Ammonia Attenuates LPS-Induced Upregulation of Pro-Inflammatory Cytokine mRNA in Co-Cultured Astrocytes and Microglia

Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/β, IL-6 and TNF-α. NH₄Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/β and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/β, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFβ_1?3 mRNA was attenuated by NH₄Cl. LPS-induced upregulation of IL-1α/β, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH₄Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

Regulatory Mechanisms of Vitamin D<Subscript>3</Subscript> on Production of Nitric Oxide and Pro-inflammatory Cytokines in Microglial BV-2 Cells

Inhibition of pro-inflammatory functions of microglia has been considered a promising strategy to prevent pathogenic events in the central nervous system under neurodegenerative conditions. Here we examined potential inhibitory effects of nuclear receptor ligands on lipopolysaccharide (LPS)-induced inflammatory responses in microglial BV-2 cells. We demonstrate that a vitamin D receptor agonist 1,25-dihydroxyvitamin D₃ (VD3) and a retinoid X receptor agonist HX630 affect LPS-induced expression of pro-inflammatory factors. Specifically, both VD3 and HX630 inhibited expression of mRNAs encoding inducible nitric oxide synthase (iNOS) and IL-6, whereas expression of IL-1β mRNA was inhibited only by VD3. The inhibitory effect of VD3 and HX630 on expression of iNOS and IL-6 mRNAs was additive. Effect of VD3 and HX630 was also observed for inhibition of iNOS protein expression and nitric oxide production. Moreover, VD3 and HX630 inhibited LPS-induced activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor κB (NF-κB). PD98059, an inhibitor of ERK kinase, attenuated LPS-induced nuclear translocation of NF-κB and induction of mRNAs for iNOS, IL-1β and IL-6. These results indicate that VD3 can inhibit production of several pro-inflammatory molecules from microglia, and that suppression of ERK activation is at least in part involved in the anti-inflammatory effect of VD3.     

Carnitine/Organic Cation Transporter OCTN1 Negatively Regulates Activation in Murine Cultured Microglial Cells

Brain immune cells, i.e., microglia, play an important role in the maintenance of brain homeostasis, whereas chronic overactivation of microglia is involved in the development of various neurodegenerative disorders. Therefore, the regulation of microglial activation may contribute to their treatment. The aim of the present study was to clarify the functional expression of carnitine/organic cation transporter OCTN1/SLC22A4, which recognizes the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo, in microglia and its role in regulation of microglial activation. Primary cultured microglia derived from wild-type mice (WT-microglia) and mouse microglial cell line BV2 exhibited time-dependent uptake of [³H]- or d9-labeled ERGO. The uptake was markedly decreased in cultured microglia from octn1 gene knockout mice (octn1 ^?/?-microglia) and BV2 cells transfected with small interfering RNA targeting the mouse octn1 gene (siOCTN1). These results demonstrate that OCTN1 is functionally expressed in murine microglial cells. Exposure of WT-microglia to ERGO led to a significant decrease in cellular hypertrophy by LPS-stimulation with concomitant attenuation of intracellular reactive oxygen species (ROS), suggesting that OCTN1-mediated ERGO uptake may suppress cellular hypertrophy via the inhibition of ROS production with microglial activation. The expression of mRNA for interleukin-1β (IL-1β) after LPS-treatment was significantly increased in octn1 ^?/?-microglia and siOCTN1-treated BV2 cells compared to the control cells. Meanwhile, treatment of ERGO minimally affected the induction of IL-1β mRNA by LPS-stimulation in cultured microglia and BV2 cells. Thus, OCTN1 negatively regulated the induction of inflammatory cytokine IL-1β, at least in part, via the transport of unidentified substrates other than ERGO in microglial cells.     

Ethanol-Induced TLR4/NLRP3 Neuroinflammatory Response in Microglial Cells Promotes Leukocyte Infiltration Across the BBB

We reported that the ethanol-induced innate immune response by activating TLR4 signaling triggers gliosis and neuroinflammation. Ethanol also activates other immune receptors, such as NOD-like-receptors, and specifically NLRP3-inflammasome in astroglial cells, to stimulate caspase-1 cleavage and IL-1β and IL-18 cytokines production. Yet, whether microglia NLRs are also sensitive to the ethanol effects that contribute to neuroinflammation is uncertain. Using cerebral cortexes of the chronic alcohol-fed WT and TLR4^?/? mice, we demonstrated that chronic ethanol treatment enhanced TLR4 mediated-NLRP3/Caspase-1 complex activation, and up-regulated pro-inflammatory cytokines and chemokines levels. Ethanol-induced NLRP3-inflammasome activation and mitochondria-ROS generation were also observed in cultured microglial cells. The up-regulation of CD45^high/CD11b⁺ cell populations and matrix metalloproteinase-9 levels was also noted in the cortexes of the ethanol-treated WT mice. Notably, elimination of the TLR4 function abolished most ethanol-induced neuroinflammatory effects. Thus, our results demonstrate that ethanol triggers TLR4-mediated NLRP3-inflammasome activation in glial cells, and suggest that microglia stimulation may compromise the permeability of blood–brain barrier events to contribute to ethanol-induced neuroinflammation and brain damage.     

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.     

Glycogen synthase kinase-3 regulates microglial migration,inflammation, and inflammation-induced neurotoxicity

Microglia play a prominent role in the brain's inflammatory response to injury or infection by migrating to affected locations, secreting inflammatory molecules, and phagocytosing damaged tissue. However, because severe or chronic neuroinflammation exacerbates many neurological conditions, controlling microglia actions may provide therapeutic benefits in a diverse array of diseases. Since glycogen synthase kinase-3 (GSK3) promotes inflammatory responses in peripheral immune cells, we investigated if inhibitors of GSK3 attenuated microglia responses to inflammatory stimuli. Treatment of BV-2 microglia with GSK3 inhibitors greatly reduced the migration of microglia in both a scratch assay and in a transwell migration assay. Treatment of BV-2 microglia with lipopolysaccharide (LPS) stimulated the production of interleukin-6 and increased the expression of inducible nitric oxide synthase (iNOS) and NO production. Each of these microglia responses to inflammatory stimulation were greatly attenuated by GSK3 inhibitors. However, GSK3 inhibitors did not cause a general impairment of microglia functions, as the LPS-induced stimulated expression of cylcooxygenase-2 was unaltered. Regulation of microglia functions were also evident in cultured mouse hippocampal slices where GSK3 inhibitors reduced cytokine production and microglial migration, and provided protection from inflammation-induced neuronal toxicity. These findings demonstrate that GSK3 promotes microglial responses to inflammation and that the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia.     

Narciclasine inhibits LPS-induced neuroinflammation by modulating the Akt/IKK/NF-κB and JNK signaling pathways

BackgroundNeuroinflammation is defined as innate immune system activation in the central nervous system, and is a complex response involved in removing pathogens, toxic components, and dead cells by activating microglial cells. However, over-activated microglia have been implicated in the pathogenesis of neurodegenerative diseases, because they release large amounts of neurotoxic factors. Thus, inhibiting microglial activation may represent an attractive approach for preventing neuroinflammatory disorders. The objective of this study was to investigate the effect of narciclasine (NA) on lipopolysaccharide (LPS)-induced neuroinflammation by evaluating related markers and neurotoxic factors.MethodsBV-2 cells were pre-incubated with NA at 0.1, 0.2, and 0.3 µM for 1h, and then co-treated with LPS for 12 h. Cellular medium and lysates were measured using a nitric oxide assay, enzyme-link immunosorbent assay (ELISA), western blotting, kinase activity assay, luciferase assay, and immunofluorescence assay. C57BL/6N mice were orally administered NA and intraperitoneally injected with LPS, and the cerebral cortex was examined using western blotting and immunofluorescence assays.ResultsNA showed novel pharmacological activity, inhibiting pro-inflammatory factors, including TNF-α, IL-6, IL-18, NO, and PGE₂, but increasing the anti-inflammatory cytokines IL-10 and TGF-β1 in LPS-induced microglial cells. Moreover, NA also attenuated the LPS-induced mRNA and proteins of iNOS and COX-2. The mechanistic study indicated that NA attenuates the secretion of pro-inflammatory factor by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways, and directly inhibits the catalytic activity of IKKα/β. Furthermore, we found that NA also reduced the expression of the microglial markers Iba-1, COX-2, and TNF-α in the mouse brain.ConclusionNA inhibits the over-expression of pro-inflammatory factors but it promotes anti-inflammatory cytokines by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways in experimental models. Thus, NA may be a potential candidate for relieving neuroinflammation.     

Minocycline selectively inhibits M1 polarization of microglia

Minocycline is commonly used to inhibit microglial activation. It is widely accepted that activated microglia exert dual functions, that is, pro-inflammatory (M1) and anti-inflammatory (M2) functions. The in vivo status of activated microglia is probably on a continuum between these two extreme states. However, the mechanisms regulating microglial polarity remain elusive. Here, we addressed this question focusing on minocycline. We used SOD1^G93A mice as a model, which exhibit the motor neuron-specific neurodegenerative disease, amyotrophic lateral sclerosis. Administration of minocycline attenuated the induction of the expression of M1 microglia markers during the progressive phase, whereas it did not affect the transient enhancement of expression of M2 microglia markers during the early pathogenesis phase. This selective inhibitory effect was confirmed using primary cultured microglia stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, which induced M1 or M2 polarization, respectively. Furthermore, minocycline inhibited the upregulation of NF-κB in the LPS-stimulated primary cultured microglia and in the spinal cord of SOD1^G93A mice. On the other hand, IL-4 did not induce upregulation of NF-κB. This study indicates that minocycline selectively inhibits the microglia polarization to a proinflammatory state, and provides a basis for understanding pathogeneses of many diseases accompanied by microglial activation.     

Direct Pro-Inflammatory Effects of Prorenin on Microglia

Neuroinflammation has been implicated in hypertension, and microglia have been proposed to play an important role in the progression of this disease. Here, we have studied whether microglia are activated within cardiovascular regulatory area(s) of the brain during hypertension, especially in high blood pressure that is associated with chronic activation of the renin-angiotensin-system. In addition, we determined whether prorenin, an essential component of the renin-angiotensin-system, exerts direct pro-inflammatory effects on these microglia. Our data indicate that two rodent models which display neurogenic hypertension and over activation of the renin-angiotensin-system in the brain (sRA mice and spontaneously hypertensive rats) exhibit microglial activation, and increased levels of pro-inflammatory cytokines, in the paraventricular nucleus of the hypothalamus, an area crucial for regulation of sympathetic outflow. Further, the renin-angiotensin-system component prorenin elicits direct activation of hypothalamic microglia in culture and induction of pro-inflammatory mechanisms in these cells, effects that involve prorenin receptor-induced NFκB activation. In addition, the prorenin-elicited increases in cytokine expression were fully abolished by microglial inhibitor minocycline, and were potentiated by pre-treatment of cells with angiotensin II. Taken together with our previous data which indicate that pro-inflammatory processes in the paraventricular nucleus are involved in the hypertensive action of renin-angiotensin-system, the novel discovery that prorenin exerts direct stimulatory effects on microglial activation and pro-inflammatory cytokine production provides support for the idea that renin-angiotensin-system -induced neurogenic hypertension is not restricted to actions of angiotensin II alone.     

Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: implications for death of activated microglia

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.     

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice

Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.     

Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated microglia. LPS treatment increased intracellular ROS in rat microglia dose-dependently. Pre-treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS-induced release in PGE2. Diphenylene iodonium (DPI), a non-specific NADPH oxidase inhibitor, decreased LPS-induced PGE2 production. In addition, microglia from NADPH oxidase-deficient mice produced less PGE2 than those from wild-type mice following LPS treatment. Furthermore, LPS-stimulated expression of COX-2 (determined by RT-PCR analysis of COX-2 mRNA and western blot for its protein) was significantly reduced by pre-treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX-2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS-induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX-2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX-2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation-mediated neurodegenerative diseases.     

Murine microglial cells produce and respond to interleukin-18

Interleukin (IL)-18 (interferon-gamma-inducing factor or IL-1gamma) belongs structurally to the IL-1 cytokine family and shares biological properties with IL-12. Expression, intracellular signaling, and functional relevance of IL-18 within the CNS are mostly unknown. We show that IL-18 protein is synthesized within mouse brain, preferentially during early postnatal stages, and that microglial cells but not astrocytes are a potential source. IL-18 is produced by cultured microglia on exposure to lipopolysaccharide (LPS). Microglia also express major components of the IL-1/IL-18 receptor system. On IL-18 stimulation, microglial IL-1 receptor-associated kinase (IRAK) can be coprecipitated with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) but not with IL-1 receptor type I, indicating that IRAK recruits TRAF6 during IL-18 signaling. IL-18 inhibits the LPS-induced release of IL-12 and attenuates that of TNF-alpha, whereas the production of IL-6 and macrophage inflammatory protein-1alpha is only marginally affected. IL-18 may play a role during CNS development and can be produced by activated microglia, thus probably contributing to immune and inflammatory processes in the brain.     

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.