Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages

In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome–lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1β, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1β, TNF-α, interferon (IFN)-β and CXCL-10 mRNAs. We also observed that pro-IL-1β protein was expressed and efficiently cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.     

Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.     

The axis of the receptor for advanced glycation endproducts in asthma and allergic airway disease

Asthma is a generalized term that describes a scope of distinct pathologic phenotypes of variable severity, which share a common complication of reversible airflow obstruction. Asthma is estimated to affect almost 400 million people worldwide, and nearly ten percent of asthmatics have what is considered “severe” disease. The majority of moderate to severe asthmatics present with a “type 2-high” (T2-hi) phenotypic signature, which pathologically is driven by the type 2 cytokines Interleukin-(IL)-4, IL-5, and IL-13. However, “type 2-low” (T2-lo) phenotypic signatures are often associated with more severe, steroid-refractory neutrophilic asthma. A wide range of clinical and experimental studies have found that the receptor for advanced glycation endproducts (RAGE) plays a significant role in the pathogenesis of asthma and allergic airway disease (AAD). Current experimental data indicates that RAGE is a critical mediator of the type 2 inflammatory reactions which drive the development of T2-hi AAD. However, clinical studies demonstrate that increased RAGE ligands and signaling strongly correlate with asthma severity, especially in severe neutrophilic asthma. This review presents an overview of the current understandings of RAGE in asthma pathogenesis, its role as a biomarker of disease, and future implications for mechanistic studies, and potential therapeutic intervention strategies.     

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8⁺ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.     

Role of phospholipase D in laminin-induced production of gelatinase A (MMP-2) in metastatic cells

Metastatic spread depends critically upon the invasiveness of tumor cells, i.e. their ability to breach basement membranes by elaborating and secreting specific proteolytic enzymes such as gelatinase A (MMP-2). Laminin is a major constituent of the extracellular matrix that can trigger production of MMP-2 in metastatic cells, but not in non-metastatic cells. The present study was designed to examine the role of phospholipase D (PLD) and its product, phosphatidic acid, in the intracellular signal transduction mechanisms that mediate induction of MMP-2 by laminin. Here we show that stimulation of tumor cells with laminin results in a time- and dose-dependent activation of PLD. Laminin-induced production of MMP-2 is attenuated by 1-butanol, a competitive substrate of PLD that reduces PLD-catalyzed production of PA. Moreover, phosphatidic acid itself can induce production of MMP-2 in metastatic tumor cells. MMP-2 can also be induced by exposing the cells to exogenous bacterial PLD. Elevated cellular phosphatidic acid induces MMP-2 in metastatic ras-transformed 3T3 fibroblasts but, like laminin, fails to do so in normal cells. These data indicate that laminin-induced activation of PLD and consequent generation of phosphatidic acid are involved in a signal propagation pathway leading to induction of MMP-2 and enhanced invasiveness of metastatic tumor cells.     

Interleukin‐10 and interferon‐γ modulate surface expression of fractalkine‐receptor (CX3CR1) via PI3K in monocytes

The membrane‐anchored form of the chemokine fractalkine (CX₃CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX₃CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX₃CL1 can be cleaved from the cell membrane to induce chemotaxis of CX₃CR1‐expressing leucocytes. Recently, marked variations in CX₃CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX₃CR1 in monocytes during basal or inflammatory/anti‐inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX₃CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX₃CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin‐10 and interferon‐γ treatment abrogated CX₃CR1 down‐modulation, through a phosphatidylinositol 3 kinase‐dependent pathway. Most importantly, CX₃CR1 membrane expression correlated with monocyte CX₃CL1‐dependent function. Taken together, our data demonstrate that CX₃CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX₃CL1‐dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.     

Ageing and Toll-like receptor expression by innate immune cells in chronic human schistosomiasis

There has been no systematic study of the immune response of individuals aged over 60 years living in Schistosomiasis mansoni-endemic areas, although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals aged over 60 years with chronic schistosomiasis is no longer negligible. Moreover, several elderly individuals who have always lived in these endemic areas stay protected from infection. An important question for studies of ageing and disease control in developing countries is which differences in the immunological profile of these negatively tested (non-infected) individuals can account for their resistance to either infection or reinfection. We show, in the present study, that non-infected (negative) elderly individuals develop innate immune mechanisms of protection that replace the age-associated decline in T cell function. Non-infected elderly individuals from endemic areas of schistosome infection present an increase in the frequency of the natural killer (NK) CD56(low) subset of NK cells expressing Toll-like receptors (TLR)-1, -2, -3 and -4 as determined by flow cytometry analysis. In addition, the proportion of dendritic cells expressing TLR-1 is elevated as well as the frequency of monocytes expressing TLR-1 and -4. These results suggest that TLR expression by cells of the innate immune system may be related to the negative status of infection in some elderly individuals who are constantly exposed to S. mansoni. Developing mechanisms of protection from infection may represent a biomarker for healthy ageing in this population.     

CD2 and TCR synergize for the activation of phospholipase Cgamma1/calcium pathway at the immunological synapse

Upon conjugation with cognate antigen-presenting cells (APCs), T lymphocytes undergo a sustained [Ca(2+)](i) increase resulting from the engagement of TCR and of accessory molecules with ligands expressed on the surface of APCs. We investigated the contribution of the accessory molecule CD2 to the activation of phospholipase Cgamma1 (PLCgamma1)/calcium pathway in antigen-stimulated T cells. We show that CD2 binding with its ligand CD58 expressed on the surface of APCs augments and sustains antigen-induced [Ca(2+)](i) increase in individual T cells interacting with APCs. We also show that in conditions in which CD2-CD58 interaction is impeded, the recruitment of PLCgamma1 to the immunological synapse (IS) is reduced. Interestingly, in these conditions PLCgamma1 phosphorylation in the regulatory tyrosine 783 is also defective. Our results indicate that TCR- and CD2-derived signals converge for the recruitment and activation of PLCgamma1 at the IS and shed new light on the accessory function of CD2 in T cell activation by specific antigen.     

The role of M2-2 PDZ-binding motifs in pulmonary innate immune responses to human metapneumovirus

Human metapneumovirus (HMPV) is a leading cause of lower respiratory tract infection (LRTI) in pediatric and geriatric populations. We recently found that two PDZ-binding motifs of the M2-2 protein, 29-DEMI-32 and 39-KEALSDGI-46, play a significant role in mediating HMPV immune evasion in airway epithelial cells (AECs). However, their role in the overall pulmonary responses to HMPV infection has not been investigated. In this study, we found that two recombinant HMPVs (rHMPV) lacking the individual M2-2 PDZ-binding motif are attenuated in mouse lungs. Mice infected with mutants produce more cytokines/chemokines in bronchoalveolar lavage (BAL) fluid compared to mice infected with wild-type rHMPV. In addition, both mutants are able to enhance the pulmonary recruitment of dendritic cells (DCs) and T cells and induce effective protections against the HMPV challenge. The DC maturation is also significantly improved by the motif mutation. Taken together, our data provide proof-of-principle for two live-attenuated M2-2 mutants to be promising HMPV vaccine candidates that are effective in inducing higher pulmonary innate immunity and generating protection against HMPV infection.     

Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells

Using human mast cells (MC) derived by culture of CD34+ peripheral blood precursors, a comprehensive study was performed of expression of 11 known Siglecs. Analysis was initially performed at the mRNA level using gene arrays. Positive results were then validated at the protein level using indirect immunofluorescence and flow cytometry, and for some Siglecs, Western blot analysis was also used. Culture-derived MC expressed mRNA for CD22 (Siglec-2), CD33 (Siglec-3), Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for CD22 and Siglec-10, where levels were low or undetectable. However, Western blotting was able to detect MC expression of CD22 and Siglec-10, suggesting that these proteins were mostly cytoplasmic. CD34+ precursor cells from peripheral blood constitutively expressed surface CD33, Siglec-5 and Siglec-10. As they matured into MC, their constitutive levels of CD33 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 appeared de novo, all in parallel with accumulation of histamine and other MC markers, such as surface expression of FcepsilonRIalpha, and CD51. Phenotypic analysis of LAD-2 MC yielded a similar pattern of Siglec expression except that CD22 expression was particularly prominent. Finally, immunohistochemistry confirmed expression of these same Siglecs by mature tryptase-positive MC in human lung tissues. These data demonstrate an extensive and previously unappreciated pattern of Siglec expression on human MC. Whether engagement and signaling through these inhibitory Siglecs can impact MC biology will require further investigation.