Inflammatory Biomarker Profiles in Very Preterm Infants within the Context of Preeclampsia, Chorioamnionitis, and Clinically Diagnosed Postnatal Infection.

Preterm delivery can be precipitated by preeclampsia or infection, and preterm infants are at heightened risk of postnatal infection. Little is known about the ontogeny of inflammatory biomarkers in extremely preterm infants. We hypothesized that suspected prenatal infection (clinical chorioamnionitis or spontaneous preterm labor) and clinically diagnosed postnatal infection would be associated with unique biomarker signatures, and those patterns would be influenced by the degree of prematurity. Venous blood was collected daily for the first week and weekly for up to 14 additional weeks from 142 neonates born at 22-32 weeks gestation. A custom array was utilized to measure monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). C-reactive protein (CRP) levels were obtained from the electronic medical record. Independent of gestational age, MCP-1 was significantly increased (p < 0.001) in association with maternal preeclampsia, but MCP-1 was decreased (p < 0.01), and CRP was increased (p < 0.01) in the presence of chorioamnionitis with funisitis. IL-6 and CRP were both increased in infants diagnosed with postnatal infection, with peak levels observed on days 2 and 3, respectively. In conclusion, suspected prenatal and postnatal infections and non-infectious complications of pregnancy are associated with unique biomarker profiles, independent of gestational age, including over a 2-fold increase in MCP-1 among newborns of mothers with preeclampsia. Further, in those clinically diagnosed with a postnatal infection in the absence of antenatal infection concerns, IL-6 increases before CRP, emphasizing a potential role for expanded biomarker screening if antibiotics are initially avoided in infants delivered for maternal indications.

Sex-associated early-life viral innate immune response is transcriptionally associated with chromatin remodeling of type-I IFN-inducible genes.

This study investigates sex-associated systemic innate immune differences by examining bone marrow-derived dendritic cells (BMDCs). BMDC grown from 7-day-old mice show enhanced type-I interferon (IFN) signaling in female compared to male BMDC. Upon respiratory syncytial virus (RSV) infection of 7-day-old mice, a significantly altered phenotype of BMDC at 4 weeks post-infection is observed in a sex-dependent manner. The alterations include heightened Ifnb/ interleukin (Il12a) and enhanced IFNAR1+ expression in BMDC from early-life RSV-infected female mice that leads to increased IFN-γ production by T cells. Phenotypic differences were verified upon pulmonary sensitization whereby EL-RSV male-derived BMDC promoted enhanced T helper 2/17 responses and exacerbated disease upon RSV infection while EL-RSV/F BMDC sensitization was relatively protective. Assay for transposase-accessible chromatin using sequencing analysis (ATAC-seq) demonstrated that EL-RSV/F BMDC had enhanced chromatin accessibility near type-I immune genes with JUN, STAT1/2, and IRF1/8 transcription factors predicted to have binding sites in accessible regions. Importantly, ATAC-seq of human cord blood-derived monocytes displayed a similar sex-associated chromatin landscape with female-derived monocytes having more accessibility in type-I immune genes. These studies enhance our understanding of sex-associated differences in innate immunity by epigenetically controlled transcriptional programs amplified by early-life infection in females via type-I immunity.

Chorioamnionitis-exposure alters serum cytokine trends in premature neonates.

OBJECTIVES: Determine if chronologic age and/or chorioamnionitis exposure alter normal serum cytokine and chemokine levels in uninfected preterm neonates during their initial NICU stay. STUDY DESIGN: A 7-plex immunoassay measured levels of serum IL-1ß, IL-6, IL-8, IL-10, TNF-α, CCL2, and CCL3 longitudinally from chorioamnionitis-exposed and unexposed preterm neonates under 33 weeks' gestation. RESULTS: Chorioamnionitis-exposed and unexposed preterm neonates demonstrated differences in the trends of IL-1ß, IL-6, IL-8, IL-10, TNF-α, and CCL2 over the first month of life. The unexposed neonates demonstrated elevated levels of these inflammatory markers in the first two weeks of life with a decrease by the third week of life, while the chorioamnionitis-exposed neonates demonstrated differences over time without a predictable pattern. Chorioamnionitis-exposed and unexposed neonates demonstrated altered IL-10 and TNF-α trajectories over the first twelve weeks of life. CONCLUSION: Chorioamnionitis induces a state of immune dysregulation in preterm neonates that persists beyond the immediate neonatal period.

Differences in H3K4me3 and chromatin accessibility contribute to altered T-cell receptor signaling in neonatal naïve CD4 T cells.

Neonatal CD4+ T cells have reduced or delayed T-cell receptor (TCR) signaling responses compared with adult cells, but the mechanisms underlying this are poorly understood. This study tested the hypothesis that human neonatal naïve CD4+ TCR signaling and activation deficits are related to differences in H3K4me3 patterning and chromatin accessibility. Following initiation of TCR signaling using anti-CD3/anti-CD28 beads, adult naïve CD4+ T cells demonstrated increased CD69, phospho-CD3ε and interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ and IL-17A compared with neonatal cells. By contrast, following TCR-independent activation using phorbol myristate acetate (PMA)/ionomycin, neonatal cells demonstrated increased expression of CD69, IL-2 and TNF-α and equivalent phospho-ERK compared with adult cells. H3K4me3 chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on separate cohorts of naïve CD4+ T cells from term neonates and adults, and RNA-seq data from neonatal and adult naïve CD4+ T cells were obtained from the Blueprint Consortium. Adult cells demonstrated overall increased chromatin accessibility and a higher proportion of H3K4me3 sites associated with open chromatin and active gene transcription compared with neonatal cells. Adult cells demonstrated increased mRNA expression of the TCR-associated genes FYN, ITK, CD4, LCK and LAT, which was associated with increased H3K4me3 at the FYN and ITK gene loci and increased chromatin accessibility at the CD4, LCK and LAT loci. These findings indicate that neonatal TCR-dependent defects in activation are epigenetically regulated and provide a potentially targetable mechanism to enhance neonatal CD4+ T-cell responses.

Hyperglycemia and Cytopenias as Signs of SARS-CoV-2 Delta Variant Infection in Preterm Infants.

Information regarding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in premature infants remains limited. Early in the pandemic, several studies reported that the risk of infection in infants was relatively small and that affected infants had a milder disease than what was seen in adults. Since the increase of the delta variant (SARS-CoV-2 B.1.617.2) within the population, there have been increased reports of more severe disease in infants. We present 3 cases of premature, very low birth weight infants with confirmed SARS-CoV-2 infection who presented with significant hyperglycemia and bone marrow dysfunction. Two infants had presumed vertical transmission, and 1 infant was infected by respiratory transmission. Despite the mode of transmission, symptom onset and duration were similar in all infants. All resolved with symptomatic management. In the context of the continuing pandemic, evaluation for SARS-CoV-2 infection should be considered in premature very low birth weight infants who demonstrate certain patterns of acute metabolic and hematologic abnormalities.

Elevated Notch ligands in serum are associated with HIV/TB coinfection.

OBJECTIVE: There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although numerous tests can identify the existence of both of these microbes within the host, a parallel assessment of the host response to HIV/TB coinfection may prove as useful confirmation in cases where microbiological tests are inconclusive. To this end we assessed the levels of Notch ligands found in serum samples of patients with TB, HIV or HIV/TB coinfection. The Notch system is involved in almost every stage of development, including the maturation of the immune response. Upon exposure to a pathogen, the innate immune system will increase expression of Notch ligands Delta-like 1 and Delta-like 4. Previous research has demonstrated that Notch ligand expression is increased on monocytes from patients diagnosed with tuberculosis. We hypothesized that if Notch ligands were present in the peripheral blood of individuals diagnosed with TB, they may serve as a novel marker for infection.Design: Serum samples from patients with HIV, TB or HIV/TB coinfection were compared to serum from uninfected individuals to determine levels of DLL1 and DLL4 in a case controlled study. METHODS: DLL1 and DLL4 were measured by ELISA. Linear regression with post tests were used to determine if levels of DLL1 and DLL4 were increased in individuals with HIV/TB coinfection as compared to individuals infected with either HIV or TB or healthy controls. RESULTS: Delta-like 1 and Delta-like 4 were significantly increased in the serum of patients with HIV and HIV/ M. tuberculosis coinfection compared to other groups. CONCLUSIONS: Assessment of Notch ligands in peripheral blood may enhance the diagnosis of individuals with active TB that are co-infected with HIV. The study will also need to be validated in in a larger cohort.

Fetal hemoglobin levels in premature newborns. Should we reconsider transfusion of adult donor blood?

PURPOSE: The aim of this study was to assess the percent decrease in fetal hemoglobin (HbF) after transfusion of adult-derived donor packed red blood cell (pRBC) units in extremely low gestational age newborns (ELGANs). METHODS: Control percent fetal hemoglobin (%HbF) levels were measured in newborn cord blood or peripheral blood samples in non-transfused patients prior to elective surgery. ELGANs were followed prospectively and %HbF was measured on residual post-test complete blood count (CBC) specimens. ELGAN %HbF values were compared to the control population and transfusions were recorded. RESULTS: Initial mean %HbF in ELGANs (n=16) was 92.2±1.3% (range 90.2-95.1%), which is similar to the control group (n=25). Mean levels dropped to 61.1±11.1% (range 34.2-73.2%) after a single pRBC transfusion (n=9) and to a mean of 35.6±6.3% after an additional transfusion (n=5). %HbF levels trended upwards if no additional transfusions were given, but levels still remained lower than expected for gestational age through discharge (n=85 samples). CONCLUSIONS: Percent fetal hemoglobin concentrations in ELGANs decrease precipitously after transfusion with adult donor pRBCs. Further studies are needed to evaluate the benefit of maintaining higher fetal hemoglobin concentrations in these patients and whether administration of HbF rather than adult donor pRBCs would improve patient outcomes.

IL-1ß prevents ILC2 expansion, type 2 cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice.

BACKGROUND: Early-life wheezing-associated respiratory infection with human rhinovirus (RV) is associated with asthma development. RV infection of 6-day-old immature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with the expansion of IL-13-producing type 2 innate lymphoid cells (ILC2s) and dependent on IL-25 and IL-33. We examined regulation of this asthma-like phenotype by IL-1ß. METHODS: Six-day-old wild-type or NRLP3-/- mice were inoculated with sham or RV-A1B. Selected mice were treated with IL-1 receptor antagonist (IL-1RA), anti-IL-1ß, or recombinant IL-1ß. RESULTS: Rhinovirus infection induced Il25, Il33, Il4, Il5, Il13, muc5ac, and gob5 mRNA expression, ILC2 expansion, mucus metaplasia, and airway hyperresponsiveness. RV also induced lung mRNA and protein expression of pro-IL-1ß and NLRP3 as well as cleavage of caspase-1 and pro-IL-1ß, indicating inflammasome priming and activation. Lung macrophages were a major source of IL-1ß. Inhibition of IL-1ß signaling with IL-1RA, anti-IL-1ß, or NLRP3 KO increased RV-induced type 2 cytokine immune responses, ILC2 number, and mucus metaplasia, while decreasing IL-17 mRNA expression. Treatment with IL-1ß had the opposite effect, decreasing IL-25, IL-33, and mucous metaplasia while increasing IL-17 expression. IL-1ß and IL-17 each suppressed Il25, Il33, and muc5ac mRNA expression in cultured airway epithelial cells. Finally, RV-infected 6-day-old mice showed reduced IL-1ß mRNA and protein expression compared to mature mice. CONCLUSION: Macrophage IL-1ß limits type 2 inflammation and mucous metaplasia following RV infection by suppressing epithelial cell innate cytokine expression. Reduced IL-1ß production in immature animals provides a mechanism permitting asthma development after early-life viral infection.

The Role of Iron in the Susceptibility of Neonatal Mice to Escherichia coli K1 Sepsis.

Sepsis from Escherichia coli expressing the K1 antigen is a leading cause of death in neonates. In a murine model, E. coli K1 grew rapidly in the peritoneal cavity of neonatal mice, causing fatal disease. In contrast, adult mice cleared the infection. Neonatal mice mounted a rapid and equivalent antimicrobial immune response compared to adult mice. Interestingly, peritoneal fluid from neonatal mice contained significantly more total iron than that of adult mice, which was sufficient to support enhanced E. coli growth. Transient iron overload in adult mice infected with E. coli resulted in 100% mortality. Maternal diet-induced mild iron deficiency decreased offspring peritoneal iron, decreased bacterial growth, and conferred protection against sepsis. Taken together, neonatal susceptibility to E. coli K1 sepsis is enhanced by a localized excess of peritoneal iron that allows for unchecked bacterial growth. Targeting this excess iron may provide a new therapeutic target in human patients.

Neonatal monocytes exhibit a unique histone modification landscape.

BACKGROUND: Neonates have dampened expression of pro-inflammatory cytokines and difficulty clearing pathogens. This makes them uniquely susceptible to infections, but the factors regulating neonatal-specific immune responses are poorly understood. Epigenetics, including histone modifications, can activate or silence gene transcription by modulating chromatin structure and stability without affecting the DNA sequence itself and are potentially modifiable. Histone modifications are known to regulate immune cell differentiation and function in adults but have not been well studied in neonates. RESULTS: To elucidate the role of histone modifications in neonatal immune function, we performed chromatin immunoprecipitation on mononuclear cells from 45 healthy neonates (gestational ages 23-40 weeks). As gestation approached term, there was increased activating H3K4me3 on the pro-inflammatory IL1B, IL6, IL12B, and TNF cytokine promoters (p < 0.01) with no change in repressive H3K27me3, suggesting that these promoters in preterm neonates are less open and accessible to transcription factors than in term neonates. Chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) was then performed to establish the H3K4me3, H3K9me3, H3K27me3, H3K4me1, H3K27ac, and H3K36me3 landscapes in neonatal and adult CD14+ monocytes. As development progressed from neonate to adult, monocytes lost the poised enhancer mark H3K4me1 and gained the activating mark H3K4me3, without a change in additional histone modifications. This decreased H3K4me3 abundance at immunologically important neonatal monocyte gene promoters, including CCR2, CD300C, ILF2, IL1B, and TNF was associated with reduced gene expression. CONCLUSIONS: These results provide evidence that neonatal immune cells exist in an epigenetic state that is distinctly different from adults and that this state contributes to neonatal-specific immune responses that leaves them particularly vulnerable to infections.