Th1 Phenotype Research Articles

Combined immunotherapeutic effect of Leishmania-derived recombinant aldolase and Ambisome against experimental visceral leishmaniasis.

Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. Combining these proteins, particularly rLdAld with Ambisome (2.5mg/kg), has significantly reduced the parasitic load (∼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-β expression, an indication towards the generation of protective immunity against experimental VL. Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.

Dynamics of immune responses are inconsistent when trauma patients are grouped by injury severity score and clinical outcomes

The injury severity score (ISS) is used in daily practice to evaluate the severity of trauma patients; however, the score is not always consistent with the prognosis. After injury, systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) are related to the prognosis of trauma patients. We aimed to evaluate the associations between the immune response and prognosis in trauma patients. Patients who admitted to the Trauma Intensive Care Unit (ICU) were eligible. Whole blood samples were collected at admission, and then 6, 12, 24, 48 and 72 h after admission. Natural killer (NK) cells, lymphocyte subset population and cytokines release were identified using flow cytometry. We grouped patients by their ISS (≤ 25 and > 25 as very severe injury) and ICU stay (≤ 10 days as a short ICU stay and > 10 days as a long ICU stay) for evaluation. Fifty-three patients were enrolled. ICU stay but not ISS was close correlated with activity daily living (ADL) at discharge. Patients with a long ICU stay had an immediate increase in NK cells followed by lymphopenia which persisted for 48 h. Immediate activation of CD8+ T cells and then exhaustion with a higher programmed cell death-1 (PD-1) expression and suppression of CD4+ T cells with a shift to an anti-inflammatory Th2 phenotype were also observed in the patients with a long ICU stay. When the patients were grouped by ISS, the dynamics of immune responses were inconsistent to those when the patients were grouped by ICU stay. Immune responses are associated with the prognosis of trauma patients, however the currently used clinical parameters may not accurately reflect immune responses. Further investigations are needed to identify accurate predictors of prognosis in trauma patients.

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers.

We investigated the direct effect of PEGylated graphene oxide (P-GO) nanoparticles on the differentiation, viability, and cytokine profile of activated T helper type 17 (Th17) in vitro. The subject of the study were cultures of "naive" T-helpers (CD4+) isolated by immunomagnetic separation and polarized into the Th17 phenotype with a TCR activator and cytokines. It was found that P-GO at low concentrations (5 µg/mL) had no effect on the parameters studied. The presence of high concentrations of P-GO in T-helper cultures (25 μg/mL) did not affect the number and viability of these cells. However, the percentage of proliferating T-helpers in these cultures was reduced. GO nanoparticles modified with linear polyethylene glycol (PEG) significantly increased the percentage of Th17/22 cells in cultures of Th17-polarized T helpers and the production of IFN-γ, whereas those modified with branched PEG suppressed the synthesis of IL-17. Thus, a low concentration of PEGylated GO nanoparticles (5 μg/mL), in contrast to a concentration of 25 μg/mL, has no effect on the Th17-polarization of T helpers, allowing their further use for in-depth studies of the functions of T lymphocytes and other immune cells. Overall, we have studied for the first time the direct effect of P-GO nanoparticles on the conversion of T helper cells to the Th17 phenotype.

Understanding the development of Th2 cell-driven allergic airway disease in early life.

Allergic diseases, including atopic dermatitis, allergic rhinitis, asthma, and food allergy, are caused by abnormal responses to relatively harmless foreign proteins called allergens found in pollen, fungal spores, house dust mites (HDM), animal dander, or certain foods. In particular, the activation of allergen-specific helper T cells towards a type 2 (Th2) phenotype during the first encounters with the allergen, also known as the sensitization phase, is the leading cause of the subsequent development of allergic disease. Infants and children are especially prone to developing Th2 cell responses after initial contact with allergens. But in addition, the rates of allergic sensitization and the development of allergic diseases among children are increasing in the industrialized world and have been associated with living in urban settings. Particularly for respiratory allergies, greater susceptibility to developing allergic Th2 cell responses has been shown in children living in urban environments containing low levels of microbial contaminants, principally bacterial endotoxins [lipopolysaccharide (LPS)], in the causative aeroallergens. This review highlights the current understanding of the factors that balance Th2 cell immunity to environmental allergens, with a particular focus on the determinants that program conventional dendritic cells (cDCs) toward or away from a Th2 stimulatory function. In this context, it discusses transcription factor-guided functional specialization of type-2 cDCs (cDC2s) and how the integration of signals derived from the environment drives this process. In addition, it analyzes observational and mechanistic studies supporting an essential role for innate sensing of microbial-derived products contained in aeroallergens in modulating allergic Th2 cell immune responses. Finally, this review examines whether hyporesponsiveness to microbial stimulation, particularly to LPS, is a risk factor for the induction of Th2 cell responses and allergic sensitization during infancy and early childhood and the potential factors that may affect early-age response to LPS and other environmental microbial components.

Role of omega-3 endocannabinoids in the modulation of T-cell activity in a multiple sclerosis experimental autoimmune encephalomyelitis (EAE) model

Epidemiological studies show that omega-3 fatty acid consumption is associated with improved conditions in neurodegenerative diseases such as multiple sclerosis (MS). However, the mechanism of this association is not well understood. Emerging evidence suggests that parent molecules such as docosahexaenoic acid are converted into downstream metabolites that are capable of directly modulating immune responses. Invitro, we found that docosahexaenoyl ethanolamide (DHEA), another dietary component and its epoxide metabolite, reduced the polarization of naïve T-cells toward proinflammatory Th1 and Th17 phenotypes. Furthermore, we identified that DHEA and related endocannabinoids are changing during the disease progression in mice undergoing relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE). In addition, daily administration of DHEA to mice delayed the onset of disease, the rate of relapse, and the severity of clinical scores at relapse in RR-EAE, an animal model of MS. Collectively, these data indicate that DHEA and their downstream metabolites reduce the disease severity in the RR-EAE model of MS and can be potential dietary adjuvants to existing MS therapeutics.

Oral pretreatment with β-lactoglobulin derived peptide and CpG co-encapsulated in PLGA nanoparticles prior to sensitizations attenuates cow's milk allergy development in mice.

Cow's milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected β-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo, 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions.

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin’s lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin’s lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

AW-NI-1: ANGIOTENSIN II EXAGGERATES SARS-COV-2 SPECIFIC T-CELL RESPONSE IN CONVALESCENT INDIVIDUALS FOLLOWING COVID-19

Background: Coronavirus disease 2019 (COVID-19) is an emerging respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent studies have suggested numerous hypotheses that may explain multi-organ dysfunction during a COVID-19 infection. One possible hypothesis posits that the renin-angiotensin system dysregulation before SARS-CoV-2 infection could exacerbate disease symptoms and severity, especially in COVID-19 patients with underlying comorbidities. Objective: This study sought to investigate the effect of exogenous angiotensin II (Ang II) on peripheral blood mononuclear cells (PBMCs) stimulated with SARS-CoV-2 peptide pool. Methods: PBMCs from recovered COVID-19 patients (n = 18) were used in this study. SARS-CoV-2 specific t-cell responses were measured using activation induced cell marker assay and intracellular cytokine staining (ICS) assay, while enzyme-linked immunosorbent assay (ELISA) and ICS assays determined functional capability and polarization. Additionally, the relative level of protein phosphorylation in PBMCs was measured using a phosphokinase array. Results: Our results showed that in vitro Ang II treatment significantly increased the magnitude of SARS-CoV-2 specific t-cell response in stimulated PBMCs with SARS-CoV-2 peptide pool. Moreover, the phosphorylation level of numerous proteins implicated in cardiovascular diseases, inflammation, and viral infection showed significant increase in the presence of Ang II. Mitogenic stimulation of PBMCs after Ang II and SARS-CoV-2 peptide pool stimulation showed functional polarization of CD4+ and CD8+ t-cells toward Th1/Th17 and Th17 phenotypes, respectively. Meanwhile, ELISA showed an increased production of IL-1b and IL-6 in Ang II-stimulated PBMCs without affecting the reduction of IL-10 level resulting from SARS-CoV-2 peptide pool stimulation. Conclusion: To our knowledge, the present study is the first to demonstrate that Ang II exaggerates SARS-CoV-2 specific t-cells response. Therefore, during COVID-19 infection Ang II may aggravate the inflammatory response and change the immune response toward a more inflammatory profile against SARS-CoV-2 infection, leading to serious complications and worse outcomes during COVID-19 infection.

Human Chorionic Gonadotropin in the Regulation of T-Helpers Type 17

Chorionic gonadotropin (hCG) is a key pregnancy hormone that regulates steroidogenesis and has immunomodulatory activity. We studied the effects of native and recombinant hCG on the differentiation, proliferation, and production of IL-17 and IFN-ɣ by T-helper cells induced into the phenotype of T-helper type 17 (Th17) in vitro. We found that hCG had no significant effects on the level of Th17 cells, as assessed by RORɣτ expression, and the proliferation of these cells (Ki-67+). In addition, no effects of hCG on the production of IL-17 and IFN-ɣ by T-helpers induced in the Th17 phenotype were found. At the same time, recombinant hCG (100 IU/mL) increased the number of non-Th17 T-helpers (RORγt-Ki-67+). Thus, hCG did not modulate Th17 cells in our experimental model.

A Multi-Center Study of the Prevalence and Characteristics of Eosinophilic Phenotype and High IgE Levels Among Chinese Patients with Severe Asthma.

Patients with severe asthma have higher total- and asthma-related health burden than those whose disease is not severe. Recent medical advances in biologic therapies allow better control of asthma characterized by type 2 inflammation. To study the prevalence of eosinophilic phenotype and IgE levels in Chinese with severe asthma, and the relationship of these type 2 characteristics with asthma control, exacerbations and lung function. This was a multicenter cross-sectional observational study in Hong Kong, in Chinese adults with asthma on Step 4 or 5 of GINA treatment. Their blood eosinophil counts and total IgE levels were measured, and the relationship of these phenotypic parameters to the number of exacerbations in the past 12 months, and to symptom control in the past 4 weeks, were investigated. A total of 232 subjects were recruited from 6 centers. The mean age was 53.9±12.9 years, with 86 (37.1%) male, and the duration of diagnosed asthma was 26.2±15.7 years. A T-helper 2 (Th2) phenotype indicated by elevated eosinophils and/or IgE was present in 169 (72.8%) of patients. Of 232 patients, 43% had an eosinophilic phenotype (blood eosinophil count ≥300 cell/mm3), while 59% had high total IgE levels of >100 IU/mL (overlap with eosinophilic phenotype in 30%) and 44% had IgE levels of >150 IU/mL (overlap with eosinophilic phenotype in 22%). Subjects with eosinophilic phenotype and IgE >150 IU/mL had a higher rate (1.8 times) of uncontrolled asthma compared with those without such a combination. In Chinese adults with severe asthma defined by the use of conventional maintenance medication regimens, the prevalence of Th2 inflammation is comparable to that reported from other ethnic populations. Those with both eosinophil count ≥300 cell/mm3 and high IgE levels >150 IU/mL had a higher rate of uncontrolled asthma compared with those without a combination of these features.

Cervical Microbiota Influences Cytokine Diversity in Cervical Intraepithelial Neoplasia among Rural Women in the Akyemansa District of Ghana.

In recent times, cervical dysbiosis which mostly causes and aggravates infections is highlighted for its role in immune modulation in cervical dysplasia, which promotes the shifting of Th1 phenotype immunity to Th2 phenotype immunity. This study therefore estimated and compared the levels of circulatory IL-4, IL-6, IL-10, TNF-α, and IFN-γ cytokines among adult women identified to have different grades of cervical intraepithelial neoplasia (CIN) and with cervicovaginal infection. A total of 157 participants were recruited from the Akyemansa District of Ghana, and cervical swabs and blood samples were taken. The Pap smear test, microbiological culture, and ELISA were employed for cytology analysis, bacteria isolation, and identification and estimation of IL-4, IL-6, IL-10, TNF-α, and IFN-γ cytokines, respectively. Overall, 14/157 (8.9%) had CIN with 7.6% having CIN 1 and 1.3% having CIN 2. The main predictor for CIN was age above 46 years (OR 11.16, 95% CI: 2.4-51.8). Bacterial vaginosis (p = 0.003) and Candida infection (p = 0.012) were significantly higher in CIN. Again, Staphylococcus aureus (60% vs. 17.6%, p = 0.005), Citrobacter sp. (40.0% vs. 13.2%, p = 0.017), and Morganella morganii (40.0% vs. 4.4%, p = 0.002) isolates were significantly higher in CIN-positive participants. IL-10 and TNF-α concentrations were elevated in participants with CIN 1+ (TNF-α NIL vs. CIN 1+ only, p < 0.05) while IL-6 was decreased among participants with CIN 1+. In the presence of vaginal infection, TNF-α decreased among CIN 1+ participants while IL-10 remained elevated. The findings of this study suggest that cervical dysbiosis causes immune suppression, which creates a suitable microenvironment for the development of CIN.

NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR–HIF–1α-axis mediated glycolysis and CPT1a-driven β-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC−/−mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.

Full-length IL-33 augments pulmonary fibrosis in an ST2- and Th2-independent, non-transcriptomic fashion

Mature IL-33 (MIL33) acting through its receptor, ST2, is known to regulate fibrosis. The precursor, full-length IL-33 (FLIL33), may function differently from MIL33 and independently of ST2. Here we report that genetic deletion of either IL-33 or ST2 attenuates pulmonary fibrosis in the bleomycin model, as does Cre-induced IL-33 deficiency in response to either acute or chronic bleomycin challenge. However, adenovirus-mediated gene delivery of FLIL33, but not MIL33, to the lungs of either wild-type or ST2-deficient mice potentiates the profibrotic effect of bleomycin without inducing a Th2 phenotype. In cultured mouse lung cells, FLIL33 overexpression induces moderate and distinct transcriptomic changes compared with a robust response induced by MIL33, whereas ST2 deletion abrogates the effects of both IL-33 forms. Thus, FLIL33 may contribute to fibrosis in an ST2-independent, Th2-independent, non-transcriptomic fashion, suggesting that pharmacological targeting of both FLIL33 and MIL33 may prove efficacious in patients with pulmonary fibrosis.

Helminth-associated changes in host immune phenotype connect top-down and bottom-up interactions during co-infection.

1. Within-host parasite interactions can be mediated by the host and changes in host phenotypes often serve as indicators of the presence or intensity of parasite interactions. 2. Parasites like helminths induce a range of physiological, morphological, and immunological changes in hosts that can drive bottom-up (resource-mediated) or top-down (immune-mediated) interactions with co-infecting parasites. Although top-down and bottom-up interactions are typically studied in isolation, the diverse phenotypic changes induced by parasite infection may serve as a useful tool for understanding if, and when, these processes act in concert. 3. Using an anthelmintic treatment study of African buffalo (Syncerus caffer), we tracked changes in host immunological and morphological phenotypes during helminth-coccidia co-infection to investigate their role in driving independent and combinatorial bottom-up and top-down parasite interactions. We also examined repercussions for host fitness. 4. Clearance of a blood-sucking helminth, Haemonchus, from the host gastrointestinal tract induced a systemic Th2 immune phenotype, while clearance of a tissue-feeding helminth, Cooperia, induced a systemic Th1 phenotype. Furthermore, the Haemonchus-associated systemic Th2 immune phenotype drove simultaneous top-down and bottom-up effects that increased coccidia shedding by changing the immunological and morphological landscapes of the intestine. 5. Higher coccidia shedding was associated with lower host body condition, a lower chance of pregnancy, and older age at first pregnancy, suggesting that coccidia infection imposed significant condition and reproductive costs on the host. 6. Our findings suggest that top-down and bottom-up interactions may commonly co-occur and that tracking key host phenotypes that change in response to infection can help uncover complex pathways by which parasites interact.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor β (TGF-β). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of β-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of β-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic β-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via β-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Antileishmanial Effects of Bunium Persicum Crude Extract, Essential Oil, and Cuminaldehyde on Leishmania Major: In Silico and In Vitro Properties.

Cuminaldehyde (CA), an oxidized aldehyde monoterpene, is a major essential oil component in cumin seeds, which has shown different promising medical effects. In this study, we comprehensively evaluated the antileishmanial potential of Bunium persicum (Boiss) B. Fedtsch (Apiaceae) and one of its main essential oil constituents, CA, focus on the mechanisms of action. We used a molecular docking approach to examine the capability of CA for binding to IL-12P40 and TNF-α. The colorimetric assay was performed to assess the effect of B. persicum crude extract, essential oil, and CA, against Leishmania major promastigotes and intracellular amastigotes. The expression of IFN-γ, IL-12P40, TNF-α, and IL-10 genes was detected using quantitative real-time polymerase chain reaction qPCR. Docking analyses in the current study indicated CA binds to IL-12P40 and TNF-α. These products were safe, extremely antileishmanial, and significantly promoted Th1-related cytokines (IFN-γ, IL-12P40, TNF-α), while downregulating the Th2 phenotype (IL-10). Cumin essential oil and its major component, CA, possessed powerful antileishmanial activity. The primary mechanism of activity involves an immunomodulatory role toward Th1 cytokine response. Therefore, cumin essential oil and CA deserve further explorations as promising medications for treating leishmaniasis.

MiRNA-21, which disrupts metabolic reprogramming to facilitate CD4+ T cell polarization toward the Th2 phenotype, accelerates arsenite-induced hepatic fibrosis

Elevated levels of arsenic may be present in groundwater, and long-term exposure to arsenic increases hepatic fibrosis. T helper 2 (Th2) cells are involved in the fibrotic cascade, and cell metabolism is a regulatory factor participating in CD4+ T cell differentiation and function. However, the mechanism for Th2 cell regulation of arsenite-induced hepatic fibrosis is not fully understood. In present study, for arsenite-fed mice, activated hepatic stellate cells may be involved in the infiltration of CD4+ T cells, accompanied by up-regulation of GATA3, a transcription factor, and IL-13, the major Th2 cytokine. Exposed to arsenite, Jurkat cells had increased aerobic glycolysis to promote the cell cycle and cell proliferation. Further, this process elevated levels of marker molecules, including those of the Th2 paradigm characterized by GATA3, IL-4, and IL-13. LX-2 cells were activated when treated with culture medium from Jurkat cells exposed to arsenite. miR-21 may be a therapeutic target for arsenite-induced hepatic fibrosis. In vitro, miR-21 knock-down caused inhibition of the PTEN/PI3K/AKT pathway induced by arsenite. It also reversed the elevated glycolysis and the accelerated cell cycle and cell proliferation. Indeed, this alteration led to diminished expression of GATA3, IL-4, and IL-13 in T cells differentiated under Th2 conditions, which inhibits activation of LX-2 cells. Consistent with the results in vitro, miR-21 knock-out in mice reversed hepatic fibrosis and attenuated the levels of GATA3 and IL-13 induced by arsenite. These findings indicate that miR-21 regulates the glycolysis of CD4+ T cells through the PTEN/PI3K/AKT pathway to accelerate the cell cycle, thereby facilitating CD4+ T cell polarization toward Th2 and releasing the fibrogenic factor IL-13, which participates in arsenite-associated hepatic fibrosis. Inhibition of Th2 polarization of CD4+T cells or miR-21 could be a therapeutic strategy to combat hepatic fibrosis caused by exposure to arsenic.

462 Gametocyte Specific Factor 1 contributes to the Th phenotype in Cutaneous T-Cell Lymphomas

Ectopic expression of Gametocyte Specific Factor 1 (GTSF1) in Cutaneous T-Cell Lymphoma (CTCL) patients has been reported by several groups. Moreover, it has been associated with a worse prognosis and late-stage CTCL. GTSF1 is a gamete gene that participates in the piRNA pathway to silence retrotransposons on the genome. Despite the established association with a worse prognosis the molecular function of GTSF1 expression remains unknown. Therefore, we established shRNA-mediated GTSF1 knockdown CTCL cell lines. Using qRT-PCR, Western blot and a retrotransposition assay, we demonstrated that GTSF1 knockdown does not lead to mobilization of the retrotransposon LINE-1. These data suggest GTSF1 has a novel role in CTCL. Interestingly, after GTSF1 knockdown in the Mac2A cell line, we observed striking growth inhibition, suggesting this gene is a key player in CTCL carcinogenesis. To further explore this role, we performed RNA-Seq, comparing GTSF1 knockdown to control cells. Differential gene expression analysis using DESeq2, revealed 12496 differentially expressed genes (p adjusted <0.05). Pathway enrichment analysis was performed via GO, KEGG, Reactome and WikiPathways. We observed an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, transcriptome analysis showed an increased expression of cytokine induced genes, such as XAF1, IFI27 and IFI44L, and genes associated with a Th1 phenotype, IFNG and TNF. Research shows that CTCL progression leads to defects in Th1 cytokine production (IFN-γ and TNF-α) by antitumor immune cells and an enhanced Th2 cytokine production (IL-4 and IL-5) by malignant T cells. The chronic production of Th2 cytokines by malignant cells leads to immune deficiency in patients, allowing malignant cells to proliferate and the disease to progress. Taken together, these data suggest GTSF1 as a novel regulator of the Th phenotype on CTCL, thereby placing it as a potential treatment target for regulating disease progression.

Gata3 ZsG and Gata3 ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.