Marit W. Anthonsen

Toll‐like receptor 3 associates with c‐Src tyrosine kinase on endosomes to initiate antiviral signaling

Double‐stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll‐like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA‐TLR3‐mediated signaling are largely unknown. Here we show that c‐Src tyrosine kinase is activated by dsRNA in human monocyte‐derived dendritic cells, and is recruited to TLR3 in a dsRNA‐dependent manner. DsRNA‐induced activation of interferon‐regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase‐deficient cells, and restored by adding back c‐Src, suggesting a central role of c‐Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA‐containing endosomes in response to dsRNA, and colocalizes with c‐Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3‐mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.

Preparative and analytical size-exclusion chromatography of chitosans

Abstract Two chitosan samples (fraction of acetylated units (FA) 0.15 and 0.52) were fractionated by preparative size exclusion chromatography (SEC). The molecular weights and molecular weight distributions of the fractions were analyzed by analytical size exclusion chromatography coupled to an on-line low angle laser light scattering detector and a differential refractive index detector (SEC-LALLS-DRI), and their intrinsic viscosities were determined. The exponent (a) of the Mark-Houwink-Kuhn-Sakurada (MHKS) equation was found to be 0.92 ± 0.07 and 1.1 ± 0.1, respectively, at I = 0.1 and pH 4.5. No variation in FA related to molecular weight was found. Reversible interaction between chitosans and different column packings strongly influenced the log M-V relationships. This interaction was generally most pronounced for the low-FA chitosan, suggesting that the protonated amino groups are involved. Ammonium acetate buffer reduced this effect and the use of a new type of SEC-packing seemed to eliminate it. The more highly acetylated chitosan also had a more pronounced tendency towards concentration dependent self-association, which most probably involve intermolecular hydrophobic interactions between the acetyl groups.

Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A2 in Human Monocyte–Derived Macrophages

Phospholipase A(2)s (PLA(2)s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA(2)s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA(2) has been shown to specifically hydrolyze low density lipoprotein (LDL)-associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA(2)s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA(2) (snpPLA(2)) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription-polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA(2) was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA(2) was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA(2) was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA(2) in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA(2) expression in human monocyte-derived macrophages. The mRNA levels of cytosolic PLA(2) group IV and snpPLA(2) group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA(2) enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA(2) enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.

Atypical /PKC Conveys 5-Lipoxygenase/Leukotriene B 4 -mediated Cross-talk between Phospholipase A 2 s Regulating NF-B Activation in Response to Tumor Necrosis Factor- and Interleukin-1*

The transcription factor nuclear factor κB (NF-κB) plays crucial roles in a wide variety of biological functions such as inflammation, stress, and immune responses. We have shown previously that secretory nonpancreatic (snp) and cytosolic (c) phospholipase A2 (PLA2) regulate NF-κB activation in response to tumor necrosis factor (TNF)-α or interleukin (IL)-1β activation and that a functional coupling mediated by the 5-lipoxygenase (5-LO) metabolite leukotriene B4 (LTB4) exists between snpPLA2and cPLA2 in human keratinocytes. In this study, we have further investigated the mechanisms of PLA2-modulated NF-κB activation with respect to specific kinases involved in TNF-α/IL-1β-stimulated cPLA2phosphorylation and NF-κB activation. The protein kinase C (PKC) inhibitors RO 31-8220, Gö 6976, and a pseudosubstrate peptide inhibitor of atypical PKCs attenuated arachidonic acid release, cPLA2 phosphorylation, and NF-κB activation induced by TNF-α or IL-1β, thus indicating atypical PKCs in cPLA2regulation and transcription factor activation. Transfection of a kinase-inactive mutant of λ/ιPKC in NIH-3T3 fibroblasts completely abolished TNF-α/IL-1β-stimulated cellular arachidonic acid release and cPLA2 activation assayed in vitro, confirming the role of λ/ιPKC in cPLA2 regulation. Furthermore, λ/ιPKC and cPLA2 phosphorylation was attenuated by phosphatidyinositol 3-kinase (PI3-kinase) inhibitors, which also reduced NF-κB activation in response to TNF-α and IL-1β, indicating a role for PI3-kinase in these processes in human keratinocytes. TNF-α- and IL-1β-induced phosphorylation of λ/ιPKC was attenuated by inhibitors toward snpPLA2 and 5-LO and by an LTB4 receptor antagonist, suggesting λ/ιPKC as a downstream effector of snpPLA2 and 5-LO/LTB4 the LTB4 receptor. Hence, λ/ιPKC regulates snpPLA2/LTB4-mediated cPLA2 activation, cellular arachidonic acid release, and NF-κB activation induced by TNF-α and IL-1β. In addition, our results demonstrate that PI3-kinase and λ/ιPKC are involved in cytokine-induced cPLA2 and NF-κB activation, thus identifying λ/ιPKC as a novel regulator of cPLA2.

The Tyrosine Kinase c-Src Enhances RIG-I (Retinoic Acid-inducible Gene I)-elicited Antiviral Signaling

Antiviral immune responses are initiated through Toll-like receptors (TLRs) and RIG-I (retinoic acid-inducible gene-I)-like RNA helicases that recognize nucleic acids from distinct viruses. In this study, we show that the tyrosine kinase c-Src participates in antiviral responses induced by the cytoplasmic RNA helicase RIG-I. Sendai virus (SV), which is recognized by RIG-I, induced c-Src phosphorylation. Functional impairment of c-Src through chemical inhibition or transient expression of a c-Src kinase-inactive mutant attenuated production of endogenous antiviral proteins after SV infection or after expression of RIG-I or its adapter protein MAVS. Importantly, SV-stimulated synthesis of antiviral proteins was significantly impaired in cells treated with c-Src small interfering RNA and in cells from c-Src-deficient mice. In addition, we found that c-Src interacted with components of the RIG-I pathway: RIG-I, MAVS, and TRAF3 (tumor necrosis factor receptor-associated factor-3). The interaction between c-Src and TRAF3 was found to occur within the RING domain of TRAF3. Taken together, our results suggest that c-Src enhances RIG-I-mediated signaling, acting at the level of TRAF3.

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity.

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [3H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA2 (sPLA2) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA2 or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA2 (cPLA2) assayed in vitro as well as increased monocyte tumor necrosis factor-α secretion. The AA liberation was attenuated by inhibitors toward cPLA2 and sPLA2, indicating that both PLA2 enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA2 or SMase potentiates cellular AA release and cPLA2 activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA2 and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.

Role of secretory and cytosolic phospholipase A2 enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA2) activity, has been analyzed. LysoPC increased AA release in THP‐1 and Mono Mac6 cells in a time‐ and concentration‐dependent manner. The monocytes expressed both secretory and cytosolic PLA2 enzymes and AA release was strongly reduced by cellular pretreatment with different PLA2 inhibitors and by pertussis toxin, an inhibitor of Gi‐protein activation. This indicates that both cytosolic and secretory PLA2 enzymes regulate specific lysoPC receptor‐induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.

Identification of a novel in vivo virus-targeted phosphorylation site in interferon regulatory factor-3 (IRF3).

The transcription factor interferon regulatory factor-3 (IRF3) regulates expression of type I interferon-β and plays an important role in antiviral immunity. Despite the biological importance of IRF3, its in vivo phosphorylation pattern has not been reported. In this study, we have identified residues in IRF3 that are phosphorylated in vivo after infection with Sendai virus. We found that Sendai virus induced phosphorylation of the C-terminal residues Thr390 and Ser396, in addition to either Ser385 or Ser386. Moreover, Ser173 and Ser175 were constitutively phosphorylated. Ser396 has previously been suggested to be the major target of the IRF3-activating kinase TBK1 (TANK-binding kinase-1), whereas Thr390 has not previously been implicated in IRF3 regulation. Mutagenesis studies indicated that phosphorylation of Thr390 promotes Ser396 phosphorylation and binding to the coactivator cAMP-response element-binding protein. Taken together, our results show that IRF3 is subject to multiple interdependent phosphorylations, and we identify Thr390 as a novel in vivo phosphorylation site that modulates the phosphorylation status of TBK1-targeted Ser396.

Hepatitis C virus triggers Golgi fragmentation and autophagy through the immunity-related GTPase M

Significance Without a vaccine or cost-effective antivirals against hepatitis C virus (HCV) there is a need to understand better the molecular mechanisms underlying the establishment of productive HCV infection and chronic liver disease. Recently, the Crohns disease and tuberculosis risk factor immunity-related GTPase M (IRGM) was found to promote HCV replication, but the mechanisms involved are unknown. Here we provide mechanistic insight into how IRGM stimulates the two membrane-remodeling pathways, Golgi fragmentation and autophagy, to facilitate HCV replication. Our findings offer insights into the replication strategies used by HCV that should be useful for antiviral approaches. Also, these findings might aid understanding how IRGM functions in infection and inflammation in the setting of other diseases, e.g., in Crohn’s disease. Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.

LysoPC and PAF Trigger Arachidonic Acid Release by Divergent Signaling Mechanisms in Monocytes

Oxidized low-density lipoproteins (LDLs) play an important role during the development of atherosclerosis characterized by intimal inflammation and macrophage accumulation. A key component of LDL is lysophosphatidylcholine (lysoPC). LysoPC is a strong proinflammatory mediator, and its mechanism is uncertain, but it has been suggested to be mediated via the platelet activating factor (PAF) receptor. Here, we report that PAF triggers a pertussis toxin- (PTX-) sensitive intracellular signaling pathway leading to sequential activation of sPLA2, PLD, cPLA2, and AA release in human-derived monocytes. In contrast, lysoPC initiates two signaling pathways, one sequentially activating PLD and cPLA2, and a second parallel PTX-sensitive pathway activating cPLA2 with concomitant activation of sPLA2, all leading to AA release. In conclusion, lysoPC and PAF stimulate AA release by divergent pathways suggesting involvement of independent receptors. Elucidation of monocyte lysoPC-specific signaling mechanisms will aid in the development of novel strategies for atherosclerosis prevention, diagnosis, and therapy.