Research paper report:This paper is relatedto a study focus on Pneumococcuspneumoniae, a worldwide pathogen that infect humans in their first mountsof life.
The bacteria induce a pneumococcal pneumonia, when the infectionspread from the nasopharynx area to the deep respiratory tract and the alveoli.The first encounter with the immune system will be with the alveolarmacrophages. Because they encounter the bacteria first, they play a key role inthe activation of the innate immune response. This activation is linked to thetranscription factor nuclear factor ?B (NF- ?B). The production of NF- ?B isessential for the expression of cytokines, which will induce many innateimmunity mediators. Another study shown that mice with defection of NF- ?B inmacrophages are more likely to develop severe respiratory infection.
By thisobservation, the researcher wants to attempt to link the macrophage NF- ?Bactivation and the population of pneumococci. Furthermore, if the first link isfind, study if less triggered macrophage NF- ?B lead to a more severe infectionin the lugs. The authors decided tostudy the question because the possible outcomes could be the development of anew drug that could help the immune system to respond to an infection from Pneumococcus pneumoniae, especially toprotect children and elderly people. Firstexperiment: The researcher notice that macrophages infected withEF3030 strain were more likely to show a RelA nuclear translocation that ifthey were infected with 6303 strains.
To show that effect, the researcherinfected mice or in vitro raw alveolar macrophages with EF3030 or 6303. And toshow the RelA nuclear translocation, the cells were taken from the mice by bronchoalveolar,washed 2 hours after intratracheal instillation of EF3030 or 6303 to C57BL/6mice. In that way the NF- ?B RelA can be revealed by usingimmunohistochemistry. Same things for the raw cells in vitro, and both sampleswere observe using confocal microscopy.
A second step of that experiment were focusedon the clonal cells line from RAW294.7 cells. The aim of that study was tomodulate the concentration of 6303 and EF3030 in multiple clonal cells linesand link that concentration to the NF- ?B activation. The results support thehypothesis where a low NF- ?B activation typify a severe pneumococcalinfection. Secondexperiment: By screening pneumococcal isolates from truepositive and false positive patient, researcher found that the response frommacrophage NF- ?B activation levels were ascertain, which means that the NF- ?Btriggering is very different following the different isolates.
The results areshown by a histogram. To study this relation, the researcher recorded the NF- ?Bactivation for different serotype. They observed that the serotype made adifference between themselves, but there is a important variation among theserotype. To conclude on that experiment, the researcher established that theserotype-related capsular polysaccharide heterogeneity may be one of themultiple variables that influence macrophage NF- ?B activation. Thirdexperiment: Because macrophage NF- ?B is important for lungdefence against pneumococcus, the researcher asses the relation between thepercentage of macrophage NF- ?B triggering and the severity of pneumococcalinfection. The researcher takes out sample from different group of patients,symptomatic and asymptomatic for pneumococcal.
The area chosen were thenasopharynx, the blood, the pleural fluid or the CSF (cerebrospinal fluid). Thedata collected from the graph suggest that a low NF- ?B activation might becritical for pneumococcal survival with special settings. To compare theabilities and low NF- ?B-activating isolates of pneumococcus to survive insidethe lungs, the researcher used an animal model. By using mice, they comparedisolates which matched, but not for 2 different serotypes, 19A and 19F.
Tostudy this difference, they focused on TNF-a and CXCL2, which are macrophagederived and NF- ?B dependent during pneumococcal pneumonia. The results,presented through histograms, support the idea that avoiding or subvertingmacrophage NF- ?B activation during acute pneumonia enhances S. pneumoniae survival in the lung.Fourth experiment: To study the necrosis that occurwhen the macrophages are infected, the researcher exited RAW264.7 cells withhigh or low NF- ?B-activator S.
pneumoniae or vehicle control. The cells were put 2 hours in coculture, andthe cells with low NF- ?B activators shown an increasing of the plasma membranepermeability, as shown by pictures of the cells with a membrane-impermeable fluorescentdye. This increasing in permeability is conjunct to the cellular oncosis andexcess ROS production, which are indicative of the programmed necrosis (shownas histograms). As necroptosis is linked to the necrosome, it’s include theactivity of the kinase RIPK1. And when the activity of RIPK1 were inhibited inlow concentration of NF- ?B, the data show that the percentage of cellular oncosisad of high oxidative activity, link to the ROS production, decreasesignificantly. These result support the concept that the altered response ofthe macrophage by low NF- ?B activators is related to the pneumococcalinduction of the programmed necrosis.
Fifthexperiment: NF- ?B activity and necroptosis are correlated, and toincrease the necroptosis within the macrophages responding to pneumococci, researchersinhibited NF- ?B. the BAY 11-7082 is an inhibitor of NF- ?B and has been usedin this study. The data, presented through a histogram, show that the inhibitorincreased necroptosis among RAW264.7 cells, while they were responding to ahigh NF- ?B activator pneumococcus, and same thing for the number of cellularoncosis. These results suggest that stimulating NF- ?B activity helps to avoidthe triggering of programmed necroptosis among macrophages. Sixth experiment: The studies show that macrophages can have two faiths: NF- ?B-mediatedgene expression or necroptosis. The researchers designed a series of costimulationand coinfection to see if it’s possible to reverse a necroptosis and a lunginfection by the presence of high NF- ?B activators. Through histograms, theresult show that presence of high NF- ?B-activating pneumococcus (EF3030) in aninfection made by a low NF- ?B-activating pneumococcus (6303) prevented theeffect of 6303.
In vitro, these data show that the adding of high NF- ?B-activatingpneumococcus in an infection provoke by a low NF- ?B-activating pneumococcusallow to stimulate a pathway that overcome the necroptosis pathways. The sameobservation was possible in vivo, where the induction of EF3030 enhance the immuneresponse from a host that was experimenting a severe infection from 6303. Theseresults support the concept that the activation of the appropriate macrophagesincluding NF- ?B RelA enhance the elimination of a high necroptosis-inducingisolate of S.
pneumoniae while a lunginfection. Those results, allcumulate, show a new way to fight severe infection. Not by using drug andvaccine, the use of pathogens can trigger and enhance the immune system when heis disrupted by a severe pathogen which target him. The researcher noted that,while they were studying the different serotype of pneumococci, more isolateswere low activators than they expected. For further studies, theresearcher suggests that new therapy, as immunostimulatory, would be possible totreat pneumonia, for drug-resistant bacteria.