Transcriptional approach to study porcine tracheal epithelial cells individually or dually infected with swine influenza virus and Streptococcus suis.

BACKGROUND: Swine influenza is a highly contagious viral infection in pigs affecting the respiratory tract that can have significant economic impacts. Streptococcus suis serotype 2 is one of the most important post-weaning bacterial pathogens in swine causing different infections, including pneumonia. Both pathogens are important contributors to the porcine respiratory disease complex. Outbreaks of swine influenza virus with a significant level of co-infections due to S. suis have lately been reported. In order to analyze, for the first time, the transcriptional host response of swine tracheal epithelial (NPTr) cells to H1N1 swine influenza virus (swH1N1) infection, S. suis serotype 2 infection and a dual infection, we carried out a comprehensive gene expression profiling using a microarray approach. RESULTS: Gene clustering showed that the swH1N1 and swH1N1/S. suis infections modified the expression of genes in a similar manner. Additionally, infection of NPTr cells by S. suis alone resulted in fewer differentially expressed genes compared to mock-infected cells. However, some important genes coding for inflammatory mediators such as chemokines, interleukins, cell adhesion molecules, and eicosanoids were significantly upregulated in the presence of both pathogens compared to infection with each pathogen individually. This synergy may be the consequence, at least in part, of an increased bacterial adhesion/invasion of epithelial cells previously infected by swH1N1, as recently reported. CONCLUSION: Influenza virus would replicate in the respiratory epithelium and induce an inflammatory infiltrate comprised of mononuclear cells and neutrophils. In a co-infection situation, although these cells would be unable to phagocyte and kill S. suis, they are highly activated by this pathogen. S. suis is not considered a primary pulmonary pathogen, but an exacerbated production of proinflammatory mediators during a co-infection with influenza virus may be important in the pathogenesis and clinical outcome of S. suis-induced respiratory diseases.

Deregulated balance of omega-6 and omega-3 polyunsaturated fatty acids following infection by the zoonotic pathogen Streptococcus suis.

Streptococcus suis is an important swine pathogen and an emergent zoonotic pathogen. Excessive inflammation caused by S. suis is responsible for early high mortality in septic shock-like syndrome cases. Polyunsaturated fatty acids (PUFAs) may contribute to regulating inflammatory processes. This study shows that mouse infection by S. suis is accompanied by an increase of arachidonic acid, a proinflammatory omega-6 (ω-6) PUFA, and by a decrease of docosahexaenoic acid, an anti-inflammatory ω-3 PUFA. Macrophages infected with S. suis showed activation of mitogen-activated protein kinase pathways and cyclooxygenase-2 upregulation. Fenretinide, a synthetic vitamin A analog, reduced in vitro expression of inflammatory mediators. Pretreatment of mice with fenretinide significantly improved their survival by reducing systemic proinflammatory cytokines during the acute phase of an S. suis infection. These findings indicate a beneficial effect of fenretinide in diminishing the expression of inflammation and improving survival during an acute infection by a virulent S. suis strain.

Capsular sialic acid of Streptococcus suis serotype 2 binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells.

Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.

Fenretinide corrects the imbalance between omega-6 to omega-3 polyunsaturated fatty acids and inhibits macrophage inflammatory mediators via the ERK pathway.

We previously identified Fragile X-related protein 1 (FXR1) as an RNA-binding protein involved in the post-transcriptional control of TNF and other cytokines in macrophages. Macrophages derived from FXR1-KO mice overexpress several inflammatory cytokines including TNF. Recently, we showed that fenretinide (4HPR) is able to inhibit several inflammatory cytokines in the lungs of cystic fibrosis mice, which also have abnormal immune responses. Therefore, we hypothesized that 4HPR might also be able to downregulate excessive inflammation even in macrophages with ablated FXR1. Indeed, our results demonstrate that 4HPR inhibited the excessive production of inflammatory mediators, including TNF, IL-6, CCL2 and CCL-5 in LPS-stimulated FXR1-KO macrophages, by selectively inhibiting phosphorylation of ERK1/2, which is naturally more phosphorylated in FXR1-KO cells. We also found that LPS stimulation of FXR1-KO macrophages led to significantly higher ratio of arachidonic acid/docosahexaenoic acid than observed in FXR1-WT macrophages. Interestingly, treatment with 4HPR was associated with the normalization of arachidonic acid/docosahexaenoic acid ratio in macrophages, which we found to impact phosphorylation of ERK1/2. Overall, this study shows for the first time that 4HPR modulates inflammatory cytokine expression in macrophages by correcting a phospholipid-bound fatty acid imbalance that impacts the phosphorylation of ERK1/2.

Group B Streptococcus and Streptococcus suis capsular polysaccharides induce chemokine production by dendritic cells via Toll-like receptor 2- and MyD88-dependent and -independent pathways.

Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1ß (IL-1ß), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.

Characterization of porcine dendritic cell response to Streptococcus suis.

Streptococcus suis is a major swine pathogen and important zoonotic agent causing mainly septicemia and meningitis. However, the mechanisms involved in host innate and adaptive immune responses toward S. suis as well as the mechanisms used by S. suis to subvert these responses are unknown. Here, and for the first time, the ability of S. suis to interact with bone marrow-derived swine dendritic cells (DCs) was evaluated. In addition, the role of S. suis capsular polysaccharide in modulation of DC functions was also assessed. Well encapsulated S. suis was relatively resistant to phagocytosis, but it increased the relative expression of Toll-like receptors 2 and 6 and triggered the release of several cytokines by DCs, including IL-1ß, IL-6, IL-8, IL-12p40 and TNF-α. The capsular polysaccharide was shown to interfere with DC phagocytosis; however, once internalized, S. suis was readily destroyed by DCs independently of the presence of the capsular polysaccharide. Cell wall components were mainly responsible for DC activation, since the capsular polysaccharide-negative mutant induced higher cytokine levels than the wild-type strain. The capsular polysaccharide also interfered with the expression of the co-stimulatory molecules CD80/86 and MHC-II on DCs. To conclude, our results show for the first time that S. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2.

Fenretinide corrects newly found ceramide deficiency in cystic fibrosis.

Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry. Our results demonstrate that patients with CF display significantly lower levels of several ceramide sphingolipid species, specifically C14:0, C20:1, C22:0, C22:1, and C24:0 ceramides, and dihydroxy ceramide (DHC16:0). We report that Cftr-knockout mice display diminished ceramide levels in CF-related organs (lung, pancreas, ileum, and plasma) compared with their littermate controls. Since it has been previously reported that in vitro treatment with fenretinide induced ceramide in neuroblastoma cell lines, we decided to test this drug in vivo using our Cftr-knockout mice in an attempt to correct this newly identified defect in ceramide levels. We demonstrate that treatment with fenretinide is able to increase ceramide concentrations in CF-related organs. We further assessed the biological effect of fenretinide on the ability of Cftr-knockout mice to combat lung infection with P. aeruginosa. Our data show dramatic improvement in the ability of Cftr-knockout mice to control P. aeruginosa infection. Overall, these findings not only document a novel deficiency in several ceramide species in patients with CF, but also demonstrate a pharmacologic means to correct this defect in Cftr-knockout mice. Our data provide a strong rationale for clinical intervention that may benefit patients with CF suffering from CF lung disease.

Posttranscriptional gene expression regulation in CpG-activated macrophages depends on FXR1P RNA-binding protein.

An RNA-binding protein (RBP) was recently identified, FXR1P, which regulates tumour necrosis factor (TNF) gene expression at the posttranscriptional level in response to lipopolysaccharide, was recently identified resulting in higher TNF production in macrophages from FXR1 knockout (KO) mice compared with wild-type (WT) macrophages. In this study, the importance of FXR1P in the induction of TNF by toll-like receptor 7 (TLR7) ligand S28463 and TLR9 ligand CpG is evaluated. The results clearly reveal a much higher level of TNF protein expression in FXR1-KO than in WT macrophages following stimulation with CpG but not with S28463. To better understand the molecular mechanism, both the steady-state levels and the stability of TNF mRNA were assessed. It was found that the TNF mRNA steady-state level was more elevated in CpG-stimulated FXR1-KO macrophages, while the stability of TNF mRNA was not affected in CpG-stimulated FXR1-KO macrophages. It was also established that FXR1P is involved in regulating the expression of several other inflammatory cytokines and chemokines. Together, the data clearly demonstrate the importance of FXR1P RBP in the regulation of a wide spectrum of inflammatory genes and suggest an important role of MAP signalling in the response of macrophages to selected TLR ligands, including CpG.

Distinct role of MAPKAPK-2 in the regulation of TNF gene expression by Toll-like receptor 7 and 9 ligands.

Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3' untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.

Fragile X-related protein FXR1P regulates proinflammatory cytokine tumor necrosis factor expression at the post-transcriptional level.

Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3'-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation.

Influence of gender and interleukin-10 deficiency on the inflammatory response during lung infection with Pseudomonas aeruginosa in mice.

Cystic fibrosis females have a worse prognosis compared to male patients. Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and lower levels of IL-10 are found in the bronchoalveolar lavage fluid compared to healthy controls. The present study was aimed at investigating the importance of gender and IL-10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa. We found that wildtype females were more susceptible than males to infection, as we observed greater weight loss, higher bacterial load, and inflammatory mediators in their lungs. IL-10 knockout mice, both females and males, had higher levels of TNF-alpha in the lungs compared to wildtype mice and maintained higher levels of polymorphonuclear cells and lower levels of macrophages for a longer period of time. Our results demonstrate that the number of bacteria recovered from the lungs of IL-10 knockout male mice was significantly higher than that observed in their wildtype male counterparts and we show that neutralization of IL-10 in infected female mice for a prolonged period of time leads to increased susceptibility to infection. Results reported in this study clearly demonstrate that females, both wildtype and IL-10 knockout mice are more susceptible to Pseudomonas aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs.