AhR Participates in COVID-19’s Immune-Inflammatory Imbalance: Comparison
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The comprehension of AhR’s role in the COVID-19 framework must consider its participation in human physiology and, in particular, in inflammatory and immune processes, where AhR is involved in the regulation of both innate and adaptive immunity, as it influences both DCs and T lymphocytes. In DCs, it decreases the expression of the Major Histocompatibility Complex II (MHC II). It also regulates the production of inflammatory cytokines, such as IL-6, IL12, IL15, and IL18, which are usually produced during DC differentiation. DCs differentiation takes place upon their exposure to T cells, viral/bacterial components or pro-inflammatory molecules, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNα, and inflammatory cytokines such as IL-6 and TNFα. This point is of pivotal importance, due to the crosstalk between several inflammatory molecules such as IL-6, TNFα and the AhR pathway. This interplay also reverberates on the differentiation of Th17 and Treg, in particular in type 1 regulatory T cell (TR1) stabilization. AhR can deeply affect T cells metabolism, which can be modulated depending on the AhR ligand. It has been demonstrated that the differentiation of TR1 cells is the result of the sequential collaboration of Hypoxia Inducible Factor-1α (HIF-1α) and AhR. In physiologic settings, AhR endows the degradation of HIF-1α, while in inflammation, which is frequently associated with hypoxia, HIF-1α inactivates AHR, thus interfering with TR1 cell differentiation. AhR is also abundantly expressed in DCs. 

  • aryl hydrocarbon receptor
  • inflammation
  • SARS-CoV-2

1. Interferons

Interferons are a group of proteins that, as suggested by their name, interfere with the replication and spreading of viruses. They stimulate the transcription of a myriad of ISGs, whose products are an extremely diversified panel of molecules. Some of them actively participate in the antiviral response, in the setting of a long-lasting adaptive immunity and the control of proliferation in repaired tissues. Early stages of the innate immune response against viral infections are mediated by Type I IFNs, which in COVID-19, are referred to as “weak” and transitory [1]. Among IFNs, IFN-Is are a large family of structurally related cytokines including IFN-α (13 different subtypes), IFN-β, IFN-ε, IFN-κ and IFN-ω. They are produced mainly by plasmacytoid Dendritic Cells (pDCs) when Pathogen-Associated Molecular Patterns (PAMPs) associated with viral nucleic acids are detected by cytoplasmic sensors belonging to the family of Retinoic acid-Inducible Gene I (RIG-1)-like receptors (RLSs) and Melanoma Differentiation-Associated gene 5 (MDA-5). This interaction engenders a complex pathway that culminates in the production and secretion of IFN-I family molecules. In SARS-CoV-2 infection, secreted IFN-Is—mainly IFNα and IFNβ—bind interferon-alpha and beta receptors (IFNARα and IFNARβ) in target cells, including type II pneumocytes, cardiomyocytes, endothelial cells, enterocytes, hepatocytes, and astrocytes [2]. IFNAR binding originates an intracellular pathway where phosphorylated STAT1 and STAT2 heterodimerize and associate with a DNA-binding protein called IFN regulatory factor 9 (IRF9) [3][1]. This complex, named IFN-stimulated growth factor 3 (ISGF3), moves to the nucleus and targets interferon-stimulated response elements (ISREs) in the promoters of ISG. [4]. ACE-2 has been described as an ISG in an in vitro model of human airway cells [5]. ISGs also include antiviral effectors and positive/negative IFN-I regulators. In some cell types, IFN-Is induce the expression of AhR; as described by Yamada’s group, through a negative feedback mechanism, AhR inhibits IFN-Is response [6]. This point is of particular interest, as IFN-Is operate on two sides: (1) they induce IDO-1, which catalyzes the transformation of Trp in the immune-suppressive Kyn, which binds and activates AhR; and (2) they also induce the expression of AhR, which as anticipated, limits IFN-Is action via negative feedback. Thus, we can hypothesize that AhR could facilitate COVID-19 infection not only through Kyn-driven immune suppression, but also through the mitigation of IFN-Is response. 

2. NF-κB

AHR activity is negatively regulated by the TCDD-inducible poly-ADP-ribose polymerase (TIPARP), an enzyme that is induced after the binding between AhR and TCDD. Therefore, the TIPARP gene belongs to the AhR gene battery [7]. TiPARP is a mono-ADP-ribosyl transferase that transcriptionally inhibits AhR through the ribosylation of its core histones [8] (Figure 1). TIPARP catalyzes the ADP-ribosylation of other targets, including TANK- Binding Kinase (TBK1). This serine/threonine-protein kinase has a key role in innate immunity, in particular during viral infections, as it coordinates the activation state of IRF3 (Interferon Regulatory Factor 3) and NF-κB. As this pathway is involved in IFN-I production and exocytosis, its inhibition by TIPARP-mediated ADP-ribosylation could indirectly link AhR to IFN-I inhibition in viral infections [6]. As anticipated, low levels of IFN-I have been reported in viral infections and, in particular, in COVID-19-affected patients [1]. A low capacity to induce IFN-I response is reported not only for SARS-CoV-2 but, more generally, for coronaviruses infections. These viruses have a set of proteins that can interfere with IFN-I production and functioning in target cells. Successful viral infection usually leads to the repression of IFN-Is, thus limiting the setting of the innate immunity. The inhibition of TBK1 mediated by TiPARP is not the only mechanism that can link AhR activation with IFN-Is inhibition. As anticipated, in 2020, Sa Ribero’s group [1] described that in SARS-CoV-2-infected cells, the RNA coronavirus interferes in IFNs production and signalling through different mechanisms that can target a) in infected cells: the intracellular sensing of the virus, the activation of cytoplasmic IFNs gene regulators, the induction of IFN-I (α/β) genes and their expression; and b) in surrounding cells, the IFN-Is binding to the interferon α and β Receptor (IFNAR) and the downstream pathway, which culminates in the induction and expression of ISGs. Some of these steps could potentially intersect with the AhR pathway: for example, after IFNAR activation, the downstream Jak/STAT pathway can recruit AhR [9]. In 2016, long before the COVID-19 outbreak, Rothhammer’s group described the involvement of AhR in the anti-inflammatory effect of IFN-Is in experimental models of CNS autoimmunity and in patients suffering from multiple sclerosis (MS). Here, it also described that AhR instigates the inhibition of NF-κB by inducing its inhibitor, SOCS2. AhR’s ability to modulate NF-κB during viral infections has been described in a paper by Giovannoni’s group [10]. Here, they showed that the activation of AhR from the Flavivirus Zika (ZIKV) negatively affects the production of IFN-I and the promyelocytic leukaemia protein (PML), which drives the intrinsic immunity to ZIKV. In particular, AHR targets NF-κB, thus limiting its crosstalk with IFN-Is and PML, whose expression and antiviral functions are seriously affected. It has long been known that NF-κB is involved in the expression of TNFα and IL-6 induced by the Spike protein of SARS coronaviruses [11] in murine macrophages; in particular, according to Liao, the nucleocapsid S protein activates NF-κB [12]. This transcription factor is increasingly gaining a central role in the so-called COVID-19 “cytokine storm”, a cascade of inflammatory events that culminates in the systemic release of proinflammatory cytokines [13][14][15].
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Figure 1. The AhR management of inflammation in COVID-19. Concept map summarizing AhR’s role in the inflammatory response and in the modulation of innate and adaptive immunity. The right side of the diagram summarizes the effects of AhR on innate (TiPARP-mediated inhibition of IFN-I) and acquired immunity (shift in Treg/Th17 balance). The left side of the diagram summarizes the IL-6_STAT-3_IDO_Kyn_AhR-driven auto inflammatory loop and AhR’s influence on ACE-2 and B0AT1 expression. The map was drawn using the freeware software CmapTools, developed by the Florida Institute for Human and Machine Cognition (IHMC) (Cmap | CmapTools (ihmc.us), accessed on 10 February 2022).
As described by a plethora of reports, AhR interacts with NF-κB and its family members RelA and RelB in various contexts, including the inflammatory context [16][17]. As evidenced by Poppe, in human CoVs infections, the NF-κB pathway can be prompted or suppressed. This complex strategy, which targets the host chromatin, is commonly present in RNA virus infections and is aimed at optimizing viral replication through the reprogramming of large sets of genes [18]. On this basis, it is possible to hypothesize that AhR and some key factors in the inflammatory response could be involved in this reprogramming. This hypothesis has been confirmed in a recent paper by Giovannoni’s group [19]. Here, considering previous studies, they used human and mouse cells infected with α and β Coronavirus. Here, they showed AHR activation by the increased expression of its cellular targets, including CYP1A1 and CYP1B1. They also highlighted the rise in the expression of AHR and other genes linked to its pathway in mouse bone marrow-derived macrophages infected with an M(murine)-CoV β coronavirus and evidenced that SARS-CoV-2 replication is halted by the antagonists-mediated inhibition of AHR. These data were confirmed in cells from infected patients. The authors also detected an increase in IDO-1 and AHR gene expression in cells from patients with medium/high gravity pathology, thus linking Kyn production to AhR activation and IDO-1-dependent tolerogenesis. More importantly, they evidenced a positive correlation between AHR  gene expression and the viral load of infected cells. They showed that AHR regulates the transcription of a panel of genes linked to the inflammatory response, including NF-κB. In particular, they suggested that the activation of the AhR pathway limits the NF-κB-mediated immune strategy. Therefore, we can hypothesize that CoVs-mediated infections are facilitated from an inadequate immune response, due in part to the AhR-negative modulation of both IFN-I and NF-κB. This complex strategy is commonly present in RNA virus infections and is aimed at optimizing viral replication. 
However, it must be considered that the progression of COVID-19 is rapid. The insufficient response by IFN-I allows for the evasion of the innate immune response for seven to ten days and for an increase in the viral load. Thus, monocytes accumulate in respiratory parenchyma and an increasingly intense and widespread inflammation develops, i.e., the so-called “cytokine storm”. According to Pallotta and Orabona [20][21], as anticipated, an increase in IL-6 drives IDO-1 to proteasomal SOCS-3-induced degradation; consequently, Kyn levels and Kyn-induced immune suppression decrease. In this IL-6-induced inflammatory environment, NF-κB is strongly induced by inflammatory mediators and plays an inflammatory role [11]. These issues encourage us to hypothesize a bi-modal interaction between AhR and NF-ĸB: a first phase, which allows for SARS-CoV-2 entry and replication, where AhR activates ACE-2 and has an inhibitory action on IFN-I. In this framework, the pp60src activation of IDO-1 favours the establishment of the IDO-1–Kyn–AhR circuit that prolongs the activation of AhR, whereas NF-κB plays an anti-inflammatory and immunosuppressive role. The protracted activation of AhR originates a proinflammatory loop, which sustains endogenous IL-6 production (Figure 2). The increase in IL-6 gradually involves other additional inflammatory pathways including the one sustained by NF-κB, whose proinflammatory influence gradually rises, playing a substantial role in the “cytokine storm”.[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]
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Figure 2. Bi-modal activation of AhR. Kyn activates AhR, which in an inflammatory context, binds to its response element in the promoter of IL-6, thus participating in IL-6′s endogenous production and the amplification of the inflammatory state. IL-6 binding to its receptor IL-6R results in STAT-3 activation, which binds to IDO-1 and AhR promoters, thus endowing IDO-1 and AhR gene expression. In the cytoplasm, IDO-1 catalyzes the formation of Kyn from Trp; Kyn binds and activates AhR, which similarly to STAT-3, binds to the IDO-1 promoter and activates IDO-1 expression. IDO-1 enzymatic activation is also obtained by pp60src, which detaches from the AhR inactivating complex and phosphorylates IDO-1, thus completing this inflammatory loop. High levels of IL-6 favor the upregulation of SOCS3, which engages the E3 ubiquitin ligase complex (E3-ULC), thus driving cytoplasmic IDO-1 to proteasomal degradation. In parallel, STAT-3 sustains the expression of AhR, which in turn, stimulates the expression of IL6, thus setting a self-sustaining autoinflammatory loop.

References

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