Modulation of immune responses by opioids through toll-like and P2X receptor signaling in COVID-19

Abstract

The pathogenesis of COVID-19 involves complex interactions between viral replication and host immune dysregulation, mediated by toll-like receptors (TLRs) and P2X receptors (P2XRs). These receptors detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), triggering inflammatory cascades. Opioids, beyond their analgesic role, modulate innate and adaptive immune responses via opioid receptors and indirectly through TLR and P2X signaling. This narrative review integrates experimental, clinical, and bioinformatic evidence to explore the mechanistic crosstalk between opioid signaling, TLRs (notably TLR2, TLR4, TLR9), and P2XRs (notably P2X4 and P2X7) in COVID-19 immunopathology. Chronic opioid exposure may either enhance or suppress inflammation depending on dose, duration, and immune context. In COVID-19, the hyperactivation of TLR4, TLR7, and TLR9 drives cytokine storms, while the release of ATP from damaged cells activates P2X7, thereby amplifying the inflammatory response. ATP breakdown into adenosine further modulates immunity via A2A and A2B receptors. Targeting TLR4 and P2X7 offers a promising therapeutic strategy to mitigate hyperinflammation and improve outcomes in COVID-19 and related inflammatory diseases. In addition, we outline how the Contact System, the Kallikrein–Kinin System (KKS), the Renin–Angiotensin System (RAS), and the NLRP3 inflammasome provide the innate inflammatory backdrop through which opioids and P2 receptor signaling may shape immune dysregulation in COVID-19. Notably, direct clinical evidence for opioid–P2 receptor interactions in COVID-19 remains limited, highlighting the need for targeted translational studies.

Introduction

The emergence of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Wuhan, China, in December 2019, triggered a global pandemic, resulting in over seven million deaths worldwide [1–3]. Despite advances in vaccines and antiviral therapies, which have alleviated the disease burden, dysregulated immune responses, particularly in the respiratory system, remain central to COVID-19 pathophysiology [4–6]. Excessive inflammation, driven by pattern recognition receptors such as toll-like receptors (TLRs) and by damage-associated molecular pattern (DAMP) receptors such as P2X7 and P2X4, contributes to severe outcomes including cytokine storms [6, 7]. Emerging evidence highlights the role of opioid signaling, traditionally recognized for analgesia, in modulating immune responses during COVID-19 [8]. Opioids interact with TLRs (e.g., TLR4) and purinergic receptors (P2XRs) (e.g., P2X7), influencing the inflammatory milieu and potentially exacerbating or mitigating disease severity. Understanding these molecular interactions may uncover novel therapeutic targets for managing COVID-19-related immune dysregulation. Although significant progress has been made in disease control, the absence of a definitive cure underscores the need to explore underexplored pathways. This review examines the interplay between TLRs, P2XRs, and the opioidergic system in COVID-19 pathogenesis, highlighting therapeutic challenges and opportunities. Beyond TLRs and P2XRs, this review also situates opioid–P2X crosstalk within key innate systems, including the Contact System, the Kallikrein–Kinin System (KKS), the Renin–Angiotensin System (RAS), and the NLRP3 inflammasome, to present a coherent mechanistic framework relevant to COVID-19 immunopathology.

Pathophysiological background of COVID-19

COVID-19, caused by the SARS-CoV-2 virus, primarily targets the respiratory system, with the majority of symptoms and clinical signs reflecting respiratory involvement. Computed tomography (CT) imaging frequently reveals interstitial lung syndrome, while histopathological studies demonstrate pulmonary edema resulting from increased vascular permeability, along with alveolar epithelial cell necrosis, diffuse alveolar damage, intra-alveolar fibrin deposition, and type II pneumocyte hyperplasia, confirming severe lung injury [9, 10]. In addition to respiratory manifestations, a broad spectrum of symptoms has been reported, with fever, cough, dyspnea, myalgia, sore throat, and diarrhea among the most prevalent [11]. Neurological manifestations, including olfactory and gustatory dysfunctions, have been observed in nearly half of the patients [11–13].

Furthermore, some cases present with hypotension, likely secondary to systemic inflammation and endothelial dysfunction. Coagulopathy is another hallmark of severe COVID-19, characterized by thrombi in the pulmonary arteries and extensive microthrombi in alveolar capillaries [14]. Elevated inflammatory markers are common, and a significant correlation between interleukin-6 (IL-6) concentrations and disease severity has been consistently reported [15, 16]. Variability in inflammatory mediator amounts and coagulation profiles suggests the existence of distinct biochemical and clinical phenotypes of COVID-19. Nonetheless, a strong association persists between clinical symptoms, inflammatory changes, and coagulopathy, underscoring the central role of immune dysregulation in the pathogenesis of COVID-19.

Innate immune mechanisms in COVID-19

The contact system and KKS

The contact system represents a key component of the innate immune response, acting as a frontline defense mechanism against external agents, including microbial pathogens, artificial surfaces, and viral particles. It contributes to host defense through its involvement in inflammatory and coagulation pathways, especially in infections such as COVID-19 [17]. This system is composed primarily of Factor XII (FXII), prekallikrein (PK), and high-molecular-weight kininogen (HK). Upon contact with negatively charged surfaces—such as microbial membranes or viral RNA—these components become activated, triggering a cascade of proteolytic events. Eukaryotic and prokaryotic RNA can activate this cascade by promoting the enzymatic activity of FXII and FXI, thereby enhancing coagulation [18]. In COVID-19, physiological stress conditions such as hypoxia, hyperthermia, ischemia, and oxidative stress lead to the release of alarmins or damage-associated molecular patterns (DAMPs) from necrotic cells. These molecules activate pattern-recognition receptors (especially TLRs), which serve as key sensors of pathogen-associated molecular patterns (PAMPs) like viral RNA [19]. Notably, both DAMPs and PAMPs can activate FXII and initiate the contact system cascade.

Structurally, the HK–PK complex contains binding domains (notably domains 5 and 6) that facilitate attachment to synthetic surfaces. To FXI, promoting the initiation of the intrinsic coagulation pathway [20], PK is first converted into kallikrein (KAL) by FXIIa, which subsequently amplifies FXII activation in a positive feedback loop [21]. Additionally, this conversion can occur via prolylcarboxypeptidase, a vessel wall-associated serine protease [20]. FXIIa, as the initiator of the intrinsic coagulation cascade, promotes fibrin formation. Conversely, KAL may promote fibrinolysis by converting plasminogen to plasmin [22], which in turn degrades fibrin, generating D-dimer fragments. These D-dimers serve as biomarkers for both thrombosis and fibrinolysis [23]. In addition, factor FXIIa can activate plasminogen [24]. Conversely, plasmin also activates FXII [25]. Targeting FXII and its cofactors, such as polyphosphate, presents a promising approach to modulating immunothrombosis in COVID-19. Anti-FXII, anti-FXI, and anti-KAL monoclonal antibodies have shown efficacy in reducing thrombosis in experimental models [26]. FXII also functions as a growth factor, promoting angiogenesis and tissue repair [27]; however, its overactivation may contribute to pathological fibroblast proliferation, particularly in the lungs, leading to pulmonary fibrosis—a finding frequently reported in severe COVID-19 cases [28]. Clinical evidence of widespread microthrombosis and elevated D-dimer concentrations in COVID-19 patients supports the role of uncontrolled activation of the contact system in disease severity [14]. Consequently, low-molecular-weight heparins (LMWHs) have been adopted as a mainstay therapy to prevent venous thromboembolism and manage thrombotic complications in affected patients.

Finally, emerging evidence indicates that the SARS-CoV-2 virus may exploit components of the contact system to suppress innate immunity. Specifically, viral non-structural proteins such as nsp3 may inhibit innate immune signaling, promoting viral persistence and pathogenicity [29]. Therefore, the contact system is not only a participant in coagulation but also a potential mediator of immune evasion and a key target for therapeutic intervention in COVID-19.

KKS, as a subset of the contact system, plays a pivotal role in initiating potent inflammatory responses. Upon activation, the KKS leads to the generation of bradykinin, a powerful pro-inflammatory mediator, although it does not directly participate in coagulation processes [20]. Bradykinin is produced when plasma kallikrein (KAL), activated by FXIIa, cleaves high-molecular-weight kininogen (HK), releasing bradykinin into the circulation. This process is amplified under inflammatory conditions, leading to increased bradykinin production [30].

Bradykinin exerts its biological functions through two G protein-coupled receptors: B1 and B2. While bradykinin primarily binds to B2 receptors, its metabolite des-Arg9-bradykinin (DABK) has a higher affinity for B1 receptors [31]. B2 receptors are constitutively expressed on endothelial and smooth muscle cells [21], whereas B1 receptors are inducible and upregulated during inflammation and infection [32]. Activation of B1 receptors promotes vasodilation, increased vascular permeability, and hypotension—clinical features commonly observed in COVID-19 patients [32]. Bradykinin-mediated stimulation of B2 receptors activates intracellular signaling cascades that further enhance vasodilation [17] and promote the release of inflammatory mediators. These include cytokines such as IL-1, TNF-α, and notably IL-6 [32, 33]. The B2 receptor antagonist icatibant has been shown to inhibit IL-6 release [34]. Similarly, chloroquine, previously investigated for COVID-19 treatment, has demonstrated IL-6-lowering effects [34]. Moreover, bradykinin enhances oxidative and nitrosative stress by inducing nitric oxide (NO) and superoxide radical formation [32]. Through B2 receptor activation on endothelial cells, bradykinin also upregulates nitric oxide synthase activity [20] and increases the release of tissue plasminogen activator (tPA), contributing to both inflammation and fibrinolysis [35]. The FXII/KAL-mediated pathway appears to be upregulated during viral infections, including COVID-19, promoting HK cleavage and excessive bradykinin production. This results in increased endothelial permeability and tissue inflammation. Inhibition of this pathway, through FXII or KAL inhibitors, or bradykinin receptor antagonists, has been shown to reduce endothelial leakage [36]. For example, B2 receptor blockade reduced airway hyperresponsiveness and pulmonary inflammation in models of parainfluenza virus infection [37]. While the precise contribution of bradykinin dysregulation in SARS-CoV-2 infection remains to be fully elucidated, there is compelling evidence that KKS activation plays a substantial role in the inflammatory vascular responses observed in COVID-19. Consequently, therapeutic modulation of the contact system and the KKS may offer promising strategies for managing severe inflammatory manifestations of the disease.

The role of the RAS

The RAS is a crucial regulatory axis involved in cardiovascular homeostasis, fluid balance, and vascular tone. All components of this system, including angiotensin-converting enzyme (ACE), are expressed in various tissues, notably in endothelial cells [38]. ACE primarily functions as a peptidyl-dipeptidase that converts angiotensin I (Ang I) to angiotensin II (Ang II), a potent vasoconstrictor. Additionally, ACE acts as kininase II, metabolizing and inactivating bradykinin and kallidin [38].

The inhibition of ACE by ACE inhibitors (ACEIs) leads to increased bradykinin amounts, a phenomenon associated with side effects such as dry cough. Notably, similar symptoms—such as dry cough and olfactory dysfunction—are observed in COVID-19 patients, suggesting a potential role of bradykinin accumulation in disease pathology [39].

In contrast, ACE2, which is highly expressed in alveolar epithelial cells, endothelial cells, and enterocytes, serves as a regulatory counterpart to ACE. ACE2 converts Ang I to angiotensin 1–9 and Ang II to angiotensin 1–7, both of which possess vasodilatory, anti-inflammatory, anti-fibrotic, and anti-hypertrophic properties [40]. Unlike ACE, ACE2 does not metabolize bradykinin and is not inhibited by ACEIs. The protective role of ACE2 in lung injury has been well documented, with studies showing that downregulation of ACE2 expression leads to neutrophil infiltration, pulmonary inflammation, and increased vascular permeability [31].

During acute lung injury, ACE2 expression declines, resulting in increased Ang II concentrations and enhanced AT1 receptor activity, promoting vasoconstriction and pulmonary damage. In contrast, stimulation of AT2 receptors appears to confer protective effects [41]. Angiotensin 1–9, generated via ACE2-mediated conversion of Ang I, contributes to cardiovascular protection by reducing pulmonary hypertension and mitigating inflammation and fibrosis via AT2 receptor signaling [42]. Importantly, ACE2 is the cellular entry receptor for SARS-CoV-2, facilitating viral attachment through its N-terminal domain. This interaction leads to ACE2 internalization and proteolytic cleavage, disrupting its physiological function [40, 43]. Consequently, ACE2 downregulation contributes to the pathogenesis of COVID-19 by impairing its protective roles and promoting unopposed Ang II activity. Accordingly, chloroquine, which has been reported to interact with ACE2, was initially proposed to reduce the symptoms of COVID-19 [44].

In addition, the bradykinin metabolite des-Arg9-bradykinin (DABK) is a substrate for ACE2. Loss of ACE2 activity during SARS-CoV-2 infection results in reduced DABK degradation, leading to increased B1 receptor signaling and inflammation. Observational studies have suggested that long-term treatment with ACEIs or angiotensin receptor blockers (ARBs) may upregulate ACE2 expression in the heart and lungs. This has raised concerns regarding increased susceptibility to severe COVID-19 outcomes in these patients [45]. However, this hypothesis remains controversial, and to date, no definitive clinical evidence has confirmed a causal relationship between ACEI/ARB use and COVID-19 severity [46–48]. Further investigation is warranted to clarify this issue and guide clinical practice.

Hyperinflammation and the role of NLRP3 inflammasome

COVID-19 presents with a wide range of clinical manifestations. Approximately 45% of patients are asymptomatic or exhibit only mild symptoms, while a subset progresses to severe disease requiring intensive care unit (ICU) admission [49]. Clinical data suggest that about 10% of infected individuals require hospitalization, and of those, up to 20% may develop life-threatening complications with a high mortality rate [50, 51].

In the early phase of infection, SARS-CoV-2 enters host cells and replicates, eliciting symptoms such as fever, fatigue, myalgia, anorexia, and cough, typically within the first seven days. In some patients, disease progression results in hypoxemia and dyspnea, indicative of acute respiratory distress syndrome (ARDS) [21]. At this stage, hyperinflammation or “cytokine storm” becomes a defining feature of severe COVID-19 [52, 53]. Elevated amounts of pro-inflammatory cytokines, especially IL-1, IL-6, IL-18, and GM-CSF, are commonly observed in the bloodstream [54–57]. Notably, SARS-CoV-2 has been shown to preferentially activate the IL-1/IL-6 axis more robustly than other coronaviruses [58].

A key molecular mechanism underlying hyperinflammation is the activation of the NLRP3 inflammasome, which leads to increased production of IL-1β and IL-18. These cytokines correlate strongly with disease severity and poor clinical outcomes [59, 60].

Elevated ferritin concentrations are also frequently reported in severe cases and are positively associated with disease severity, resembling findings seen in macrophage activation syndrome [61, 62].

Bronchoalveolar lavage fluid (BALF) analysis from critically ill patients has revealed a distinct inflammatory cell profile characterized by increased numbers of CD68⁺ infiltrating macrophages and neutrophils in the lungs of patients with severe COVID-19. In contrast, the frequencies of plasmacytoid dendritic cells, myeloid dendritic cells, and T lymphocytes remain relatively unchanged. In addition to these recruited immune cells, lung-resident macrophages (particularly alveolar macrophages) are thought to amplify pulmonary inflammation by producing large amounts of pro-inflammatory cytokines—most notably IL-6, IL-8, and IL-1β—and chemokines such as CCL2, CCL3, CCL4, and CCL7. These mediators are markedly elevated in the pulmonary environment of patients with severe disease compared to those with milder forms [63].

These findings underscore the critical role of lung-resident macrophages in initiating and perpetuating the inflammatory response, ultimately driving the hyperinflammatory state that characterizes severe COVID-19 [64].

Opioid medications and immune modulation

Classical and non-classical opioid receptors

Opioids are among the most potent analgesic agents used in clinical medicine to manage moderate to severe pain. Their pharmacological effects are mediated primarily through interaction with opioid receptors, which are distributed not only in the central and peripheral nervous systems but also in various non-neuronal tissues such as bone, joints, and immune cells.

Accumulating evidence indicates that opioids modulate both innate and adaptive immune responses. In particular, TLRs play a significant role in the immune system and may serve as key molecular interfaces in opioid-mediated immunomodulation [65].

In this review, we examine the immunological effects of opioid compounds, with a specific focus on their potential therapeutic and immunomodulatory roles in the context of COVID-19.

Opioid receptors are a subclass of G protein–coupled receptors (GPCRs) widely expressed in various tissues, including the brain, spinal cord, gastrointestinal tract, and skin. These receptors mediate the pharmacological effects of opioid compounds, such as analgesia, euphoria, respiratory depression, tolerance, and dependence [66].

Opioid receptors are broadly classified into two main groups: classical receptors, including the mu-opioid receptor (MOR), kappa-opioid receptor (KOR), and delta-opioid receptor (DOR), and non-classical receptors, such as the nociceptin/orphanin FQ peptide receptor (NOP) [67]. The classical opioid receptors, MOR, KOR, and DOR, have been well characterized for their roles in analgesia, euphoria, sedation, and other physiological functions. More recently, additional opioid-related receptors such as the nociceptin/orphanin FQ receptor (NOR) and zeta (ZOR) receptors, as well as non-opioid targets including TLRs and receptors like P2X4, have been implicated in mediating opioid effects beyond pain relief.

These receptors play central roles not only in pain modulation and reward mechanisms but also in regulating respiratory and cardiovascular functions. Moreover, a growing body of evidence suggests their involvement in modulating immune responses, highlighting their relevance in inflammation, infection, and immune homeostasis [68].

Expression of opioid receptors in immune cells

The immunomodulatory effects of opioids are multifaceted and are partially mediated through the expression of opioid receptors on immune cells. Constitutive and inducible expression patterns have been observed, particularly in response to inflammatory stimuli.

In humans, classical (MOR, KOR, and DOR) and non-classical (NOP) opioid receptors are expressed on a variety of immune cell subsets. T lymphocytes, B lymphocytes, and natural killer (NK) cells express multiple opioid receptor subtypes, while polymorphonuclear leukocytes (PMNs) and monocytes/macrophages predominantly express MOR and NOP receptors [67].

These findings demonstrate that opioid compounds can modulate immune cell activity and signaling, contributing to either pro-inflammatory or anti-inflammatory outcomes depending on receptor subtype, dosage, and the immunological context (Table 1).

Table 1.

Expression of opioid receptors in human immune cells

Opioid receptor	Immune cell types with confirmed expression
MOR	Polymorphonuclear cells (PMNs), monocytes, macrophages, CD4⁺ T cells, peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), T and B lymphocytes, natural killer (NK) cells
KOR	PBMCs, CD4⁺ T cells, PBLs, monocytes, macrophages, T and B lymphocytes, NK cells
DOR	PBLs, NK cells
NOP	PBLs, PBMCs, monocytes, PMNs, NK cells

Effects of opioids on innate immunity

Opioids exert significant immunomodulatory effects on the innate immune system, particularly through their impact on monocytes, neutrophils, and macrophages. In vitro studies have demonstrated that opioid compounds impair the chemotactic and phagocytic functions of monocytes and neutrophils. Additionally, opioids promote apoptosis in these phagocytic cells, thereby weakening the first line of host defense [68].

High-dose morphine administration has been shown to induce macrophage apoptosis via activation of TLR4 and the p38 mitogen-activated protein kinase (MAPK) signaling pathway [69]. In the context of gram-negative bacterial infections, morphine impairs effective pathogen clearance by reducing phagocytosis, an effect mediated through TLR dysregulation [70].

These findings highlight the potential immunosuppressive role of opioids in innate immunity and underscore their relevance in infection-prone conditions such as sepsis or viral respiratory illnesses like COVID-19.

Effects of opioids on adaptive immunity

Opioids have been shown to impair adaptive immune responses by directly affecting T and B lymphocytes. In vitro studies demonstrate that opioids reduce the activation and function of these cells, while in vivo evidence indicates that opioid exposure diminishes lymphocyte proliferative capacity [71].

The expression of opioid receptors in immune cells is generally lower than in central nervous system (CNS) cells; however, certain receptors, such as the nociceptin/orphanin FQ (NOP) receptor, exhibit comparable expression concentrations in both immune and CNS tissues. Upon immune cell activation, the expression of opioid receptors increases, suggesting a role in immunoregulatory feedback mechanisms. Chronic opioid use has been associated with further upregulation of opioid receptors on immune cells [65]. Signaling through these receptors exerts immunomodulatory and anti-inflammatory effects, depending on the receptor subtype, cellular context, and duration of exposure [72].

Collectively, these observations suggest that the immunological effects of opioids are mediated through both direct interactions with immune cells and indirect modulation via neuroendocrine and CNS pathways.

Suppression or activation of TLR/NF-κB pathways

Opioids modulate innate immune responses through interactions with TLRs and downstream signaling pathways such as nuclear factor kappa B (NF-κB). In monocytes, opioid administration has been shown to suppress tumor necrosis factor-alpha (TNF-α) expression via inhibition of the NF-κB pathway [73].

In murine models of peritonitis, morphine reduced inflammatory responses by altering the expression levels of TLRs, NF-κB, and pro-inflammatory cytokines, including TNF-α [74]. Notably, in infections caused by Streptococcus pneumoniae, morphine-mediated suppression of the NF-κB pathway appears to be modulated through TLR9 signaling [75].

Interestingly, opioids may exert differential effects depending on the immunological context. In a Mycobacterium tuberculosis infection model, morphine stimulated TLR9 activation, contributing to enhanced resistance in the lungs of mice [75].

Additionally, opioids can bind to TLR4 expressed on microglial cells, key components of the innate immune system within the CNS, triggering the activation of NF-κB and subsequent release of pro-inflammatory cytokines such as TNF-α and IL-1β [76, 77]. This dual regulatory capacity underscores the complex and context-dependent nature of opioid-mediated modulation of TLR and NF-κB pathways.

Anti-inflammatory effects of opioids

Since the late twentieth century, numerous studies have investigated the immunomodulatory properties of opioid compounds. A substantial body of evidence supports the immunosuppressive potential of opioids, particularly their inhibitory effects on various components of the innate and adaptive immune systems [67].

Opioid administration has been shown to impair multiple immune functions, including antibody production, natural killer (NK) cell cytotoxicity, cytokine expression, and phagocytic activity [78]. In animal models, acute morphine administration significantly suppresses immune responses, a finding that has also been validated in human studies.

Chronic opioid exposure is associated with persistent suppression of NK cell activity and dysregulation of macrophage function, creating a favorable environment for opportunistic infections, particularly in the lungs and peritoneal cavity [79].

Mechanistically, opioids can stimulate the hypothalamic–pituitary–adrenal (HPA) axis, leading to increased cortisol secretion. This neuroendocrine response further contributes to the suppression of NK cell cytotoxicity and overall immune competency [80, 81].

Several in vivo studies have demonstrated that opioids possess anti-inflammatory properties, primarily through the suppression of pro-inflammatory cytokines and inhibition of leukocyte migration. Two key markers commonly used to assess inflammation, plasma extravasation and neutrophil infiltration, are modulated by opioid administration [82].

During respiratory syncytial virus (RSV) infection, opioids selectively reduce neutrophil recruitment into the bronchoalveolar space without significantly affecting lymphocyte or monocyte populations [82]. Activation of peripheral KORs mediates anti-inflammatory effects through multiple mechanisms: partial KOR agonists have been reported to suppress prostaglandin E2 (PGE2) production in rodent models without eliciting CNS effects [82]. Additionally, opioids reduce the expression of adhesion molecules on endothelial cells, thereby decreasing leukocyte accumulation and TNF-α secretion. This mechanism has been implicated in opioid-mediated reduction of inflammation in arthritis models [83].

Ji and Wang (2019) further demonstrated that endorphins, acting as MOR agonists, attenuate lung injury in a murine model of acute respiratory distress syndrome (ARDS) by downregulating TNF-α, IL-1β, and endothelial adhesion molecules via the PI3K/Akt signaling pathway [84].

Moreover, opioid receptor signaling has been shown to mitigate lipopolysaccharide (LPS)-induced ARDS. Sufentanil, a high-affinity MOR agonist, was reported by Kwan et al. (2020) to significantly reduce pulmonary inflammation by lowering concentrations of TNF-α, IL-6, and IL-1β [85]. These findings support the potential of opioids as modulators of systemic and pulmonary inflammation.

TLRs and COVID-19: key players in innate immunity

TLRs are a class of pattern recognition receptors (PRRs) that play a pivotal role in the initiation of innate immune responses through the recognition of pathogen-associated molecular patterns (PAMPs) [86]. These receptors are expressed on a wide range of immune cells, including dendritic cells (DCs), macrophages, natural killer (NK) cells, and lymphocytes, as well as on non-immune cells such as fibroblasts and epithelial cells [87]. TLRs are located on either the plasma membrane (e.g., TLR1, TLR2, TLR4, TLR6) or within endosomes (e.g., TLR3, TLR7, TLR8, TLR9) [88]. TLRs play an important role in people’s susceptibility to COVID-19 [89].

Upon ligand recognition, TLRs initiate intracellular signaling cascades that culminate in the activation of transcription factors such as NF-κB, interferon regulatory factors (IRFs), and MAP kinases. These pathways lead to the production of pro-inflammatory cytokines, chemokines, and type I interferons, orchestrating the immune response against pathogens [90]. Ten functional TLRs have been identified in humans, each utilizing different adaptor proteins, primarily MyD88 and TRIF, to mediate downstream signaling [91].

Among these receptors, TLR2, TLR4, and TLR9 have been particularly implicated in the recognition of SARS-CoV-2 and in triggering hyperinflammatory cytokine responses during infection. The innate immune system utilizes TLRs to detect viral components such as double-stranded RNA (TLR3) and single-stranded RNA (TLR7, TLR8), as well as viral glycoproteins (recognized by TLR4) [92]. Activation of these receptors triggers MyD88-dependent and TRIF-dependent signaling pathways, leading to the expression of pro-inflammatory mediators, including IL-1β, IL-6, TNF-α, and interferon-β [91, 93].

In viral infections such as COVID-19, hyperactivation of TLR pathways can lead to an exaggerated inflammatory response, often described as a “cytokine storm.” This cascade has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS), systemic inflammation, and multiorgan failure in severe cases of SARS-CoV-2 infection [94, 95].

Plasmacytoid dendritic cells (pDCs) are uniquely equipped to detect viral nucleic acids through endosomal toll-like receptors, particularly TLR7 and TLR9. These receptors are highly expressed in pDCs and serve as primary sensors for exogenous single-stranded RNA (via TLR7) and unmethylated CpG DNA motifs (via TLR9) [96, 97]. Upon activation, these TLRs initiate robust type I interferon (IFN-I) responses via two distinct but overlapping signaling cascades: the MyD88–NF-κB and MyD88–IRF7 pathways.

The specific pathway engaged depends largely on the subcellular localization of the TLR and the nature of the ligand involved [98, 99]. For example, multimeric CpG-A oligonucleotides localize to early endosomes in pDCs and preferentially trigger the MyD88–IRF7 axis, resulting in high amounts of IFN-I production. In contrast, monomeric CpG-B oligonucleotides localize to late endolysosomal compartments, where they primarily activate the MyD88–NF-κB pathway, leading to the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) [100, 101].

Interestingly, in pDCs, but not in conventional dendritic cells (cDCs), the trafficking of CpG-A to early endosomes and subsequent IFN-I production is dependent on Src family kinases, specifically Lyn and Fyn [102]. This spatial regulation of ligand trafficking and signaling has been proposed as a mechanistic basis for the differential cytokine profiles observed between CpG-A and CpG-B stimulation [103]. While these patterns are characteristic, they are not absolute; some overlap in cytokine output exists depending on the cellular context and stimulation conditions.

Multiple studies have suggested that SARS-CoV-2 components, particularly the spike (S) protein, interact with TLRs to modulate host immunity. In silico molecular docking studies have demonstrated strong binding affinities between the S protein and TLR1, TLR4, and TLR6, with TLR4 showing the highest predicted interaction potential [104]. This interaction may involve hydrogen bonding and hydrophobic contacts and could contribute to excessive TLR4 activation and inflammation [105].

SARS-CoV-2 is also known to activate endosomal TLRs, such as TLR3, TLR7, TLR8, and TLR9, thereby inducing the production of inflammatory cytokines, including IL-1, IL-6, IL-2, and IL-4. This contributes to increased vascular permeability, epithelial damage, and pulmonary edema, culminating in ARDS [94]. TLR4, traditionally associated with bacterial LPS recognition, has emerged as a potential viral sensor through accessory proteins like CD14 and MD-2 [106–108].

Host genetic studies have revealed that rare loss-of-function mutations in TLR7 are associated with impaired type I interferon responses and increased severity of COVID-19 in young male patients [109]. Similarly, murine studies have shown that deficiency in MyD88 or TRIF exacerbates disease severity in coronavirus infections, including SARS-CoV-1 and MERS-CoV [101, 110].

Pharmacologically, statins have been reported to stabilize MYD88 expression and attenuate NF-κB activation, suggesting a potential role in mitigating COVID-19-related inflammation [111, 112]. These findings support the exploration of TLR-targeted therapy, such as TLR4 antagonists or TLR7 agonists, for immune modulation in COVID-19 [113].

TLRs are key regulators of innate immunity with dual roles in pathogen defense and inflammatory pathology. While TLR activation is essential for initiating antiviral responses, overactivation, particularly of TLR4 and TLR7/8, may contribute to hyperinflammation and tissue damage in COVID-19. The interaction between viral components and specific TLRs underpins much of the disease’s immunopathology, especially in severe cases [114]. It has recently been shown that patients with COVID-19 exhibit impaired TLR-mediated cytokine and chemokine production [115].

Targeted modulation of TLR signaling, through selective agonists or antagonists depending on the receptor subtype, may offer promising therapeutic strategies. Future studies should aim to further delineate the context-specific roles of individual TLRs in COVID-19 to enable precision immunotherapy.

TLRs and opioids: immunological and neurological crosstalk

TLRs, particularly TLR4, have emerged as critical mediators not only in host defense but also in the pharmacological and immunological effects of opioids. Studies have demonstrated that TLRs are involved in modulating various opioid-induced responses, including analgesia, tolerance, and neuroinflammation [116].

Chronic administration of morphine has been shown to disrupt both innate and adaptive immune responses [71]. One of the key mechanisms involves the activation of the TLR4/NF-κB signaling axis within the CNS, particularly in microglial cells. This leads to the upregulation of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-18 (IL-18), which in turn increase the expression of MOR on neurons, potentially amplifying opioid sensitivity and reinforcing dependence-related pathways [117].

Beyond TLR4, morphine has also been reported to interact with TLR2 and TLR9, suggesting that opioid-induced immunomodulation involves a broader range of TLR family members [71]. Furthermore, opioids may exert dual effects on the immune system—suppressing protective immune functions while simultaneously promoting neuroinflammatory signaling via both classical opioid receptors (e.g., MOR) and TLR-dependent pathways.

These findings underscore a complex bidirectional relationship between opioid pharmacodynamics and innate immune signaling, with implications for both opioid-based analgesia and the pathogenesis of opioid-induced immune dysfunction.

P2XRs: ATP-mediated modulators of inflammation

ATP release from various cells is induced by tissue damage or pathogens; it is a strong immunomodulator. ATP regulates the release of inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-10, and TNF-α by activating different P2 receptors (P2Rs) [118]. The incidence of pathological events related to cell damage causes the accumulation of large amounts of ATP, which in turn activates the inflammatory cascade. In inflamed tissues, both ATP and adenosine are elevated, indicating a key role for P2 and P1 receptor signaling in inflammation [119]. P2X receptors (P2X1-7) are ligand-gated ion channels located in the cell membrane. These receptors are widely distributed in different tissues, including the central nervous system, smooth muscle cells, and the immune system [120]. Among purinergic receptors, P2X7 receptors (P2X7) have been studied more than other receptors. Activation of these receptors has been shown to release inflammatory cytokines such as IL1β, NO, and reactive oxygen species (ROS) [121]. It has been shown that ATP release from damaged cells increases the inflammatory response by stimulating P2X7 and by increasing prostaglandin E2 synthesis [122]. As illustrated in Fig. 1, opioids interact not only with classical opioid receptors but also activate TLR4 and P2X7 pathways, leading to pro-inflammatory signaling in immune cells. While these mechanistic links are biologically plausible, direct evidence specifically connecting opioids to P2 receptor signaling in COVID-19 patients remains limited and largely preclinical.

Fig. 1.

A mechanistic link between opioid signaling and immune modulation via TLR4 and P2X7. Opioid compounds, such as morphine, modulate immune responses not only through classical MOR but also by engaging pattern recognition receptors such as TLR4 and P2X7 on immune cells. Activation of these pathways leads to downstream signaling through NF-κB and MAPK cascades, resulting in the release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Chronic opioid exposure may therefore contribute to immunosuppression or immune overactivation, depending on receptor context and cellular environment

Expression and functions of P2X receptors in the immune system

P2XRs are widely distributed across multiple tissues, including neural, epithelial, muscular, and immune systems. Among them, P2X4 and P2X7 are the most extensively expressed subtypes in both immune and neural cells [123, 124]. P2X4 receptors (P2X4) are found not only in the central and peripheral nervous systems but also in ductal epithelia, smooth muscle cells of the gastrointestinal tract and bladder, uterine endometrium, and adipose tissue [125].

Under inflammatory conditions, the expression of P2XRs, particularly P2X4 and P2X7, is upregulated on innate immune cells such as monocytes, macrophages, and mast cells [122]. These receptors are also prominently expressed in pulmonary tissues, where P2X4 and P2X7 dominate over other P2X subtypes [126]. Notably, P2X7 is expressed by almost all cells of the innate and adaptive immune systems [127], highlighting its central role in host defense and immune regulation.

The functional significance of these receptors varies by subtype. P2X7 mediates a range of immune responses, including the release of inflammatory cytokines (e.g., IL-1β), chemokine secretion, regulation of T cell differentiation and survival, activation of transcription factors, and induction of programmed cell death [127].

In contrast, P2X4 activation has been implicated in the regulation of pulmonary epithelial function, promoting mucin and surfactant secretion, important for respiratory barrier integrity [126]. Blocking P2X4 in neuronal cells has also been shown to alleviate inflammation-related neural damage, suggesting its potential as an anti-inflammatory target [123].

Therapeutically, genetic deletion or pharmacological inhibition of P2X7 has shown promise in models of acute lung injury, asthma, and pulmonary fibrosis. In P2X7 knockout mice, survival following adenoviral infection improved due to reduced IL-1β production. These mice also exhibited protection against smoke-induced pneumonia and emphysema, further underscoring the receptor’s role in pulmonary inflammation and injury [128, 129].

P2XRs and COVID-19 infection

SARS-CoV-2 infection has been shown to stimulate the release of extracellular ATP in the lungs, primarily via the activation of mononuclear phagocytes such as alveolar macrophages. This ATP surge activates P2X7 on antigen-presenting cells (APCs) and macrophages, leading to enhanced secretion of inflammatory cytokines, chemokines, and further ATP release, thereby amplifying the local immune response [128].

Emerging evidence suggests that inhibition of P2X7 may represent a promising therapeutic strategy to mitigate hyperinflammation associated with severe COVID-19 cases [130]. In infected cells, ATP is released through Pannexin-1 channels, where it acts in an autocrine and paracrine fashion to sustain immune activation.

Once in the extracellular space, ATP undergoes enzymatic degradation by ectonucleotidases CD39 and CD73, yielding adenosine. This adenosine then binds to A2B adenosine receptors, which are highly expressed on macrophages and dendritic cells in inflamed tissues, stimulating the production of interleukin-6 (IL-6), a key cytokine in the COVID-19-related cytokine storm [131].

During viral infection, extracellular release of ATP has been shown to activate P2 receptors, particularly P2X7 and P2Y1. Stimulation of these receptors promotes the production of proinflammatory cytokines, including IL-1β and TNF-α, which in turn contribute to severe lung injury [132].

These observations highlight the central role of P2 signaling, particularly the ATP–P2X7–IL-1β and ATP–adenosine–A2B–IL-6 axes, in the pathogenesis of COVID-19 and suggest that targeting these pathways may offer novel avenues for therapeutic intervention. As shown in Fig. 2, ATP released from infected cells activates P2X7 and is metabolized to adenosine, which further stimulates inflammatory signaling via A2B receptors.

Fig. 2.

Schematic representation of P2 receptor signaling in COVID-19 inflammation. During SARS-CoV-2 infection, ATP is released from damaged or infected cells via Pannexin-1 channels. Extracellular ATP activates P2X7 on macrophages and dendritic cells, promoting the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-18. Concurrently, ATP is enzymatically converted to adenosine via CD39 and CD73. Adenosine binds to A2B receptors, further enhancing IL-6 production in inflamed tissues. This dual purinergic axis contributes to the cytokine storm and pulmonary dysfunction observed in severe COVID-19

Opioid–P2 receptor crosstalk: preclinical evidence and current gaps

Preclinical studies indicate that opioid exposure can influence ATP-driven immune signaling. For instance, microglial P2X4 contributes to morphine tolerance and neuroinflammation, and its inhibition attenuates morphine-induced changes in glial markers and MOR expression, suggesting a functional interface between opioid and P2 receptors [133, 134]. Moreover, ATP release through pannexin-1 (PANX1) channels can amplify P2X7 activation and downstream cytokine production, providing a mechanistic route by which opioids could modulate P2X receptor signaling in inflamed tissues [135, 136].

Although these observations support biological plausibility, direct evidence in COVID-19 remains scarce and is largely inferred from models of infection, lung injury, or neuroinflammation.

Collectively, experimental and disease-model data linking P2X7 to acute lung injury/ARDS and to COVID-19-associated neuropathology underscore the therapeutic rationale for P2X7 antagonism in hyperinflammatory settings [130–132, 137, 138]. However, targeted studies are needed to test whether chronic or acute opioid exposure modifies P2X4/P2X7 activity and clinical outcomes in SARS-CoV-2 infection.

P2XRs and opioids: intersection of neuroimmune and P2 receptor signaling

Interactions between opioid and P2 receptor systems play a significant role in shaping immune responses, particularly in the context of inflammation, infection, and tissue injury. Notably, MOR and P2X4 have been shown to co-regulate microglial migration via the PI3K/Akt pathway, illustrating a functional convergence between opioid and ATP-mediated signaling in the central nervous system [133].

ATP, released during infection, inflammation, hypoxia, or trauma, acts as a potent immunomodulatory molecule by engaging P2XRs and triggering downstream immune responses. In T lymphocytes, ATP is released through Pannexin-1 channels following antigen stimulation, activating P2X1 and P2X4, elevating intracellular Ca²⁺ concentrations, and ultimately activating NFAT, which promotes IL-2 production [134].

P2XR expression varies across immune cells. Neutrophils express P2X1 and P2X7. P2X1 tends to suppress chemotaxis, while P2X7 promotes pulmonary infiltration during inflammation [135, 137]. Macrophages express P2X1, P2X4, P2X5, and P2X7, but only P2X7 mediates calcium influx and caspase-1 activation, driving the maturation and release of IL-1β and IL-18 [138]. Dendritic cells (DCs) express mRNA for P2X1, P2X4, P2X5, and P2X7; their activation by ATP induces the release of IL-1β and TNF-α [138].

Recent studies suggest that P2X4 modulates P2X7 signaling, acting as a regulatory checkpoint for inflammatory cytokine production in macrophages [138]. In the setting of COVID-19, excessive P2X7 activation is implicated in acute lung injury and ARDS. These receptors, highly expressed on alveolar macrophages, contribute to cytokine storm development through the secretion of IL-1β, IL-6, and IL-18. Inhibition of P2X7 has been shown to mitigate this hyperinflammatory state in preclinical models [139].

In parallel, adenosine, a byproduct of ATP degradation via CD39 and CD73, modulates immune responses through P1 receptors (A1, A2A, A2B, and A3). Among these, A2A and A2B receptor activation increases intracellular cAMP, suppresses pro-inflammatory cytokine release (e.g., TNF-α, IL-6, IL-12), and promotes IL-10 production by monocytes and macrophages. Dendritic cells predominantly express A2A receptors, and A2B–ADA–CD26 complexes at DC–T cell interfaces further contribute to TNF and IFN-γ production [140]. Additionally, A3 receptor activation attenuates mast cell degranulation, relevant to bronchospasm in asthma.

Collectively, these findings underscore a complex interplay between opioid, P2 receptor, and adenosine signaling pathways, offering multiple therapeutic entry points to modulate immune activation and inflammation, particularly in diseases like COVID-19, where hyperinflammation is a central pathological feature. Therapeutic targeting of TLR4 and P2X7 may attenuate cytokine storms and pulmonary injury in COVID-19 (see Fig. 3).

Fig. 3.

Therapeutic potential of targeting TLR4 and P2X7 signaling in COVID–19–induced hyperinflammation. SARS-CoV-2 infection activates TLR4 and P2X7 on alveolar macrophages, triggering downstream inflammatory cascades via NF-κB and inflammasome activation. This results in the release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, contributing to cytokine storm and acute respiratory distress syndrome (ARDS). Pharmacological inhibition of TLR4 and P2X7 may attenuate these responses, reduce pulmonary inflammation, and prevent severe lung damage in COVID-19 patients

Conclusion

This review elucidates the critical role of opioid signaling, TLRs, and P2XRs in shaping immune responses during COVID-19, offering insights into novel therapeutic strategies. Chronic opioid exposure modulates innate and adaptive immunity through classical opioid receptors and direct interactions with TLR2, TLR4, TLR9, and P2X4 and P2X7, influencing cytokine production and immune cell recruitment. In COVID-19, hyperactivation of TLR4 and P2X7 pathways drives cytokine storms, acute lung injury, and multi-organ dysfunction, while adenosine signaling via A2A and A2B receptors provides a compensatory anti-inflammatory axis. Given the dual pro- and anti-inflammatory effects of opioids, their clinical use in COVID-19 patients requires careful consideration to avoid exacerbating immune dysregulation. Pharmacological targeting of TLR4 and P2X7, using antagonists such as eritoran or AZD9056, respectively, holds promise for mitigating hyperinflammation. Opioid and P2 receptor signaling appear to act within a broader innate inflammatory network involving the Contact System, KKS, RAS, and NLRP3 inflammasome. This integrated view may refine therapeutic approaches targeting TLR4 and P2X7 in COVID-19. Importantly, direct opioid–purinergic data in COVID-19 are still sparse, with most support derived from preclinical or non-COVID models. Future clinical and preclinical studies should prioritize delineating the context-specific roles of opioids in TLR and P2X signaling, particularly in patients with chronic opioid use, to develop precision immunotherapies for COVID-19 and related inflammatory diseases.

Mohammad Abbas Sheikholeslami

is a pharmacologist at the Department of Pharmacology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. He received his Pharm.D. degree from Tabriz University of Medical Sciences and his Ph.D. in Pharmacology from Shahid Beheshti University of Medical Sciences. His main research interests focus on pain pharmacology, opioid use and dependence, addiction-related neurobiological mechanisms, and neuroinflammation, with particular emphasis on experimental models of nociception and opioid-induced adaptations. He has authored approximately 26 peer-reviewed scientific publications, and his current research explores the interaction between opioids, glial activation, and inflammatory signaling pathways in neurological and systemic disorders.

Author contributions

M.A.S. and S.P. conducted the literature review and prepared the initial draft of the manuscript. S.G. contributed to data collection, critical analysis, and manuscript editing. M.Z. conceptualized the study, supervised the project, and finalized the manuscript. All

Funding

This work received no specific funding.

Data availability

Data available on request from the authors.

Declarations

Ethics approval and consent to participate

Not applicable.

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Not applicable.

Competing interests

The authors declare no competing interests.