The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

Unnexin is a protein subunit of a large-pore channel expressed by unicellular organisms.

Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca2+/Mg2+ levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca2+ and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in Xenopus oocytes. Downregulation of Unx1 mRNA with morpholine contains Trypanosoma cruzi invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.

Unnexins: Homologs of innexin proteins in Trypanosomatidae parasites.

Large-pore channels, including those formed by connexin, pannexin, innexin proteins, are part of a broad family of plasma membrane channels found in vertebrates and invertebrates, which share topology features. Despite their relevance in parasitic diseases such as Chagas and malaria, it was unknown whether these large-pore channels are present in unicellular organisms. We identified 14 putative proteins in Trypanosomatidae parasites as presumptive homologs of innexin proteins. All proteins possess the canonical motif of the innexin family, a pentapeptide YYQWV, and 10 of them share a classical membrane topology of large-pore channels. A sequence similarity network analysis confirmed their closeness to innexin proteins. A bioinformatic model showed that a homolog of Trypanosoma cruzi (T. cruzi) could presumptively form a stable octamer channel with a highly positive electrostatic potential in the internal cavities and extracellular entrance due to the notable predominance of residues such as Arg or Lys. In vitro dye uptake assays showed that divalent cations-free solution increases YO-PRO-1 uptake and hyperosmotic stress increases DAPI uptake in epimastigotes of T. cruzi. Those effects were sensitive to probenecid. Furthermore, probenecid reduced the proliferation and transformation of T. cruzi. Moreover, probenecid or carbenoxolone increased the parasite sensitivity to antiparasitic drugs commonly used in therapy against Chagas. Our study suggests the existence of innexin homologs in unicellular organisms, which could be protein subunits of new large-pore channels in unicellular organisms.

Contribution of large-pore channels to inflammation induced by microorganisms.

Plasma membrane ionic channels selectively permeate potassium, sodium, calcium, and chloride ions. However, large-pore channels are permeable to ions and small molecules such as ATP and glutamate, among others. Large-pore channels are structures formed by several protein families with little or no evolutionary linkages including connexins (Cxs), pannexins (Panxs), innexin (Inxs), unnexins (Unxs), calcium homeostasis modulator (CALHMs), and Leucine-rich repeat-containing 8 (LRRC8) proteins. Large-pore channels are key players in inflammatory cell response, guiding the activation of inflammasomes, the release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß), and the release of adenosine-5'-triphosphate (ATP), which is considered a danger signal. This review summarizes our current understanding of large-pore channels and their contribution to inflammation induced by microorganisms, virulence factors or their toxins.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII.

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Anti-parasitic drugs modulate the non-selective channels formed by connexins or pannexins.

The proteins connexins, innexins, and pannexins are the subunits of non-selective channels present in the cell membrane in vertebrates (connexins and pannexins) and invertebrates (innexins). These channels allow the transfer of ions and molecules across the cell membrane or, and in many cases, between the cytoplasm of neighboring cells. These channels participate in various physiological processes, particularly under pathophysiological conditions, such as bacterial, viral, and parasitic infections. Interestingly, some anti-parasitic drugs also block connexin- or pannexin-formed channels. Their effects on host channels permeable to molecules that favor parasitic infection can further explain the anti-parasitic effects of some of these compounds. In this review, the effects of drugs with known anti-parasitic activity that modulate non-selective channels formed by connexins or pannexins are discussed. Previous studies that have reported the presence of these proteins in worms, ectoparasites, and protozoa that cause parasitic infections have also been reviewed.

Innexins: Expression, Regulation, and Functions.

The innexin (Inx) proteins form gap junction channels and non-junctional channels (named hemichannels) in invertebrates. These channels participate in cellular communication playing a relevant role in several physiological processes. Pioneer studies conducted mainly in worms and flies have shown that innexins participate in embryo development and behavior. However, recent studies have elucidated new functions of innexins in Arthropoda, Nematoda, Annelida, and Cnidaria, such as immune response, and apoptosis. This review describes emerging data of possible new roles of innexins and summarizes the data available to date.

Trypanosoma cruzi Infection Induces Pannexin-1 Channel Opening in Cardiac Myocytes.

Trypanosoma cruzi, the etiological agent of Chagas diseases, invades the cardiac tissue causing acute myocarditis and heart electrical disturbances. In T. cruzi invasion, the parasite induces [Ca2+]i transients in the host cells, an essential phenomenon for invasion. To date, knowledge on the mechanism that elicits transients of [Ca2+]i during the infection of cardiac myocytes has not been fully characterized. Pannexin1 (Panx1) channel are poorly selective channels found in all vertebrates that serve as a pathway for ATP release. In this article, we demonstrate that T. cruzi infection results in the opening of Panx1 channels in cardiac myocytes. We show that pharmacological blockade of Panx1 channels inhibits T. cruzi-induced [Ca2+]i transients and invasion in cardiac myocytes. Our results indicate that opening of Panx1 channels are required for T. cruzi invasion in cardiac myocytes, and we propose that targeting Panx1 channel could provide new potential therapeutic approaches to treat Chagas disease.