Nuclear segmentation facilitates neutrophil migration.

Neutrophils are among the fastest-moving immune cells. Their speed is critical to their function as 'first responder' cells at sites of damage or infection, and it has been postulated that the unique segmented nucleus of neutrophils functions to assist their rapid migration. Here, we tested this hypothesis by imaging primary human neutrophils traversing narrow channels in custom-designed microfluidic devices. Individuals were given an intravenous low dose of endotoxin to elicit recruitment of neutrophils into the blood with a high diversity of nuclear phenotypes, ranging from hypo- to hyper-segmented. Both by cell sorting of neutrophils from the blood using markers that correlate with lobularity and by directly quantifying the migration of neutrophils with distinct lobe numbers, we found that neutrophils with one or two nuclear lobes were significantly slower to traverse narrower channels, compared to neutrophils with more than two nuclear lobes. Thus, our data show that nuclear segmentation in primary human neutrophils provides a speed advantage during migration through confined spaces.

The gut microbiota composition has no predictive value for the endotoxin-induced immune response or development of endotoxin tolerance in humans invivo.

BACKGROUND: It is largely unknown whether the gut microbiome regulates immune responses in humans. We determined relationships between the microbiota composition and immunological phenotypes in 108 healthy volunteers, using 16S sequencing, an ex vivo monocyte challenge model, and an in vivo challenge model of systemic inflammation induced by lipopolysaccharide (LPS). RESULTS: Significant associations were observed between the microbiota composition and ex vivo monocytic cytokine responses induced by several stimuli, most notably IL-10 production induced by Pam3Cys, Pseudomonas aeruginosa and Candida albicans, although the explained variance was rather low (0.3-4.8%). Furthermore, a number of pairwise correlations between Blautia, Bacteroides and Prevotella genera and cytokine production induced by these stimuli were identified. LPS administration induced a profound transient in vivo inflammatory response. A second LPS challenge one week after the first resulted in a severely blunted response, reflecting endotoxin tolerance. However, no significant relationships between microbiota composition and in vivo parameters of inflammation or tolerance were found (explained variance ranging from 0.4 to 1.5%, ns). CONCLUSIONS: The gut microbiota composition explains a limited degree of variance in ex vivo monocytic cytokine responses to several pathogenic stimuli, but no relationships with the LPS-induced in vivo immune response or tolerance was observed.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

CytoSorb hemoperfusion markedly attenuates circulating cytokine concentrations during systemic inflammation in humans in vivo.

BACKGROUND: The CytoSorb hemoadsorption device has been demonstrated to be capable of clearing inflammatory cytokines, but has not yet been shown to attenuate plasma cytokine concentrations. We investigated the effects of CytoSorb hemoperfusion on plasma levels of various cytokines using the repeated human experimental endotoxemia model, a highly standardized and reproducible human in vivo model of systemic inflammation and immunological tolerance induced by administration of bacterial lipopolysaccharide (LPS). METHODS: Twenty-four healthy male volunteers (age 18-35) were intravenously challenged with LPS (a bolus of 1 ng/kg followed by continuous infusion of 0.5 ng/kg/hr for three hours) twice: on day 0 to quantify the initial cytokine response and on day 7 to quantify the degree of endotoxin tolerance. Subjects either received CytoSorb hemoperfusion during the first LPS challenge (CytoSorb group), or no intervention (control group). Plasma cytokine concentrations and clearance rates were determined serially. This study was registered at ClinicalTrials.gov (NCT04643639, date of registration November 24th 2020). RESULTS: LPS administration led to a profound increase in plasma cytokine concentrations during both LPS challenge days. Compared to the control group, significantly lower plasma levels of tumor necrosis factor (TNF, - 58%, p < 0.0001), interleukin (IL)-6 ( - 71%, p = 0.003), IL-8 ( - 48%, p = 0.02) and IL-10 ( - 26%, p = 0.03) were observed in the CytoSorb group during the first LPS challenge. No differences in cytokine responses were observed during the second LPS challenge. CONCLUSIONS: CytoSorb hemoperfusion effectively attenuates circulating cytokine concentrations during systemic inflammation in humans in vivo, whereas it does not affect long-term immune function. Therefore, CytoSorb therapy may be of benefit in conditions characterized by excessive cytokine release.

The impact of ADRB2 polymorphisms on immune responses and norepinephrine-induced immunosuppression.

RATIONALE: To evaluate whether common nonsynonymous variants [single-nucleotide polymorphisms (SNPs) or SNP haplotypes] in the ß2-adrenergic receptor render subjects more susceptible to norepinephrine-induced immunosuppression and whether they are associated with dysregulated ex vivo and in vivo inflammatory responses. METHODS: Peripheral blood mononuclear cells from healthy volunteers (main cohort: n = 106, secondary cohort: n = 408) were ex vivo stimulated with various stimuli and production of cytokines was assessed. Additionally, ex vivo modulation of cytokine production by norepinephrine was evaluated in the main cohort. Volunteers from the main cohort also underwent experimental endotoxemia (administration of 1 ng/kg lipopolysaccharide), during which in vivo plasma cytokine concentrations and clinical inflammatory parameters were measured. Subjects were genotyped, common SNPs in the ADRB2 gene were extracted (rs1042711, rs1042713, and rs1042714), and the presence of haplotypes was identified (CysGlyGln, CysArgGln, and ArgGlyGlu). RESULTS: In both cohorts, presence of ADRB2 SNPs or haplotypes was not associated with altered ex vivo cytokine responses. Norepinephrine attenuated production of the proinflammatory cytokines TNF and IL-6 [-26% (-22% to -30%) and -14% (-9% to -18%), respectively, both P < 0.0001] and enhanced release of the anti-inflammatory IL-10 [+9% (+3% to +15%), P = 0.003]. These effects were not modulated by the presence of ADRB2 SNPs or haplotypes (all P values >0.37). In addition, no influence of SNPs or haplotypes on in vivo cytokine concentrations or clinical inflammatory parameters was observed (P values >0.14). CONCLUSIONS: Common nonsynonymous variants in the ADRB2 gene influence neither ex vivo cytokine production or norepinephrine-mediated immunosuppression nor the systemic in vivo inflammatory response induced by lipopolysaccharide administration in healthy volunteers.

Clinical sepsis phenotypes in critically ill COVID-19 patients.

BACKGROUND: A greater understanding of disease heterogeneity may facilitate precision medicine for coronavirus disease 2019 (COVID-19). Previous work identified four distinct clinical phenotypes associated with outcome and treatment responses in non-COVID-19 sepsis patients, but it is unknown if and how these phenotypes are recapitulated in COVID-19 sepsis patients. METHODS: We applied the four non-COVID-19 sepsis phenotypes to a total of 52,274 critically ill patients, comprising two cohorts of COVID-19 sepsis patients (admitted before and after the introduction of dexamethasone as standard treatment) and three non-COVID-19 sepsis cohorts (non-COVID-19 viral pneumonia sepsis, bacterial pneumonia sepsis, and bacterial sepsis of non-pulmonary origin). Differences in proportions of phenotypes and their associated mortality were determined across these cohorts. RESULTS: Phenotype distribution was highly similar between COVID-19 and non-COVID-19 viral pneumonia sepsis cohorts, whereas the proportion of patients with the δ-phenotype was greater in both bacterial sepsis cohorts compared to the viral sepsis cohorts. The introduction of dexamethasone treatment was associated with an increased proportion of patients with the δ-phenotype (6% vs. 11% in the pre- and post-dexamethasone COVID-19 cohorts, respectively, p < 0.001). Across the cohorts, the α-phenotype was associated with the most favorable outcome, while the δ-phenotype was associated with the highest mortality. Survival of the δ-phenotype was markedly higher following the introduction of dexamethasone (60% vs 41%, p < 0.001), whereas no relevant differences in survival were observed for the other phenotypes among COVID-19 patients. CONCLUSIONS: Classification of critically ill COVID-19 patients into clinical phenotypes may aid prognostication, prediction of treatment efficacy, and facilitation of personalized medicine.

Ex vivo and in vitro Monocyte Responses Do Not Reflect in vivo Immune Responses and Tolerance.

Cytokine production by ex vivo (EV)-stimulated leukocytes is commonly used to gauge immune function and frequently proposed to guide immunomodulatory therapy. However, whether EV cytokine production capacity accurately reflects the in vivo (IV) immune status is largely unknown. We investigated relationships between EV monocyte cytokine responses and IV cytokine responses in a large cohort of healthy volunteers using a highly standardized IV model of short-lived LPS-induced systemic inflammation, which captures hallmarks of both hyperinflammation and immunological tolerance. Therefore, 110 healthy volunteers were intravenously challenged with 1 ng/kg LPS twice: on day 0 to determine the extent of the IV (hyper)inflammatory response and on day 7 to determine the degree of IV endotoxin tolerance. Baseline EV monocyte cytokine production capacity was assessed prior to LPS administration. Short-term and long-term EV tolerance was assessed in monocytes isolated 4 h and 7 days after LPS administration, respectively. No robust correlations were observed between baseline EV cytokine production capacity and IV cytokine responses following LPS administration. However, highly robust inverse correlations were observed between IV cytokine responses and EV cytokine responses of monocytes isolated 4 h after IV LPS administration. No correlations between IV and EV tolerance were found. In conclusion, attenuated EV cytokine production capacity reflects ongoing IV inflammation rather than immune suppression. Results of EV assays should be interpreted with caution at the risk of improper use of immuno-stimulatory drugs.

Treatment With Acetylsalicylic Acid Reverses Endotoxin Tolerance in Humans In Vivo: A Randomized Placebo-Controlled Study.

OBJECTIVE: To investigate immunostimulatory effects of acetylsalicylic acid during experimental human endotoxemia and in sepsis patients. DESIGN: Double-blind, randomized, placebo-controlled study in healthy volunteers and ex vivo stimulation experiments using monocytes of septic patients. SETTING: Intensive care research unit of an university hospital. SUBJECTS: Thirty healthy male volunteers and four sepsis patients. INTERVENTIONS: Healthy volunteers were challenged IV with endotoxin twice, at a 1-week interval, with each challenge consisting of a bolus of 1 ng/kg followed by continuous administration of 1 ng/kg/hr during 3 hours. Volunteers were randomized to acetylsalicylic acid prophylaxis (80 mg acetylsalicylic acid daily for a 14-d period, starting 7 d before the first endotoxin challenge), acetylsalicylic acid treatment (80 mg acetylsalicylic acid daily for the 7-d period in-between both endotoxin challenges), or the control group (receiving placebo). Furthermore, monocytes of sepsis patients were incubated with acetylsalicylic acid preexposed platelets and were subsequently stimulated with endotoxin. MEASUREMENTS AND MAIN RESULTS: Acetylsalicylic acid prophylaxis enhanced plasma tumor necrosis factor-α concentrations upon the first endotoxin challenge by 50% compared with the control group (p = 0.02) but did not modulate cytokine responses during the second endotoxin challenge. In contrast, acetylsalicylic acid treatment resulted in enhanced plasma levels of tumor necrosis factor-α (+53%; p = 0.02), interleukin-6 (+91%; p = 0.03), and interleukin-8 (+42%; p = 0.02) upon the second challenge, whereas plasma levels of the key antiinflammatory cytokine interleukin-10 were attenuated (-40%; p = 0.003). This proinflammatory phenotype in the acetylsalicylic acid treatment group was accompanied by a decrease in urinary prostaglandin E metabolite levels (-27% ± 7%; p = 0.01). Ex vivo exposure of platelets to acetylsalicylic acid increased production of tumor necrosis factor-α (+66%) and decreased production of interleukin-10 (-23%) by monocytes of sepsis patients. CONCLUSIONS: Treatment, but not prophylaxis, with low-dose acetylsalicylic acid, partially reverses endotoxin tolerance in humans in vivo by shifting response toward a proinflammatory phenotype. This acetylsalicylic acid-induced proinflammatory shift was also observed in septic monocytes, signifying that patients suffering from sepsis-induced immunoparalysis might benefit from initiating acetylsalicylic acid treatment.

Quasinormal Modes and Strong Cosmic Censorship.

The fate of Cauchy horizons, such as those found inside charged black holes, is intrinsically connected to the decay of small perturbations exterior to the event horizon. As such, the validity of the strong cosmic censorship (SCC) conjecture is tied to how effectively the exterior damps fluctuations. Here, we study massless scalar fields in the exterior of Reissner-Nordström-de Sitter black holes. Their decay rates are governed by quasinormal modes of the black hole. We identify three families of modes in these spacetimes: one directly linked to the photon sphere, well described by standard WKB-type tools; another family whose existence and time scale is closely related to the de Sitter horizon; finally, a third family which dominates for near-extremally charged black holes and which is also present in asymptotically flat spacetimes. The last two families of modes seem to have gone unnoticed in the literature. We give a detailed description of linear scalar perturbations of such black holes, and conjecture that SCC is violated in the near extremal regime.

Holographic Equilibration of Nonrelativistic Plasmas.

We study far-from-equilibrium physics of strongly interacting plasmas at criticality and zero charge density for a wide range of dynamical scaling exponents z in d dimensions using holographic methods. In particular, we consider homogeneous isotropization of asymptotically Lifshitz black branes with full backreaction. We find stable evolution and equilibration times that exhibit small dependence of z and are of the order of the inverse temperature. Performing a quasinormal mode analysis, we find a corresponding narrow range of relaxation times, fully characterized by the fraction z/(d-1). For z≥d-1, equilibration is overdamped, whereas for z