The Inflammatory Cytokines From Macrophages Downregulated By Group A Streptococcus

Objective: The bacterial pathogen group A Streptococcus (GAS; Streptococcus pyogenes) causes many distinct human diseases. The most common GAS infections are those of the upper respiratory tract, leading to development of pharyngitis (strep throat). Although pharyngeal infections are self-limiting, postinfection sequelae such as acute glomerular nephritis, acute rheumatic fever and rheumatic heart disease may develop if left untreated. Occasionally, GAS can cause severe invasive diseases, such as streptococcal toxic shock syndrome, and necrotizing fasciitis. The severity and outcome of the infections caused by S. pyogenes are likely to depend on the ability of host innate immune mechanisms to control bacterial growth and to limit further spread of the pathogen beyond the site of infection. Macrophages have a central role in initiating the innate immune response, which leads to activation of the adaptive response in the later phase. Moreover, previous studies examining host responses to S. pyogenes in a mouse model of infection also have shown the importance of resident macrophages for controlling infection. Macrophages are capable of recognizing, phagocytosing, and destroying S. pyogenes in order to eliminate the invading pathogen, while also producing cytokines and chemokines that are crucial in controlling the recruitment and activation of inflammatory cells at the site of infection. Therefore, the functional activities of macrophages during S. pyogenes infection may greatly influence the character, course, and outcome of the pathogenic process.However, GAS has been evolved multiple strategies to overcome host immune defences. A common observation of severe necrotizing GAS infections is the paucity of front-line defence cells at the site of infection. In the vivo infectional model, it is well-known that the GAS M protein is one powerful inducer of inflammation. M protein and other virulence factors have been shown to trigger the release of the cytokines including IL-6, IL-1β, tumor necrosis factor alpha (TNF-α), and transforming growth factorβ(TGF-β) following recognition by pattern recognized receptors (PRRs) on the surface of monocytes. It should be noted that cytokine release not only be able to induce inflammation, but also may influence mechanisms such as streptococcal invasion, for example, GAS-mediated TGF-βrelease was found to upregulate integrinα5 expression, namely, GAS is able to manipulate host cytokine production to increase levels of expression of target receptors and so promote its own adherence and internalization. Aside from the described above, GAS induces apoptosis of macrophages by inducing cytokine release such as the ligation of cell-death receptors on the cell surface. The ability to induce apoptosis or cell death may be beneficial to bacteria, enabling escape from phagocytic activity or damaging host tissues and so promoting deeper penetration and dissemination.This study aims to further improve the understanding of the complex response of macrophages to GAS thereby contributing to adaptive fitness and persisting infections, to further investiage the evasion strategy of GAS in mouse mcrophage, and to further explore the pathogenic mechanism of GAS. For this purpose, we are going to perform these experiments: 1. The global inflammatory molecules expression spectrum of murine macrophages cells were analyzed after in vivo/ vitro infection with GAS by protein array technology,and the comparative kinetics of the inflammatory molecules were verified by Real-time PCR, Escherichia coli (E.coli) and staphylococci aureus (SA) as G- and G+ pathogen-infected Mcrophage control, respectively. 2. The dynamic change of IL-6, the mediator of the inflammatory network, was detcted by in-cell western and ELISA, and the activiation of IL-6-specific nuclear factor C/EBPβwas measured by western blot. 3. TLRs, the adapter MyD88 and NF-κB of murine mcrophage following infection with bacteria were detected by flow cytometry experiment, Real-time PCR and Western blot to get some ideas on the mcrophage signaling transduction after sitimulation by bacteria. 4. Animal assay were performed to verify whether the results from anmals were consisted with those from cells in vitro. We hope this research could provide a novel sight in the study of pathogenesis and immunotherapy of GAS.Methods:1 The global inflammatory molecules expression spectrum of murine peritoneal macrophages and mouse mcrophage RAW264.7 cells were analyzed after in vivo/ vitro infection with GAS by protein array technology,E.coli and SA as G- pathogens control and G+ pathogens control, respectively.2 The comparative kinetics of the pro-inflammary cytokines (such as IL-6,IL-1β,TNF-α) and the chemokines (such as MCP-1,Rantes,GRO-α) produced by murine peritoneal macrophages and mouse mcrophage RAW264.7 cells following infections with GAS/SA/E.coli were identified by Real-time PCR.3 The kinetic analysis of IL-6, the major pro-inflammary cytokine, produced by mouse mcrophage RAW264.7 cells following infections with GAS/SA/E.coli were measured by ELISA and in-cell Western.4 The TIR-domain-containing adapter MyD88 of murine peritoneal macrophages and mouse mcrophage RAW264.7 cells following infections with GAS/SA/E.coli was identified by Real-time PCR.5 The difference in the transcription factor NF-kB translocation of mouse RAW264.7 cells following infections with GAS/SA/E.coli were detected by Western blot and EMSA.6 The transcription factor C/EBPβexpression of mouse RAW264.7 cells following infections with GAS/SA/E.coli were detected by Western blot.Results:1 The data from protein array technology show that: the highest one of inflammatory molecules, compared with normal control, secreted by RAW264.7 cells with GAS/SA/E.coli infections at the day 3 was TNF-α(Ratio:6.07). BLC(Ratio:4.2),IL-1α(Ratio:2.85),Rantes(Ratio:2.68),MCP-1(2.17),LIX (Ratio:1.98),G-CSF(Ratio:1.88),IL-6(Ratio:1.71),M-CSF(Ratio:1.59),and IL-1β(Ratio:1.55)were ordinally decreased. The highest one screted by murine peritoneal macrophages after infections with the above bacteria at the day 3 was BLC(5.0), KC(Ratio:4.49),IL-1α(Ratio:4.3),TNF-α(2.49),LIX(Ratio:2.25),M-CSF(1.93),IL-1β(1.69),IL-6(1.5)were ordinally decreased.2 The comparative kinetics of the cytokines produced by mcrophages following infections with GAS/SA/E.coli by Real-time PCR show that the major moleculars expressed by RAW264.7 cells within 2 hours were GRO-α,IL-1βand TNF-α, after 2 hours the major secreted moleculars were IL-1β,IL-6,IL-12,MCP-1 and IL-10. the major moleculars expressed by murine peritoneal macrophages within 2 hours were GRO-α,IL-23,TNF-α,Rantes, after 2 hours the major secreted moleculars were GRO-α,IL-1β,IL-12,MCP-1,MIP-1α.3 The levels of the cytokines secreted not only by RAW264.7 cells but also by murine peritoneal macrophages with GAS infections were lower than those secreted by the two type macrophages with SA or E.coli infections, except for IL-10 and TGF-β.4 The expressions of TLRs:The results from FCM or Real-time PCR show that: the levels of TLR2 on both RAW264.7 cells and murine peritoneal macrophages with GAS or SA infection were higher than that on the cells infected with E.coli or that on the normal control, while although the level of TLR2 on those cells with E.coli was slight higher than that of normal control, the difference is not significant. The levels of TLR4 on both RAW264.7 cells and murine peritoneal macrophages with GAS or SA or E.coli infections were higher than that of normal control; however, the differece in the three groups was not significant.5 The mRNA levels of MyD88 in the murine peritoneal mcrophages following GAS or SA or E.coli infections within 2 hours were higher than that of normal control, while the differece in the three groups had no significant difference, although the level of MyD88 in the GAS-, SA- or E.coli- infected RAW264.7 cells had no significant difference from normal control.6 NF-κB in the nucleus of RAW264.7 cells with GAS infection within 2 days was progressively increased, and that of RAW264.7 cells with SA infection was kept in a high level, but that of the cells with E.coli infection was kept in a low lever, and became gradually less by western blot analysis. EMSA showed that: NF-κB in the nucleus of RAW264.7 cells reached to the highest level at the first day after infection of GAS, and about 2h~1d after the cells infected with SA, NF-κB in the nucleus kept in a high level, especially at 6h, while NF-κB in the nucleus of the cells after infection with E.coli kept in a low level, only at 2h~6h after infection a few NF-κB was discoverable. These results were consisted with those detected by western blot.The level of C/EBPβin the nucleus of RAW264.7 cells after infections with GAS or SA was higher than that infected with E.coli or normal control, which was consistent with the level of IL-6 secreted by the cells with G+ pathogens infections (including GAS and SA) by the above variou detection methods.Conclusions:1 GAS/SA/E.coli induces RAW264.7 cells and murine peritoneal microphages to produce various degrees of inflammatory response. Macrophages, in the early stage of infections, produce flammatory moleculars like GRO-α, IL-1βand TNF-α, which mainly recruit neutrophil and induce acute phase protein, and, at the third day after infections, produce flammatory moleculars like IL-1β,IL-6,IL-12,MCP-1 which chiefly induce and sustain the flammatory response.2 GAS elicits macrophage to produce a retarded and mild inflammatory cytokine response in vivo or in vitro experiments. S. aureus elicits RAW264.7 cells to trigger a retarded but stronger inflammtory cytokine response in vitro, while elicits murine peritoneal macrophage to produce a weaker inflammatory cytokine response than in RAW264.7 cells. E. coli causes a faster and stronger inflammatory cytokine response not only in vitro but also in vivo, especially in vivo.3 The macrophage-inflammatory cytokine response induced by GAS/SA/E.coli is related with TLR2 or TLR4. E.coli induces macrophage high-expressing TLR4, while GAS and SA induce macrophage high-expressing both TLR2 and TLR4.4 Adapter MyD88 is involved in the signaling transduction of pathogen-infected macrophage, but there is no necessary relation between fast or slow and strong or week of the inflammatory reaction induced by GAS/SA/E.coli. There may be other adapters taken part in the signaling transduction to lead to the difference between when and how many NF-kB entering into nucleus, which - in part - might explain the different responses induced by GAS and SA; while the inflammatory genes of E.coli-infected mcrophages were promoted not chiefly by the activation of NF-kB.