The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice.

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.

Activation of the NLRP3-CASP-1 inflammasome is restrained by controlling autophagy during Glaesserella parasuis infection.

Infection with Glaesserella parasuis, the primary pathogen behind Glässer's disease, is often associated with diverse clinical symptoms, including serofibrinous polyserositis, arthritis, and meningitis. Autophagy plays a dual role in bacterial infections, exerting either antagonistic or synergistic effects depending on the nature of the pathogen. Our previous studies have demonstrated that autophagy serves as a defense mechanism, combating inflammation and invasion caused by infection of highly virulent G. parasuis. However, the precise mechanisms remain to be elucidated. Pathogens exhibit distinct interactions with inflammasomes and autophagy processes. Herein, we explored the effect of autophagy on inflammasomes during G. parasuis infection. We found that G. parasuis infection triggers NLRP3-dependent pro-CASP-1-IL-18/IL-1ß processing and maturation pathway, resulting in increased release of IL-1ß and IL-18. Inhibition of autophagy enhances NLRP3 inflammasome activity, whereas stimulation of autophagy restricts it during G. parasuis infection. Furthermore, assembled NLRP3 inflammasomes undergo ubiquitination and recruit the autophagic adaptor, p62, facilitating their sequestration into autophagosomes during G. parasuis infection. These results suggest that the induction of autophagy mitigates inflammation by eliminating overactive NLRP3 inflammasomes during G. parasuis infection. Our research uncovers a mechanism whereby G. parasuis infection initiates inflammatory responses by promoting the assembly of the NLRP3 inflammasomes and activating NLRP3-CASP-1, both of which processes are downregulated by autophagy. This suggests that pharmacological manipulation of autophagy could be a promising approach to modulate G. parasuis-induced inflammatory responses.

Mechanism of Vaginal Epithelial Cell Pyroptosis Induced by the NLRP3 Inflammasome in Vulvovaginal Candidiasis.

PROBLEM: Vulvovaginal candidiasis (VVC) is a common mucosal fungal infection, and Candida albicans is the main causative agent. The NLRP3 inflammasome plays an important role in VVC, but the underlying mechanism is unknown. METHOD OF STUDY: Vaginal epithelial cells were divided into three groups: control, C. albicans strain SC5314 (wild-type, WT), and WT+ Matt Cooper Compound 950 (MCC950, a specific NLRP3 inhibitor). After human vaginal epithelial cells were pretreated with 1 µmol/L MCC950 for 2 h, C. albicans (MOI = 1) was cocultured with the human vaginal epithelial cells for 12 h. The cell supernatants were collected, LDH was detected, and the IL-1ß and IL-18 levels were determined by ELISA. The expression of the pyroptosis-related proteins NLRP3, Caspase-1 p20 and GSDMD was measured by Western blotting analysis. The protein expression of the pyroptosis-related N-terminus of GSDMD (GSDMD-N) was detected by immunofluorescence. RESULTS: In this study, we showed that the WT C. albicans strain induced pyroptosis in vaginal epithelial cells, as indicated by the LDH and proinflammatory cytokine levels and the upregulated levels of the pyroptosis-related proteins NLRP3, Caspase-1 p20, and GSDMD-N. MCC950 reversed the changes in the expression of these proteins and proinflammatory cytokines in vaginal epithelial cells. CONCLUSION: C. albicans activated the NLRP3 inflammasome to induce vaginal epithelial cell pyroptosis. MCC950 inhibited the NLRP3 inflammasome, reduced vaginal epithelial cell pyroptosis, and decreased the release of inflammatory cytokines.

Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens (C. perfringens) infection, an extracellular bacterial pathogen. In a pathogenic C. perfringens gas gangrene model, Aim2-/- mice are more susceptible to pathogenic C. perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C. perfringens. Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C. perfringens-derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C. perfringens infections.

Elucidating the molecular landscape of tendinitis: the role of inflammasome-related genes and immune interactions.

Tendinitis, characterized by the inflammation of tendons, poses significant challenges in both diagnosis and treatment due to its multifaceted etiology and complex pathophysiology. This study aimed to dissect the molecular mechanisms underlying tendinitis, with a particular focus on inflammasome-related genes and their interactions with the immune system. Through comprehensive gene expression analysis and bioinformatics approaches, we identified distinct expression profiles of inflammasome genes, such as NLRP6, NLRP1, and MEFV, which showed significant correlations with immune checkpoint molecules, indicating a pivotal role in the inflammatory cascade of tendinitis. Additionally, MYD88 and CD36 were found to be closely associated with HLA family molecules, underscoring their involvement in immune response modulation. Contrary to expectations, chemokines exhibited minimal correlation with inflammasome genes, suggesting an unconventional inflammatory pathway in tendinitis. Transcription factors like SP110 and CREB5 emerged as key regulators of inflammasome genes, providing insight into the transcriptional control mechanisms in tendinitis. Furthermore, potential therapeutic targets were identified through the DGidb database, highlighting drugs that could modulate the activity of inflammasome genes, offering new avenues for targeted tendinitis therapy. Our findings elucidate the complex molecular landscape of tendinitis, emphasizing the significant role of inflammasomes and immune interactions, and pave the way for the development of novel diagnostic and therapeutic strategies.

Pulmonary inflammation and viral replication define distinct clinical outcomes in fatal cases of COVID-19.

COVID-19 has affected more than half a billion people worldwide, with more than 6.3 million deaths, but the pathophysiological mechanisms involved in lethal cases and the host determinants that determine the different clinical outcomes are still unclear. In this study, we assessed lung autopsies of 47 COVID-19 patients and examined the inflammatory profiles, viral loads, and inflammasome activation. Additionally, we correlated these factors with the patient's clinical and histopathological conditions. Robust inflammasome activation was detected in the lungs of lethal cases of SARS-CoV-2. Experiments conducted on transgenic mice expressing hACE2 and infected with SARS-CoV-2 showed that Nlrp3-/- mice were protected from disease development and lethality compared to Nlrp3+/+ littermate mice, supporting the involvement of this inflammasome in disease exacerbation. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2. Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, viral loads, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation led to different clinical outcomes. We provide important information to understand clinical variations in severe COVID-19, a process that is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

Anti-Inflammatory and Immunomodulatory Effects of 0.1 Sub-Terahertz Irradiation in Collagen-Induced Arthritis Mice.

The primary emphasis of photoimmunology is the impact of nonionizing radiation on the immune system. With the development of terahertz (THz) and sub-terahertz (sub-THz) technology, the biological effects of this emerging nonionizing radiation, particularly its influence on immune function, remain insufficiently explored but are progressively attracting attention. Here, we demonstrated that 0.1 sub-THz radiation can modulate the immune system and alleviate symptoms of arthritis in collagen-induced arthritis (CIA) mice through a nonthermal manner. The application of 0.1 sub-THz irradiation led to a decrease in proinflammatory factors within the joints and serum, reducing the levels of blood immune cells and the quantity of splenic CD4+ T cells. Notably, 0.1 sub-THz irradiation restored depleted Treg cells in CIA mice and re-established the Th17/Treg equilibrium. These findings suggested that sub-THz irradiation plays a crucial role in systemic immunoregulation. Further exploration of its immune modulation mechanisms revealed the anti-inflammatory properties of 0.1 sub-THz on LPS-stimulated skin keratinocytes. Through the reduction in NF-κB signaling and NLRP3 inflammasome activation, 0.1 sub-THz irradiation effectively decreased the production of inflammatory factors and immune-active substances, including IL-1ß and PGE2, in HaCaT cells. Consequently, 0.1 sub-THz irradiation mitigated the inflammatory response and contributed to the maintenance of immune tolerance in CIA mice. This research provided significant new evidence supporting the systemic impacts of 0.1 sub-THz radiation, particularly on the immune system. It also enhanced the field of photoimmunology and offered valuable insights into the potential biomedical applications of 0.1 sub-THz radiation for treating autoimmune diseases.

NLRP3 promotes allergic responses to birch pollen extract in a model of intranasal sensitization.

Introduction & Objective: Allergic sensitization is an essential step in the development of allergic airway inflammation to birch pollen (BP); however, this process remains to be fully elucidated. Recent scientific advances have highlighted the importance of the allergen context. In this regard, microbial patterns (PAMPs) present on BP have attracted increasing interest. As these PAMPs are recognized by specialized pattern recognition receptors (PRRs), this study aims at investigating the roles of intracellular PRRs and the inflammasome regulator NLRP3. Methods: We established a physiologically relevant intranasal and adjuvant-free sensitization procedure to study BP-induced systemic and local lung inflammation. Results: Strikingly, BP-sensitized Nlrp3-deficient mice showed significantly lower IgE levels, Th2-associated cytokines, cell infiltration into the lung, mucin production and epithelial thickening than their wild-type counterparts, which appears to be independent of inflammasome formation. Intriguingly, bone-marrow chimera revealed that expression of NLRP3 in the hematopoietic system is required to trigger an allergic response. Conclusion: Overall, this study identifies NLRP3 as an important driver of BP-induced allergic immune responses.

The critical role of NLRP3 inflammasome activation in Streptococcus suis-induced blood-brain barrier disruption.

Streptococcus suis (S. suis) type 2 (SS2) is an important zoonotic pathogen causing severe neural infections in pigs and causes serious threat to public health. Inflammasome activation plays an important role in the host against microbial infection but the role of inflammasome activation in the blood-brain barrier (BBB) integrity during S. suis infection is rarely studied. This study investigated the mechanism by which S. suis-induced NLRP3 inflammasome activation led to BBB disruption. Our results showed that S. suis infection activated NLRP3 inflammasome in brain microvascular endothelial cells (BMECs) leading to the secretion of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and chemokines (CCL-2 and CXCL-2) as well as the cleavage of Gasdermin D (GSDMD) which were significantly attenuated by inflammasome inhibitor MCC950. Furthermore, S. suis infection significantly downregulated expression of tight junctions (TJs) proteins and trans-endothelial electrical resistance (TEER) while NLRP3 inhibition rescued S. suis-induced degradation of TJs proteins and significantly reduced the number of S. suis crossing BBB in transwell infection model. Moreover, recombinant IL-1ß exacerbated the reduction of TJs proteins in BMECs. In murine S. suis-infection model, MCC950 reduced the bacterial load and the excessive inflammatory response in mice brain. In addition, the integrity of the BBB was protected with increased TJ proteins expression and decreased pathological injury after the inhibition of NLRP3 inflammasome, indicating NLRP3 inflammasome plays a destructive role in meningitis induced by S. suis. Our study expands the understanding on the role of NLRP3 inflammasome in bacterial meningitis, which provide the valuable information for the development of anti-infective agents targeting NLRP3 to treat bacterial meningitis.

In vitro analysis of the expression of inflammasome, antiviral, and immune genes in an Oreochromis niloticus liver cell line following stimulation with bacterial ligands and infection with tilapia lake virus.

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1ß and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 106 TCID50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1ß showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent.

Overexpression of NLRP12 enhances antiviral immunity and alleviates herpes simplex keratitis via pyroptosis/IL-18/IFN-γ signaling.

Herpes simplex keratitis (HSK) is a blinding disease caused by herpes simplex virus type 1 (HSV-1) infection, and rapid eradication of the virus from the affected cornea is imperative. Nod-like receptors (NLRs) are intracellular innate immune sensors closely associated with cell death, inflammation and immune responses. In this study, we investigated the role of NLRP12 in the antiviral immunology in HSK and the underlying mechanisms. We found that NLRP12 expression was significantly decreased in HSV-1-infected human corneal epithelial cells (HCE-Ts) and HSK mouse corneas. Overexpression of NLRP12 significantly reduced viral replication in infected HCE-Ts and functioned through inflammasome-mediated pyroptosis and downstream IL-18-IFN-γ axis. In HSK mouse models, overexpression of NLRP12 reduced viral replication in the cornea and alleviated HSK symptoms. This resulted from enhanced antiviral immune responses including the activation of specific immune cells in both the cornea and the draining lymph nodes. Specifically, the NLRP12-IL-18-IFN-γ axis regulated the interaction between infected corneal epithelial cells and macrophages. In conclusion, our study identified a role of NLRP12 in mediating pyroptosis and regulating antiviral immune responses. This novel finding opens the possibilities of NLRP12 as a viable target in the therapeutic strategies for HSV-1 infection.

Inhibition of NLRP3 inflammasome alleviates cognitive deficits in a mouse model of anti-NMDAR encephalitis induced by active immunization.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neurological disorder, characterized by cognitive deficits as one of its vital features. The nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome is a key contributor to neuroinflammation and cognitive deficits in neurological diseases. However, the underlying mechanism of anti-NMDAR encephalitis remains unclear, and the biological function of the NLRP3 inflammasome in this condition has not been elucidated. In this study, a mouse model of anti-NMDAR encephalitis was induced by active immunization with the GluN1356-385 peptide (NEA model). The NLRP3 inflammasome in the hippocampus and temporal cortex was investigated using real-time quantitative PCR (RT-qPCR), western blotting, and immunofluorescence staining. The impact of MCC950 on cognitive function and NLRP3 inflammation was assessed. Confocal immunofluorescence staining and Sholl analysis were employed to examine the function and morphology of microglia. In the current study, we discovered overactivation of the NLRP3 inflammasome and an enhanced inflammatory response in the NEA model, particularly in the hippocampus and temporal cortex. Furthermore, significant cognitive dysfunction was observed in the NEA model. While, MCC950, a selective inhibitor of the NLRP3 inflammasome, sharply attenuated the inflammatory response in mice, leading to mitigated cognitive deficits of mice and more regular arrangements of neurons and reduced number of hyperchromatic cells were also observed in the hippocampus area. In addition, we found that the excess elevation of NLRP3 inflammasome was mainly expressed in microglia accompanied with the overactivation of microglia, while MCC950 treatment significantly inhibited the increased number and activated morphological changes of microglia in the NEA model. Altogether, our study reveals the vital role of overactivated NLRP3 signaling pathway in aggravating the inflammatory response and cognitive deficits and the potential protective effect of MCC950 in anti-NMDAR encephalitis. Thus, MCC950 represents a promising strategy for anti-inflammation in anti-NMDAR encephalitis and our study lays a theoretical foundation for it to become a clinically targeted drug.

DUSP1 Mitigates MSU-Induced Immune Response in Gouty Arthritis Reinforcing Autophagy.

BACKGROUND: Persistent hyperuricemia can lead to the generation and deposition of monosodium urate (MSU) crystals. This can trigger gouty arthritis (GA), which in turn induces inflammation. Activation of the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the onset and progression of GA. Autophagy may have a dual effect on GA with regard to the NLRP3 inflammasome. Therefore, the present study aimed to gain a deeper comprehension of the interaction between autophagy and NLRP3 inflammasome activation is imperative for developing more efficacious treatments for GA. METHODS: Peripheral blood monocytes (PBMCs) were first isolated from GA patients and healthy controls and underwent bulk RNA sequencing analysis. Overexpression and knockdown of dual specificity phosphatase 1 (DUSP1) was performed in THP-1 monocytes to investigate its role in the immune response and mitochondrial damage. The luciferase assay and Western blot analysis were used to study the interaction between autophagy and NLRP3 inflammasome activation. RESULTS: Bulk RNA sequencing analysis showed significant upregulation of DUSP1 expression in PBMCs from GA patients compared to healthy controls. This result was subsequently verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). DUSP1 expression in human THP-1 monocytes was also shown to increase after MSU treatment. Downregulation of DUSP1 expression increased the secretion of inflammatory cytokines after MSU treatment, whereas the overexpression of DUSP1 decreased the secretion levels. Lipopolysaccharides (LPS) combined with adenosine-triphosphate (ATP) led to mitochondrial damage, which was rescued by overexpressing DUSP1. DUSP1 overexpression further increased the level of autophagy following MSU treatment, whereas downregulation of DUSP1 decreased autophagy. Treatment with the autophagy inhibitor 3-Methyladenine (3-MA) restored inflammatory cytokine secretion levels in the DUSP1 overexpression group. MSU caused pronounced pathological ankle swelling in vivo. However, DUSP1 overexpression significantly mitigated this phenotype, accompanied by significant downregulation of inflammatory cytokine secretion levels in the joint tissues. CONCLUSIONS: This study revealed a novel function and mechanism for DUSP1 in promoting autophagy to mitigate the MSU-induced immune response in GA. This finding suggests potential diagnostic biomarkers and anti-inflammatory targets for more effective GA therapy.

The non-pathogenic protozoon Leishmania tarentolae interferes with the activation of NLRP3 inflammasome in human cells: new perspectives in the control of inflammation.

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.

Unmasking the NLRP3 inflammasome in dendritic cells as a potential therapeutic target for autoimmunity, cancer, and infectious conditions.

Proper and functional immune response requires a complex interaction between innate and adaptive immune cells, which dendritic cells (DCs) are the primary actors in this coordination as professional antigen-presenting cells. DCs are armed with numerous pattern recognition receptors (PRRs) such as nucleotide-binding and oligomerization domain-like receptors (NLRs) like NLRP3, which influence the development of their activation state upon sensation of ligands. NLRP3 is a crucial component of the immune system for protection against tumors and infectious agents, because its activation leads to the assembly of inflammasomes that cause the formation of active caspase-1 and stimulate the maturation and release of proinflammatory cytokines. But, when NLRP3 becomes overactivated, it plays a pathogenic role in the progression of several autoimmune disorders. So, NLRP3 activation is strictly regulated by diverse signaling pathways that are mentioned in detail in this review. Furthermore, the role of NLRP3 in all of the diverse immune cells' subsets is briefly mentioned in this study because NLRP3 plays a pivotal role in modulating other immune cells which are accompanied by DCs' responses and subsequently influence differentiation of T cells to diverse T helper subsets and even impact on cytotoxic CD8+ T cells' responses. This review sheds light on the functional and therapeutic role of NLRP3 in DCs and its contribution to the occurrence and progression of autoimmune disorders, prevention of diverse tumors' development, and recognition and annihilation of various infectious agents. Furthermore, we highlight NLRP3 targeting potential for improving DC-based immunotherapeutic approaches, to be used for the benefit of patients suffering from these disorders.

Metabolism-inflammasome crosstalk shapes innate and adaptive immunity.

Inflammasomes are a central component of innate immunity and play a vital role in regulating innate immune response. Activation of inflammasomes is also indispensable for adaptive immunity, modulating the development and response of adaptive immunity. Recently, increasing studies have shown that metabolic alterations and adaptations strongly influence and regulate the differentiation and function of the immune system. In this review, we will take a holistic view of how inflammasomes bridge innate and adaptive (especially T cell) immunity and how inflammasomes crosstalk with metabolic signals during the immune responses. And, special attention will be paid to the metabolic control of inflammasome-mediated interactions between innate and adaptive immunity in disease. Understanding the metabolic regulatory functions of inflammasomes would provide new insights into future research directions in this area and may help to identify potential targets for inflammasome-associated diseases and broaden therapeutic avenues.