Expression Of Dectin-1 Research Articles

A novel experimental model to investigate fungal involvement shows expression of Dectin-1 in periapical lesion pathogenesis.

Candida albicans is linked to persistent endodontic lesions. However, the recognition receptor that identifies it is not explored previously. The aim of this study was to (1) establish a zymosan-induced model of apical periodontitis in mouse, (2) observe the expression of Dectin-1 and its possible relationship with toll-like receptor (TLR) 2 and (3) observe relationship between Osteopontin (OPN) and inflammatory cytokines. A total of 138 Naval Medical Research Institute (NMRI) mice were randomly divided into; Experimental Group n = 69 and Zymosan Group n = 69. Periapical periodontitis was developed in right maxillary molar. The animals were sacrificed at 7, 21 and 42 days. Bone blocks containing the mesial root (n = 15 for qRT-PCR, n = 45 for enzyme-linked immune sorbent assay (ELISA)) were collected for mRNA expression and ELISA. While whole maxilla (n = 3 from each time interval) were used for histology and immunohistochemical analysis. One way analysis of variance (ANOVA) and Tuckey's posthoc was used for statistical analysis at p ≤ .05. TLR-2, Dectin-1 and TLR4-positive cells was detected at all time intervals in both groups. A strong positive correlation was observed between TLR-2 and Dectin-1 in both lesions (regular r = .680, p = .015, zymosan (r = .861, p < .001)). A significant correlation was found between OPN and tumour necrosis factor-alpha (TNF-α) in zymosan lesion (r = .827, p = .001). Immune cells of inflamed periapical tissue expressed Dectin-1 receptor in response to the microbial challenge from infected root canals and showed positive correlation with TLR-2 and OPN suggesting a possible receptor collaboration mediated by OPN. The expression of OPN and TNF-α showed positive correlation in response to fungal antigen, indicating a possible relationship.

SOCS3 Regulates Dectin-2-Induced Inflammation in PBMCs of Diabetic Patients

Abstract The C-type lectin receptors (CLRs) Dectin-1 and Dectin-2 are involved in several innate immune responses and are expressed mainly in dendritic cells, monocytes, and macrophages. This study aims to evaluate the expression and function of Dectin-2 in peripheral blood mononuclear cells (PBMCs) isolated from diabetic patients and non-diabetic controls. Our results show that Dectin-2 expression was the highest in monocytes compared with other leukocyte subpopulations. The expression of Dectin-2 was also found to be significantly higher in T2D patients monocytes compared with non-diabetic controls. The expression of Dectin-2 was found to be positively correlated with markers of glucose homeostasis, including HOMA-IR and HbA1c. Interestingly, SOCS3, a negative regulator of inflammation, was expressed significantly lower in the PBMCs of T2D patients. Moreover, SOCS3 expression was negatively correlated with Dectin-2 expression level. Further analysis of inflammatory signaling pathways showed a persistent activation of the Dectin-2-Syk-NFkB pathway that was instigated by the diminished expression of SOCS3. Dectin-2 activation failed to induce SOCS3 expression and suppress subsequent inflammatory responses in the PBMCs of diabetic patients. siRNA-mediated knockdown of SOCS3 in PBMCs displayed a similar inflammatory phenotype to diabetic PBMCs when exposed to Dectin-2 ligands. Altogether, our findings suggest that elevated Dectin-2 and its relationship with SOCS3 could be involved in the abnormal immune response observed in T2D patients. This work was supported and funded by the Kuwait University Research Grant No. [SL01/21] and by the Kuwait Foundation for the Advancement of Sciences (KFAS) grant No. (RA MoH-2022-002).

Dectin-1 deficiency alleviates diabetic cardiomyopathy by attenuating macrophage-mediated inflammatory response

Cardiovascular diseases are the primary cause of mortality in patients with diabetes and obesity. Hyperglycemia and hyperlipidemia in diabetes alters cardiac function, which is associated with broader cellular processes such as aberrant inflammatory signaling. Recent studies have shown that a pattern recognition receptor called Dectin-1, expressed on macrophages, mediates pro-inflammatory responses in innate immunity. In the present study, we examined the role of Dectin-1 in the pathogenesis of diabetic cardiomyopathy. We observed increased Dectin-1 expression in heart tissues of diabetic mice and localized the source to macrophages. We then investigated the cardiac function in Dectin-1-deficient mice with STZ-induced type 1 diabetes and high-fat-diet-induced type 2 diabetes. Our results show that Dectin-1 deficient mice are protected against diabetes-induced cardiac dysfunction, cardiomyocyte hypertrophy, tissue fibrosis, and inflammation. Mechanistically, our studies show that Dectin-1 is important for cell activation and induction of inflammatory cytokines in high-concentration glucose and palmitate acid (HG + PA)-challenged macrophages. Deficiency of Dectin-1 generate fewer paracrine inflammatory factors capable of causing cardiomyocyte hypertrophy and fibrotic responses in cardiac fibroblasts. In conclusion, this study provides evidence that Dectin-1 mediates diabetes-induced cardiomyopathy through regulating inflammation. Dectin-1 may be a potential target to combat diabetic cardiomyopathy.

Abstract 2964: Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042

Abstract Tumor-associated macrophages (TAMs) are the largest immune cell population in many cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. To this end, we developed BDC-3042, an agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A). Dectin-2 is a C-type lectin receptor (CLR) known best for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We previously demonstrated that Dectin-2 agonism with natural ligands stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models. Here we present our preclinical studies demonstrating the therapeutic potential of the Dectin-2 agonistic antibody, BDC-3042, as a novel TAM-directed immunotherapy for diverse human cancers. BDC-3042 exhibits strong binding to Dectin-2-expressing macrophages generated in vitro and to primary human TAMs from a range of solid tumor types. In contrast, BDC-3042 binds weakly to peripheral monocytes and minimally to other immune cells in blood and tumor tissues. Macrophages exposed to cytokines and growth factors commonly found in the TME exhibit increased Dectin-2 expression and BDC-3042 binding. Treatment with BDC-3042 activates in vitro-generated macrophages and primary TAMs to produce an array of pro-inflammatory cytokines and chemokines associated with anti-tumor immunity. Consistent with low target expression, BDC-3042 elicits little to no activation of peripheral monocytes or cells in whole blood. BDC-3042 activity is dependent on both Dectin-2 and FcγRs, as indicated through studies utilizing Fc variants with enhanced or attenuated effector function. In mice with humanized immune systems, BDC-3042 elicits activation of TAMs, as evidenced by modulation of key activation markers. Systemically administered BDC-3042 mediates anti-tumor efficacy as a monotherapy, and combination with checkpoint blockade therapy enhances anti-tumor efficacy. The data presented demonstrate the therapeutic potential of targeting Dectin-2 expressed by TAMs with the agonistic antibody BDC-3042 as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Fang Xiao, Po Y. Ho, Jess L. Nolin, Rishali K. Gadkari, Laughing Bear Torrez, David T. Omstead, Katelynn A. McEachin, Jason Ptacek, Rachel Grgich, Cecelia I. Pearson, Stefan Chun, Cindy L. Kreder, Karla A. Henning, Han K. Kim, Lu Xu, Steven J. Chapin, Michael N. Alonso, Shelley E. Ackerman. Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2964.

Glabridin Inhibits Aspergillus fumigatus Growth and Alleviate Inflammation Mediated by Dectin-2 and NLRP3 Inflammasome

Purpose The research was used to uncover the mechanism of glabridin in Aspergillus fumigatus keratitis in anti-fungus and anti-inflammation. Methods In vitro, RAW 264.7 cells were infected with A. fumigatus with incubation of glabridin in different concentrations. Real-time quantitative polymerase chain reaction (RT‑qPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA) were used to assess the inflammatory severe and alternation with the intervention of Dectin-2 siRNA and glabridin. In vivo, A. fumigatus keratitis mouse models were established by spore intra-stromal injection and treated with glabridin or PBS. And disease scores, inflammatory mediators, and periodic acid-schiff (PAS) staining were exhibited to demonstrate the therapeutic efficiency of glabridin in vivo. Morphological interference assay monitored fungal germination. Scanning and transmission electron microscopy were used to observe the growth of fungi. Results In RAW 264.7 cells and mouse keratitis models, noncytotoxic 16 μg/mL glabridin showed significant inhibition in the expression of Dectin-2, NLRP3, Caspase-1, IL-1β, and TNF-α after A. fumigatus infection, almost similar to the intervention of Dectin-2 siRNA. PAS staining illustrated the reduced hyphal distribution in cornea stroma with glabridin treatment. Glabridin remarkably inhibited A. fumigatus growth through delaying germination and disrupting the integrity of the hyphae membrane. Conclusion Glabridin plays an anti-inflammatory role in A. fumigatus challenge via suppression of the Dectin-2 and NLRP3 inflammasome, and plays an anti-fungal role through delaying germination and changing the hyphal integrity. KEY MESSAGES Glabridin plays an anti-inflammatory role in A. fumigatus infection of RAW264.7 cells in a concentration-dependent manner and through Dectin-2 mediation. Glabridin decreases fungal distribution and inflammation in mouse A. fumigatus keratitis. Glabridin inhibits A. fumigatus growth by delaying germination and disrupting cellular structure in vitro.

The BCG Moreau Vaccine Upregulates In Vitro the Expression of TLR4, B7-1, Dectin-1 and EP2 on Human Monocytes.

Background: Tuberculosis (TB) is currently the second greatest killer worldwide and is caused by a single infectious agent. Since Bacillus Calmette−Guérin (BCG) is the only vaccine currently in use against TB, studies addressing the protective role of BCG in the context of inducible surface biomarkers are urgently required for TB control. Methods: In this study, groups of HIV-negative adult healthy donors (HD; n = 22) and neonate samples (UCB; n = 48) were voluntarily enrolled. The BCG Moreau strain was used for the in vitro mononuclear cell infections. Subsequently, phenotyping tools were used for surface biomarker detection. Monocytes were assayed for TLR4, B7-1, Dectin-1, EP2, and TIM-3 expression levels. Results: At 48 h, the BCG Moreau induced the highest TLR4, B7-1, and Dectin-1 levels in the HD group only (p-value < 0.05). TIM-3 expression failed to be modulated after BCG infection. At 72 h, BCG Moreau equally induced the highest EP2 levels in the HD group (p-value < 0.005), and higher levels were also found in HD when compared with the UCB group (p-value < 0.05). Conclusions: This study uncovers critical roles for biomarkers after the instruction of host monocyte activation patterns. Understanding the regulation of human innate immune responses is critical for vaccine development and for treating infectious diseases.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans

Ethnopharmacological relevancePulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC.Aim of the study: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. Materials and methodsUltra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1β and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. ResultsAfter administration of BEPD-containing serum, the levels of IL-1β, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. ConclusionsBEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

Innate immune response of human periodontal ligament fibroblasts via the Dectin-1/Syk pathway.

Introduction. A diverse microbiota including fungi exists in the subgingival sites of patients with chronic periodontitis. The cell wall of Candida albicans, the most abundant fungal species, contains β-glucan. Dectin-1 binds β-glucan and participates in fungal recognition.Gap statement. Human periodontal ligament fibroblasts (PDLFs) are present in the periodontal ligament and synthesize immunomodulatory cytokines that influence the local response to infections. However, the expression and role of Dectin-1 in PDLFs have not been explored.Aim. This study aimed to determine if PDLFs express Dectin-1 and induce innate immune responses through Dectin-1 and the signalling molecule Syk.Methodology. The expression of Dectin-1 in PDLFs was determined by flow cytometry, western blotting and confocal microscopy. Real-time PCR and Western blotting were used to determine the immune response of PDLFs stimulated with β-glucan-rich zymosan and C. albicans.Results. Dectin-1 was constitutively expressed in PDLFs. Zymosan induced the expression of cytokines, including IL6, IL1B and IL17A, and the chemokine IL8. Zymosan also induced the expression of the antimicrobial peptide β-defensin-1 (DEFB1). Further, the phosphorylation of Syk and NF-κB occurred upon Dectin-1 activation. Notably, heat-killed C. albicans induced the expression of IL6, IL17A, IL8 and DEFB1, and this activation was suppressed by the Syk inhibitor, R406.Conclusion. These findings indicate that the Dectin-1/Syk pathway induces an innate immune response of PDLFs, which may facilitate the control of oral infections such as candidiasis and periodontitis.

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2.

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing β-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

Gene expression analysis of toll like receptor 2 and 4, Dectin-1, Osteopontin and inflammatory cytokines in human dental pulp ex-vivo

BackgroundToll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation.ObjectiveThe aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls.MethodsA total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05.ResultsGene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis.ConclusionDectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship.

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

BackgroundAsymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known.MethodsThree mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system.ResultsFor macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor.ConclusionOur results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

Effect and Mechanism of Cotrimoxazole Against Talaromyces marneffei in vitro.

Talaromyces marneffei (formerly Penicillium marneffei) is an important thermally dimorphic fungus endemic which is characterized by one of the most frequent opportunistic infections in HIV/AIDS patients, mainly prevalent in Southeast Asia, southern China, and northeastern India. Cotrimoxazole(CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi, thereby commonly used to prevent several opportunistic infections among HIV/AIDS patients. In addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis are considered to have the potential to prevent T. marneffei infection in HIV/AIDS patients receiving antiretroviral therapy (ART). However, the effect of cotrimoxazole towards T. marneffei fungus in vitro remains unclear. Human THP-1 macrophages were used as cell model in vitro to explore the effect and mechanism of cotrimoxazole resistance towards T. marneffei. Cell viability assay and drug sensitivity colony forming units (CFU) experiments were conducted to determine the minimum inhibitory concentration (MIC) of cotrimoxazole inside and outside THP-1 macrophages respectively. Enzyme-linked immunosorbent assay (Elisa) was used to measure the concentration of Dihydropteroic acid synthetase (DHPS), Dihydrofolate synthetase (DHFS) and Dihydrofolate reductase (DHFR) between T. marneffei adding TMP/SMX and without adding TMP/SMX group respectively. Real-time fluorescence quantitative PCR(qPCR) was performed to detect the mRNA expression levels in Dectin-1 mediated signaling pathway and downstream inflammatory cytokines including IL-6, IL-10, IL-23A, CXCL8 and TNF-α released by T. marneffei-infected macrophages between adding TMP/SMX and without adding TMP/SMX group respectively. Cotrimoxazole can inhibit the proliferation of T. marneffei within safe concentration inside and outside THP-1 macrophages. Drug susceptibility results showed the minimal inhibit concentration(MIC) of 1:5 TMP/SMX was ranging from 14/70 to 68/340μg/ml. The MIC of SMX was ranging from 100 to 360μg/ml. The MIC of TMP was ranging from 240 to 400μg/ml outside macrophages. The MIC of TMP/SMX was ranging from 36/180 to 68/340μg/ml. The MIC of SMX was ranging from 340 to 360μg/ml. The MIC of TMP was ranging from 320 to 400μg/ml inside macrophages. The synergistic interaction of 1:5 TMP/SMX was more effective in inhibiting T. marneffei than separate SMX and TMP. DHPS, DHFS and DHFR can be inhibited by cotrimoxazole within safe and effective concentration. Dectin-1 expression is increased following T. marneffei infection, leading to the increase of IL-6, IL-10, IL-23A and the decrease of CXCL8 and TNF-α. Conversely, cotrimoxazole decrease the levels of Dectin-1, IL-6, IL-10, IL-23A and increase the levels of CXCL8 and TNF-α, thereby enhancing the intracellular killing-T. marneffei capacity of macrophages. Our findings indicated that cotrimoxazole directly inhibited T. marneffei growth by blocking DHPS, DHFS and DHFR and indirectly inhibited T. marneffei growth perhaps by regulating the Dectin-1 signaling pathway, which may effectively interfere with the defense ability of the host against T. marneffei infection.

Expression of Toll-like receptor 2, Dectin-1, and Osteopontin in murine model of pulpitis.

This in vivo animal study aimed to develop a murine model of pulpitis induced by pulp exposure with or without application of zymosan in Naval Medical Research Institute (NMRI) mice and observe expressions of Toll-like receptor (TLR)-2, TLR-4, Dectin-1, Osteopontin (OPN), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1ß. A total of 168 NMRI mice were divided into two groups, i.e., group A (n = 84) (pulpitis induced by pulp exposure only) and group B (n = 84) (pulpitis induced by pulp exposure and zymosan application). Right maxillary molar pulps were exposed with ¼ round bur, and animals were sacrificed at 0, 6, 9, 12, 24, 48, and 72h. The exposed teeth were obtained for real-time polymerase chain reaction (qRT-PCR) analysis and histological and immunohistochemistry (IHC) analysis. Histological evaluation revealed a time-dependent steady increase in inflammation. Similar time-dependent increase in the expression of inflammatory cytokines was noted. Group A exhibited an increase in TLR-4, Dectin-1, and OPN at 6h, while TLR-2 was expressed at 24h. Group B expressed TLR-2, Dectin-1, and OPN at 9, 48, and 72h, respectively (p ≤ 0.05). Expression of OPN and TNF-α exhibited a similar pattern in both groups. IHC also detected expression of TLR-2, Dectin-1, TLR4, and CD68 in some cells at 6 and 9h. NMRI mice provided for a stable pulp inflammation model. Zymosan may be used to develop pulp inflammation model and study inflammatory response towards fungal antigens. Dental pulp expressed Dectin-1 receptor. OPN and TNF-α exhibited a similar expression pattern. Innate immunity of dental pulp is capable of detecting fungal pathogens.

Inhibition of Dectin-1 Alleviates Neuroinflammatory Injury by Attenuating NLRP3 Inflammasome-Mediated Pyroptosis After Intracerebral Hemorrhage in Mice: Preliminary Study Results.

BackgroundNeuroinflammation plays an important role following intracerebral hemorrhage (ICH). NLRP3 inflammasome-mediated pyroptosis contributes to the mechanism of neuroinflammation. It has been reported that dendritic cell-associated C-type lectin-1 (Dectin-1) activation triggers inflammation in neurological diseases. However, the role of Dectin-1 on NLRP3 inflammasome-mediated pyroptosis after ICH remains unclear. Here, we aimed to explore the effect of Dectin-1 on NLRP3 inflammasome-mediated pyroptosis and neuroinflammation after ICH.MethodsAdult male C57BL/6 mice were used to establish the ICH model. Laminarin, an inhibitor of Dectin-1, was administered for intervention. Expression of Dectin-1 was evaluated by Western blot and immunofluorescence. Brain water content and neurobehavioral function were tested to assess brain edema and neurological performance. Western blot was conducted to evaluate the level of GSDMD-N. ELISA kits were used to measure the levels of IL-1β and IL-18. qRT–PCR and Western blot were performed to evaluate the expressions of NLRP3 inflammasome, IL-1β, and IL-18.ResultsThe expression of Dectin-1 increased following ICH, and Dectin-1 was expressed on microglia. In addition, inhibition of Dectin-1 by laminarin decreased brain edema and neurological impairment after ICH. Moreover, inhibition of Dectin-1 decreased the expression of pyroptosis-related protein, GSDMD-N, and inflammatory cytokines (IL-1β and IL-18). Mechanistically, Dectin-1 blockade inhibits NLRP3 inflammasome activation, thereby alleviating neuroinflammatory injury by attenuating NLRP3 inflammasome-mediated pyroptosis both in vivo and in vitro.ConclusionOur study indicates that the inhibition of Dectin-1 alleviates neuroinflammation by attenuating NLRP3 inflammasome-mediated pyroptosis after ICH.

P126 Effects of Histoplasma capsulatum infection on activation and proliferation of hematopoietic stem cells

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackgroundHematopoietic stem cells (HSCs) are considered a multipotent population with high proliferative potential, and are widely used in the treatment of leukemias, multiple myeloma, and some lymphomas. In the context of infectious diseases, some microorganisms have been reported to induce changes in the expression of surface markers in HSCs by a direct effect or through the induction of cytokines. Systemic infections are characterized by inducing stress on the bone marrow, which is reflected in an increase or decrease in leukocytes and platelets in peripheral blood, a process known as ‘emergency hematopoiesis’. Histoplasmosis is a systemic mycosis caused by Histoplasma spp., which occurs mainly in immunosuppressed individuals; this mycosis can present a severe clinical picture with dissemination to various organs, including the bone marrow, and is associated with anemia and pancytopenia. So far, the effect of a possible interaction of Histoplasma with HSCs is unknown.ObjectivesTo evaluate, in vitro, the effects of Histoplasma capsulatum infection on activation and proliferation of HSCs.MethodsHSCs were obtained from bone marrow of C57BL/6 male mice; after isolation and purification, they were characterized by flow cytometry. Later, the basal expression of toll-like receptor (TLR)-2, TLR4, and Dectin-1 was determined using flow cytometry. HSCs were infected with H. capsulatum yeasts in a multiplicity of infection (MOI) of 5 and incubated for 24 h. In addition, some of the co-cultures were previously treated with specific blocking antibodies for TLR2 and TLR4 or with a blocking peptide specific for Dectin-1 (CLEC7A). Furthermore, phagocytosis, microbicidal, and cell proliferation assays were done, and the expression of the genes encoding the cytokines IL-1β, IL-6, IL-10, IL-17, TNF-α, and TGF-β as well as arginase-1 and iNOS were assessed.ResultsWe observed that H. capsulatum has the capability to adhere and internalize within these HSCs; nonetheless, this process did not affect the survival of the fungus. The interaction of H. capsulatum with HSCs induced a significantly increased expression of TLR2 and Dectin-1 but not TLR4. In addition, this fungal interaction significantly induced an augmented expression of IL-6, IL-1β, IL-10, IL-17, TNF-α, TGF-β, as well as the immune mediators Arg-1 and iNOS. Interestingly, blockade of these receptors significantly decreased the phagocytosis process as well the expression of all inflammatory mediators evaluated, especially when blocking TLR4 and Dectin-1. Of note, H. capsulatum induced apoptosis but did not inhibit the proliferation of these stem cells.ConclusionsThese results indicate that HSCs are capable of phagocytosing H. capsulatum but do not affect its survival; moreover, this fungal pathogen could induce changes in the expression of pattern-recognition receptors (PRRs), especially TLR2 and Dectin-1, and could subsequently activate the HSCs leading to the expression of inflammatory mediators as well as affecting the viability of these stem cells. Altogether, these findings indicate that H. capsulatum could affect the hematopoiesis process as reflected in an increase or decrease in leukocytes, erythrocytes, and platelets as observed in patients with severe and disseminated disease, especially in those with dissemination to bone marrow.

Beta-Glucan from S. cerevisiae Protected AOM-Induced Colon Cancer in cGAS-Deficient Mice Partly through Dectin-1-Manipulated Macrophage Cell Energy.

Although the impacts of Saccharomyces cerevisiae on cancers are mentioned, data on its use in mice with cyclic GMP-AMP synthase deficiency (cGAS-/-) are even rarer. Here, 12 weeks of oral administration of S. cerevisiae protected cGAS-/- mice from azoxymethane (AOM)-induced colon cancers, partly through dysbiosis attenuation (fecal microbiome analysis). In parallel, a daily intralesional injection of a whole glucan particle (WGP; the beta-glucan extracted from S. cerevisiae) attenuated the growth of subcutaneous tumor using MC38 (murine colon cancer cell line) in cGAS-/- mice. Interestingly, the incubation of fluorescent-stained MC38 with several subtypes of macrophages, including M1 (using Lipopolysaccharide; LPS), M2 (IL-4), and tumor-associated macrophages (TAM; using MC38 supernatant activation), could not further reduce the tumor burdens (fluorescent intensity) compared with M0 (control culture media). However, WGP enhanced tumoricidal activities (fluorescent intensity), the genes of M1 pro-inflammatory macrophage polarization (IL-1β and iNOS), and Dectin-1 expression and increased cell energy status (extracellular flux analysis) in M0, M2, and TAM. In M1, WGP could not increase tumoricidal activities, Dectin-1, and glycolysis activity, despite the upregulated IL-1β. In conclusion, S. cerevisiae inhibited the growth of colon cancers through dysbiosis attenuation and macrophage energy activation, partly through Dectin-1 stimulation. Our data support the use of S. cerevisiae for colon cancer protection.

Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity.

Candida albicans is the most frequent pathogen of fungal sepsis associated with substantial mortality in critically ill patients and those who are immunocompromised. Identification of novel immune-based therapeutic targets from a better understanding of its molecular pathogenesis is required. Here, we reported that the production of progranulin (PGRN) levels was significantly increased in mice after invasive C.albicans infection. Mice that lacked PGRN exhibited attenuated kidney injury and increased survival upon a lethal systemic infection with C. albicans. In mice, PGRN deficiency protected against systemic candidiasis by decreasing aberrant inflammatory reactions that led to renal immune cell apoptosis and kidney injury, and by enhancing antifungal capacity of macrophages and neutrophils that limited fungal burden in the kidneys. PGRN in hematopoietic cell compartment was important for this effect. Moreover, anti-PGRN antibody treatment limited renal inflammation and fungal burden and prolonged survival after invasive C. albicans infection. In vitro, PGRN loss increased phagocytosis, phagosome formation, reactive oxygen species production, neutrophil extracellular traps release, and killing activity in macrophages or neutrophils. Mechanistic studies demonstrated that PGRN loss up-regulated Dectin-2 expression, and enhanced spleen tyrosine kinase phosphorylation and extracellular signal-regulated kinase activation in macrophages and neutrophils. In summary, we identified PGRN as a critical factor that contributes to the immunopathology of invasive C.albicans infection, suggesting that targeting PGRN might serve as a novel treatment for fungal infection.

Dectin-1 as a Potential Inflammatory Biomarker for Metabolic Inflammation in Adipose Tissue of Individuals with Obesity.

In obesity, macrophage activation and infiltration in adipose tissue (AT) underlie chronic low-grade inflammation-induced insulin resistance. Although dectin-1 is primarily a pathogen recognition receptor and innate immune response modulator, its role in metabolic syndromes remains to be clarified. This study aimed to investigate the dectin-1 gene expression in subcutaneous AT in the context of obesity and associated inflammatory markers. Subcutaneous AT biopsies were collected from 59 nondiabetic (lean/overweight/obese) individuals. AT gene expression levels of dectin-1 and inflammatory markers were determined via real-time reverse transcriptase-quantitative polymerase chain reaction. Dectin-1 protein expression was assessed using immunohistochemistry. Plasma lipid profiles were measured by ELISA. AT dectin-1 transcripts and proteins were significantly elevated in obese as compared to lean individuals. AT dectin-1 transcripts correlated positively with body mass index and fat percentage (r ≥ 0.340, p ≤ 0.017). AT dectin-1 RNA levels correlated positively with clinical parameters, including plasma C-reactive protein and CCL5/RANTES, but negatively with that of adiponectin. The expression of dectin-1 transcripts was associated with that of various proinflammatory cytokines, chemokines, and their cognate receptors (r ≥ 0.300, p ≤ 0.05), but not with anti-inflammatory markers. Dectin-1 and members of the TLR signaling cascade were found to be significantly associated, suggesting an interplay between the two pathways. Dectin-1 expression was correlated with monocyte/macrophage markers, including CD16, CD68, CD86, and CD163, suggesting its monocytes/macrophage association in an adipose inflammatory microenvironment. Dectin-1 expression was independently predicted by CCR5, CCL20, TLR2, and MyD88. In conclusion, dectin-1 may be regarded as an AT biomarker of metabolic inflammation in obesity.

SOCS3 Regulates Dectin-2-Induced Inflammation in PBMCs of Diabetic Patients.

The C-type lectin receptors (CLRs) Dectin-1 and Dectin-2 are involved in several innate immune responses and are expressed mainly in dendritic cells, monocytes, and macrophages. Dectin-1 activation exacerbates obesity, inflammation, and insulin resistance/type 2 diabetes (T2D). However, the role of Dectin-2 is not clear in T2D. This study aims to evaluate the expression and function of Dectin-2 in peripheral blood mononuclear cells (PBMCs) isolated from diabetic patients and non-diabetic controls. Flow-cytometry and qRT-PCR were performed to evaluate the expression of Dectin-2 in different leukocyte subpopulations isolated from T2D patients (n = 10) and matched non-diabetic controls (n = 11). The functional activity of Dectin-2 was identified in PBMCs. CRP, IL-1β, and TNF-α concentrations were determined by ELISA. siRNA transfection and Western blotting were performed to assess p-Syk and p-NF-kB expression. siRNA transfection was performed to knock down the gene of interest. Our results show that Dectin-2 expression was the highest in monocytes compared with other leukocyte subpopulations. The expression of Dectin-2 was significantly increased in the monocytes of T2D patients compared with non-diabetic controls. Dectin-2 expression positively correlated with markers of glucose homeostasis, including HOMA-IR and HbA1c. The expression of inflammatory markers was elevated in the PBMCs of T2D patients. Interestingly, SOCS3, a negative regulator of inflammation, was expressed significantly lowlier in the PBMCs of T2D patients. Moreover, SOCS3 expression was negatively correlated with Dectin-2 expression level. The further analysis of inflammatory signaling pathways showed a persistent activation of the Dectin-2-Syk-NFkB pathway that was instigated by the diminished expression of SOCS3. Dectin-2 activation failed to induce SOCS3 expression and suppress subsequent inflammatory responses in the PBMCs of diabetic patients. siRNA-mediated knockdown of SOCS3 in PBMCs displayed a similar inflammatory phenotype to diabetic PBMCs when exposed to Dectin-2 ligands. Altogether, our findings suggest that elevated Dectin-2 and its relationship with SOCS3 could be involved in the abnormal immune response observed in T2D patients.

Human Plasmacytoid Dendritic Cells Express C-Type Lectin Receptors and Attach and Respond to Aspergillus fumigatus.

Plasmacytoid dendritic cells (pDCs) have been implicated as having a role in antifungal immunity, but mechanisms of their interaction with fungi and the resulting cellular responses are not well understood. In this study, we identify the direct and indirect biological response of human pDCs to the fungal pathogen Aspergillus fumigatus and characterize the expression and regulation of antifungal receptors on the pDC surface. Results indicate pDCs do not phagocytose Aspergillus conidia, but instead bind hyphal surfaces and undergo activation and maturation via the upregulation of costimulatory and maturation markers. Measuring the expression of C-type lectin receptors dectin-1, dectin-2, dectin-3, and mannose receptor on human pDCs revealed intermediate expression of each receptor compared with monocytes. The specific dectin-1 agonist curdlan induced pDC activation and maturation in a cell-intrinsic and cell-extrinsic manner. The indirect activation of pDCs by curdlan was much stronger than direct stimulation and was mediated through cytokine production by other PBMCs. Overall, our data indicate pDCs express various C-type lectin receptors, recognize and respond to Aspergillus hyphal Ag, and serve as immune enhancers or modulators in the overarching fungal immune response.