- Open Access
Inhibition of store-operated calcium entry in microglia by helminth factors: implications for immune suppression in neurocysticercosis
Journal of Neuroinflammation volume 11, Article number: 210 (2014)
Neurocysticercosis (NCC) is a disease of the central nervous system (CNS) caused by the cestode Taenia solium. The infection exhibits a long asymptomatic phase, typically lasting 3 to 5 years, before the onset of the symptomatic phase. The severity of the symptoms is thought to be associated with the intensity of the inflammatory response elicited by the degenerating parasite. In contrast, the asymptomatic phase shows an absence of brain inflammation, which is presumably due to immunosuppressive effects of the live parasites. However, the host factors and/or pathways involved in inhibiting inflammation remain largely unknown. Recently, using an animal model of NCC in which mice were intracranially inoculated with a related helminth parasite, Mesocestoides corti, we reported that Toll-like receptor (TLR)-associated signaling contributes to the development of the inflammatory response. As microglia shape the initial innate immune response in the CNS, we hypothesized that the negative regulation of a TLR-induced inflammatory pathway in microglia may be a novel helminth-associated immunosuppressive mechanism in NCC.
Methods and results
Here we report that helminth soluble factors (HSFs) from Mesocestoides corti inhibited TLR ligation-induced production of inflammatory cytokines in primary microglia. This was correlated with an inhibition of TLR-initiated upregulation of both phosphorylation and acetylation of the nuclear factor κB (NF-κB) p65 subunit, as well as phosphorylation of JNK and ERK1/2. As Ca2+ influx due to store-operated Ca2+ entry (SOCE) has been implicated in induction of downstream signaling, we tested the inhibitory effect of HSFs on agonist-induced Ca2+ influx and specific Ca2+ channel activation. We discovered that HSFs abolished the lipopolysaccharide (LPS)- or thapsigargin (Tg)-induced increase in intracellular Ca2+ accumulation by blocking the ER store release and SOCE. Moreover, electrophysiological recordings demonstrated HSF-mediated inhibition of LPS- or Tg-induced SOCE currents through both TRPC1 and ORAI1 Ca2+ channels on plasma membrane. This was correlated with a decrease in the TRPC1-STIM1 and ORAI1-STIM1 clustering at the plasma membrane that is essential for sustained Ca2+ entry through these channels.
Inhibition of TRPC1 and ORAI1 Ca2+ channel-mediated activation of NF-κB and MAPK pathways in microglia is likely a novel helminth-induced immunosuppressive mechanism that controls initiation of inflammatory response in the CNS.
Neurocysticercosis (NCC) is the most common parasitic infection of the central nervous system (CNS) caused by the cestode Taenia solium. It affects 50 million to 100 million people worldwide . As many as 50% of adult-onset seizures and 10% of the stroke cases in endemic areas are attributed to NCC ,. Treatment of NCC remains a major challenge, as the severity of the symptoms is thought to be due mainly to the inflammatory response elicited by the degenerating larvae resulting from therapeutic treatment or normal attrition ,. Interestingly, a long asymptomatic phase (lasting for years) precedes the onset of the symptomatic phase. This asymptomatic phase is characterized by little to no sign of inflammation detected around the live cysts ,,. One possible explanation could be that viable cysticerci induce immunosuppressive effects to evade the host immune response. Furthermore, loss of these effects (when the parasite dies) could lead to uncontrolled hyperinflammatory responses that contribute to tissue pathology and clinical signs and symptoms such as severe headaches, epilepsy, intracranial hypertension, focal deficit and cognitive impairment ,,. However, the mechanisms underlying the immunosuppressive events remain poorly understood.
The regulation of inflammatory responses is mediated mainly via the innate immune system through myeloid antigen-presenting cells, such as macrophages and dendritic cells. In the CNS, microglia are the native myeloid cells that shape the initial innate immune response . These cells recognize external stimuli through a wide variety of surface receptors, which culminates in release of proinflammatory cytokines and/or chemokines and effector molecules such as the antimicrobial molecules and reactive oxygen and reactive nitrogen intermediates –. Generally, the induced inflammatory response promotes the destruction of pathogens, but it can cause widespread tissue damage as a result of their overactivation ,. Thus, regulation of microglial activation may play an important role in enabling the parasite to (1) suppress host inflammation and immunity as well as the development of the asymptomatic phase and (2) contain damaging inflammation in the symptomatic phase of NCC.
Recognition of external stimuli by cells is mediated through a wide variety of pattern recognition receptors (PRRs). The Toll-like receptor (TLR) family of molecules is the major class of PRRs instrumental in the regulation of the host immune response –. Indeed, our findings using a murine experimental model of NCC suggest that the TLR signaling pathway plays a prominent role in the development of the initial hyperinflammatory response, which in turn contributes to the neuropathology and disease severity in NCC . Important unanswered questions are (1) whether neurocysticercal antigens inhibit TLR signaling-induced inflammatory cytokine production in microglia and (2) what are the mechanisms involved in this process. Helminth-induced modulation of microglial activation and regulation of production of inflammatory mediators from these brain cells is poorly understood. In this regard, the first step in agonist-induced activation of downstream signaling pathways by the pathogen or pathogen-associated molecular patterns involves immediate release of Ca2+ from intracellular endoplasmic reticulum (ER) stores, followed by subsequent store-operated Ca2+ entry (SOCE) across the plasma membrane (PM) . Such Ca2+ influx could in turn culminate in the induction of the host inflammatory response . The present study is focused on determining the immunosuppressive effect of helminth secretory or soluble factors (HSFs) to regulate TLR ligand-induced activation of the Ca2+ signaling pathway in microglia. Our results indicate that HSFs downregulate the agonist-induced inflammatory response and the activation of SOCE channels (transient receptor potential channel 1 (TRPC1) and ORAI Ca2+ release-activated Ca2+ modulator 1 (ORAI1)) and associated signaling pathways in microglia. This process likely plays an important role in regulating the initiation of the inflammatory responses in the nervous tissue during pathogenic conditions involving helminth parasites.
Animals and antigens
Maintenance of the animals and tissue collection for performing experiments were conducted according to the guidelines of the University of North Dakota system Institutional Animal Care and Use Committee, the US Department of Agriculture and the National Institutes of Health. The female BALB/c or C57BL/6 mice used in this study were purchased from Charles River Laboratories (Wilmington, MA, USA). Mesocestoides corti (M. corti) was kindly provided by Dr. De’Broski R Herbert (Division of Immunobiology, Cincinnati Children’s Research Foundation, Cincinnati, OH, USA). M. corti metacestodes were maintained in the peritoneal cavity of BALB/c mice by serial intraperitoneal infection for propagation ,. HSFS consisting of M. corti soluble factors was prepared from M. corti metacestodes in phosphate-buffered saline (PBS). Briefly, isolated metacestodes from the peritoneal cavity of infected mice were washed with 8 volumes of ice-cold Hanks’ Balanced Salt solution eight to ten times. M. corti metacestodes were suspended in 5 volumes of PBS, incubated on ice for 10 minutes and pelleted by centrifugation at 500 × g for 5 minutes, and then the supernatant was aspirated. The pelleted metacestodes were suspended in 5 volumes of PBS with protease inhibitors, and the above-mentioned procedure was repeated two more times, but additionally with freezing and thawing of the metacestodes. The supernatants were pooled together, passed through 0.2-μm filters for sterilization and used as HSFs.
Primary microglia maturation and activation
Microglia were derived from postnatal day 1 (P1) C57BL/6 mouse brains as previously described ,. Briefly, the cortex (free of meninges) was taken from P1 mice and then isolated and trypsinized. Cells were plated onto tissue culture plastic in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 with l-glutamine (DMEM/F-12; Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum and 5% heat-inactivated horse serum, and half the medium changed every third day. After about 14 days, the cultures were shaken vigorously for 30 minutes at 120 rpm on a rotary shaker to remove microglia. Microglial purity was routinely determined to be approximately 90% cells by immunofluorescence microscopy using a specific marker (Iba1 or CD11b) and by negative staining for glial fibrillary acidic protein, a marker for astrocytes. To test the inhibitory effect of HSFs on the secretion of proinflammatory cytokines, the cells were plated at 8 × 104 cells per well in 96-well flat-bottomed plates and were stimulated with medium alone or in the presence of HSFs, TLR ligands or HSF/TLR ligands. Culture supernatants were collected 24 hours after stimulation, and tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions (BD OptEIA; BD Biosciences, San Jose, CA, USA).
Assessment of microglia by MTT assay
Primary microglia were treated with medium alone, HSF, lipopolysaccharide (LPS), or HSF and LPS, and cell viability was measured using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay according to the manufacturer’s instructions (Sigma-Aldrich, St Louis, MO, USA).
Microglia were plated at 1 × 106 cells on collagen- and poly-d-lysine–coated, 35-mm, glass-bottomed culture dishes (MatTek, Ashland, MA, USA) at 37°C. After a 2- to 4-hour incubation, cells were washed twice with standard extracellular solution (SES) (145 mM NaCl, 5 mM CsCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10 mM glucose, pH 7.4, with NaOH, containing 0.02% soybean trypsin inhibitor and 0.1% bovine serum albumin and collagenase P (2.5 mg/8 ml of buffer)) for 15 to 20 minutes at 37°C. Cells were incubated in SES buffer containing 2 μM Fura-2 acetoxymethyl ester (Fura-2 AM) for 45 to 60 minutes at 37°C ,. Before we performed Ca2+ measurements, the culture dishes were washed with and placed in Ca2+-free SES buffer. Cells were stimulated with medium alone or with 25 μg/ml HSF for 20 minutes before addition of thapsigargin (Tg) or LPS. Tg is a sarco(endo)plasmic reticulum Ca2+ ATPase pump blocker that does not engage any PRRs on the cell membrane. Fluorescence measurements were performed by imaging the Fura-2 AM–loaded microglia using an Olympus IX50 inverted microscope (Olympus America, Center Valley, PA, USA) with excitation light provided by a Polychrome IV monochromator (TILL Photonics, Hillsboro, OR, USA) ,. Images were acquired using a Photometrics CoolSNAP HQ charge-coupled device camera (Photometrics, Tucson, AZ, USA) and MetaFluor software (Molecular Devices, Sunnyvale, CA, USA). Relative average intracellular Ca2+ concentration values are from at least 30 to 40 microglia and are representative of results obtained in at least three or four individual experiments.
All electrophysiological experiments were performed according to a previously published protocol –. Coverslips with freshly isolated microglia were transferred to the recording chamber and perfused continually through a custom-designed, gravity-driven, speed-controlled system at a rate of 5 ml/min with an external Ringer’s solution (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.4, with NaOH). The patch pipette had resistances between 3 mΩ and 5 mΩ after filling with the standard intracellular solution (145 mM cesium methane sulfonate, 8 mM NaCl, 10 mM MgCl2, 10 mM HEPES, 10 mM ethylene glycol tetraacetic acid, pH 7.2, with CsOH). Osmolarity for all solutions was adjusted with d-mannitol to 305 ± 5 mmol/kg using a VAPRO pressure osmometer (Wescor/ELITech Group, Princeton, NJ, USA). Patch-clamp experiments were performed in the tight-seal, whole-cell configuration at room temperature (22°C to 25°C) using an Axopatch 200B amplifier (Molecular Devices). Voltage ramps ranging from −90 to 90 mV over a period of 1 second were imposed every 4 seconds from a holding potential of 0 mV and digitized at a rate of 1 kHz. A liquid junction potential of <8 mV was not corrected, and capacitive currents and series resistance were determined and minimized. For analysis, the first ramp was used for leak subtraction for the subsequent current records. Currents were normalized to the initial size of the cell to obtain current densities (pA/pF).
Microglia at 1.5 × 106 per well in 12-well plate were pulsed with medium alone or with HSF for 20 minutes, followed by addition of LPS or medium alone. Cells were solubilized in 100 μl of 2× SDS-PAGE sample buffer, and 30 μl of extracts were resolved on 12% SDS-PAGE, transferred onto an Immobilon polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA) for immunostaining. Blots were incubated with primary antibodies (Abs) against phosphorylated nuclear factor κB (NF-κB; p65 subunit), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) or acetylated NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA). For loading controls, blots were incubated with primary Abs against a ubiquitously expressed protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Bands were visualized using corresponding secondary horseradish peroxidase–conjugated Abs and standard enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA).
Coimmunoprecipitation and Western blot analyses
Coimmunoprecipitation and Western blot analyses were carried out as described elsewhere ,. Microglia were pulsed with medium alone or with HSF at 25 μg/ml for 20 minutes, followed by addition of 2 μM Tg, LPS or dimethyl sulfoxide (0.1% vol/vol) for 5 minutes at 37°C, and then they were washed with ice-cold PBS and lysed in 1× radioimmunoprecipitation assay buffer supplemented with 0.1% SDS, 1% Triton X-100, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride and 1× protease and phosphatase inhibitors. Protein concentrations were adjusted to 1 mg/ml and immunoprecipitated with Abs against stromal interaction molecule 1 (anti-STIM1) . Immunocomplexes were separated using Protein A Agarose Plus beads (Pierce Biotechnology, Rockford, IL, USA), eluted with 50 μl of 1× SDS dye and resolved in 4–12% SDS-PAGE gels (Bio-Rad Laboratories), followed by Western blotting as described previously using anti-TRPC1, anti-ORAI1 and anti-STIM1 Abs ,.
We used the Student’s t-test and one-way analysis of variance for comparison of mean values in different groups (SigmaPlot 8.0 software (Systat Software, San Jose, CA, USA)). A P-value <0.05 was considered to be statistically significant.
Helminth soluble factors inhibit Toll-like receptor ligand-induced activation of microglia
The effect of HSFs on activation of primary microglia was tested in vitro. HSFs alone did not modulate expression of the inflammatory cytokines IL-6 and TNF-α (Figure 1 and data not shown). We then tested their immunosuppressive effects on agonist-induced activation of microglia. As expected, exposure of microglia to the TLR ligands Pam3Cys4 (TLR1/2 ligand), double-stranded RNA (dsRNA; TLR3 ligand), LPS (TLR4 ligand), single-stranded RNA (ssRNA; TLR7/8 ligand) and CpG site DNA (TLR9 ligand) led to upregulated secretion of the inflammatory cytokines IL-6 and TNF-α (Figure 1A and 1B). This was correlated with complete inhibition of TLR activation-induced mRNA expression by HSFs in microglia cells (data not shown). Interestingly, coexposure with HSFs led to complete inhibition of TLR ligand-induced secretion of IL-6 and TNF-α cytokines by microglia (Figure 1A and 1B). HSFs inhibited LPS-induced cytokine secretion in a concentration-dependent manner (Figure 1C). This inhibition by HSFs was abolished when glycosidase was used to modify the glycan constituent (Figure 1D), indicating the specificity of this effect and possible involvement of glycan moieties in this process. To further exclude nonspecific effects of HSFs on cell death, which may indirectly contribute to HSF-induced inhibition of agonist-elicited activation, the cell viability of microglia was assessed by MTT assay. A similar level of cell viability was observed after stimulation of microglia with HSFs alone or upon exposure to LPS in the presence or absence of HSFs (Figure 1E). This further attested to the specificity of the immunosuppressive effect of HSFs. These results demonstrate that, though HSF alone does not elicit microglial activation, it efficiently inhibits TLR ligand-induced production of inflammatory mediators in microglia.
HSF inhibits activation of LPS-induced NF-κB and MAPK activity
The cascade of events that follows the TLR activation that leads to NF-κB and mitogen-activated protein kinase (MAPK) activation culminates in the production of inflammatory mediators ,. We examined the effect of HSF on LPS-induced NF-κB and MAPK activity by measuring phosphorylation and acetylation of NF-κB p65, as well as phosphorylation of JNK and ERK1/2, all of which are important downstream signaling components activated by TLR ligation . LPS stimulation increased both the phosphorylation and acetylation of p65 (Figure 2A and 2C), which was inhibited by HSF (Figure 2B and 2C). Similarly, LPS stimulation increased the phosphorylation of both JNK and ERK1/2 (Figure 2A and 2B), which was decreased by HSF exposure (Figure 2B and 2C). Further, the GAPDH level was unaffected in control cells or in those stimulated with LPS, HSF or LPS and HSF together (Figure 2A and 2B). Thus, HSF-induced suppression of the LPS-initiated inflammatory response correlated with an inhibition of induced NF-κB and MAPK activity.
Helminth soluble factor inhibits activation induced Ca2+entry
Ca2+ entry through SOCE channels has been implicated in the activation of signaling pathways essential for the inflammatory response elicited after cellular exposure to stimuli ,. Thus, we performed Ca2+ imaging and electrophysiological experiments to determine whether HSFs affect the Ca2+ signaling pathway in primary microglia. Exposure to HSFs alone did not produce any substantial changes in cytosolic Ca2+ levels in microglia (data not shown). However, LPS stimulation of microglia in a Ca2+-free buffer resulted in an increase in cytosolic Ca2+ levels due to the release of internal ER Ca2+ (left peak (arrow) in Figure 3A; quantitative data are shown in Figure 3B) and in terms of Ca2+ influx through the PM (right peak (arrow) in Figure 3A; quantitative data are shown in Figure 3B) due to ER store depletion-induced SOCE. HSF-treated microglia showed a significant decrease in the cytosolic Ca2+ level in response to LPS stimulation (Figure 3). This was evidenced by a reduced LPS-stimulated cytosolic Ca2+ influx without a significant change in the ER Ca2+ levels (Figure 3), suggesting that HSFs block activation-induced Ca2+ channel activation in the PM and associated Ca2+ influx. Next, to test the specificity of HSFs affecting induced Ca2+ influx and/or channel activation, we examined if HSFs have any effect on Tg-induced activation of intracellular Ca2+ turnover. Stimulation of microglia with Tg induced both Ca2+ release at the ER and Ca2+ influx at the PM (left peak (arrow) in Figure 3C). Interestingly, exposure of microglia to HSFs abolished this Tg-induced Ca2+ turnover (right peak (arrow) in Figure 3C). This inhibition of Tg-induced SOCE through the PM by treatment with HSFs was statistically significant (Figure 3D).
Next, electrophysiological recordings were performed to identify the SOCE channel involved in microglial cells. As shown in Figure 4A and 4B, addition of LPS initiated a nonselective Ca2+ current that reversed between 0 to 5 mV and was partially inward-rectifying. Furthermore, the Ca2+ current was inhibited in cells that were pretreated with the HSFs (Figure 4A and 4B), a finding which was statistically significant (Figure 4C). Additionally, the current-voltage (I-V) properties of the channel were different, as observed for both TRPC1-dependent store-operated current (I SOC) and ORAI1-mediated Ca2+ release-activated Ca2+ current , suggesting that the channel is probably a mixed channel that consists of TRPC1 and ORAI1. Exposure of cells to HSF not only decreased Ca2+ currents (Figure 4A, 4B and 4C) but also made the current slightly inward-rectifying (Figure 4B), again suggesting that this might be a mixed channel. Furthermore, addition of Tg, which also initiates ER Ca2+ release and induces SOCE, was significantly decreased in microglial cells exposed to HSF (Figure 4D, 4E and 4F). Furthermore, similarly to LPS, the I-V properties of the channel-activated/Ca2+ currents induced by Tg and inhibited by HSF in microglia are consistent with both TRPC1- and ORAI1-dependent Ca2+ current. To confirm the inhibition of TRPC1 and ORAI1 functions in the Ca2+ influx by HSF, biochemical analyses were performed. In this regard, interaction of STIM1 (the ER Ca2+ sensor protein) with channels such as TRPC1 and ORAI1 on PM is essential for their activation and subsequent SOCE . Thus, coimmunoprecipitation was performed using STIM1 Abs to pull down PM proteins bound to it in microglia stimulated with LPS or Tg with or without HSF. This was followed by immunoblot analysis in which we specific Abs against TRPC1 and ORAI1. As shown in Figure 5, the unstimulated cells and the cells exposed to HSF alone did not display any difference in TRPC1-STIM1 or ORAI1-STIM1 association. On the other hand, microglial stimulation with LPS (Figure 5A and 5B) or Tg (Figure 5C and 5D) alone led to a significant increase in TRPC1-STIM1 or ORAI1-STIM1 association. However, upon treatment with HSF, both LPS- and Tg-induced TRPC1-STIM1 or ORAI1-STIM1 association reverted back to basal control levels (Figure 5). Further, immunoblot analysis revealed that the stimulation had no measurable effect on STIM1 levels, as compared to the untreated microglia (Figure 5A and 5C). Together, these data confirm that HSF inhibits agonist-induced increases in TRPC1- and ORAI1-dependent Ca2+ influx through PM in microglia.
Inflammatory disorders can be triggered by overactive immune responses directed against host and/or self-tissues. It is now evident that a wide range of neuroinflammation-associated disorders, such as infections of the CNS, stroke and cognitive disorders, and even classic mental illnesses, such as schizophrenia and depression, can be triggered and/or exacerbated by overactive immune responses against nervous tissues –. Accordingly, agents that suppress inflammatory responses may provide a means by which to ameliorate the pathologies associated with many of these disorders. In this regard, it is remarkable that parasitic helminths are master regulators of host inflammatory response and pathology . In the case of NCC, it is presumed that factors associated with the live stage of helminths likely inhibit CNS inflammation and hence induce an asymptomatic phase lasting 3 to 5 years ,. In contrast, the loss of immunosuppressive effects when the parasite dies is thought to lead to an uncontrolled, detrimental, hyperinflammatory response that contributes to tissue pathology and clinical manifestations such as epileptic seizures, strokes and severe neurological symptoms in NCC ,,,. However, the molecular mechanisms by which the helminths and their products inhibit CNS inflammation are not well understood. In this study, we investigated the mechanism of helminth-induced inhibition of innate immune pathway activation in microglia. To our knowledge, this study is the first to demonstrate helminth-mediated inhibition of activation of ORAI1 and TRPC1 Ca2+ channel function in microglia. The results reported here strongly support the hypothesis that the downregulation of Ca2+ channel activation and associated signaling by helminths is involved in blocking innate immune pathway activation-induced inflammatory response in microglia and in the CNS in general.
The CNS is regarded as an immune-privileged site because of the absence of a defined lymphatic drainage system and the presence of the blood–brain barrier (BBB) . However, the CNS possesses active immune processes and regulatory mechanisms. In this context, among all the nervous tissue cell types, microglia are thought to be the main cell type of the innate immune system in the brain and are commonly associated with immune reactions. During infection, recognition of microbial molecules by microglial PRRs leads to production of inflammatory mediators such as chemokines and cytokines ,. Thus, activation of microglia plays an important role in leukocyte trafficking into the brain through its effects on the BBB, eventuating in pathogen-specific adaptive immune responses in the CNS . However, aberrant regulation of microglia-elicited inflammatory responses has been implicated in the majority of neurodegenerative disorders –. In this regard, increasing evidence also indicates that, among the PRRs, the TLR family of molecules plays a major role in several inflammatory CNS pathologies –. TLR signaling leads to production of host inflammatory mediators, which in turn plays a significant role in leukocyte trafficking into the CNS . Indeed, induction of TLRs in the infected and/or diseased brain for extended periods is integral to persistent inflammation and its associated pathology ,,. The results of our previous studies in murine models of NCC suggest that TLRs 1 through 13 exhibit differential expression and regulation in normal and NCC mouse brains, with TLRs 2, 3, 4, 6, 7, 8 and 9 expressed on CD11b + myeloid cells ,. Moreover, MyD88-deficient mice display a reduction in infiltration by immune cells as well as proinflammatory cytokine responses in the CNS that coincides with reduced neuropathology and disease severity .
To the best of our knowledge, we are the first to report clear results showing that helminth factors directly inhibit both MyD88-dependent (exposure to LPS, Pam3Cys4, ssRNA or CpG site DNA) and MyD88-independent (exposure to dsRNA) TLR signaling pathway activation-associated inflammatory cytokine production in microglia. This inhibitory effect is dependent on the concentration of the immunosuppressive helminth factors and TLR ligands used. Interestingly, the inhibition of the LPS-induced effect by HSF was abrogated when was exposed to glycosidase, suggesting the specificity of the effect as well as the possible involvement of glycan factors in the parasite-induced immune modulation. Helminths (worms), in their pursuit to establish long-term infections and survival in the host, use glycans, which are abundant on their surface (tegument) and in their excretory and/or secretory products ,, to regulate and suppress host immune responses. Indeed, both M. corti and the human parasite Taenia solium are complex organisms, and, during the infection process, they release many molecules, including glycans –. Interestingly, in the human brain, the presence of glycan antigens in CD68+ myeloid cells (presumably macrophages and/or microglia) has been shown to correlate with a lack of surface expression of the activation and/or maturation marker major histocompatibility complex (MHC) class II ,. During both human and murine NCC, the glycan antigens are released from the parasite and are taken up by immune cells (in areas around the metacestode) that typically display a lack of maturation of antigen-presenting cells by way of reduced expression of MHC class II . We expect that novel immunosuppressive molecules in HSF (for example, glycans) are released from live parasites and have profound effects on the net proinflammatory response in NCC ,,. In contrast, our unpublished observations indicate that somatic antigen preparations from M. corti can induce proinflammatory cytokine production. This is supported by earlier studies showing that DNA and lipid antigens of parasites such as Trypanosoma cruzi and Plasmodium falciparum induce proinflammatory cytokine production ,. In NCC patients, T. solium DNA could be detected in the cerebrospinal fluid of symptomatic NCC patients ,.
Characterization of the specific neurocysticercal antigens and host PRR interactions, as well as subsequent development of both inflammatory and immunosuppressive mechanisms, is a major focus of the current research in our laboratory. Taking our findings in toto, we speculate that the HSF-associated immunosuppressive helminth molecules are released from live larvae in the brain which interact with receptors on innate cells such as microglia and activate inhibitory signaling events, leading to failure of induction of the host inflammatory response. As the infection progresses, some of the parasites die, and the somatic antigens thus released activate a TLR-dependent inflammatory response. It is likely that the intensity of the polarized response elicited by these factors from live/dead parasites shapes the net inflammatory response in the CNS and determines the overall neuropathology and disease symptoms, or the lack thereof.
Cellular activation invariably involves Ca2+ signaling to regulate functions such as proliferation, migration, phagocytosis and gene transcription of inflammatory mediators . Ca2+ entry across the PM is mediated by various ion channels, including SOCE and voltage-gated Ca2+ channels . However, Ca2+ entry through SOCE channels seems to play an important role in activation of nonexcitable cells such as microglia ,. We and others have previously demonstrated that the ORAI1–STIM1 and TRPC1–STIM1 interactions on PM mediate opening of the ORAI1 and TRPC1 channels and Ca2+ influx ,–. In our present study, we demonstrate that, though helminth factors themselves do not modulate basal levels of cytosolic Ca2+ turnover or channel activation, they can abolish TLR ligand (LPS)- and Tg-induced increases in Ca2+ influx as well as TRPC1–STIM1 and ORAI1–STIM1 interactions on PM of microglia. To the best of our knowledge, this report is the first to describe the involvement of TRPC1 SOCE channel activation in Ca2+ turnover in activated microglia. Moreover, the regulation of SOCE Ca2+ channel activation in microglia by parasites is likely a novel immunosuppressive mechanism that blocks the initiation of the inflammatory pathway in the CNS.
There is convincing evidence that Ca2+ entry through SOCE channels differentially activates different downstream signaling pathways. This is supported by our earlier studies showing contributions of TRPC1-mediated SOCE induced by TRPC1-STIM1 interactions to NF-κB activation ,. The data we report here demonstrate that HSF-stimulated microglia exhibited significantly reduced levels of LPS-induced phosphorylation and acetylation of NF-κB (p65 subunit), as well as phosphorylation of ERK1/2 and JNK, as determined by immunoblot analysis. HSF efficiently inhibits both MyD88-dependent and MyD88-independent production of inflammatory mediators after TLR ligation. As a large body of literature indicates that TLR ligands activate protein kinase C (PKC) as well as other Ca2+-sensitive signaling mediators upstream of MAPK and NF-κB activation –, one possibility is that HSF-induced inhibition of TRPC1 or ORAI1 SOCE may directly mediate downregulation of MAPK and NF-κB activation elicited by TLR ligands in both a MyD88-dependent and MyD88-independent manner, as a result of an upstream inhibition of the kinases, such as by the PKC that activates them. An additional possibility could be that downregulation of MAPK activities indirectly inhibit SOCE channel function. This hypothesis is supported by findings that the phosphorylation of STIM1 by ERK1/2 at residues Ser575, Ser608 and Ser621 was required to trigger full ORAI1 SOCE . Along the same line, our results in the present study suggest that HSF-mediated inhibition of ERK1/2 activation and STIM1 phosphorylation may further augment the inhibitory effect on Ca2+ entry, particularly through the ORAI1 channel. Another interesting possibility could be that HSF directly binds to SOCE channels and inhibits their function, as our results from electrophysiological analysis show a HSF-induced instant decrease in I SOC-like Ca2+ current associated with TRPC1 SOCE channels in LPS- or Tg-stimulated cells (data not shown). However, further studies are required to characterize specific immunosuppressive parasitic molecules that might directly interact with SOCE channels to inhibit agonist-induced activation and Ca2+ entry. This is a major focus of the current research in our laboratory.
Collectively, the results of the present study demonstrate that HSFs suppress TLR ligand-induced MAPK and NF-κB activation, as well as inflammatory cytokine production, in primary microglia, which might have implications for the manifestation of the asymptomatic phase in NCC. Importantly, the negative regulation of TRPC1 SOCE and ORAI1 SOCE channel activation by HSFs in microglia represents a novel immunosuppressive mechanism to block the initiation of the inflammatory pathway in the CNS and is a completely unexplored area of research. Further characterization of immunosuppressive mechanisms involved could lead to novel therapeutic targets, as Ca2+-mediated, deregulated inflammatory response underlies the pathophysiological processes of many chronic inflammatory and autoimmune diseases.
Central nervous system
Extracellular signal-regulated kinase 1/2
Fura-2 acetoxymethyl ester
glyceraldehyde 3-phosphate dehydrogenase
Helminth secretory or soluble factor
c-Jun N-terminal kinase
Mitogen-activated protein kinase
Nuclear factor κB
ORAI Ca2+ release-activated Ca2+ modulator 1
Store-operated Ca2+ entry
Stromal interaction molecule 1
Tumor necrosis factor α
Transient receptor potential channel 1
Nash TE, Singh G, White AC, Rajshekhar V, Loeb JA, Proaño JV, Takayanagui OM, Gonzalez AE, Butman JA, DeGiorgio C, Del Brutto OH, Delgado-Escueta A, Evans CA, Gilman RH, Martinez SM, Medina MT, Pretell EJ, Teale J, Garcia HH: Treatment of neurocysticercosis: current status and future research needs. Neurology. 2006, 67: 1120-1127. 10.1212/01.wnl.0000238514.51747.3a.
White AC: Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management. Annu Rev Med. 2000, 51: 187-206. 10.1146/annurev.med.51.1.187.
White AC: Neurocysticercosis: a major cause of neurological disease worldwide. Clin Infect Dis. 1997, 24: 101-115. 10.1093/clinids/24.2.101.
Robinson P, Atmar RL, Lewis DE, White AC: Granuloma cytokines in murine cysticercosis. Infect Immun. 1997, 65: 2925-2931.
Restrepo BI, Alvarez JI, Castaño JA, Arias LF, Restrepo M, Trujillo J, Colegial CH, Teale JM: Brain granulomas in neurocysticercosis patients are associated with a Th1 and Th2 profile. Infect Immun. 2001, 69: 4554-4560. 10.1128/IAI.69.7.4554-4560.2001.
White AC, Robinson P, Kuhn R: Taenia solium cysticercosis: host–parasite interactions and the immune response. Chem Immunol. 1997, 66: 209-230. 10.1159/000058663.
Rivest S: Regulation of innate immune responses in the brain. Nat Rev Immunol. 2009, 9: 429-439. 10.1038/nri2565.
Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol. 2003, 21: 335-376. 10.1146/annurev.immunol.21.120601.141126.
Dabbagh K, Lewis DB: Toll-like receptors and T-helper-1/T-helper-2 responses. Curr Opin Infect Dis. 2003, 16: 199-204. 10.1097/00001432-200306000-00003.
Cherry JD, Olschowka JA, O’Banion MK: Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation. 2014, 11: 98-10.1186/1742-2094-11-98.
Geijtenbeek TB, van Vliet SJ, Engering A, ‘t Hart BA, van Kooyk Y: Self- and nonself-recognition by C-type lectins on dendritic cells. Annu Rev Immunol. 2004, 22: 33-54. 10.1146/annurev.immunol.22.012703.104558.
Medzhitov R: Toll-like receptors and innate immunity. Nat Rev Immunol. 2001, 1: 135-145. 10.1038/35100529.
Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature. 1998, 392: 245-252. 10.1038/32588.
Mishra BB, Gundra UM, Wong K, Teale JM: MyD88-deficient mice exhibit decreased parasite-induced immune responses but reduced disease severity in a murine model of neurocysticercosis. Infect Immun. 2009, 77: 5369-5379. 10.1128/IAI.00455-09.
Pani B, Bollimuntha S, Singh BB: The TR (i)P to Ca2+ signaling just got STIMy: an update on STIM1 activated TRPC channels. Front Biosci. 2012, 17: 805-823. 10.2741/3958.
Putney JW, Broad LM, Braun FJ, Lievremont JP, Bird GS: Mechanisms of capacitative calcium entry. J Cell Sci. 2001, 114: 2223-2229.
Alvarez JI, Mishra BB, Gundra UM, Mishra PK, Teale JM: Mesocestoides corti intracranial infection as a murine model for neurocysticercosis. Parasitology. 2010, 137: 359-372. 10.1017/S0031182009991971.
Floden AM, Li S, Combs CK: β-amyloid-stimulated microglia induce neuron death via synergistic stimulation of tumor necrosis factor α and NMDA receptors. J Neurosci. 2005, 25: 2566-2575. 10.1523/JNEUROSCI.4998-04.2005.
Nagamoto-Combs K, Combs CK: Microglial phenotype is regulated by activity of the transcription factor, NFAT (nuclear factor of activated T cells). J Neurosci. 2010, 30: 9641-9646. 10.1523/JNEUROSCI.0828-10.2010.
Liu X, Cheng KT, Bandyopadhyay BC, Pani B, Dietrich A, Paria BC, Swaim WD, Beech D, Yildrim E, Singh BB, Birnbaumer L, Ambudkar IS: Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1−/− mice. Proc Natl Acad Sci U S A. 2007, 104: 17542-17547. 10.1073/pnas.0701254104.
Pani B, Cornatzer E, Cornatzer W, Shin DM, Pittelkow MR, Hovnanian A, Ambudkar IS, Singh BB: Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier’s disease. Mol Biol Cell. 2006, 17: 4446-4458. 10.1091/mbc.E06-03-0251.
Singh BB, Zheng C, Liu X, Lockwich T, Liao D, Zhu MX, Birnbaumer L, Ambudkar IS: Trp1-dependent enhancement of salivary gland fluid secretion: role of store-operated calcium entry. FASEB J. 2001, 15: 1652-1654.
Pani B, Ong HL, Liu X, Rauser K, Ambudkar IS, Singh BB: Lipid rafts determine clustering of STIM1 in endoplasmic reticulum–plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE). J Biol Chem. 2008, 283: 17333-17340. 10.1074/jbc.M800107200.
Pani B, Ong HL, Brazer SC, Liu X, Rauser K, Singh BB, Ambudkar IS: Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1. Proc Natl Acad Sci U S A. 2009, 106: 20087-20092. 10.1073/pnas.0905002106.
Pani B, Liu X, Bollimuntha S, Cheng KT, Niesman IR, Zheng C, Achen VR, Patel HH, Ambudkar IS, Singh BB: Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1. J Cell Sci. 2013, 126: 667-675. 10.1242/jcs.118943.
Mishra BB, Gundra UM, Teale JM: Toll-like receptors in CNS parasitic infections. Curr Top Microbiol Immunol. 2009, 336: 83-104.
Palm NW, Medzhitov R: Pattern recognition receptors and control of adaptive immunity. Immunol Rev. 2009, 227: 221-233. 10.1111/j.1600-065X.2008.00731.x.
Izquierdo JH, Bonilla-Abadía F, Cañas CA, Tobón GJ: Calcium, channels, intracellular signaling and autoimmunity. Reumatol Clin. 2014, 10: 43-47. 10.1016/j.reuma.2013.05.008.
Feske S: Calcium signalling in lymphocyte activation and disease. Nat Rev Immunol. 2007, 7: 690-702. 10.1038/nri2152.
Debnath M, Venkatasubramanian G: Recent advances in psychoneuroimmunology relevant to schizophrenia therapeutics. Curr Opin Psychiatry. 2013, 26: 433-439. 10.1097/YCO.0b013e328363b4da.
Morales I, Guzmán-Martínez L, Cerda-Troncoso C, Farías GA, Maccioni RB: Neuroinflammation in the pathogenesis of Alzheimer’s disease: a rational framework for the search of novel therapeutic approaches. Front Cell Neurosci. 2014, 8: 112.
Amor S, Puentes F, Baker D, van der Valk P: Inflammation in neurodegenerative diseases. Immunology. 2010, 129: 154-169. 10.1111/j.1365-2567.2009.03225.x.
Hewitson JP, Grainger JR, Maizels RM: Helminth immunoregulation: the role of parasite secreted proteins in modulating host immunity. Mol Biochem Parasitol. 2009, 167: 1-11. 10.1016/j.molbiopara.2009.04.008.
Ransohoff RM, Kivisäkk P, Kidd G: Three or more routes for leukocyte migration into the central nervous system. Nat Rev Immunol. 2003, 3: 569-581. 10.1038/nri1130.
Chavarria A, Alcocer-Varela J: Is damage in central nervous system due to inflammation?. Autoimmun Rev. 2004, 3: 251-260. 10.1016/j.autrev.2003.09.006.
Ransohoff RM, Liu L, Cardona AE: Chemokines and chemokine receptors: multipurpose players in neuroinflammation. Int Rev Neurobiol. 2007, 82: 187-204. 10.1016/S0074-7742(07)82010-1.
Nguyen MD, Julien JP, Rivest S: Innate immunity: the missing link in neuroprotection and neurodegeneration?. Nat Rev Neurosci. 2002, 3: 216-227. 10.1038/nrn752.
Qian L, Flood PM, Hong JS: Neuroinflammation is a key player in Parkinson’s disease and a prime target for therapy. J Neural Transm. 2010, 117: 971-979. 10.1007/s00702-010-0428-1.
Lull ME, Block ML: Microglial activation and chronic neurodegeneration. Neurotherapeutics. 2010, 7: 354-365. 10.1016/j.nurt.2010.05.014.
McGeer PL, McGeer EG: Glial reactions in Parkinson’s disease. Mov Disord. 2008, 23: 474-483. 10.1002/mds.21751.
Orr CF, Rowe DB, Halliday GM: An inflammatory review of Parkinson’s disease. Prog Neurobiol. 2002, 68: 325-340. 10.1016/S0301-0082(02)00127-2.
Block ML, Hong JS: Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol. 2005, 76: 77-98. 10.1016/j.pneurobio.2005.06.004.
Kielian T, Esen N, Bearden ED: Toll-like receptor 2 (TLR2) is pivotal for recognition of S. aureus peptidoglycan but not intact bacteria by microglia. Glia. 2005, 49: 567-576. 10.1002/glia.20144.
Koedel U, Rupprecht T, Angele B, Heesemann J, Wagner H, Pfister HW, Kirschning CJ: MyD88 is required for mounting a robust host immune response to Streptococcus pneumoniae in the CNS. Brain. 2004, 127: 1437-1445. 10.1093/brain/awh171.
Turrin NP, Rivest S: Innate immune reaction in response to seizures: implications for the neuropathology associated with epilepsy. Neurobiol Dis. 2004, 16: 321-334. 10.1016/j.nbd.2004.03.010.
Mishra BB, Mishra PK, Teale JM: Expression and distribution of Toll-like receptors in the brain during murine neurocysticercosis. J Neuroimmunol. 2006, 181: 46-56. 10.1016/j.jneuroim.2006.07.019.
Cummings RD, Nyame AK: Glycobiology of schistosomiasis. FASEB J. 1996, 10: 838-848.
Khoo KH, Dell A: Glycoconjugates from parasitic helminths: structure diversity and immunobiological implications. Adv Exp Med Biol. 2001, 491: 185-205. 10.1007/978-1-4615-1267-7_14.
Alvarez JI, Rivera J, Teale JM: Differential release and phagocytosis of tegument glycoconjugates in neurocysticercosis: implications for immune evasion strategies. PLoS Negl Trop Dis. 2008, 2: e218-10.1371/journal.pntd.0000218.
Sciutto E, Chavarria A, Fragoso G, Fleury A, Larralde C: The immune response in Taenia solium cysticercosis: protection and injury. Parasite Immunol. 2007, 29: 621-636. 10.1111/j.1365-3024.2007.00967.x.
Tsang VC, Brand JA, Boyer AE: An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J Infect Dis. 1989, 159: 50-59. 10.1093/infdis/159.1.50.
Restrepo BI, Obregón-Henao A, Mesa M, Gil DL, Ortiz BL, Mejía JS, Villota GE, Sanzón F, Teale JM: Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int J Parasitol. 2000, 30: 689-696. 10.1016/S0020-7519(00)00057-6.
Haslam SM, Restrepo BI, Obregón-Henao A, Teale JM, Morris HR, Dell A: Structural characterization of the N-linked glycans from Taenia solium metacestodes. Mol Biochem Parasitol. 2003, 126: 103-107. 10.1016/S0166-6851(02)00250-5.
Correa D, Dalma D, Espinoza B, Plancarte A, Rabiela MT, Madrazo I, Gorodezky C, Flisser A: Heterogeneity of humoral immune components in human cysticercosis. J Parasitol. 1985, 71: 535-541. 10.2307/3281420.
Grewal JS, Kaur S, Bhatti G, Sawhney IM, Ganguly NK, Mahajan RC, Malla N: Cellular immune responses in human neurocysticercosis. Parasitol Res. 2000, 86: 500-503. 10.1007/s004360050701.
Bafica A, Santiago HC, Goldszmid R, Ropert C, Gazzinelli RT, Sher A: Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection. J Immunol. 2006, 177: 3515-3519. 10.4049/jimmunol.177.6.3515.
Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, Visintin A, Halmen KA, Lamphier M, Olivier M, Bartholomeu DC, Gazzinelli RT, Golenbock DT: Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A. 2007, 104: 1919-1924. 10.1073/pnas.0608745104.
Hernández M, Gonzalez LM, Fleury A, Saenz B, Parkhouse RM, Harrison LJ, Garate T, Sciutto E: Neurocysticercosis: detection of Taenia solium DNA in human cerebrospinal fluid using a semi-nested PCR based on HDP2. Ann Trop Med Parasitol. 2008, 102: 317-323. 10.1179/136485908X278856.
Almeida CR, Ojopi EP, Nunes CM, Machado LR, Takayanagui OM, Livramento JA, Abraham R, Gattaz WF, Vaz AJ, Dias-Neto E: Taenia solium DNA is present in the cerebrospinal fluid of neurocysticercosis patients and can be used for diagnosis. Eur Arch Psychiatry Clin Neurosci. 2006, 256: 307-310. 10.1007/s00406-006-0612-3.
Farber K, Kettenmann H: Functional role of calcium signals for microglial function. Glia. 2006, 54: 656-665. 10.1002/glia.20412.
Sun Y, Sukumaran P, Bandyopadhyay BC, Singh BB: Physiological function and characterization of TRPCs in neurons. Cells. 2014, 3: 455-475. 10.3390/cells3020455.
Parekh AB, Putney JW: Store-operated calcium channels. Physiol Rev. 2005, 85: 757-810. 10.1152/physrev.00057.2003.
Ohana L, Newell EW, Stanley EF, Schlichter LC: The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia. Channels (Austin). 2009, 3: 129-139. 10.4161/chan.3.2.8609.
Muik M, Frischauf I, Derler I, Fahrner M, Bergsmann J, Eder P, Schindl R, Hesch C, Polzinger B, Fritsch R, Kahr H, Madl J, Gruber H, Groschner K, Romanin C: Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem. 2008, 283: 8014-8022. 10.1074/jbc.M708898200.
Navarro-Borelly L, Somasundaram A, Yamashita M, Ren D, Miller RJ, Prakriya M: STIM1–Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy. J Physiol. 2008, 586: 5383-5401. 10.1113/jphysiol.2008.162503.
Wu MM, Buchanan J, Luik RM, Lewis RS: Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol. 2006, 174: 803-813. 10.1083/jcb.200604014.
Liou J, Fivaz M, Inoue T, Meyer T: Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion. Proc Natl Acad Sci U S A. 2007, 104: 9301-9306. 10.1073/pnas.0702866104.
Park CY, Hoover PJ, Mullins FM, Bachhawat P, Covington ED, Raunser S, Walz T, Garcia KC, Dolmetsch RE, Lewis RS: STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell. 2009, 136: 876-890. 10.1016/j.cell.2009.02.014.
Luik RM, Wang B, Prakriya M, Wu MM, Lewis RS: Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature. 2008, 454: 538-542. 10.1038/nature07065.
Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J, Pani B, Gwack Y, Srikanth S, Singh BB, Gill DL, Ambudkar IS: Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx: evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem. 2007, 282: 9105-9116. 10.1074/jbc.M608942200.
Chano F, Descoteaux A: Modulation of lipopolysaccharide-induced NF-IL6 activation by protein kinase C-α in a mouse macrophage cell line. Eur J Immunol. 2002, 32: 2897-2904. 10.1002/1521-4141(2002010)32:10<2897::AID-IMMU2897>3.0.CO;2-J.
Chen BC, Hsieh SL, Lin WW: Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages. J Leukoc Biol. 2001, 69: 280-288.
Chen CC, Wang JK, Lin SB: Antisense oligonucleotides targeting protein kinase C-α, -βI, or -δ but not -η inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor κΒ-dependent mechanism. J Immunol. 1998, 161: 6206-6214.
Fujihara M, Connolly N, Ito N, Suzuki T: Properties of protein kinase C isoforms (βII, ε, and ζ) in a macrophage cell line (J774) and their roles in LPS-induced nitric oxide production. J Immunol. 1994, 152: 1898-1906.
Giroux M, Descoteaux A: Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-α. J Immunol. 2000, 165: 3985-3991. 10.4049/jimmunol.165.7.3985.
Goodridge HS, Harnett W, Liew FY, Harnett MM: Differential regulation of interleukin-12 p40 and p35 induction via Erk mitogen-activated protein kinase-dependent and -independent mechanisms and the implications for bioactive IL-12 and IL-23 responses. Immunology. 2003, 109: 415-425. 10.1046/j.1365-2567.2003.01689.x.
Kontny E, Kurowska M, Szczepańska K, Maśliński W: Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in human monocytes. J Leukoc Biol. 2000, 67: 249-258.
Novotney M, Chang ZL, Uchiyama H, Suzuki T: Protein kinase C in tumoricidal activation of mouse macrophage cell lines. Biochemistry. 1991, 30: 5597-5604. 10.1021/bi00236a037.
Shinji H, Akagawa KS, Tsuji M, Maeda M, Yamada R, Matsuura K, Yamamoto S, Yoshida T: Lipopolysaccharide-induced biphasic inositol 1,4,5-trisphosphate response and tyrosine phosphorylation of 140-kilodalton protein in mouse peritoneal macrophages. J Immunol. 1997, 158: 1370-1376.
St-Denis A, Chano F, Tremblay P, St-Pierre Y, Descoteaux A: Protein kinase C-α modulates lipopolysaccharide-induced functions in a murine macrophage cell line. J Biol Chem. 1998, 273: 32787-32792. 10.1074/jbc.273.49.32787.
Langlet C, Springael C, Johnson J, Thomas S, Flamand V, Leitges M, Goldman M, Aksoy E, Willems F: PKC-α controls MYD88-dependent TLR/IL-1R signaling and cytokine production in mouse and human dendritic cells. Eur J Immunol. 2010, 40: 505-515. 10.1002/eji.200939391.
Pozo-Guisado E, Campbell DG, Deak M, Alvarez-Barrientos A, Morrice NA, Alvarez IS, Alessi DR, Martín-Romero FJ: Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry. J Cell Sci. 2010, 123: 3084-3093. 10.1242/jcs.067215.
This work was supported by grant RDC21418401001905 and a seed grant (to BBM) under National Institutes of Health (NIH) grant P30GM103329 (principal investigator: Jonathan Geiger), NIH grant R01 DE017102 (to BBS), and NIH grants 1R21AI107457-01A1 and 1R21AI101644-01 (to JS).
The authors declare that they have no competing interests.
BBM conceived of and designed the experiments. YS, AC and PS performed the experiments. BBM and BBS analyzed the data. BBM, BBS and JS contributed the reagents, materials and analytical tools. BBM, BBS and JS wrote the manuscript. All authors read and approved the final manuscript.
Yuyang Sun, Arun Chauhan contributed equally to this work.
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Sun, Y., Chauhan, A., Sukumaran, P. et al. Inhibition of store-operated calcium entry in microglia by helminth factors: implications for immune suppression in neurocysticercosis. J Neuroinflammation 11, 210 (2014). https://doi.org/10.1186/s12974-014-0210-7
- Calcium signaling
- Immune suppression
- Store-operated calcium entry