Structural Evaluations of a Selective Human STINGA230 Agonist and Its Use in Macrophage Immunotherapies.

Previously we identified a non-nucleotide agonist BDW568 that selectively activates the human STINGA230 allele. Here, we further characterized the mechanism of BDW568 and highlighted its potential use for selectively controlling the activation of engineered macrophages that constitutively express STINGA230 as a genetic adjuvant. We obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite) at 1.95 Å resolution. Structure-activity relationship studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining activity. We demonstrated that BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230. In contrast, BDW568 could not stimulate innate immune responses in human primary peripheral blood mononuclear cells in healthy donors in the absence of a STINGA230 allele. This high STING variant specificity suggested a promising application of STINGA230 agonists in macrophage-based therapeutic approaches.

Structural and Biological Evaluations of a Non-Nucleoside STING Agonist Specific for Human STINGA230 Variants.

Previously we identified a non-nucleotide tricyclic agonist BDW568 that activates human STING (stimulator of interferon genes) gene variant containing A230 in a human monocyte cell line (THP-1). STINGA230 alleles, including HAQ and AQ, are less common STING variants in human population. To further characterize the mechanism of BDW568, we obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite of BDW568) at 1.95 Å resolution and found the planar tricyclic structure in BDW-OH dimerizes in the STING binding pocket and mimics the two nucleobases of the endogenous STING ligand 2',3'-cGAMP. This binding mode also resembles a known synthetic ligand of human STING, MSA-2, but not another tricyclic mouse STING agonist DMXAA. Structure-activity-relationship (SAR) studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining the compound's activity. BDW568 could robustly activate the STING pathway in human primary peripheral blood mononuclear cells (PBMCs) with STINGA230 genotype from healthy individuals. We also observed BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230, suggesting its potential use to selectively activate genetically engineered macrophages in macrophage-based approaches, such as chimeric antigen receptor (CAR)-macrophage immunotherapies.

Autophagy inducer rapamycin treatment reduces IFN-I-mediated Inflammation and improves anti-HIV-1 T cell response in vivo.

A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4+ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1-infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I-mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.

Cannabidiol modulates expression of type I IFN response genes and HIV infection in macrophages.

Cannabis (Cannabis sativa) is a widely used drug in the United States and the frequency of cannabis use is particularly high among people living with HIV (PLWH). One key component of cannabis, the non-psychotropic (-)-cannabidiol (CBD) exerts a wide variety of biological actions, including anticonvulsive, analgesic, and anti-inflammatory effects. However, the exact mechanism of action through which CBD affects the immune cell signaling remains poorly understood. Here we report that CBD modulates type I interferon responses in human macrophages. Transcriptomics analysis shows that CBD treatment significantly attenuates cGAS-STING-mediated activation of type I Interferon response genes (ISGs) in monocytic THP-1 cells. We further showed that CBD treatment effectively attenuates 2'3-cGAMP stimulation of ISGs in both THP-1 cells and primary human macrophages. Interestingly, CBD significantly upregulates expression of autophagy receptor p62/SQSTM1. p62 is critical for autophagy-mediated degradation of stimulated STING. We observed that CBD treated THP-1 cells have elevated autophagy activity. Upon 2'3'-cGAMP stimulation, CBD treated cells have rapid downregulation of phosphorylated-STING, leading to attenuated expression of ISGs. The CBD attenuation of ISGs is reduced in autophagy deficient THP-1 cells, suggesting that the effects of CBD on ISGs is partially mediated by autophagy induction. Lastly, CBD decreases ISGs expression upon HIV infection in THP-1 cells and human primary macrophages, leading to increased HIV RNA expression 24 hours after infection. However, long term culture with CBD in infected primary macrophages reduced HIV viral spread, suggesting potential dichotomous roles of CBD in HIV replication. Our study highlights the immune modulatory effects of CBD and the needs for additional studies on its effect on viral infection and inflammation.

Neutrophil dysfunction predicts 90-day survival in patients with acute on chronic liver failure: A longitudinal case-control study.

BACKGROUND AND AIM: Innate immune disarray is a key component in the development and progression of acute on chronic liver failure (ACLF) and predisposition to infections. We evaluated the neutrophil dysfunction and its impact on outcomes in patients with ACLF. METHODS: Forty patients with acute decompensation of cirrhosis (10 each of grades 0, 1, 2, and 3 ACLF) and 10 healthy controls were prospectively evaluated for neutrophil immunophenotype (NP), neutrophil phagocytic capacity (NPC), and oxidative burst (OB) in both resting and stimulated conditions. The patients were followed up for 90 days or until death or transplant, whichever was earlier. RESULTS: NP was normal (in %) and NPC (in mean fluorescence intensity [MFI]) was better in controls compared to patients with ACLF (83.74 ± 12.38 vs 63.84 ± 22.98; P = 0.007 and 98.33 ± 130.60 vs 18.73 ± 17.88, P = 0.001, respectively). Resting OB was higher in patients with ACLF compared to controls (97 ± 4.9% vs 91 ± 9%; P = 0.034), but it failed to increase further after stimulation, suggesting an immune exhaustion. NP was normal (in %) and NPC (in MFI) was better in 90-day survivors compared to nonsurvivors (78 ± 11.9 vs 62.2 ± 24.11, P = 0.02 and 33.3 ± 22.7 vs 16.36 ± 13.3; P = 0.004, respectively). Phenotypically normal neutrophils >71.7% had 78.6% sensitivity and 65.4% specificity with an area under receiver operating curve (AUROC) of 0.70 (95% confidence interval [CI]: 0.55-0.90); P = 0.017, and NPC >17.32. MFI had 71.4% sensitivity and 69.6% specificity with an AUROC of 0.73 (95% CI: 0.54-0.86), P = 0.035, in predicting 90-day survival. CONCLUSION: Neutrophils have impaired bactericidal function in patients with ACLF compared to healthy adults. Neutrophil phenotype and phagocytic capacity may be used to predict 90-day survival in patients with ACLF.

A juggernaut of innate & adaptive immune cells in chronic hepatitis C.

Hepatitis C virus (HCV) is a small positive-sense, single-stranded RNA virus, the causal organism for chronic hepatitis. Chronic hepatitis leads to inflammation of liver, causing cirrhosis, fibrosis and steatosis, which may ultimately lead to liver cancer in a few cases. Innate and adaptive immune responses play an important role in the pathogenesis of HCV infection, thus acting as an important component in deciding the fate of the disease. Numerous studies have indicated that the derangement of these immune responses results in the persistence of infection leading to chronic state of the disease. Interactions between virus and host immune system generally result in the elimination of virus, but as the virus evolves with different evading mechanisms, it makes environment favourable for its survival and replication. It has been reported that HCV impairs the immune system by functional modulation of the cells of innate as well as adaptive immune responses, resulting in chronic state of the disease, influencing the response to antiviral therapy in these patients. These defects in the immune system lead to suboptimal immune responses and therefore, impaired effector functions. This review highlights the involvement or association of different immune cells such as natural killer cells, B cells, dendritic cells and T cells in HCV infection and how the virus plays a role in manipulating certain regulatory mechanisms to make these cells dysfunctional for its own persistence and survival.

Reversal of endothelial dysfunction post-immunosuppressive therapy in adult-onset podocytopathy and primary membranous nephropathy.

BACKGROUND AND AIMS: The effect of nephrotic syndrome (NS) and its treatment on endothelial dysfunction is not evident. This study assessed endothelial dysfunction in adult-onset NS and its impact of immunosuppressive therapy. METHODS: Newly diagnosed patients with adult-onset NS (podocytopathy and primary membranous nephropathy (PMN)) and normal renal function were enrolled. Flow mediated vasodilatation (FMD) assessed endothelial function and CD4+CD28null T cells, E-selectin and pulse wave velocities (PWV) were measured at baseline and after treatment to characterize this further. Monitoring included monthly proteinuria, serum albumin, creatinine and lipid profile at baseline and post-treatment. The healthy control (HC) included 25 voluntary kidney donors who were assessed for markers of endothelial dysfunction. RESULTS: Fifty participants with new-onset NS were studied. Amongst the NS group, 26 (52%) patients had PMN, while the remaining 24 (48%) had podocytopathy. Twenty-one (88%) patients in the podocytopathy and 18 (69%) patients in the PMN cohort were in either complete or partial remission at the end of 8 months. FMD at baseline in NS patients was significantly lower as compared to HC (p = 0.002) while PWV (p = 0.007), E-selectin (p < 0.001) and CD4+CD28null T cells (p = 0.003) were significantly higher as compared with HC. Following treatment with immunosuppressive medication, FMD increased from 3 to 8% (p < 0.001). PWV also improved from a baseline of 7.70 to 6.65 m/s (p = 0.001). At the end of 8 months, E-selectin decreased significantly from 127 to 82 ng/ml (p = 0.002) while the CD4+CD28null T cell population reduced from 5.20 to 3.70% (p = 0.032) of total CD4+ cells. In the PMN cohort, despite significant reduction, E-selectin and CD4+CD28null T cells at follow-up remained higher than in healthy controls. CONCLUSION: Immunosuppressive treatment contributes substantially to the improvement of endothelial dysfunction present at baseline in NS patients. Persistent subtle endothelial dysfunction remains in the sub-group of patients with PMN.

Change in serum levels of inflammatory markers reflects response of percutaneous catheter drainage in symptomatic fluid collections in patients with acute pancreatitis.

BACKGROUND: Percutaneous catheter drainage (PCD) is used as the first step in the management of symptomatic fluid collections in patients with acute pancreatitis (AP). There are limited data on the effect of PCD on inflammatory markers. AIM: To study the effects of PCD on serum levels of C-reactive protein (CRP), IL-6, and IL-10 and its correlation with the outcome. METHODS: Consecutive patients of AP with symptomatic fluid collections undergoing PCD were evaluated for serum levels of CRP, IL-6, and IL-10 before PCD and at 3 and 7 days after PCD. Resolution of organ failure (OF), sepsis, and pressure symptoms was considered to demonstrate the success of PCD. Changes in levels following PCD were correlated with outcome. RESULTS: Indications of PCD in 59 patients (age 38.9 ± 13.17 years, 49 male) were suspected/documented infected pancreatic necrosis (n = 45), persistent OF (n = 40), and pressure symptoms (n = 7). A total of 49 (83.1%) patients improved with PCD, five patients required surgery, and six died. A significant difference was noted between baseline levels of CRP (P = 0.026) and IL-6 (P = 0.013) among patients who improved compared to those who worsened following PCD. Significant decrease (P < 0.01) of all three markers on day 3 of PCD insertion, with further decrease (P < 0.01) on day 7, was noted. The percentage of the decrease of IL-6 levels on day 3 and of CRP on day 7 correlated with the outcome. CONCLUSION: PCD is associated with a significant decrease in CRP, IL-6, and IL-10 levels. Percentage decrease in IL-6 on day 3 and CRP on day 7 correlated with the outcome of patients managed with PCD.

Monocyte human leukocyte antigen - Antigen D related, neutrophil oxidative burst and cytokine analysis in patients of decompensated cirrhosis with and without acute-on chronic liver failure.

BACKGROUND AND AIM: Due to a dysregulated immune response, patients with acute-on-chronic liver failure (ACLF) have increased risk of infection and multi organ failure in comparison to compensated cirrhosis. The comparative data on the presence of 'immune paresis' in patients with ACLF and decompensated cirrhosis without ACLF is not available. Aim of the present study was to compare the immunological parameters in patients with decompensated cirrhosis with and without ACLF. METHODOLOGY: In a prospective study, 76 patients with decompensated cirrhosis with (n = 38) and without (n = 38) ACLF and 10 healthy controls (HC) were evaluated for monocytic human leukocyte antigen-antigen D Related (HLA-DR) expression, mean density of HLA-DR expressed on the surface of these cells, neutrophil oxidative burst (NOB) capacity and serum levels of cytokines (IL-1, IL-6, IL-8, IL10, IL-12, and TNF-α). RESULTS: Patients of decompensated cirrhosis with and without ACLF demonstrated significantly lower mean percentage of monocytes expressing HLA-DR and quantitative increase in the NOB after stimulation with PMA when compared to HC. However there was no difference in mean percentage of monocytes with HLA-DR expression (43.61±26.56% vs. 43.10±20.98%) (p = 0.91), mean density of HLA-DR expression on the surface (30.34±29.32 vs. 41.71±52.13) (p = 0.42) and quantitative increase in NOB after stimulation with PMA (16.55±11.91 vs. 17.24±16.18) (p = 0.47) amongst patients with decompensated cirrhosis with and without ACLF. Patients with ACLF had significantly higher pro-inflammatory and anti-inflammatory cytokines in comparison to patients with decompensated cirrhosis without ACLF. CONCLUSION: Patients with decompensated cirrhosis demonstrate a component of immune-paresis, however there is similar impairment in HLA-DR expression and NOB capacity in patients with and without ACLF. Both inflammatory and anti-inflammatory cytokines are increased in patients with ACLF in comparison to patients with decompensated cirrhosis without ACLF.

Dominating expression of negative regulatory factors downmodulates major histocompatibility complex Class-II expression on dendritic cells in chronic hepatitis C infection.

AIM: To elucidate the molecular mechanisms leading to development of functionally impaired dendritic cells (DCs) in chronic hepatitis C (CHC) patients infected with genotype 3 virus. METHODS: This prospective study was conducted on the cohorts of CHC individuals identified as responders or non-responders to antiviral therapy. Myeloid DCs were isolated from the peripheral blood of each subject using CD1c (BDCA1)(+) DC isolation Kit. Monocytes from healthy donor were cultured with DC growth factors such as IL-4 and GM-CSF either in the presence or absence of hepatitis C virus (HCV) viral proteins followed by LPS stimulation. Phenotyping was done by flowcytometry and gene expression profiling was evaluated by real-time PCR. RESULTS: Non-responders [sustained virological response (SVR)-ve] to conventional antiviral therapy had significantly higher expression of genes associated with interferon responsive element such as IDO1 and PD-L1 (6-fold) and negative regulators of JAK-STAT pathway such as SOCS (6-fold) as compared to responders (SVR+ve) to antiviral therapy. The down-regulated genes in non-responders included factors involved in antigen processing and presentation mainly belonging to major histocompatibility complex (MHC) Class-II family as HLA-DP, HLA-DQ (2-fold) and superoxide dismutase (2-fold). Cells grown in the presence of HCV viral proteins had genes down-regulated for factors involved in innate response, interferon signaling, DC maturation and co-stimulatory signaling to T-cells, while the genes for cytokine signaling and Toll-like receptors (4-fold) were up-regulated as compared to cells grown in absence of viral proteins. CONCLUSION: Underexpressed MHC class-II genes and upregulated negative regulators in non-responders indicate diminished capacity to present antigen and may constitute mechanism of functionally defective state of DCs.