Comparative analysis of antibody- and lipid-based multiplexing methods for single-cell RNA-seq.

BACKGROUND: Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called "hashing." RESULTS: Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects. CONCLUSIONS: Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung.

Microglia Reduce Herpes Simplex Virus 1 Lethality of Mice with Decreased T Cell and Interferon Responses in Brains.

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and can induce encephalitis, which is the most common cause of sporadic, fatal encephalitis. An increase of microglia is detected in the brains of encephalitis patients. The issues regarding whether and how microglia protect the host and neurons from HSV-1 infection remain elusive. Using a murine infection model, we showed that HSV-1 infection on corneas increased the number of microglia to outnumber those of infiltrating leukocytes (macrophages, neutrophils, and T cells) and enhanced microglia activation in brains. HSV-1 antigens were detected in brain neurons, which were surrounded by microglia. Microglia depletion increased HSV-1 lethality of mice with elevated brain levels of viral loads, infected neurons, neuron loss, CD4 T cells, CD8 T cells, neutrophils, interferon (IFN)-ß, and IFN-γ. In vitro studies demonstrated that microglia from infected mice reduced virus infectivity. Moreover, microglia induced IFN-ß and the signaling pathway of signal transducer and activator of transcription (STAT) 1 to inhibit viral replication and damage of neurons. Our study reveals how microglia protect the host and neurons from HSV-1 infection.

Tpl2 Ablation Leads to Hypercytokinemia and Excessive Cellular Infiltration to the Lungs During Late Stages of Influenza Infection.

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase known to promote inflammation in response to various pathogen-associated molecular patterns (PAMPs), inflammatory cytokines and G-protein-coupled receptors and consequently aids in host resistance to pathogens. We have recently shown that Tpl2-/- mice succumb to infection with a low-pathogenicity strain of influenza (x31, H3N2) by an unknown mechanism. In this study, we sought to characterize the cytokine and immune cell profile of influenza-infected Tpl2-/- mice to gain insight into its host protective effects. Although Tpl2-/- mice display modestly impaired viral control, no virus was observed in the lungs of Tpl2-/- mice on the day of peak morbidity and mortality suggesting that morbidity is not due to virus cytopathic effects but rather to an overactive antiviral immune response. Indeed, increased levels of interferon-ß (IFN-ß), the IFN-inducible monocyte chemoattractant protein-1 (MCP-1, CCL2), Macrophage inflammatory protein 1 alpha (MIP-1α; CCL3), MIP-1ß (CCL4), RANTES (CCL5), IP-10 (CXCL10) and Interferon-γ (IFN-γ) was observed in the lungs of influenza-infected Tpl2-/- mice at 7 days post infection (dpi). Elevated cytokine and chemokines were accompanied by increased infiltration of the lungs with inflammatory monocytes and neutrophils. Additionally, we noted that increased IFN-ß correlated with increased CCL2, CXCL1 and nitric oxide synthase (NOS2) expression in the lungs, which has been associated with severe influenza infections. Bone marrow chimeras with Tpl2 ablation localized to radioresistant cells confirmed that Tpl2 functions, at least in part, within radioresistant cells to limit pro-inflammatory response to viral infection. Collectively, this study suggests that Tpl2 tempers inflammation during influenza infection by constraining the production of interferons and chemokines which are known to promote the recruitment of detrimental inflammatory monocytes and neutrophils.

Neutrophil Extracellular Traps in Fatal COVID-19-Associated Lung Injury.

An excess formation of neutrophil extracellular traps (NETs), previously shown to be strongly associated with cytokine storm and acute respiratory distress syndrome (ARDS) with prevalent endothelial dysfunction and thrombosis, has been postulated to be a central factor influencing the pathophysiology and clinical presentation of severe COVID-19. A growing number of serological and morphological evidence has added to this assumption, also in regard to potential treatment options. In this study, we used immunohistochemistry and histochemistry to trace NETs and their molecular markers in autopsy lung tissue from seven COVID-19 patients. Quantification of key immunomorphological features enabled comparison with non-COVID-19 diffuse alveolar damage. Our results strengthen and extend recent findings, confirming that NETs are abundantly present in seriously damaged COVID-19 lung tissue, especially in association with microthrombi of the alveolar capillaries. In addition, we provide evidence that low-density neutrophils (LDNs), which are especially prone to NETosis, contribute substantially to COVID-19-associated lung damage in general and vascular blockages in particular.

A Fragile Balance: Does Neutrophil Extracellular Trap Formation Drive Pulmonary Disease Progression?

Neutrophils act as the first line of defense during infection and inflammation. Once activated, they are able to fulfil numerous tasks to fight inflammatory insults while keeping a balanced immune response. Besides well-known functions, such as phagocytosis and degranulation, neutrophils are also able to release "neutrophil extracellular traps" (NETs). In response to most stimuli, the neutrophils release decondensed chromatin in a NADPH oxidase-dependent manner decorated with histones and granule proteins, such as neutrophil elastase, myeloperoxidase, and cathelicidins. Although primarily supposed to prevent microbial dissemination and fight infections, there is increasing evidence that an overwhelming NET response correlates with poor outcome in many diseases. Lung-related diseases especially, such as bacterial pneumonia, cystic fibrosis, chronic obstructive pulmonary disease, aspergillosis, influenza, and COVID-19, are often affected by massive NET formation. Highly vascularized areas as in the lung are susceptible to immunothrombotic events promoted by chromatin fibers. Keeping this fragile equilibrium seems to be the key for an appropriate immune response. Therapies targeting dysregulated NET formation might positively influence many disease progressions. This review highlights recent findings on the pathophysiological influence of NET formation in different bacterial, viral, and non-infectious lung diseases and summarizes medical treatment strategies.

Neutrophil Extracellular Traps (NETs) in Severe SARS-CoV-2 Lung Disease.

Neutrophil extracellular traps (NETs), built from mitochondrial or nuclear DNA, proteinases, and histones, entrap and eliminate pathogens in the course of bacterial or viral infections. Neutrophils' activation and the formation of NETs have been described as major risk factors for acute lung injury, multi-organ damage, and mortality in COVID-19 disease. NETs-related lung injury involves both epithelial and endothelial cells, as well as the alveolar-capillary barrier. The markers for NETs formation, such as circulating DNA, neutrophil elastase (NE) activity, or myeloperoxidase-DNA complexes, were found in lung specimens of COVID-19 victims, as well as in sera and tracheal aspirates obtained from COVID-19 patients. DNA threads form large conglomerates causing local obstruction of the small bronchi and together with NE are responsible for overproduction of mucin by epithelial cells. Various components of NETs are involved in the pathogenesis of cytokine storm in SARS-CoV-2 pulmonary disease. NETs are responsible for the interplay between inflammation and thrombosis in the affected lungs. The immunothrombosis, stimulated by NETs, has a poor prognostic significance. Better understanding of the role of NETs in the course of COVID-19 can help to develop novel approaches to the therapeutic interventions in this condition.

Transmission of cell-associated human cytomegalovirus isolates between various cell types using polymorphonuclear leukocytes as a vehicle.

Polymorphonuclear leukocytes (PMNs) are regarded as vehicles for the hematogenous dissemination of human cytomegalovirus (HCMV). In cell culture, this concept has been validated with cell-free laboratory strains but not yet with clinical HCMV isolates that grow strictly cell-associated. We, therefore, aimed to evaluate whether PMNs can also transmit such isolates from initially infected fibroblasts to other cell types, which might further clarify the role of PMNs in HCMV dissemination and provide a model to search for potential inhibitors. PMNs, which have been isolated from HCMV-seronegative individuals, were added for 3 h to fibroblasts infected with recent cell-associated HCMV isolates, then removed and transferred to various recipient cell cultures. The transfer efficiency in the recipient cultures was evaluated by immunofluorescence staining of viral immediate early antigens. Soluble derivatives of the cellular HCMV entry receptor PDGFRα were analyzed for their potential to interfere with this transfer. All of five tested HCMV isolates could be transferred to fibroblasts, endothelial and epithelial cells with transfer rates ranging from 2 to 9%, and the transferred viruses could spread focally in these recipient cells within 1 week. The PDGFRα-derived peptides IK40 and GT40 reduced transfer by 40 and 70% when added during the uptake step. However, when added during the transfer step, only IK40 was effective, inhibiting transmission by 20% on endothelial cells and 50-60% on epithelial cells and fibroblasts. These findings further corroborate the assumption of cell-associated HCMV dissemination by PMNs and demonstrate that it is possible to inhibit this transmission mode.

Maternal selenium status plays a crucial role on clinical outcomes of pregnant women with COVID-19 infection.

Adequate maternal selenium level is essential for immune response and healthy pregnancy. This study aimed to shed light on the selenium status of pregnant women with COVID-19 and the effects of potential deficiency in serum selenium levels. Totally 141 pregnant women, 71 of them were COVID-19 patients, in different trimesters were included in the study. Maternal serum selenium levels, demographic and clinical parameters were determined. Serum selenium levels of pregnant women in the second (p: .0003) and third (p: .001) trimesters with COVID-19 were significantly lower than in the healthy group. Maternal selenium level was found to be negatively correlated with gestational week (p < .0001, r: -.541), D-dimer (p: .0002, r: -.363) and interleukin-6 (IL-6) level (p: .02, r: -.243). In the second trimester, serum selenium level positively correlated with white blood cell (p: .002, r: .424), neutrophil (p: .006, r: .39), lymphocyte (p: .004, r: .410) count and hemoglobin (p: .02, r: .323), hematocrit (p: .008, r: .38) status. In the third trimester, it was found that maternal selenium level positively correlated with monocyte (p: .04, r: .353) and negatively correlated with C-reactive protein level (p: .03, r: -.384). Serum selenium level was gradually decreased during the pregnancy period, however, this natural decrease was enhanced together with COVID-19 infection. The reason might be increased selenium needs depended on the immune response against infection. The decrease in maternal selenium level was found to be related to IL-6 and D-dimer levels, which indicate selenium's role in disease progression.

Effect of the coronavirus pandemic on tumor markers.

The new type of coronavirus could cause severe acute respiratory syndrome and injuries in other systems as well. Multiple organ damage can occur rapidly in patients infected with coronavirus disease 2019 (COVID-19). Previous studies have shown that many laboratory biomarkers were not within the normal ranges in COVID-19 patients. We aimed to summarize laboratory parameters and the tumor markers in COVID-19 patients. This is a retrospective cohort study conducted on 53 women between the ages of 19-85 years infected with COVID-19 at a training and research hospital between May 2020 and August 2020. Of the 53 women, 16 (30.2%) had leukopenia. The mean C-reactive protein level was 18.42 ± 59.33 mg/L. The mean procalcitonin level was 0.1 ± 0.21 µg/L. The liver function tests were within normal limits. The mean creatinine level was 0.58 ± 0.37 mg/dl. Elevated levels of α-fetoprotein (AFP) in 1 patient, elevated levels of carcinoembryonic antigen (CEA) in 2 patients, elevated levels of cancer antigen 125 (CA125) in 4 patients, elevated levels of CA19-9 in 2 patients, and elevated levels of CA15-3 in 2 patients were detected. One of 4 patients who were taken to the intensive care unit had elevated levels of AFP. In addition, 2 of 4 patients who were taken to the intensive care unit had elevated levels of CA125 and CA15-3. Except for AFP, levels of all tumor markers of the patient who died were high. We found that COVID-19 had no effect on tumor markers (CA125, CA19-9, CA15-3, AFP, and CEA).

Interactions of HIV and Antiretroviral Therapy With Neutrophils and Platelets.

Neutrophils are important components of the innate immune system that mediate pathogen defense by multiple processes including phagocytosis, release of proteolytic enzymes, production of reactive oxygen species, and neutrophil extracellular trap formation. Abnormalities of neutrophil count and function have been described in the setting of HIV infection, with the majority of antiretroviral agents (ARVs), excluding zidovudine, having been reported to correct neutropenia. Questions still remain, however, about their impact on neutrophil function, particularly the possibility of persistent neutrophil activation, which could predispose people living with HIV to chronic inflammatory disorders, even in the presence of virally-suppressive treatment. In this context, the effects of protease inhibitors and integrase strand transfer inhibitors, in particular, on neutrophil function remain poorly understood and deserve further study. Besides mediating hemostatic functions, platelets are increasingly recognized as critical role players in the immune response against infection. In the setting of HIV, these cells have been found to harbor the virus, even in the presence of antiretroviral therapy (ART) potentially promoting viral dissemination. While HIV-infected individuals often present with thrombocytopenia, they have also been reported to have increased platelet activation, as measured by an upregulation of expression of CD62P (P-selectin), CD40 ligand, glycoprotein IV, and RANTES. Despite ART-mediated viral suppression, HIV-infected individuals reportedly have sustained platelet activation and dysfunction. This, in turn, contributes to persistent immune activation and an inflammatory vascular environment, seemingly involving neutrophil-platelet-endothelium interactions that increase the risk for development of comorbidities such as cardiovascular disease (CVD) that has become the leading cause of morbidity and mortality in HIV-infected individuals on treatment, clearly underscoring the importance of unraveling the possible etiologic roles of ARVs. In this context, abacavir and ritonavir-boosted lopinavir and darunavir have all been linked to an increased risk of CVD. This narrative review is therefore focused primarily on the role of neutrophils and platelets in HIV transmission and disease, as well as on the effect of HIV and the most common ARVs on the numbers and functions of these cells, including neutrophil-platelet-endothelial interactions.

Bacteriophages immunomodulate the response of monocytes.

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.

P2Y14 Receptor as a Target for Neutrophilia Attenuation in Severe COVID-19 Cases: From Hematopoietic Stem Cell Recruitment and Chemotaxis to Thrombo-inflammation.

The global SARS-CoV-2 pandemic starting in 2019 has already reached more than 2.3 million deaths. Despite the scientific community's efforts to investigate the COVID-19 disease, a drug for effectively treating or curing patients yet needs to be discovered. Hematopoietic stem cells (HSC) differentiating into immune cells for defense express COVID-19 entry receptors, and COVID-19 infection hinders their differentiation. The importance of purinergic signaling in HSC differentiation and innate immunity has been recognized. The metabotropic P2Y14 receptor subtype, activated by UDP-glucose, controls HSC differentiation and mobilization. Thereon, the exacerbated activation of blood immune cells amplifies the inflammatory state observed in COVID-19 patients, specially through the continuous release of reactive oxygen species and extracellular neutrophil traps (NETs). Further, the P2Y14 subtype, robustly inhibits the infiltration of neutrophils into various epithelial tissues, including lungs and kidneys. Here we discuss findings suggesting that antagonism of the P2Y14 receptor could prevent the progression of COVID-19-induced systemic inflammation, which often leads to severe illness and death cases. Considering the modulation of neutrophil recruitment of extreme relevance for respiratory distress and lung failure prevention, we propose that P2Y14 receptor inhibition by its selective antagonist PPTN could limit neutrophil recruitment and NETosis, hence limiting excessive formation of oxygen reactive species and proteolytic activation of the kallikrein-kinin system and subsequent bradykinin storm in the alveolar septa of COVID-19 patients.

Neutrophil-to-lymphocyte ratio, a critical predictor for assessment of disease severity in patients with COVID-19.

INTRODUCTION: Monitoring of laboratory indicators is important for predicting changes in disease severity and clinical outcomes. We aimed to identify the critical predictors that can effectively assess the disease conditions of patients with COVID-19 by analyzing the clinical characteristics and laboratory findings of patients with SARS-CoV-2 infection. METHODS: All consecutive patients (n = 294) with confirmed SARS-CoV-2 infection admitted to the General Hospital of Central Theater Command of the PLA from February 6 to February 21, 2020, were enrolled. These patients were divided into the severe group and the nonsevere group according to disease severity during hospitalization. RESULTS: The median neutrophil-to-lymphocyte ratio (NLR) value of the severe patients was dramatically higher than that of the nonsevere patients (10.4 vs 2.6; P < .001). The NLR value equal to 5 was a boundary value worthy of reference, because more than 80% severe patients had an NLR value greater than 5 and over 80% nonsevere patients had an NLR value less than 5. The NLR value of these COVID-19 patients was positively and respectively correlated with the values of C-reactive protein (R = .5921, P < .001), lactate dehydrogenase (R = .4509, P < .001), procalcitonin (R = .5504, P < .001), fibrinogen (R = .4710, P < .001), and D-dimers (R = .4425, P < .001). However, the NLR value was merely and positively correlated with the interleukin-6 value (R = .3594, P < .05), but had no correlations with the values of interleukin-10, interleukin-4, interleukin-17, interferon-γ, and tumor necrosis factor-α (P > .05). DISCUSSION: Neutrophil-to-lymphocyte ratio is a critical predictor for assessment of disease severity in patients with COVID-19, and it has a close relation with the laboratory indicators related to disease conditions.

Clotting disorder in severe acute respiratory syndrome coronavirus 2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human respiratory viral infection that has rapidly progressed into a pandemic, causing significant morbidity and mortality. Blood clotting disorders and acute respiratory failure have surfaced as the major complications among the severe cases of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection. Remarkably, more than 70% of deaths related to COVID-19 are attributed to clotting-associated complications such as pulmonary embolism, strokes and multi-organ failure. These vascular complications have been confirmed by autopsy. This study summarizes the current understanding and explains the possible mechanisms of the blood clotting disorder, emphasizing the role of (1) hypoxia-related activation of coagulation factors like tissue factor, a significant player in triggering coagulation cascade, (2) cytokine storm and activation of neutrophils and the release of neutrophil extracellular traps and (3) immobility and ICU related risk factors.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Neutrophil extracellular traps from healthy donors and HIV-1-infected individuals restrict HIV-1 production in macrophages.

Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 ß-chemokines Rantes and MIP-1ß, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.

Early changes in immune cell subsets with corticosteroids in patients with solid tumors: implications for COVID-19 management.

BACKGROUND: The risk-benefit calculation for corticosteroid administration in the management of COVID-19 is complex and urgently requires data to inform the decision. The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation associated with poor prognosis in both COVID-19 and cancer. Investigating NLR as an inflammatory marker and lymphocyte levels as a critical component of antiviral immunity may inform the dilemma of reducing toxic hyperinflammation while still maintaining effective antiviral responses. METHODS: We performed a retrospective analysis of NLR, absolute neutrophil counts (ANCs) and absolute lymphocyte counts (ALCs) in patients with cancer enrolled in immunotherapy trials who received moderate-dose to high-dose corticosteroids. We compared paired presteroid and available poststeroid initiation values daily during week 1 and again on day 14 using the Wilcoxon signed-rank test. Associated immune subsets by flow cytometry were included where available. RESULTS: Patients (n=48) with a variety of solid tumors received prednisone, methylprednisolone, or dexamethasone alone or in combination in doses ranging from 20 to 190 mg/24 hours (prednisone equivalent). The median NLR prior to steroid administration was elevated at 5.0 (range: 0.9-61.2). The corresponding median ANC was 5.1 K/µL (range: 2.03-22.31 K/µL) and ALC was 1.03 K/µL (0.15-2.57 K/µL). One day after steroid administration, there was a significant transient drop in median ALC to 0.54 K/µL (p=0.0243), driving an increase in NLR (median 10.8, p=0.0306). Relative lymphopenia persisted through day 14 but was no longer statistically significant. ANC increased steadily over time, becoming significant at day 4 (median: 7.31 K/µL, p=0.0171) and remaining significantly elevated through day 14. NLR was consistently elevated after steroid initiation, significantly at days 1, 7 (median: 8.2, p=0.0272), and 14 (median: 15.0, p=0.0018). Flow cytometry data from 11 patients showed significant decreases in activated CD4 cells and effector memory CD8 cells. CONCLUSIONS: The early drop in ALC with persistent lymphopenia as well as the prolonged ANC elevation seen in response to corticosteroid administration are similar to trends associated with increased mortality in several coronavirus studies to include the current SARS-CoV-2 pandemic. The affected subsets are essential for effective antiviral immunity. This may have implications for glucocorticoid therapy for COVID-19.

CXCL10 Signaling Contributes to the Pathogenesis of Arthritogenic Alphaviruses.

Emerging and re-emerging arthritogenic alphaviruses, such as Chikungunya virus (CHIKV) and O'nyong nyong virus, cause acute and chronic crippling arthralgia associated with inflammatory immune responses. Approximately 50% of CHIKV-infected patients suffer from rheumatic manifestations that last 6 months to years. However, the physiological functions of individual immune signaling pathways in the pathogenesis of alphaviral arthritis remain poorly understood. Here, we report that a deficiency in CXCL10, which is a chemoattractant for monocytes/macrophages/T cells, led to the same viremia as wild-type animals, but fewer immune infiltrates and lower viral loads in footpads at the peak of arthritic disease (6-8 days post infection). Macrophages constituted the largest immune cell population in footpads following infection, and were significantly reduced in Cxcl10-/- mice. The viral RNA loads in neutrophils and macrophages were reduced in Cxcl10-/- compared to wild-type mice. In summary, our results demonstrate that CXCL10 signaling promotes the pathogenesis of alphaviral disease and suggest that CXCL10 may be a therapeutic target for mitigating alphaviral arthritis.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.