Nonhuman primate models that closely emulate the disease course, pathogenesis, and supportive care provided to human patients in the modern intensive care unit with bacterial sepsis are urgently needed to study pathogenesis and assess novel therapies. We therefore developed a non-human primate model of septic shock that includes supportive care akin to a modern intensive care unit. In this study, we characterized pathogen kinetics and evaluated the physiologic, immunologic, and pathologic responses in this model of septic shock induced by the clinically relevant pathogen Klebsiella pneumoniae across a three-log dose range. We observed dose-dependent bacteremia and circulating levels of Klebsiella pneumoniae DNA and endotoxin. Tachycardia and hypotension occurred in all animals and the study endpoint occurred in 8 of 12 animals that were euthanized. The infused bacterial dose was significantly associated with the severity of renal insufficiency and coagulopathy. Neutrophil activation evidenced by increased CD11b expression, decreased CD62L expression, and increased circulating levels of myeloperoxidase, lactoferrin, and neutrophil extracellular traps; monocyte activation evidenced by increased circulating levels of interleukin-6, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1; and endothelial activation evidenced by increased circulating levels of syndecan-1 and angiopoietin-II were all consistent with human sepsis. Our model provides an opportunity to study pathogenesis and investigate novel therapeutics for the treatment of bacterial sepsis in the setting of modern supportive care. IMPORTANCE There is currently a disconnect between the efficacy of sepsis therapies in pre-clinical animal models and human clinical trials. Therefore, developing nonhuman primate models that closely mimic human sepsis pathogenesis to study novel host-targeted therapeutics is a priority. In this study, we developed a model of septic shock with a clinically relevant bacteria (Klebsiella pneumoniae) that provides standard supportive care including mechanical ventilation, invasive hemodynamic monitoring, volume resuscitation, vasopressors, antibiotics, and steroids. In a dose-dependent manner, we observed that this model closely emulates the hemodynamic, end-organ dysfunction, and cellular and soluble responses associated with human sepsis. This validated model provides a unique opportunity to study the pathogenesis of acute septic shock and evaluate host-directed therapeutics in a large animal model that closely emulates the modern-day intensive care unit and supportive critical care.

Supplementary Materials & Methods, Supplementary Figures 1-10 with Figure Legends

Objective The aim of this study was to examine whether colchicine's anti-inflammatory effects would improve measures of lipolysis and distribution of leukocyte populations in subcutaneous adipose tissue (SAT). Methods A secondary analysis was conducted for a double-blind, randomized, placebo-controlled pilot study in which 40 adults with obesity and metabolic syndrome (MetS) were randomized to colchicine 0.6 mg or placebo twice daily for 3 months. Non-insulin-suppressible (l(0)), insulin-suppressible (l(2)), and maximal (l(0)+l(2)) lipolysis rates were calculated by minimal model analysis. Body composition was determined by dual-energy x-ray absorptiometry. SAT leukocyte populations were characterized by flow cytometry analysis from biopsied samples obtained before and after the intervention. Results Colchicine treatment significantly decreased l(2) and l(0)+l(2) versus placebo (p < 0.05). These changes were associated with a significant reduction in markers of systemic inflammation, including high-sensitivity C-reactive protein, resistin, and circulating monocytes and neutrophils (p < 0.01). Colchicine did not significantly alter SAT leukocyte population distributions (p > 0.05). Conclusions In adults with obesity and MetS, colchicine appears to improve insulin regulation of lipolysis and reduce markers of systemic inflammation independent of an effect on local leukocyte distributions in SAT. Further studies are needed to better understand the mechanisms by which colchicine affects adipose tissue metabolic pathways in adults with obesity and MetS.

Strich, Jeffrey1; Ramos-Benitez, Marcos2; Connelly, Mark3; Swerczek, Joanna4; Kendall, Heather4; Babyak, Ashley2; Stein, Sydney2; Valencia, Luis Perez2; Warner, Seth2; Ramelli, Sabrina2; Platt, Andrew5; Vannella, Kevin M2; Herbert, Richard4; Chertow, Daniel5 Author Information

Ramos-Benitez, Marcos; Connelly, Mark; Strich, Jeffrey; Swerczek, Joanna; Stein, Sydney; Babyak, Ashley; Valencia, Luis Perez; Sun, Junfeng; Vannella, Kevin M; Moore, Ian; Herbert, Richard; Chertow, Daniel Author Information

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-κB signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-κB response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-κB responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-κB responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Increased risk of premature cardiovascular disease (CVD) is well recognized in systemic lupus erythematosus (SLE). Aberrant type I-Interferon (IFN)-neutrophil interactions contribute to this enhanced CVD risk. In lupus animal models, the Janus kinase (JAK) inhibitor tofacitinib improves clinical features, immune dysregulation and vascular dysfunction. We conducted a randomized, double-blind, placebo-controlled clinical trial of tofacitinib in SLE subjects (ClinicalTrials.gov NCT02535689). In this study, 30 subjects are randomized to tofacitinib (5 mg twice daily) or placebo in 2:1 block. The primary outcome of this study is safety and tolerability of tofacitinib. The secondary outcomes include clinical response and mechanistic studies. The tofacitinib is found to be safe in SLE meeting study’s primary endpoint. We also show that tofacitinib improves cardiometabolic and immunologic parameters associated with the premature atherosclerosis in SLE. Tofacitinib improves high-density lipoprotein cholesterol levels ( p = 0.0006, CI 95%: 4.12, 13.32) and particle number ( p = 0.0008, CI 95%: 1.58, 5.33); lecithin: cholesterol acyltransferase concentration ( p = 0.024, CI 95%: 1.1, −26.5), cholesterol efflux capacity ( p = 0.08, CI 95%: −0.01, 0.24), improvements in arterial stiffness and endothelium-dependent vasorelaxation and decrease in type I IFN gene signature, low-density granulocytes and circulating NETs. Some of these improvements are more robust in subjects with STAT4 risk allele.

Abstract Background: Obesity-associated inflammation promotes adipose tissue (AT) dysfunction and contributes to the progression of type 2 diabetes and cardiovascular disease. Recent clinical studies have demonstrated that colchicine may improve metabolic and cardiovascular outcomes; however, colchicine’s effects on metabolic and inflammatory measures within AT remain unclear. Methods: The aim of this study was to examine if colchicine’s anti-inflammatory effects would improve measures of lipolysis and immune cell populations in subcutaneous AT (SAT). This is a secondary analysis of a double-blind, randomized, placebo-controlled pilot study in which 40 nondiabetic adults with obesity and metabolic syndrome (MetS) were randomized to colchicine 0.6mg or placebo twice daily for 3 months. Blood samples for insulin, glucose, and free fatty acids were collected in the fasted state and during a frequently-sampled intravenous glucose tolerance test. Noninsulin-suppressible (l0), insulin-suppressible (l2), and maximal (l0+l2) lipolysis rates were calculated by minimal model analysis. Body composition was determined by DXA. SAT immune cell populations were characterized by flow cytometry fluorescence-activated single cell sorting of the stromovascular fractions obtained after collagenase digestion of SAT samples obtained using a mini-liposuction technique pre- and post-intervention. Results: Data from 18 subjects in the colchicine group (Mean ± SD: age 48.4 ± 13.5 y; BMI 39.3 ± 6.3 kg/m2; sex: female 72.2%) and 18 subjects in the placebo group (age 44.7 ± 10.2 y; BMI 41.8 ± 8.2 kg/m2; sex: female 77.8%) were available for this study. Colchicine treatment significantly reduced l2 (p = 0.04) and l0+l2 (p = 0.04) versus placebo. These changes were significantly associated with reductions in systemic inflammation, including the changes in high-sensitivity C-reactive protein concentrations, white blood cell count, circulating monocyte and neutrophil populations, and the neutrophil-lymphocyte ratio (p’s < 0.015). Colchicine did not significantly alter SAT immune cell population distributions (p’s > 0.05). Conclusions: In adults with obesity and MetS, colchicine may improve insulin action at the level of AT. These improvements were positively associated with the suppression of systemic inflammation. However, no local AT inflammatory cell populations were significantly affected by colchicine use in our study, suggesting that colchicine’s systemic, rather than local, anti-inflammatory effects may be more consequential in ameliorating AT metabolic pathways in MetS. Further studies are warranted to elucidate the biological mechanisms underlying colchicine’s effects in AT, as these investigations could potentially shed light on treatments to improve metabolic outcomes in human obesity.

Abstract Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand–dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8–dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. Significance: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies.

Neutrophil extracellular traps (NETs) contribute to immunothrombosis and have been associated with mortality in coronavirus disease 2019 (COVID-19). We stimulated donor neutrophils with plasma from patients with COVID-19 and demonstrated that R406 can abrogate the release of NETs. These data provide evidence for how fostamatinib may mitigate neutrophil-associated mechanisms contributing to COVID-19 immunopathogenesis.

A 26-year-old otherwise healthy man died of fulminant myocarditis. Nasopharyngeal specimens collected premortem tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Histopathological evaluation of the heart showed myocardial necrosis surrounded by cytotoxic T-cells and tissue-repair macrophages. Myocardial T-cell receptor (TCR) sequencing revealed hyper-dominant clones with highly similar sequences to TCRs that are specific for SARS-CoV-2 epitopes. SARS-CoV-2 RNA was detected in the gut, supporting a diagnosis of multisystem inflammatory syndrome in adults (MIS-A). Molecular targets of MIS-associated inflammation are not known. Our data indicate that SARS-CoV-2 antigens selected high-frequency T-cell clones that mediated fatal myocarditis.

There is limited information about the impact of Zika virus (ZIKV) exposure in utero on the anti-ZIKV immune responses of offspring. We infected six rhesus macaque dams with ZIKV early or late in pregnancy and studied four of their offspring over the course of a year postpartum. Despite evidence of ZIKV exposure in utero, we observed no structural brain abnormalities in the offspring. We detected infant-derived ZIKV-specific immunoglobulin A antibody responses and T cell memory responses during the first year postpartum in the two offspring born to dams infected with ZIKV early in pregnancy. Critically, although the infants had acquired some immunological memory of ZIKV, it was not sufficient to protect them against reinfection with ZIKV at 1 year postpartum. The four offspring reexposed to ZIKV at 1 year postpartum all survived but exhibited acute viremia and viral tropism to lymphoid tissues; three of four reexposed offspring exhibited spinal cord pathology. These data suggest that macaque infants born to dams infected with ZIKV during pregnancy remain susceptible to postnatal infection and consequent neuropathology.

Changes in maternal immunity during pregnancy can result in an altered immune state, and as a natural perturbation, this provides an opportunity to understand functional interactions of the immune system in vivo. We report characterization of maternal peripheral immune phenotypes for 33 longitudinally sampled normal pregnancies, using clinical measurements of complete blood counts and major immune cell populations, as well as high parameter flow cytometry for 30 leukocyte antigens characterizing 79 cell populations, and monitoring of 1305 serum proteins using the SomaLogic platform. Cellular analyses characterized transient changes in T cell polarization and more persistent alterations in T and B cell subset frequencies and activation. Serum proteomic analysis identified a potentially novel set of 7 proteins that are predictive of gestational age: DDR1, PLAU, MRC1, ACP5, ROBO2, IGF2R, and GNS. We further show that gestational age can be predicted from the parameters obtained by complete blood count tests and clinical flow cytometry characterizing 5 major immune cell populations. Inferring gestational age from this routine clinical phenotyping data could be useful in resource-limited settings that lack obstetric ultrasound. Overall, both the cellular and proteomic analyses validate previously reported phenotypic immunological changes of pregnancy and uncover potentially new alterations and predictive markers.

Background A pharmacologic intervention that modulates JAK/STAT signaling pathways represents a novel approach for the treatment of Systemic Lupus Erythematosus (SLE). In animal models of SLE, tofacitinib improved clinical features, immune dysregulation and vascular dysfunction. The STAT4 risk allele is associated with higher risk of severe manifestations in SLE. We hypothesized that immune modulation in response to JAK/STAT inhibition would be more robust in SLE subjects that carry the STAT4 risk allele. Methods We conducted a phase 1b/2a randomized, double-blind, placebo-controlled clinical trial of oral tofacitinib, 5 mg twice daily, in 30 SLE subjects (2:1 drug to placebo ratio) with mild to moderate disease activity, stratified by the presence or absence of STAT4 risk allele. Study duration was 84 days (56 days of active treatment ; 28 days of off drug). In addition to recording adverse events (AEs), lipoprotein profile, non-invasive vascular function studies, immuno-phenotyping, and gene expression studies were performed. Results Tofacitinib was well tolerated with no worsening of SLE disease activity, and no severe AEs, opportunistic infections or liver function abnormalities. A total of 43 AEs (mostly mild respiratory infections) occurred in the treated group compared to 28 AEs in placebo. There was a significant increase in HDL-C and HDL particle size in tofacitinib-treated patients at day 56 (p=0.006) accompanied by significant improvements in plasma protein lecithin: cholesterol acyltransferase (LCAT) concentration. There were also trends for improvements in vascular stiffness in the tofacitinib-treated group. The Interferon response genes (type I IFN), the levels of low- density granulocytes (LDGs) and neutrophil extracellular trap (NET remnants) significantly decreased in the tofacitinib treated group who were STAT 4 risk allele positive but not in the placebo group at day 56, accompanied by significant changes in pSTAT phosphorylation of different immune cells. Levels of activation and checkpoint markers CD103, CXCR3, ICOS, and PD-1 were significantly decreased on multiple T cell subsets, in tofacitinib treated individuals that lack the STAT4 risk allele.Abstract 183 Figure 1 Adverse Events by Organ System. Adverse Events in Subjects on Tofacitinib vs Placebo Conclusions In a short-term trial, tofacitinib was well tolerated in SLE subjects with mild-moderate disease activity. Use of tofacitinib resulted in improvements in lipoprotein profile and HDL function and decreases in the type I IFN and aberrant neutrophil responses characteristic of SLE. Long-term studies are needed to determine the efficacy of tofacitinib in the various manifestations of SLE. Funding Source(s): Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health and in part by Pfizer Inc. Adverse Events in Subjects on Tofacitinib vs Placebo

Investigation of aeroponically grown Physalis peruviana resulted in the isolation of 11 new withanolides, including perulactones I-L (1-4), 17-deoxy-23β-hydroxywithanolide E (5), 23β-hydroxywithanolide E (6), 4-deoxyphyperunolide A (7), 7β-hydroxywithanolide F (8), 7β-hydroxy-17-epi-withanolide K (9), 24,25-dihydro-23β,28-dihydroxywithanolide G (10), and 24,25-dihydrowithanolide E (11), together with 14 known withanolides (12-25). The structures of 1-11 were elucidated by the analysis of their spectroscopic data, and 12-25 were identified by comparison of their spectroscopic data with those reported. All withanolides were evaluated for their cytotoxic activity against a panel of tumor cell lines including LNCaP (androgen-sensitive human prostate adenocarcinoma), 22Rv1 (androgen-resistant human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal human foreskin fibroblast cells. Of these, the 17β-hydroxywithanolides (17-BHWs) 6, 8, 9, 11-13, 15, and 19-22 showed selective cytotoxic activity against the two prostate cancer cell lines LNCaP and 22Rv1, whereas 13 and 20 exhibited selective toxicity for the ACHN renal carcinoma cell line. These cytotoxicity data provide additional structure-activity relationship information for the 17-BHWs.