Eva Särndahl

Incorporation of Mycobacterium tuberculosis Lipoarabinomannan into Macrophage Membrane Rafts Is a Prerequisite for the Phagosomal Maturation Block

ABSTRACT Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

Gene Polymorphisms in the NALP3 Inflammasome Are Associated With Interleukin-1 Production and Severe Inflammation : Relation to Common Inflammatory Diseases?

OBJECTIVE NALP3, ASC, and TUCAN are components of the NALP3 inflammasome, which triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Activating mutations in the gene encoding NALP3 (NLRP3) have recently been linked to familial periodic fever syndromes. We undertook this study to determine whether a patient with arthritis and antibiotic-resistant fever carried mutations in the genes encoding the NALP3 inflammasome. METHODS Genetic analysis of NLRP3 and the gene encoding TUCAN (CARD-8) was performed on genomic DNA from the patient and from a population-based collection of DNA (806 subjects). For in vitro studies of IL-1beta production and caspase 1 activity, blood was obtained from the patient at different time points after administration of anakinra, an IL-1 receptor antagonist, as well as from 5 healthy age- and sex-matched control subjects. RESULTS Mutation analysis of the patients genes encoding NALP3, ASC, and TUCAN revealed variations in the NLRP3 (Q705K) and CARD-8 (C10X) genes. The allele frequencies of these single-nucleotide polymorphisms (SNPs) in the population were 6.5% and 34%, respectively. The elevated activity of caspase 1 and the high levels of IL-1beta measured in samples from the patient returned to normal levels after treatment with anakinra. CONCLUSION Our results indicate that the patients symptoms were due to elevated levels of IL-1beta, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1beta levels and inflammatory symptoms observed, but further studies are needed to reveal a functional relationship. The prevalence of the polymorphisms (4% of the population carry both SNPs) in the general population may suggest a genetic predisposition for common inflammatory disorders.

The Q705K Polymorphism in NLRP3 Is a Gain-of-Function Alteration Leading to Excessive Interleukin-1β and IL-18 Production

Background The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system. Principal Findings NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner. Conclusions Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.

Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho-GTPases and Akt

In addition to direct activation of caspase‐1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella‐mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild‐type Salmonella typhimurium but not by phagocytosed, serum‐opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol‐3 kinase (PI‐3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild‐type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI‐298, a geranylgeranyltransferase‐1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella‐induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant‐negative Cdc42 or Rac1 significantly inhibited Salmonella‐induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine‐ or PI‐3K. Moreover, Salmonella infection activated Akt protein in a tyrosine‐kinase or PI‐3K‐dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella‐mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI‐3K during receptor‐mediated phagocytosis protects cells from apoptosis.

Lipoxin A4 Inhibits Porphyromonas gingivalis-Induced Aggregation and Reactive Oxygen Species Production by Modulating Neutrophil-Platelet Interaction and CD11b Expression

ABSTRACT Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA4. Furthermore, we found that LXA4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts.

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galbeta1,4Manalpha-PO(4) attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.

Activation of cAMP‐dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis

We have investigated the role of cAMP and cAMP‐dependent protein kinase (cAPK) in neutrophil phagocytosis. Inhibition of cAPK with H‐89 reduced complement‐ and IgG‐dependent phagocytosis to 83 and 46%, respectively. Fluorescence intensity measurements of phalloidin‐stained actin in neutrophils showed a reduced amount of filamentous actin (F‐actin) in pseudopods and around the phagosome in cells treated with H‐89 or cAMP‐elevating agents (forskolin and rolipram). The amount of phosphotyrosine‐containing proteins was also reduced in pseudopods and around the phagosome. Taken together, the data show that cAMP/cAPK regulates F‐actin reorganization during receptor‐mediated phagocytosis, particularly triggered by IgG‐FcR interaction. Our results support the hypothesis that active subcortical reorganization of F‐actin is a prerequisite for FcR‐mediated phagocytosis, but is less important during CR3‐mediated ingestion. J. Leukoc. Biol. 67: 520–528; 2000.

Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: Effect of an association of the ligand-receptor complex to the cytoskeleton

The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15 degrees C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.

Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils

Phagocytosis is a complex process engaging a concerted action of signal‐transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi‐mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcγR)‐mediated activation is Rac‐dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcγR and CR3‐ and FcγR‐mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor‐engagement of the CR3 or FcγRs, a downstream target of these small GTPases, p21‐activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N‐acetyl‐S‐farnesyl‐L‐cysteine and N‐acetyl‐S‐geranylgeranyl‐L‐cysteine, we found that the phagocytic uptake of bacteria was not Rac2‐ or Cdc42‐dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR‐ and CR3‐induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis

Activation of the NLRP3 inflammasome and subsequent generation of interleukin 1β is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequent infection of the cells with virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.