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IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

ABSTRACT: Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a globally important zoonotic disease for which arthritis is the most common focal complication in humans. Wild-type mice infected systemically with Brucella typically do not exhibit arthritis, but mice lacking IFN-γ develop arthritis regardless of the route of Brucella infection. Here, we investigated mechanisms by which IFN-γ suppresses Brucella-induced arthritis. Several cell types, including innate lymphoid cells, contributed to IFN-γ production and suppression of joint swelling. IFN-γ deficiency resulted in elevated joint IL-1β levels, and severe joint inflammation that was entirely inflammasome dependent, and in particular, reliant on the NLRP3 inflammasome. IFN-γ was vital for induction of the nitric oxide producing enzyme, iNOS, in infected joints, and nitric oxide directly inhibited IL-1β production and inflammasome activation in Brucella-infected macrophages in vitro. During in vivo infection, iNOS deficiency resulted in an increase in IL-1β and inflammation in Brucella-infected joints. Collectively, this data indicate that IFN-γ prevents arthritis both by limiting Brucella infection, and by inhibiting excessive inflammasome activation through the induction of nitric oxide.

SUBMITTER: Lacey CA

PROVIDER: S-EPMC6597291 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

Lacey Carolyn A CA Chambers Catherine A CA Mitchell William J WJ Skyberg Jerod A JA

Journal of leukocyte biology 20190212 1

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a globally important zoonotic disease for which arthritis is the most common focal complication in humans. Wild-type mice infected systemically with Brucella typically do not exhibit arthritis, but mice lacking IFN-γ develop arthritis regardless of the route of Brucella infection. Here, we investigated mechanisms by which IFN-γ suppresses Brucella-induced arthritis. Several cell types, including innate lymphoid cells, contr ...[more]

PMID: 30748031

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Project description:IntroductionThe COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified.MethodsGiven that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1β) on the IFN-g-induced NO production and its antiviral efficacy.ResultsTreatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1β enhanced IFN-γ induction of NO, but it had little effect on antiviral activity.DiscussionGiven that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

Project description:BackgroundInflammatory and immune responses in the brain that contribute to various neuropsychiatric disorders may begin as microglial "priming". Interferon (IFN)-γ is known to cause microglial priming, but the mechanism is unclear.MethodsWe examined the effects of IFN-γ on gene expression, microglial activation, inflammatory and immune responses and activity of the NLRP3 inflammasome in primary microglia and in the brains of mice.ResultsOur results showed that treating microglial cultures with IFN-γ induced a hedgehog-like morphology and upregulated markers of microglial activation (CD86, CD11b) and pro-inflammatory molecules (IL-1β, IL-6, TNF-α, iNOS), while downregulating markers of microglial homeostasis (CX3CR1, CD200R1), anti-inflammatory molecules (MCR1, Arg-1) and neurotrophic factors (IGF-1, BDNF). IFN-γ also upregulated markers of NLRP3 inflammasome activation (NLRP3, caspase-1, gasdermin D, IL-18). This particular transcriptional profiling makes IFN-γ-primed microglia with exaggerated responses upon lipopolysaccharide (LPS) stimulation. The level of NLRP3, caspase-1, gasdermin D, IL-1β, IL-18, TNF-α and iNOS in microglia cultures treated with both IFN-γ and LPS were highest than with either one alone. Injecting IFN-γ into the lateral ventricle of mice induced similar morphological and functional changes in hippocampal microglia as in primary microglial cultures. The effects of IFN-γ on NLRP3 inflammasome and microglia from cultures or hippocampus were abolished when STAT1 was inhibited using fludarabin. Injecting mice with IFN-γ alone or together with LPS induced anxiety- and depression-like behaviors and impaired hippocampus-dependent spatial memory; these effects were mitigated by fludarabin.ConclusionsIFN-γ primes microglia by activating STAT1, which upregulates genes that activate the NLRP3 inflammasome. Inhibiting the IFN-γ/STAT1 axis may be a way to treat neurodegenerative diseases and psychiatric disorders that involve microglial priming.

Project description:Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the nitric oxide-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (dynasore, Dyngo 4a, Dynole 34-2, and MiTMAB) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate V-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore or Dyngo 4a blocked the NO-dependent shift in EGABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To further resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores which may in part be due to depletion of synaptic vesicles.

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IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

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IFN-γ-dependent nitric oxide suppresses Brucella-induced arthritis by inhibition of inflammasome activation.

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