Objective—: Platelets modulate hemostasis and immune responses via interactions with immune cells through secretion of immunemodulators and cell–cell interactions. The P2Y ₁₂ receptor mediates ADP-induced aggregation and secretion in platelets. Approach and Results—: Using a mouse model of intra-abdominal sepsis and acute lung injury, we investigated the role of the P2Y ₁₂ receptor in neutrophil migration and lung inflammation in P2Y ₁₂ null mice and in mice pretreated with the P2Y ₁₂ antagonist clopidogrel. Our data show a decrease in circulating white blood cells and a decrease in platelet activation and platelet–leukocyte interactions in treated mice compared with untreated mice. Additionally, lung injury and platelet sequestration were diminished in clopidogrel-treated mice compared with their untreated septic littermates. Similar results were observed in P2Y ₁₂ null mice: platelet activation and platelet–leukocyte aggregates were decreased in septic P2Y ₁₂ null mice compared with wild-type mice. P2Y ₁₂ null mice were refractory to lung injury compared with wild-type mice. Finally, to evaluate P2Y ₁₂ -independent effects of clopidogrel, we pretreated P2Y ₁₂ null mice. Interestingly, the number of circulating neutrophils was reduced in treated septic P2Y ₁₂ null mice, suggesting neutrophils as a target for clopidogrel pleiotropic effects. No difference was observed in P2Y ₁ null mice during sepsis, indicating that the P2Y ₁₂ receptor is responsible for the effects. Conclusions—: P2Y ₁₂ null mice are refractory to sepsis-induced lung injury, suggesting a key role for activated platelets and the P2Y ₁₂ receptor during sepsis.

Thienopyridines are a class of antiplatelet drugs that are metabolized in the liver to several metabolites, of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have also been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. However, it is not clear whether it is caused only by the P2Y12 antagonism or whether off-target effects of other metabolites also intervene. To address this question, we investigated P2Y12 KO mice during a LPS-induced model of systemic inflammation, and we treated these KO mice with a thienopyridine drug (clopidogrel). Contrary to the reported effects of clopidogrel, numbers of circulating WBCs and plasma levels of cytokines were increased in LPS-exposed KO mice compared with WT in this inflammation model. Moreover, both spleen and bone marrow show an increase in cell content, suggesting a role for P2Y12 in regulation of bone marrow and spleen cellular composition. Finally, the injury was more severe in the lungs of KO mice compared with WT. Interestingly, clopidogrel treatments also exerted protective effects in KO mice, suggesting off-target effects for this drug. In conclusion, the P2Y12 receptor plays an important role during LPS-induced inflammation, and this signaling pathway may be involved in regulating cell content in spleen and bone marrow during LPS systemic inflammation. Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade.

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. While iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce β-1,3-glucan masking in C. albicans. We show here that iron-induced changes in β-1,3-glucan exposure are lactate-dependent; and high iron causes β-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response.

Platelets play an important role not only during thrombosis, but also in modulating immune responses through their interaction with immune cells and by releasing inflammatory mediators upon activation. The P2Y₁₂ receptor is a Gicoupled receptor that not only regulates ADP-induced aggregation but can also dramatically potentiate secretion, when platelets are activated by other stimuli. Considering the importance of P2Y₁₂ receptor in platelet function, a class of antiplatelet drugs, thienopyridines, have been designed and successfully used to prevent thrombosis. This review will focus on the role of activated platelets in inflammation and the effects that P2Y₁₂ antagonism exerts on the inflammatory process. A change in platelet functions was noted in patients treated with thienopyridines during inflammatory conditions, suggesting that platelets may modulate the inflammatory response. Further experiments in a variety of animal models of diseases, such as sepsis, rheumatoid arthritis, myocardial infarction, pancreatitis and pulmonary inflammation have also demonstrated that activated platelets influence the inflammatory state. Platelets can secrete inflammatory modulators in a P2Y₁₂–dependent manner, and, as a result, directly alter the inflammatory response. P2Y₁₂ receptor may also be expressed in other cells of the immune system, indicating that thienopyridines could directly influence the immune system rather than only through platelets. Overall the results obtained to date strongly support the notion that activated platelets significantly contribute to the inflammatory process and that antagonizing P2Y₁₂ receptor can influence the immune response.

P2Y₁₂ is a G-protein-coupled receptor that is activated upon ADP binding. Considering its well-established role in platelet activation, blocking P2Y₁₂ has been used as a therapeutic strategy for antiplatelet aggregation in cardiovascular disease patients. However, receptor studies have shown that P2Y₁₂ is functionally expressed not only in platelets and the microglia but also in other cells of the immune system, such as in monocytes, dendritic cells, and T lymphocytes. As a result, studies were carried out investigating whether therapies targeting P2Y₁₂ could also ameliorate inflammatory conditions, such as sepsis, rheumatoid arthritis, neuroinflammation, cancer, COVID-19, atherosclerosis, and diabetes-associated inflammation in animal models and human subjects. This review reports what is known about the expression of P2Y₁₂ in the cells of the immune system and the effect of P2Y₁₂ activation and/or inhibition in inflammatory conditions. Lastly, we will discuss the major problems and challenges in studying this receptor and provide insights on how they can be overcome.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2^-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2^-/- mice, we observed decreased hypoxia in the gut tissue of Stat2^-/- mice. Neutrophil numbers were similar in wild-type and Stat2^-/- mice, yet Stat2^-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2^-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2^-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2^-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2^-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2^-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium. The spread of invading microbes is frequently contained by an inflammatory response. Yet, some pathogenic microbes have evolved to utilize inflammation for niche generation and to support their metabolism. Here, we demonstrate that S. Typhimurium exploits type I IFN signaling, a prototypical anti-viral response, to foster its own growth in the inflamed gut. In the absence of STAT2-dependent type I IFN, production of neutrophil reactive oxygen species was impaired, and epithelial metabolism returned to homeostatic hypoxia. Consequently, S. Typhimurium was unable to respire in the absence of type I IFN, and failed to expand in the gut lumen. Furthermore, perturbation of the gut microbiota was dependent on type I IFN signaling. Taken together, our work suggests that S. Typhimurium utilizes STAT2-dependent type I IFN signaling to generate a niche in the inflamed intestinal tract and outcompete the gut microbiota.