Stimulation Of Macrophages Research Articles

TLR2 dependent enhancement of bacterial clearance in macrophages via TNFR2

Abstract The public health crisis of antimicrobial resistance is especially challenging due to the rapid pace of bacterial adaptation to antibiotics. Thus, development of alternatives to antibiotic-based therapies are crucial. We found that stimulation of murine macrophage TLR2/6 heterodimer with Pam2CSK4 (PAM2), a synthetic bacterial lipoprotein, enhanced clearance of subsequent Staphylococcus aureus infection. Whole lung RNA sequencing identified tumor necrosis factor receptor superfamily member 1B (TNFRSF1B, TNFR2) and its cognate cytokine, TNFα, to be upregulated in response to PAM2. ELISA and qPCR data showed both bioactive forms of TNFα, transmembrane (tmTNFα) and soluble (sTNFα), to be upregulated. To mechanistically address the role that TNFR2 could play in PAM2 induced enhanced bacterial clearance (EBC) ability, antibody blocking of TNFR2 led to reduced macrophage S. aureus clearance. In contrast, sTNFα reduction by small molecule inhibition of ADAM17, the enzyme that cleaves tmTNFα to sTNFα, did not reduce macrophage EBC ability. These data suggest tmTNFα signaling via TNFR2 may have a protective effect which enhances S. aureus clearance. Our work aims to elucidate TLR signaling events through which enhancement of existing host bacterial resolution mechanisms can be achieved. As such, we are investigating whether/how tmTNFα:TNFR2 initiates intracellular signaling pathways that may contribute to upregulation of macrophage mechanisms to enhance pathogen clearance. An in-depth understanding of these signaling events could facilitate development of novel treatment strategies designed to modulate and enhance hostimmune responses against pathogens, which could be leveraged to circumvent antimicrobial resistance.

Adjuvant effects of peptidoglycan on CD4 +T cell activation by MHC-II +fibroblast-like synoviocytes

Abstract Lyme arthritis (LA) is the predominant late-stage manifestation of Lyme disease, caused by tick-borne spirochete Borrelia burgdorferi. LA can resolve with antibiotic therapy or persist after treatment in a subset of patients, known as post-antibiotic LA. Post-antibiotic LA is characterized by proliferative synovitis, dysregulated Th1/ IFNɣ responses and MHC-II+ fibroblast-like synoviocytes (FLS), a common cell type in inflamed joints. High levels of B. burgdorferi peptidoglycan (PG) have been found in LA patient synovia, yet the effects of PG on host immune responses are not well characterized. We hypothesize that PG acts as an adjuvant to enhance antigen presentation and CD4 +T cell activation in the joint. To test this, primary murine macrophages and FLS, both abundant in LA synovia, were treated in vitro with IFNɣ, PG, or both. Stimulation of macrophages led to upregulation of MHC-II in an IFNɣ-dependent manner, as expected. FLS stimulated with IFNɣ led to upregulation of MHC-II and production of proinflammatory cytokines, which was enhanced by PG as well as the muramyl-dipeptide subunit of PG, a known NOD2 ligand. To examine T cell responses, IFNɣ-primed MHC-II +FLS were co-cultured with OT-II mouse T cells (all with identical antigen-specific T cell receptors) and supplemented with the cognate OVA 323–339peptide. MHC-II +FLS activated naïve CD4 +T cell proliferation, which was enhanced by PG stimulation. Co-culture supernatants showed upregulated IFNɣ and IL-12 production, indicating a Th1 response. Negative controls using an alternative OVA 257–264peptide or NOD2-deficient FLS failed to induce T cell responses. These data show that PG acts as an immune adjuvant enhancing peptide-specific CD4 +T cell activation by MHC-II +FLS.

Induction of macrophages matriptase and activation of macrophage stimulating protein 1 in sickle cell anemia - related chronic kidney disease

BACKGROUND: More than 50% of sickle cell anemia (SCA) patients develop chronic kidney disease (CKD). The mechanism of CKD is multifactorial and is not yet fully understood. SCA is a vasculopathy associated with multiple mechanisms driven by cell-free mutated hemoglobin (HbS). We have demonstrated that activation of recepteur d'origine nantais (RON) kinase contributes to the renal endothelial injury in SCA mouse model. Inhibition of RON reduces endothelial injury and ameliorates renal disease. Macrophage-stimulating protein 1 (MSP1) is the only known ligand for RON. MSP1 is synthesized by the liver and released in the circulation as biologically inactive pro-MSP1 protein that is activated by proteolytic cleavage. Macrophage membrane matriptase (MT-SP1) cleaves MSP1, making it able to bind RON receptor. The levels of plasma MSP1 and macrophage MT-SP1 in SCA have not been investigated. OBJECTIVES: To evaluate MT-SP1 expression and serum MSP1 levels in SCA patients and mouse model. HYPOTHESIS: The level of circulating MSP1 is reduced in SCA patients and mice due to its accumulation in organs. METHODS: The study was approved by Howard University IRB.All subjects provided written informed consent. Whole blood samples were obtained from 44 SCA patients enrolled at HU CSCD Registry study and 6 healthy control individuals. SCA mice (Townes) were housed in Howard University; the animal protocol was approved by the IACUC. ELISA kits were used to test human and mouse MSP1. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 μM). Total RNA was used for RNA Seq. Comparisons were conducted using Dragen differential expression software v. 3.6.3 (Illumina), and gene counts were normalized using DESeq2 package from Bioconductor. RESULTS: RNA Seq results demonstrated increased levels of MT-SP1 in THP-1-derived macrophages after treatment with HbS compared to treatment with normal hemoglobin (HbA) (p=0.036). The results were further confirmed by real-time RT-PCR. The levels of plasma MSP1 were about 7-fold lower in SCD patients compared to non-SCD controls (146.8±111.7 ng/ml, N=6 in control vs. 20.6 ng/ml, N=44 in SCD, p=3.97x10-10). Log2 (MSP1) slightly correlated with albuminuria log2 (ALB/CRE) (r=0.1243, R2=0.0154, p=0.4216, N=44). This correlation was more profound in females (r=0.3158, R2=0.0997, p=0.1632, N=21). No correlation was found in males. We found moderate inverted correlation between eGFR and log2 (MSP1) (r=-0.3313, R2=0.1094, p=0.0280, N=44). In SCA patients with renal disease, MSP1 levels positively correlated with stages of CKD (r=0.3990, R2=0.1592, p=0.0320, N=29). In mouse, serum MSP1 levels were about 2.3-fold reduced in SCA compared to control mice (441.0±97.98 ng/ml, N=5 in control vs. 192.2 ±27.02, N=7 in SCD, p=0.0173). Immunostaining demonstrated the high levels of MSP1 accumulation on the surface of endothelial cells in the ki This work was supported by NIH Research Grants SC1HL150685, P50HL118006, R01HL125005, and G12MD007597. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macrophage to Myofibroblast Transition in Diastolic Dysfunction Development

Heart failure is the top killer in the U.S and worldwide, often caused by hypertension, aging, or cardiac toxicity. Diastolic Dysfunction (DD), the often the first step of heart failure, occurs when the chambers of the heart are not able to expand during the filling phase. In addition, hypertension is the leading cause of DD. Though this is known, the progression of hypertension into DD remains poorly understood. Of note is the ability of M2 macrophages to differentiate into a myofibroblast-like phenotype, known as macrophage to myofibroblast transition (MMT). This transition has been observed in interstitial fibrosis in chronic renal allograft injury. The researchers noted that MMT account for 50% of the myofibroblast population. To our knowledge, the extent of fibrotic remodeling due to MMT in the heart has not been ardently investigated. Therefore, we aim to answer whether MMT plays a causal role in the progression of hypertension to DD and the pathogenesis of heart failure.Utilizing the uninephrectomized, DOCA-salt model, mice were subjected to this treatment and hypertension was induced. As a negative control, another group of mice received a uninephrectomy and the same salt in their drinking water, however, DOCA was not administered (Sham group). MMT prevalence, identified as CD68+ α-SMA+ expressions, was observed inside the hearts of DOCA and Sham mice. The hearts were isolated and homogenized via gentleMACS tissue Dissociator, the monocytes were purified via Percoll isolation, and flow cytometry was used to identify the population of CD68+ α-SMA+ expressing cells.As soon as 10 days post DOCA implantation, the DOCA group exhibited worsening diastolic function as it took longer for the pressure to change in the hearts of DOCA mice than in Sham (-dp/dt). These data were gathered via left ventricular (LV) catheter insertion. At a later time, utilizing a different cohort of mice preliminary data show an increase of monocyte (CD68+) and fibroblast (α-SMA+) double-positive expressing cells in the hearts of hypertensive mice.MMT has been observed in the hearts of hypertensive mice. This transition may exacerbate Diastolic Dysfunction. More research must be performed to investigate the wide variety of functions of macrophages (i.e., M1 to M2 ratio and transition, macrophage residence or infiltration, and macrophage stimulation of myofibroblasts), as well as the specific interactions between macrophages and cardiomyocytes, or other immune cells, that might mediate this transition. These data provide a preliminary, but interesting glimpse, into the role of macrophages in exacerbating Diastolic Dysfunction and mediating Heart Failure. NIH RO1-HL146713 and AHA 15BGIA25730047 (Shengyu Mu) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Self-assembling paclitaxel-mediated stimulation of tumor-associated macrophages for postoperative treatment of glioblastoma

The unique cancer-associated immunosuppression in brain, combined with a paucity of infiltrating T cells, contributes to the low response rate and poor treatment outcomes of T cell-based immunotherapy for patients diagnosed with glioblastoma multiforme (GBM). Here, we report on a self-assembling paclitaxel (PTX) filament (PF) hydrogel that stimulates macrophage-mediated immune response for local treatment of recurrent glioblastoma. Our results suggest that aqueous PF solutions containing aCD47 can be directly deposited into the tumor resection cavity, enabling seamless hydrogel filling of the cavity and long-term release of both therapeutics. The PTX PFs elicit an immune-stimulating tumor microenvironment (TME) and thus sensitizes tumor to the aCD47-mediated blockade of the antiphagocytic "don't eat me" signal, which subsequently promotes tumor cell phagocytosis by macrophages and also triggers an antitumor T cell response. As adjuvant therapy after surgery, this aCD47/PF supramolecular hydrogel effectively suppresses primary brain tumor recurrence and prolongs overall survivals with minimal off-target side effects.

Enhancing anti-tumor immunity through liposomal oxaliplatin and localized immunotherapy via STING activation

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence suggests that chemotherapy-induced DNA damage can directly induce dendritic cell (DC) maturation and recruitment, which synergizes with STING activation to enhance anti-tumor effects. As an immunogenic cell death (ICD) inducer, oxaliplatin generates massive double-stranded DNA (dsDNA) crosslinks, release of tumor-associated antigens and promoting the “eat me” signal. STING activation improves antigen immunogenicity, which can promote T cell activation and infiltration. In this study, we developed liposomes encapsulating oxaliplatin and combine this formulation with a STING agonist (ADU-S100) for treating colorectal cancer. The liposomes efficiently inhibited the proliferation of tumor cells while induced ICD in CT26 colorectal cancer cells, which enhanced dendritic cell maturation and phagocytosis in vitro. The liposome-based immunochemotherapy exhibited the strongest efficacy, resulting in complete remission upon tumor inoculation. Mechanistic studies showed this potent anti-cancer effect was related to the significant recruitment of infiltrating CD8 and CD4 T cells, reduction of suppressive Treg cells, and a shift in the phenotype of tumor-associated suppressive macrophages that promote cancer to immune stimulating macrophages. Thus, our study demonstrated the potential of combining oxaliplatin-loaded liposomes with a STING agonist to reduce tumor growth by regulating the immunosuppressive state in the tumor.

Antioxidant, Immunostimulatory, and Anticancer Properties of Hydrolyzed Wheat Bran Mediated through Macrophages Stimulation.

Previous studies demonstrated that enzymatic hydrolysis enhances wheat bran (WB) biological properties. This study evaluated the immunostimulatory effect of a WB hydrolysate (HYD) and a mousse enriched with HYD (MH) before and after in vitro digestion on murine and human macrophages. The antiproliferative activity of the harvested macrophage supernatant on colorectal cancer (CRC) cells was also analyzed. MH showed significantly higher content than control mousse (M) in soluble poly- and oligosaccharides (OLSC), as well as total soluble phenolic compounds (TSPC). Although in vitro gastrointestinal digestion slightly reduced the TSPC bioaccessibility of MH, ferulic acid (FA) levels remained stable. HYD showed the highest antioxidant activity followed by MH, which demonstrated a greater antioxidant activity before and after digestion as compared with M. RAW264.7 and THP-1 cells released the highest amounts of pro-inflammatory cytokines after being treated with 0.5 mg/mL of digested WB samples. Treatment with digested HYD-stimulated RAW264.7 supernatant for 96 h showed the most anticancer effect, and spent medium reduced cancer cell colonies more than direct WB sample treatments. Although a lack of inner mitochondrial membrane potential alteration was found, increased Bax:Bcl-2 ratio and caspase-3 expression suggested activation of the mitochondrial apoptotic pathway when CRC cells were treated with macrophage supernatants. Intracellular reactive oxygen species (ROS) were positively correlated with the cell viability in CRC cells exposed to RAW264.7 supernatants (r = 0.78, p < 0.05) but was not correlated in CRC cells treated with THP-1 conditioned media. Supernatant from WB-stimulated THP-1 cells may be able to stimulate ROS production in HT-29 cells, leading to a decrease of viable cells in a time-dependent manner. Therefore, our present study revealed a novel anti-tumour mechanism of HYD through the stimulation of cytokine production in macrophages and the indirect inhibition of cell proliferation, colony formation, and activation of pro-apoptotic proteins expression in CRC cells.

Rapid Profiling of Protein Complex Reorganization in Perturbed Systems.

Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition, or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information. Methods based on cofractionation paired with mass spectrometry have demonstrated the capability for deep biological insight, but the scope of studies using this approach has been limited by the large measurement time per biological sample and challenges with data analysis. There has been little uptake of this strategy into the broader life science community despite its rich biological information content. We present a rapid integrated experimental and computational workflow to assess the reorganization of protein complexes across multiple cellular states. The workflow combines short gradient chromatography and DIA/SWATH mass spectrometry with a data analysis toolset to quantify changes in a complex organization. We applied the workflow to study the global protein complex rearrangements of THP-1 cells undergoing monocyte to macrophage differentiation and subsequent stimulation of macrophage cells with lipopolysaccharide. We observed substantial proteome reorganization on differentiation and less pronounced changes in macrophage stimulation. We establish our integrated differential pipeline for rapid and state-specific profiling of protein complex organization.

Timing of Interleukin-4 Stimulation of Macrophages Determines Their Anti-Microbial Activity during Infection with Salmonella enterica Serovar Typhimurium.

Priming of macrophages with interferon-gamma (IFNγ) or interleukin-4 (IL-4) leads to polarisation into pro-inflammatory or anti-inflammatory subtypes, which produce key enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), respectively, and in this way determine host responses to infection. Importantly, L-arginine is the substrate for both enzymes. ARG1 upregulation is associated with increased pathogen load in different infection models. However, while differentiation of macrophages with IL-4 impairs host resistance to the intracellular bacterium Salmonella enterica serovar Typhimurium (S.tm), little is known on the effects of IL-4 on unpolarised macrophages during infection. Therefore, bone-marrow-derived macrophages (BMDM) from C57BL/6N, Tie2Cre+/-ARG1fl/fl (KO), Tie2Cre-/-ARG1fl/fl (WT) mice were infected with S.tm in the undifferentiated state and then stimulated with IL-4 or IFNγ. In addition, BMDM of C57BL/6N mice were first polarised upon stimulation with IL-4 or IFNγ and then infected with S.tm. Interestingly, in contrast to polarisation of BMDM with IL-4 prior to infection, treatment of non-polarised S.tm-infected BMDM with IL-4 resulted in improved infection control whereas stimulation with IFNγ led to an increase in intracellular bacterial numbers compared to unstimulated controls. This effect of IL-4 was paralleled by decreased ARG1 levels and increased iNOS expression. Furthermore, the L-arginine pathway metabolites ornithine and polyamines were enriched in unpolarised cells infected with S.tm and stimulated with IL-4. Depletion of L-arginine reversed the protective effect of IL-4 toward infection control. Our data show that stimulation of S.tm-infected macrophages with IL-4 reduced bacterial multiplication via metabolic re-programming of L-arginine-dependent pathways.

Abstract 377: RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy

Abstract Background: Although the epidermal growth factor receptor (EGFR) is overexpressed in 80-90% of Head and Neck Squamous Cell Carcinomas (HNSCCs), response to anti-EGFR targeted therapy remains low. Trans-activation of other receptor tyrosine kinases is a common mechanism of resistance to EGFR directed therapy. The RON receptor tyrosine kinase (Macrophage stimulating 1-receptor/MST1R) is a member of the c-MET oncogene family. While limited, prior studies by our group and others have reported elevated RON expression in over 60% of primary HNSCC samples and that receptor cross talk exists with RON and EGFR. Based on this information, we hypothesize that the pro-tumorigeneic role of RON and its crosstalk with EGFR contributes to HNSCC growth and to anti-EGFR therapeutic failure. To test this hypothesis, we examined the expression level of RON and EGFR in human and mouse HNSCC cell lines and in HNSCC patient samples from publicly available databases. Further, we investigated the role of RON in radiation resistance as radiation is a primary treatment modality for HNSCC. Results: Immunoblotting demonstrated strong RON expression in eight of nine human HNSCC cell lines tested and strong EGFR expression in six HNSCC cell lines. In exploring mRNA expression of RON using the Kaplan-Meier Plotter HNSCC RNA-seq dataset, high RON expression was associated with poorer overall survival (P = 0.054) in patients with Stage 3 HNSCC and low mutation burden. To assess the relationship between RON and EGFR, mRNA expression was mined in the GSE6631 dataset from the NCBI gene expression omnibus (GEO). Our analysis found RON to be significantly overexpressed in HNSCC samples compared to normal matched controls (P = 0.029). Interestingly, EGFR mRNA expression was negatively correlated (r = -0.2) with RON mRNA expression from the same patients. This inverse correlation between RON and EGFR expression was corroborated by immunoblotting data of HNSCC lines, UM-SCC-090 and 93-UV-147T which demonstrated low levels of EGFR and high levels of RON. As ionizing radiation is a pillar of HNSCC treatment and is often delivered concurrent with anti-EGFR therapy, we next examined the relative radiosensitivity in multiple murine and human cancer cell lines with and without RON expression using colony formation assays. Preliminary studies demonstrated significantly increased colony formation in high RON-expressing cells relative to low RON-expressing cells. Conclusion: RON is expressed at high levels in multiple HNSCC cell lines and in human samples. Protein expression data and in silica analyses support an inverse association between EGFR and RON. RON expression is associated with radiation resistance. Future studies will investigate interactions between RON and EGFR, and whether RON inhibition can increase anti-EGFR treatment and radiation sensitivity in HNSCC cells. Citation Format: Zhixin Lu, Vinita Takiar, Susan E. Waltz. RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy [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 377.

ATG2B upregulated in LPS-stimulated BMSCs-derived exosomes attenuates septic liver injury by inhibiting macrophage STING signaling.

Pretreated mesenchymal stem cells (MSCs)-derived exosomes have shown great potential in the treatment of various inflammatory diseases. Recent evidence suggests that macrophage stimulator of interferon genes (STING) signal activation plays a critical role in sepsis and septic liver injury. Here, we aimed to investigate the role and effects of lipopolysaccharide (LPS)-pretreated bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (L-Exo) on macrophage STING signaling in septic liver injury. Exosomes were collected from the BMSCs medium via ultracentrifugation. Liver injury, intrahepatic inflammation, and the activation of macrophage STING signaling were analyzed. Mitophagy and the release of mitochondrial DNA (mtDNA) into the cytosol were investigated. Through in vivo and in vitro experiments, L-Exo could markedly attenuate cecal ligation and puncture-induced septic liver injury and inhibit macrophage STING signaling. Mechanistically, L-Exo inhibited macrophage STING signaling by enhancing mitophagy and inhibiting the release of mtDNA into the cytosol. Furthermore, autophagy-related protein 2 homolog B (ATG2B) may be a major factor involved in this effect of L-Exo. These findings reveal that macrophage STING signaling plays an important role in septic liver injury and may be a therapeutic target. In addition, LPS pretreatment is an effective and promising approach for optimizing the therapeutic efficacy of MSCs-derived exosomes in septic liver injury, providing new strategies for treatment.

NLRP3 Plays a Key Role in Antihelminth Immunity in the Enteral and Parenteral Stages of Trichinella spiralis-Infected Mice.

Trichinellosis is an important foodborne zoonosis, and no effective treatments are yet available. Nod-like receptor (NLR) plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis infection. The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, the role that NLRP3 plays during T. spiralis infection of mice was evaluated using enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, histopathological evaluation, bone marrow-derived macrophage (BMDM) stimulation, and immunofluorescence. The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult and migrating stages and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (interferon gamma [IFN-γ]) and suppressed the release of anti-inflammatory factors (interleukin 4 [IL-4]). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1β and IL-18 release. This study revealed that NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis.

Immune Response Evaluation in Commercial Turkeys Affected with Clostridial Dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum and Clostridium perfringens, is an economically important emerging disease of turkeys characterized by sudden deaths and necrotic dermatitis. Immune responses in CD-affected commercial turkeys are poorly understood. In the present study, C. septicum was isolated from CD-affected commercial turkeys during a recent outbreak, and the tissues (skin, muscle, and spleen) were collected and analyzed for immune gene expression, along with samples from clinically healthy birds. The results showed that CD-affected turkeys had significantly higher levels of IL-1β, IL-6, IFNγ, and iNOS transcripts in the skin, muscle, and spleen tissues compared to healthy birds. Affected turkeys also had a significantly elevated transcription of toll-like receptor (TLR21) gene in the skin and spleen tissues, suggesting a role for this receptor in the immune recognition. The expression of IL-4 and IL-13 genes in the spleen and muscle was also significantly higher in the affected birds. Additional birds from the same affected and healthy farms examined for serology revealed that the CD-affected turkeys had significantly higher levels of serum IgM and IgY antibodies. Furthermore, in vitro stimulation of MQ-NCSU macrophages with C. septicum led to a significant transcriptional upregulation of IL-1β and IFNγ genes, while the IL-10 gene expression was downregulated. The surface expression of MHC-II protein and cellular production of nitric oxide were also significantly increased in the C. septicum-stimulated macrophages, indicating cellular activation. Collectively, our findings suggest that the host responses in CD-affected turkeys involve a robust inflammatory response as well as a response mediated by IL4/IL-13 cytokines that may aid in antibody-mediated immunity.

The Effects of Ginkgo biloba Extract on Autophagy in Human Macrophages Stimulated by Cigarette Smoke Extract.

To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Therapeutic targets for inflammatory bowel disease: proteome-wide Mendelian randomization and colocalization analyses.

Identifying new drug targets for inflammatory bowel disease (IBD) is urgently needed. The proteome is a major source of therapeutic targets. We conducted a proteome-wide Mendelian randomization (MR) and colocalization analyses to identify possible targets for IBD. Summary-level data of 4907 circulating protein levels were extracted from a large-scale protein quantitative trait loci study including 35,559 individuals. Genetic associations with IBD and its subtypes were obtained from the Inflammatory Bowel Disease Genetics Consortium (25,024 cases and 34,915 controls), the FinnGen study (7206 cases and 253,199 controls), and the UK Biobank study (7045 cases and 449,282 controls). MR analysis was conducted to estimate the associations between protein and IBD risk. The colocalization analysis was used to examine whether the identified proteins and IBD shared casual variants. Genetically predicted levels of 3, and 5 circulating proteins were associated with IBD and ulcerative colitis (UC), respectively. With high supporting evidence of colocalization, genetically predicted MST1 (macrophage stimulating 1) and HGFAC (hepatocyte growth factor activator) levels were inversely associated with IBD risks. The associations of STAT3 (signal transducer and activator of transcription 3), MST1, CXCL5 (C-X-C motif chemokine ligand 5), and ITPKA (inositol-trisphosphate 3-kinase A) with the risk of UC were supported by colocalization analysis. The proteome-wide MR investigation identified many proteins associated with the risk of IBD. MST1, HGFAC, STAT3, ITPKA, and CXCL5 deserve further investigation as potential therapeutic targets for IBD. SCL is supported by research grants from the Swedish Research Council for Health, Working Life and Welfare (Forte; grant no. 2018-00123) and the Swedish Research Council (Vetenskapsrådet; grant no. 2019-00977). XYW is supported by research grants from the National Natural Science Foundation of China (81970494) and Key Project of Research and Development Plan of Hunan Province (2019SK2041). XL is supported by research grants from the Natural Science Fund for Distinguished Young Scholars of Zhejiang Province (LR22H260001).

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.

Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions

Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, the effect of chalcones 1-18 against the metabolic viability of cervical (HeLa) and prostate (PC-3 and LNCaP) tumor cell lines was tested, to compare the activity against solid and liquid tumor cells. Their effect was also evaluated on the Jurkat cell line. Chalcone 16 showed the highest inhibitory effect on the metabolic viability of the tested tumor cells and was selected for further studies. Recent antitumor therapies include compounds with the ability to influence immune cells on the tumor microenvironment, with immunotherapy being one actual goal in cancer treatment. Therefore, the effect of chalcone 16 on the expression of mTOR, HIF-1α, IL-1β, TNF-α, IL-10, and TGF-β, after THP-1 macrophage stimulation (none, LPS or IL-4), was evaluated. Chalcone 16 significantly increased the expression of mTORC1, IL-1β, TNF-α, and IL-10 of IL-4 stimulated macrophages (that induces an M2 phenotype). HIF-1α and TGF-β were not significantly affected. Chalcone 16 also decreased nitric oxide production by the RAW 264.7 murine macrophage cell line, this effect probably being due to an inhibition of iNOS expression. These results suggest that chalcone 16 may influence macrophage polarization, inducing the pro-tumoral M2 macrophages (IL-4 stimulated) to adopt a profile closer to the antitumor M1 profile.

MLKL deficiency attenuated hepatocyte oxidative DNA damage by activating mitophagy to suppress macrophage cGAS-STING signaling during liver ischemia and reperfusion injury

Mixed-lineage kinase domain-like protein (MLKL)-mediated necroptosis has been implicated in aggravating liver ischemia and reperfusion (IR) injury. However, the precise role and mechanism of MLKL in regulating oxidative DNA damage of hepatocytes and subsequent activation of macrophage stimulator of interferon genes (STING) signaling remains unclear. In this study, we investigated the role of MLKL in regulating the interplay between hepatocyte injury and macrophage pro-inflammatory responses during liver IR injury. We found that IR increased MLKL expression in liver tissues of wild type (WT) mice. MLKL knockout (KO) attenuated liver IR injury and suppressed the activation of cGAS-STING signaling in intrahepatic macrophages, which was abrogated by STING activation with its agonist. Mechanistically, IR induced oxidative DNA damage in hepatocytes, leading to cGAS-STING activation in macrophages, which was suppressed by MLKL KO. Moreover, increased PTEN-induced kinase 1 (PINK1)-mediated mitophagy contributed to reduced oxidative DNA damage in hepatocytes and subsequent decreased activation of STING signaling in macrophages in MLKL KO mice. Our findings demonstrated a non-canonical role of MLKL in the pathogenesis of liver IR. MLKL deficiency significantly promoted PINK1-mediated mitophagy activation to inhibit oxidative DNA damage in hepatocytes, which in turn suppressed macrophage cGAS-STING activation and inflammatory liver IR injury.

Disturbance of suprachiasmatic nucleus function improves cardiac repair after myocardial infarction by IGF2-mediated macrophage transition.

Suprachiasmatic nucleus (SCN) in mammals functions as the master circadian pacemaker that coordinates temporal organization of physiological processes with the environmental light/dark cycles. But the causative links between SCN and cardiovascular diseases, specifically the reparative responses after myocardial infarction (MI), remain largely unknown. In this study we disrupted mouse SCN function to investigate the role of SCN in cardiac dysfunction post-MI. Bilateral ablation of the SCN (SCNx) was generated in mice by electrical lesion; myocardial infarction was induced via ligation of the mid-left anterior descending artery (LAD); cardiac function was assessed using echocardiography. We showed that SCN ablation significantly alleviated MI-induced cardiac dysfunction and cardiac fibrosis, and promoted angiogenesis. RNA sequencing revealed differentially expressed genes in the heart of SCNx mice from D0 to D3 post-MI, which were functionally associated with the inflammatory response and cytokine-cytokine receptor interaction. Notably, the expression levels of insulin-like growth factor 2 (Igf2) in the heart and serum IGF2 concentration were significantly elevated in SCNx mice on D3 post-MI. Stimulation of murine peritoneal macrophages in vitro with serum isolated from SCNx mice on D3 post-MI accelerated the transition of anti-inflammatory macrophages, while antibody-mediated neutralization of IGF2 receptor blocked the macrophage transition toward the anti-inflammatory phenotype in vitro as well as the corresponding cardioprotective effects observed in SCNx mice post-MI. In addition, disruption of mouse SCN function by exposure to a desynchronizing condition (constant light) caused similar protective effects accompanied by elevated IGF2 expression on D3 post-MI. Finally, mice deficient in the circadian core clock genes (Ckm-cre; Bmal1f/f mice or Per1/2 double knockout) did not lead to increased serum IGF2 concentration and showed no protective roles inpost-MI, suggesting that the cardioprotective effect observed in this study was mediated particularly by the SCN itself, but not by self-sustained molecular clock. Together, we demonstrate that inhibition of SCN function promotes Igf2 expression, which leads to macrophage transition and improves cardiac repair post-MI.