Involvement of Interleukin-1 Receptor-Associated Kinase 4 and Interferon Regulatory Factor 5 in the Immunopathogenesis of SARS-CoV-2 Infection: Implications for the Treatment of COVID-19.

Interleukin-1 receptor-associated kinase 4 (IRAK4) and interferon regulatory factor 5 (IRF5) lie sequentially on a signaling pathway activated by ligands of the IL-1 receptor and/or multiple TLRs located either on plasma or endosomal membranes. Activated IRF5, in conjunction with other synergistic transcription factors, notably NF-κB, is crucially required for the production of proinflammatory cytokines in the innate immune response to microbial infection. The IRAK4-IRF5 axis could therefore have a major role in the induction of the signature cytokines and chemokines of the hyperinflammatory state associated with severe morbidity and mortality in COVID-19. Here a case is made for considering IRAK4 or IRF5 inhibitors as potential therapies for the "cytokine storm" of COVID-19.

Fusogenic porous silicon nanoparticles as a broad-spectrum immunotherapy against bacterial infections.

Bacterial infections are re-emerging as substantial threats to global health due to the limited selection of antibiotics that are capable of overcoming antibiotic-resistant strains. By deterring such mutations whilst minimizing the need to develop new pathogen-specific antibiotics, immunotherapy offers a broad-spectrum therapeutic solution against bacterial infections. In particular, pathology resulting from excessive immune response (i.e. fibrosis, necrosis, exudation, breath impediment) contributes significantly to negative disease outcome. Herein, we present a nanoparticle that is targeted to activated macrophages and loaded with siRNA against the Irf5 gene. This formulation is able to induce >80% gene silencing in activated macrophages in vivo, and it inhibits the excessive inflammatory response, generating a significantly improved therapeutic outcome in mouse models of bacterial infection. The versatility of the approach is demonstrated using mice with antibiotic-resistant Gram-positive (methicillin-resistant Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) muscle and lung infections, respectively. Effective depletion of the Irf5 gene in macrophages is found to significantly improve the therapeutic outcome of infected mice, regardless of the bacteria strain and type.

miR-31 from adipose stem cell-derived extracellular vesicles promotes recovery of neurological function after ischemic stroke by inhibiting TRAF6 and IRF5.

Ischemic stroke affects many people in the world, but the underlying mechanism is not completely understood. In this study, we investigated the effect of microRNA (miR)-31 on ischemic stroke. We also determined downstream signaling pathway of miR-31 in recovery of neurological function in ischemic stroke. Middle cerebral artery occlusion (MCAO) in mice was used to mimic human stroke. Foot fault test and mNSS were used to evaluate neurological deficits in mice after stroke. TTC staining in brain tissues was used for determining infarct volume. We extracted and identified extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) to study the impact of miR-31 and TRAF6 by miR-31 overexpression or TRAF6 knockdown on stroke recovery. Primary mouse neuron exposed to oxygen-glucose deprivation (OGD) was used to mimic neuronal ischemic injury. RT-qPCR and Western blot analysis were used for determination of mRNA and protein expression, respectively. MTT assay was used for studying cell survival. TUNEL staining was sued for neuron apoptosis. Starbase website and dual luciferase reporter gene assay were utilized to predicted and verify binding relationship between miR-31 and TRAF6. Neurological functions were improved by miR-31 from ADSC-derived EVs, as suggested by improved foot fault and mNSS. miR-31 from ADSC-derived EVs also reduced infarct volume and neuronal cell apoptosis after stroke in mice. Similarly, in neuronal cell culture, miR-31 from ADSC-derived EVs reduced the expression of apoptosis-related factors cleaved caspase-3 and Bax, increased the survival, and reduced apoptosis of neuronal cells after OGD. miR-31 was found to downregulate the expression of TRAF6 by binding to the 3'-untranslated region (3'-UTR) of TRAF6, which in turn upregulated IRF5 expression. Increased expression of IRF5 led to increased neuron apoptosis after OGD. In conclusion, miR-31 from ADSC-derived EVs can downregulate expression of TRAF6 and IRF5, leading to reduced neuronal damage induced by ischemic stroke.

Exploiting Protein Translation Dependence in Multiple Myeloma with Omacetaxine-Based Therapy.

PURPOSE: The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen-specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine. EXPERIMENTAL DESIGN: We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis. RESULTS: Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival. CONCLUSIONS: Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

Inhibition of IRF5 hyperactivation protects from lupus onset and severity.

The transcription factor IFN regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 are associated with a risk of systemic lupus erythematosus (SLE), and mice lacking Irf5 are protected from lupus onset and severity, but how IRF5 functions in the context of SLE disease progression remains unclear. Using the NZB/W F1 model of murine lupus, we show that murine IRF5 becomes hyperactivated before clinical onset. In patients with SLE, IRF5 hyperactivation correlated with dsDNA titers. To test whether IRF5 hyperactivation is a targetable function, we developed inhibitors that are cell permeable, nontoxic, and selectively bind to the inactive IRF5 monomer. Preclinical treatment of NZB/W F1 mice with an inhibitor attenuated lupus pathology by reducing serum antinuclear autoantibodies, dsDNA titers, and the number of circulating plasma cells, which alleviated kidney pathology and improved survival. Clinical treatment of MRL/lpr and pristane-induced lupus mice with an inhibitor led to significant reductions in dsDNA levels and improved survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation and reversed basal IRF5 hyperactivation in SLE immune cells. We believe this study provides the first in vivo clinical support for treating patients with SLE with an IRF5 inhibitor.

Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway.

Tick-borne encephalitis virus (TBEV) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia and causes encephalitis in humans. In this study, we demonstrate TBEV appears to activate the interferon (IFN)-ß dependent on RIG-I/MDA5. Both the IFN-ß accumulation and the IFN stimulated genes (ISGs) transcription greatly delay. Further studies reveal that TBEV NS4A could block the phosphorylation and dimerization of STAT1/STAT2 to affect type I and II IFN-mediated STAT signalling. Additional data indicate that the residue at K132 of TBEV NS4A could be modified by ubiquitination and this modification is necessary for the interaction of NS4A with STAT1. Dynamic ubiquitination of the NS4 protein during TBEV infection might account for delayed activation of the ISGs. These results define the TBEV NS4A as an antagonist of the IFN response, by demonstrating a correlation between the association and STAT interference. Our findings provide a foundation for further understanding how TBEV evade innate immunity and a potential viral target for intervention.

Mechanism of berberine in treating Helicobacter pylori induced chronic atrophic gastritis through IRF8-IFN-γ signaling axis suppressing.

AIMS: In this study, we will investigate the therapeutic effects of berberine (BBR) in Helicobacter pylori (H. pylori) induced chronic atrophic gastritis (CAG). Furthermore, potential mechanisms of BBR in regulating IRF8-IFN-γ signaling axis will also be investigated. MATERIALS AND METHODS: H. pylori were utilized to establish CAG model of rats. Therapeutic effects of BBR on serum supernatant indices, and histopathology of stomach were analyzed in vivo. Moreover, GES-1 cells were infected by H. pylori, and intervened with BBR in vitro. Cell viability, morphology, proliferation, and quantitative analysis were detected by high-content screening (HCS) imaging assay. To further investigate the potential mechanisms of BBR, relative mRNA, immunohistochemistry and protein expression in IRF8-IFN-γ signaling axis were measured. KEY FINDINGS: Results showed serum supernatant indices including IL-17, CXCL1, and CXCL9 were downregulated by BBR intervention, while, G-17 increased significantly. Histological injuries of gastric mucosa induced by H. pylori also were alleviated. Moreover, cell viability and morphology changes of GES-1 cells were improved by BBR intervention. In addition, proinflammatory genes and IRF8-IFN-γ signaling axis related genes, including Ifit3, Upp1, USP18, Nlrc5, were suppressed by BBR administration in vitro and in vivo. The proteins expression related to IRF8-IFN-γ signaling axis, including Ifit3, IRF1 and Ifit1 were downregulated by BBR intervention.

Inhibition of IRF4 in dendritic cells by PRR-independent and -dependent signals inhibit Th2 and promote Th17 responses.

Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.

Differential Roles of the mTOR-STAT3 Signaling in Dermal γδ T Cell Effector Function in Skin Inflammation.

Dermal γδT cells play critical roles in skin homeostasis and inflammation. However, the underlying molecular mechanisms by which these cells are activated have not been fully understood. Here, we show that the mechanistic or mammalian target of rapamycin (mTOR) and STAT3 pathways are activated in dermal γδT cells in response to innate stimuli such as interleukin-1ß (IL-1ß) and IL-23. Although both mTOR complex 1 (mTORC1) and mTORC2 are essential for dermal γδT cell proliferation, mTORC2 deficiency leads to decreased dermal γδT17 cells. It appears that mitochondria-mediated oxidative phosphorylation is critical in this process. Notably, although the STAT3 pathway is critical for dermal Vγ4T17 effector function, it is not required for Vγ6T17 cells. Transcription factor IRF-4 activation promotes dermal γδT cell IL-17 production by linking IL-1ß and IL-23 signaling. The absence of mTORC2 in dermal γδT cells, but not STAT3, ameliorates skin inflammation. Taken together, our results demonstrate that the mTOR-STAT3 signaling differentially regulates dermal γδT cell effector function in skin inflammation.

Identification of lenalidomide resistance pathways in myeloma and targeted resensitization using cereblon replacement, inhibition of STAT3 or targeting of IRF4.

To understand immunomodulatory drug (IMiD) resistance in multiple myeloma (MM), we created isogenic human multiple myeloma cell lines (HMCLs) sensitive and resistant to lenalidomide, respectively. Four HMCLs were demonstrated to be resistant to all IMiDs including lenalidomide, pomalidomide, and CC-220, but not to Bortezomib. In three HMLCs (MM.1.SLenRes, KMS11LenRes and OPM2LenRes), CRBN abnormalities were found, including chromosomal deletion, point mutation, and low CRBN expression. The remaining HMCL, XG1LenRes, showed no changes in CRBN but exhibited CD147 upregulation and impaired IRF4 downregulation after lenalidomide treatment. Depletion of CD147 in XG1LenRes and three additional HMCLs had no significant impact on MM viability and lenalidomide response. Further analysis of XG1LenRes demonstrated increased IL6 expression and constitutive STAT3 activation. Inhibition of STAT3 with a selective compound (PB-1-102) re-sensitized XG1LenRes to lenalidomide. Since XG1LenRes harbors a truncated IRF4 that is not downregulated by lenalidomide, we targeted IRF4/MYC axis with a selective inhibitor of the bromodomain of CBP/EP300 (SGC-CBP30), which restored lenalidomide response in XG1LenRes. This strategy also appeared to be more broadly applicable as SGC-CBP30 could re-sensitize two resistant HMCLs with low but detectable CRBN expression to lenalidomide, suggesting that targeting CBP/E300 is a promising approach to restore IMiD sensitivity in MM with detectable CRBN expression.

Advances and challenges in targeting IRF5, a key regulator of inflammation.

Interferon regulatory factor 5 (IRF5) belongs to a family of transcription factors, originally implicated in antiviral responses and interferon production. However, studies conducted in different laboratories over the last decade have placed IRF5 as a central regulator of the inflammatory response. It has become clear that IRF5 contributes to the pathogenesis of many inflammatory and autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus. Given the role of IRF5 in physiology and disease, IRF5 represents a potential therapeutic target. However, despite a significant interest from the pharmaceutical industry, inhibitors that interfere with the IRF5 pathway remain elusive. Here, we review the advances made by various studies in targeting multiple steps of signalling leading to IRF5 activation with their therapeutic potential, and the possible complications of such strategies are discussed.

How Does Vaccinia Virus Interfere With Interferon?

Interferons (IFNs) are secreted glycoproteins that are produced by cells in response to virus infection and other stimuli and induce an antiviral state in cells bearing IFN receptors. In this way, IFNs restrict virus replication and spread before an adaptive immune response is developed. Viruses are very sensitive to the effects of IFNs and consequently have evolved many strategies to interfere with interferon. This is particularly well illustrated by poxviruses, which have large dsDNA genomes and encode hundreds of proteins. Vaccinia virus is the prototypic poxvirus and expresses many proteins that interfere with IFN and are considered in this review. These proteins act either inside or outside the cell and within the cytoplasm or nucleus. They function by restricting the production of IFN by blocking the signaling pathways leading to transcription of IFN genes, stopping IFNs binding to their receptors, blocking IFN-induced signal transduction leading to expression of interferon-stimulated genes (ISGs), or inhibiting the antiviral activity of ISG products.

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

Interferon regulatory factor 4 (IRF4) is overexpressed in human non­small cell lung cancer (NSCLC) and activates the Notch signaling pathway.

The transcription factor, interferon regulatory factor 4 (IRF4), serves an essential role in the regulation of immune responses, and has been reported to act as a diagnostic and prognostic marker for various hematological malignancies. The present study aimed to investigate whether IRF4 could exert effects on human non­small cell lung cancer (NSCLC) and to explore the underlying mechanism. The mRNA and protein expression of IRF4 was detected in NSCLC tissues using reverse­transcription quantitative polymerase chain reaction and western blotting, respectively. In the in vitro experiment, IRF4 expression was knocked down or overexpressed using lentivirus in human lung adenocarcinoma A549 and lung squamous cell carcinoma LC­AI cell lines. Cell proliferation and colony number were analyzed using MTT and colony formation assays, respectively. The expression levels of IRF4 mRNA and protein were significantly higher in NSCLC tissues (n=54) compared with that in adjacent non­tumor tissues. Similarly, the expression levels of Notch1 and Notch2 mRNA were significantly higher in NSCLC tissues. Furthermore, the expression level of IRF4 mRNA was positively correlated with the levels of Notch1 and Notch2 mRNA in NSCLC tissues. Consequently, using NSCLC cell lines, it was demonstrated that the knockdown of IRF4 expression significantly reduced the cell proliferation rate and colony formation, whereas IRF4­overexpression significantly increased them. Notably, the IRF4 knockdown significantly decreased the expression levels of Notch1 and Notch2 mRNA, and phosphorylated protein kinase B (AKT), whereas IRF4 overexpression resulted in the opposite. The results of the present study indicate that IRF4 is overexpressed and serves as a tumor promoter in human NSCLC, at least partially, through activating the Notch­Akt signaling pathway.

BCG Increased Membrane Expression of TRIM59 Through the TLR2/ TLR4/IRF5 Pathway in RAW264.7 Macrophages.

OBJECTIVE: In our previous study, we showed that Bacillus Calmette-Guerin (BCG)- activated macrophages have the ability to directly kill tumor cells. One of the main properties of these macrophages is the high expression of tripartite motif family protein 59 (TRIM59). This study was conducted to investigate the mechanism of BCG-induced TRIM59 expression on macrophages and to identify the subcellular localization of TRIM59. METHODS: TRIM59 expression and TNF-α secretion were compared in RAW264.7 macrophage cells that were stimulated using BCG with or without Toll-like receptor 2/4 (TLR2/4)-neutralizing antibodies. Next, small interfering RNA (siRNA) was used to down-regulated interferon regulatory factor 5 (IRF5) gene expression in RAW264.7 cells. Transfected cells were stimulated with BCG, after which TRIM59 expression and TNF-α secretion were evaluated in cells pre-treated with siRNA or scramble control. After treatments, supernatants were co-cultured with MCA207, and cell viabilities were determined. Moreover, BCG-stimulated RAW264.7 cells were stained for TRIM59 and F4/80 expression. RESULTS: In this study, we showed that TRIM59 was expressed on the membrane of RAW264.7 cells. After blocking TLR2/4, treatment with BCG failed to induce the expression of TRIM59, IRF5, and TNF-α on RAW264.7 cells. In addition, down-regulation of IRF5 inhibited TRIM59 and TNF-α expression. CONCLUSION: Our study showed that TRIM59 is a membrane protein, and that BCG treatment upregulated TRIM59 expression on macrophages via TLR2/4 and IRF5 pathways.

An oligodeoxynucleotide with AAAG repeats significantly attenuates burn-induced systemic inflammatory responses via inhibiting interferon regulatory factor 5 pathway.

Previously, we showed that an oligodeoxynucleotide with AAAG repeats (AAAG ODN) rescued mice from fatal acute lung injury (ALI) induced by influenza virus and inhibited production of tumor necrosis factor-α (TNF-α) in the injured lungs. However, the underlying mechanisms remain to be elucidated. Upon the bioinformatic analysis revealing that the AAAG ODN is consensus to interferon regulatory factor 5 (IRF5) binding site in the cis-regulatory elements of proinflammatory cytokines, we tried to explore whether the AAAG ODN could attenuate burn injury induced systemic inflammatory responses via inhibiting IRF5 pathway. Using the mouse model with sterile systemic inflammation induced by burn injury, we found that AAAG ODN prolonged the life span of the mice, decreased the expression of IRF5 at injured skin, reduced the production of TNF-α and IL-6 in blood and injured skin, and attenuated the ALI. Furthermore, AAAG ODN could bind IRF5 and inhibit the nuclear translocation of IRF5 in THP-1 cells. The data suggested that the AAAG ODN could act as a cytoplasmic decoy capable of interfering the function of IRF5, and be developed as a drug candidate for the treatment of inflammatory diseases.

An AAAG-Rich Oligodeoxynucleotide Rescues Mice from Bacterial Septic Peritonitis by Interfering Interferon Regulatory Factor 5.

A previous study found that an AAAG-rich Oligodeoxynucleotide (ODN), designated as MS19, could lessen the acute lung inflammatory injury (ALII) in mice infected by influenza viruses. Bioinformatics analysis found that MS19 is consensus with the binding site of interferon regulatory factor 5 (IRF5) in the regulatory elements of pro-inflammatory genes. This study established a septic peritonitis model in Institute of Cancer Research (ICR) mice infected with Escherichia coli (E. coli), and found that MS19 prolonged the survival of the mice and down-regulated the expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). In cultured RAW264.7 cells, MS19 significantly reduced the expression of iNOS, IRF5, IL-6, and TNF-α and inhibited the nuclear translocation of IRF5. This data may provide a new insight for understanding how MS19 reduces the excessive inflammatory responses in sepsis.

Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence.

The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater effect on the IRF8 nuclear localization versus total fluorescence assessment. The data thus indicate that IRF8 can have cytoplasmic presence and that during disease its nuclear-cytoplasmic distribution may be altered, which may provide a basis for potential myeloid defects during certain pathologies.

Inhibition of IRF8 Negatively Regulates Macrophage Function and Impairs Cutaneous Wound Healing.

The inflammatory response is essential for normal cutaneous wound healing. Macrophages, as critical inflammatory cells, coordinate inflammation and angiogenesis phases during wound healing. It has been reported that the transcription factor interferon regulatory factor 8 (IRF8), a member of the IRF family, plays a critical role in the development and function of macrophages and is associated with inflammation. However, the role of IRF8 in cutaneous wound healing and its underlying mechanism remain elusive. Through immunohistochemical (IHC) staining, we showed that IRF8 is involved in the wound repair process in mice and patients. Furthermore, we ascertain that the repression of IRF8 by small interfering RNA (siRNA) leads to delayed wound healing. To explore the mechanism by which IRF8 impacts wound healing, we observed its effect on macrophage-related mediators by IHC or real-time PCR. The results demonstrated that the inhibition of IRF8 decreases the mRNA expression of inflammatory mediators associated with M1 macrophage (il-1b, il-6, inos, and tnf-a) but no impact on M2 macrophage-related mediators (arg-1, mrc-1, and il-10) and the number of macrophages in the wounds. Furthermore, the inhibition of IRF8 induced apoptosis in the wounds. In summary, this study demonstrates that the down-regulation of IRF8 in the wound leads to impaired wound healing possibly through the regulation of macrophage function and apoptosis in skin wound.

AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes.

Autophagy is essential in physiological and pathological processes, however, the role of autophagy in cutaneous wound healing and the underlying molecular mechanism remain elusive. We hypothesized that autophagy plays an important role in regulating wound healing. Here, we show that enhanced autophagy negatively impacts on normal cutaneous healing process and is related to chronic wounds as demonstrated by the increased LC3 in diabetic mice skin or patients' chronic wounds. In addition, inhibition of autophagy by 3-MA restores delayed healing in C57BL/6 or db/db mice, demonstrating that autophagy is involved in regulating wound healing. Furthermore, we identify that macrophage is a major cell type underwent autophagy in wounds and increased autophagy induces macrophages polarization into M1 with elevated CD11c population and gene expressions of proinflammatory cytokines. To explore the mechanism underlying autophagy-impaired wound healing, we tested the role of IRF8, a regulator of autophagy, in autophagy-modulated macrophages polarization. IRF8 activation is up-regulating autophagy and M1 polarization of macrophages after AGEs (advanced glycation endproducts) treatment, blocking the IRF8 with shIRF8 inhibits autophagic activity and M1 polarization. In summary, this study elucidates that AGEs induces autophagy and modulates macrophage polarization to M1 via IRF8 activation in impairment of cutaneous wound healing.