The role of connexin and pannexin containing channels in the innate and acquired immune response

Abstract

Connexin (Cx) and pannexin (Panx) containing channels – gap junctions (GJs) and hemichannels (HCs) – are present in virtually all cells and tissues. Currently, the role of these channels under physiological conditions is well defined. However, their role in the immune response and pathological conditions has only recently been explored. Data from several laboratories demonstrates that infectious agents, including HIV, have evolved to take advantage of GJs and HCs to improve viral/bacterial replication, enhance inflammation, and help spread toxicity into neighboring areas. In the current review, we discuss the role of Cx and Panx containing channels in immune activation and the pathogenesis of several infectious diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

1. Introduction

The innate immune response, a critical step in the defense against infectious agents, is composed of the epithelial and endothelial barriers, neutrophils, macrophages and monocytes, dendritic cells, granulocytes, natural killer cells and mast cells. Normally this type of immune response is transient and not specific to any given pathogen [1–4]. In contrast, the adaptive (or acquired) immune response, which is also composed of specialized cells, is highly specific to a particular pathogen and generate immunological memory. The generation of long lasting immunological memory requires cell-to-cell contact to maintain the specificity, proliferation, and differentiation of distinct cellular subsets [1–4]. Connexin (Cx) and pannexin (Panx) containing channels – gap junctions (GJ) and hemichannels (HC) – have been proposed to participate in these processes.

HCs are Cx hexamers formed by identical (homomeric) or different (heteromeric) Cx subunits. GJ channels are formed by the docking of two HCs positioned on the surface of two adjacent cells (Fig. 1A). GJs can be homotypic, i.e. formed by two identical HCs, or heterotypic, i.e. formed by different HCs [2,5–7]. The multiple potential combinations of different Cxs result in the formation of channels with different biophysical properties and permeability [8,9]. The large internal diameter of the pore of these channels is around 12 Å, allowing ions and intracellular messengers less than 1.2 kDa to diffuse between connected cells, including IP₃, calcium, cyclic nucleotides, metabolites, toxic molecules, neurotransmitters, viral peptides, antigens, and electrical signals [2,5–7]. Non-docking HCs can open on the surface of the cells enabling the release of signaling molecules into the extracellular space including ATP, PGE₂, S1L and NAD+ (Fig. 1B).

Fig. 1.

Schematic representation of Cx channels: gap junctions (GJs) and hemichannels (HCs). a) Representation of GJ channel communication from cell 1 to cell 2. GJ enable intracellular molecules to diffuse between connected cells. Some of these signaling molecules are ATP, IP₃, Ca²⁺, toxic molecules and antigens (second messengers are represented as green balls) diffuse through cell 1 to cell 2 to coordinate several physiological and pathological functions. b) Representation of HCs on the surface of cells. Upon opening of these channels, intracellular signaling molecules such as ATP, NAD⁺, ions and other small molecules (represented as blue balls) are released into extracellular media.

Panxs are a family of proteins similar to Cxs, which consists of three members: Panx-1, -2 and -3 [10]. Panxs are structurally and topologically similar to Cxs, although they share no sequence homology. Like Cxs, Panxs consist of a cytosolic N-terminal domain, four transmembrane domains with two extracellular loops and a cytosolic C-terminal domain [11,12]. Also like Cxs, Panx proteins form large pore channels located on the plasma membrane. Initially, it was speculated that Panx channels could form GJ channels between adjacent cells [13–15]. However, ultra-structural analysis by electron microscopy indicated that Panx-1 junctional areas do not appear as canonical GJs [12]. In support of this conclusion, asparagine residues found in the extracellular domains of Panxs are glycosylated and therefore make docking between two Panxs unlikely due to structural/sterical hindrance [11,12].

Panx-1 is ubiquitously expressed in many cell types, including those in the eye, thyroid, prostate, kidney, liver, immune system, and CNS [16]. Panx-2 is mainly expressed in the central nervous system (CNS) while Panx-3 is localized in the skin, osteoblasts, and chondrocytes [17,18]. While Panx channels remain in a closed state under physiological conditions, they open during pathological conditions such as membrane depolarization, increased intracellular Ca²⁺ signaling, changes in blood flow, airway defense, tumorigenesis, cellular differentiation, cell death and during activation of innate and adaptive immune responses [19–21]. Upon Panx channel opening, small signaling molecules including NAD⁺, PGE₂, glutamate, and ATP are released into the extracellular space resulting in autocrine and paracrine stimulation [22–25].

Cxs and Panxs are present in all immune cells and related tissues. Cx43 is the most well studied Cx isoform in the immune system and is expressed by macrophages, neutrophils, lymphocytes and mast cells during hematopoiesis, differentiation, and inflammatory reactions [26, 27]. However, for a long time, the expression and function of GJs and HCs in the immune system were highly controversial. Initially, lack of reliable evidence was mostly related to the fast degradation of Cxs/ Panx proteins in activated immune cells. Only recently, most laboratories and publications agreed that in parenchymal cells, Cxs are negatively affected by inflammatory conditions [27,28]. However, during the last 10 years, it became evident that the regulation of these channels is different in immune cells: increased protein expression, channel formation and opening are detected during immune activation and inflammation to coordinate the innate and adaptive immune responses [26,27]. In this review, we will describe the Cx/Panx expression profiles of several types of immune cells and the role of these channels in the generation, amplification, and consolidation of the innate and adaptive immune responses. Briefly, Cxs and Panxs are expressed in different immune cells and play a key role during the innate immune response. Table 1 resumes the different stimulus who improves the expression of GJ or HC. Below we will discuss the role of GJ and HC in particular immune cells.

Table 1.

Brief summary of the expression of different of Connexins (Cxs) and Pannexins (Panxs), and their role in the innate immune response as GJs or HCs.

Cell type	Cx/Panx expression	Specific stimuli	Gap junction (GJ)/hemichannel (HC)	Ref.
Monocytes/macrophages	Cx43	LPS, INF-γ, and TNF-α, oxidative stress, tissue-specific conditions present in atherosclerosis, communication with dendritic cells.	GJ	[38], [39], [47–50], [66–68]
	Cx32, Cx37, Cx43, Panx-1	Sepsis, calcium, inflammatory diseases, HIV.	HC	[25], [70], [74], [80,81], [84]
Microglia	Cx29, Cx32, Cx36, Cx43, Cx45	LPS, granulocyte-macrophage colony-stimulating factor, INF-γ, and TNF-α, injury border or penumbra, Staphylococcus aureus-derived peptidoglycan.	GJ	[36], [37], [57–59]
	Panx-1, Panx-2	INF-γ, TNF-α, ATP, amyloid-β peptide, IL-1β	HC	[85–91]
Kupffer's cells	Cx43	Proinflammatory agent (LPS and INF-γ)	GJ	[35]
Mouse Macrophage cell line J774	Cx43	ATP	GJ	[40–43]
Astrocytes	Cx43	Quinine, metabolic inhibition	HC	[71,73]
B lymphocyte	Cx43	Chemokines	GJ	[44], [45]
T-cells	Cx31.1, Cx32,Cx43, Cx45 and Cx46	cAMP, INF-γ, TNF-α	GJ	[54], [92], [97–100], [103–105], [107]
	Cx43	Cytokines, inflammation	HC	[93], [92]
	Panx-1	Pathological conditions, calcium, inflammation	HC	[93], [92], [112–116]
NK cells	Cx43	LPS plus INF-γ, LPS plus TNF-α	GJ	[107], [62,110,111]
Neutrophils	Cx40, Cx43	LPS, INF-γ	GJ	[43]
	Panx-1	Inhibition by hypertonic salines	HC	[120]
Granulocytes	Cx43	Inflammation	GJ	[43]
Eosinophils	Cx43	Allergic inflammation	GJ	[118]
Mast cells	Cx32, Cx43	Inflammation	GJ	[123,124]
	Panx-2	Inflammation	HC	[125]

2. Expression of connexins and pannexins in immune cells

2.1. Role of GJs and HCs in monocyte/macrophage function

Monocytes are members of the mononuclear phagocyte system that originate in the bone marrow. They circulate for three days and then infiltrate different tissues to become resident macrophages [29,30]. Macrophages survive in tissues for weeks to years [31–34], and upon local activation or non-inflammatory surveillance, they migrate into damaged or apoptotic areas where they aggregate, present antigens, clear pathogens and help to eliminate damaged cells, thereby improving tissue recovery, renewal and coordinating the immune response during inflammatory conditions. Based on our publications and those of others, we propose that due to their physical proximity or aggregation, macrophages express Cxs and form GJs as well as open Cxs and Panx-1 HCs to enable an efficient local and systemic immune response.

In the 90s it was highly controversial and unclear whether macrophages expressed Cxs, formed GJs or HCs, or both. Ten years later, it was demonstrated that several immune cells including monocyte/macrophages, microglia, and Kupffer's cells express low levels of Cx43 under resting conditions, but upon specific stimulation Cx43 expression is induced [35–39]. Pioneering experiments using J774 cells, a mouse macrophage cell line, indicated that in response to ATP treatment macrophages can form a Cx43 containing pore that associated with purinergic receptor signaling [40–43]. This was one of the first indications that Cx43 containing channels (probably hemichannels) in concert with secreted ATP and purinergic receptors participate in the immune response. However, how immune cells activate Cx expression and functional communication is still poorly understood. Nevertheless, this data indicates that endothelial activation results in secretion of factors that induce Cx43 expression and promotes the formation of functional GJs in circulating cells. In agreement, in vivo, Cx43 expression has been detected in activated peritoneal macrophages; however, freshly isolated circulating monocytes (CD14⁺CD16⁻ cells) were negative for Cx43 mRNA and protein. Furthermore, it was demonstrated that Cx43 expression was required for monocyte activation and adhesion to the endothelium and that increased expression of Cx43 in monocytes contributed to the enhanced release of metalloproteinases MMP-2 and MMP-9 to enable the transmigration of monocytes across the blood-brain barrier (BBB) [38]. In agreement, cell migration is critical for immune cell differentiation, tissue formation, and organ development [44]. Recent findings have shown that GJs, and specifically Cx43 containing channels, play an important role in these processes, impacting adhesion and cytoskeletal rearrangements, transendothelial migration and direct migration of B cells [45,46]. In this model, Cx43 containing channels were able to show membrane distribution, influenced by the F-actin cytoskeleton, rearrangement, and these channels were necessary for B cell motility, CXCL12-mediated (chemokine) and Rap1 (GTPase protein) activation, which regulate B cell migration [45].

Different stimulus can activate the generation and amplification of the immune response by GJs. It was found that the activation of PKC by PMA and increasing intracellular calcium using a calcium ionophore [38], lipopolysaccharide (LPS) or TNF-α in combination with IFN-γ, acts cooperatively to induce the expression of Cx43 and formation of functional GJ channels in monocytes/macrophages and microglia [38]. Other pathological conditions that induce expression of Cx43 in monocytes and macrophages are oxidative stress, general inflammation, and during the stabilization of atheromatous plaques [47]. For example, foam cells in the atherosclerotic lesions in the carotid artery were found to contain Cx43 mRNA [38,48]. Nevertheless, a definitive demonstration of functional macrophage-macrophage or macrophage-polymorphonuclear or epithelial cell GJ communication [49,50] in these types of lesions has been controversial [51,52]. One complicating factor is that expression of Cxs does not always translate into GJ communication. For example, in cancer, the expression of Cxs occasionally does not associate with functional GJ communication. Instead, Cxs serve as proto-oncogenes or “sinks” to accumulate proteins in the cytoplasm due to the multifunctional binding sites present in connexins [53,54]. Thus, a clear demonstration of functional coupling or HC opening is still required to prove diffusion of second messengers in these pathologies.

Microglia, one of the major components of the innate immune system in the brain and the primary immune cell in the central nervous system (CNS) [55], express Cx36 and Cx43 [36,37]. A higher expression of Cx36 was to found to be related to a possible mechanism for cellular hyperexcitability [56]. In vivo, our data demonstrated that upon a stab wound injury, microglia becomes activated and migrate to the damaged area. Upon microglia aggregation around the lesion, Cx43 expression and functional GJ channels were required for an effective immune response, clearing of debris and promoting partial regeneration [37,57]. Furthermore, after cryo-traumatic brain injury, activated microglia started expressing Cx29 and Cx32 in the injury border or penumbra regions [58]. The treatment of microglia with Staphylococcus aureus-derived peptidoglycan also resulted in efficient expression of Cx43 and functional inter-cellular communication [59]. Thus, cellular damage, inflammation and regeneration trigger expression of Cxs and functional GJ communication among aggregated microglia.

GJs also play an essential role in dendritic cell activation and the amplification of antigen presentation. Specifically, antigens can diffuse from the cell processing the antigen into GJ connected cells that have never been exposed to the pathogen. This process, called cross-presentation [60–65], enables coupled cells to share viral peptides (antigens) and trigger an effective cytotoxic T lymphocyte (CTL) response, even among cells never directly exposed to the pathogen [64]. Recently, it was demonstrated that oral tolerance could be established by sharing antigens via GJs between macrophages and dendritic cells [66]. We believe this mechanism of antigen signaling amplification explains a critical point mostly ignored in immunology: how cell-to-cell activation can result in a broad and systemic activation that cannot be explained by cell to cell contact with the antigen-presenting cell [67,68]. Currently, the challenge is understanding how this mechanism occurs in vivo.

In addition to GJs, Cxs also form HCs that upon opening enable intra-cellular metabolites to diffuse into the extracellular environment (Fig. 1B). Normally, Cx containing HC exist in a closed state. However, during cell volume changes in response to stress, apoptosis, and metabolic inhibition, these channels open [13,69–74]. In macrophages, the opening of HCs has been proposed to serve as a diffusion pathway for the release of intracellular messengers like ATP, NAD⁺ or Ca²⁺ into the extracellular space to mediate autocrine and paracrine communication [75]. In agreement, changes in extracellular ATP and intracellular calcium concentration can retro-regulate Cx HC opening and closing [75–79]. In inflammatory diseases, such as atherosclerosis, Cx37 HC (present in primary monocytes and macrophages) may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion to the endothelium [80]. The HIV-induced opening of Cx43 HC was also found to dysregulate the secretion of the dickkopf-1 protein [81]. The data indicate that HC containing Cxs or Panxs are critical in the generation, amplification, and resolution of inflammatory responses and contribute to the pathogenesis of several diseases.

Panxs only form HCs and are expressed in several immune cells including macrophages. During physiological conditions, the Panx-1 HCs remain in a closed state. However, upon Panx-1 HC opening, several in-tracellular factors such as ATP are released into the extracellular space resulting in activation of several purinergic and adenosine receptors [19,82]. The cross-talk between these channels and ATP/adenosine receptors initiates an intracellular signaling cascade which results in the rearrangement of the F-actin microfilament network in C6 cells [83]. The opening of Panx-1 HCs can also be induced by several chemokines in a transient manner to control directed chemotaxis in normal and pathological conditions, including migration of T cells into the spinal cord in an animal model of multiple sclerosis [25]. We showed that Panx-1 channels concentrate in the leading edge of the cell to control FAK activation and actin rearrangement [25]. Thus, the opening of Panx-1 HC is essential to control cell migration. Furthermore, only recently, our laboratory demonstrated that macrophages exposed to HIV release ATP via the opening of the Panx-1 HC, but not Cx43 HC, facilitating the autocrine activation of purinergic receptors, which plays a significant role in HIV entry and replication in human macrophages [25,84] (see model in Fig. 2). Reducing the synthesis or opening of Panx-1 channels resulted in the nearly complete blocking of HIV entry and replication [25,84]. Thus, these channels constitute an attractive potential therapeutic target to block or reduce HIV infection and replication.

Fig. 2.

Proposed mechanism of release ATP by opening Panx-1 HCs and subsequent activation of purinergic and adenosine signaling during the immune response. The release of ATP from intracellular space to extracellular media it is mediated by the opening of Panx-1 HCs on the surface of immune cells by stress-related stimuli. Upon opening ATP is released through the channel into the extracellular space to activate purinergic receptors (P2X and P2Y). In addition, secreted ATP is subjected to degradation mainly by ecto-ATPases resulting in the hydrolysis of ATP to ADP and ADP to AMP, which promotes the formation of adenosine and activation of adenosine receptors (P1).

Microglia under resting conditions express detectable levels of Panx-1 but not Panx-2 [85–87]. Activation of microglia with different pro-inflammatory agents including IFN-γ [88], amyloid-β peptide [89,90], TNF-α plus ATP [91], TNF-α plus IFN-γ [91], and TNF-α plus IL-1β [91] increases the expression as well as the opening of Panx-1 HCs. For example, treatment of microglia with amyloid-β increased Cx and Panx-1 expression as well as the opening of these channels to promote glutamate release, suggesting their participation in the pathogenesis of Alzheimer's disease [90]. In summary, GJs and HCs play key roles in monocyte/macrophage activation, differentiation, and transmigration as well as to coordinate their immune response.

2.2. The role of GJ and HC in T cell differentiation and immune response consolidation

T lymphocytes play a fundamental role in cell-mediated immunity. T-cells originate in the bone marrow, but their differentiation and maturation occurs in the thymus. Cxs and Panxs have been identified in several lymphoid tissues and cells, and are proposed to play a role in the generation, amplification, and consolidation of the immune response. For example, blocking Cx containing GJs and HCs by specific extracellular peptides disrupts immunoglobulin release and cytokine production by lymphocytes [92–94]. siRNA against Cx43 in bone marrow-derived dendritic cells leads to diminished T cell stimulation, suggesting that Cx43-mediated communication is essential to trigger efficient T cell proliferation [95]. Additionally, Th1 cells can downregulate Cx43 expression and communication in astrocytes via microglia activation [96], but the soluble factors controlling these changes in expression are unknown. In T cell differentiation, the expression of Cxs has been proposed to coordinate the exchange of nutrients, peptides, and second messengers including cyclic adenosine monophosphate (cAMP), an essential metabolite for T cell differentiation. High levels of cAMP can inhibit T cell function during the immune response, e.g. blunting CD4⁺ T cell activation, cell proliferation, and production of certain cytokines, such as IFN-γ and TNF-α [97–100]. Similarly, a high concentration of cAMP induces immunosuppressive effects and stimulates inflammation by promoting IL-17 production and Th17 cell expansion [101,102]. An increase in the intracellular concentration of cAMP also induces a higher expression of Cx31.1, Cx32, Cx43, Cx45, and Cx46 in T-cells, including Tregs [103–105], but the role of these Cxs in T cell differentiation is still under active investigation. Currently, the profile of Cx expression in T cell subpopulations, including Th1, Th2, Th17, Th0, and Tregs is unknown; however, it is likely that Cx containing channels participate in T cell differentiation and associated cytokine release. For instance, it has been reported that Cx43 HCs and GJs are required to sustain the proliferation and therefore clonal expansion of T cells to initiate an effective immune response [39].

The role of GJs and HCs in T cells has been investigated during pathological conditions including cancer. In human melanoma biopsies, it has been demonstrated that Cx43 is expressed between tumor and endothelial cells and between T-cells and cancer cells, indicative of GJ formation [54]. This study proposed that GJs between these cells regulate the formation of an immunological synapse among T lymphocytes and autologous melanoma cells [106]. In this model, the opening of Cx43 HCs has been associated with cytokine release by T-cells and regulation of Ca²⁺ oscillations among T cells. Furthermore, it has been reported that cell conjugates forming an immunological synapse may target melanoma cells [107]. A key cytokine involved in T cell activation is IFN-γ, and Cx43 in T cells is essential for its secretion; blocking Cx43 GJs substantially diminished IFN-γ secretion by primed T cells [107]. The secretion of these key cytokines is also essential to mediate proper T cell differentiation (surface markers) and to regulate all the downstream events of the immune response [28,108,109]. Thus, Cx43 GJs may contribute to sustained communication between T cell and antigen presenting cells, allowing optimal T cell activation [107].

In the context of cancer, Cx43 expression by NK cells plays a key role in mediating bidirectional and functional intercellular communication between NK cells and dendritic cells [107,110,111]. Moreover blocking Cx43 inhibited NK cell activation and reduced the influx of Ca⁺² and cytokine release by T cells [107]. Using mimetic peptides for as a blocker of Cx43 reduced CD69 and CD25 (activation markers for T cells) expression and INF-γ release by dendritic stimulated NK cells [110]. Furthermore, cell-to-cell communication between dendritic cells is required for efficient immune activation including expression of costimulatory molecules such as CD80 and CD86, MHC class II and allostimulatory capacity [62].

Upon αβT-cell receptor engagement, T-cells use HCs to release ATP, resulting in subsequent activation of a purinergic receptor and cellular activation [112]. γδT cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or an intermediate of the isoprenoid synthesis pathway (IPP), resulting in cell activation. Pretreatment of γδT cells with Panx-1 blocker, carbenoxolone [(3β, 20β)-3-(3-carboxy-1-oxopropoxy)-11-oxoolean-12-en-29-oic acid disodium] (CBX), or bafilomycin A (BFA) reverses the stimulation and induces an increase in extracellular ATP concentration, indicating that Panx-1 HC, Cx HC, and gap junctions contribute to the controlled release of ATP into the extracellular space [112]. Furthermore, T cell receptor (TCR) stimulation results in the influx of Ca²⁺, which is buffered by the mitochondria and promotes ATP synthesis. ATP released from activated T-cells through Panx-1 HCs was found to activate purinergic P₂XR to sustain mitogen-activated protein kinase (MAPK) signaling [113]. In vivo administration of oATP, a purinergic receptor blocker reduces the onset of diabetes mediated by anti-islet TCR transgenic T cells and impairs the development of colitogenic T cells in inflammatory bowel disease [114–116]. In conclusion, HCs in concert with secreted ATP are not the only key to controlling the immune response and T-cell activation, but also play key roles in several diseases involving anergy, exhaustion and over-proliferation and activation often observed in several cancers and immune compromised individuals.

2.3. The role of GJs and HCs in granulocytes

Granulocytes (neutrophils, eosinophils, mast cells) are an essential part of the innate immune response. Cx and Panx containing channels are present in granulocytes to enhance inflammation and to promote cellular activation [117–119]. In particular, it has been shown that neutrophils aggregate and communicate with each other via GJ channels [43]. The demonstration of Cx expression in these cells was extremely difficult due to the high content of proteases that compromised the integrity of the Cxs proteins. Despite these problems, it has been shown that treatment with LPS or TNF-α induced the formation of aggregates of neutrophils that can communicate with endothelial cells via GJs [43] containing Cx43 and Cx40, but not Cx32 [43]. In addition to Cxs, granulocytes also express Panxs [43,120]. This area of research is still open, with a further examination required to understand the functions of Cx and Panx containing channels in neutrophils.

Eosinophils are a rich source of enzymes, chemokines, cytokines, and lipid mediators that participate in allergic inflammation [121,122]. Eosinophils from atopic individuals express Cx43 mRNA and the protein, but not Cx32, and Cx43 is localized not only in the cytoplasm but also to the plasma membrane [118]. The expression of Cx43 by eosinophils facilitates the transfer of second messengers to epithelial and endothelial cells. Cx43 also play a significant role in eosinophil transendothelial migration, enabling these cells to migrate to sites of inflammation [118]. Formation of GJs between eosinophils and tissue resident cells may provide another mechanism of interaction during inflammatory reactions, which could lead to limited and specific activation of communicating cells, as opposed to the broad activation obtained through the paracrine effects of released cytokines and chemokines [118].

Mast cells, also called mastocytes, express Cx43, Cx32 [123–125], and Panx-2 in myenteric and submucosal ganglia [126]. Cx43 and Cx32 expression have also been detected in murine bone marrow cultured mast cells and the mast cell line C57 [123]. Mast cells have important functions in the epidermis, wound healing, and diabetes, where Cx43 plays a key role [127]. However, whether these proteins form GJs or HCs in mast cells is still unknown.

3. Role of GJ and HC in the generation, amplification, and consolidation of inflammation

Inflammation is a first-line mechanism in the innate immune response to protect the body against pathogens and associated damage [128–130]. The purpose of inflammation is to eliminate the initial injury and to clear the damaged or dead cells from the tissue to enable tissue repair and regeneration [128]. The inflammatory process is a complex and multistep process, requiring the recruitment of immune cells, vasodilation, increased permeability, immune activation, apoptosis/necrosis, clearance of the pathogen/damage and regeneration [128]. The inflammatory response includes cell types such as fibroblasts, endothelial cells, and adipocytes, in addition to the typical circulating and resident leukocytes and mast cells, as well as the production of inflammatory cytokines and chemokines [128,131].

Changes in Cx expression and GJ communication in response to inflammation have been found in several cells and tissues. For example, in the presence of lipopolysaccharide (LPS), a potent inflammatory mediator, Cx expression decreases after inflammation in the heart [132–137], the liver [138,139], and the brain [140,141]. LPS treatment also reduces the expression of Cx40 and associated GJ communication in endothelial cells [142]. In pulmonary inflammatory diseases, expression of Cx37 and Cx40 decreases during lung inflammation [143]. In the brain, IL-1β decreases the expression of Cx43, inhibiting GJ coupling and conductance in human astrocytes [144]. In the liver, the cytokine IL-1β also reduces the expression of Cx32 and Cx43 [145,146]. These examples indicate that systemic inflammation decreases GJ expression and communication in parenchymal cells.

In contrast, in the liver, Kupffer's cells treated with LPS and IFN-γ show increased expression of Cx43 and functional GJ communication among aggregated liver macrophages [35,147]. Activation of Kupffer's cells in vivo and in vitro results in enhanced Cx43 expression and formation of GJ that regulate secretion of several cytokines and metalloproteases. LPS results in up-regulation of Cx40 expression in the aorta associated with reduced endothelium-dependent relaxation and omega-3 fatty acids supplementation decreases the expression of Cx40 in aortic tissue [148]. Thus, understanding the profiles of expression and function of these channels in inflammatory conditions could illuminate new therapeutic opportunities for these pathologies.

In the context of HCs, cell or tissue damage has been shown to cause the release of ATP via HC from damaged cells, resulting in P₂ receptor-mediated purinergic signaling and the initiation of inflammation [25, 149]. Secreted ATP is one of the most potent inflammatory molecules and serves as a “find me signal” to coordinate the recruitment of monocytes, macrophages and dendritic cells into the damaged areas. As described previously, chemokines that bind CCR5 and CXCR4 transiently open Panx-1 channels, allowing the release of ATP to extracellular media, which also controls actin rearrangement for directed migration [150]. Thus, ATP and chemokines are highly integrated with HC function to support inflammation and regeneration.

Interestingly, Panx-1 HCs in inflammatory bowel diseases including colitis and Crohn's disease show a cell protective function, but the mechanism of this protection is unknown [151]. In the colon, there is a high density and broad cellular distribution of Panx-1, suggesting an important role of this protein in the gut. In an animal model of colitis, Panx-1 HC are required for P2X₇ receptor-mediated enteric neuron cell death and associated intestinal inflammation [152,153]. Inhibition of Panx-1 HCs protects neurons and maintains proper control of the colonic muscles, preserving mobility [152]. Thus, Panx-1 HCs could be a novel therapeutic target for the treatment of dysfunction of inflammatory bowel diseases and possibly other gastrointestinal disorders with an inflammatory component.

4. The connection between HCs, ATP and purinergic receptors

As described above, HCs in concert with extracellular ATP are an important component of the inflammatory cascade, serving as a danger signal that causes activation of the inflammasome, immune cell infiltration, and tissue repair [154–157]. Also, GJs and HCs play a critical role in the resolution of pathogenic agents or spinal cord injury. For example, activation of P2X₇ receptors by extracellular ATP released upon the opening of HCs results in enhanced intracellular bacterial killing and prevention of sepsis [74]. A similar mechanism has been described in macrophages infected with Bacillus anthracis [158]. A lethal B. anthracis toxin that blocks kinases p38, MAPK and AKT also results in the opening of Cx43 HCs, ATP release, and induction of macrophage death [158]. In the CNS, the opening of HCs has been described in astrocytes, neuronal precursors, neutrophils, and myocytes in response to different stimuli such as hypoxia, ischemia, as well as pathogen-associated molecular patterns (PAMPs) [159–166]. Experiments using Cx43 or Panx-1 knocked down astrocytes indicate that downregulation of Panx-1, but not Cx43, prevents the release of ATP from astrocytes [167]. In contrast, experiments using conditional Cx43 knockout demonstrate that reduction of Cx43 protein expression reduces animal recovery time and inflammation in response to spinal cord injury, suggesting a role for Cx43 HCs in inflammation and recovery [168].

Additionally, extracellular ATP participates in the activation of the inflammasome (NLPR3) [23,155,169]. Inflammasomes are large multiprotein complexes, leading to caspase-1-activated maturation of cytokines, including interleukin-1β (IL-1β) [170,171], by a P2X₇ receptor-mediated mechanism [172]. The activation of P2X₄ and P2X₇ in response to ATP released by Panx-1 HCs has been related to induced ROS production and inflammasome activation in gingival epithelial cells [173]. The data further supports the involvement of Panx-1 HCs, purinergic receptors and extracellular ATP in inflammasome activation. Together, HCs, ATP, and purinergic receptors play a critical role during the inflammatory response and thus represent a promising target for new therapies.

Extracellular ATP also controls cellular and tissue defense/repair processes via signaling through P1, P2X, and P2Y purinergic receptors, with P2X₇ signaling recently associated with tumor growth and metastasis [174]. For example, P2Y₁ and P2Y₂ receptors are involved in cell proliferation, P2X₄ receptors are involved in differentiation (and are therefore anti-proliferative), and P2X₇ receptors are involved in cell death [175–177]. Human melanomas express functional P2X₇ receptors that mediate the apoptotic functions of ATP, whereas P2Y₁ and P2Y₂ receptor agonists cause a decrease and increase in cell numbers, respectively [175–177]. Thus, extracellular ATP can function as a prototypical danger signal that activates a potent immune response, but can also promote cancer progression [174]. Thus, cancer cells can use the ATP/ HC/purinergic system and autophagy to survive, metastasize and avoid the immune response.

5. The unexplored role of Cx and Panx containing channels in HIV infection, replication, and associated pathogenesis

Human immunodeficiency virus type-1 (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV infection is a major public health problem that is partially controlled by the use of antiretroviral agents [178–180]. HIV infects a variety of immune and non-immune cells, including CD4⁺ T cells, monocyte/macrophages, microglia, and astrocytes [84,181–183].

HIV entry into the CNS is an early event after infection [184], resulting in neurological dysfunction in a significant number of individuals. In 50–60% of the infected population, the neurological manifestation of HIV infection produces a number of debilitating clinical disorders collectively termed HIV-Associated Neurocognitive Disorders (HANDs) [185]. The HIV-induced CNS damage is dependent on HIV infection, but not on replication [186–188]. Our laboratory has proposed that GJ channels, chemical synapses and alternative mechanisms of cell-cell communication, such as HCs, amplify HIV-associated CNS damage (see diagram in Fig. 3A) [184]. Our results have shown that HIV-infected astrocytes and macrophages are protected from apoptosis, which possibly contributes to the persistence of HIV within the CNS [189–191]. Despite the low to undetectable HIV replication in these surviving cells, they are extremely well coupled by GJs and express functional Cx HCs on their surface. In the last couple of years, we demonstrated that GJ channels play a key role in transmitting and thereby amplifying toxic signals originating from HIV-infected astrocytes to uninfected astrocytes [190]. Furthermore, it was demonstrated that a few HIV-infected astrocytes (4.7 ± 2.8% in vitro and 8.2 ± 3.9% in vivo) compromise the BBB integrity by a mechanism involving activation of purinergic receptors, BK channels, and others [192]. We also demonstrated that GJs and HCs are essential to allow the spread of apoptotic signals into uninfected cells, including neurons, astrocytes and endothelial cells [193].

Fig. 3.

Cell to cell spread of apoptotic stimuli during HIV infection via GJs and HCs. a) GJ channels (in red), composed mainly of Cx43, remain expressed and open during HIV infection resulting in the transfer of toxic or apoptotic signal to uninfected cells resulting in apoptosis. Surprisingly, HIV-infected cells survive apoptosis generating viral reservoirs within the CNS. b) HCs (in blue), composed by Cx or Panx-1, allow the release of ATP into extracellular media and activate purinergic receptor in the targeted cell to facilitate HIV entry and infection but also ATP diffuse to surrounding uninfected cells to promote inflammation.

Recently we showed that HIV-tat (HIV-transactivator of the virus) enhances Cx43 expression in primary human astrocytes [184,194]. The data indicate that the presence of a single HIV protein, HIV-tat, expressed by an early HIV gene, increases Cx43 expression in astrocytes to maintain communication between the few HIV-infected cells and surrounding uninfected cells. This mechanism of promoting intercellular communication by a pathogen is unique and is not blocked by any anti-retroviral therapy. Our results show that a small number of HIV-infected astrocytes result in extensive alterations in glutamate metabolism and secretion of key chemokines (CCL2) involved in NeuroAIDS [193,195]. For instance, CCL2 alone can compromise the BBB, but if CCL2 reaches the circulation, it can also specifically recruit HIV-infected circulating cells into the CNS [196,197]. Thus, functional GJs in HIV-infected astrocytes contribute to the spread of toxic signals among cells and the recruitment of infected and inflammatory cells into the CNS.

The role of HCs in HIV infection was only recently described. HIV mostly infects immune cells by binding the viral envelope glycoprotein gp120 to the host extracellular proteins CD4, CCR5, and CXCR4, resulting in fusion of the viral envelope with the cell membrane [198]. No other host plasma proteins have been identified that participate directly in the process of viral entry [198]. However, we recently discovered that Panx-1 HCs are essential to support HIV entry into immune cells. Our data indicate that HIV binding to CD4 and CCR5 or CXCR4 results in an extended (up to 1 h) opening of Panx-1 HCs. This opening enables a local release of ATP that results first in P2X₁ receptor activation, due to its high sensitivity to low amounts of ATP. P2X₁ receptors are essential for HIV entry because they are necessary for actin rearrangement on the surface of the immune cells, which allows the virus to fuse with the plasma membrane. Later on, when the concentrations of ATP reach higher levels, P2X₇ receptors become activated, but their role the HIV cell cycle is still under active investigation. Extracellular ATP is subjected to ectoATPases that produce ADP, AMP and later on adenosine. P2Y₁ receptors also participate in HIV replication, but this receptor is mostly activated by ADP, and we are still working in the viral functions targeted by this receptor.

In addition, we found that Panx-1 HCs become transiently (in the range of second) open in response to chemokines binding to their receptors, CCR5 and CXCR4. Chemokine receptors normally activate upon binding of their endogenous ligands, chemokines, to regulate physiological and pathological cellular trafficking of particular cell populations into different tissues [199]. Thus, we propose that under normal conditions the transient opening of these HCs helps regulate cell trafficking. However, we also propose that several pathogens have adapted to take advantage of these channels to improve their infectivity and replication.

Recently it was demonstrated that HIV infection leads to the opening of Cx43 HCs in human astrocytes, and we found that this opening did not affect viral replication or bystander apoptosis. Instead, HIV-induced opening of Cx43 HCs led to dysregulated secretion of dickkopf-1 protein (DKK1), a soluble WNT pathway inhibitor [81]. DKK1 is an embryonic developmental protein involved in the anterior visceral endoderm development, resulting in heart, head and forelimb formation. However, HIV infection activates this embryonic program to spread toxicity and inflammation. Indeed, treatment of mixed cultures of neurons and astrocytes with DKK1 in the absence of HIV infection results in the collapse of neuronal processes [81]. These experiments demonstrate that HIV infection of astrocytes induces dysregulation of DKK1 by an HC-dependent mechanism, contributing to synaptic compromise observed in HIV-infected individuals. This data provides a novel bystander mechanism of damage in NeuroAIDS and indicate potential new targets for therapeutic interventions, such as DKK1 inhibitors, to reduce the ongoing CNS effects of HIV.

Our findings are in concordance with observations of Séror et al., who reported that ATP rapidly is released from HIV-1 target cells through Panx-1 channels upon interaction with the HIV-1 envelope protein and activation of specific target cell purinergic receptors (including P2Y₂), activated proline-rich tyrosine kinase 2 (Pyk2 kinase, an enzyme related to IFN-α, IL-6, IL-10 and IL-12 signaling), and transient plasma membrane depolarization, generating a cascade that involves Panx-1 → ATP → P2Y₂ → Pyk2 [181]. They also demonstrated that ATP not only acts as a danger signal but also contributes to viral uptake. Therefore, Panx-1 HCs and purinergic receptors are physically recruited to the site of the infection to facilitate HIV infection [181]. Thus, Panx-1 HCs, ATP, and purinergic receptors accelerate HIV infection and replication (Fig. 3B). Consistent with this conclusion, pharmacological inhibition of the biological activity of the Panx-1 HCs has also been associated with reduced HIV infection and viral replication.

Experiments in human macrophages indicate that HIV infection requires secretion of ATP and activation of purinergic receptors. Our work and the work of others showed that HIV binding to CD4⁺ T lymphocytes and macrophages also results in activation of Panx-1 HCs, the release of ATP, and activation of specific purinergic receptors [84, 181,182]. Our laboratory demonstrated the participation of P2X₁, P2X₇, and P2Y₁ in HIV replication and showed that P2X₁ is necessary for HIV entry into human macrophages. We also demonstrated that interaction of the HIV surface protein gp120 with macrophages stimulates an increase in ATP release and plays a critical role in HIV infection and replication in immune cells by contributing to viral entry and possibly in other steps of the viral lifecycle [84].

ATP in solution is extremely unstable, resulting in ADP, AMP, and adenosine [200,201]. Activation of adenosine receptors (also called P1 receptors) is protective against HIV-tat-induced toxicity in neurons [202,203], suggesting that adenosine may play an important role in preventing HIV-tat neurotoxicity within the CNS. Thus, the opening of HC could also contribute to neuroprotection in some conditions. Additionally, activation of adenosine receptors downregulates the surface expression of CXCR4 and CCR5 on CD4⁺ T lymphocytes [204], suggesting that adenosine is protective against HIV infection and that blocking Panx-1 HCs can suppress viral replication. Further studies are required to design new therapeutic approaches to reduce and prevent HIV infection and replication in immune and CNS cells.

6. Conclusion

In this review, we have summarized the currently available data about the expression and function of Cx and Panx containing channels, GJs and HCs, in immune cells and their contribution to the immune response to several pathogens. The emerging understanding and discoveries regarding the role of these channels in immune cells and tissues are providing important insight contributing to the development of novel therapeutic targets and approaches to limit the devastating consequences of uncontrolled immune responses involved in multiple pathologies.