Investigation of RIG-I responses under hypoxic conditions in microglia

Abstract

The immune system constitutes a highly sophisticated and multifaceted network that functions to defend the host against a wide spectrum of threats ranging from microbial infections to endogenous cellular damage. This system is broadly divided into the innate and adaptive arms, with the innate immune system serving as the first responder to pathogenic invasion and cellular stress. Unlike adaptive immunity, which requires antigen-specific memory, innate immunity relies on germline-encoded pattern recognition receptors (PRRs) that detect conserved microbial motifs called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) released from damaged or dying cells. Among the diverse families of PRRs, RIG-I-like receptors (RLRs) play a pivotal role in cytoplasmic sensing of viral patterns. Retinoic acid-inducible gene I (RIG-I) is a cytosolic helicase that recognizes 5'-triphosphate bearing single-stranded RNA and short double-stranded RNA molecules typically derived from RNA viruses, initiating a cascade of antiviral signaling. Upon ligand binding, RIG-I undergoes conformational changes facilitating interaction with the mitochondrial antiviral-signaling protein (MAVS), which in turn triggers downstream activation of transcription factors including interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). These transcription factors orchestrate the production of type I interferons and pro-inflammatory cytokines that mediate innate immune defenses. While RIG-I is predominantly recognized for its role in antiviral defense within peripheral immune cells, its function within the central nervous system (CNS), particularly in resident immune cells such as microglia, remains an emerging area of investigation that highlights the intersection of innate immunity and neuroinflammation. The CNS, comprising the brain and spinal cord, is considered an immune-privileged site due to anatomical and physiological barriers such as the blood-brain barrier (BBB) that restrict immune cell entry from circulation and tightly regulate immune responses to protect neural tissue from excessive inflammation. Therefore, the CNS harbors a specialized local immune system, mainly composed of resident glial cells. Microglia are principal mediators of neuroinflammation in the CNS. Originating from yolk sac progenitors, microglia are long-lived, self-renewing cells that perform surveillance, phagocytosis, and cytokine production. Their activation can be broadly categorized into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. M1 microglia contribute to host defense but may also promote neurotoxicity when excessively activated. In contrast, M2 microglia support tissue repair, homeostasis, and resolution of inflammation. Given their sensitivity to environmental changes, microglia are significantly influenced by various stressors such as hypoxia. Hypoxia, defined as the pathological reduction of oxygen availability, is a critical stressor implicated in a wide array of neurological disorders including ischemic stroke, traumatic brain injury, and neurodegenerative diseases. In the CNS, hypoxic conditions, collectively termed cerebral hypoxia, activate intricate cellular adaptation mechanisms primarily mediated by hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α is a transcription factor stabilized under low oxygen conditions that regulates genes involved in angiogenesis, erythropoiesis, metabolism, and survival pathways. The molecular and cellular consequences of hypoxia are complex and often intersect with innate immune signaling, fostering a neuroinflammatory milieu that contributes to tissue damage and disease progression. Therefore, unraveling the molecular mechanisms by which microglia detect and respond to hypoxia—especially involving innate immune receptors such as RIG-I—is crucial for developing novel interventions to prevent hypoxia-related CNS injury. This study thus aims to investigate the contribution of RIG-I to microglial activation and inflammation under chemically induced hypoxic conditions. This approach may also help us identify overlapping molecules that are involved in tissue injuries resulting from microbial infections and hypoxia-like conditions. To model hypoxic stress in vitro, cobalt chloride (CoCl₂) was used to chemically induce stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in HMC3 human microglial cells. This hypoxia-mimetic approach effectively replicated key molecular features of oxygen deprivation, as evidenced by the robust, dose- and time-dependent accumulation of HIF-1α protein. Importantly, 7-AAD viability staining confirmed that CoCl₂ treatment did not compromise cellular integrity, ruling out the possibility that the observed molecular changes were secondary to cytotoxic effects. Having established a reliable model of chemical hypoxia, we then aimed to investigate the role of RIG-I in modulating microglial adaptation to this stress. Interestingly, CoCl₂ exposure did not significantly alter RIG-I protein levels in HMC3 cells, suggesting that its contribution might not depend on transcriptional upregulation, but rather on functional engagement within stress-related signaling pathways. To explore this possibility, RIG-I was silenced using a siRNA-based knockdown approach, and the functional consequences were assessed under both normoxic and hypoxic conditions. For this, we initially focused on the AKT/mTOR signaling axis, which is a central pathway governing cellular survival, proliferation, and energy homeostasis. AKT, also known as protein kinase B, is activated via phosphorylation and promotes a variety of downstream processes, including inhibition of apoptosis and stimulation of protein synthesis through activation of mechanistic target of rapamycin (mTOR). mTOR, particularly through its complex mTORC1, integrates environmental cues such as nutrient levels, oxygen availability, and stress signals to regulate the balance between growth and autophagy. In line with this understanding, CoCl₂ treatment led to a notable decrease in phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) levels in HMC3 cells, reflecting inhibition of the AKT/mTOR axis. However, in cells lacking RIG-I, both p-AKT and p-mTOR levels were significantly elevated under normoxic and hypoxic conditions, indicating that RIG-I may contribute to the suppression of this pathway in response to hypoxia. This reversal suggests a functional role for RIG-I in maintaining cellular adaptation by limiting survival-promoting signals that could otherwise be maladaptive under stress conditions. These findings reveal RIG-I as a potential upstream modulator of stress-responsive intracellular signaling in microglia. Given the close relationship between mTOR activity and autophagy, our study also evaluated the canonical markers of autophagy, a catabolic process essential for cellular survival during stress. Autophagy enables the recycling of damaged organelles and misfolded proteins via autophagosome formation and lysosomal degradation, and is particularly upregulated during hypoxia. This process is initiated by Beclin-1 and monitored through the conversion of LC3-I to LC3-II, which are widely used as biochemical markers of autophagic flux. In response to CoCl₂-induced hypoxia, HMC3 cells exhibited increased Beclin-1 expression, indicating effective autophagy induction. This autophagic response was consistent with the observed suppression of mTOR activity, supporting the notion that hypoxia promotes autophagy as an adaptive mechanism. Upon RIG-I knockdown, however, this response was markedly impaired. Beclin-1 level was significantly declined under hypoxic conditions. These findings suggest that RIG-I facilitates autophagy in human microglia, likely through its influence on the AKT/mTOR axis. In the absence of RIG-I, microglial cells appear to favor survival and growth signaling over stress-responsive degradation pathways, potentially compromising their ability to manage hypoxic insult. The modulation of autophagy by RIG-I adds a new layer of complexity to our understanding of how innate immune receptors contribute to cellular homeostasis under pathological conditions. To gain further insight into the inflammatory dimension of this regulatory network, we assessed interleukin-6 (IL-6) levels, a pro-inflammatory cytokine commonly associated with neuroinflammation and microglial activation. Secreted IL-6 levels did not change in response to CoCl₂ treatment in HMC3 cells. Moreover, IL-6 secretion decreased with RIG-I knockdown under both normoxia and hypoxia. These findings suggest that RIG-I may help preserve or enhance microglial inflammatory capacity under hypoxic stress. Rather than amplifying inflammation, RIG-I appears to act as a stabilizing factor that sustains a basal level of immune responsiveness in microglia when oxygen availability is limited. This observation challenges simplistic assumptions that RIG-I uniformly drives inflammation and instead points to a context-dependent regulatory role, particularly in the CNS. In conclusion, our study provided evidence that RIG-I functions as a key regulator of microglial adaptation to hypoxic stress. Although RIG-I expression levels remain unchanged under CoCl₂-induced hypoxia, its silencing suggested critical roles in modulating intracellular signaling and stress response pathways. Specifically, RIG-I contributes to the suppression of AKT/mTOR signaling, facilitates the induction of autophagy via Beclin-1 processing, and supports the maintenance of IL-6 expression under hypoxic conditions. The loss of RIG-I disrupts this balance, leading to enhanced AKT/mTOR activity, impaired autophagic flux, and attenuated inflammatory signaling. Collectively, these findings highlight RIG-I as a multifaceted mediator of microglial resilience, integrating immune sensing with metabolic and inflammatory adaptation in the context of cerebral hypoxia. Understanding this regulatory role opens new avenues for therapeutic strategies aimed at modulating microglial behavior in neurological disorders characterized by oxygen deprivation and immune dysregulation.