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Introduction
Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections.
Objectives
Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection.
Methods
C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining.
Results
We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen.
Conclusions
Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.
To induce an appropriate immune response, pathogen- or damage-associated molecular
patterns (PAMPs and DAMPs) are sensed by innate immune cells. One of the first
mechanisms after recognition is the assembly of an inflammasome leading to the cleavage of
pro-IL-1β and pro-IL-18 into the active cytokines IL-1β and IL-18, respectively. Hyperinflammatory
conditions and excessive activation of the inflammasome, however, are drivers
of inflammatory and autoimmune diseases. The NLR family pyrin domain-containing 3
(NLRP3) inflammasome is suggested to be involved in the development of gout,
artherosclerosis, type II diabetes, cryopyrin-associated periodic syndrome (CAPS), various
types of cancer, and inflammatory bowel disease (IBD). To tackle these diseases, drugs
interfering with IL-1 signaling are of great value. Anakinra, an IL-1 receptor antagonist,
inhibits binding of both IL-1α and IL-1β. Rilonacept consists of the Fc portion of human IgG1
with two IL-1 receptors grafted onto it. This allows neutralization of IL-1α and IL-1β. The
monoclonal IgG1 antibody Canakinumab specifically blocks IL-1β. Although well tolerated,
patients undergoing a therapy with these drugs are at a higher risk of developing bacterial
respiratory tract infections.
The human upper respiratory tract (URT) is commonly colonized asymptomatically by
bacteria like Staphylococcus aureus and Streptococcus pneumoniae. Only a limited number of
studies have so far addressed the role of the NLRP3 inflammasome during pneumococcal
infections. Imbalances in the immune system enhance the dissemination of these bacteria into
the lungs thereby causing life-threatening infections. Asides from immune-compromising
therapies such as IL-1 inhibition, viral infections are common factors leading to the
development of a secondary, bacterial infection. Influenza A virus infection of C57BL/6J
mice was characterized by a bi-phasic disease progression with a complete remission around
day 16 irrespectively of a previous Streptococcus pneumoniae 19F colonization. At this time
point, low amounts of bacteria were recovered from the lungs of 50% of co-infected mice.
This co-infection model was used for in-depth analyses of the innate immune responses.
Bioactive lipids, such as eicosanoids, are host-derived metabolites, which can heavily impact
the cellular immune response and skew it towards a pro- or anti-inflammatory phenotype.
Eicosanoid analyses revealed increased levels of different hydroxydocosahexaenoic acids
(HDHAs) and hydroxyeicosatetraenoic acids (HETEs) in the lung after single viral infection.
Staphylococcal pneumonia mainly resulted in high expression of 20-HETE. During a coinfection
with S. pneumoniae and influenza A virus (IAV), reduction of HETEs and HDHAs
was measured. Furthermore, ceramide-1-phosphate (C1P) was elevated in the lung, whereas
sphingosine-1-phosphate (S1P) was reduced. The tight control of an inflammatory response
by bioactive lipid mediators might be protective during later stages of an infection.
Cytokine analyses revealed high expression of inflammatory cytokines in lungs and plasma of
single bacteria- and virus infected as well as co-infected mice at early stages of an infection.
In all three infections monocyte chemoattractant protein-1 (MCP-1) dependent monocyte
recruitment was noted. The elevated expression of CC chemokine receptor 2 (CCR2) and
major histocompatibility complex II (MHCII) in the majority of analysed myeloid cells
indicated an activation of these cells. In single virus and co-infections, we further observed a
mainly neutrophil-independent immune response. During pneumococcal pneumonia, an influx
of neutrophils was observed.
Neutrophils, as first recruited responders during an inflammation, can release their granule
contents after activation in order to eliminate extracellular pathogens. However, excessive
neutrophil activation correlates with tissue damage and detrimental outcome of an infection.
As a result of excessive cell death, large amounts of ATP are released into the extracellular
space. Pneumolysin (Ply), a pore-forming toxin and major virulence factor of S. pneumoniae,
is a potent activator of human neutrophils. Stimulation of neutrophils with sublytic
concentrations of Ply readily resulted in the release of resistin and other granule proteins into
the supernatant. Microscale Thermophoresis (MST) analyses revealed that ATP neutralizes
Ply by binding it in the extracellular space. Co-stimulation of neutrophils with both lytic and
sublytic amounts of Ply and physiologically relevant concentrations of ATP resulted in
diminished neutrophil activation. We speculate that during hyper-inflammation (caused by
excessive neutrophil recruitment and activation), tissue injury results in the release of large
amounts of extracellular ATP which, in turn, will bind to Ply. Ultimately, binding of ATP to
Ply inhibits further neutrophil activation and might mitigate immunopathology.