NLRP6 Serves as a Negative Regulator of Neutrophil Recruitment and Function During Streptococcus pneumoniae Infection

Streptococcus pneumoniae is an invasive pathogen with high morbidity and mortality in the immunocompromised children and elderly. NOD-like receptor family pyrin domain containing 6 (NLRP6) plays an important role in the host innate immune response against pathogen infections. Our previous studies have shown that NLRP6 plays a negative regulatory role in host defense against S. pneumoniae, but the underlying mechanism is still unclear. The further negative regulatory role of NLRP6 in the host was investigated in this study. Our results showed that NLRP6(-/-) mice in the lung had lower bacterial burdens after S. pneumoniae infection and expressed higher level of tight junction (TJ) protein occludin compared to WT mice, indicating the detrimental role of NLRP6 in the host defense against S. pneumoniae infection. Transcriptome analysis showed that genes related to leukocytes migration and recruitment were differentially expressed between wild-type (WT) and NLRP6 knockout (NLRP6(-/-)) mice during S. pneumoniae infection. Also, NLRP6(-/-) mice showed higher expression of chemokines including C-X-C motif chemokine ligand 1 (CXCL1) and 2 (CXCL2) and lower gene expression of complement C3a receptor 1 (C3aR1) and P-selectin glycoprotein ligand-1 (PSGL-1) which are the factors that inhibit the recruitment of neutrophils. Furthermore, NLRP6(-/-) neutrophils showed increased intracellular bactericidal ability and the formation of neutrophil extracellular traps (NETs) during S. pneumoniae infection. Taken together, our study suggests that NLRP6 is a negative regulator of neutrophil recruitment and function during S. pneumoniae infection. Our study provides a new insight to develop novel strategies to treat invasive pneumococcal infection

Impact of CRAMP-34 on Pseudomonas aeruginosa biofilms and extracellular metabolites

Biofilm is a structured community of bacteria encased within a self-produced extracellular matrix. When bacteria form biofilms, they undergo a phenotypic shift that enhances their resistance to antimicrobial agents. Consequently, inducing the transition of biofilm bacteria to the planktonic state may offer a viable approach for addressing infections associated with biofilms. Our previous study has shown that the mouse antimicrobial peptide CRAMP-34 can disperse Pseudomonas aeruginosa (P. aeruginosa) biofilm, and the potential mechanism of CRAMP-34 eradicate P. aeruginosa biofilms was also investigated by combined omics. However, changes in bacterial extracellular metabolism have not been identified. To further explore the mechanism by which CRAMP-34 disperses biofilm, this study analyzed its effects on the extracellular metabolites of biofilm cells via metabolomics. The results demonstrated that a total of 258 significantly different metabolites were detected in the untargeted metabolomics, of which 73 were downregulated and 185 were upregulated. Pathway enrichment analysis of differential metabolites revealed that metabolic pathways are mainly related to the biosynthesis and metabolism of amino acids, and it also suggested that CRAMP-34 may alter the sensitivity of biofilm bacteria to antibiotics. Subsequently, it was confirmed that the combination of CRAMP-34 with vancomycin and colistin had a synergistic effect on dispersed cells. These results, along with our previous findings, suggest that CRAMP-34 may promote the transition of PAO1 bacteria from the biofilm state to the planktonic state by upregulating the extracellular glutamate and succinate metabolism and eventually leading to the dispersal of biofilm. In addition, increased extracellular metabolites of myoinositol, palmitic acid and oleic acid may enhance the susceptibility of the dispersed bacteria to the antibiotics colistin and vancomycin. CRAMP-34 also delayed the development of bacterial resistance to colistin and ciprofloxacin. These results suggest the promising development of CRAMP-34 in combination with antibiotics as a potential candidate to provide a novel therapeutic approach for the prevention and treatment of biofilm-associated infections

Chicken respiratory infections: Protective roles of macrophages and cathelicidins

Avian pathogenic E. coli (APEC) is one of most prevalent pathogens causing systemic diseases called avian colibacillosis. During APEC infection, epithelial cells in the lung are the first cellular defense line against APEC infection and will produce cytokines and chemokines to initiate the immune response. Similarly, macrophages and heterophils will be quickly recruited to the site of infection to fight against the infection in the respiratory tract. Nevertheless, the exact role of these cells in controlling APEC infection is still unclear and therefore investigated in the studies described in this thesis. So far, the treatment of an APEC infection in chicken mainly depends on antibiotics, but increasing antibiotic resistance makes this less effective. Therefore, new anti-infectives are needed and host defense peptides (HDPs) such as cathelicidins are considered as a promising alternatives. In this thesis, we investigated the interaction of chicken macrophages and epithelial cells with avian pathogenic E.coli to establish an appropriate APEC infection model in vitro. Then, we investigated the immunomodulatory effect of CATH-B1 on the chicken macrophages. Furthermore, antiviral activity of chicken cathelicidins against influenza A virus was studied. In chapter 2, the interactions of chicken lung epithelial cells with APEC is described to understand the role of epithelial cells in APEC infections. The results showed APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Moreover, APEC infection resulted in a significant increase in IL-8 gene expression. In chapter 3, the interactions of macrophage HD11 cells with APEC is described to understand the role of macrophages in controlling APEC. The results showed APEC was phagocytosed and subsequenlt killed by HD11 cells, finally resulting in activation of HD11 cells by the release of NO and cytokines expression. In chapter 4, the establishment of a standardized culturing method of monocyte-derived macrophages is described. A 3-day culture was optimal to obtain pro-inflammatory M1-like macrophages. These cells were a homogenous population of flat, ‘fried-egg’ like shaped cells, similar to human M1 macrophages. Furthermore, LPS stimulation of the cultured cells induced gene expression of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8 after 3 days of culture. Finally, it was shown that 3-day-cultured macrophages were able to phagocytose avian pathogenic E. coli (APEC) and respond by nitric oxide production. In chapter 5, the immunomodulatory functions of CATH-B1 are described increasing our understanding of the function of this cathelicidin. CATH-B1 showed anti-inflammatory effect on APEC-infected or LPS-stimulated macrophages. In chapter 6, the anti-IAV activity of CATH-B1 is described exploring the possible use of cathelicidins in the development of anti-infective therapies. CATH-B1 has good antiviral activity against IAV by binding to the viral particle and thereby blocking viral entry

Chicken respiratory infections: Protective roles of macrophages and cathelicidins

Avian pathogenic E. coli (APEC) is one of most prevalent pathogens causing systemic diseases called avian colibacillosis. During APEC infection, epithelial cells in the lung are the first cellular defense line against APEC infection and will produce cytokines and chemokines to initiate the immune response. Similarly, macrophages and heterophils will be quickly recruited to the site of infection to fight against the infection in the respiratory tract. Nevertheless, the exact role of these cells in controlling APEC infection is still unclear and therefore investigated in the studies described in this thesis. So far, the treatment of an APEC infection in chicken mainly depends on antibiotics, but increasing antibiotic resistance makes this less effective. Therefore, new anti-infectives are needed and host defense peptides (HDPs) such as cathelicidins are considered as a promising alternatives. In this thesis, we investigated the interaction of chicken macrophages and epithelial cells with avian pathogenic E.coli to establish an appropriate APEC infection model in vitro. Then, we investigated the immunomodulatory effect of CATH-B1 on the chicken macrophages. Furthermore, antiviral activity of chicken cathelicidins against influenza A virus was studied. In chapter 2, the interactions of chicken lung epithelial cells with APEC is described to understand the role of epithelial cells in APEC infections. The results showed APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Moreover, APEC infection resulted in a significant increase in IL-8 gene expression. In chapter 3, the interactions of macrophage HD11 cells with APEC is described to understand the role of macrophages in controlling APEC. The results showed APEC was phagocytosed and subsequenlt killed by HD11 cells, finally resulting in activation of HD11 cells by the release of NO and cytokines expression. In chapter 4, the establishment of a standardized culturing method of monocyte-derived macrophages is described. A 3-day culture was optimal to obtain pro-inflammatory M1-like macrophages. These cells were a homogenous population of flat, ‘fried-egg’ like shaped cells, similar to human M1 macrophages. Furthermore, LPS stimulation of the cultured cells induced gene expression of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8 after 3 days of culture. Finally, it was shown that 3-day-cultured macrophages were able to phagocytose avian pathogenic E. coli (APEC) and respond by nitric oxide production. In chapter 5, the immunomodulatory functions of CATH-B1 are described increasing our understanding of the function of this cathelicidin. CATH-B1 showed anti-inflammatory effect on APEC-infected or LPS-stimulated macrophages. In chapter 6, the anti-IAV activity of CATH-B1 is described exploring the possible use of cathelicidins in the development of anti-infective therapies. CATH-B1 has good antiviral activity against IAV by binding to the viral particle and thereby blocking viral entry

The Roles of c-Jun N-Terminal Kinase (JNK) in Infectious Diseases

c-Jun N-terminal kinases (JNKs) are among the most crucial mitogen-activated protein kinases (MAPKs) and regulate various cellular processes, including cell proliferation, apoptosis, autophagy, and inflammation. Microbes heavily rely on cellular signaling pathways for their effective replication; hence, JNKs may play important roles in infectious diseases. In this review, we describe the basic signaling properties of MAPKs and JNKs in apoptosis, autophagy, and inflammasome activation. Furthermore, we discuss the roles of JNKs in various infectious diseases induced by viruses, bacteria, fungi, and parasites, as well as their potential to serve as targets for the development of therapeutic agents for infectious diseases. We expect this review to expand our understanding of the JNK signaling pathway’s role in infectious diseases and provide important clues for the prevention and treatment of infectious diseases

A Comparison of Pseudorabies Virus Latency to Other α-Herpesvirinae Subfamily Members

Pseudorabies virus (PRV), the causative agent of Aujeszky’s disease, is one of the most important infectious pathogens threatening the global pig industry. Like other members of alphaherpesviruses, PRV establishes a lifelong latent infection and occasionally reactivates from latency after stress stimulus in infected pigs. Latent infected pigs can then serve as the source of recurrent infection, which is one of the difficulties for PRV eradication. Virus latency refers to the retention of viral complete genomes without production of infectious progeny virus; however, following stress stimulus, the virus can be reactivated into lytic infection, which is known as the latency-reactivation cycle. Recently, several research have indicated that alphaherpesvirus latency and reactivation is regulated by a complex interplay between virus, neurons, and the immune system. However, with those limited reports, the relevant advances in PRV latency are lagging behind. Therefore, in this review we focus on the regulatory mechanisms in PRV latency via summarizing the progress of PRV itself and that of other alphaherpesviruses, which will improve our understanding in the underlying mechanism of PRV latency and help design novel therapeutic strategies to control PRV latency

Avian pathogenic Escherichia coli-induced activation of chicken macrophage HD11 cells

Avian pathogenic Escherichia coli (APEC) can cause severe respiratory diseases in poultry. The initial interaction between APEC and chicken macrophages has not been characterized well and it is unclear how effective chicken macrophages are in neutralizing APEC. Therefore, the effect of APEC on activation of chicken macrophage HD11 cells was studied. Firstly, the effect of temperature (37 vs 41 °C) on phagocytosis of APEC by HD11 cells was determined. The results showed that APEC was more susceptible to being phagocytosed by HD11 cells at 41 °C than 37 °C. Subsequently, the capacity of HD11 cells to kill APEC was shown. In addition, HD11 cells produced nitric oxide (NO) at 18 h post-infection and a strong increase in the mRNA expression of IL-8, IL-6, IL-1β and IL-10 was detected, while IFN-β gene expression remained unaffected. Finally, it was shown that the response of HD11 was partially dependent on viability of APEC since stimulation of HD11 cells with heat-killed APEC resulted in a reduced expression level of these cytokines. In conclusion, APEC induces an effector response in chicken macrophages by enhanced NO production and cytokines gene expression

RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

Abstract Pasteurella multocida is a gram-negative bacterium that causes serious diseases in a wide range of animal species. Inflammasomes are intracellular multimolecular protein complexes that play a critical role in host defence against microbial infection. Our previous study showed that bovine P. multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation. However, the exact mechanism underlying PmCQ2-induced NLRP3 inflammasome activation is not clear. Here, we show that NLRP3 inflammasome activation is positively regulated by a scaffold protein called receptor for activated C kinase 1 (RACK1). This study shows that RACK1 expression was downregulated by PmCQ2 infection in primary mouse peritoneal macrophages and mouse tissues, and overexpression of RACK1 prevented PmCQ2-induced cell death and reduced the numbers of adherent and invasive PmCQ2, indicating a modulatory role of RACK1 in the cell death that is induced by P. multocida infection. Next, RACK1 knockdown by siRNA significantly attenuated PmCQ2-induced NLRP3 inflammasome activation, which was accompanied by a reduction in the protein expression of interleukin (IL)-1β, pro-IL-1β, caspase-1 and NLRP3 as well as the formation of ASC specks, while RACK1 overexpression by pcDNA3.1-RACK1 plasmid transfection significantly promoted PmCQ2-induced NLRP3 inflammasome activation; these results showed that RACK1 is essential for NLRP3 inflammasome activation. Furthermore, RACK1 knockdown decreased PmCQ2-induced NF-κB activation, but RACK1 overexpression had the opposite effect. In addition, the immunofluorescence staining and immunoprecipitation results showed that RACK1 colocalized with NLRP3 and that NEK7 and interacted with these proteins. However, inhibition of potassium efflux significantly attenuated the RACK1-NLRP3-NEK7 interaction. Our study demonstrated that RACK1 plays an important role in promoting NLRP3 inflammasome activation by regulating NF-κB and promoting NLRP3 inflammasome assembly

Avian pathogenic Escherichia coli infection of a chicken lung epithelial cell line

International audienceVirulent strains of Escherichia coli (Avian Pathogenic E. Coli: APEC) can cause initial infection of the respiratory tract in chickens potentially leading to systemic infection called colibacillosis, which remains a major cause of economic losses in the poultry industry. The role of epithelial lung cells as first targets of APEC and in initiating the innate immune response is unclear and was investigated in this study. APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Invasion was confirmed using confocal microscopy after infection with GFP-labelled APEC. Moreover, the infection resulted in a significant increase in IL-8 gene expression, a chemo-attractant of macrophages and heterophils. Gene expression of interferon α and β were not significantly upregulated and chicken Surfactant Protein A, also did not show a significant upregulation on either gene or protein level. The immune response of CLEC213 cells towards APEC was shown to be similar to stimulation with E. coli LPS. These results establish CLEC213 cells as a novel model system for studying bacterial infection of the lung epithelium and show that these cells may play a role in the initial innate response towards bacterial pathogens