Development of a new HISCL automated CXCL9 immunoassay.

C-X-C motif chemokine ligand 9 (CXCL9), a candidate biomarker, reflects type 1 (T1) inflammation pathology. Here, we report the analytical performance and clinical characteristics of a new CXCL9 reagent for a fully automated immunoassay device. We evaluated the limits of blank, detection, and quantitation (LoQ) along with other efficacy parameters, and the ability of the assay to report patient health, COVID-19 status, and the presence of asthma and/or interstitial lung diseases (ILDs). The coefficient of variation for 5-day total precision using two instruments was 7% across two controls, serum, and plasma panels. LoQ of 2.2 pg/mL suggested the efficacy of the assay in detecting T1 inflammation in plasma or serum; no cross-reactivity or interference was observed. We identified high serum CXCL9 levels in samples from patients with acute COVID-19 infections (n = 57), chronic bird-related hypersensitivity pneumonitis (n = 61), asthma (n = 194), and ILDs (n = 84) compared to healthy individuals (< 39.0 pg/mL). Furthermore, CXCL9 levels increased with age in asthma patients, and an opposite trend was observed for T2 inflammatory factors. These results suggest the utility of the automated CXCL9 immunoassay for measuring CXCL9 in clinical samples and reflect its role in T1 inflammation.

The Chemokine MIG is Associated with an Increased Risk of COVID-19 Mortality in Mexican Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emergent viral disease in which the host inflammatory response modulates the clinical outcome. Severe outcomes are associated with an exacerbation of inflammation in which chemokines play an important role as the attractants of immune cells to the tissues. OBJECTIVE: To evaluate the relationship of the chemokines IL-8, RANTES, MIG, MCP-1, and IP-10 with COVID-19 severity and outcomes in Mexican patients. METHODS: We analyzed the serum levels of IL-8, RANTES, MIG, MCP-1 and IP-10 in 148 COVID-19 hospitalized patients classified as mild (n=20), severe (n=61), and critical (n=67), as well as in healthy individuals (n=10), by flow cytometry bead array assay. RESULTS: Chemokine levels were higher in patients than in the healthy individuals, but only MIG, MCP-1, and IP-10 increased according to the disease severity, showing the highest levels in the critical group. MIG, MCP-1, and IP-10 levels were also higher in COVID-19 patients with comorbidities such as renal disease, type 2 diabetes, and hypertension. Moreover, elevated MIG levels seem to be related to organic failure/shock, and an increased risk of death. CONCLUSIONS: Our results suggest that the increased levels of MCP-1, IP-10, and especially MIG might be useful in predicting severe COVID-19 outcomes and could be promising therapeutic targets.

Proteomic profiling of MIS-C patients indicates heterogeneity relating to interferon gamma dysregulation and vascular endothelial dysfunction.

Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in pediatric patients. Weeks after an often mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MIS-C present with varying degrees of cardiovascular and hyperinflammatory symptoms. Here we perform a comprehensive analysis of the plasma proteome of more than 1400 proteins in children with SARS-CoV-2. We hypothesize that the proteome would reflect heterogeneity in hyperinflammation and vascular injury, and further identify pathogenic mediators of disease. We show that protein signatures demonstrate overlap between MIS-C, and the inflammatory syndromes macrophage activation syndrome (MAS) and thrombotic microangiopathy (TMA). We demonstrate that PLA2G2A is an important marker of MIS-C that associates with TMA. We find that IFNγ responses are dysregulated in MIS-C patients, and that IFNγ levels delineate clinical heterogeneity.

Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Plasmacytoid Dendritic Cells Depletion and Elevation of IFN-γ Dependent Chemokines CXCL9 and CXCL10 in Children With Multisystem Inflammatory Syndrome.

Background: SARS-CoV-2 occurs in the majority of children as COVID-19, without symptoms or with a paucisymptomatic respiratory syndrome, but a small proportion of children develop the systemic Multi Inflammatory Syndrome (MIS-C), characterized by persistent fever and systemic hyperinflammation, with some clinical features resembling Kawasaki Disease (KD). Objective: With this study we aimed to shed new light on the pathogenesis of these two SARS-CoV-2-related clinical manifestations. Methods: We investigated lymphocyte and dendritic cells subsets, chemokine/cytokine profiles and evaluated the neutrophil activity mediators, myeloperoxidase (MPO), and reactive oxygen species (ROS), in 10 children with COVID-19 and 9 with MIS-C at the time of hospital admission. Results: Patients with MIS-C showed higher plasma levels of C reactive protein (CRP), MPO, IL-6, and of the pro-inflammatory chemokines CXCL8 and CCL2 than COVID-19 children. In addition, they displayed higher levels of the chemokines CXCL9 and CXCL10, mainly induced by IFN-γ. By contrast, we detected IFN-α in plasma of children with COVID-19, but not in patients with MIS-C. This observation was consistent with the increase of ISG15 and IFIT1 mRNAs in cells of COVID-19 patients, while ISG15 and IFIT1 mRNA were detected in MIS-C at levels comparable to healthy controls. Moreover, quantification of the number of plasmacytoid dendritic cells (pDCs), which constitute the main source of IFN-α, showed profound depletion of this subset in MIS-C, but not in COVID-19. Conclusions: Our results show a pattern of immune response which is suggestive of type I interferon activation in COVID-19 children, probably related to a recent interaction with the virus, while in MIS-C the immune response is characterized by elevation of the inflammatory cytokines/chemokines IL-6, CCL2, and CXCL8 and of the chemokines CXCL9 and CXL10, which are markers of an active Th1 type immune response. We believe that these immunological events, together with neutrophil activation, might be crucial in inducing the multisystem and cardiovascular damage observed in MIS-C.

Chronic Obstructive Pulmonary Disease Patients Have Increased Levels of Plasma Inflammatory Mediators Reported Upregulated in Severe COVID-19.

Background: Chronic obstructive pulmonary disease (COPD) is associated with increased risk of severe COVID-19, but the mechanisms are unclear. Besides, patients with severe COVID-19 have been reported to have increased levels of several immune mediators. Methods: Ninety-two proteins were quantified in 315 plasma samples from 118 asthmatics, 99 COPD patients and 98 healthy controls (age 40-90 years), who were recruited in Colombia before the COVID-19 pandemic. Protein levels were compared between each disease group and healthy controls. Significant proteins were compared to the gene signatures of SARS-CoV-2 infection reported in the "COVID-19 Drug and Gene Set Library" and with experimentally tested protein biomarkers of severe COVID-19. Results: Forty-one plasma proteins showed differences between patients and controls. Asthmatic patients have increased levels in IL-6 while COPD patients have a broader systemic inflammatory dysregulation driven by HGF, OPG, and several chemokines (CXCL9, CXCL10, CXCL11, CX3CL1, CXCL1, MCP-3, MCP-4, CCL3, CCL4 and CCL11). These proteins are involved in chemokine signaling pathways related with response to viral infections and some, were found up-regulated upon SARS-CoV-2 experimental infection of Calu-3 cells as reported in the COVID-19 Related Gene Sets database. An increase of HPG, CXCL9, CXCL10, IL-6, MCP-3, TNF and EN-RAGE has also been experimentally detected in patients with severe COVID-19. Conclusions: COPD patients have altered levels of plasma proteins that have been reported increased in patients with severe COVID-19. Our study suggests that COPD patients have a systemic dysregulation in chemokine networks (including HGF and CXCL9) that could make them more susceptible to severe COVID-19. Also, that IL-6 levels are increased in some asthmatic patients (especially in females) and this may influence their response to COVID-19. The findings in this study depict a novel panel of inflammatory plasma proteins in COPD patients that may potentially associate with increased susceptibility to severe COVID-19 and might be useful as a biomarker signature after future experimental validation.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Immunological and inflammatory profiles during acute and convalescent phases of severe/ critically ill COVID-19 patients.

BACKGROUND: The 2019 Coronavirus (COVID-19) pandemic poses a huge threat internationally; however, the role of the host immune system in the pathogenesis of COVID-19 is not well understood. METHODS: Cytokine and chemokine levels and characterisation of immune cell subsets from 20 COVID-19 cases after hospital admission (17 critically ill and 3 severe patients) and 16 convalescent patients were determined using a multiplex immunoassay and flow cytometry, respectively. RESULTS: IP-10, MCP-1, MIG, IL-6, and IL-10 levels were significantly higher in acute severe/critically ill patients with COVID-19, whereas were normal in patients who had reached convalescence. CD8 T cells in severe and critically ill COVID-19 patients expressed high levels of cytotoxic granules (granzyme B and perforin)and was hyperactivated as evidenced by the high proportions of CD38. Furthermore, the cytotoxic potential of natural killer (NK) cells, and the frequencies of myeloid dendritic cells and plasmacytoid dendritic cells was reduced in patients with severe and critical COVID-19; however, these dysregulations were found to be restored in convalescent phases. CONCLUSION: Thus, elicitation of the hyperactive cytokine-mediated inflammatory response, dysregulation of CD8 T and NK cells, and deficiency of host myeloid and plasmacytoid DCs, may contribute to COVID-19 pathogenesis and provide insights into potential therapeutic targets and strategies.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

Factors of Severity in Patients with COVID-19: Cytokine/Chemokine Concentrations, Viral Load, and Antibody Responses.

The severity of COVID-19 ranges from mild to critical diseases. However, limited data have been published on the detailed kinetics of viral load and host immune response throughout the disease course depending on disease severity. In this study, we comprehensively analyzed viral load, antibody responses to SARS-CoV-2, and cytokines/chemokines during the disease course, and identified the factors related to severity. Nasopharyngeal (NP) and plasma specimens were obtained from 31 patients with COVID-19 during hospitalization. Viral RNA in NP specimens was quantified by reverse transcription-PCR. Anti-SARS-CoV-2 antibodies and cytokines/chemokines in plasma specimens were analyzed by ELISA and cytometric bead array. The viral load in patients with COVID-19 peaked at the early stage of the disease and continuously decreased. Severe and critical cases showed higher viral load and prolonged viral shedding than asymptomatic and mild cases. Whereas plasma IgG was gradually increased and maintained during hospitalization, plasma IgM peaked at 3 weeks after symptom onset and dissipated. The antibody response in severe and critical cases was slightly delayed but stronger than those in others. High levels of interferon (IFN)-α, IFN-γ-induced protein-10, monokine induced by IFN-γ, and interleukin-6 at 5-10 days from symptom onset were associated with the severity of COVID-19. Our data indicate that high viral load in the respiratory tract and excessive production of cytokines and chemokines between 1 and 2 weeks from the symptom onset were significantly associated with the severity of COVID-19.

Immune response in children with COVID-19 is characterized by lower levels of T-cell activation than infected adults.

Study of immunological features of immune response in 14 children (aged from 12 days up to 15 years) and of 10 adults who developed COVID-19 show increased number of activated CD4 and CD8 cells expressing DR and higher plasmatic levels of IL-12 and IL-1ß in adults with COVID-19, but not in children. In addition, plasmatic levels of CCL5/RANTES are higher in children and adults with COVID-19, while CXCL9/MIG was only increased in adults. Higher number of activated T cells and expression of IL-12 and CXCL9 suggest prominent Th1 polarization of immune response against SARS-CoV2 in infected adults as compared with children.