STAT5 Activation Research Articles

The PAX5‐JAK2 translocation acts as dual‐hit mutation that promotes aggressive B‐cell leukemia via nuclear STAT5 activation

While PAX5 is an important tumor suppressor gene in B‐cell acute lymphoblastic leukemia (B‐ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5‐JAK2 encodes a protein consisting of the PAX5 DNA‐binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5‐JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B‐ALL in the absence of another cooperating exogenous gene mutation. The DNA‐binding function and kinase activity of Pax5‐Jak2 as well as IL‐7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild‐type Pax5 allele, allowing efficient DNA‐binding of Pax5‐Jak2. While we could not find evidence for a nuclear role of Pax5‐Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B‐ALL tumors, implying that nuclear Pax5‐Jak2 phosphorylates STAT5. Together, these data reveal Pax5‐Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

Indoleamine 2,3-dioxygenase 1 regulates breast cancer tamoxifen resistance through interleukin-6/signal transducer and activator of transcription

Breast cancer is the primary cause of cancer-related deaths in women. Tamoxifen (TAM) is the preferred drug for treating premenopausal luminal-type breast cancer, but TAM resistance restricts its ability to benefit patients. To date, the mechanism of this resistance remains unclear, and there is currently no effective treatment for reversing it. The expression of indoleamine 2,3-dioxygenase 1 (IDO1) has been shown to be elevated in various malignancies. Here, we aimed to investigate the role of IDO1 in TAM-resistant breast cancer. We confirmed that IDO1 is strongly expressed in TAM-resistant breast cancer, and it mediates drug-resistant cell proliferation, metastasis, and TAM resistance in vivo and in vitro through interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3). We also found that the mechanism by which TAM upregulates IDO1 is dependent on STAT1 activation. In summary, IDO1 regulates TAM resistance and can serve as a novel target for treatment of TAM-resistant breast cancer.

Inhibition of the Na+/K+-ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2′,3′-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.

POS-379 TO INVESTIGATE THE EFFECT OF HERBA LYSIMACHIAE IN THE TREATMENT OF OBSTRUCTIVE NEPHROPATHY BASED ON STAT-1/TREM-1 PATHWAY

Obstructive nephropathy is a major cause of chronic renal failure in children and adults.Clinical and animal experiments have found that renal damage still exists after obstruction is relieved, which is mainly related to irreversible fibrosis changes caused by persistent inflammatory infiltration, and there is no specific medicine for western medicine treatment. Herba lysimachiae can detoxify detumescence, diuresis tonglin, has a certain kidney protection. However, there is still a lack of systematic knowledge about the herba lysimachiae components and mechanism of s. chinensis in the treatment of obstructive nephropathy. Twenty SD rats were randomly divided into Sham operation group (Sham), unilateral ureteral obstruction operation group (UUO), Pennyswort intervention Sham operation group (Sham+Lys), herba lysimachiae intervention model group (UUO+ Lys).From the 1st day after surgery, the decoction of herba lysimachiae was given 24g/kg, once a day, for continuous 2w.Or intragastric administration of normal saline at the same time.Routine biochemical kit was used to detect renal function 2 weeks later.Renal tubule dilatation, mesangial hyperplasia and renal interstitial fibrosis were detected by HE, PAS and Masson staining.Immunohistochemistry and immunofluorescence were used to detect the activation of STAT-1/ TreM-1 pathway, macrophage aggregation (CD68+ iNOS+), renal tubular injury markers (KIM-1), and fibrosis related molecules (α-SMA, TGF-beta) in renal tissues. In the model group, the renal tubules were oppressed by urine retention on the obstructed side. Pathological staining showed that the epithelial cells of the renal tubules were necrotic to varying degrees, and the interstitial was infiltrated by acute and chronic inflammatory cells. Some of the necrotic renal tubules were gradually replaced by fibrous scar, and the formation of renal interstitial fibrosis was visible.Biochemical tests showed that the creatinine and renal injury scores were significantly higher than those of the normal group.Immunohistochemistry/immunofluorescence assay showed increased stat-1 /TREM-1 expression.Abnormal aggregation of M1-type macrophages in renal interstitium, accompanied by renal injury and fibrosis, showed increased expressions of KIM-1, α-SMA and TGF-beta (all P<0.05).In contrast, stat-1 /TREM-1 expression was decreased in renal tissue after herba lysimachiae intervention, and the above inflammatory and fibrosis related indicators were improved (P<0.05). Herba lysimachiae can protect renal function and inhibit renal inflammation and fibrosis in UUO rats, and this mechanism is related to the STAT-1/TREM-1 pathway.

Abstract 20: Targeted Ablation Of STAT1 Enhances Resolution Of Inflammation By Microglia/macrophages And Promotes Long-term Recovery After Ischemic Stroke

Introduction: Identification of key molecules that control conversion of resting microglia/macrophages (Mi/MΦ) to a detrimental or beneficial phenotype may help develop novel therapies to ischemic stroke. The transcription factor STAT1 contributes to acute (&lt; 24h) neuronal death after brain ischemia/reperfusion (I/R), but its role in Mi/MΦ and impact on long term stroke outcome remain unknown. Hypothesis: Activation of STAT1 dictates proinflammatory responses of Mi/MΦ at subacute stage (3-5 d) after I/R. Selective deletion of STAT1 reprograms Mi/MΦ into an inflammation-resolving phenotype and improves long term stroke outcome. Methods: We generated mice with tamoxifen-induced, Mi/MΦ-specific knockout (mKO) of STAT1. Brain injury, inflammation and various behavioral deficits were assessed after 1 h MCAO and 1-35 d reperfusion. Mi/MΦ phenotype was examined in vivo (FACS followed by RNA-seq) and in vitro (primary microglia). Results: STAT1 was activated (phosphorylated) in Mi/MΦ 3 d after I/R in WT but not in STAT1 mKO mice. STAT1 mKO did not alter 24-h infarct size (TTC; p&gt;0.05, n=8), but attenuated Mi/MΦ release of HMGB1 and increased arginase 1-producing Mi/MΦ 3d after I/R (immunostain, n=6), suggesting boosted inflammation-resolving responses of Mi/MΦ. RNA-seq further revealed downregulated proinflammatory genes and a concomitant elevation of phagocytosis-related genes in STAT1 mKO Mi/MΦ 3d after I/R (n=3, FDR&lt;0.05). Consistently, phagocytosis of dead neurons was enhanced in STAT1 mKO mice 3d after I/R (n=7) or in cultured STAT1 KO microglia (n=4). As a result, STAT1 mKO mitigated brain inflammation 5d after I/R (ELISArray; 11 out of 27 markers, p&lt;0.05, n=6), and improved motor/cognitive functions for 35d in mice of both sexes (p&lt;0.01 vs WT, n=10-11). Conclusions: Mi/MΦ-targeted STAT1 KO does not offer acute neuroprotection but primes Mi/MΦ toward an inflammation-resolving phenotype, thereby facilitating long term functional recovery after stroke.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Mouse Modeling Dissecting Macrophage-Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor‐associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL‐4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro‐tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

Targeting pancreatic cancer by TAK-981: a SUMOylation inhibitor that activates the immune system and blocks cancer cell cycle progression in a preclinical model

ObjectivePancreatic ductal adenocarcinoma (PDAC) has the characteristics of high-density desmoplastic stroma, a distinctive immunosuppressive microenvironment and is profoundly resistant to all forms of chemotherapy and immunotherapy, leading to a 5-year...

P030 Distinct molecular profiles between idiopathic cryptoglandular and Crohn-related perianal fistulas

Abstract Background Perianal fistula can originate idiopathically (cryptoglandular fistula) or can be a representation of underlying Crohn’s disease (CD). In case of idiopathic fistula, the cryptoglandular theory suggests their development from the anal glands. In contrast, the pathophysiology of CD-related fistula is very poorly understood. Hence, we molecularly characterised and compared the fistula tract in both conditions. Methods We collected surgical biopsies from the fistula tract in 70 CD patients with active draining perianal fistula and in 12 patients with cryptoglandular non-CD related, active draining fistula, all requiring surgical examination under anaesthesia. RNA was sequenced using Illumina HiSeq4000, and these data were analysed through differential gene expression analysis (DESeq2). A false discovery rate (FDR) of 0.05 and a |log 2-fold change (log2FC)| &amp;gt;1 was considered significant. Pathway analysis was performed using IPA (Qiagen). In addition, cellular deconvolution methods (xCell) were applied to study the cellular composition. Results Gene expression analysis identified 1087 genes being differentially expressed between CD-related and cryptoglandular fistula (716 up, 371 down). Top differentially expressed genes encode proteins implicated in IBD pathogenesis including CARD18 (log2FC= 23.2, p=3.9E-21), BATF2 (log2FC=2.8, p=1.8E-8), ETV7 (log2FC=2.1, p=2.0E-7), DSG1 (log2FC=7.8, p=2.7E-6) and IL22RA1 (log2FC=5.3, p=4.6E-6). Additional pathway analysis highlighted various proinflammatory processes in CD fistula (as compared to cryptoglandular fistula), including upregulation of antigen presentation and Th1/Th2 activation (p&amp;lt;1.0E-9). Intriguingly, CD fistula showed a significant downregulation of wound healing signaling pathways (p=6.5E-6), emphasising the refractory character of this debilitating condition. Upstream analyses showed an increased activation of top regulators IFNγ, TNF, LPS and STAT1 (p&amp;lt;1.0E-24). Cellular deconvolution identified significant differences between both fistula types, with a predominance of inflammatory cells in CD including Th1 cells, dendritic cells, naïve CD4 T cells, memory B cells and central memory CD8 and CD4 T cells (p&amp;lt;5.0E-2). In contrast, cryptoglandular fistula were characterised by a significant enrichment of myocytes, smooth muscle cells and neurons (p&amp;lt;3.0E-3). Presence of neutrophils, wound healing and pro-inflammatory macrophages did not differ between both fistula types. Conclusion CD fistulas have a strong proinflammatory fingerprint, but seem to have lower wound healing capacity as compared to cryptoglandular fistula. An aggressive medical and surgical treatment is therefore required, including the search for novel, powerful anti-inflammatory and wound healing compounds.

STAT6/VDR Axis Mitigates Lung Inflammatory Injury by Promoting Nrf2 Signaling Pathway.

Lung inflammatory injury is a global public health concern. It is characterized by infiltration of diverse inflammatory cells and thickening of pulmonary septum along with oxidative stress to airway epithelial cells. STAT6 is a nuclear transcription factor that plays a crucial role in orchestrating the immune response, but its function in tissue inflammatory injury has not been comprehensively studied. Here, we demonstrated that STAT6 activation can protect against particle-induced lung inflammatory injury by resisting oxidative stress. Specifically, genetic ablation of STAT6 was observed to worsen particle-induced lung injury mainly by disrupting the lungs' antioxidant capacity, as reflected by the downregulation of the Nrf2 signaling pathway, an increase in malondialdehyde levels, and a decrease in glutathione levels. Vitamin D receptor (VDR) has been previously proved to positively regulate Nrf2 signals. In this study, silencing VDR expression in human bronchial epithelial BEAS-2B cells consistently suppressed autophagy-mediated activation of the Nrf2 signaling pathway, thereby aggravating particle-induced cell damage. Mechanically, STAT6 activation promoted the nuclear translocation of VDR, which increased the transcription of autophagy-related genes and induced Nrf2 signals, and silencing VDR abolished these effects. Our research provides important insights into the role of STAT6 in oxidative damage and reveals its potential underlying mechanism. This information not only deepens the appreciation of STAT6 but also opens new avenues for the discovery of therapies for inflammatory respiratory system disorders.

MRNA Profiling Involved in Triggering of STAT1 with Regulatory Involvement of IRF7, PTPRF, and miR-145p in Patients Suffering from Gall Bladder Carcinoma.

Background Gall Bladder Cancer (GBC) is a type of extremely malignant tumor, which has high incidences of mortality. There is rare information about its mechanisms of invasion and gene expression regulations. microRNA-155 (miR-155) has mostly been reported to be over expressed in cases of solid tumors and hematopoietic malignancies. In this study, we have investigated the role and clinical significance of miR-155 in a Chinese population suffering from GBC and compared the results with nonneoplastic inflammation. Methods Tissue specimens were collected on 50 patients of Gall Bladder Carcinoma and 10 patients suffering from nonneoplastic inflammation who have undergone surgeries at the Department of Pathology, Renji Hospital, Shanghai, from January 2019 to January 2020. We performed profiling of miR-155 expression in both nonneoplastic and gall bladder carcinoma tissues by QRT-PCR. Results Expression levels of miR-155 were found to be extremely high in GBC patients in comparison to the nonneoplastic tissues (∗P < 0.05), as high miRNA is correlated with TNM stages. Further results noted were that miR-145-5p expressed genes mimic the gene expression of STAT1, a downregulation of IRF7 was noted in the GBC, and an activation of STAT1 was significantly noted in carcinoma cells of the gallbladder. Downregulation of PTPRF was also noted during the expression of miR-145. Conclusions As downregulation of IRF7 is linked with low rates of survival, it was found that gall bladder carcinoma patients may face high mortality. The STAT-1 expression of unregulated in GBC patients was also noted.

Nanoparticle-Induced Airway Eosinophilia Is Independent of ILC2 Signaling but Associated With Sex Differences in Macrophage Phenotype Development.

The majority of lung diseases occur with a sex bias in terms of prevalence and/or severity. Previous studies demonstrated that, compared with males, female mice develop greater eosinophilic inflammation in the airways after multiwalled carbon nanotube (MWCNT) exposure. However, the mechanism by which this sex bias occurs is unknown. Two immune cells that could account for the sex bias are type II innate lymphoid cells (ILC2s) and alveolar macrophages (AMs). In order to determine which immune cell type was responsible for MWCNT-induced airway eosinophil recruitment and subsequent sex differences in inflammation and disease, male and female C57BL/6 mice were exposed to MWCNTs (2 mg/kg) via oropharyngeal aspiration, and the respiratory immune response was assessed 7 d later. Greater eosinophilia and eotaxin 2 levels were observed in MWCNT-treated females and corresponded with greater changes in airway hyperresponsiveness than those in MWCNT-treated males. In MWCNT-treated females, there was a significant increase in the frequency of ILC2s within the lungs compared with control animals. However, depletion of ILC2s via α-CD90.2 administration did not decrease eosinophil recruitment 24 h and 7 d after MWCNT exposure. AMs isolated from control and MWCNT-treated animals demonstrated that M2a macrophage phenotype gene expression, ex vivo cytokine production, and activation of (p)STAT6 were upregulated to a significantly greater degree in MWCNT-treated females than in males. Our findings suggest that sex differences in AM phenotype development, not ILC2 signaling, are responsible for the observed female bias in eosinophilic inflammation after MWCNT inhalation.

In silico Prediction on the PI3K/AKT/mTOR Pathway of the Antiproliferative Effect of O. joconostle in Breast Cancer Models.

The search for new cancer treatments from traditional medicine involves developing studies to understand at the molecular level different cell signaling pathways involved in cancer development. In this work, we present a model of the PI3K/Akt/mTOR pathway, which plays a key role in cell cycle regulation and is related to cell survival, proliferation, and growth in cancer, as well as resistance to antitumor therapies, so finding drugs that act on this pathway is ideal to propose a new adjuvant treatment. The aim of this work was to model, simulate and predict in silico using the Big Data-Cellulat platform the possible targets in the PI3K/Akt/mTOR pathway on which the Opuntia joconostle extract acts, as well as to indicate the concentration range to be used to find the mean lethal dose in in vitro experiments on breast cancer cells. The in silico results show that, in a cancer cell, the activation of JAK and STAT, as well as PI3K and Akt is related to the effect of cell proliferation, angiogenesis, and inhibition of apoptosis, and that the extract of O. joconostle has an antiproliferative effect on breast cancer cells by inhibiting cell proliferation, regulating the cell cycle and inhibiting apoptosis through this signaling pathway. In vitro it was demonstrated that the extract shows an antiproliferative effect, causing the arrest of cells in the G2/M phase of the cell cycle. Therefore, it is concluded that the use of in silico tools is a valuable method to perform virtual experiments and discover new treatments. The use of this type of model supports in vitro experimentation, reducing the costs and number of experiments in the real laboratory.

Histone Deacetylase 1 Is Required for the Protective Immunity Against Mycobacterium tuberculosis Infection Through Regulation of STAT1 Activation

Histone Deacetylase 1 Is Required for the Protective Immunity Against Mycobacterium tuberculosis Infection Through Regulation of STAT1 Activation

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

IL-27 induces IFN/STAT1-dependent genes and enhances function of TIGIT+ HIVGag-specific T cells

SummaryHIV-specific T cells have diminished effector function and fail to control/eliminate the virus. IL-27, a member of the IL-6/IL-12 cytokine superfamily has been shown to inhibit HIV replication. However, whether or not IL-27 can enhance HIV-specific T cell function is largely unknown. In the present manuscript, we investigated the role of IL-27 signaling in human T cells by evaluating the global transcriptional changes related to the function of HIV-specific T cells. We found that T cells from people living with HIV (PLWH), expressed higher levels of STAT1 leading to enhanced STAT1 activation upon IL-27 stimulation. Observed IL-27 induced transcriptional changes were associated with IFN/STAT1-dependent pathways in CD4 and CD8 T cells. Importantly, IL-27 dependent modulation of T-bet expression promoted IFNγ secretion by TIGIT+HIVGag-specific T cells. This new immunomodulatory effect of IL-27 on HIV-specific T cell function suggests its potential therapeutic use in cure strategies.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling.

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

IRF3 inhibits IFN-γ-mediated restriction of intracellular pathogens in macrophages independently of IFNAR.

Macrophages use an array of innate immune sensors to detect intracellular pathogens and to tailor effective antimicrobial responses. In addition, extrinsic activation with the cytokine IFN-γ is often required as well to tip the scales of the host-pathogen balance toward pathogen restriction. However, little is known about how host-pathogen sensing impacts the antimicrobial IFN-γ-activated state. It was observed that in the absence of IRF3, a key downstream component of pathogen sensing pathways, IFN-γ-primed macrophages more efficiently restricted the intracellular bacterium Legionella pneumophila and the intracellular protozoan parasite Trypanosoma cruzi. This effect did not require IFNAR, the receptor for Type I IFNs known to be induced by IRF3, nor the sensing adaptors MyD88/TRIF, MAVS, or STING. This effect also did not involve differential activation of STAT1, the major signaling protein downstream of both Type 1 and Type 2 IFN receptors. IRF3-deficient macrophages displayed a significantly altered IFN-γ-induced gene expression program, with up-regulation of microbial restriction factors such as Nos2. Finally, we found that IFN-γ-primed but not unprimed macrophages largely excluded the activated form of IRF3 from the nucleus following bacterial infection. These data are consistent with a relationship of mutual inhibition between IRF3 and IFN-γ-activated programs, possibly as a component of a partially reversible mechanism for modulating the activity of potent innate immune effectors (such as Nos2) in the context of intracellular infection.

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1-STAT1 loop in lung adenocarcinoma.

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c‐MYC). Additionally, KPNA2 knockdown regulated CSC‐related gene expression in radioresistant cells. Next‐generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2‐mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1‐STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.