Priming Of Alveolar Macrophages Research Articles

SARS‐CoV‐2 evades immune detection in alveolar macrophages

Respiratory infections, like the current COVID‐19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue‐resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS‐CoV‐2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non‐infectious donors are challenged with SARS‐CoV‐2. We demonstrate that challenged AMs are incapable of sensing SARS‐CoV‐2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS‐CoV‐2 could explain the initial asymptotic phase observed during COVID‐19 and argues against AMs being the sources of pro‐inflammatory cytokines later during infection.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due toenhanced bacterial clearance, which was causedby IFNɣ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

Role of K+ Channels in Alveolar Macrophages-Mediated Inflammatory Response upon Anthrax Lethal Toxin Stimulation

The causes of rapid death in anthrax infection both in animals and humans are unknown and concern over the use for biological warfare has renewed interest in elucidating the mechanisms of anthrax induced inflammation. Interleukin-1β (IL-1β) secretion is an important inflammatory response against anthrax lethal toxin (LeTx) a virulence factor of Bacillus anthracis. Here, we report that LeTx induces a significant increase in inwardly-rectifying K+ (Kir) and voltage-gated K+ (Kv) currents in mouse and human macrophages. Furthermore, we also show that blocking either Kir or Kv channels significantly inhibits LeTx-induced IL-1β secretion suggesting that activation of macrophage K+ channels plays an important role in LeTx-induced inflammatory response. In addition, we also investigated the role of macrophage K+ channels in macrophage priming, a well-known macrophage infection model involving pre-exposure of the cells to a low level of antigen that augments the response to subsequent challenge. Specifically, priming of alveolar macrophages by either Lipopolysaccharides (LPS), an endotoxin of all gram-negative bacteria, or Bacillus spores, augments inflammatory response upon LeTx stimulation as compared to unprimed cells challenged with LeTx alone. Our study shows that pre-exposure to low levels of LPS or to the spores also significantly augments LeTx-induced activation of macrophage K+ channels suggesting that activation of K+ channels might be part of the priming mechanism.

Priming of Alveolar Macrophages upon Instillation of Lipopolysaccharide in the Human Lung

The airways are continuously exposed to respiratory pathogens, which may result in bacterial pneumonia, one of the most common infectious diseases and the leading cause of sepsis. Considering that recurrent exposure to microbial products can lead to tolerance of immune cells, and that this might contribute to the susceptibility to nosocomial infection, we investigated the effect of in vivo lipopolysaccharide (LPS) instillation on the responsiveness of alveolar macrophages. In eight healthy humans, sterile saline was instilled into a lung segment by bronchoscope, followed by instillation of LPS into the contralateral lung; 6 hours later, a bilateral bronchoalveolar lavage was performed, and purified alveolar macrophages were ex vivo stimulated with LPS or lipoteichoic acid (LTA), triggering Toll-like receptor (TLR)-4 and -2, respectively. In vivo LPS-exposed alveolar macrophages were primed, as reflected by increased ex vivo LPS- and LTA-induced IL-1 beta and IL-6 gene expression and production compared with in vivo saline-exposed alveolar macrophages. LPS instillation did not influence the surface expression of TLR4 or TLR2. Furthermore, LPS instillation did not impact on the expression of a number of extracellular and intracellular regulators of TLR signaling. However, p38 mitogen-activated protein kinase remained phosphorylated in alveolar macrophages upon LPS instillation. The current data demonstrate that LPS instillation in the human lung primes alveolar macrophages for further stimulation with either LPS or LTA, possibly by sustained p38 mitogen-activated protein kinase activation.

DO CIRCULATING MONOCYTES UNDERGO FUNCTIONAL CHANGES FOLLOWING BLUNT CHEST TRAUMA?

We have previously demonstrated that alveolar macrophages (AM) are primed to release cytokines following lung contusion. So far, it has not been elucidated whether circulating monocytes (CM) undergo functional changes as well and whether posttraumatic alterations are limited to the bronchoalveolar space. We therefore sought to clarify (I) the function of CM following lung contusion and (II) the release of mediators - which predominately act on macrophages into the bronchoalveolar lavage (BAL) and blood plasma. Methods: Wistar rats were randomly allotted to 4 groups, n = 8 each. Three groups were subjected to a blast wave injury and sacrificed 10 min (TX-10 m), 6 hours (TX-6 h), and 24 hours (TX-24 h) after the trauma; another group served as control. The CM were isolated and cultured for 24 hours in the presence of 1000 ng LPS /ml. The release of TNF-α, IL-1β, MIP-2, and IL-10 was determined (ELISA) in the supernatant. In a second series, the concentrations of IL-1β, MIP-2, IL-10, and PGE2 were analyzed in the BAL and blood plasma. Results: Blunt chest trauma resulted in an excessive mediator release into the bronchoalveolar space. As soon as 10 min following trauma there was an increase (P < 0.05) of IL-10 and PGE2 in trauma animals which returned to control levels at TX-24 h. The mediator release into BAL showed a delayed kinetic with a twofold increase of IL-1β and MIP-2 (P < 0.05) in TX-24 h animals compared to controls. In sharp contrast there was no difference in the mediator concentration in the blood plasma between trauma or control animals. In agreement with these findings, there were no differences between CM of trauma and control animals with regard to the release of pro-or anti-inflammatory cytokines into the supernatant. Conclusion: The priming of AM but not CM and the excessive release of mediators which are produced by and act upon macrophages into the BAL suggests autocrine mechanism in the activation macrophages following blunt chest trauma.

Stilbazulenyl nitrone (STAZN), a novel azulenyl nitrone antioxidant prevents lung injury following hemorrhagic shock and alters TLR4 distribution in alveolar macrophages

Introduction: Oxidants (OX) produced during shock/resuscitation (SR) contribute to organ failure by priming alveolar macrophages (AM) for increased responsiveness to subsequent inflammatory stimuli such as LPS. STAZN is a second-generation azulenyl nitrone – a potent antioxidant. Since our initial studies showed that OX/SR induces priming of AM by colocalization of LPS receptor - TLR4 within cell signaling domains – the lipid rafts (LR), we hypothesize that STAZN can protect lungs from injury by preventing macrophage LR recruitment of TLR4. Methods: Rats were bled to MAP of 40mmHg, 1h later resuscitated with blood and Ringer’s Lactate (RL) plus intraperitoneal STAZN (30 mg/kg × 2 doses) or vehicle (dimethyl sulfoxide). 1hr post resuscitation intratracheal LPS (3ug/10gm) or saline (SAL) were administered and 4hrs later bronchoalveolar lavage (BAL) fluid was used for differential cell counts. Alternatively, BAL AM were recovered 1hr after resuscitation, LRs were isolated by sucrose gradient ultracentrifugation and presence of TLR4 or GM-1 (LR marker) were detected by dot blotting. Results: Treatment with STAZN and not vehicle alone significantly attenuated LPS induced lung neutrophilia following RL resuscitation by 67% (n = 3, p<0.05). Shock and RL resuscitation induced TLR4 migration into the AM lipid raft fractions (marked by presence of GM-1), an effect inhibited by STAZN. Conclusions: Therapy with STAZN is effective at attenuating lung injury after shock resuscitation through preventing AM lipid raft recruitment of the LPS receptor. Azulenyl nitrones represent a novel and promising class of potent antioxidants for combating organ failure following trauma.

Pulmonary surfactant phospholipids modulate priming of rabbit alveolar macrophages for oxidative responses.

We investigated the effect of individual phospholipids contained in pulmonary surfactant (PS) on the macrophage-activating factor (MAF)-induced priming of rabbit alveolar macrophages (AMs) for oxidative responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). AMs were incubated with MAF with or without phospholipids for 18 h. After incubation, oxidative responses were elicited with PMA (0.5 micrograms/ml) or Op-Zym (250 micrograms/ml) and monitored by chemiluminescence (CL) assays. The data indicate that natural surfactant inhibited MAF-induced priming of rabbit AMs for PMA- or Op-Zym-elicited oxidative responses. Artificial surfactant inhibited PMA-elicited CL responses but enhanced Op-Zym-elicited CL responses. Individual phospholipids differed in modulative activities. Dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), and phosphatidylinositol (PI) inhibited MAF-induced priming when the oxidative responses were elicited with PMA. Whereas DPPG inhibited Op-Zym-elicited oxidative responses, dipalmitoyl phosphatidylcholine (DPPC) and DOPC primed AMs for increased Op-Zym-elicited oxidative responses. DOPC did not affect the binding of phorbol dibutyrate to AMs, which suggests that reduced cell binding of phorbol ester was not responsible for the inhibition of PMA-elicited oxidative responses in AMs treated with DOPC. Similarly, DPPC, DOPC, and DPPG did not affect the number of zymosan particles phagocytosed by AMs compared to the control, which suggested that enhanced or reduced Op-Zym-elicited oxidative responses by phospholipids were not due to altered phagocytic activity of AMs. In conclusion, our data indicate that individual surfactant phospholipid differently modulates priming of AMs for oxidative responses, and the effect of individual phospholipids does not account for the effect of complete PS on priming of AMs.

Pulmonary surfactant inhibits priming of rabbit alveolar macrophage. Evidence that surfactant suppresses the oxidative burst of alveolar macrophage in infant rabbits.

We have investigated the possible role of pulmonary surfactant in mediating a deficiency in alveolar macrophages (AM) from infant animals to produce chemiluminescent (CL) responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). Freshly harvested AM from rabbits 7 to 28 days of age produced much reduced PMA-elicited CL responses compared to 5- to 6-month-old rabbits. Initially we observed that AM from both infant and adult rabbits exhibited increasing PMA- or Op-Zym-elicited CL responses in a time-dependent manner when they were incubated in RPMI 1640 medium without serum for more than 3 h. In addition, AM from infant rabbits acquired the capacity to become primed with macrophage activation factor (MAF) containing supernatants or fetal bovine serum (FBS) during an 18-h incubation period that was basically equivalent to that of AM from adult rabbits. Our studies reveal that natural and artificial surfactant inhibited "spontaneous" priming as well as priming of AM induced by MAF-containing supernatant or FBS for producing PMA- or OP-Zym-elicited CL responses from both normal infant and adult animals. In addition, these surfactant preparations also suppressed the response of AM from adult BCG-sensitized rabbits during the course of an 18-h incubation period. It is especially noteworthy that AM from infant rabbits appeared to be more susceptible to the inhibitory activity of surfactant than AM from adult animals. Our data indicate that freshly harvested AM from infant rabbits are under greater suppression than AM from adult rabbits in terms of their capacities to produce oxidative responses.(ABSTRACT TRUNCATED AT 250 WORDS)