Cross-species single-cell analysis uncovers the immunopathological mechanisms associated with IgA nephropathy progression.

IgA nephropathy (IgAN) represents the main cause of renal failure, while the precise pathogenetic mechanisms have not been fully determined. Herein, we conducted a cross-species single-cell survey on human IgAN and mouse and rat IgAN models to explore the pathogenic programs. Cross-species single-cell RNA sequencing (scRNA-Seq) revealed that the IgAN mesangial cells (MCs) expressed high levels of inflammatory signatures CXCL12, CCL2, CSF1, and IL-34 and specifically interacted with IgAN macrophages via the CXCL12/CXCR4, CSF1/IL-34/CSF1 receptor, and integrin subunit alpha X/integrin subunit alpha M/complement C3 (C3) axes. IgAN macrophages expressed high levels of CXCR4, PDGFB, triggering receptor expressed on myeloid cells 2, TNF, and C3, and the trajectory analysis suggested that these cells derived from the differentiation of infiltrating blood monocytes. Additionally, protein profiling of 21 progression and 28 nonprogression IgAN samples revealed that proteins CXCL12, C3, mannose receptor C-type 1, and CD163 were negatively correlated with estimated glomerular filtration rate (eGFR) value and poor prognosis (30% eGFR as composite end point). Last, a functional experiment revealed that specific blockade of the Cxcl12/Cxcr4 pathway substantially attenuated the glomerulus and tubule inflammatory injury, fibrosis, and renal function decline in the mouse IgAN model. This study provides insights into IgAN progression and may aid in the refinement of IgAN diagnosis and the optimization of treatment strategies.

Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

The Immunomodulatory effect of exosomes in diabetes: a novel and attractive therapeutic tool in diabetes therapy.

Exosomes carry proteins, metabolites, nucleic acids and lipids from their parent cell of origin. They are derived from cells through exocytosis, are ingested by target cells, and can transfer biological signals between local or distant cells. Therefore, exosomes are often modified in reaction to pathological processes, including infection, cancer, cardiovascular diseases and in response to metabolic perturbations such as obesity and diabetes, all of which involve a significant inflammatory aspect. Here, we discuss how immune cell-derived exosomes origin from neutrophils, T lymphocytes, macrophages impact on the immune reprogramming of diabetes and the associated complications. Besides, exosomes derived from stem cells and their immunomodulatory properties and anti-inflammation effect in diabetes are also reviewed. Moreover, As an important addition to previous reviews, we describes promising directions involving engineered exosomes as well as current challenges of clinical applications in diabetic therapy. Further research on exosomes will explore their potential in translational medicine and provide new avenues for the development of effective clinical diagnostics and therapeutic strategies for immunoregulation of diabetes.

Sevoflurane blocks KLF5-mediated transcriptional activation of ITGB2 to inhibit macrophage infiltration in hepatic ischemia/reperfusion injury.

BACKGROUND: Sevoflurane (Sevo) preconditioning and postconditioning play a protective role against injury induced by hepatic ischemia/reperfusion (I/R). At the same time, the involvement of macrophage infiltration in this process and the precise mechanisms are unclear. Here, we designed this research to elucidate the protective effects of Sevo against hepatic I/R injury and the molecules involved. METHODS: The alleviating effect of Sevo on the liver injury was analyzed by liver function analysis, hematoxylin and eosin staining, Masson trichrome staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling, western blot analysis and an enzyme-linked immunosorbent assay. An in vitro cell model was developed using alpha mouse liver 12 (AML12) cells, and the cell model was treated with oxygen-glucose deprivation and reoxygenation and Sevo. Multiple bioinformatics databases were used to screen transcriptional regulators related to hepatic I/R injury and the targets of Krueppel-like factor 5 (KLF5). KLF5 expression was artificially upregulated alone or with integrin beta-2 (ITGB2) knockdown to substantiate their involvement in Sevo-mediated hepatoprotection. RESULTS: Sevo protected the liver against I/R injury by reducing cell apoptosis and inflammatory response. KLF5 was upregulated in liver tissues following I/R injury, whereas KLF5 overexpression aggravated macrophage infiltration and liver injury induced by I/R injury. KLF5 bound to the promoter of ITGB2 to enhance ITGB2 transcription. Knockdown of ITGB2 reversed the aggravation of injury caused by KLF5 overexpression in mice and AML12 cells. CONCLUSIONS: Sevo blocked KLF5-mediated transcriptional activation of ITGB2, thereby inhibiting macrophage infiltration in hepatic I/R injury.

Hyphal Als proteins act as CR3 ligands to promote immune responses against Candida albicans.

Patients with decreased levels of CD18 (ß2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMß2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the ß-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the "outside-in signaling" for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1ß release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.

Generation of Eroded Nanoplastics from Domestic Wastes and Their Impact on Macrophage Cell Viability and Gene Expression.

This study reports an innovative approach for producing nanoplastics (NP) from various types of domestic waste plastics without the use of chemicals. The plastic materials used included water bottles, styrofoam plates, milk bottles, centrifuge tubes, to-go food boxes, and plastic bags, comprising polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP), high-density polyethylene (HDPE), and Poly (Ethylene-co-Methacrylic Acid) (PEMA). The chemical composition of these plastics was confirmed using Raman and FTIR spectroscopy, and they were found to have irregular shapes. The resulting NP particles ranged from 50 to 400 nm in size and demonstrated relative stability when suspended in water. To assess their impact, the study investigated the effects of these NP particulates on cell viability and the expression of genes involved in inflammation and oxidative stress using a macrophage cell line. The findings revealed that all types of NP reduced cell viability in a concentration-dependent manner. Notably, PS, HDPE, and PP induced significant reductions in cell viability at lower concentrations, compared to PEMA and PET. Moreover, exposure to NP led to differential alterations in the expression of inflammatory genes in the macrophage cell line. Overall, this study presents a viable method for producing NP from waste materials that closely resemble real-world NP. Furthermore, the toxicity studies demonstrated distinct cellular responses based on the composition of the NP, shedding light on the potential environmental and health impacts of these particles.

Structural Characterization of Polygonatum Cyrtonema Polysaccharide and Its Immunomodulatory Effects on Macrophages.

A neutral Polygonatum cyrtonema polysaccharide (NPCP) was isolated and purified from Polygonatum cyrtonema by various chromatographic techniques, including DEAE-52 and Sephadex-G100 chromatography. The structure of NPCP was characterized by HPLC, HPGPC, GC-MS, FT-IR, NMR, and SEM. Results showed that NPCP is composed of glucose (55.4%) and galactose (44.6%) with a molecular weight of 3.2 kDa, and the sugar chain of NPCP was →1)-α-D-Glc-(4→1)-ß-D-Gal-(3→. In vitro bioactivity experiments demonstrated that NPCP significantly enhanced macrophages proliferation and phagocytosis while inhibiting the M1 polarization induced by LPS as well as the M2 polarization induced by IL-4 and IL-13 in macrophages. Additionally, NPCP suppressed the secretion of IL-6 and TNF-α in both M1 and M2 cells but promoted the secretion of IL-10. These results suggest that NPCP could serve as an immunomodulatory agent with potential applications in anti-inflammatory therapy.

Application of Cinnamomum burmannii Essential Oil in Promoting Wound Healing.

Skin wounds, leading to infections and death, have a huge negative impact on healthcare systems around the world. Antibacterial therapy and the suppression of excessive inflammation help wounds heal. To date, the application of wound dressings, biologics and biomaterials (hydrogels, epidermal growth factor, stem cells, etc.) is limited due to their difficult and expensive preparation process. Cinnamomum burmannii (Nees & T. Nees) Blume is an herb in traditional medicine, and its essential oil is rich in D-borneol, with antibacterial and anti-inflammatory effects. However, it is not clear whether Cinnamomum burmannii essential oil has the function of promoting wound healing. This study analyzed 32 main components and their relative contents of essential oil using GC-MS. Then, network pharmacology was used to predict the possible targets of this essential oil in wound healing. We first proved this essential oil's effects in vitro and in vivo. Cinnamomum burmannii essential oil could not only promote the proliferation and migration of skin stromal cells, but also promote M2-type polarization of macrophages while inhibiting the expression of pro-inflammatory cytokines. This study explored the possible mechanism by which Cinnamomum burmannii essential oil promotes wound healing, providing a cheap and effective strategy for promoting wound healing.

Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile.

BACKGROUND: Inhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials. RESULTS: We investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators. CONCLUSIONS: Straight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

Inflammatory corpuscle AIM2 facilitates macrophage foam cell formation by inhibiting cholesterol efflux protein ABCA1.

The inflammatory corpuscle recombinant absents in melanoma 2 (AIM2) and cholesterol efflux protein ATP binding cassette transporter A1(ABCA1) have been reported to play opposing roles in atherosclerosis (AS) plaques. However, the relationship between AIM2 and ABCA1 remains unclear. In this study, we explored the potential connection between AIM2 and ABCA1 in the modulation of AS by bioinformatic analysis combined with in vitro experiments. The GEO database was used to obtain AS transcriptional profiling data; screen differentially expressed genes (DEGs) and construct a weighted gene co-expression network analysis (WGCNA) to obtain AS-related modules. Phorbol myristate acetate (PMA) was used to induce macrophage modelling in THP-1 cells, and ox-LDL was used to induce macrophage foam cell formation. The experiment was divided into Negative Control (NC) group, Model Control (MC) group, AIM2 overexpression + ox-LDL (OE AIM2 + ox-LDL) group, and AIM2 short hairpin RNA + ox-LDL (sh AIM2 + ox-LDL) group. The intracellular cholesterol efflux rate was detected by scintillation counting; high-performance liquid chromatography (HPLC) was used to detect intracellular cholesterol levels; apoptosis levels were detected by TUNEL kit; levels of inflammatory markers (IL-1ß, IL-18, ROS, and GSH) were detected by ELISA kits; and levels of AIM2 and ABCA1 proteins were detected by Western blot. Bioinformatic analysis revealed that the turquoise module correlated most strongly with AS, and AIM2 and ABCA1 were co-expressed in the turquoise module with a trend towards negative correlation. In vitro experiments demonstrated that AIM2 inhibited macrophage cholesterol efflux, resulting in increased intracellular cholesterol levels and foam cell formation. Moreover, AIM2 had a synergistic effect with ox-LDL, exacerbating macrophage oxidative stress and inflammatory response. Silencing AIM2 ameliorated the above conditions. Furthermore, the protein expression levels of AIM2 and ABCA1 were consistent with the bioinformatic analysis, showing a negative correlation. AIM2 inhibits ABCA1 expression, causing abnormal cholesterol metabolism in macrophages and ultimately leading to foam cell formation. Inhibiting AIM2 may reverse this process. Overall, our study suggests that AIM2 is a reliable anti-inflammatory therapeutic target for AS. Inhibiting AIM2 expression may reduce foam cell formation and, consequently, inhibit the progression of AS plaques.

Mechanical stretch preconditioned adipose-derived stem cells elicit polarization of anti-inflammatory M2-like macrophages and improve chronic wound healing.

Transplantation of adipose-derived stem cells (ASCs) is a promising option in the field of chronic wounds treatment. However, the effectiveness of ASCs therapies has been hampered by highly inflammatory environment in chronic wound areas. These problems could be partially circumvented using efficient approaches that boost the survival and anti-inflammatory capacity of transplanted ASCs. Here, by application of mechanical stretch (MS), we show that ASCs exhibits increased survival and immunoregulatory properties in vitro. MS triggers the secretion of macrophage colony stimulating factor (M-CSF) from ASCs, a chemokine that is linked to anti-inflammatory M2-like macrophages polarization. When the MS-ASCs were transplanted to chronic wounds, the wound area yields significantly faster closure rate and lower inflammatory mediators, largely due to macrophages polarization driven by transplanted MS-ASCs. Thus, our work shows that mechanical stretch can be harnessed to enhance ASCs transplantation efficiency in chronic wounds treatment.

Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation.

BACKGROUND: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. METHODS: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. RESULTS: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. CONCLUSION: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.

Tectorigenin inhibits inflammatory responses in murine inflammatory bowel disease and LPS-stimulated macrophages via inactivating MAPK signaling pathway.

BACKGROUND: Considering the antihepatitis effects of Tectorigenin (TEC), and the same adenosine mitogen-activated protein kinase (MAPK) pathway in both hepatitis and inflammatory bowel disease (IBD) models, exploring the role of TEC in IBD is contributive to develop a new treatment strategy against IBD. METHODS: The IBD mouse model was constructed by feeding with dextran sodium sulfate (DSS) and injection of TEC. Afterward, the mouse body weight, colon length, and disease activity index (DAI) were tested to assess the enteritis level. Mouse intestine lesions were detected by hematoxylin and eosin staining. Murine macrophages underwent lipopolysaccharide (LPS) induction to establish an inflammation model. Cell viability was determined by cell counting kit-8 assay. Enzyme-linked immunosorbent assay was performed to measure interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) levels. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions were quantified via quantitative reverse transcription polymerase chain reaction. Levels of MAPK pathway-related proteins (p-P38, P38, p-Jun N-terminal kinase (JNK), JNK, signal-regulated kinase (ERK), p-ERK), COX-2 and iNOS were quantitated by Western blot. RESULTS: TEC improved the inflammatory response through ameliorating weight loss, shortening colon, and increasing DAI score in IBD mouse. Expressions of intestinal inflammatory factors (IL-6, TNF-α, iNOS and COX-2) and MAPK pathway-related proteins (p-P38, p-JNK, and p-ERK) were increased both in DSS-induced mouse intestinal tissue, but TEC inhibited expressions of inflammatory factors. The same increased trend was identified in LPS-induced macrophages, but TEC improved macrophage inflammation, as evidenced by downregulation of inflammatory factors. CONCLUSION: TEC mitigates IBD and LPS-induced macrophage inflammation in mice via inhibiting MAPK signaling pathway.

Uncovering a Novel Functional Interaction Between Adult Hepatic Progenitor Cells, Inflammation and EGFR Signaling During Bile Acids-Induced Injury.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-ß regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

The Effect Mechanism of N6-adenosine Methylation (m6A) in Melatonin Regulated LPS-induced Colon Inflammation.

Colon inflammation is characterized by disturbances in the intestinal microbiota and inflammation. Melatonin (Mel) can improve colon inflammation. However, the underlying mechanism remains unclear. Recent studies suggest that m6A methylation modification may play an important role in inflammatory responses. This study aimed to explore the effects of melatonin and LPS-mediated m6A methylation on colon inflammation. Our study found that melatonin inhibits M1 macrophages, activates M2 macrophages, inhibit the secretion of pro-inflammatory factors, maintain colon homeostasis and improves colon inflammation through MTNR1B. In addition, the increased methylation level of m6A is associated with the occurrence of colon inflammation, and melatonin can also reduce the level of colon methylation to improve colon inflammation. Among them, the main methylated protein METTL3 can be inhibited by melatonin through MTNR1B. In a word, melatonin regulates m6A methylation by improving abnormal METTL3 protein level to reshape the microflora and activate macrophages to improve colon inflammation, mainly through MTNR1B.

Aberrant Expression of SLC7A11 Impairs the Antimicrobial Activities of Macrophages in Staphylococcus Aureus Osteomyelitis in Mice.

Staphylococcus aureus (S. aureus) persistence in macrophages, potentially a reservoir for recurrence of chronic osteomyelitis, contributes to resistance and failure in treatment. As the mechanisms underlying survival of S. aureus in macrophages remain largely unknown, there has been no treatment approved. Here, in a mouse model of S. aureus osteomyelitis, we identified significantly up-regulated expression of SLC7A11 in both transcriptomes and translatomes of CD11b+F4/80+ macrophages, and validated a predominant distribution of SLC7A11 in F4/80+ cells around the S. aureus abscess. Importantly, pharmacological inhibition or genetic knockout of SLC7A11 promoted the bactericidal function of macrophages, reduced bacterial burden in the bone and improved bone structure in mice with S. aureus osteomyelitis. Mechanistically, aberrantly expressed SLC7A11 down-regulated the level of intracellular ROS and reduced lipid peroxidation, contributing to the impaired bactericidal function of macrophages. Interestingly, blocking SLC7A11 further activated expression of PD-L1 via the ROS-NF-κB axis, and a combination therapy of targeting both SLC7A11 and PD-L1 significantly enhanced the efficacy of clearing S. aureus in vitro and in vivo. Our findings suggest that targeting both SLC7A11 and PD-L1 is a promising therapeutic approach to reprogram the bactericidal function of macrophages and promote bacterial clearance in S. aureus osteomyelitis.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites ß-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

Quantifying NF-κB Activation by Flow Cytometry of IκBα Degradation.

Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.