Macrophages play a key role in responding to pathogens and initiate an inflammatory response to combat microbe multiplication. Deactivation of macrophages facilitates resolution of the inflammatory response. Deactivated macrophages are characterized by an immunosuppressive phenotype, but the lack of unique markers that can reliably identify these cells explains the poorly defined biological role of this macrophage subset. We identified lipocalin 2 (LCN2) as both a marker of deactivated macrophages and a macrophage deactivator. We show that LCN2 attenuated the early inflammatory response and impaired bacterial clearance, leading to impaired survival of mice suffering from pneumococcal pneumonia. LCN2 induced IL-10 formation by macrophages, skewing macrophage polarization in a STAT3-dependent manner. Pulmonary LCN2 levels were tremendously elevated during bacterial pneumonia in humans, and high LCN2 levels were indicative of a detrimental outcome from pneumonia with Gram-positive bacteria. Our data emphasize the importance of macrophage deactivation for the outcome of pneumococcal infections and highlight the role of LCN2 and IL-10 as determinants of macrophage performance in the respiratory tract.
Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.
Interactions of bacterial and host products in activating the innate immune system is an important area to address. The role of lipoteichoic acid (LTA) in these interactions is particularly important because it is understudied in comparison to other factors. This study evaluated the effect of cationic peptides (CPs) on LTA-induced proinflammatory cytokine production in human whole blood and on purified leukocytes. Four different CPs of truncated derivatives from the known peptides LL37, BPI, and CP207 were used. Two of the CPs (IG33 and LL33), derivatives from LL37, potentiated S. aureus LTA induced TNF?, IL-6 and IL-1? production in whole blood. The release of TNF? was increased 30-fold after 16 hours incubation. Intact LL37 also increased LTA-induced TNF? and IL-1? in a time dependent manner. LTA in combination with either LL33 or IG23 demonstrated a synergistic enhanced TNF? and IL-1? secretion on isolated leukocytes but not on purified monocytes. When complexed with IG23 and LL33, the electrophoretic mobility of LTA was altered in a non-denaturating gel electrophoresis. LTA was disaggregated and migrated more rapidly, suggesting an amphiphilic effect of CPs on LTA. In conclusion, LTA synergizes with LL37 and its truncated derivatives and this may lead to proinflammatory cytokine production and cause problems in sepsis therapy.
Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.
Proinflammatory cytokines are centrally involved in tumor progression and survival in non-small cell lung cancer, and both the presence of infiltrating neutrophils and bacterial infection in the lung may indicate a poor prognosis. Against this background, we investigated the effect of the bacterial cell wall component lipopolysaccharide (LPS) on interleukin (IL)-6 and IL-8 synthesis in the non-small cell lung cancer line A549 and in A549-neutrophil cocultures. The LPS induced a dose-dependent and time-dependent release of IL-8 from A549 cells, whereas IL-6 could not be detected. Interestingly, in A549-neutrophil cocultures, IL-8 synthesis was massively amplified and IL-6 was also released, compared with the respective monocultures. The A549 cells were identified as the primary cellular source of these cytokines, as enhanced cytokine mRNA transcription was detected in this cell type, although not in neutrophils in the coculture system. Experiments done in transwells indicated that direct cell-cell contact was a prerequisite for the increased cytokine generation. Inhibition of tumor necrosis factor-alpha bioactivity by neutralizing antibodies and blocking cyclooxygenase-2 activity blunted the enhanced cytokine generation in the coculture system. Amplification of LPS-induced cytokine secretion could be reproduced when the small cell lung cancer cell line H69 was cocultured with neutrophils. When the Gram-positive cell wall component lipoteichoic acid was used instead of LPS, cytokine synthesis was also amplified in A549-neutrophil cocultures, to a similar extent to that observed with LPS. These data indicate that interaction between bacterial pathogens, neutrophils, and tumor cells might amplify the release of proinflammatory cytokines which may promote tumor growth in vivo.
Plasma lipoproteins such as LDL (low-density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram-positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size-exclusion chromatography and solid-phase-binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA-induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100-directed immuno-apheresis confirmed that ApoB100 inhibited LTA-induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.