Neuronal GPCR NMUR-1 regulates energy homeostasis in response to pathogen infection.

A key question in current immunology is how the innate immune system generates high levels of specificity. Our previous study in Caenorhabditis elegans revealed that NMUR-1, a neuronal G protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U (NMU), regulates distinct innate immune responses to different bacterial pathogens. Here, by using quantitative proteomics and functional assays, we discovered that NMUR-1 regulates F1FO ATP synthase and ATP production in response to pathogen infection, and that such regulation contributes to NMUR-1-mediated specificity of innate immunity. We further demonstrated that ATP biosynthesis and its contribution to defense is neurally controlled by the NMUR-1 ligand CAPA-1 and its expressing neurons ASG. These findings indicate that NMUR-1 neural signaling regulates the specificity of innate immunity by controlling energy homeostasis as part of defense against pathogens. Our study provides mechanistic insights into the emerging roles of NMU signaling in immunity across animal phyla.

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

BACKGROUND: Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. METHODS: In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. RESULTS: The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. CONCLUSIONS: The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

Molecular insights into STAT1a protein in rohu (Labeo rohita): unveiling expression profiles, SRC homology domain recognition, and protein-protein interactions triggered by poly I: C.

Introduction: STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a). Methods: The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations. Results: The full-length CDS of Labeo rohita STAT1a (LrSTAT1a) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a. Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106. Discussion: This study provides molecular insights into the STAT1a protein in Rohu (Labeo rohita) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC.

Infection and the Glycome─New Insights into Host Response.

Glycans play critical roles in the host-pathogen interactions leading to infection. However, we still understand very little about the dynamic nature of glycosylation in response to infection and its function in modulating host immunity. Many of the host proteins involved in immune defense are glycoproteins. Furthermore, the innate immune system recognizes glycans. The glycoform of a protein can impact proteolytic stability, receptor interactions, serum half-life, and other aspects. New, cutting-edge chemical biology tools are shedding light on the interplay between infection and the host glycome. In this review, we highlight new work on the importance of dynamic glycosylation of host proteins in the innate and adaptive immune pathways in response to infection. These include recent findings on altered glycoprofiles of mucins, complement components, and antibodies.

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

Diminazene aceturate inhibits the SARS-CoV-2 spike protein-induced inflammation involving leukocyte migration and DNA extracellular traps formation.

AIMS: To investigate the SARS-CoV-2 Spike protein (Spk)-induced inflammatory response and its downmodulation by diminazene aceturate (DIZE). MATERIALS AND METHODS: Through inducing Spk inflammation in murine models, leukocyte migration to the peritoneum, levels of myeloperoxidase (MPO), malondialdehyde (MDA), rolling and adhesion of mesenteric leukocytes, and vascular permeability were investigated. Extracellular DNA traps (DETs) induced by Spk and the production of IL-6 and TNF-α were analyzed using human neutrophils, monocytes, and macrophages. In silico assays assessed the molecular interaction between DIZE and molecules related to leukocyte migration and DETs induction. KEY FINDINGS: Spk triggered acute inflammation, demonstrated by increasing leukocyte migration. Oxidative stress was evidenced by elevated levels of MPO and MDA in the peritoneal liquid. DIZE attenuated cell migration, rolling, and leukocyte adhesion, improved vascular barrier function, mitigated DETs, and reduced the production of Spk-induced pro-inflammatory cytokines. Computational studies supported our findings, showing the molecular interaction of DIZE with targets such as ß2 integrin, PI3K, and PAD2 due to its intermolecular coupling. SIGNIFICANCE: Our results outline a novel role of DIZE as a potential therapeutic agent for mitigating Spk-induced inflammation.

Weep and sweep and the broom: tuft cell acetylcholine limits the worm.

Two studies defined how tuft cell acetylcholine promotes parasite expulsion. Billip et al. demonstrated that acetylcholine increases water secretion, to promote the 'weep' response. Ndjim et al. found that tuft cell acetylcholine has a direct effect on worm fecundity. Both processes are only effective in the remodeled epithelium when the rare tuft cells have become abundant.

Activation of autoreactive lymphocytes in the lung by STING gain-of-function mutation expressing radioresistant cells.

Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation STING V154M (VM) similarly develop ILD, but exhibit severe T and B cell lymphopenia, low serum Ig titers, and lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with wildtype (WT) stem cells (WT→VM) still develop ILD. In this study, we find that WT→VM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in SAVI patients. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+Tg→VM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WTVM chimeras were adoptively transferred to naïve Rag1-deficient 2º hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.

Longitudinal multiomic profiling and corticosteroid modulation of the immediate innate immune response to an adenovirus-vector vaccine.

Among new vaccine technologies contributed to the control of the COVID-19 pandemic, ChAdOx1 nCoV-19, a chimpanzee adenovirus (ChAd)-vector vaccine expressing the SARS-CoV-2 spike protein, could be administered globally owing to its low production cost and lack of a requirement for frozen storage. Despite its benefits, most recipients have reported immediate inflammatory reactions after the initial dose vaccination. We comprehensively examined the immune landscape following ChAdOx1 nCoV-19 vaccination based on the single-cell transcriptomes of immune cells and epigenomic profiles of monocytes. Monocyte and innate-like activated T cell populations expressing interferon-stimulated genes (ISGs) increased 1 day post-vaccination with appearance of distinct subtype of ISG-activated cells, returning to baseline by day 14. Pre-treatment with oral corticosteroids effectively curtailed these ISG-associated inflammatory responses by decreasing chromatin accessibility of major ISGs, without hampering vaccine immunogenicity. Our findings provide insights into the human immune response following ChAd-based vaccination and propose a method to reduce inflammatory side effects.

CLADIN- CLADribine and INnate immune response in multiple sclerosis - A phase IV prospective study.

Cladribine (Mavenclad®) is an oral treatment for relapsing remitting MS (RRMS), but its mechanism of action and its effects on innate immune responses in unknown. This study is a prospective Phase IV study of 41 patients with RRMS, and aims to investigate the mechanism of action of cladribine on peripheral monocytes, and its impact on the P2X7 receptor. There was a significant reduction in monocyte count in vivo at week 1 post cladribine administration, and the subset of cells being most impacted were the CD14lo CD16+ 'non-classical' monocytes. Of the 14 cytokines measured in serum, CCL2 levels increased at week 1. In vitro, cladrabine induced a reduction in P2X7R pore as well as channel activity. This study demonstrates a novel mechanism of action for cladribine. It calls for studying potential benefits of cladribine in progressive forms of MS and other neurodegenerative diseases where innate immune related inflammation is implicated in disease pathogenesis.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge.

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Histone deacetylase 8 promotes innate antiviral immunity through deacetylation of RIG-I.

Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.

Effect on neutrophil migration and antimicrobial functions by the Bruton's tyrosine kinase inhibitors tolebrutinib, evobrutinib and fenebrutinib.

Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease that is still incurable. Nowadays, a variety of new drugs are being developed to prevent excessive inflammation and halt neurodegeneration. Among these are the inhibitors of Bruton's tyrosine kinase (BTK). Being indispensable for B cells, this enzyme became an appealing therapeutic target for autoimmune diseases. Recognizing the emerging importance of BTK in myeloid cells, we investigated the impact of upcoming BTK inhibitors on neutrophil functions. Although adaptive immunity in MS has been thoroughly studied, unanswered questions about the pathogenesis can be addressed by studying the effects of candidate MS drugs on innate immune cells such as neutrophils, previously overlooked in MS. In this study, we used three BTK inhibitors (evobrutinib, fenebrutinib and tolebrutinib), and found that they reduce neutrophil activation by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine and the chemokine interleukin 8/CXCL8. Furthermore, they diminished the production of reactive oxygen species and release of neutrophil extracellular traps. Additionally, the production of CXCL8 and interleukin-1ß in response to inflammatory stimuli was decreased. Inhibitory effects of the drugs on neutrophil activation were not related to toxicity. Instead, BTK inhibitors prolonged neutrophil survival in an inflammatory environment. Finally, treatment with BTK inhibitors decreased neutrophil migration towards CXCL8 in a Boyden chamber assay but not in a trans endothelial set-up. Also, in vivo CXCL1-induced migration was unaffected by BTK inhibitors. Collectively, this study provides novel insights into the impact of BTK inhibitors on neutrophil functions, thereby holding important implications for autoimmune or hematological diseases where BTK is crucial.

Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis.

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon ß and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

Novel ApeC-containing protein mediates the recognition and internalization of Vibrio splendidus in Apostichopus japonicus.

Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery.

Background: Gingipains are important virulence factors present in Porphyromonas gingivalis. Arginine-specific gingipains (RgpA and RgpB) are critically associated with increased proteolytic activity and immune system dysfunction, including neutrophilic activity. In this study, we assessed the impact of gingipains (RgpA and RgpB) on neutrophil function. Methods: Peripheral blood samples were obtained; neutrophils were isolated and incubated with P. gingivalis A7436, W50, and the double RgpA/RgpB double knockout mutant E8 at MOI 20 for 2 hours. Neutrophil viability was assessed by Sytox staining. Phagocytic capacity and apoptosis were measured by flow cytometry. Superoxide release was measured by superoxide dismutase and cytochrome c reduction assay. Gene expression of TLR2, p47-phox, p67-phox, and P2 × 7was measured by qPCR. Inflammatory cytokine and chemokine production was measured by IL-1ß, IL-8, RANTES, and TNF-α in cell supernatants. Results: Neutrophil TLR2 gene expression was reduced in the absence of RgpA/RgpB (p < 0.05), while superoxide production was not significantly impacted. RgpA/RgpB-/- significantly impaired neutrophil phagocytic function (p < 0.05) and increased TNF-α production when compared with the wild-type control (p < 0.05). Neutrophil apoptosis was not altered when exposed to RgpA/RgpB-/- E8 (p > 0.05). Conclusion: These data suggest that arginine-specific gingipains (RgpA/RgpB) can modulate neutrophil responses against P. gingivalis infection.

P. gingivalis-derived arginine-specific gingipains impaired the phagocytic and apoptotic function in neutrophils.

Microbiome-derived bacterial lipids regulate gene expression of proinflammatory pathway inhibitors in systemic monocytes.

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study, our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides-the serine-glycine (S/G) lipids, which are TLR2 ligands, access the systemic circulation, and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity, we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses, decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion, our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2, S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells, such as microglia, in diseases such as Alzheimer's disease.

In Fanconi Anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress.

Fanconi Anemia (FA) is an inherited disorder of DNA-repair due to mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF. Since termination of emergency granulopoiesis is triggered by accumulation of bone marrow neutrophils, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of a sustained inflammation that is experienced by Fancc-/- mice. In the current work, we found that blocking neutrophil accumulation during emergency granulopoiesis led to BMF in Fancc-/-Tp53+/- mice, consistent with this hypothesis. Blocking neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice (but not WT) impaired cell cycle checkpoint activity, also found in Fancc-/- mice. Mechanisms for loss of cell cycle checkpoints during infections challenges may define molecular markers of FA progression, or suggest therapeutic targets for bone marrow protection in this disorder.

Inhibiting SIRT-2 by AK-7 restrains airway inflammation and oxidative damage promoting lung resurgence through NF-kB and MAP kinase signaling pathway.

Introduction: Chronic obstructive pulmonary disease (COPD) is a major global cause of mortality with limited effective treatments. Sirtuins (SIRT) are histone deacetylases that are involved in the regulation of redox and inflammatory homeostasis. Hence, the present study aims to investigate the role of SIRT-2 in modulating inflammation in a murine model of COPD. Methods: COPD in mice was established by cigarette smoke (CS) exposure for 60 days, and AK-7 was used as the specific SIRT-2 inhibitor. AK-7 (100 µg/kg and 200 µg/kg body weight) was administered intranasally 1 h before CS exposure. Molecular docking was performed to analyze the binding affinity of different inflammatory proteins with AK-7. Results: Immune cell analysis showed a significantly increased number of macrophages (F4/80), neutrophils (Gr-1), and lymphocytes (CD4+, CD8+, and CD19+) in the COPD, group and their population was declined by AK-7 administration. Total reactive oxygen species, total inducible nitric oxide synthase, inflammatory mediators such as neutrophil elastase, C-reactive protein, histamine, and cytokines as IL4, IL-6, IL-17, and TNF-α were elevated in COPD and declined in the AK-7 group. However, IL-10 showed reverse results representing anti-inflammatory potency. AK-7 administration by inhibiting SIRT-2 decreased the expression of p-NF-κB, p-P38, p-Erk, and p-JNK and increased the expression of Nrf-2. Furthermore, AK-7 also declined the lung injury by inhibiting inflammation, parenchymal destruction, emphysema, collagen, club cells, and Kohn pores. AK-7 also showed good binding affinity with inflammatory proteins. Discussion: The current study reveals that SIRT-2 inhibition mitigates COPD severity and enhances pulmonary therapeutic interventions, suggesting AK-7 as a potential therapeutic molecule for COPD medication development.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

Objective: This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target. Methods: Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1, the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target. Results: Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice. Conclusion: This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating AMD.