Role of capsular modified heptose in the virulence of Campylobacter jejuni

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

The role of SOCS3/pSTAT3 pathway in the development of diabetic retinopathy

Purpose: Suppressor of cytokine signalling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 critically controls STAT3 activation, cytokine signalling and inflammatory gene expression in macrophages and microglia. In this study, we investigated the role of SOCS3/STAT3 in myeloid cells in the initiation and progression of diabetic retinopathy (DR). 
Methods: Mice with a conditional deletion of SOCS3 in myeloid cells (LysMCre-SOCS3 fl/fl) and C57BL/6J (as control) were rendered diabetic by a low-dose multiple intraperitoneal injections of Stroptozocine. Diabetes related retinal changes, including leukostasis, acellular capilliaries, and microglial activation were assessed at different stages of disease. Bone marrow derived macrophages (BMDMs) from LysMCreSOCS3 fl/fl and C57BL/6J mice were cultured in high glucose (HG) medium, and cell activation was evaluated by real-time RT-PCR.
Results: In C57BL/6J diabetic mice the expression of phosphorylated STAT3 (pSTAT3) was increased and SOCS3 was decreased in the retina. Interleukin 6 (IL-6), the main cytokine that stimulates STAT3 activation, was increased in the plamsa in diabetic mice. Although blood glucose levels and Hbac-1 were comparable between LysMCre-SOCS3fl/fl and WT mice after STZ injection, the LysMCreSOCS3 fl/fl diabetic mice developed severe retinal vasculopathy, including increased leukostasis and microglial activation at one month and enhanced acellular capillary formation at 6 months after diabetes induction. 
Conclusions: our study suggests that the JAK/STAT3 pathway is involved in the initiation and progression of DR, and uncontrolled STAT3 activation results in accelerated DR progression. Targeting the STAT3 pathway may be a novel approach for the management of DR.

Regulating macrophage phenotype and function by retinal pigment epithelial cells

Purpose: We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase-1 in the aging eye. Subretinal macrophages are in close contact with retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may play an important role in regulating macrophage/microglial phenotype and function. The aim of this study was to investigate the effect of RPE cells on the phenotype and function of bone marrow–derived macrophages (BM-DMs).
Methods: BM-DM from C57BL/6J mice were cultured in DMEM supplemented with 20%L929 cell supernatant for 5 days. The phenotype of BM-DMs was confirmed by flow cytometry as CD11b+F4/80+. Primary RPE cells were cultured from C57BL/6J mice and confirmed by RPE65 and cytokeratin staining. BMDMs were co-cultured with different types of RPE cells (healthy, oxidized, and apoptotic RPE) and then isolated from the co-culture system for phenotypic and functional assays.
Results: Co-culture of BM-DMs with RPE cells results in a time-dependent down-regulation of MHC-II expression and the generation of CD11b+F4/80+Ly6G+ myeloid-derived suppressor cells (MDSC). qRT-PCR analysis showed that RPE-induced MDSCs expressed high levels of IL-6, IL-1β, and Arginase-1, but lower levels of IL-12p40 and TNF-a compared to naïve BM-DMs. The expression levels of iNOS, TGF-β and Ym1 did not differ 207 between naive BMDMs and RPE-induced MDSCs. Furthermore, functional studies showed that these cells had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre-treated with oxidized photoreceptor outer segments before co-culturing with BMDMs, the expression of IL-1β and IL-6 in BMDMs was increased whereas the expression of Arginase-1 was decreased. 
Conclusion: Our results suggest that healthy RPE cells can convert BMDMs into myeloid-derived suppressor cells under in vitro culture conditions, RPE-induced myeloid-derived suppressor cells are CD11b+F4/80+Ly6G+MHCIIlowIL6+IL1b+Arg-1+. The ability of RPE cells is reduced when suffering from oxidative insults.

Semi-interpenetrating polymer networks incorporating polygalacturonic acid for medical device applications

Background Over 20 million people in the US are living with an implantable medical device [ADDIN RW.CITE{{3114 Higgins,DavidM 2009}}1], with similar figures anticipated for Europe. Complications in the use of medical implants include the Foreign Body Response (FBR) characterised by macrophage adherence and fusion, and device-related infection due to bacterial biofilm formationADDIN RW.CITE{{3124 Harding,JacquelineL 2014}}2. Both can have detrimental consequences on the structural and functional integrity of the medical device [ADDIN RW.CITE{{3101 Anderson,JamesM 2008; 3124 Harding,JacquelineL 2014}}2,3], often necessitating removal; a painful and expensive procedure [ADDIN RW.CITE{{3121 Mah,Thien-FahC 2001}}4]. Materials are sought to attenuate both the FBR and device-related infection, leading to medical devices with improved biocompatibility and performance. Objectives The present work involves development of a semi-interpenetrating network (SIPN) hydrogel containing polygalacturonic acid (PGA), a biopolysaccharide similar in structure to hyaluronic acid. We aim to synthesise, characterise and determine the in vitro biocompatibility of the developed SIPN. Results & Discussion We have successfully incorporated PGA into a poly(HEMA) based hydrogel, which shows favourable swelling and wettability. The surface topography appears altered in comparison to the control material, with pronounced micrometer-scale features. In terms of in vitro performance, the SIPN showed increased protein adsorption, and biofilm formation (Staphylococcus epidermidis and Escherichia coli, up to 1 Log CFU/sample greater than control). However the SIPN displayed minimal cytotoxicity towards L929 fibroblasts, and was resistant to the adherence of RAW 264.7 macrophages. Conclusions The PGA incorporated SIPN lacks cytotoxicity and shows reduced macrophage adherence, however the increased biofilm formation highlights a concern regarding possible device related infection in clinical use. Future work will focus on strategies to reduce bacterial adherence, while maintaining biocompatibility.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.

Intracellular survival of Burkholderia cepacia complex in phagocytic cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance, and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with advances to genetically manipulate these bacteria have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of B. cenocepacia within macrophages.

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.

Molecular mechanisms of virulence of Burkholderia cepacia complex bacteria: Survival in Phagocytic Cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that chronically infect the airways of cystic fibrosis patients, but they can also infect patients with various types of immunosuppressive disorders. Bcc members are multidrug resistant bacteria that have the ability to persist in the infected host and also elicit robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with dramatic advances in the ability to genetically manipulate these microorganisms have contributed to increase our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of the cell host responses triggering inflammation. This chapter reviews our understanding of the pathogenic mechanisms used by Bcc to establish an intracellular niche in phagocytic cells and modulate host cell responses that ultimately end up in cell death and a proinflammatory response.

Targeting Siglecs with a sialic acid-decorated nanoparticle abrogates inflammation

Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2→8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E-dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.

CCL2 is transcriptionally controlled by the lysosomal protease cathepsin S in a CD74-dependent manner

Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.

The role of antimicrobial peptides in periodontal disease.

Objectives Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.