Specific IL-2 Research Articles

Effect of parasympathetic inhibition on expression of ILC2 cells in a mouse model of allergic rhinitis

BackgroundWe wanted to investigate whether parasympathetic inhibition affected the expression of type 2 innate lymphoid cells (ILC2s) in the nasal mucosa of a mouse model of allergic rhinitis (AR). MethodsThirty male C57BL/6 mice were randomly divided into 3 groups: control group, AR group, AR-treated group. AR nasal symptoms were assessed on a semi-quantitative scale according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The expression of cytokines protein in serum was detected by enzyme linked immunosorbent assay (ELISA). The number of ILC2s in nasal mucosa was detected by immunofluorescence double staining assay. Quantitative real-time Polymerase Chain Reaction (qPCR) was used to detect the expression of ILC2-associated factor in nasal mucosa. ResultsThe symptom scores of the AR group were significantly higher than those of the control group and AR-treated group. The expression levels of mouse ovalbumin (OVA) specific IgE, IL4, IL5, and IL13 in the serum of AR group were significantly higher than those in the control group and AR-treated group. The number of ILC2s and the gene expression of ILC2s related factors GATA3, CD25 and CD90 (Thy1) in the nasal mucosa of the AR group were significantly higher than those of the control group and AR-treated group. ConclusionsWe found that parasympathetic inhibition relieved AR symptoms and inhibited immune response of AR mice. ILC2s play an important role in the occurrence and development of AR, and parasympathetic nerve inhibition reduced the number of ILC2s and inhibited the cytokines expression by ILC2s. Our data provide information on the mechanism of action of parasympathetic inhibition in AR.

SERUM LEVEL OF IL-33 IN GOUTY RENAL PATIENTS

Background: Gout is a form of inflammatory arthritis and the most common metabolic disease in humans. It may result in tophi, kidney stones, or even kidney damage. It has become more common in recent decades due to increasing risk factors in the population, such as metabolic syndrome. Interleukin-33, the latest discovery in IL-1 cytokine family, acts as an early alerter of inflammation, it has been linked to several human pathologies including autoimmune diseases, sepsis, and allergy through its specific IL-1 receptor ST2 (suppression of tumorigenicity 2). However, there is little information regarding the role of IL-33 in gout.Aim of the Work: Our study aimed to examine the clinical utility of IL-33 level in serum among gouty patients and correlating its concentration with renal affection.Patients and Methods: Our case-control study was conducted on 90 subjects divided into three groups. Group I: included thirty gout patients, with no evidence of kidney affection, Group II: included thirty gout patients with kidney affection, their diagnosis based on the American College of Rheumatology classification criteria, Group III: thirty age and sex-matched, apparently healthy subjects included in the study as a control group. All individuals were subjected to detailed medical history, clinical examination including weight measurement and body mass index (BMI), and laboratory parameters including serum uric acid, creatinine, urea, lipid profile, and serum level of IL-33 measured by ELISA.Results: Serum IL-33 was predominantly increased in gout patients (with or without kidney affection) compared to healthy controls and was even higher in patients without kidney injury compared to gout patients with kidney injury. IL-33 was positively correlated with serum HDL, BMI, although it was negatively correlated with LDL and cholesterol.In Conclusion: IL-33 might have a protective role in kidney injury among gout patients.

Naturally Oxidized Olive Oil Promotes Active Cutaneous Anaphylaxis and Th2 Cytokine Production.

The excessive ingestion of oxidized dietary oils may exacerbate some allergic diseases. We previously reported that oxidized olive oil exacerbates active cutaneous anaphylaxis (ACA), one of the immediate allergic reactions. This study was conducted to clarify the effects of oxidized olive oil on the T cell response during ACA. BALB/c female mice were orally administered naturally oxidized olive oil once every 2 d for 2 weeks after ovalbumin (OVA)/aluminum hydroxide gel sensitization, after which ACA was elicited by intracutaneous administration of OVA into the ear auricles. Compared with fresh olive oil, oxidized olive oil administration increased the antigen-specific immunoglobulin E (IgE) antibody titer 2 weeks after OVA-sensitization and vascular hyperpermeability increased due to ACA. In the oxidized olive oil-administered mice, the mRNA expression levels of T-helper 2 (Th2) cytokines, interleukin (IL)-4, -5, -6, and -10, in the lymph nodes increased, as did the proportion of cluster designation (CD)3+CD4+ cells in the spleen and lymph nodes. In CD3+CD4+ cells, the mRNA expression levels of IL-4 and GATA-binding protein 3 (GATA3), the master regulator of Th2, were higher in the oxidized olive oil-group. Antigen-stimulated specific IL-4 production was promoted in CD3+CD4+ cells of oxidized olive oil-administered mice. This suggests that oxidized olive oil exacerbates ACA by promoting Th2 dominance in immediate allergic diseases.

Poly (D, L-lactide-co-glycolide) delivery system improve the protective efficacy of recombinant antigen TA4 against Eimeria tenella infection

Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles carrying recombinant TA4 protein of E. tenella (rEtTA4) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 (PLGA-rEtTA4) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8+ T lymphocytes. However, no significant differences were observed in the proportion of CD4+ T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella. Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles–immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8+ T lymphocytes compared with rEtTA4 subunit vaccine–immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.

Abstract PO066: Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses

Abstract Background: Oral cancer is a malignant, neoplastic disorder most commonly presenting as squamous cell carcinoma (HNSCC). It is responsible for nearly 300,000 new diagnoses and 145,000 deaths globally each year, and in the United States alone, approximately one person will die from oral cancer every hour every day in 2020. Although primary surgical resection remains the standard of care for these patients, tumor recurrence is common (40%) and late stage five year survival rate is low (<50%). As a result, ionizing radiation has gained traction as an adjunctive therapy to improve oral cancer clinical outcomes. However, the effects of radiation on oral cancer cell viability and subsequent anti-tumor T cell responses to oral cancer are not fully understood. Objective: To examine the effects of ionizing radiation on HNSCC cell viability and determine the ability of irradiated HNSCC cells to elicit antigen specific T-cell immune responses. Methods: A metastatic mouse HNSCC cell line, LY2, was seeded and exposed to increasing doses of ionizing radiation (0, 20, 50, 100 200 Gy) generated via an RS2000 Irradiator, a ten minute exposure to ultraviolet (UV) light, freeze/thaw cycling, or a combination of these treatments. Following exposure, cells were cultured for 96 hours. Cell viability was assessed by microscopic imaging using a Leica microscope, and analysis was performed in ImageJ. Proteins were extracted from the cells, and protein concentration was quantified using a BCA assay. Next, single cell suspensions from the lymph nodes of LY2 tumor bearing, immunocompetent BALB/c mice were obtained and stimulated using various concentrations (0, 1.6, 8, and 40 µg) of proteins obtained from the LY2 cells. Antigen specific T cell proliferative responses, as determined by Alamar Blue assay, were compared between different concentrations of irradiated LY2 protein as well as the different exposure types. Cell supernatants were also harvested for analysis of the T cell specific cytokines, IL-4, IL-2, and IFNg. Results: LY2—HNSCC showed reduced viability in response ionizing radiation in a dose-dependent manner. The ideal, immunogenic range for harvested protein between 1.6µg and 8µg elicited an optimal T cell immune response. Additionally, Alamar Blue assay analysis demonstrated that LY2 proteins harvested from cells exposed to ionizing radiation or freeze/thaw cycling exhibited a proliferative response that was both dose-dependent and antigen specific, while cells exposed to UV light displayed no significant T cell proliferation. Conclusion: These results confirm a dose-dependent, cytotoxic effect of ionizing radiation on HNSCC cells. Proteins from cells exposed to high doses of ionizing radiation and freeze/thaw cycles can be successfully extracted with preserved antigenic profiles. Future work will allow characterization of the cellular response and enhance the study of the anti-tumor immune mechanisms in new and exciting ways. Citation Format: Travis Pero, Nathan Ryan, Kelvin Anderson, Steve Oghumu. Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO066.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans–induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Postoperative cognitive dysfunction is made persistent with morphine treatment in aged rats

Postoperative cognitive dysfunction (POCD) is the collection of cognitive impairments, lasting days to months, experienced by individuals following surgery. Persistent POCD is most commonly experienced by older individuals and is associated with a greater vulnerability to developing Alzheimer’s disease, but the underlying mechanisms are not known. It is known that laparotomy (exploratory abdominal surgery) in aged rats produces memory impairments for 4 days. Here we report that postsurgical treatment with morphine extends this deficit to at least 2 months while having no effects in the absence of surgery. Indeed, hippocampal-dependent long-term memory was impaired 2, 4, and 8 weeks postsurgery only in aged, morphine-treated rats. Short-term memory remained intact. Morphine is known to have analgesic effects via μ-opioid receptor activation and neuroinflammatory effects through Toll-like receptor 4 activation. Here we demonstrate that persistent memory deficits were mediated independently of the μ-opioid receptor, suggesting that they were evoked through a neuroinflammatory mechanism and unrelated to pain modulation. In support of this, aged, laparotomized, and morphine-treated rats exhibited increased gene expression of various proinflammatory markers (IL-1β, IL-6, TNFα, NLRP3, HMGB1, TLR2, and TLR4) in the hippocampus at the 2-week time point. Furthermore, central blockade of IL-1β signaling with the specific IL-1 receptor antagonist (IL-1RA), at the time of surgery, completely prevented the memory impairment. Finally, synaptophysin and PSD95 gene expression were significantly dysregulated in the hippocampus of aged, laparotomized, morphine-treated rats, suggesting that impaired synaptic structure and/or function may play a key role in this persistent deficit. This instance of long-term memory impairment following surgery closely mirrors the timeline of persistent POCD in humans and may be useful for future treatment discoveries.

Age-related changes in the immunomodulatory effects of human dental pulp derived mesenchymal stem cells on the CD4+ T cell subsets

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells. The effects of the aging on these abilities of MSCs have not been adequately clarified. In this study, alterations in immunomodulatory abilities of MSCs caused by aging were investigated. For this, dental pulp (DP) MSCs and peripheral blood mononuclear cells (PBMCs) of elderly and young donors were co-cultured age-matched and cross. We detected that the effects of DP-MSCs on Th1 and Th2 cells and their specific cytokines IFN-γ and IL-4 are not affected by aging. However, we observed that young and elderly DP-MSCs have different effects on Th17 and Treg cells. Th17 frequencies of young and elderly PBMCs were significantly increased only by young DP-MSCs, in contrast, Treg frequencies were significantly increased by elderly DP-MSCs. IL-6, IL-17a and HGF levels of both young and elderly PBMCs showed a significant increase only by young DP-MSCs, but TGF-β levels were significantly increased only by elderly DP-MSCs. The oral cavity is home to a rich microflora. The interactions of dental tissues with this microflora can lead them to acquire different epigenetic modifications. Aging can affect the microflora composition of the oral cavity and change this process in different directions. According to our findings, DP-MSCs are effective cells in the regulation of CD4+ T cells, and their effects on Th1 and Th2 cells were not affected by aging. However, pleiotropic molecules IL-6 and HGF expressions, which are important in dental and bone tissue regeneration, decreased significantly in elderly DP-MSCs. This situation may have indirectly made a difference in the modulation effects of young and elderly DP-MSCs on the Th17 and Treg cells.

Activation-Induced Marker Expression Identifies Mycobacterium tuberculosis-Specific CD4 T Cells in a Cytokine-Independent Manner in HIV-Infected Individuals with Latent Tuberculosis.

HIV infection is a significant risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease, yet the mechanisms whereby HIV impairs T cell immunity to M. tuberculosis have not been fully defined. Evaluation of M. tuberculosis–specific CD4 T cells is commonly based on IFN-γ production, yet increasing evidence indicates the immune response to M. tuberculosis is heterogeneous and encompasses IFN-γ–independent responses. We hypothesized that upregulation of surface activation-induced markers (AIM) would facilitate detection of human M. tuberculosis–specific CD4 T cells in a cytokine-independent manner in HIV-infected and HIV-uninfected individuals with LTBI. PBMCs from HIV-infected and HIV-uninfected adults in Kenya were stimulated with CFP-10 and ESAT-6 peptides and evaluated by flow cytometry for upregulation of the activation markers CD25, OX40, CD69, and CD40L. Although M. tuberculosis–specific IFN-γ and IL-2 production was dampened in HIV-infected individuals, M. tuberculosis–specific CD25+OX40+ and CD69+CD40L+ CD4 T cells were detectable in the AIM assay in both HIV-uninfected and HIV-infected individuals with LTBI. Importantly, the frequency of M. tuberculosis–specific AIM+ CD4 T cells was not directly impacted by HIV viral load or CD4 count, thus demonstrating the feasibility of AIM assays for analysis of M. tuberculosis–specific CD4 T cells across a spectrum of HIV infection states. These data indicate that AIM assays enable identification of M. tuberculosis–specific CD4 T cells in a cytokine-independent manner in HIV-uninfected and HIV-infected individuals with LTBI in a high-tuberculosis burden setting, thus facilitating studies to define novel T cell correlates of protection to M. tuberculosis and elucidate mechanisms of HIV-associated dysregulation of antimycobacterial immunity.

Characterization of immune responses and the lung transcriptome in a murine model of IL-33 challenge

IL-33 induces airway inflammation and hyper-responsiveness in respiratory diseases. Although defined as a therapeutic target, there are limited studies that have comprehensively investigated IL-33-mediated responses in the lungs in vivo. In this study, we characterized immunological and physiological responses induced by intranasal IL-33 challenge, in a mouse model. We identified specific cytokines, IL-4, IL-5, IL-6, IL-10, IP-10 and MIP1-α, that are increased in bronchoalveolar lavage and lung tissues by IL-33. Using transcriptomics (RNA-Seq) we demonstrated that 2279 transcripts were up-regulated and 1378 downregulated (≥ 2-fold, p < 0.01) in lung tissues, in response to IL-33. Bioinformatic interrogation of the RNA-Seq data was used to predict biological pathways and upstream regulators involved in IL-33-mediated responses. We showed that the mRNA and protein of STAT4, a predicted upstream regulator of IL-33-induced transcripts, was significantly enhanced in the lungs following IL-33 challenge. Overall, this study provides specific IL-33-induced molecular targets and endpoints that can be used as a resource for in vivo studies, e.g. in preclinical murine models examining novel interventions to target downstream effects of IL-33.

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease ‘botulism’ in human and animals. Their potential use as biological weapon led to their classification in category ‘A’ biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni–NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

The inflammatory cytokine, interleukin‐1‐beta, drives central immune cell infiltration, changes in sympathetic nerve activity and blood pressure

IntroductionThe area postrema (AP) is a brain region that lacks a blood brain barrier allowing blood‐borne factors to access the brain. Since the AP is connected to numerous brain regions that play a key role blood pressure regulation, we hypothesised that blood‐borne inflammatory factors such as interleukin‐1 beta (IL‐1β) might play a role in the development of hypertension. In support of this hypothesis, plasma Interleukin‐1 beta is increased in hypertensive patients (Dalekos et al., 1997, J Lab Clin Med,129: 300–8). Furthermore, the receptors for IL‐1β are abundantly expressed in the AP. Here we test whether IL‐1β can act on the AP to cause an increase in blood pressure and renal sympathetic nerve activity (RSNA) in rats.MethodsBlood pressure and renal sympathetic nerve activity (RSNA) were recorded and analyzed after microinjection of different doses of IL‐1β into the AP (10ng and 25ng) of anaesthetised rats. In order to show that IL‐1β, specifically causes an increase in blood pressure, specific IL‐1 receptor blocking antibodies (IL‐1Ra) were injected into the AP prior to a 25ng IL‐1β injection. The AP was extracted for fluorescent activated cell sorting (FACS).ResultsExperiments showed that microinjection of 25ng IL‐1β caused a significant (p&lt;0.01; Mann‐Whitney test) increase in both systolic blood pressure (19.6 ± 3.6 mmHg) and heart rate (55 ±10 bpm) compared with control injections of saline (SBP: 5.47 ± 1.58; HR 12 ± 6). Pre‐treatment with IL‐1Ra attenuated the response to IL‐1β, and was comparable with saline controls. IL‐1β also caused an increase in RSNA (137% ± 43%) and FACS revealed an increase in immune cell infiltration. Immunohistochemistry also revealed that IL‐1β injection results in Fos activation in the AP.ConclusionsIL‐1β injection appears to activate neurons in the area postrema which leads to an increase in blood pressure. By providing new insight into the actions of IL‐1β in the brain, we show that IL‐1β might be a potential target for future treatments for hypertension.Support or Funding InformationAustralian Research Council (FT170100363)National Health and Medical Research Council (GNT 1079680)Rebecca L Cooper Medical Research Foundation

The immunogenicity and protective immunity of multi-epitopesDNA prime-proteinboost vaccines encoding Amastin-Kmp-11, Kmp11-Gp63 and Amastin-Gp63 against visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis if left untreated and 50,000 to 90,000 new cases of VL occur worldwide each year. Although various vaccines had been studied in animal models, none of them was eligible to prevent human from infections. In this study, according to the silico analysis of Leishmania Amastin, Kmp-11 and Gp63 protein, dominant epitope sequences of these proteins were selected and linked to construct dominant multi-epitopes DNA and protein vaccines (Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63) against VL. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a new Leishmania parasite strain isolated from a VL patient. After immunization, the results including specific antibody titers, IL-4 and TNF-α levels, and CD4 and CD8 T cell proportion suggested the potent immunogenicity of the three vaccines. After infection, the results of spleen parasite burdens in the three vaccine groups were significantly lower than those of control groups, and the parasite reduction rates of Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 groups were 89.38%, 91.01% and 88.42%, respectively. Spleen smear observation and liver histopathological changes showed that all vaccine groups could produce significant immunoprotection against VL and Amastin-Gp63 vaccine was the best. In conclusion, our work demonstrated that the three dominant multi-epitopes Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 DNA prime-protein boost vaccines might be new vaccine candidates for VL, and the Amastin-Gp63 vaccine have best efficacy.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Interleukin-17 Inhibition in Spondyloarthritis Is Associated With Subclinical Gut Microbiome Perturbations and a Distinctive Interleukin-25-Driven Intestinal Inflammation.

To characterize the ecological effects of biologic therapies on the gut bacterial and fungal microbiome in psoriatic arthritis (PsA)/spondyloarthritis (SpA) patients. Fecal samples from PsA/SpA patients pre- and posttreatment with tumor necrosis factor inhibitors (TNFi; n = 15) or an anti-interleukin-17A monoclonal antibody inhibitor (IL-17i; n = 14) underwent sequencing (16S ribosomal RNA, internal transcribed spacer and shotgun metagenomics) and computational microbiome analysis. Fecal levels of fatty acid metabolites and cytokines/proteins implicated in PsA/SpA pathogenesis or intestinal inflammation were correlated with sequence data. Additionally, ileal biopsies obtained from SpA patients who developed clinically overt Crohn's disease (CD) after treatment with IL-17i (n = 5) were analyzed for expression of IL-23/Th17-related cytokines, IL-25/IL-17E-producing cells, and type 2 innate lymphoid cells (ILC2s). There were significant shifts in abundance of specific taxa after treatment with IL-17i compared to TNFi, particularly Clostridiales (P = 0.016) and Candida albicans (P = 0.041). These subclinical alterations correlated with changes in bacterial community co-occurrence, metabolic pathways, IL-23/Th17-related cytokines, and various fatty acids. Ileal biopsies showed that clinically overt CD was associated with expansion of IL-25/IL-17E-producing tuft cells and ILC2s (P < 0.05), compared to pre-IL-17i treatment levels. In a subgroup of SpA patients, the initiation of IL-17A blockade correlated with features of subclinical gut inflammation and intestinal dysbiosis of certain bacterial and fungal taxa, most notably C albicans. Further, IL-17i-related CD was associated with overexpression of IL-25/IL-17E-producing tuft cells and ILC2s. These results may help to explain the potential link between inhibition of a specific IL-17 pathway and the (sub)clinical gut inflammation observed in SpA.

Immunization with the recombinant myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant elicits a Th1-biased immune response and a reduction of parasite burden in Fasciola hepatica infected rats

The aim of this study was to assess the immune response and the protective efficacy elicited by the vaccination with the recombinant Fasciola hepatica myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant against the infection with F. hepatica in rats. Four groups of 15 animals each were used for the study, one group was immunized with the recombinant F. hepatica MRLC in Adjuplex® adjuvant and the other groups remained as adjuvant, positive and negative control groups. The parasitological study showed that a statistically significant reduction of 65.1% and 82.1% in fluke burden and fecal egg count, respectively, was detected in vaccinated animals. In addition, vaccination with FhrMRLC induced a well-defined humoral and cellular immune response characterized by a significant production of specific IgG and IL-2, IL-12, TNF-α and IFN-γ; which confirms the immunogenic capacity of the FhrMRLC.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.)

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-γ in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae.

Inflammasome signaling and regulation of interleukin-1 family cytokines.

Specific IL-1 family cytokines are expressed by cells as cytosolic pro-forms that require cleavage for their activity and cellular release. IL-1β, IL-18, and IL-37 maturation and secretion is governed by inflammatory caspases within signaling platforms called inflammasomes. By inducing pyroptosis, inflammasomes can also drive the release of the alarmin IL-1α. Recent advances have transformed our mechanistic understanding of inflammasome signaling, cell death decisions, and cytokine activation and secretion. Here, we provide an updated view of inflammasome signaling; mechanisms underpinning IL-1α, IL-1β, IL-18, and IL-37 maturation and release; and the functions of these cytokines in protective and pathological inflammation.

Enhanced IFN-\u03b3, but not IL-2, response to Mycobacterium tuberculosis antigens in HIV/latent TB co-infected patients on long-term HAART

BackgroundHIV-infected individuals with latent TB infection are at increased risk of developing active TB. HAART greatly reduces the incidence rate of TB in HIV-infected patients and reconstitutes Mycobacterium tuberculosis (M. tuberculosis)-specific immune response in the first 12 months of therapy. The durability of the anti-mycobacterial immune restoration after a year of HAART however remains less investigated.MethodA cross-sectional study was conducted to evaluate M. tuberculosis-specific functional immune responses in HIV/latent TB co-infected patients who were on HAART for at least 1.5 up to 9 years as compared to HAART-naïve patients. Three-hundred sixteen HIV-infected patients without active TB were screened by tuberculin skin testing for M. tuberculosis infection and peripheral blood mononuclear cells (PBMCs) were isolated from 61 HIV/latent TB co-infected patients (30 HAART-naïve and 31 HAART-treated). IFN-γ and IL-2 ELISPOT as well as CFSE cell proliferation assays were performed after stimulation with M. tuberculosis antigens PPD and ESAT-6.ResultThe median frequency of PPD and ESAT-6 specific IFN-γ secreting cells was significantly higher in the HAART-treated patients as compared to HAART-naïve patients, p = 0.0021 and p = 0.0081 respectively. However, there was no significant difference in the median frequency of IL-2 secreting cells responding to PPD (p = 0.5981) and ESAT-6 (p = 0.3943) antigens between HAART-naïve and-treated groups. Both IFN-γ and IL-2 responses were independent of CD4+ T cell count regardless of the HAART status. Notably, the frequency of PPD and ESAT-6 specific IL-2 secreting cells was positively associated with CD4+ T cell proliferation while inversely correlated with duration of HAART, raising the possibility that M. tuberculosis-specific IL-2 response that promote the antigen-specific CD4+ T cell proliferation diminish with time on antiretroviral therapy in HIV/latent TB co-infected patients.ConclusionThis study shows an increased M. tuberculosis-specific IFN-γ, but not IL-2, response in HIV/latent TB co-infected patients with long-term HAART, consistent with only partial immune restoration. Future studies should, therefore, be done to prospectively define the rate and extent to which functional immune responses to M. tuberculosis are restored after long-term HAART.