Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

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 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 (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. 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 ELISA 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.

Cell-Free Hemoglobin in the Pathophysiology of Trauma: A Scoping Review.

OBJECTIVES: Cell-free hemoglobin (CFH) is a potent mediator of endothelial dysfunction, organ injury, coagulopathy, and immunomodulation in hemolysis. These mechanisms have been demonstrated in patients with sepsis, hemoglobinopathies, and those receiving transfusions. However, less is known about the role of CFH in the pathophysiology of trauma, despite the release of equivalent levels of free hemoglobin. DATA SOURCES: Ovid MEDLINE, Embase, Web of Science Core Collection, and BIOSIS Previews were searched up to January 21, 2023, using key terms related to free hemoglobin and trauma. DATA EXTRACTION: Two independent reviewers selected studies focused on hemolysis in trauma patients, hemoglobin breakdown products, hemoglobin-mediated injury in trauma, transfusion, sepsis, or therapeutics. DATA SYNTHESIS: Data from the selected studies and their references were synthesized into a narrative review. CONCLUSIONS: Free hemoglobin likely plays a role in endothelial dysfunction, organ injury, coagulopathy, and immune dysfunction in polytrauma. This is a compelling area of investigation as multiple existing therapeutics effectively block these pathways.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

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.

Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γELISpot: A Multicenter, Prospective Observational Study.

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.

The use of machine learning and artificial intelligence within pediatric critical care.

The field of pediatric critical care has been hampered in the era of precision medicine by our inability to accurately define and subclassify disease phenotypes. This has been caused by heterogeneity across age groups that further challenges the ability to perform randomized controlled trials in pediatrics. One approach to overcome these inherent challenges include the use of machine learning algorithms that can assist in generating more meaningful interpretations from clinical data. This review summarizes machine learning and artificial intelligence techniques that are currently in use for clinical data modeling with relevance to pediatric critical care. Focus has been placed on the differences between techniques and the role of each in the clinical arena. The various forms of clinical decision support that utilize machine learning are also described. We review the applications and limitations of machine learning techniques to empower clinicians to make informed decisions at the bedside. IMPACT: Critical care units generate large amounts of under-utilized data that can be processed through artificial intelligence. This review summarizes the machine learning and artificial intelligence techniques currently being used to process clinical data. The review highlights the applications and limitations of these techniques within a clinical context to aid providers in making more informed decisions at the bedside.

COVID-19 disease and immune dysregulation.

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.

High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

Dysregulation of the leukocyte signaling landscape during acute COVID-19.

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.

Overlapping but Disparate Inflammatory and Immunosuppressive Responses to SARS-CoV-2 and Bacterial Sepsis: An Immunological Time Course Analysis.

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.

Interleukin-7 Reverses Lymphopenia and Improves T-Cell Function in Coronavirus Disease 2019 Patient With Inborn Error of Toll-Like Receptor 3: A Case Report.

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.

IL-7 Immunotherapy in a Nonimmunocompromised Patient With Intractable Fungal Wound Sepsis.

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.

In Vitro-Administered Dexamethasone Suppresses T Cell Function With Reversal by Interleukin-7 in Coronavirus Disease 2019.

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.

Severe Acute Respiratory Syndrome-Associated Coronavirus 2 Infection and Organ Dysfunction in the ICU: Opportunities for Translational Research.

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.

A Whole Blood Enzyme-Linked Immunospot Assay for Functional Immune Endotyping of Septic Patients.

Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies.

Platelet-activating factor antagonists decrease follicular dendritic-cell stimulation of human B lymphocytes.

Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF). In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs), which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA) gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig) G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA). WEB 2170 (10-6 to 10-8 M) inhibited [3H]-thymidine incorporation by up to 35% +/- 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA) from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.