Ketamine affects in vitro differentiation of monocyte into immature dendritic cells.

BACKGROUND: Monocytes (MOs) have the unique ability to differentiate into immature dendritic cells (iDCs) (MO→iDC) under the influence of interleukin-4 and granulocyte-monocyte colony-stimulating factor (IL-4&GM-CSF). In this study, the authors investigated the influence of ketamine on the process of MO→iDC. METHODS: iDCs were cultured from MO obtained from 36 subjects in the presence of IL-4 and GM-CSF and ketamine at 100, 10, and 1 µg/ml for 5 days. In some of the experiments, the authors used nonspecific N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, NMDA, or a neutralizing antibody for transforming growth factor ß (TGFß). The expression of surface markers and functional assays were used to assess the effect of ketamine on IL-4&GM-CSF-stimulated MO. IL-4&GM-CSF-stimulated MO's supernatants were assessed for cytokine levels. RESULTS: Ketamine at 10 µg/ml, and higher concentrations, diminished the expression of CD1a on IL-4&GM-CSF-stimulated MO and retarded both their ability to process DQ ovalbumin and mixed lymphocyte reaction stimulation. The addition of ketamine to IL-4&GM-CSF-differentiated MO resulted in the persistent expression of CD14 and unchanged expression of CD86 and CD206. The phagocytic abilities of IL-4&GM-CSF-differentiated MO were not changed by ketamine. MK-801, a nonselective NMDA agonist, mimicked ketamine's effect on MO→iDC differentiation. Adding exogenous NMDA to IL-4&GM-CSF-stimulated MO in the presence of ketamine partially restored the level of CD1a. TGFß was elevated in supernatants of IL-4&GM-CSF-stimulated MO in the presence of ketamine. Adding neutralizing TGFß antibody or TGFßR1 blocker (SB431542) resulted in the full recovery of MO→iDC, despite the presence of ketamine. CONCLUSIONS: Ketamine diminishes the process of MO→iDC in vitro. This is mediated via NMDA-dependent mechanisms and TGFß.

IL-18R1 and IL-18RAP SNPs may be associated with bronchopulmonary dysplasia in African-American infants.

INTRODUCTION: The genetic contribution to the development of bronchopulmonary dysplasia (BPD) in prematurely born infants is substantial, but information related to the specific genes involved is lacking. RESULTS: Genotype analysis revealed, after multiple comparisons correction, two significant single-nucleotide polymorphism (SNPs), rs3771150 (IL-18RAP) and rs3771171 (IL-18R1), in African Americans (AAs) with BPD (vs. AAs without BPD; q < 0.05). No associations with Caucasian (CA) BPD, AA or CA respiratory distress syndrome (RDS), or prematurity in either AAs or CAs were identified with these SNPs. Respective frequencies were 0.098 and 0.093 in infants without BPD and 0.38 for each SNP in infants with BPD. In the replication set (82 cases; 102 controls), the P values were 0.012 for rs3771150 and 0.07 for rs3771171. Combining P values using Fisher's method, overall P values were 8.31 × 10(-7) for rs3771150 and 6.33 × 10(-6) for rs3771171. DISCUSSION: We conclude that IL-18RAP and IL-18R1 SNPs identify AA infants at risk for BPD. These genes may contribute to AA BPD pathogenesis via inflammatory-mediated processes and require further study. METHODS: We conducted a case-control SNP association study of candidate genes (n = 601) or 6,324 SNPs in 1,091 prematurely born infants with gestational age <35 weeks, with or without neonatal lung disease including BPD. BPD was defined as a need for oxygen at 28 days.

Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation.

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.

Loss of circadian protection against influenza infection in adult mice exposed to hyperoxia as neonates.

Adverse early-life exposures have a lasting negative impact on health. Neonatal hyperoxia that is a risk factor for bronchopulmonary dysplasia confers susceptibility to influenza A virus (IAV) infection later in life. Given our previous findings that the circadian clock protects against IAV, we asked if the long-term impact of neonatal hyperoxia vis-à-vis IAV infection includes circadian disruption. Here, we show that neonatal hyperoxia abolishes the clock-mediated time of day protection from IAV in mice, independent of viral burden through host tolerance pathways. We discovered that the lung intrinsic clock (and not the central or immune clocks) mediated this dysregulation. Loss of circadian protein, Bmal1, in alveolar type 2 (AT2) cells recapitulates the increased mortality, loss of temporal gating, and other key features of hyperoxia-exposed animals. Our data suggest a novel role for the circadian clock in AT2 cells in mediating long-term effects of early-life exposures to the lungs.

Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice.

Inflammation plays a critical role in the development of severe neonatal morbidities. Myeloid-derived suppressor cells (MDSCs) were recently implicated in the regulation of immune responses in newborns. Here, we report that the presence of MDSCs and their functional activity in infants are closely associated with the maturity of newborns and the presence of lactoferrin (LF) in serum. Low amounts of MDSCs at birth predicted the development of severe pathology in preterm infants - necrotizing enterocolitis (NEC). In vitro treatment of newborn neutrophils and monocytes with LF converted these cells to MDSCs via the LRP2 receptor and activation of the NF-κB transcription factor. Decrease in the expression of LRP2 was responsible for the loss of sensitivity of adult myeloid cells to LF. LF-induced MDSCs (LF-MDSCs) were effective in the treatment of newborn mice with NEC, acting by blocking inflammation, resulting in increased survival. LF-MDSCs were more effective than treatment with LF protein alone. In addition to affecting NEC, LF-MDSCs demonstrated potent ability to control ovalbumin-induced (OVA-induced) lung inflammation, dextran sulfate sodium-induced (DSS-induced) colitis, and concanavalin A-induced (ConA-induced) hepatitis. These results suggest that cell therapy with LF-MDSCs may provide potent therapeutic benefits in infants with various pathological conditions associated with dysregulated inflammation.

Long-term Monocyte Dysfunction after Sepsis in Humanized Mice Is Related to Persisted Activation of Macrophage-Colony Stimulation Factor (M-CSF) and Demethylation of PU.1, and It Can Be Reversed by Blocking M-CSF In Vitro or by Transplanting Naïve Autologous Stem Cells In Vivo.

The duration of post-sepsis long-term immune suppression is poorly understood. Here, we focused on the role of monocytes (MO) as the pivotal cells for long-term regulation of post-sepsis milieu. Lost ability of MO to adapt is seen in several acute conditions, but it is unclear for how long MO aberrancy post-sepsis can persist. Interestingly, the positive feedback loop sustaining secretion of macrophage-colony stimulation factor (M-CSF) can persist even after resolution of sepsis and significantly alters performance of MO. Here, we investigated the activation of M-CSF, and it as critical regulator of PU.1 in mice surviving 28 days after sepsis. Our primary readout was the ability of MO to differentiate into dendritic cells (DCs; MO→iDC) in vitro since this is one of the critical processes regulating a successful transition from innate to acquired immunity. We utilized a survival modification of the cecal ligation and puncture (CLP) model of sepsis in humanized mice. Animals were sacrificed 28 days after CLP (tCLP+28d). Untouched (CONTR) or sham-operated (SHAM) animals served as controls. Some animals received rescue from stem cells originally used for grafting 2 weeks after CLP. We found profound decrease of MO→iDC in the humanized mice 28 days after sepsis, demonstrated by depressed expression of CD1a, CD83, and CD209, diminished production of IL-12p70, and depressed ability to stimulate T cells in mice after CLP as compared to SHAM or CONTR. In vitro defect in MO→iDC was accompanied by in vivo decrease of BDCA-3+ endogenous circulating DC. Interestingly, post-CLP MO had persistent activation of M-CSF pathway, shown by exaggerated secretion of M-CSF, activation of PU.1, and demethylation of SPII. Neutralization of the M-CSF in vitro reversed the post-CLP MO→iDC aberration. Furthermore, transplantation of naïve, autologous stem cell-derived MO restored CLP-deteriorated ability of MO to become DC, measured as recovery of CD1a expression, enhanced production of IL-12p70, and ability of IL-4 and GM-CSF MO to stimulate allogeneic T cells. Our results suggest the role of epigenetic mediated M-CSF aberration in mediating post-sepsis immune system recovery.

The clinical and immunological performance of 28 days survival model of cecal ligation and puncture in humanized mice.

Sepsis triggers a coordinated and thorough immune system response with long-term unfavorable sequelae after the initial insult. Long-term recovery from sepsis has garnered increasing attention recently, but a lack of suitable animal models impairs progress in this area. Our study, therefore, aimed to address the performance of the immune system in a survivable model of sepsis (cecal ligation and sepsis; CLP) for up to 28 d after the initial injury in humanized mice. Our model mimics human sepsis with weight loss and post-sepsis hypothermia. Within the first 7 d of sepsis, the M1 inflammatory cell subtype predominated, as evidenced by increased CD16 expression, but at 28 d, a mixed population of M1 and M2 inflammatory cells emerged, as evidenced by increased secretion of transforming growth factor TGFß and CD206 expression. This change was accompanied by normalized production of interleukin (IL)-6, tumor necrosis factor TNFα and IL-10 at 28 d. Furthermore, the ability of MO to become regulatory DC or the frequency of endogenous DC were severely affected at 28 days. Thus, sepsis results in profound and persistent changes in the function of myeloid cells up to 28 days after CLP demonstrating the persistence of the new acquired immunological features long after resolution of the sepsis.