The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

A Methodological Approach Using rAAV Vectors Encoding Nanobody-Based Biologics to Evaluate ARTC2.2 and P2X7 In Vivo.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Diphenyleneiodonium enhances P2X7 dependent non-opsonized phagocytosis and suppresses inflammasome activation via blocking CX43-mediated ATP leakage.

The beneficial effects of antioxidants against oxidative stress have been well described. However, the pharmacological impacts of antioxidants other than inhibiting the production of reactive oxygen species (ROS) remain less understood. This study demonstrated that diphenyleneiodonium (DPI), a canonical NADPH oxidase 2 (NOX2) inhibitor, effectively promoted non-opsonized bacterial phagocytosis. Indeed, DPI abrogated the elevation in the extracellular ATP level of Escherichia coli (E. coli) -infected murine peritoneal macrophages, thereby restoring the association of the purinergic receptor P2X7 with non-muscle myosin heavy chain 9 (MYH9) to upregulate the P2X7 -dependent phagocytosis of E. coli. DPI also suppressed inflammasome activation and reduced necroptosis in E. coli-infected macrophages by decreasing extracellular ATP levels. Mechanistically, DPI upregulated p38 MAPK phosphorylation to suppress the expression and activity of the hemichannel protein connexin 43 (CX43), leading to the inhibition of CX43-mediated ATP efflux in E. coli-infected macrophages. In a murine E. coli infection model, DPI effectively reduced ATP release, decreased bacterial load and inhibited inflammasome activation, thereby improving survival and ameliorating organ injuries in model mice. In summary, our study demonstrates a previously unknown function of DPI in conferring protection against bacterial infection and suggests a putative antimicrobial strategy of modulating CX43 -dependent ATP leakage.

P2RX7 at the Host-Pathogen Interface of Infectious Diseases.

The P2X7 receptor (P2RX7) is an important molecule that functions as a danger sensor, detecting extracellular nucleotides from injured cells and thus signaling an inflammatory program to nearby cells. It is expressed in immune cells and plays important roles in pathogen surveillance and cell-mediated responses to infectious organisms. There is an abundance of literature on the role of P2RX7 in inflammatory diseases and the role of these receptors in host-pathogen interactions. Here, we describe the current knowledge of the role of P2RX7 in the host response to a variety of pathogens, including viruses, bacteria, fungi, protozoa, and helminths. We describe in vitro and in vivo evidence for the critical role these receptors play in mediating and modulating immune responses. Our observations indicate a role for P2X7 signaling in sensing damage-associated molecular patterns released by nearby infected cells to facilitate immunopathology or protection. In this review, we describe how P2RX7 signaling can play critical roles in numerous cells types in response to a diverse array of pathogens in mediating pathogenesis and immunity to infectious agents.

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

Inherent P2X7 Receptors Regulate Macrophage Functions during Inflammatory Diseases.

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1ß (pro-IL-1ß)-degrading caspase-1 to lead to IL-1ß release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn's disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

Purinergic Signaling in Pancreas-From Physiology to Therapeutic Strategies in Pancreatic Cancer.

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling.

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca2+ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca2+ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca2+ increase is specifically triggered by the autoreactive CD4+ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca2+ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.

20S-Protopanaxatriol Ameliorates Hepatic Fibrosis, Potentially Involving FXR-Mediated Inflammatory Signaling Cascades.

Ginseng has been used as a functional food and tonic for enhancing immune power. Here, the potential protective effect of 20S-protopanaxatriol (M4), the metabolite of protopanaxatriol, against hepatic fibrosis is investigated, which could provide nutritional interventions for disease treatment. M4 could inhibit extracellular matrix (ECM) deposition and reduce the levels of proinflammatory cytokines such as caspase 1, interleukin 1 ß (IL-1ß), interleukin 1 receptor type 1 (IL1R1), and interleukin 6 (IL-6). M4 also significantly increased the expression of farnesoid X receptor (FXR), suppressed the purinergic ligand-gated ion channel 7 receptor (P2X7r) signaling pathway, and works as an FXR agonist, GW4064. In thioacetamide (TAA)-induced mice, M4 could attenuate the histopathological changes and significantly regulate the expression levels of FXR and P2X7r. M4 ameliorated TAA-induced hepatic fibrosis due to the reduction of P2X7r secretion, inhibition of hepatic stellate cell (HSCs) activation, and inflammation, which were all associated with FXR activation. Hence, M4 might be useful a nutritional preventive approach in antihepatic fibrosis and antihepatic inflammation.

The P2X7 Receptor as Regulator of T Cell Development and Function.

Unique structural features characterize the P2X7 receptor with respect to other P2X family members. Dual gating by eATP and regulated expression of P2X7 can imprint distinct outcomes to the T cell depending on the metabolic fitness and/or developmental stage. In the thymus, signaling by P2X7 contributes to γδ T cell lineage choice. In secondary lymphoid organs, P2X7 stimulation promotes Th1/Th17 polarization of CD4+ naïve cells, Tregs conversion to Th17 cells and cell death of Tfh cells that are not stimulated by cognate antigen. Moreover, P2X7 stimulation in eATP rich microenvironments, such as damaged and/or inflamed tissues as well as tumors, induces cell death of various T cell effector subsets.

Long-lived T follicular helper cells retain plasticity and help sustain humoral immunity.

CD4+ memory T cells play an important role in protective immunity and are a key target in vaccine development. Many studies have focused on T central memory (Tcm) cells, whereas the existence and functional significance of long-lived T follicular helper (Tfh) cells are controversial. Here, we show that Tfh cells are highly susceptible to NAD-induced cell death (NICD) during isolation from tissues, leading to their underrepresentation in prior studies. NICD blockade reveals the persistence of abundant Tfh cells with high expression of hallmark Tfh markers to at least 400 days after infection, by which time Tcm cells are no longer found. Using single-cell RNA-seq, we demonstrate that long-lived Tfh cells are transcriptionally distinct from Tcm cells, maintain stemness and self-renewal gene expression, and, in contrast to Tcm cells, are multipotent after recall. At the protein level, we show that folate receptor 4 (FR4) robustly discriminates long-lived Tfh cells from Tcm cells. Unexpectedly, long-lived Tfh cells concurrently express a distinct glycolytic signature similar to trained immune cells, including elevated expression of mTOR-, HIF-1-, and cAMP-regulated genes. Late disruption of glycolysis/ICOS signaling leads to Tfh cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of Tfh and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct long-lived T cell subsets and establish Tfh cells as an attractive target for the induction of durable adaptive immunity.

Production, characterization, and application of a monoclonal antibody specific for the extracellular domain of human P2X7R.

This paper focuses on the production of a high-affinity monoclonal antibody (mAb) that can efficiently detect and block purinergic ligand-gated ion channel 7 receptor (P2X7R). To achieve this goal, the extracellular domain of human P2X7R, P2X7R-ECD, was used as an immunogen for BALB/c mice, inducing them to produce spleen lymphocytes that were subsequently fused with myeloma cells. Screening of the resultant hybridoma clones resulted in the selection of one stable positive clone that produced a qualified mAb, named 4B3A4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purity of the purified 4B3A4 mAb was above 85%, with prominent bands corresponding to molecular weights of 55 kDa (heavy chain) and 25 kDa (light chain), and the BCA assay showed that the concentration of the purified 4B3A4 mAb was 0.3 mg/mL. Western blot analysis revealed that the 4B3A4 mAb could specifically recognize and bind both P2X7R-ECD and the full-length P2X7R protein. Laser scanning confocal microscopy (LSCM) revealed that the 4B3A4 mAb specifically bound to P2X7R on the membrane of human peripheral blood mononuclear cells (PBMCs). P2X7R expression was significantly different between healthy individuals and people with certain cancers as determined by flow cytometry (FCM). In addition, the 4B3A4 mAb significantly reduced ATP-stimulated Ca2+ entry and YO-PRO-1 uptake, which indicated that the 4B3A4 mAb effectively blocked P2X7R activity. These data indicate that the 4B3A4 mAb can be further used as not only an antibody to detect cell surface P2X7R but also as a therapeutic antibody to target P2X7R-related signaling pathways.

Toll-Like Receptor-Mediated Activation of CD39 Internalization in BMDCs Leads to Extracellular ATP Accumulation and Facilitates P2X7 Receptor Activation.

Toll-like receptors (TLRs) trigger innate immune responses through their recognition of conserved molecular ligands of either endogenous or microbial origin. Although activation, function, and signaling pathways of TLRs were already well-studied, their precise function in specific cell types, especially innate immune cells, needs to be further clarified. In this study, we showed that when significantly decreased amounts of membrane CD39, an adenosine triphosphate (ATP)-degrading enzyme, were detected in lipopolysaccharide (LPS)-treated bone marrow-derived dendritic cells (BMDCs), Cd39 mRNA expression, and whole-cell CD39 expression were at the same levels as those in untreated BMDCs. Further experiments demonstrated that the downregulation of membrane CD39 expression in LPS-treated BMDCs was mediated by endocytosis, leading to membrane-exposed CD39 downregulation, which was positively associated with decreased enzymatic activity in ATP metabolism and increased extracellular ATP accumulation. The accumulated ATP promoted intracellular calcium accumulation and IL-1ß production in BMDCs through P2X7 signaling activation. Further research revealed that not only LPS but also other TLR ligands, excluding polyI:C, induced CD39 internalization in BMDCs and that the MyD88 pathway was critical in this process. The results suggested that the activation of CD39 internalization in DCs induced by a TLR ligand caused increased ATP accumulation, leading to P2X7 receptor activation that mediated a proinflammatory effect. Considering the strong modulatory effect of extracellular ATP accumulation on the immune response and inflammation, the manipulation of membrane CD39 expression on DCs may have implications on the regulation and treatment of inflammatory responses.

ATP and adenosine: Role in the immunopathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a classic inflammatory autoimmune disease. Local joint destruction and extra-articular manifestations of RA deeply compromise the life quality of the affected patients. RA immunopathogenesis depends on continuous immunogenic activation in which the purinergic system participates. The purinergic system comprises the signaling and metabolism of purines such as adenosine triphosphate (ATP) and adenosine. ATP signaling is involved in the activation and maintenance of the inflammatory state of RA through the activation of P2X7 and the production of cytokines, which orchestrate the pathogenesis of RA. The breakdown of ATP through the CD39/CD73 axis produces adenosine, which mostly inhibits the inflammatory process through activation of specific P1 receptors. Adenosine is hydrolyzed by adenosine deaminase (ADA) that interacts with other molecules playing additional roles in this disease. This review explores the release, metabolism, and the effects of binding of ATP and adenosine to their respective receptors in the context of RA, as well as their potential use as biomarkers and therapeutic targets.

Non-canonical NLRP3 inflammasome activation and IL-1ß signaling are necessary to L. amazonensis control mediated by P2X7 receptor and leukotriene B4.

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1ß. It was demonstrated that NLRP3 inflammasome activation and IL-1ß signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1ß signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1ß, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1ß also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1ß, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1ß signaling.

P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis.

Sepsis is characterized by a systemic inflammatory response followed by immunosuppression of the host. Metabolic defects and mitochondrial failure are common in immunocompromised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection.

P2X7 receptor: A potential therapeutic target for autoimmune diseases.

P2X7 receptor (P2X7R), a distinct ligand-gated ion channel, is a member of purinergic type 2 receptor family with ubiquitous expression in human body. Previous studies have revealed a pivotal role of P2X7R in innate and adaptive immunity. Once activated, it will meditate some vital cascaded responses including the assembly of nucleotide-binding domain (NOD) like receptor protein 3 (NLRP3) inflammasome, non-classical secretion of IL-1ß, modulation of cytokine-independent pathways in inflammation such as P2X7R- transglutaminase-2 (TG2) and P2X7R-cathepsin pathway, activation and regulation of T cells, etc. In fact, above responses have been identified to be involved in the development of autoimmunity, specifically, the NLRP3 inflammasome could promote inflammation in massive autoimmune diseases and TG2, as well as cathepsin may contribute to joint destruction and degeneration in inflammatory arthritis. Recently, numerous evidences further suggested the significance of P2X7R in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), etc. In this review, we will succinctly discuss the biological characteristics and summarize the recent progress of the involvement of P2X7R in the development and pathogenesis of autoimmune diseases, as well as its clinical implications and therapeutic potential.

Inflammasome gene expression is associated with immunopathology in human localized cutaneous leishmaniasis.

Localized cutaneous leishmaniasis (LCL) can ultimately progress to chronic ulcerated lesions with strong local inflammatory reactions. The functional role of certain inflammasomes in mediating inflammation caused by Leishmania braziliensis needs to be addressed. By combining PCR-array, quantitative real-time PCR and immunohistochemical analysis, we identified inflammasome genes, such as IL-1ß, NLRP3, NLRP1, NLRC5, AIM2 and P2RX7, that were upregulated in LCL patients. Temporal gene expression studies showed that the early phase of LCL displayed increased NLRP3 and reduced AIM2 and NLRP1 expression, while the late stages showed increased AIM2 and NLRP1 and lower NLRP3 expression. Our findings also showed that AIM2, NLRP1, and P2RX7 promoted susceptibility to experimental L. braziliensis infection. These results highlight the importance of inflammasome machinery in human LCL and suggest that inflammasome machinery plays a role in the acute and chronic phases of the disease.