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UNLABELLED: Antagonism of the PD-1/PD-L1 axis is a critical therapeutic strategy for patients with advanced bladder cancer. IFNγ functions as a key regulator of PD-L1 in both immune as well as cancer cells. Forkhead box P3 (FOXP3) is a transcription factor synonymous in T regulatory cell function but with increasingly described functions in cancer cells. Here, we investigated the relationship between FOXP3 and PD-L1 in bladder cancer. We showed that FOXP3 is critical in the ability for IFNγ to activate PD-L1 in bladder cancer cells. FOXP3 can bind to the PD-L1 promoter and induces a gene program that leads to regulation of multiple immune-related genes and genes involved in epithelial-to-mesenchymal transition (EMT). Using in vitro and in vivo human and murine models, we showed that FOXP3 can influence bladder cancer EMT as well as promote cancer metastases. Furthermore, FOXP3 may be a convergent factor for multiple activators of PD-L1, including the chemotherapeutic drug cisplatin. SIGNIFICANCE: Historically a key transcription factor driving T regulatory cell function, FOXP3 has an increasingly recognized role in cancer cells. In bladder cancer, we defined a novel mechanism whereby FOXP3 mediates the activation of the immune checkpoint PD-L1 by the cytokine IFNγ. We also showed that FOXP3 induces other immune checkpoints as well as genes involved in EMT, promoting immune resistance and cancer metastases.

The efficacy of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on active and stable lesions was evaluated in patients with active non-segmental vitiligo in a phase 2b trial (NCT03715829). Patients were randomized to placebo or daily ritlecitinib 50 mg (with or without 4-week 100-mg or 200-mg loading dose), 30 mg, or 10 mg for 24 weeks. Active lesions showed greater baseline expression of inflammatory/immune markers IFNG and CCL5, levels of CD103, and T-cell infiltrates than stable lesions. Patients with more active than stable vitiligo lesions showed higher baseline serum levels of CXCL9 and PD-L1, while patients with more stable than active lesions showed higher baseline serum levels of HO-1. At Week 24, ritlecitinib 50 mg significantly stabilized mean percent change from baseline in depigmentation extent in both active lesions and stable lesions vs. placebo-response, with stable lesions showing greater repigmentation. After 24 weeks of treatment, ritlecitinib 50 mg increased expression of melanocyte markers in stable lesions, while Th1/Th2-related and co-stimulatory molecules decreased significantly in both stable and active lesions. Serum from patients with more active than stable lesions showed decreased levels of ICOS and NK cell activation markers. These data, confirmed at transcription/protein levels, indicate that stable lesion repigmentation occurs early with ritlecitinib, while active lesions require stabilization of inflammation first. ClinicalTrials.gov: NCT03715829.

Microglial neuroinflammation appears to be neuroprotective in the early pathological stage, yet neurotoxic, which often precedes neurodegeneration in Alzheimer's disease (AD). However, it remains unclear how the microglial activities transit to the neurotoxic state during AD progression, due to complex neuron-glia interactions. Here, the mechanism of detrimental microgliosis in AD by employing 3D human AD mini-brains, brain tissues of AD patients, and 5XFAD mice is explored. In the human and animal AD models, amyloid-beta (Aβ)-overexpressing neurons and reactive astrocytes produce interferon-gamma (IFNγ) and excessive oxidative stress. IFNγ results in the downregulation of mitogen-activated protein kinase (MAPK) and the upregulation of Kelch-like ECH-associated Protein 1 (Keap1) in microglia, which inactivate nuclear factor erythroid-2-related factor 2 (Nrf2) and sensitize microglia to the oxidative stress and induces a proinflammatory microglia via nuclear factor kappa B (NFκB)-axis. The proinflammatory microglia in turn produce neurotoxic nitric oxide and proinflammatory mediators exacerbating synaptic impairment, phosphorylated-tau accumulation, and discernable neuronal loss. Interestingly, recovering Nrf2 in the microglia prevents the activation of proinflammatory microglia and significantly blocks the tauopathy in AD minibrains. Taken together, it is envisioned that IFNγ-driven Nrf2 downregulation in microglia as a key target to ameliorate AD pathology.

Objective: In this systematic review, we determined the association of selected inflammatory cytokines in blood and urine with diabetic nephropathy (DN). Methods: A literature search using the key terms "tumor necrosis factor-alpha," "interferon-gamma," and "diabetic nephropathy" was conducted in PubMed from December 2020 to January 2021. Relevant findings from these studies were collated for qualitative synthesis. Results: Based on the synthesis of results, TNF-α and IFN-γ levels were consistently higher among DN patients than T2DM patients with no renal complications and healthy individuals. A negative correlation with the estimated glomerular filtration rate (eGFR) and a positive correlation with the albumin-to-creatinine ratio (ACR) were also noted by most studies which assessed their relationship with renal markers. Conclusion: The findings of this systematic review suggest the association of serum and urinary TNF-α and IFN-γ levels with diabetic nephropathy. However, further studies must be conducted to confirm these findings. [ABSTRACT FROM AUTHOR]

It has been demonstrated that CXCL12 inhibits hair growth via CXCR4, and its neutralizing antibody (Ab) increases hair growth in alopecia areata (AA). However, the molecular mechanisms have not been fully elucidated. In the present study, we further prepared humanized CXCL12 Ab for AA treatment and investigated underlying molecular mechanisms using single-cell RNA sequencing. Subcutaneous injection of humanized CXCL12 Ab significantly delayed AA onset in mice, and dorsal skin was analyzed. T cells and dendritic cells/ macrophages were increased in the AA model, but decreased after CXCL12 Ab treatment. Pseudobulk RNA sequencing identified 153 differentially expressed genes that were upregulated in AA model and downregulated after Ab treatment. Gene ontology analysis revealed that immune cell chemotaxis and cellular response to type II interferon were upregulated in AA model but downregulated after Ab treatment. We further identified key immune cellrelated genes such as Ifng, Cd8a, Ccr5, Ccl4, Ccl5, and Il21r, which were colocalized with Cxcr4 in T cells and regulated by CXCL12 Ab treatment. Notably, CD8+ T cells were significantly increased and activated via Jak/Stat pathway in the AA model but inactivated after CXCL12 Ab treatment. Collectively, these results indicate that humanized CXCL12 Ab is promising for AA treatment via immune modulatory effects. [ABSTRACT FROM AUTHOR]

Background: Inflammatory and immune responses in the brain that contribute to various neuropsychiatric disorders may begin as microglial "priming". Interferon (IFN)‐γ is known to cause microglial priming, but the mechanism is unclear. Methods: We examined the effects of IFN‐γ on gene expression, microglial activation, inflammatory and immune responses and activity of the NLRP3 inflammasome in primary microglia and in the brains of mice. Results: Our results showed that treating microglial cultures with IFN‐γ induced a hedgehog‐like morphology and upregulated markers of microglial activation (CD86, CD11b) and pro‐inflammatory molecules (IL‐1β, IL‐6, TNF‐α, iNOS), while downregulating markers of microglial homeostasis (CX3CR1, CD200R1), anti‐inflammatory molecules (MCR1, Arg‐1) and neurotrophic factors (IGF‐1, BDNF). IFN‐γ also upregulated markers of NLRP3 inflammasome activation (NLRP3, caspase‐1, gasdermin D, IL‐18). This particular transcriptional profiling makes IFN‐γ‐primed microglia with exaggerated responses upon lipopolysaccharide (LPS) stimulation. The level of NLRP3, caspase‐1, gasdermin D, IL‐1β, IL‐18, TNF‐α and iNOS in microglia cultures treated with both IFN‐γ and LPS were highest than with either one alone. Injecting IFN‐γ into the lateral ventricle of mice induced similar morphological and functional changes in hippocampal microglia as in primary microglial cultures. The effects of IFN‐γ on NLRP3 inflammasome and microglia from cultures or hippocampus were abolished when STAT1 was inhibited using fludarabin. Injecting mice with IFN‐γ alone or together with LPS induced anxiety‐ and depression‐like behaviors and impaired hippocampus‐dependent spatial memory; these effects were mitigated by fludarabin. Conclusions: IFN‐γ primes microglia by activating STAT1, which upregulates genes that activate the NLRP3 inflammasome. Inhibiting the IFN‐γ/STAT1 axis may be a way to treat neurodegenerative diseases and psychiatric disorders that involve microglial priming. [ABSTRACT FROM AUTHOR]

The contribution of immunoregulatory cytokines to the formation of cold airway hyperresponsiveness in asthma patients is important for understanding approaches to therapy. Aim of the study was to investigate the interaction of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) with bronchial phagocytes in non-allergic asthma patients depending on the airway response to cold air. Material and methods. In 41 patients with asthma, standard monitoring of clinical symptoms of the disease was performed with assessment of the level of asthma control using the Asthma Control Test (ACT) questionnaire, basic spirometry, collection of induced and spontaneously produced sputum, as well as exhaled breath condensate (EBC), and a bronchoprovocation test of isocapnic hyperventilation with cold (–20 ºС) air (IHCA), the content of TNF-α, IFN-γ in EBC and cellular composition of sputum. Results. Cold airway hyperresponsiveness was revealed in 15 patients (group 1), and in 26 patients (group 2) bronchial reaction to IHCA was absent. There were no intergroup differences in the level of asthma control (17.1 ± 0.89 and 18.7 ± 0.63 ACT points, respectively; p > 0.05) and indices of lung function (FEV1 91.2 ± 4.07 and 98.8 ± 2.61 %, respectively; p > 0.05). In response to IHCA in sputum of group 1 patients, the number of neutrophils significantly increased, the number of macrophages, structurally integral epithelial cells decreased, proportionally to this, the level of IFN-γ increased significantly in EBC, TNF-α content did not change. Conclusions. The response to isocapnic hyperventilation with cold air in non-allergic asthma patients with cold airway hyperresponsiveness is characterized by an increase in the level of IFN-γ in the exhaled breath condensate with no significant changes in the content of TNF-α. The increase in the proportion of neutrophils in sputum under the influence of cold stimulus is accompanied by a decrease in the number of macrophages and structurally integral epithelial cells because of inflammatory damage, destruction and cytolysis.

Abstract This study examined the associations between tobacco smoking and serum cotinine levels, an objective biochemical measure of tobacco smoke exposure, with markers of inflammation, i.e., interferon-gamma (IFN-γ), interleukin 10 (IL-10), interleukin 2 (IL-2) and tumour necrosis factor-alpha (TNF-α) in people living with HIV (PLWH).These specific markers were selected because of their hypothesised associations with smoking, PLWH and their outcomes. In a random sample of ≥ 18-year-old PLWH receiving care at 17 public healthcare facilities across the Western Cape Province in South Africa, data collection included self-reported smoking history, and serum levels of cotinine and selected inflammatory markers. The inflammatory marker data were log transformed because of the skewedness of their distribution. Linear regression models (1) adjusted for age and gender, and (2) fully adjusted for age, gender, current alcohol use, body mass index and CD4 counts were used to examine the associations between smoking tobacco or serum cotinine and inflammatory markers. Level of significance was p

ObjectivesTo assess associations between disease severity in index TB patients and QuantiFERON-TB Gold Plus (QFT-Plus) results in contacts, and predictors for QFT-Plus conversion in contacts over 6-12 months.MethodsTB patients (n = 295) and the contacts (n = 1051) were enrolled during 2018-2021 with QFT-Plus performed at baseline and months 6 and 12. A strong CD8 response was defined as TB2 interferon gamma (IFN-γ) response minus TB1 >0.6 IU/ml and stringent conversion as change from QFT-plus negative to high-positive QFT-Plus (TB1 or TB2 IFN-γ responses >0.7 IU/ml).ResultsContacts with index TB patients with sputum smear >1+ was associated with positive QFT-Plus compared to those without (p

Background and Objectives: IFNγ is a pleiotropic cytokine and through the regulation of immunologically relevant genes, they coordinate a wide range of cellular programs.[1] As a potent pro-inflammatory cytokine, it plays a pivotal role to induce depigmentation in vitiligo. In this study, we aim to assess the role of IFNγ in the activity, duration and extent of the disease and the antagonistic action of IL-10 is also assessed in vitiligo. Materials and Methods: A case-control study was conducted with 100 study participants with 50 cases clinically diagnosed as Vitiligo and 50 controls. All patients underwent complete evaluation with detailed demographic parameters, history and physical examination. The severity of the disease was assessed clinically by Vitiligo Area Scoring Index (VASI) and Vitiligo Disease Activity Score (VIDA). And blood investigations done were IFN-γ and IL-10. Results: We observed significantly higher levels of serum IFNγ levels in the patient group when compared with those of the normal controls (p=0.002) and showed a positive correlation with the activity and severity of the disease with a significant VASI (p=0.05) and VIDA score (p=

Background and Aim: In chronic viral infections, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) significantly suppress immune responses. The CTLA-4 receptor abundance in regulatory T cells showed a positive association with viral load and a negative association with interferon-gamma (IFN-γ) production in bovine leukemia virus (BLV)-infected cattle. Blocking this receptor boosted IFN-γ production, recovering immune response against this illness. In human cancer patients, not everyone responded positively to non-immunotherapy using CTLA-4 receptor antibodies. The present study analyzed the synergistic effects of CTLA-4 and PD-L1 receptor blockade on IFN-γ production in BLV+ cattle in vitro. Materials and Methods: The genes for bovine CTLA-4 and PD-L1 were artificially produced. The amino acid sequences of the extracellular receptor domains were sourced from the National Center for Biotechnology Information PubMed database. The western blotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were employed for the characterization of recombinant CTLA-4 (rCTLA-4) and recombinant PD-L1 (rPD-L1) proteins. The immunoinhibitory effects of recombinant proteins in Staphylococcus enterotoxin B (SEB)-stimulated cattle peripheral blood mononuclear cells (PBMCs) were investigated. Enzyme-linked immunosorbent assay (ELISA) was used to analyze monoclonal antibodies against rCTLA-4 and rPD-L1. Antibodies generated from peripheral blood mononuclear cells of healthy and BLV-seropositive cows were employed to evaluate their blocking capabilities. Results: The resulting recombinant proteins specifically reacted with commercial homogeneous monoclonal antibodies (mAbs) using ELISA and anti-His-tag mAbs using western blotting. Analysis of the proteins using LC-MS/MS revealed correspondence with the sequences of rCTLA-4 and rPD-L1 located in the Mascot database. rCTLA-4 and rPD-L1 proteins inhibited IFN-γ production in bovine PBMCs of activated SEB. When PBMCs from cows were cultured with activated SEB containing rCTLA-4 and rPD-L1, the mAbs increased IFN-γ production in PBMCs. The combined cultivation of mAbs and PBMCs from BLV+ cattle enhanced IFN-γ production in the cells. Conclusion: These findings suggest that the combined blockade of bovine CTLA-4 and PD-L1 receptors can be used as a therapy for bovine leukemia. However, it was shown that a single PBMC sample from a BLV-positive donor did not amplify the synergistic effect. Therefore, it is necessary to perform further studies on a larger population and assessing a wider range of cytokines.

Abstract Background and objectives Oral lichen planus (OLP) is a common, prevalent, immune-mediated, inflammatory disease affecting both the skin and oral mucosa and is considered one of the potentially malignant diseases. Since OLP is regarded as an immunologically mediated disease, some studies suggest the use of vitamin D (VD) for its management as it exhibits immune-modulatory, anti-inflammatory, and antimicrobial properties, as well as anti-proliferative, pro-differentiative, and anti-angiogenic effects. VD has demonstrated a suppressive effect on TH1 pro-inflammatory cytokines, including IFN-γ while augmenting the secretion of anti-inflammatory cytokines. At the same time, VD deficiency is a prevalent public issue. Therefore, the present study aimed to investigate the role of VD as an adjunct to steroids in the management of VD-deficient OLP patients as well as its inhibitory effect on IFN-γ through measurement of salivary and tissue IFN-γ levels in OLP patients. Methods A total of 40 patients with ulcerative or erythematous OLP, diagnosed according to the World Health Organization’s (WHO) modified criteria for OLP, were randomly allocated into one of the two study groups to receive either systemic steroids in addition to VD supplements (Group A) or systemic steroids only (Group B). Blood samples were collected for the measurement of serum VD level (SVDL) using the enzyme-linked immunosorbent assay (ELISA) to involve only patients with VD deficiency or insufficiency (≤ 30 ng/ml). Clinical evaluation of the lesion involved objective signs and subjective symptoms. Also, changes in salivary and tissue INF-γ levels (in pg/mL and pg/mg, respectively) were determined using the ELISA technique. All parameters were measured at baseline and after 4 weeks of treatment. The clinical pharmacy team devised a checklist to record all team interventions. The interventions were categorized into six domains, including drug interactions and/or adverse reactions, medication dose issues, drug selection issues, support with medication history, patient-related concerns, and suggestions for dental medication. Results After one month of treatment, a significantly greater number of patients in group A showed complete pain relief and resolution of clinical lesions, as well as a greater number of patients showing a reduction in the clinical severity of lesions than in group B (P = 0.005). Also, there was a statistically significant reduction in average VAS pain scores and clinical scores in group A compared to group B after 1 month of treatment (P = 0.001 and 0.002, respectively). Furthermore, there was a statistically significant greater reduction in salivary and tissue IFN-γ levels in group A than in group B (P ≤ 0.001 and 0.029, respectively) after 1 month of treatment. Conclusion Current evidence suggests a significant preventive and therapeutic role for VD as an adjunct to standard therapies indicated for OLP lesions. These protective and therapeutic functions are achieved through the suppressive effect of VD on pro-inflammatory cytokines, particularly IFN-γ. Also, salivary IFN-γ appears to be a valuable prognostic marker for monitoring the progression of OLP. In addition, the inter-professional collaboration between dentists and clinical pharmacists helped to deliver complete, patient-centered primary care and ensured the quality of the medications included in patient kits, thus improving patient treatment and management. Nevertheless, further studies with larger sample sizes, longer follow-ups, and standardized designs may still be needed.

Background/Aims The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. Methods Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors. The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. Results We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. Conclusions Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.

We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors. [ABSTRACT FROM AUTHOR]

Background and Aim: In chronic viral infections, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) significantly suppress immune responses. The CTLA-4 receptor abundance in regulatory T cells showed a positive association with viral load and a negative association with interferon-gamma (IFN-γ) production in bovine leukemia virus (BLV)-infected cattle. Blocking this receptor boosted IFN-γ production, recovering immune response against this illness. In human cancer patients, not everyone responded positively to non-immunotherapy using CTLA-4 receptor antibodies. The present study analyzed the synergistic effects of CTLA-4 and PD-L1 receptor blockade on IFN-γ production in BLV+ cattle in vitro. Materials and Methods: The genes for bovine CTLA-4 and PD-L1 were artificially produced. The amino acid sequences of the extracellular receptor domains were sourced from the National Center for Biotechnology Information PubMed database. The western blotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were employed for the characterization of recombinant CTLA-4 (rCTLA-4) and recombinant PD-L1 (rPD-L1) proteins. The immunoinhibitory effects of recombinant proteins in Staphylococcus enterotoxin B (SEB)-stimulated cattle peripheral blood mononuclear cells (PBMCs) were investigated. Enzyme-linked immunosorbent assay (ELISA) was used to analyze monoclonal antibodies against rCTLA-4 and rPD-L1. Antibodies generated from peripheral blood mononuclear cells of healthy and BLV-seropositive cows were employed to evaluate their blocking capabilities. Results: The resulting recombinant proteins specifically reacted with commercial homogeneous monoclonal antibodies (mAbs) using ELISA and anti-His-tag mAbs using western blotting. Analysis of the proteins using LC-MS/MS revealed correspondence with the sequences of rCTLA-4 and rPD-L1 located in the Mascot database. rCTLA-4 and rPD-L1 proteins inhibited IFN-γ production in bovine PBMCs of activated SEB. When PBMCs from cows were cultured with activated SEB containing rCTLA-4 and rPD-L1, the mAbs increased IFN-γ production in PBMCs. The combined cultivation of mAbs and PBMCs from BLV+ cattle enhanced IFN-γ production in the cells. Conclusion: These findings suggest that the combined blockade of bovine CTLA-4 and PD-L1 receptors can be used as a therapy for bovine leukemia. However, it was shown that a single PBMC sample from a BLV-positive donor did not amplify the synergistic effect. Therefore, it is necessary to perform further studies on a larger population and assessing a wider range of cytokines. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease that is characterized by the infiltration of peripheral immune cells into the central nervous system (CNS), secretion of inflammatory factors, demyelination, and axonal degeneration. Inflammatory mediators such as cytokines alter cellular function and activate resident CNS cells, including astrocytes. Notably, interferon (IFN)γ is a prominent pleiotropic cytokine involved in MS that contributes to disease pathogenesis. Astrocytes are dynamic cells that respond to changes in the cellular microenvironment and are highly responsive to many cytokines, including IFNγ. Throughout the course of MS, intrinsic cell stress is initiated in response to inflammation, which can impact the pathology. It is known that cell stress is pronounced during MS; however, the specific mechanisms relating IFNγ signaling to cell stress responses in astrocytes are still under investigation. This review will highlight the current literature regarding the impact of IFNγ signaling alone and in combination with other immune mediators on astrocyte synthesis of free oxygen radicals and cell death, and cover what is understood regarding astrocytic mitochondrial dysfunction and endoplasmic reticulum stress. [ABSTRACT FROM AUTHOR]

Endometrial hyperplasia (EH), an abnormal proliferation of the endometrial cells, is considered as one of the most common causes of abnormal uterine bleeding. Previous studies have reported that melatonin plays a fundamental role in disease treatment. This study aimed the comparison of the effects of progesterone, as the most common therapeutic approach, and melatonin with progesterone alone in improvement of non-atypical endometrial hyperplasia (NEH) and changes in pro-inflammatory cytokine levels. Study population consisted of 40 patients with NEH. Patients were divided into two groups, including 20 subjects treated with melatonin and progesterone and 20 individuals treated with progesterone alone. The blood and endometrial sampling was performed from participants before and after a three-month treatment. The histological examination was microscopically done. The serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were measured using ELISA. There was no significant difference in the diabetes status and mean age between patients treated with progesterone and melatonin and those treated with progesterone alone. The improvement rate in the EH was significantly higher in individuals treated with progesterone and melatonin than those treated with progesterone alone (p < 0.05). Additionally, the patients treated with progesterone and melatonin showed significant increases inIFN-γ and TNF-αlevels compared to the control group (p < 0.001-P < 0.05). Melatonin supplementation has a beneficial effect in the treatment of EH due perhaps to enhance the level of IFN-γ and TNF-α. [ABSTRACT FROM AUTHOR]

In this study, we investigated the anti-microbial and anti-inflammatory activities of the cosmopolite macrolichens Usnea articulata (L.) Hoffm., Umbilicaria crustulosa (Ach.) Lamy and Bryoria fuscescens (Gyeln.) Brodo & D.Hawksw hydroalcoholic extracts to contribute the potential pharmacological uses of lichens. In vitro antimicrobial activities of ethanol extracts against Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus, and the yeast Candida albicans were presented using the Broth microdilution method. The most effective lichen extract against gram-positive bacteria S. aureus was U. articulata ethanol extract with a MIC value of 0.125 mg/ml. U. articulata and B. fuscences extracts have similar anti-fungal activities despite having MIC values of 0.5 mg/ml. The anti-inflammatory effects of the extracts on Lipopolysaccharide/Interferon-gamma (LPS/IF-γ) induced macrophage-like cellular systems (BV-2 microglia and RAW 264,7 macrophages) were evaluated by measuring P38 mitogen-activated protein kinase phosphorylation (P38MAPK), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase 2 (NOS2) mRNA and protein expression. Especially, Usnea and Umbilicaria extracts also attenuated the LPS/IF-γ induced increase in P38MAPK phosphorylation, COX-2, and NOS2 expression in both macrophage-like cells without any cytotoxicity. According to the results of our study, we suggest that the anti-inflammatory mechanism of lichen extracts might result from the inhibition of P38MAPK phosphorylation through a reduction in COX-2 and NOS2 expressions. [ABSTRACT FROM AUTHOR]

Near‐infrared photoimmunotherapy (NIR‐PIT) is a new type of cancer therapy that employs antibody‐IRDye700DX conjugates (AbPCs) and near‐infrared (NIR) light at a wavelength of 689 nm, the excitation wavelength of IR700. Administered intravenously, injected AbPCs bind specifically to cells expressing the target antigen, whereupon NIR light exposure causes rapid, selective killing. This process induces an anticancer T cell response, leading to sustained anticancer host immune response. Programmed cell death ligand‐1 (PD‐L1) is a major inhibitory immune checkpoint molecule expressed in various cancers. In this study, we first assessed the efficacy of PD‐L1‐targeted NIR‐PIT (αPD‐L1‐PIT) in immune‐competent tumor mouse models. αPD‐L1‐PIT showed a significant therapeutic effect on the tumor models with high PD‐L1 expression. Furthermore, αPD‐L1‐PIT induced an abscopal effect on distant tumors and long‐term immunological memory. In contrast, αPD‐L1‐PIT was not as effective for tumor models with low PD‐L1 expression. To improve the efficacy of PD‐L1‐targeted NIR‐PIT, PEGylated interferon‐gamma (IFNγ) was administered with αPD‐L1‐PIT. The combination therapy improved the treatment efficacy by increasing PD‐L1 expression leading to more efficient cell killing by αPD‐L1‐PIT. Furthermore, the PEGylated IFNγ led to a CD8+ T cell‐dominant tumor microenvironment (TME) with an enhanced anticancer T cell response after αPD‐L1‐PIT. As a result, even so‐called cold tumors exhibited complete responses after αPD‐L1‐PIT. Thus, combination therapy of PEGylated IFNγ and PD‐L1‐targeted NIR‐PIT has the potential to be an important future strategy for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

ObjectiveResearch has established links between social isolation and heightened levels of proinflammatory cytokines (e.g., interleukin-6 [IL-6], tumour necrosis factor alpha [TNF-α]). Recent advances allow for the examination of cytokines that may also play a role in antiviral immunity (interferon-gamma [IFN-γ]). The present work explored how various features of social experience relate to circulating cytokines in breast cancer survivors, as inflammation has been tied to cancer recurrence and mortality.DesignFemale breast cancer survivors (N = 43) completed a blood draw to assess circulating levels of proinflammatory cytokines (IL-6, TNF-α) and levels of a cytokine that also relates to antiviral immunity (IFN-γ).Main outcome measuresWe examined associations between cytokines and different aspects of social experience, including household size, psychosocial well-being, and social threat anxiety.ResultsCirculating levels of IFN-γ were associated with larger household size (r = 0.32, p = 0.04) and higher levels of psychosocial well-being (r = 0.33, p = 0.04). Additionally, heightened levels of IL-6 were associated with social threat anxiety (r = 0.38, p = 0.01). Heightened IL-6 was also associated with household size (r = 0.33, p = 0.03).ConclusionThese findings are consistent with work suggesting that antiviral immunity and inflammation may have distinct contributions to the links between social experience and health, particularly for those previously diagnosed with breast cancer.

Immune checkpoint blockade (ICB) treatment has demonstrated excellent medical effects in oncology, and it is one of the most sought after immunotherapies for tumors. However, there are several issues with ICB therapy, including low response rates and a lack of effective efficacy predictors. Gasdermin-mediated pyroptosis is a typical inflammatory death mode. We discovered that increased expression of gasdermin protein was linked to a favorable tumor immune microenvironment and prognosis in head and neck squamous cell carcinoma (HNSCC). We used the mouse HNSCC cell lines 4MOSC1 (responsive to CTLA-4 blockade) and 4MOSC2 (resistant to CTLA-4 blockade) orthotopic models and demonstrated that CTLA-4 blockade treatment induced gasdermin-mediated pyroptosis of tumor cells, and gasdermin expression positively correlated to the effectiveness of CTLA-4 blockade treatment. We found that CTLA-4 blockade activated CD8+ T cells and increased the levels of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) cytokines in the tumor microenvironment. These cytokines synergistically activated the STAT1/IRF1 axis to trigger tumor cell pyroptosis and the release of large amounts of inflammatory substances and chemokines. Collectively, our findings revealed that CTLA-4 blockade triggered tumor cells pyroptosis via the release of IFN-γ and TNF-α from activated CD8+ T cells, providing a new perspective of ICB.

IntroductionCortisol rhythm disruptions predict early mortality in renal, colorectal, lung, and metastatic breast cancer. In head and neck cancer (HNC), various cortisol indices are known to correlate with adverse psychological and biological (e.g., inflammatory) outcomes, but links to mortality have yet to be demonstrated. We hypothesize that the prognostic value of diurnal cortisol aberrations will hold in HNC. Prior work leads us to predict that flattened or elevated diurnal cortisol profiles will be associated with elevations of serum inflammatory and tumor-promoting cytokines in this population, and that these immune markers would themselves predict poor progression-free survival.MethodWe prospectively recruited a pilot sample of HNC patients (N=40) at a multidisciplinary HNC clinic. Most patients presented with late-stage oral/oropharyngeal cancer, were older than 50, male, and subsequently received combined-modality (surgery and/or radiotherapy with or without chemotherapy) treatment with curative intent. Saliva was collected twice daily for six days to assess diurnal slope, mean, waking, and evening cortisol levels. Serum was assayed for an exploratory panel of inflammatory and tumor-promoting cytokines. Two years post study-entry, disease progression and survivorship status were abstracted from medical records. Bivariate correlations, linear regressions, and Cox Proportional Hazards models tested hypotheses.ResultsElevations of evening cortisol and diurnal mean levels were each associated with shorter progression-free survival (evening: Hazard Ratio [HR]=1.848, 95% Confidence Interval [CI]=1.057-3.230, p=.031; diurnal mean: HR=2.662, 95% CI=1.115-6.355, p=.027). Bivariate correlations revealed that higher levels of the serum inflammatory marker interferon (IFN)-γ were linked with elevated evening (r=.405, p=.014) and mean (r=.459, p=.004) cortisol. Higher expression of IFN-γ also predicted poorer progression-free survival (HR=4.671, 95% CI=1.409-15.484, p=.012).DiscussionElevated evening and diurnal mean cortisol were both prognostic; suggesting cortisol secretion is both dysregulated and elevated among patients who subsequently experienced accelerated disease progression. These exploratory data from 40 HNC patients mirror relationships between cortisol and survival identified among patients with numerous other tumor types. This pilot study highlights the need for research on effects of cortisol rhythm disruption among HNC patients. Future research in larger samples should also examine the role of inflammatory and tumor-promoting factors–both systemically and within the tumor microenvironment–as potential mediators of cortisol rhythm disruption.

Summary: Background: Endotype classification becomes the cornerstone of understanding sepsis pathogenesis. Macrophage activation-like syndrome (MALS) and immunoparalysis are the best recognized major endotypes, so far. Interferon-gamma (IFNγ) action on tissue macrophages stimulates the release of the cytotoxic chemokine CXCL9. It was investigated if this mechanism may be an independent sepsis endotype. Methods: In this cohort study, 14 patient cohorts from Greece, Germany and Italy were studied. The cohorts were 2:1 randomly split into discovery and validation sets. Sepsis was defined by the Sepsis-3 definitions and blood was sampled the first 24 h from meeting the Sepsis-3 definitions. Concentrations of IFNγ, CXCL9, IP-10 (IFNγ induced protein-10), soluble CD163 and ferritin were measured. The endotype of IFNγ-driven sepsis (IDS) was defined in the discovery set as the combination of a) blood IFNγ above a specified cut-off associated with the minimal risk for immunoparalysis (defined as ≥8000 HLA-DR receptors on CD45/CD14-monoytes); and b) increase of CXCL9. Results were compared to the validation set. Findings: 5503 patients were studied; 3670 in the discovery set and 1833 in the validation set. IDS was defined as IFNγ more than 3 pg/ml and CXCL9 more than 2200 pg/ml. The frequency of IDS in the discovery set was 19.9% (732 patients; 95% confidence intervals-CIs 18.7–21.3%) and in the validation set 20.0% (366 patients; 95% CIs 18.2–21.9%). Soluble CD163, a marker of macrophage activation, was greater in IDS and IDS had features distinct from MALS. The mortality in IDS patients was 43.0% (315 patients; 95% CIs 39.5–46.6%) in the discovery set and 40.4% in the validation set (148 patients; 95% CIs 35.5–45.5%) (p = 0.44 compared to patients of the discovery set). IDS was an independent risk factor for death in the presence of other endotypes, severity scores and organ dysfunctions of the multivariate model [hazard ratio 1.71 (95% CIs 1.45–2.01) in the discovery set and 1.70 (95% CIs 1.34–2.16) in the validation set]. Decreases of IFNγ and CXCL9 blood levels within the first 72 h were associated with better outcome. Interpretation: IDS is a new sepsis endotype independently associated with unfavorable outcome. Funding: Hellenic Institute for the Study of Sepsis; Horizon 2020 project ImmunoSep; Swedish Orphan BioVitrum AB (publ) and German Federal Ministry of Education and Research.

Summary: Background: Influenza-associated pulmonary aspergillosis (IAPA) is a severe fungal superinfection in critically ill influenza patients that is of incompletely understood pathogenesis. Despite the use of contemporary therapies with antifungal and antivirals, mortality rates remain unacceptably high. We aimed to unravel the IAPA immunopathogenesis as a means to develop adjunctive immunomodulatory therapies. Methods: We used a murine model of IAPA to investigate how influenza predisposes to the development of invasive pulmonary aspergillosis. Immunocompetent mice were challenged with an intranasal instillation of influenza on day 0 followed by an orotracheal inoculation with Aspergillus 4 days later. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (μCT) and fungal burden with bioluminescence imaging (BLI). At endpoint, high parameter immunophenotyping, spatial transcriptomics, histopathology, dynamic phagosome biogenesis assays with live imaging, immunofluorescence staining, specialized functional phagocytosis and killing assays were performed. Findings: We uncovered an early exuberant influenza-induced interferon-gamma (IFN-γ) production as the major driver of immunopathology in IAPA and delineated the molecular mechanisms. Specifically, excessive IFN-γ production resulted in a defective Th17-immune response, depletion of macrophages, and impaired killing of Aspergillus conidia by macrophages due to the inhibition of NADPH oxidase-dependent activation of LC3-associated phagocytosis (LAP). Markedly, mice with partial or complete genetic ablation of IFN-γ had a restored Th17-immune response, LAP-dependent mechanism of killing and were fully protected from invasive fungal infection. Interpretation: Together, these results identify exuberant viral induced IFN-γ production as a major driver of immune dysfunction in IAPA, paving the way to explore the use of excessive viral-induced IFN-γ as a biomarker and new immunotherapeutic target in IAPA. Funding: This research was funded by the Research Foundation Flanders (FWO), project funding under Grant G053121N to JW, SHB and GVV; G057721N, G0G4820N to GVV; 1506114 N to KL and GVV; KU Leuven internal funds (C24/17/061) to GVV, clinical research funding to JW, Research Foundation Flanders (FWO) aspirant mandate under Grant 1186121N/1186123 N to LS, 11B5520N to FS, 1SF2222N to EV and 11M6922N/11M6924N to SF, travel grants V428023N, K103723N, K217722N to LS. FLvdV was supported by a Vidi grant of the Netherlands Association for Scientific Research. FLvdV, JW, AC and GC were supported by the Europeans Union’s Horizon 2020 research and innovation program under grant agreement no 847507 HDM-FUN. AC was also supported by the Fundação para a Ciência e a Tecnologia (FCT), with the references UIDB/50026/2020, UIDP/50026/2020, PTDC/MED-OUT/1112/2021 (https://doi.org/10.54499/PTDC/MED-OUT/1112/2021), and 2022.06674.PTDC (http://doi.org/10.54499/2022.06674.PTDC); and the “la Caixa” Foundation under the agreement LCF/PR/HR22/52420003 (MICROFUN).

Replication complexes (RCs), formed by positive-strand (+) RNA viruses through rearrangements of host endomembranes, protect their replicating RNA from host innate immune defenses. We have shown that two evolutionarily conserved defense systems, autophagy and interferon (IFN), target viral RCs and inhibit viral replication collaboratively. However, the mechanism by which autophagy proteins target viral RCs and the role of IFN-inducible GTPases in the disruption of RCs remains poorly understood. Here, using murine norovirus (MNV) as a model (+) RNA virus, we show that the guanylate binding protein 1 (GBP1) is the human GTPase responsible for inhibiting RCs. Furthermore, we found that ATG16L1 mediates the LC3 targeting of MNV RC by binding to WIPI2B and CAPRIN1, and that IFN gamma-mediated control of MNV replication was dependent on CAPRIN1. Collectively, this study identifies a novel mechanism for the autophagy machinery-mediated recognition and inhibition of viral RCs, a hallmark of (+) RNA virus replication. IMPORTANCE Replication complexes provide a microenvironment important for (+) RNA virus replication and shield it from host immune response. Previously we have shown that interferon gamma (IFNG) disrupts the RC of MNV via evolutionarily conserved autophagy proteins and IFN-inducible GTPases. Elucidating the mechanism of targeting of viral RC by ATG16L1 and IFN-induced GTPase will pave the way for development of therapeutics targeting the viral replication complexes. Here, we have identified GBP1 as the sole GBP targeting viral RC and uncovered the novel role of CAPRIN1 in recruiting ATG16L1 to the viral RC.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Toxoplasma virulence depends on its ability to evade or survive the toxoplasmacidal mechanisms induced by interferon gamma (IFNγ). While many Toxoplasma genes involved in the evasion of the murine IFNγ response have been identified, genes required to survive the human IFNγ response are largely unknown. In this study, we used a genome-wide loss-of-function screen to identify Toxoplasma genes important for parasite fitness in IFNγ-stimulated primary human fibroblasts. We generated gene knockouts for the top six hits from the screen and confirmed their importance for parasite growth in IFNγ-stimulated human fibroblasts. Of these six genes, three have homology to GRA32, localize to dense granules, and coimmunoprecipitate with each other and GRA32, suggesting they might form a complex. Deletion of individual members of this complex leads to early parasite egress in IFNγ-stimulated cells. Thus, prevention of early egress is an important Toxoplasma fitness determinant in IFNγ-stimulated human cells. IMPORTANCE Toxoplasma infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth. While most Toxoplasma parasites are cleared by this immune response, some can survive by blocking or evading the IFNγ-induced restrictive environment. Many Toxoplasma genes that determine parasite survival in IFNγ-activated murine cells are known but parasite genes conferring fitness in IFNγ-activated human cells are largely unknown. Using a Toxoplasma adapted genome-wide loss-of-function screen, we identified many Toxoplasma genes that determine parasite fitness in IFNγ-activated human cells. The gene products of four top hits play a role in preventing early parasite egress in IFNγ-stimulated human cells. Understanding how IFNγ-stimulated human cells inhibit Toxoplasma growth and how Toxoplasma counteracts this, could lead to the development of novel therapeutics.

Background Chemotherapy is a crucial treatment for patients with acute myeloid leukemia (AML), with bone marrow suppression as the most common toxic side effect. The development, proliferation, and differentiation of various types of blood cells are regulated by a variety of hematopoietic factors. However, it is not clear whether there is a difference in the expression level of hematopoietic factors after chemotherapy in patients with AML at different altitudes and the change pattern has not been clarified. Objective To investigate whether there are differences in the expression levels of hematopoietic factors in bone marrow and serum before and after chemotherapy and their changes in patients with AML at varying altitudes. Methods A total of 28 AML patients (non-M3 type) treated for the first time in the Department of Hematology at Qinghai Provincial People's Hospital (1 501 to 2 500 m in the middle altitude area) and the Department of Hematology of Xi-Jing Hospital, Air Force Medical University of PLA (500 to 1 500 m in low altitude area) from 2021 to 2022 were selected as the research subjects, and according to the altitude of the hospitals they visited, the AML patients were divided into the middle altitude group (13 cases) and the low altitude group (15 cases). The WHO grading criteria for acute and subacute toxicity of anticancer drugs was used to evaluate the myelosuppressive scores of the patients on days 8, 14, and 28 of chemotherapy. The expression levels of erythropoietin (EPO), FMS-like tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (TPO), and interferon γ (IFN-γ) in serum and bone marrow before chemotherapy and on day 8, day 28 of chemotherapy were detected and compared by enzyme-linked immunosorbent assay. Results On days 14 and 28 of chemotherapy, the myelosuppressive scores of patients in the low-altitude group were higher than those in the middle-altitude group (Z=-1.975, P=0.048; Z=-2.049, P=0.040). On day 28 of chemotherapy, the serum and bone marrow EPO expression levels in the low-altitude group were higher than those in the middle-altitude group (P0.05) . Conclusion The expression levels of haemopoietic factors including EPO, Flt3-L, and TPO were higher during the recovery of myelosuppression after chemotherapy in AML patients at low altitude, compared to those at middle altitude. There was no significant difference between the two groups in terms of the hematopoietic inhibitory factor IFN-γ. Myelosuppression grades and degree after chemotherapy are more severe in AML patients at middle altitude than at low altitude.