<i>Background:</i> Neonatal neutrophil dysfunction contributes to inflammatory tissue damage in newborn infants. Toll-like receptors (TLRs) activate the innate immune response through recognition of pathogen-associated molecular patterns. Expression and function of TLRs by neonatal neutrophils has not well been characterized. <i>Objective:</i> We hypothesized that, compared to polymorphonuclear leukocytes (PMNs) isolated from adults, neonatal PMNs isolated from either term or preterm infants express and release different levels of inflammatory cytokines and chemokines in response to stimulation with TLR1–9 agonists. <i>Methods:</i> We stimulated PMNs isolated from preterm (n = 12) and term (n = 10) infants as well as adults (n = 10) with agonists recognized by TLRs1–9 and quantified chemokine and cytokine expression and secretion by ELISA and Luminex® multiplex quantification assay. <i>Results:</i> Neonatal and adult PMNs stimulated with agonists recognized by TLRs1–9 differentially secrete inflammatory products. Signaling via TLR2 heterodimers is a potent mechanism for release of interleukin-8, a critical proinflammatory chemokine, by neonatal PMNs – a previously unrecognized facet of neonatal inflammation. Following TLR1/2 (PAM3CSK4) stimulation, interleukin-8 secretion by neonatal PMNs, whether term or preterm, substantially exceeds that of adult PMNs assayed in parallel. <i>Conclusions:</i> These studies provide new insights relevant to the inflammatory biology of neonates, both term and preterm, and implicate exaggerated PMN recruitment in neonatal syndromes of dysregulated inflammation such as necrotizing enterocolitis or neonatal chronic lung disease.
Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface-expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.
Background The neonatal myocardium is more sensitive to volatile anesthetics compared with adults. The greater myocardial sensitivity of neonates may be attributable to greater anesthetic effect on force regulation at the level of the cross-bridge. In the current study, the authors compared the effects of 1 and 2 minimum alveolar concentration (MAC) halothane and sevoflurane on cardiac muscle from 0- to 3-day-old (neonate) and 84-day-old (adult) rats. Methods Triton X-100-skinned muscle strips were maximally activated at pCa (negative logarithm of the Ca2+ concentration) of 4.0, and the following were measured in the presence or absence of anesthetic: Rate of force redevelopment after rapid shortening and restretching (ktr) and isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (alphafs) and apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp) were calculated assuming a two-state model for cross-bridge cycling. Anesthetic-induced changes in the mean stiffness per cross-bridge were also estimated from values in rigor versus maximum activation in the presence or absence of anesthetic. Results Neonatal cardiac muscle displayed significantly smaller alphafs slower ktr and slower fapp compared with adult cardiac muscle; however, gapp was not significantly different. Halothane, and sevoflurane to a significantly lesser extent, decreased alphafs, fapp, and the mean force per cross-bridge and increased gapp to a greater extent in neonates. Conclusions These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics. The authors conclude that the greater myocardial sensitivity of neonates to volatile anesthetics reflects, at least in part, a direct inhibition of cross-bridge cycling, especially the rates of cross-bridge attachment and detachment.
Background Depression of myocardial contractility as a result of isoflurane appears to be greater in myocardial hypertrophy, and the cellular basis for this difference in susceptibility is not clear. In this study we examined the effects of isoflurane and sevoflurane on contractility and intracellular calcium in an animal model of pressure-overload hypertrophy. Methods Pressure-overload hypertrophy was established in young male ferrets by banding the main pulmonary artery for 1 month and the effects of isoflurane and sevoflurane on contractility and intracellular calcium ([Ca]i) were examined in isolated right ventricular papillary muscles, trabeculae, and myocytes. Intracellular calcium was measured with the bioluminescent photoprotein aequorin in isolated papillary muscles, and also with the fluorescent indicator fluo-3 in isolated ventricular myocytes. In addition, Ca sensitivity was assessed in isolated trabeculae after disruption of the surface membrane with a nonionic detergent (skinned fibers). Results In the presence of isoflurane and sevoflurane, papillary muscles from banded animals exhibited a greater depression of contractility and isolated ventricular myocytes showed a greater decrease in peak [Ca]i. Furthermore, baseline calcium sensitivity was decreased and the slope of the relationship between [Ca] and force was increased in skinned trabeculae from banded animals. Isoflurane decreased calcium sensitivity in trabeculae from both normal and banded animals. Conclusions These results suggest that changes in [Ca]i and altered calcium sensitivity are both responsible for the exaggerated effects of some volatile anesthetics on contractility in pressure-overload hypertrophy.
Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.
Background Activated human polymorphonuclear leukocytes (PMNs) participate in the innate immune response precipitated by tissue injury or bacterial invasion. As a recently described response to bacterial invasion, PMNs secrete lattices of DNA and antimicrobial proteins termed neutrophil extracellular traps (NETs) to effect extracellular killing of bacteria. However, PMNs isolated from infants born prematurely exhibit in vitro and in vivo functional deficits in bacterial killing. Whether these deficits result from impaired NET formation in preterm PMNs remains unknown. Hypothesis We hypothesize that preterm PMNs fail to robustly form NETs following activation in vitro. Methods We therefore stimulated human PMNs isolated from adults, term infants, and preterm infants with platelet-activating factor (PAF) (10−8M), lipopolysaccharide (LPS) (100 ng/mL), or live bacteria (Escherichia coli, 0.1-10 MOI) for 30 to 120 minutes. We visualized NET formation of live and fixed PMNs stained for extracellular DNA and myeloperoxidase expression via confocal microscopy, with and without time-lapse photography. We also obtained high-resolution images of NET formation in all three cell types via scanning electron microscopy of fixed PMNs following stimulation with PAF, LPS, or E. coli. Results We visualized limited or absent NET formation via confocal microscopy at 30 and 120 minutes in LPS- and E. coli-stimulated preterm PMNs compared with robust NET formation at 30 and 120 minutes in term and adult PMNs following the same stimuli. PAF failed to induce NET formation in preterm PMNs but did induce NET formation in term and adult PMNs at 30 minutes. Time-lapse confocal microscopy allowed visualization of NET formation in term and adult PMNs but failed to show NET formation in preterm PMN preparations. Furthermore, scanning electron microscopy confirmed robust NET formation at 30 minutes in term and adult PMNs following stimulation with PAF, LPS, and E. coli but noted minimal NET formation under those conditions in preterm PMNs. Conclusion We conclude that in vitro NET formation is impaired in preterm PMNs when compared with term and adult PMNs. Speculation We speculate that impaired NET formation may contribute to bacterial killing deficits documented in preterm infants, thus contributing to the morbidity and mortality in preterm infants associated with neonatal sepsis.
Protease activated receptor (PAR) 4 (gene: F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not recognized whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice where F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared to hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P-selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of non-standard anti-platelet therapies for patients expressing PAR4 Thr120.
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