Jyotsna Nateri

Innate Immune Function and Mortality in Critically Ill Children With Influenza: A Multicenter Study*

Objective:To prospectively evaluate relationships among serum cytokine levels, innate immune responsiveness, and mortality in a multicenter cohort of critically ill children with influenza infection. Design:Prospective, multicenter, observational study. Setting:Fifteen pediatric ICUs among members of the Pediatric Acute Lung Injury and Sepsis Investigators network. Patients:Patients ⩽18 yrs old admitted to a PICU with community-acquired influenza infection. A control group of outpatient children was also evaluated. Interventions:ICU patients underwent sampling within 72 hrs of ICU admission for measurement of a panel of 31 serum cytokine levels and quantification of whole blood ex vivo lipopolysaccharide-stimulated tumor necrosis factor-&agr; production capacity using a standardized stimulation protocol. Outpatient control subjects also underwent measurement of tumor necrosis factor-&agr; production capacity. Measurements and Main Results:Fifty-two patients (44 survivors, eight deaths) were sampled. High levels of serum cytokines (granulocyte macrophage colony-stimulating factor, interleukin-6, interleukin-8, interferon-inducible protein-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1&agr;) were associated with mortality (p < 0.0016 for each comparison) as was the presence of secondary infection with Staphylococcus aureus (p = 0.007), particularly methicillin-resistant S. aureus (p < 0.0001). Nonsurvivors were immunosuppressed with leukopenia and markedly reduced tumor necrosis factor-&agr; production capacity compared with outpatient control subjects (n = 21, p < 0.0001) and to ICU survivors (p < 0.0001). This association remained after controlling for multiple covariables. A tumor necrosis factor-&agr; response <250 pg/mL was highly predictive of death and longer duration of ICU stay (p < 0.0001). Patients with S. aureus coinfection demonstrated the greatest degree of immunosuppression (p < 0.0001). Conclusions:High serum levels of cytokines can coexist with marked innate immune suppression in children with critical influenza. Severe, early innate immune suppression is highly associated with both S. aureus coinfection and mortality in this population. Multicenter innate immune function testing is feasible and can identify these high-risk children.

Pyrin Critical to Macrophage IL-1β Response to Francisella Challenge

Relative to monocytes, human macrophages are deficient in their ability to process and release IL-1β. In an effort to explain this difference, we used a model of IL-1β processing and release that is dependent upon bacterial escape into the cytosol. Fresh human blood monocytes were compared with monocyte-derived macrophages (MDM) for their IL-1β release in response to challenge with Francisella novicida. Although both cell types produced similar levels of IL-1β mRNA and intracellular pro-IL-1β, only monocytes readily released processed mature IL-1β. Baseline mRNA expression profiling of candidate genes revealed a remarkable deficiency in the pyrin gene, MEFV, expression in MDM compared with monocytes. Immunoblots confirmed a corresponding deficit in MDM pyrin protein. To determine whether pyrin levels were responsible for the monocyte/MDM difference in mature IL-1β release, pyrin expression was knocked down by nucleofecting small interfering RNA against pyrin into monocytes or stably transducing small interfering RNA against pyrin into the monocyte cell line, THP-1. Pyrin knockdown was associated with a significant drop in IL-1β release in both cell types. Importantly, M-CSF treatment of MDM restored pyrin levels and IL-1β release. Similarly, the stable expression of pyrin in PMA-stimulated THP-1-derived macrophages induces caspase-1 activation, associated with increased IL-1β release after infection with F. novicida. In summary, intracellular pyrin levels positively regulate MDM IL-1β responsiveness to Francisella challenge.

Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution

BACKGROUND: Reduced monocyte function is associated with adverse outcomes from critical illness. Red blood cells (RBCs) are thought to impair monocyte function but relationships between RBC storage solution and monocyte suppression are unknown. This study was designed to test the hypothesis that immunosuppressive effects of RBCs on monocytes are related to both storage time and preservative solution.

Innate immune function predicts the development of nosocomial infection in critically injured children.

ABSTRACT Background: Critical injury has been associated with reduction in innate immune function in adults, with infection risk being related to degree of immune suppression. This relationship has not been reported in critically injured children. Hypothesis: Innate immune function will be reduced in critically injured children, and the degree of reduction will predict the subsequent development of nosocomial infection. Methods: Children (⩽18 years old) were enrolled in this longitudinal, prospective, observational, single-center study after admission to the pediatric intensive care unit following critical injury, along with a cohort of outpatient controls. Serial blood sampling was performed to evaluate plasma cytokine levels and innate immune function as measured by ex vivo lipopolysaccharide-induced tumor necrosis factor &agr; (TNF-&agr;) production capacity. Results: Seventy-six critically injured children (and 21 outpatient controls) were enrolled. Sixteen critically injured subjects developed nosocomial infection. Those subjects had higher plasma interleukin 6 and interleukin 10 levels on posttrauma days 1–2 compared with those who recovered without infection and outpatient controls. Ex vivo lipopolysaccharide-induced TNF-&agr; production capacity was lower on posttrauma days 1–2 (P = 0.006) and over the first week following injury (P = 0.04) in those who went on to develop infection. A TNF-&agr; response of less than 520 pg/mL at any time in the first week after injury was highly associated with infection risk by univariate and multivariate analysis. Among transfused children, longer red blood cell storage age, not transfusion volume, was associated with lower innate immune function (P < 0.0001). Trauma-induced innate immune suppression was reversible ex vivo via coculture of whole blood with granulocyte-macrophage colony-stimulating factor. Conclusions: Trauma-induced innate immune suppression is common in critically injured children and is associated with increased risks for the development of nosocomial infection. Potential exacerbating factors, including red blood cell transfusion, and potential therapies for pediatric trauma-induced innate immune suppression are deserving of further study.

Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.

We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC‐derived microvesicles (MVs) were responsible for monocyte suppression.

42: IMMUNE FUNCTION AND OUTCOMES IN YOUNG CHILDREN WITH ACUTE RESPIRATORY FAILURE DUE TO VIRAL INFECTION

Learning Objectives: Critical illness-induced immune suppression is characterized by low absolute lymphocyte count (ALC), low monocyte HLA-DR (mHLA-DR) expression, and increased lymphocyte PD-1 expression. Little is known about these relationships in young children with acute respiratory failure due to viral infection. We hypothesize that immune suppression will be associated with adverse outcomes in this population. Methods: Enrollment in this prospective observational study is open to children < 2 yrs old without major co-morbidities admitted to the PICU with a new invasive or noninvasive (NIV) positive pressure ventilation (PPV) requirement and a +PCR test for a respiratory virus. Patients are sampled within 48hrs of PPV initiation then 2x/week for viral load (nasopharynx: PCR) and immune function testing (blood: ALC, mHLA-DR, PD-1) until ICU discharge. Complicated course =r emaining virus + and in the PICU after ICU day 6. Data represent median (IQR). Results: 58 subjects have been enrolled to date (age: 3.5 [0.4–9] mo, 67% male). Viruses: RSV (59%), rhino/entero (34%), hMPV (10%), corona (9%), adeno (7%), influenza (5%), parainfluenza (5%), polyviral (24%). Durations of: PPV: 81 (44–161) hrs, PICU stay: 4.4 (1.4–8.3) days, hospital stay: 7 (3.7–12) days. Intubated subjects (n = 23) had lower ALC (p = 0.004) and a trend toward lower mHLA-DR (p = 0.06) over the first week of illness vs those with NIV. Immune function was also lower in subjects who were treated for secondary bacterial pneumonia (n = 29) vs those who were not (p = 0.003 [ALC], p = 0.05 [HLA-DR]). Initial mHLADR was lower in children who went on to have a complicated course (n = 13; 8065 vs 12300 molecules/cell, p = 0.014). Initial ALC was similar between groups but ALC was lower over the first week in those with a complicated course (p = 0.01). Lower initial mHLA-DR (but not ALC) was associated with longer duration of PPV (p = 0.025), PICU stay (p = 0.024), and hospital stay (p = 0.04). Lymphocyte PD-1 expression was not associated with outcomes (p = NS for all). Immune function was similar between subjects who did (n = 16) and did not receive methylprednisolone (p = NS for all). Conclusions: Our preliminary data suggest that immune suppression is common in young children with acute respiratory failure due to viral infection, with low ALC and low mHLA-DR (but not lymphocyte PD-1 expression) predicting adverse outcomes. Interestingly, receipt of methylprednisolone did not significantly influence these immune parameters. Enrollment in this study is ongoing.

1138: VIRAL-BACTERIAL INTERACTIONS AND IMMUNE FUNCTION IN INFANTS WITH VIRAL-INDUCED RESPIRATORY FAILURE

Critical Care Medicine • Volume 46 • Number 1 (Supplement) www.ccmjournal.org Learning Objectives: Viral lower respiratory tract infections (LRTI) are a leading cause of PICU admission in infants. The frequency of nasopharyngeal (NP) colonization with potentially pathogenic bacteria (PPB) in these children and its potential influence on clinical outcomes is not well understood. We hypothesized that detection of specific PPB in the NP of children with severe LRTI was associated with impaired innate immune response and enhanced disease severity. Methods: Children < 2 years of age admitted to the PICU with viral LRTI were enrolled. NP and lower respiratory samples (ETT) were collected within 48 hours of PICU admission for detection by RT-PCR of common respiratory viruses and four PPB including gram positive (GPB: S. aureus, S. pneumoniae) and gram negative (GNB: H. influenzae and M. catarrhalis) bacteria. Blood samples were collected in parallel to evaluate monocyte (m)HLADR expression as a marker of innate immune function. Viral and bacterial detection and immune function were correlated with clinical outcomes including duration of positive pressure ventilation (PPV) and PICU length of stay (LOS). Results: From January-April, 2017, 56 patients, median age 3.4 months, were enrolled. Respiratory syncytial virus (RSV) was identified in 59% of children, rhinovirus (RV) in 20% and other respiratory viruses in 21%. NP PPB were identified in 89% of patients, with substantial/moderate agreement between NP and ETT detection in the 17 patients that required intubation for GNB [Kappa coefficient: 0.61–0.75] and S. pneumoniae [Kappa: 0.51], and poor agreement for S. aureus [Kappa 0.2]. In infants with RSV vs. RV LRTI, NP detection of GNB was more frequent (60% vs. 36%) while S. pneumoniae detection in the NP was less common (27% vs. 46% respectively; p = 0.04). However, only in children with RSV LRTI, S. pneumoniae (n = 10) vs. GNB NP detection (n = 19), was associated with lower mHLA-DR expression (p < 0.01), longer duration of PICU LOS (11.2 vs. 6.6 days; p = 0.04) and a trend for longer PPV (8.9 vs 5.5 days; p = 0.06). Conclusions: Detection of specific PPB in the nasopharynx was common, reflected detection in the lower respiratory tract for GNB and S. pneumoniae, and varied based upon the etiology of the viral LRTI. Despite that GNB were the most common PPB identified in the NP, only S. pneumoniae in infants with RSV LRTI was associated with impaired innate immune responses and worse clinical outcomes.