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Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24â¯h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4â¯h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.
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ABSTRACT: Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The enzyme-linked immunospot (ELISpot) assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture. Blood and spleens were harvested serially, and ex vivo interferon γ and TNF-α production were compared by ELISpot and enzyme-linked immunosorbent assay. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example, dexamethasone, arginine, and IL-7, in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and enzyme-linked immunosorbent assay results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.
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Imunidade Adaptativa , ELISPOT , Imunidade Inata , Sepse , Sepse/imunologia , Animais , Imunidade Inata/imunologia , Imunidade Adaptativa/imunologia , Camundongos , Masculino , Interferon gama/metabolismo , Interferon gama/imunologia , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismo , Feminino , Dexametasona/uso terapêutico , Dexametasona/farmacologiaRESUMO
Sepsis is a common and deadly condition. Within the current model of sepsis immunobiology, the framing of dysregulated host immune responses into proinflammatory and immunosuppressive responses for the testing of novel treatments has not resulted in successful immunomodulatory therapies. Thus, the recent focus has been to parse observable heterogeneity into subtypes of sepsis to enable personalised immunomodulation. In this Personal View, we highlight that many fundamental immunological concepts such as resistance, disease tolerance, resilience, resolution, and repair are not incorporated into the current sepsis immunobiology model. The focus for addressing heterogeneity in sepsis should be broadened beyond subtyping to encompass the identification of deterministic molecular networks or dominant mechanisms. We explicitly reframe the dysregulated host immune responses in sepsis as altered homoeostasis with pathological disruption of immune-driven resistance, disease tolerance, resilience, and resolution mechanisms. Our proposal highlights opportunities to identify novel treatment targets and could enable successful immunomodulation in the future.
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Resistência à Doença , Sepse , Humanos , ImunomodulaçãoRESUMO
BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.
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Interferon gama , Sepse , Humanos , Interferon gama/metabolismo , Imunoadsorventes/uso terapêutico , Estudos Prospectivos , BiomarcadoresRESUMO
BACKGROUND: We report the case of a patient with aplastic anemia and pancytopenia on immune-suppressive therapy who developed invasive pulmonary infection with mucormycosis and was treated with immune adjuvant therapy. CASE SUMMARY: Given the patient's profound lymphopenia and progressive invasive mucor despite dual antifungal drug therapy, interleukin (IL)-7, a cytokine that induces lymphocyte activation and proliferation, was instituted and resulted in normalization of absolute lymphocyte counts and was temporally associated with clearance of fungal pathogens and resolution of clinical symptoms. CONCLUSION: Patients with life-threatening fungal infections are frequently immune suppressed and immune adjuvant therapies should be considered in patients who are not responding to antifungal drugs and source control. Well-designed, double-blind, placebo-controlled trials are needed to advance the field. Although a number of immune adjuvants may be beneficial in fungal sepsis, IL-7 is a particularly attractive immune adjuvant because of its broad immunologic effects on key immunologic pathways that mediate enhanced antifungal immune system activity.
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BACKGROUND: Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS: An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS: Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS: A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.
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Sepsis is a global health priority, yet effective host-directed targeted therapies have not been identified outside of the setting of coronavirus disease 2019 (COVID-19). Lymphopenia occurs in up to ~52% of patients with sepsis and is associated with a higher mortality at both 30 and 100 days. In COVID-19, the presence of lymphopenia correlates with intensive care unit admission, acute respiratory distress syndrome and death. The mechanisms underpinning lymphopenic sepsis remain unknown, and while high rates of lymphocyte apoptosis have been implicated, the relative contributions of cellular trafficking to inflamed tissues and reduction in lymphopoiesis require investigation. Further delineation of these underlying mechanisms holds the potential to open new avenues for the development of host-directed therapies in lymphopenic sepsis. These may include recombinant cytokines (e.g. interleukin-7), monoclonal antibodies (e.g. anti-interleukin-1, anti-programmed cell death protein 1) and small interfering RNA (e.g. targeting interleukin-10, transforming growth factor beta). Applying the frontier tools of translational cellular and genomic medicine to understand lymphopenia in the setting of critical infections holds the potential to significantly reduce the excessive global burden of sepsis.
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COVID-19 , Síndromes de Imunodeficiência , Linfopenia , Sepse , Humanos , COVID-19/complicações , Linfopenia/complicações , Sepse/complicações , Anticorpos MonoclonaisRESUMO
The SARS-CoV-2 virus has complex and divergent immune alterations in differing hosts and over disease evolution. Much of the nuanced COVID-19 disease immune dysregulation was originally dominated by innate cytokine changes, which has since been replaced with a more complex picture of innate and adaptive changes characterized by simultaneous hyperinflammatory and immunosuppressive phenomena in effector cells. These intricacies are summarized in this review as well as potential relevance from acute infection to a multisystem inflammatory syndrome commonly seen in children. Additional consideration is made for the influence of variant to variant host cellular changes and the impact of potential vaccination upon these phenotypes. Finally, therapeutic benefit for immune alterations are discussed.
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COVID-19 , Humanos , SARS-CoV-2 , Imunidade Inata , CitocinasRESUMO
Low-dose interleukin-2 (LD-IL-2) treatment has been shown to effectively reverse chronic migraine-related behaviors and the sensitization of trigeminal ganglion (TG) neurons through expansion and activation of peripheral regulatory T cells (Tregs) in mice. In this study, we investigated the molecular mechanisms underlying the effects of LD-IL-2 and Treg cells. LD-IL-2 treatment increases the production of cytokines interleukin-10 (IL-10) and transforming growth factor beta-1 (TGFß1) in T cells, especially Treg cells, suggesting that they may mediate the therapeutic effect of LD-IL-2. Indeed, neutralizing antibodies against either IL-10 or TGFß completely blocked the effects of LD-IL-2 on the facial mechanical hypersensitivity as well as the sensitization of TG neurons resulting from repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration in mice, indicating that LD-IL-2 and Treg cells engage both peripheral IL-10 and TGFß signaling pathways to reverse chronic-migraine related sensitizations. In an in vitro assay, incubation of TG culture with exogenous IL-10 or TGFß1 fully reversed NTG-induced sensitization of TG neurons, suggesting that the IL-10 and TGFß1 signaling in TG neurons contribute to LD-IL-2's therapeutic effects. Collectively, these results not only elucidate the molecular mechanisms through which LD-IL-2 and Treg cells reverse chronic-migraine related sensitizations, but also suggest that the IL-10 and TGFß1 signaling pathways in TG neurons are potential targets for chronic migraine therapy.
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The global COVID-19 pandemic has claimed the lives of more than 750,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. We find that although COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations, STAT3 phosphorylation predominated in both monocytes and T cells. STAT3 phosphorylation was tightly correlated with circulating IL-6 levels and high levels of phospho-STAT3 was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-γ production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.
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COVID-19 , Humanos , Monócitos/metabolismo , Pandemias , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Linfócitos TRESUMO
INTRODUCTION: Current surgical guidelines for the treatment of intra-abdominal sepsis recommend interventional source control as the key element of therapy, alongside resuscitation and antibiotic administration. Past trials attempted to predict the success of interventional source control to assess whether further interventional therapy is needed. However, no predictive score could be developed. MATERIALS AND METHODS: We utilized an established murine abdominal sepsis model, the cecal ligation and puncture (CLP), and performed a successful surgical source control intervention after full development of sepsis, the CLP-excision (CLP/E). We then sought to evaluate the success of the source control by characterizing circulating neutrophil phenotype and functionality 24 h postintervention. RESULTS: We showed a significant relative increase of neutrophils and a significant absolute and relative increase of activated neutrophils in septic mice. Source control with CLP/E restored these numbers back to baseline. Moreover, main neutrophil functions, the acidification of cell compartments, such as lysosomes, and the production of Tumor Necrosis Factor-alpha (TNF-α), were impaired in septic mice but restored after CLP/E intervention. CONCLUSIONS: Neutrophil characterization by phenotyping and evaluating their functionality indicates successful source control in septic mice and can serve as a prognostic tool. These findings provide a rationale for the phenotypic and functional characterization of neutrophils in human patients with infection. Further studies will be needed to determine whether a predictive score for the assessment of successful surgical source control can be established.
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Neutrófilos , Sepse , Animais , Ceco/cirurgia , Modelos Animais de Doenças , Humanos , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , Sepse/patologiaRESUMO
OBJECTIVES: To test the hypothesis that forced-air warming of critically ill afebrile sepsis patients improves immune function compared to standard temperature management. DESIGN: Single-center, prospective, open-label, randomized controlled trial. SETTING: One thousand two hundred-bed academic medical center. PATIENTS: Eligible patients were mechanically ventilated septic adults with: 1) a diagnosis of sepsis within 48 hours of enrollment; 2) anticipated need for mechanical ventilation of greater than 48 hours; and 3) a maximum temperature less than 38.3°C within the 24 hours prior to enrollment. Primary exclusion criteria included: immunologic diseases, immune-suppressing medications, and any existing condition sensitive to therapeutic hyperthermia (e.g., brain injury). The primary outcome was monocyte human leukocyte antigen (HLA)-DR expression, with secondary outcomes of CD3/CD28-induced interferon gamma (IFN-γ) production, mortality, and 28-day hospital-free days. INTERVENTIONS: External warming using a forced-air warming blanket for 48 hours, with a goal temperature 1.5°C above the lowest temperature documented in the previous 24 hours. MEASUREMENTS AND MAIN RESULTS: We enrolled 56 participants in the study. No differences were observed between the groups in HLA-DR expression (692 vs 2,002; p = 0.396) or IFN-γ production (31 vs 69; p = 0.678). Participants allocated to external warming had lower 28-day mortality (18% vs 43%; absolute risk reduction, 25%; 95% CI, 2-48%) and more 28-day hospital-free days (difference, 2.6 d; 95% CI, 0-11.6). CONCLUSIONS: Participants randomized to external forced-air warming did not have a difference in HLA-DR expression or IFN-γ production. In this pilot study, however, 28-day mortality was lower in the intervention group. Future research should seek to better elucidate the impact of temperature modulation on immune and nonimmune organ failure pathways in sepsis.
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COVID-19 , Hipertermia Induzida , Sepse , Adulto , Estado Terminal/terapia , Antígenos HLA-DR , Humanos , Projetos Piloto , Estudos Prospectivos , SARS-CoV-2 , Sepse/terapiaRESUMO
Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent ß-lactam and ß-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane ß-lactamase inhibitor to restore in vitro susceptibilities in combination with ß-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 µg/mL and an MIC50/MIC90 of ≤0.06/0.25 µg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the ß-lactamase BlaMab with a relative Michaelis constant (Ki app) of 4 × 10-3 ± 0.8 × 10-3 µM and acylation rate (k2/K) of 1 × 107 M-1 s-1. Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and LdtMab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a BlaMab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with LdtMab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and LdtMab2,4 supports new therapeutic approaches using ß-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of BlaMab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with LdtMab2 and LdtMab4. The ability of DUR to protect amoxicillin and imipenem against BlaMab and its intrinsic activity along with the dual ß-lactam target redundancy can explain the rationale behind the potent activity of this combination.
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Mycobacterium abscessus , beta-Lactamas , Humanos , beta-Lactamas/farmacologia , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/farmacologia , Cefuroxima/farmacologia , Testes de Sensibilidade Microbiana , Imipenem/farmacologia , Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , beta-LactamasesRESUMO
Both severe SARS-CoV-2 infections and bacterial sepsis exhibit an immunological dyscrasia and propensity for secondary infections. The nature of the immunological dyscrasias for these differing etiologies and their time course remain unclear. In this study, thirty hospitalized patients with SARS-CoV-2 infection were compared with ten critically ill patients with bacterial sepsis over 21 days, as well as ten healthy control subjects. Blood was sampled between days 1 and 21 after admission for targeted plasma biomarker analysis, cellular phenotyping, and leukocyte functional analysis via enzyme-linked immunospot assay. We found that circulating inflammatory markers were significantly higher early after bacterial sepsis compared with SARS-CoV-2. Both cohorts exhibited profound immune suppression through 21 days (suppressed HLA-DR expression, reduced mononuclear cell IFN-gamma production), and expanded numbers of myeloid-derived suppressor cells (MDSCs). In addition, MDSC expansion and ex vivo production of IFN-gamma and TNF-alpha were resolving over time in bacterial sepsis, whereas in SARS-CoV-2, immunosuppression and inflammation were accelerating. Despite less severe initial physiologic derangement, SARS-CoV-2 patients had similar incidence of secondary infections (23% vs 30%) as bacterial sepsis patients. Finally, COVID patients who developed secondary bacterial infections exhibited profound immunosuppression evident by elevated sPD-L1 and depressed HLA-DR. Although both bacterial sepsis and SARS-CoV-2 are associated with inflammation and immune suppression, their immune dyscrasia temporal patterns and clinical outcomes are different. SARS-CoV-2 patients had less severe early inflammation and organ dysfunction but had persistent inflammation and immunosuppression and suffered worse clinical outcomes, especially when SARS-CoV-2 infection was followed by secondary bacterial infection.
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Infecções Bacterianas/imunologia , COVID-19/imunologia , Tolerância Imunológica/imunologia , Sepse/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , SARS-CoV-2RESUMO
BACKGROUND: Immunotherapy treatment for coronavirus disease 2019 combined with antiviral therapy and supportive care remains under intense investigation. However, the capacity to distinguish patients who would benefit from immunosuppressive or immune stimulatory therapies remains insufficient. Here, we present a patient with severe coronavirus disease 2019 with a defective immune response, treated successfully with interleukin-7 on compassionate basis with resultant improved adaptive immune function. CASE SUMMARY: A previously healthy 43-year-old male developed severe acute respiratory distress syndrome due to the severe acute respiratory syndrome coronavirus 2 virus with acute hypoxemic respiratory failure and persistent, profound lymphopenia. Functional analysis demonstrated depressed lymphocyte function and few antigen-specific T cells. Interleukin-7 administration resulted in reversal of lymphopenia and improved T-cell function. Respiratory function and clinical status rapidly improved, and he was discharged home. Whole exome sequencing identified a deleterious autosomal dominant mutation in TICAM1, associated with a dysfunctional type I interferon antiviral response with increased severity of coronavirus disease 2019 disease. CONCLUSIONS: Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening severe coronavirus disease 2019 infections, particularly by applying a precision medicine approach in selecting patients expressing an immunosuppressive phenotype.
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A nonimmunocompromised patient developed life-threatening soft tissue infection with Trichosporon asahii, Fusarium, and Saksenaea that progressed despite maximum antifungal therapies and aggressive debridement. Interleukin-7 immunotherapy resulted in clinical improvement, fungal clearance, reversal of lymphopenia, and improved T-cell function. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening fungal infections.
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OBJECTIVES: Corticosteroid therapy has become standard of care therapy for hospitalized patients infected with the severe acute respiratory syndrome coronavirus-2 global pandemic-causing virus. Whereas systemic inflammation is a notably important feature in coronavirus disease 2019 pathogenesis, adaptive immune suppression and the inability to eradicate effectively the virus remain significant factors as well. We sought to evaluate the in vitro effects of dexamethasone phosphate on T cell function in peripheral blood mononuclear cells derived from patients with acute, severe, and moderate coronavirus disease 2019. DESIGN: Prospective in vitro laboratory study. SETTING: Coronavirus disease 2019-specific medical wards and ICUs at a single-center, quaternary-care academic hospital between October 1, 2020, and November 15, 2020. PATIENTS: Eleven patients diagnosed with coronavirus disease 2019 admitted to either the ICU or hospital coronavirus disease 2019 unit. Three patients had received at least one dose of dexamethasone prior to enrollment. INTERVENTIONS: Fresh whole blood was collected, and peripheral blood mononuclear cells were immediately isolated and plated onto precoated enzyme-linked immunospot plates for detection of interferon-γ production. Samples were incubated with CD3/CD28 antibodies alone and with three concentrations of dexamethasone. These conditions were also stimulated with recombinant human interleukin-7. Following overnight incubation, the plates were washed and stained for analysis using Cellular Technology Limited ImmunoSpot S6 universal analyzer (ImmunoSpot by Cellular Technology Limited, Cleveland, OH). MEASUREMENTS AND MAIN RESULTS: Functional cytokine production was assessed by quantitation of cell spot number and total well intensity after calculation for each enzyme-linked immunospot well using the Cellular Technology Limited ImmunoSpot Version 7.0 professional software (CTL Analyzers, Shaker Heights, OH). Comparisons were made using t test and using a nonparametric analysis of variance Friedman test. The number of functional T cells producing interferon-γ and the intensity of the response decrease significantly with exposure to 1.2-µg/mL dexamethasone. About 0.12 µg/mL does not significantly affect the functional immune response on enzyme-linked immunospot. Interleukin-7 increases the overall number of activated T cells, including those exposed to dexamethasone. CONCLUSIONS: Further evaluation of the effect of immunomodulatory therapies is warranted in coronavirus disease 2019. A refined functional, precision medicine approach that evaluates the cellular immune function of individual patients with coronavirus disease 2019 is needed to better define which therapies could have benefit or cause harm for specific patients.
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OBJECTIVES: Since the beginning of the coronavirus disease 2019 pandemic, hundreds of thousands of patients have been treated in ICUs across the globe. The severe acute respiratory syndrome-associated coronavirus 2 virus enters cells via the angiotensin-converting enzyme 2 receptor and activates several distinct inflammatory pathways, resulting in hematologic abnormalities and dysfunction in respiratory, cardiac, gastrointestinal renal, endocrine, dermatologic, and neurologic systems. This review summarizes the current state of research in coronavirus disease 2019 pathophysiology within the context of potential organ-based disease mechanisms and opportunities for translational research. DATA SOURCES: Investigators from the Research Section of the Society of Critical Care Medicine were selected based on expertise in specific organ systems and research focus. Data were obtained from searches conducted in Medline via the PubMed portal, Directory of Open Access Journals, Excerpta Medica database, Latin American and Caribbean Health Sciences Literature, and Web of Science from an initial search from December 2019 to October 15, 2020, with a revised search to February 3, 2021. The medRxiv, Research Square, and clinical trial registries preprint servers also were searched to limit publication bias. STUDY SELECTION: Content experts selected studies that included mechanism-based relevance to the severe acute respiratory syndrome-associated coronavirus 2 virus or coronavirus disease 2019 disease. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: Not applicable. CONCLUSIONS: Efforts to improve the care of critically ill coronavirus disease 2019 patients should be centered on understanding how severe acute respiratory syndrome-associated coronavirus 2 infection affects organ function. This review articulates specific targets for further research.
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The global COVID-19 pandemic has claimed the lives of more than 450,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated local tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations. STAT3 phosphorylation predominated in both monocytes and T cells and was tightly correlated with circulating IL-6 levels. High levels of STAT3 phosphorylation was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-gamma production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.
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ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.