19 cases. In contrast, airways of patients with severe COVID-19 display hyperinflammation with elevated levels of inflammatory monocytes and monocyte-derived macrophages, and reduced levels of tissue-resident alveolar macrophages. These monocyte-derived cells contribute to excess inflammation by producing cytokines and chemokines. Here, we review the current knowledge on the role of monocytes, DCs, and M-MDSCs in COVID-19 and how alterations and the anatomical distribution of these cell populations may relate to disease severity.

Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel-Lindau syndrome.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

Comparison of Lung-Homing Receptor Expression and Activation Profiles on NK Cell and T Cell Subsets in COVID-19 and Influenza.

Respiratory viral infections with SARS-CoV-2 and influenza viruses commonly induce a strong infiltration of immune cells into the human lung, with potential detrimental effects on the integrity of the lung tissue. Despite comprising the largest fractions of circulating lymphocytes in the lung, rather little is known about how peripheral blood natural killer (NK) cell and T cell subsets are equipped for lung-homing in COVID-19 and influenza. Here, we provide a detailed comparative analysis of NK cells and T cells in patients infected with SARS-CoV-2 or influenza virus, focusing on the protein and gene expression of chemokine receptors known to be involved in recruitment to the lung. For this, we used 28-colour flow cytometry as well as re-analysis of a publicly available single-cell RNA-seq dataset from bronchoalveolar lavage (BAL) fluid. Frequencies of NK cells and T cells expressing CXCR3, CXCR6, and CCR5 were altered in peripheral blood of COVID-19 and influenza patients, in line with increased transcript expression of CXCR3, CXCR6, and CCR5 and their respective ligands in BAL fluid. NK cells and T cells expressing lung-homing receptors displayed stronger phenotypic signs of activation compared to cells lacking lung-homing receptors, and activation was overall stronger in influenza compared to COVID-19. Together, our results indicate a role for CXCR3+, CXCR6+, and/or CCR5+ NK cells and T cells that potentially migrate to the lungs in moderate COVID-19 and influenza patients, identifying common targets for future therapeutic interventions in respiratory viral infections.

Pulmonary and blood dendritic cells from sarcoidosis patients more potently induce IFNγ-producing Th1 cells compared with monocytes.

Sarcoidosis is a systemic inflammatory disease mainly affecting the lungs. The hallmark of sarcoidosis are granulomas that are surrounded by activated T cells, likely targeting the disease-inducing antigen. IFNγ-producing Th1 and Th17.1 T cells are elevated in sarcoidosis and associate with disease progression. Monocytes and dendritic cells (DCs) are antigen-presenting cells (APCs) and required for T cell activation. Several subsets of monocytes and DCs with different functions were identified in sarcoidosis. However, to what extent different monocyte and DC subsets can support activation and skewing of T cells in sarcoidosis is still unclear. In this study, we performed a transcriptional and functional side-by-side comparison of sorted monocytes and DCs from matched blood and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients. Transcriptomic analysis of all subsets showed upregulation of genes related to T cell activation and antigen presentation in DCs compared with monocytes. Allogeneic T cell proliferation was higher after coculture with monocytes and DCs from blood compared with BAL and DCs induced more T cell proliferation compared with monocytes. After coculture, proliferating T cells showed high expression of the transcription factor Tbet and IFNγ production. We also identified Tbet and RORγt coexpressing T cells that mainly produced IFNγ. Our data show that DCs rather than monocytes from sarcoidosis patients have the ability to activate and polarize T cells towards Th1 and Th17.1 cells. This study provides a useful in vitro tool to better understand the contribution of monocytes and DCs to T cell activation and immunopathology in sarcoidosis.

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

Understanding the presence and durability of antibodies against SARS-CoV-2 in the airways is required to provide insights into the ability of individuals to neutralize the virus locally and prevent viral spread. Here, we longitudinally assessed both systemic and airway immune responses upon SARS-CoV-2 infection in a clinically well-characterized cohort of 147 infected individuals representing the full spectrum of COVID-19 severity, from asymptomatic infection to fatal disease. In addition, we evaluated how SARS-CoV-2 vaccination influenced the antibody responses in a subset of these individuals during convalescence as compared with naive individuals. Not only systemic but also airway antibody responses correlated with the degree of COVID-19 disease severity. However, although systemic IgG levels were durable for up to 8 months, airway IgG and IgA declined significantly within 3 months. After vaccination, there was an increase in both systemic and airway antibodies, in particular IgG, often exceeding the levels found during acute disease. In contrast, naive individuals showed low airway antibodies after vaccination. In the former COVID-19 patients, airway antibody levels were significantly elevated after the boost vaccination, highlighting the importance of prime and boost vaccinations for previously infected individuals to obtain optimal mucosal protection.

Correction: Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

[This corrects the article DOI: 10.1371/journal.ppat.1009400.].

Shedding of infectious SARS-CoV-2 by hospitalized COVID-19 patients in relation to serum antibody responses.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic. The understanding of the transmission and the duration of viral shedding in SARS-CoV-2 infection is still limited. OBJECTIVES: To assess the timeframe and potential risk of SARS-CoV-2 transmission from hospitalized COVID-19 patients in relation to antibody response. METHOD: We performed a cross-sectional study of 36 COVID-19 patients hospitalized at Karolinska University Hospital. Patients with more than 8 days of symptom duration were sampled from airways, for PCR analysis of SARS-CoV-2 RNA and in vitro culture of replicating virus. Serum SARS-CoV-2-specific immunoglobulin G (IgG) and neutralizing antibodies titers were assessed by immunofluorescence assay (IFA) and microneutralization assay. RESULTS: SARS-CoV-2 RNA was detected in airway samples in 23 patients (symptom duration median 15 days, range 9-53 days), whereas 13 patients were SARS-CoV-2 RNA negative (symptom duration median 21 days, range 10-37 days). Replicating virus was detected in samples from 4 patients at 9-16 days. All but two patients had detectable levels of SARS-CoV-2-specific IgG in serum, and SARS-CoV-2 neutralizing antibodies were detected in 33 out of 36 patients. Total SARS-CoV-2-specific IgG titers and neutralizing antibody titers were positively correlated. High levels of both total IgG and neutralizing antibody titers were observed in patients sampled later after symptom onset and in patients where replicating virus could not be detected. CONCLUSIONS: Our data suggest that the presence of SARS-Cov-2 specific antibodies in serum may indicate a lower risk of shedding infectious SARS-CoV-2 by hospitalized COVID-19 patients.