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Chronic hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA) is the sole viral transcription template and not affected by current treatment options, constituting a key determinant of HBV persistence. Novel therapeutics with demonstrable effectiveness against cccDNA are required. Methods: Previously, we established an HBV persistence mouse model using replicon plasmid derived from a clinical isolate (termed BPS) and identified IL-21 as a potent clearance-inducer. We also described another persistence mouse model based on cccDNA mimics produced in vivo termed recombinant cccDNA (rcccDNA). In this work, effectiveness of IL-21-based gene and cellular therapies was tested using these models. Results: In both models of HBV persistence, single injections with adeno-associated virus (AAV) expressing murine IL-21 highly efficiently induced clearance of both HBV markers from serum, and more importantly, BPS DNA and rcccDNA from mouse liver. Mechanistically, IL-21-induced clearance was associated with activation and liver infiltration of CD8+ T cells, and CD8 antibody injections negatively affected AAV-IL-21 effectiveness. More notably, adoptive transfer of CD8+ T cells from AAV-IL-21-cured mice engendered clearance in acceptor HBV persistence mice. Furthermore, cured mice were protected against re-challenge with long-lived memory. Most significantly, infusion of splenocytes from treatment-naive mice stimulated ex vivo with IL-21 protein and HBV antigen could also induce clearance in treatment-naive mice. Conclusion: These data demonstrate IL-21-based gene and cellular therapies as valid candidates for treating chronic HBV infections, with potential in removing cccDNA-harboring hepatocytes via activated CD8+ T cells accompanied by long-term protective memory.

Seven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

Background Both malaria and mental disorders are associated with immune changes. We have previously reported the associations between malaria and mental disorders. We now report associations between malaria, mental disorders and immunity. Methods A household survey of malaria, mental disorders and immunity was conducted in a health and demographic surveillance system's site of 70,000 population in an area endemic for malaria in western Kenya. A random sample of 1190 adults was selected and approached for consent, blood samples and structured interview. Findings We found marginally raised CD4/CD3 ratios of participants with malaria parasites, but no difference in CD4/CD3 ratios for participants with common mental disorder (CMD) or psychotic symptoms. People with psychotic symptoms had increased levels of IL-6, IL-8, and IL-10, and lower levels of IL-1beta. People with CMD had higher levels of IL-8 and IL-10. People with malaria had higher levels of IL-10 and lower levels of TNF-alpha. At the bivariate level, CMD was associated with log TNF-α levels using unadjusted odds ratios, but not after adjusting for malaria. Psychotic symptoms were associated with log IL-10 and log TNF-α levels at the bivariate level while in the adjusted analysis, log TNF-α levels remained highly significant.. Interpretation This is the first population based study of immune markers in CMD and psychotic symptoms, and the first to examine the 3 way relationship with malaria. Our findings suggest that TNF-α may mediate the relationship between malaria and CMD. Fund The study was funded by UK Aid, Department for International Development , Kenya office.

Introduction Alopecia areata (AA) is considered an autoimmune disorder characterized by patchy loss of hair from the scalp and other body parts. Many patients develop the disease in childhood. Aim To answer the question whether abnormal production of some proinflammatory cytokines (IL-2, IL-6, IL-15, IL-17A and IFNγ) in children with AA may facilitate the development or progression of the disease. Material and methods The study group consisted of 42 children with AA, the control group – 37 healthy children. Peripheral venous blood samples were collected from patients with AA and healthy controls and the concentrations of serum cytokines, namely IL-2, IL-6, IL-15, IL-17A, IFN-γ were determined quantitatively by ELISA method. Results The serum IL-6, IL-15, IL-17A and IFNγ levels were significantly increased in patients with AA compared with control subjects (p < 0.05). The serum IL-15 level was found to be increased when the total duration of AA was increased (q = 0.30; p = 0.05). The serum cytokine level of IL-17A was found to be decreased when duration of the current episode was longer than 2 years (p < 0.05), but the correlation between IL-17A serum level and duration of the current episode was not confirmed in the Spearman test (q = –0.06; p = 0.68). The serum IL-17A level was found to be significantly decreased when the thyroiditis was present (q = –2.378; p < 0.05). Conclusions The increased levels of serum IL-6, IL-15, IL-17A and IFNγ in children suggest imbalance in the serum proinflammatory cytokines production in AA.

Infections with gastrointestinal nematodes provoke immune and inflammatory responses mediated by cytokines released from T-helper type-2 (Th2) cells. Infections with Trichinella species have been reported to differ by the host species. Previously, in rats, we observed acute liver inflammation in response to infection with Trichinella spiralis, and the rat hosts showed a series of biochemical changes characterized by a decrease in serum paraoxonase (PON) 1 activity associated with the down-regulation of hepatic PON1 synthesis. In the present study, we investigated the effect(s) of species differences on the immune response against T. spiralis infection by analyzing serum PON1 activity and the associated inflammatory/anti-inflammatory mediators in mice. There were inconsistent changes in the serum PON1 activity of mice infected with T. spiralis, and these changes were associated with significant increases in the serum levels of interleukin (IL)-2, IL-4, IL-10, IL-12 (p70), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α during the enteric phase of the infection, while the levels of IL-5 and interferon γ were significantly increased throughout the entire experimental period. Moreover, T. spiralis infection in mice was associated with little inflammatory cell infiltration in hepatic tissues. Given the zoonotic prevalence of T. spiralis, further mechanistic research in this area is warranted.

Introduction: There are reports that the TNFα gene (rs1800629) can be involved in the pathogenesis of metabolic syndrome through an increased production of pro-inflammatory cytokines. Therefore, we have decided to search for the relationship between the TNFα gene polymorphisms and serum levels of proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNFα, IFNγ) and CRP in women with metabolic syndrome. Material and methods: The study sample consisted of 416 women aged 45-60 years, including 118 with metabolic syndrome. The participants were surveyed and subjected to anthropometric, biochemical and genetic analysis. Results: We noticed that in the group meeting the criteria for metabolic syndrome, the G/G genotype of the TNFα gene was related to higher IL-6 levels than in the remainder group. The carriers of the A/G genotype in the metabolic syndrome group had significantly higher levels of IFNγ than those in the group without this syndrome. CRP was significantly higher in the group with metabolic syndrome, irrespective of the women’s genotypes. Conclusions: The upregulation of IFNγ and IL-6 and CRP suggests that autoinflammatory process may play a significant role in the pathogenesis of metabolic syndrome. However, a direct relationship between the TNFα gene polymorphisms and inflammatory biomarkers analyzed in our study was not confirmed.

Background Interleukin-24 (IL-24) binds to two kinds of receptor complexes, namely IL-20R1/IL-20R2 and IL-20R2/IL-22R1, which are also bound by IL-20. IL-20 plays a detrimental role in liver fibrosis. Due to the sharing of receptor complexes, we aimed to determine whether IL-24 also participates in liver fibrosis. Methods Clinical biopsy specimens from various stages of liver fibrosis were used to analyze IL-24 expression. IL-24 protein was administered to mice with thioacetamide (TAA)-induced liver injury. The direct effects of IL-24 on mouse primary hepatocytes or hepatic stellate cells (HSCs) were analyzed. Wild-type, IL-20R1-, and IL20R2-deficient mice were used to establish a model of acute TAA-induced liver injury. Findings Among patients with more severe liver fibrosis, there was a reduced IL-24/IL-20 ratio. Administration of IL-24 protein protected mice from TAA-induced liver injury and reduction of liver inflammation by antioxidant effects. IL-24 protected hepatocytes from TAA-induced apoptosis and prevented liver fibrosis through the inhibition of the HSCs activation. The protective role of IL-24 acted on liver cells were mainly IL-20R1-independent. IL-20R2-deficient mice exhibited more severe liver injury upon TAA treatment, thus confirming the protective role of IL-24. Interpretation IL-24 plays a key protective role in the progression of liver injury and has therapeutic potential for treating liver injuries. Funding This work was supported by the Ministry of Science and Technology of Taiwan (MOST 106–2320-B-006–024) and Taiwan Liver Disease Prevention & Treatment Research Foundation.

Although macroautophagy/autophagy has been proposed as a critical defense mechanism against HIV-1 by targeting viral components for degradation, its contribution as a catabolic process in providing optimal anti-HIV-1 immunity has never been addressed. The failure to restore proper antiviral CD8A/CD8 T-cell immunity, especially against HIV-1, is still the major limitation of current antiretroviral therapies. Consequently, it is of clinical imperative to provide new strategies to enhance the function of HIV-1-specific CD8A T-cells in patients under antiretroviral treatments (ART). Here, we investigated whether targeting autophagy activity could be an optional solution to make this possible. Our data show that, after both polyclonal and HIV-1-specific activation, CD8A T-cells from ART displayed reduced autophagy-dependent degradation of lysosomal contents when compared to naturally HIV-1 protected elite controllers (EC). We further confirmed in EC, by using specific BECN1 gene silencing and lysosomal inhibitors, the critical role of active autophagy in superior CD8A T-cell protection against HIV-1. More importantly, we found that an IL21 treatment was effective in rescuing the antiviral CD8A T-cell immunity from ART in an autophagy-dependent manner. Finally, we established that IL21-dependent rescue occurred due to the enhanced degradation of endogenous lipids via autophagy, referred to as lipophagy, which fueled the cellular rates of mitochondrial beta-oxidation. In summary, our data show that autophagy/lipophagy can be considered as a therapeutic tool to elicit functional antiviral CD8 T-cell responses. Our results also provide additional insights toward the development of improved T-cell-based prevention and cure strategies against HIV-1. Abbreviations: ART: patients under antiretroviral therapy; BaF: bafilomycin A1; BECN1: beclin 1; CEF: cytomegalo-, Epstein-Barr- and flu-virus peptide pool; Chloro.: chloroquine; EC: elite controllers; FAO: fatty acid beta-oxidation; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBMC: peripheral blood mononuclear cells; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

The maintenance of a strong IL21 production in memory CD4 T cells, especially in HIV-1-specific cells, represents a major correlate of natural immune protection against the virus. However, the molecular mechanisms underlying IL21 production during HIV-1 infection, which is only elevated among the naturally protected elite controllers (EC), are still unknown. We recently found out that lipophagy is a critical immune mediator that control an antiviral metabolic state following CD8A T cell receptor engagement, playing an important role in the natural control of HIV-1 infection. This led us to investigate whether the beneficial role of a strong macroautophagy/autophagy, could also be used to ensure effective IL21 production as well. Herein, we confirm that after both polyclonal and HIV-1-specific activation, memory CD4 T cells (Mem) from EC display enhanced activity of the autophagy-mediated proteolysis compared to ART. Our results indicate that the enhanced autophagy activity in EC was controlled by the energy-sensing PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1). We further confirmed the critical role of the autophagy-mediated proteolysis in the strong IL21 production in EC by using BECN1 gene silencing as well as protease, PRKAA1, and lysosomal inhibitors. Finally, we established that high autophagy-mediated proteolysis in EC fuels their cellular rates of mitochondrial respiration due to glutaminolysis. Our data confirm the critical role of autophagy in dictating the metabolic input, which is required not only to ensure protective cytotoxic CD8A T cell responses, but also to provide strong IL21 production among antiviral CD4 T cells. Abbreviations: AKG: alpha-ketoglutarate; ART: patients under antiretroviral therapy; ATG7: autophagy related 7; BaF: bafilomycin A1; BECN1: beclin 1; Chloro.: chloroquine; EC: elite controllers; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FOXO3: forkhead box O3; GLS: glutaminase; GLUD1: glutamate dehydrogenase 1; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MTOR: mechanistic target of rapamycin kinase; PBMC: peripheral blood mononuclear cells; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; SQSTM1: sequestosome 1; TCA: tricarboxylic acid cycle; ULK1: unc-51 like autophagy activating kinase

Obstructive sleep apnea (OSA) may lead to increased circulating concentrations of inflammatory biomarkers and treatment may change these. We aimed to assess the effect of oral appliance (OA) therapy on inflammatory biomarkers in a randomised controlled pilot trial. A total of 71 patients with OSA and systemic hypertension were randomly allocated to an active, mandible protruded (OAa) or a passive, mandible non-protruded device (OAp) treatment. Serum concentrations of the inflammatory biomarkers white blood cells, high-sensitivity C-reactive protein, interleukin 6, interleukin 10, and tumour necrosis factor-alpha were measured at baseline and after 3 months of OA treatment. The differences between treatment groups in biomarker concentration change during the treatment were presented as the Vargha and Delaney effect size and evaluated with the Wilcoxon-Mann-Whitney test. This effect size expresses the probability of a higher value in a random participant from one group compared with a random patient from the other group, and a value of 0.5 means stochastically equal groups. After 3 months of treatment, there was a significant reduction of the apnea-hypopnea index in the OAa group compared with the OAp group (effect size 0.258, 95% confidence interval 0.146-0.386, p < .001). There were no significant differences between the groups in any of the inflammatory markers' concentration changes during the treatment period (effect sizes between 0.488 and 0.524; all p values >=.737). Thus, OA treatment for 3 months did not affect circulating concentrations of some common inflammatory markers in patients with OSA and systemic hypertension.

Background Tetanus, diphtheria, acellular pertussis, inactivated polio (Tdap-IPV) vaccines administered during pregnancy protect young infants from Bordetella pertussis (B. pertussis) infection. Whilst the impact of maternal Tdap-IPV vaccination on infants’ humoral response to subsequent pertussis immunisation has been investigated, little is known about any impact on innate responses. Methods We investigated the immune response to B. pertussis in mothers and infants from Tdap-IPV-vaccinated and unvaccinated pregnancies, utilising a whole blood assay and flow cytometric phenotyping of neonatal natural killer (NK) cells, monocytes and dendritic cells. Blood was collected from mother and umbilical cord at birth, and from infants at seven weeks (one week pre-primary pertussis immunisation) and five months of age (one month post-primary pertussis immunisation). 21 mothers and 67 infants were studied. Findings Vaccinated women had elevated pro-inflammatory cytokine responses to B. pertussis. At birth, babies of vaccinated women had elevated IL-2 and IL-12 responses, elevated classical monocyte proportions, and reduced monocyte and NK cell cytokine responses. The elevated IL-2 response persisted to seven weeks-of-age, when lower IL-10 and IL-13 responses were also seen. One-month post-primary pertussis vaccination, infants from vaccinated pregnancies still had lower IL-10 responses to B. pertussis, as well as lower IL-4. Interpretation This study suggests that pertussis vaccination during pregnancy impacts infant cellular immune responses, potentially contributing to the modification of antibody responses already reported following primary immunisation against B. pertussis. Funding National Institute for Health Research Imperial Biomedical Research Centre and IMmunising PRegnant women and INfants neTwork (funded by the GCRF Networks in Vaccines R&D).

Objective: Interleukin (IL)-38 is a newly discovered member of the IL-1 cytokine family with a proposed anti-inflammatory profile. We studied the probable role of this cytokine in the pathogenesis of two autoimmune diseases: multiple sclerosis (MS) and systemic sclerosis (SSc). Subjects and Methods: A total of 87 MS patients and 86 SSc patients (40 new and recently untreated cases and 46 treated cases) were selected for this study. Eighty-seven and 80 age- and sex-matched healthy subjects were included as controls for MS and SSc, respectively. Clinical and paraclinical features of the patients were recorded at the time of sampling. Serum IL-38 was measured by ELISA. Results: Levels of serum IL-38 did not significantly differ between the total MS or SSc patients compared to controls. However, levels of IL-38 were significantly higher in newly diagnosed patients of MS (206.43 ± 38.97 pg/mL, p < 0.0001) than in those previously treated (158.04 ± 39.45 pg/mL). Similarly, new/recently untreated cases of SSc patients showed increased IL-38 levels (185.19 ± 36.27 pg/mL, p = 0.001) compared to treated patients (166.82 ± 33.08 pg/mL). IL-38 levels in newly diagnosed MS patients (p = 0.007) and new/recently untreated SSc patients (p = 0.032) were significantly higher than those in healthy controls. Conclusion: The higher serum levels of IL-38 in new or recently untreated cases of MS and SSc patients than in treated patients and healthy controls suggest the possible role of this cytokine in the development of these diseases or as part of a feedback loop to attenuate the inflammatory conditions in early stages of these diseases.

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H+ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.

IFNL2 is a potent antiviral interferon, but the regulation of its gene expression is not fully clear. Here, we report the regulation of ATG10S for IFNL2 transcription. Through sequential deletion of the IFNL2 promoter sequence, we found LP1-1, a fragment of the promoter responding to ATG10S activity. Subcellular localization and DNA immunoprecipitation assays showed ATG10S translocating into the nucleus and binding to LP1-1. Online prediction for transcription factor binding sites showed an IRF1 targeting locus in LP1-1. Luciferase assays, RT-PCR, and western blot analysis revealed a core motif (CAAGAC) existing in LP1-1, which determined ATG10S and IRF1 activity; individual nucleotide substitution showed that the functional nucleotides of ATG10S targeting were C1, A3, and C6, and the ones associated with IRF1 were A3 and G4 within the core motif. Co-immunoprecipitation assays revealed ATG10S combination with KPNA1/importin α, KPNB1/importin β, and IRF1. The knockdown of endogenous IRF1 increased ATG10S activity on IFNL2 transcription. These results indicate that ATG10S as a transcription factor competes with IRF1 for the same binding site to promote IFNL2 gene transcription. Abbreviations: ATG10: autophagy related 10; ATG10S: the shorter isoform of autophagy related 10; BD: binding domain; CM: core motif; co-IP: co-immunoprecipitation; GFP: green fluorescent protein; HCV: hepatitis C virus; IF: immunofluorescence; IFN: interferon; IRF: interferon regulatory factor; LP: lambda promoter; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RLU: relative light unit; SQSTM1: sequestosome 1.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

Background: Untreated HIV infection leads to alterations in HIV-specific CD4+ T cells including increased expression of co-inhibitory receptors (IRs) and skewing toward a T follicular helper cell (Tfh) signature. However, which changes are maintained after suppression of viral replication with antiretroviral therapy (ART) is poorly known. Methods: We analyzed blood CD4+ T cells specific to HIV and comparative viral antigens in ART-treated people using a cytokine-independent activation-induced marker assay alone or in combination with functional readouts. Findings: In intra-individual comparisons, HIV-specific CD4+ T cells were characterized by a larger fraction of circulating Tfh (cTfh) cells than CMV- and HBV-specific cells and preferentially expressed multiple IRs and showed elevated production of the Tfh cytokines CXCL13 and IL-21. In addition, HIV-specific cTfh exhibited a predominant Th1-like phenotype and function when compared to cTfh of other specificities, contrasting with a reduction in Th1-functions in HIV-specific non-cTfh. Using longitudinal samples, we demonstrate that this distinct HIV-specific cTfh profile was induced during chronic untreated HIV infection, persisted on ART and correlated with the translation-competent HIV reservoir but not with the total HIV DNA reservoir. Interpretation: Expansion and altered features of HIV-specific cTfh cells are maintained during ART and may be driven by persistent HIV antigen expression. Funding: This work was supported by the National Institutes of Health (NIH), the Canadian Institutes of Health Research (CIHR) and the FRQS AIDS and Infectious Diseases Network. Keywords: HIV, T Follicular helper T cells, HIV-specific CD4+ T cells, Antiretroviral therapy (ART)

Individuals fail to elicit protective antibody after hepatitis B vaccination remain at risk for hepatitis B virus infection. Analysis of the transcriptome of peripheral blood mononuclear cells (PBMCs) is essential to elucidate the characteristics of gene expression in non-responders. In this study, we enrolled seven responders who had received three injections and seven non-responders who had six injections of hepatitis B vaccine before. All the participants were then vaccinated with a three-dose boost regimen. Microarray analysis and Luminex assay were applied to examine mRNA expression and Th1/Th2/Th9/Th17/Th22/Treg cytokine and chemokine profiles in non-responders and responders. Differentially expressed genes in PBMCs of non-responders at 5 time points, i.e. pre-vaccination, 3(rd), 7(th), 28(th) day post the first dose vaccination and 7(th) day post the second dose vaccination indicated a dense network trend. Compared with responders, nine coding genes (BPI, DEFA1B, DEFA4, CEACAM8, MMP8, FOLR3, LTF, TCN1 and TKTL1) were significantly up-regulated in non-responders at all 5 time points, which could probably be the characteristic genes in hepatitis B vaccine non-responsiveness. Gene ontology analysis revealed that most of the DEGs were related with immune responses. Validation results of these 9 genes using quantitative real-time polymerase chain reaction were mostly consistent with the results of microarray. Cytokine analysis demonstrated that IL-27 and CXCL12 concentrations in responders were significantly higher than non-responders on the 3(rd) day after the first dose and 7(th) day after the second dose of vaccination, respectively. No significant difference was observed in other cytokine and chemokine signatures between the two groups. In conclusion, our results revealed characteristic transcriptome and cytokine changes in hepatitis B vaccine non-responders after boost immunization.

Objective: To evaluate the effect of laser irradiation at different wavelengths on the expression of selected growth factors and inflammatory mediators at particular stages of the wound healing process. Methods: Sixty-seven patients were recruited, treated, and analyzed (group A - 940 nm: 17 patients; group B - 808 nm: 18 patients; group C - 658 nm: 16 patients; group D - sham therapy: 17 patients). Patients received a basic treatment, including repositioning and mobilization, air pressure mattress and bed support surfaces, wound cleansing and drug therapy. Additionally, patients received laser therapy once a day, 5 times a week for 1 month in use of a semiconductor lasers (GaAIAs) which emitted a continuous radiation emission at separate wavelengths of 940 nm (group A), 808 nm (group B) and 658 nm (group C). In group D (sham therapy), laser therapy was applied in the same manner, but the device was off during each session (only the applicator was switched on to scan pressure ulcers using none coherent red visible light). Results: The positive changes in the measured serum (IL-2, IL-6 and TNF-alpha) and wound tissue (TNF-alpha, VEGF and TGF beta 1) parameters appeared to be connected only with the wavelength of 658 nm. The significant change in pro-inflammatory mediator levels [interleukin 2 (IL-2) with p=0.008 and interleukin 6 (IL-6) with p=0.016] was noticed after two weeks of laser therapy. In the other groups, the inflammation was also reduced, but the process was not as marked as in group C. Similarly, in the case of tumor necrosis factor (TNF-alpha) concentration, where after two weeks of treatment with irradiation at a wavelength of 658 nm, a rapid suppression was observed (p=0.001), whereas in the other groups, these results were much slower and not as obvious. Interestingly, again in the case of group C, the change in TNF-alpha concentration in wound tissue was most intensive (approximate to 75% reduction), whereas the changes in other groups were not as obvious (approximate to 50% reduction). After irradiation (658 nm), the VEGF expression increased significantly within the first two weeks, and then it decreased and maintained a stable level. In contrast, the TGF beta 1 activity remained level, but always higher in comparison to other groups Conclusions: The effective healing of pressure ulcers is connected with laser irradiation at a wavelength of 658 nm. We believe that this effect is related to the inhibition of inflammatory processes in the wound and stimulation of angiogenesis and fibroblast proliferation at this specific radiation (based both on concentration of interleukins and TNF-alpha serum level and VEGF, TGF beta 1, TNF-alpha activities in wound biopsies). Laser therapy at wavelengths of 940 and 808 nm does not significantly affect the above-mentioned repair processes, which explains its low effectiveness in the treatment of pressure ulcers.

Acute or acute-on-chronic liver failure is a leading cause of death in liver diseases without effective treatment. Interleukin-22 (IL-22) is currently in clinical trials for the treatment of severe alcoholic hepatitis, but the underlying mechanisms remain to be explored. Autophagy plays a critical role in alleviating liver injury. The aim of the current study is to explore the role of autophagy in IL-22-mediated hepato-protective effect against acetaminophen (APAP)-induced liver injury. Methods: A model of acute liver injury induced by APAP was used in vivo. IL-22 was administrated to the APAP-treated mice. Hepatocytes were pre-incubated with IL-22, followed by exposure to APAP for in vitro analyses. Results: IL-22 administration significantly reduced serum ALT and AST, hepatic reactive oxygen species, and liver necrosis in APAP-challenged mice. APAP treatment increased hepatic autophagosomes, which was further intensified by IL-22 co-treatment. Hepatic LC3-II was moderately upregulated after APAP administration without obvious alteration of phosphorylation of AMP-activated kinase (p-AMPK). IL-22 pretreatment significantly upregulated hepatic LC3-II and p-AMPK in APAP-treated mice. IL-22 also alleviated APAP-induced cytotoxicity and upregulated LC3-II and p-AMPK expression in cultured hepatocytes treated with APAP in vitro. When p-AMPK was blocked with compound C (an AMPK inhibitor), IL-22-mediated LC3-II conversion and protection against APAP-induced cytotoxicity was weakened. Conclusions: Enhanced AMPK-dependent autophagy contributes to protective effects of IL-22 against APAP-induced liver injury.

Background The initial clinical manifestations and abdominal imaging findings of neonates with necrotising enterocolitis (NEC) and food protein-induced enterocolitis syndrome (FPIES) are sometimes similar; however, their prognosis and therapies are different. We aimed to evaluate the utility of interleukin (IL)-27 as a differentiation marker between NEC and highly suspected early onset (HSEO)-FPIES. Methods All samples used in this study were obtained from the neonatal diagnosis centre of Children's Hospital of Chongqing Medical University. In the case-control study, neonates with NEC (n = 13), HSEO-FPIES (n = 9), and jaundice (control, n = 8) were enroled to determine the serum IL-27 levels using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the validation cohort study, the NEC (n = 87), HSEO-FPIES (n = 62), and jaundice (control, n = 54) groups were included to analyse the diagnostic efficiency of IL-27 for discriminating between NEC and HSEO-FPIES using a receiver operating characteristic (ROC) curve. Findings In the case-control study, IL-27 levels were higher in the NEC group than in the HSEO-FPIES group (p = 0·005). In the cohort study, the area under the ROC curve (AUC) of IL-27 for differentiating NEC from HSEO-FPIES was 0·878, which was higher than the AUCs of IL-6 (0·761), C-reactive protein (0·800), white blood cell count (0·637), neutrophils (0·765), lymphocytes (0·782), neutrophil to lymphocyte ratio (0·781), and platelet count (0·729). Interpretation Serum IL-27 is a novel biomarker that may potentially discriminate NEC from HSEO-FPIES in neonates. Funding None.

The high rate of chronic hepatitis C (CHC) was one of the key issues of global public health concern. Interferon (IFN)-λ relevant genes were in the antiviral treatment pathway, not only influenced hepatitis C virus (HCV) spontaneous clearance, but also affected the IFN-mediated viral clearance. The aim of this study was to identify the association of interleukin 28B (IL28B), myxovirus resistance A (MxA) gene polymorphisms with HCV spontaneous clearance and therapeutic response in Chinese CHC patients. IL28B and MxA gene genotypes were detected among 231 CHC carriers, 428 subjects with HCV spontaneous clearance and 662 CHC patients with pegylated IFN-α and ribavirin (pegIFN-α/RBV) treatment. Patients with MxA rs2071430 TT genotype were more likely to develop HCV infection chronicity (additive model: odds ratio (OR) 1.22, 95% confidence interval (CI) 1.01–1.48, P = 0.042). IL28B rs1298075 variant genotypes (additive model: OR 0.58, 95% CI 0.34–0.98, P = 0.040) and MxA rs17000900 variant genotypes (additive model: OR 0.54, 95% CI 0.30–0.99, P = 0.048) were less likely to achieve a sustained virological response. The life table indicated that patients with IL28B rs1298075 AG genotype were slower to achieve a viral load P = 0.018). During the treatment, the downward trend in viral load was different among each IL28B rs1298075 genotype, especially in subgroup with a baseline HCV-RNA >106 copies/ml (all P < 0.05). This study illustrated that the carriage of IL28B rs12980275 AA had a positive effect on treatment response to pegIFN-α/RBV among Chinese CHC patients.

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin‐17 (IL‐17) is key in host defence in TB but its role in TB‐driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T‐helper 17 (TH‐17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH‐17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT‐qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho‐western analysis and siRNA. IL‐17 is expressed in TB patient granulomas and MMP‐3 is expressed in adjacent pulmonary epithelial cells. IL‐17 had a divergent, concentration‐dependent effect on MMP secretion, increasing epithelial secretion of MMP‐3 (p < 0.001) over 72 h, whilst decreasing that of MMP‐9 (p < 0.0001); mRNA levels were similarly affected. Both IL‐17 and IL‐22 increased fibroblast Mtb‐dependent MMP‐3 secretion but IL‐22 did not modulate epithelial MMP‐3 expression. Both IL‐17 and IL‐22, but not IL‐23, were significantly up‐regulated in BALF from TB patients. IL‐17‐driven MMP‐3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL‐17 drives airway stromal cell‐derived MMP‐3, a mediator of tissue destruction in TB, alone and with monocyte‐dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: Genetic variation near the interferon lambda 3 (IFNL3) is known to be associated with response to pegylated interferon (pegIFN) and ribavirin combination therapy in patients with chronic hepatitis C virus (HCV) infection which is often accompanied by hepatic steatosis. Aims: We examined whether this genetic variation is associated with host lipids and treatment response. Methods: A total of 101 Japanese patients who had underwent liver biopsy before treatment with pegIFN and ribavirin for HCV genotype 1b infection were retrospectively analyzed for association between IFNL3 genotypes (rs8099917) and clinical factors including histopathological features of the liver. The presence of >5% steatosis in the liver specimen was defined as hepatic steatosis. Results: Forty patients (40%) had liver steatosis before therapy. Patients with IFNL3 minor genotype (non-TT) showed lower low-density lipoprotein cholesterol level (p=0.0045), higher γ-glutamyl transpeptidase level (p=0.0003) and higher prevalence of hepatic steatosis (p=0.0002). Advanced fibrosis [odds ratio (OR) 4.63, p=0.03] and IFNL3 major genotype (OR 0.13, p=0.001) were 2 independent factors for determining the presence of hepatic steatosis. Among the factors associated with sustained virological response, IFNL3 genotype was the most significant predictor, as per multivariate analysis. Conclusions: Our results confirmed that IFNL3 genotype is associated with hepatic steatosis as well as IFN response.

Background Patients with advanced malignancies, e.g. lung cancer, ovarian cancer or melanoma, frequently present with brain metastases. Clinical presentation and disease progression of cancer is in part shaped by the interaction of the immune system with malignant cells. Antigen-targeted immune responses have been implicated in the prolonged survival of patients with cancer. This includes the tumor-associated antigen (TAA) mature mesothelin, a 40 kDa cell surface-bound antigen that is overexpressed in several malignancies including lung ovarian and pancreatic cancer. We examined in an observational, prospective study the survival of patients with brain metastases in association with clinical parameters and cellular immune responses to molecularly defined TAAs or viral (control) target antigens. Methods Immune cells in peripheral blood obtained from thirty-six patients with brain metastases were tested for cytokine production in response to a broad panel of defined viral and TAA target antigens, including full-length mesothelin. Incubation of immune cells with antigenic targets was carried out in i) medium alone, (ii) in a cytokine cocktail of interleukin (IL)-2/IL-15/IL-21, or (iii) IL-2/IL-7. Supernatants were tested for interferon gamma (IFN-γ) production, after which univariate and multivariate analyses (Cox stepwise regression model) were performed to identify independent clinical and immunological factors associated with patient survival. Patients were followed-up for at least 500 days after surgery or until death. Findings Univariate analysis identified age, gender, radiotherapy and mutational load as clinical parameters affecting survival of patients with brain metastases. Cox multivariate analysis showed that radiotherapy (P = 0·004), age (P = 0·029) and IFN-γ responses to mature mesothelin, conditioned by IL-2/IL-7 (P = 0·045) were independent predictors of the survival of patients from surgery up to follow-up or death. Interpretation This is the first evidence that immune responses to mesothelin serve as a marker of increased overall survival in patients with brain metastases, regardless of the primary tumor origin. Analyses of immunological markers could potentially serve as prognostic markers in patients with brain metastases and help to select patients in need for adjunct, immunological, treatment strategies., Highlights • Mesothelin-specific cellular immune responses (CIR) can be detected in the blood of patients with brain metastases • This response can be amplified using cytokine cocktails i.e. interleukin (IL) 2 & IL-7 or IL-2, IL- 15 & IL-21 • Mesothelin epitope-specific CIR can predict survival of patients with brain metastases, with implications for immunotherapy Mesothelin expression, its role in metastasis, and its immunodiagnostic potential in several hard-to-treat solid tumors has been described. The present study shows that mesothelin-directed peripheral cellular immune responses can be detected in patients with brain metastases, and furthermore, may influence their survival, underlining that the host’s ‘immunological fitness’ is vital to control transformed cells. T cells recognizing mesothelin epitopes may be developed for immunotherapy of patients with brain metastases lacking anti-mesothelin responses. The current study also warrants the discovery of other biologically and clinically relevant cellular targets in patients with brain metastases, which may provide robust tools for improving healthcare decisions, complementing surgery and radiotherapy.

International audience; Angiogenesis represents a hallmark of tumor progression in Multiple Myeloma (MM), a still incurable malignancy. Here we analyzed the activity of cytokine-stimulated NK cells against tumor-associated endothelial cells isolated from bone marrow aspirates of MM patients with active disease (MMECs). We show that NK cells activated with optimal doses of IL-15 killed MMECs thanks to the concerted action of multiple activating receptors. In particular, according to the high expression of PVR and Nectin-2 on MMECs, DNAM-1 actively participated in target recognition. Interestingly, in MMECs the surface density of PVR was significantly higher than that detected in endothelium from patients with MM in complete remission or with monoclonal gammopathy of undetermined significance (MGUS). Importantly, IL-27, which unlike IL-15 does not display pro-angiogenic properties, maintained or increased the NK cell functions induced by suboptimal concentrations of IL-15. NK cell properties included killing of MMECs, IFN-γ production as well as a peculiar increase of NKp46 expression on NK cell surface. Finally, IL-27 showed a striking capability of up-regulating the expression of PD-L2 and HLA-I on tumor endothelium, whereas it did not modify that of PD-L1 and HLA-II. Our results suggest that cytokine-activated endogenous or adoptively transferred NK cells might support conventional therapies improving the outcome of MM patients.

// Jiu-Ming Zou 1, * , Jian Qin 1, * , Yong-Chao Li 1 , Yu Wang 1 , Dong Li 1 , Yu Shu 1 , Chao Luo 1 , Shan-Shan Wang 1 , Gang Chi 1 , Fang Guo 1 , Gui-Mei Zhang 1 , Zuo-Hua Feng 1 1 Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, The People’s Republic of China * These authors have contributed equally to this work Correspondence to: Zuo-Hua Feng, email: fengzhg_tj@163.com Yong-Chao Li, email: yongchaolee@outlook.com Keywords: IL-35, neutrophils, inflammation, tumor, cytokine Received: December 08, 2016 Accepted: March 24, 2017 Published: April 04, 2017 ABSTRACT IL-35 is an immunosuppressive cytokine and exerts regulatory effects on T cells, B cells, macrophages and dendritic cells. Neutrophils are important innate immune cells that play key roles in tumor development. The effect of IL-35 on neutrophils remains unknown. Here, we report that IL-35 can induce N2 neutrophil polarization (protumor phenotype) by increasing G-CSF and IL-6 production, and promote neutrophil infiltration into tumor microenvironment. The sustained expression of IL-35 could promote chronic inflammation to augment the proangiogenic and immunosuppressive function of neutrophils. IL-35 stimulated macrophages to secrete proinflammatory cytokines IL-1β and IL-6. IL-1β stimulated γδ T cells to produce IL-17, which in turn increased the production of G-CSF. By increasing the expression of G-CSF and IL-6, IL-35 could up-regulate the expression of MMP-9 and Bv8, and down-regulate TRAIL expression in neutrophils, thus augmenting the proangiogenic function of neutrophils. Moreover, G-CSF/IL-6 induced the enhanced activation of STAT3 and ERK pathways in neutrophils, thus increasing the expression of iNOS to suppress T cell activation. Our findings suggest that IL-35 can promote tumor progression by functioning as an up-stream cytokine to promote cancer-associated inflammation and control neutrophil polarization. Targeting IL-35 might be an important approach for designing new strategy of tumor therapy.

// Yulin Liu 1, * , Fan Xiang 1, * , Yongming Huang 1 , Liang Shi 2 , Chaojie Hu 1 , Yiming Yang 1 , Di Wang 1 , Nan He 1 , Kaixiong Tao 1 , Ke Wu 1 , Guobin Wang 1 1 Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China * These authors have contributed equally to this work Correspondence to: Guobin Wang, email: guobinwanguh@163.com Ke Wu, email: wuke_2015@yeah.net Keywords: interleukin-22, colon cancer, aerobic glycolysis, proliferation, hexokinase-2 Received: August 20, 2016 Accepted: February 15, 2017 Published: March 06, 2017 ABSTRACT Interleukin-22 has been explored extensively in human cancer, but its functions and underlying mechanisms are incompletely understood. Here, we show that aberrant interleukin-22 expression facilitates aerobic glycolysis in colon cancer cells. Elevated interleukin-22 mRNA expression was observed and positively correlated with hexokinase-2 in colon cancer tissues. In vitro , interleukin-22 enhanced glucose consumption and lactate production via targeting hexokinase-2 in colon cancer cells. Moreover, the transcriptional factor c-Myc and signal transducer and activator of transcription 3 were involved in interleukin-22-induced up-regulation of hexokinase-2. We further demonstrated that hexokinase-2 partly accounted for interleukin-22-mediated cellular proliferation in DLD-1 cells. In vivo , our data demonstrated that interleukin-22 significantly promoted tumor growth along with elevated expression of c-Myc and hexokinase-2 in mice. In summary, our findings provide a new perspective on the pro-inflammatory cytokine interleukin-22 in promoting aerobic glycolysis associated with tumor progression in human colon cancer cells.

// Stefania Madonna 1 , Claudia Scarponi 1 , Martina Morelli 1 , Rosanna Sestito 1, 3 , Pasqualina Liana Scognamiglio 2 , Daniela Marasco 2 , Cristina Albanesi 1 1 Laboratory of Experimental Immunology, IDI-IRCCS, Fondazione “Luigi M. Monti” (FLMM), Rome, Italy 2 Department of Pharmacy, CIRPEB, University of Naples “Federico II”, Naples, Italy 3 Current address: Preclinical Models and New Therapeutic Agents Unit, Regina Elena National Cancer Institute, Rome, Italy Correspondence to: Stefania Madonna, email: s.madonna@idi.it Cristina Albanesi, email: c.albanesi@idi.it. Keywords: SOCS1, SOCS3, non-melanoma skin cancers, IL-22 signaling, healthy and transformed keratinocytes Received: July 28, 2016 Accepted: February 15, 2017 Published: February 22, 2017 ABSTRACT Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes. In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo , in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts.

// Shiri Davidi 1, * , Ella Fremder 1, * , Tal Kan 1 , Ziv Raviv 1 , Michael Timaner 1 , Nathan Karin 2 , Dov Hershkovitz 3 , Ami Arohneim 1, * , Yuval Shaked 1, * 1 Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion, Haifa, Israel 2 Department of Immunology, Rappaport Faculty of Medicine, Technion, Haifa, Israel 3 Department of Pathology, Rambam Medical Center, Haifa, Israel * These authors contributed equally to this work Correspondence to: Yuval Shaked, email: yshaked@tx.technion.ac.il Ami Aronheim, email: aronheim@tx.technion.ac.il Keywords: metastasis, angiogenesis, cancer therapy, host response Received: December 14, 2016 Accepted: January 16, 2017 Published: January 27, 2017 ABSTRACT Pro-inflammatory cytokines in the tumor microenvironment are known for their ability to either inhibit or promote cancer progression. Here we evaluated the role of Interleukin-31 (IL31), a protein belonging to the pro-inflammatory IL-6 cytokine family which has been characterized in autoimmune disease, in tumorigenesis. We show that IL31 and its receptor, IL31RA, are highly expressed in various human and mouse cancer cell lines, as well as in tumor specimens from cancer patients. MC38 murine colon carcinoma cells depleted of IL31 exhibit an increase in invasive and migratory properties in vitro , effects that are reversed by supplementing the cells with exogenous IL31. In vivo , IL31-depleted MC38 tumor cells implanted to mice grow faster than control tumors. In contrast, MC38 tumor-bearing mice infused with recombinant IL31, exhibit a significant reduction in tumor growth than control mice. Furthermore, IL31 infusion reduces the number of metastatic lesions in the lungs of mice bearing 4T1 murine metastatic breast carcinoma. Lastly, injecting tumor-bearing, chemotherapy-treated mice with a long-lived IL31-IgG fusion protein reduces tumor growth, angiogenesis and pulmonary metastasis to a greater extent than when chemotherapy is used alone. The IL31 anti-tumor activity is explained, in part, by the anti-angiogenic effects demonstrated both in vitro and in vivo highlighting the potential use of IL31 as an anti-cancer drug.

Objective To investigate the responses of refrigerated osteochondral allograft cartilage (OCA) and fresh cartilage (FC), including cell survival and metabolism, to surgical impaction and proinflammatory cytokines. Design Osteochondral plugs (8 mm diameter) were harvested from prolonged-refrigerated (14-28 days) and fresh (≤24 hours postmortem) human femoral hemicondyles and subjected to a 0.2 N s pneumatic impaction impulse. Cartilage explants were removed from subchondral bone and randomized to 1 of 6 treatment groups: (1) Unimpacted control (UIC), (2) Impacted control (IC), (3) Impacted + interleukin (IL)-1β (0.1 ng/mL), (4) Impacted + IL-1β (0.1 ng/mL) + IL-6, (5) Impacted + IL-1β (10 ng/mL), and (6) Impacted + IL-1β (10 ng/mL) + IL-6. Samples were measured for cell viability, histology, and proteoglycan (PG) content at days 0, 2, 7, and 14 of culture. Results In UIC, cell viability was indistinguishable between OCA and FC and remained constant. Impaction alone decreased cell viability by 30% ( P < 0.01) in the OCA superficial layer and by 26% ( P < 0.01) in the entire tissue, but did not affect viability in FC. Cytokine addition did not further influence cell viability. Impaction alone did not affect PG synthesis. Addition of cytokines to impacted tissue decreased PG synthesis by ~3-fold in both tissue types in comparison with corresponding impacted controls ( P < 0.01). Throughout 2-week culture, PG release remained stable in all FC groups, but peaked at day 14 in OCA cartilage subjected to cytokines. Conclusions Mechanical impaction, mimicking surgical insertion, has a more profound effect on cell viability in OCA than in FC. Addition of proinflammatory cytokines further decreases OCA tissue metabolism and integrity.

// Dawei Cui 1,2,* , Fengyun Zhong 3,* , Jie Lin 4,* , Yidong Wu 5 , Quan Long 1,2 , Xianzhi Yang 1,2 , Qiaoyun Zhu 1,2 , Li Huang 1,2 , Qifen Mao 1,2 , Zhaoxia Huo 1,2 , Zhe Zhou 1,2 , Guoliang Xie 1,2 , Shufa Zheng 1,2 , Fei Yu 1,2 and Yu Chen 1,2 1 Department of Laboratory Medicine, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2 Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, China 3 Department of General Surgery, the Second Affiliated Hospital of Soochow University, Suzhou, China 4 Department of Clinical Laboratory, Center of Community Health Service of Qingbo Street, Hangzhou, China 5 Clinical Laboratory, Hangzhou Children’s Hospital, Hangzhou, China * These authors have contributed equally to this work Correspondence to: Yu Chen, email: // Keywords : HFMD, EV71, circulating Th22 cell, circulating Th17 cell Received : June 06, 2016 Accepted : December 16, 2016 Published : December 21, 2016 Abstract Interleukin (IL)-22 + CD4 + T (Th22) cells play crucial roles in the pathogenesis of autoimmune diseases and infectious diseases, although the role of Th22 cells remains largely unclear in children with hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71). This study aims to explore the role of circulating IL-22 + IL-17A - CD4 + T (cTh22) cells in children with EV71-associated HFMD. We found that during the acute stage of illness, the frequencies of cTh22 and circulating IL-22 + IL-17A + CD4 + T (IL-22 + cTh17) cells in CD4 + T cells infrom affected patients, and especially in severely affected patients, were significantly higher than in healthy controls (HC). The major source of IL-22 production was cTh22 cells, partially from cTh17 cells. Moreover, the protein and mRNA levels of IL-22, IL-17A, IL-23, IL-6, and TNF-α were significantly different among the mild patients, severe patients and HC, as well as AHR and RORγt mRNA levels. A positive correlation was found between plasma IL-22 levels and cTh22 cell frequencies, and cTh17 cell and IL-22 + cTh17 cell frequencies. Furthermore, the frequencies of cTh22 were significantly decreased in the convalescent patients. Our findings indicated that cTh22 cells could play critical roles in the pathogenesis of EV71 infection, and are potential therapeutic targets for patients with EV71-associated HFMD.

// Yi-Ching Lin 1, 2, 3, 4 , Yu-Chih Lin 5 , Cheng-Chin Wu 6 , Ming-Yii Huang 7 , Wen-Chan Tsai 2, 6 , Chih-Hsing Hung 1, 3, 8, 9, 10 , Po-Lin Kuo 1, 11 1 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Department of Laboratory Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Division of General Internal Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 8 Department of Pediatrics, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan 9 Department of Pediatrics, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 10 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 11 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Wen-Chan Tsai, email: d740094@cc.kmu.edu.tw Chih-Hsing Hung, email: pedhung@gmail.com Po-Lin Kuo, email: kuopolin@seed.net.tw Keywords: TNF-α, Th17, RORγt, histone acetylation, rheumatoid arthritis Received: August 31, 2016 Accepted: November 22, 2016 Published: December 03, 2016 ABSTRACT The presence of interleukin (IL)-17-related cytokines correlates with rheumatoid arthritis (RA) pathogenesis. Epigenetic modifications, including histone acetylation, regulate gene expression in RA pathogenesis. Tumour necrosis factor-alpha (TNF-α) inhibitors such as etanercept and adalimumab, represent a breakthrough in RA treatment. We aimed to investigate the effects of etanercept and adalimumab on human Th17-polarized cells and the possible intracellular regulators of these effects, including the Th17-specific transcription factors signal transducer, activator of transcription 3 (STAT3), retinoid-related orphan receptor γ-T (RORγt) and epigenetic modification. Human CD4 + T cells from healthy subjects and patients with RA were pretreated with TNF-α inhibitors and then being polarized into IL-17-producing cells. The Th17-related cytokine levels in the culture supernatants were determined with an enzyme-linked immunosorbent assay. Intracellular signalling was investigated by western blot, real-time RT-PCR, and chromatin immunoprecipitation. Th17-polarized cells from patients with RA produced more IL-17A, IL-17F and IL-22 than those from healthy subjects. Etanercept and adalimumab suppressed IL-17A, IL-17F and IL-22 levels in Th17-polarized cells from healthy subjects and patients with RA. Western blot analysis revealed that etanercept and adalimumab decreased mitogen-activated protein kinase-phospho-p38, nuclear factor-κB-phospho-p65, phospho-STAT3 and RORγt levels. Etanercept and adalimumab decreased histone (H)3 and H4 acetylation in the RORγt gene promotor region by decreasing the recruitment of the acetyltransferases p300, CBP and PCAF. The present study broadens our knowledge of the mechanisms underlying the immunomodulatory effects of TNF-α inhibitors in rheumatoid arthritis treatment.

// Fengyan Jin 1 , Hai Lin 1 , Sujun Gao 1 , Zheng Hu 2 , Song Zuo 2 , Liguang Sun 2 , Chunhui Jin 1 , Wei Li 1 , Yanping Yang 1 1 Department of Hematology, The First Bethune Hospital of Jilin University, Changchun, China 2 Institute of Translational Medicine, The First Bethune Hospital of Jilin University, Changchun, China Correspondence to: Yanping Yang, email: yangyanping_1978@163.com Keywords: natural killer cells, interleukin, interferon-γ, NKG2D, leukemia Received: December 13, 2015 Accepted: October 26, 2016 Published: November 01, 2016 ABSTRACT Although natural killer cells (NK cells) were traditionally classified as members of the innate immune system, NK cells have recently been found also to be an important player in the adaptive immune systems. In this context, in vitro activation of NK cells by cytokines leads to generation of NK cells with memory-like properties characterized by increased interferon-γ (IFNγ) production. However, it remains to be defined whether these memory-like NK cells exist in vivo after cytokine activation. Furthermore, it is also unclear whether such memory-like NK cells induced in vivo by cytokines could have effective anti-leukemia response. To address these issues, we used an in vivo pre-activation and re-stimulation system that was able to produce NK cells with increased IFNγ secretion. It was found that after in vivo pre-activation and re-stimulation with interleukins (ILs), NK cells retained a state to produce increased amount of IFNγ. Of note, whereas this intrinsic capacity of enhanced IFNγ production after in vivo IL pre-activation and re-stimulation could be transferred to the next generation of NK cells and was associated with prolonged survival of the mice with acute lymphoid leukemia. Moreover, the anti-leukemia activity of these memory-like NK cells was associated with IFNγ production and up-regulation of NK cells activation receptor-NK Group 2 member D (NKG2D). Together, these findings argue strongly that in vivo IL pre-activation and re-stimulation is capable to induce memory-like NK cells as observed previously in vitro , which are effective against acute lymphoblastic leukemia, likely via NKG2D-dependent IFNγ production, in intact animals.

// Denada Dibra 1 , Abhisek Mitra 1 , Melissa Newman 2 , Xueqing Xia 1 , Camille Keenan 3 , Jeffry J. Cutrera 1 , J. Michael Mathis 4 , Xiao-Jing Wang 5 , Jeffrey Myers 6 , Shulin Li 1 1 Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Banfield Pet Hospital, Sugar Land, TX 77479, USA 3 The University of Texas Health Science Center at Houston, Medical School, Houston, TX 77030, USA 4 Department of Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803, USA 5 Department of Pathology, University of Colorado Anschutz Medical School, Aurora, CO 80045, USA 6 Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Shulin Li, email: sli4@mdanderson.org Denada Dibra, email: ddibra@mdanderson.org Keywords: ET1/ETAR, IL27, IL27RA, KRAS, inflammation Received: December 14, 2015 Accepted: September 02, 2016 Published: October 12, 2016 ABSTRACT Establishment of a permissive pre-malignant niche in concert with mutant stem are key triggers to initiate skin carcinogenesis. An understudied area of research is finding upstream regulators of both these triggers. IL27, a pleiotropic cytokine with both pro- and anti-inflammatory properties, was found to be a key regulator of both. Two step skin carcinogenesis model and K15-KRAS G12D mouse model were used to understand the role of IL27 in skin tumors. CD11b -/- mice and small-molecule of ETAR signaling (ZD4054) inhibitor were used in vivo to understand mechanistically how IL27 promotes skin carcinogenesis. Interestingly, using in vivo studies, IL27 promoted papilloma incidence primarily through IL27 signaling in bone-marrow derived cells. Mechanistically, IL27 initiated the establishment of the pre-malignant niche and expansion of mutated stem cells in K15-KRAS G12D mouse model by driving the accumulation of Endothelin A receptor (ETAR)-positive CD11b cells in the skin—a novel category of pro-tumor inflammatory identified in this study. These findings are clinically relevant, as the number of IL27RA-positive cells in the stroma is highly related to tumor de-differentiation in patients with squamous cell carcinomas.

Fil: Cao, Qiuhua. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Cao, Qiuhua. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Gao, Xinghua. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Gao, Xinghua. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Lin, Yanting. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Lin, Yanting. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Yue, Chongxiu. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Yue, Chongxiu. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Wang, Yue. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Wang, Yue. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Quan, Fei. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Quan, Fei. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Zhang, Zixuan. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Zhang, Zixuan. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Liu, Xiaoxuan. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Liu, Xiaoxuan. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Lu, Yuan.China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Lu, Yuan. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Zhan, Yanling. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Zhan, Yanling. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Yang, Hongbao. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Yang, Hongbao. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Li, Xianjing. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Li, Xianjing. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Fil: Qin, Di. Nanjing sport institute. School of Sports and Health; China Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Laboratory of Neurobiology; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Hao, Kun. China Pharmaceutical University. Key Lab of Drug Metabolism & Pharmacokinetics; China Fil: Yang, Yong. China Pharmaceutical University. State Key Laboratory of Natural Medicines; China Fil: Yang, Yong. China Pharmaceutical University. Center for New Drug Safety Evaluation and Research; China Background: Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease, notoriously challenging to treat. Previous studies have found a positive correlation between thymic atrophy and colitis severity. It was, therefore, worthwhile to investigate the effect of thymopentin (TP5), a synthetic pentapeptide corresponding to the active domain of the thymopoietin, on colitis. Methods: Dextran sulfate sodium (DSS)-induced colitis mice were treated with TP5 by subcutaneous injection. Body weight, colon length, colon weight, immune organ index, disease activity index (DAI) score, and the peripheral blood profile were examined. The immune cells of the spleen and colon were analyzed by flow cytometry. Histology was performed on isolated colon tissues for cytokine analysis. Bacterial DNA was extracted from mouse colonic feces to assess the intestinal microbiota. Intestinal lamina propria mononuclear cells (LPMCs), HCT116, CT26, and splenocytes were cultured and treated with TP5. Results: TP5 treatment increased the body weight and colon length, decreased the DAI score, and restored colon architecture of colitic mice. TP5 also decreased the infiltration of immune cells and expression levels of pro-inflammatory cytokines such as IL-6. Importantly, the damaged thymus and compromised lymphocytes in peripheral blood were significantly restored by TP5. Also, the production of IL-22, both in innate and adaptive lymphoid cells, was triggered by TP5. Given the critical role of IL-22 in mucosal host defense, we tested the effect of TP5 on mucus barrier and gut microbiota and found that the number of goblet cells and the level of Mucin-2 expression were restored, and the composition of the gut microbiome was normalized after TP5 treatment. The critical role of IL-22 in the protective effect of TP5 on colitis was further confirmed by administering the anti-IL-22 antibody (αIL-22), which completely abolished the effect of TP5. Furthermore, TP5 significantly increased the expression level of retinoic acid receptor-related orphan receptor γ (RORγt), a transcription factor for IL-22. Consistent with this, RORγt inhibitor abrogated the upregulation of IL-22 induced by TP5. Conclusion: TP5 exerts a protective effect on DSS-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes. This study delineates TP5 as an immunomodulator that may be a potential drug for the treatment of UC.

Background: Previous studies in human subjects have mostly been confined to peripheral blood lymphocytes for Pneumocystis infection. We here aimed to compare circulating and pulmonary T-cell populations derived from human immunodeficiency virus (HIV)-uninfected immunocompromised patients with Pneumocystis jirovecii pneumonia (PCP) in order to direct new therapies. Methods: Peripheral blood and bronchoalveolar lavage samples were collected from patients with and without PCP. Populations of Th1/Tc1, Th2/Tc2, Th9/Tc9, and Th17/Tc17 CD4+ and CD8+ T cells were quantified using multiparameter flow cytometry. Results: No significant differences were found between PCP and non-PCP groups in circulating T cells. However, significantly higher proportions of pulmonary Th1 and Tc9 were observed in the PCP than in the non-PCP group. Interestingly, our data indicated that pulmonary Th1 was negatively correlated with disease severity, whereas pulmonary Tc9 displayed a positive correlation in PCP patients. Conclusions: Our findings suggest that pulmonary expansion of Th1 and Tc9 subsets may play protective and detrimental roles in PCP patients, respectively. Thus, these specific T-cell subsets in the lungs may serve as targeted immunotherapies for patients with PCP.

IBD (inflammatory bowel disease), where CD (Crohn's disease) and UC (ulcerative colitis) represent the two main forms, are chronic inflammatory conditions of the intestine. Macrophages play a central role in IBD pathogenesis and are regulated by major differentiation factors such as CSF-1 (colony-stimulating factor 1) in homoeostasis and inflammation. IL (interleukin)-34 has recently been discovered as a second ligand for CSF-1R (CSF-1 receptor). However, expression and involvement of IL-34 in IBD remain unknown. In the present paper, we investigated the expression of IL34, CSF1 and their shared receptor CSF1R in normal human ileum and colon, in inflamed and non-inflamed tissues of CD and UC patients, and in a mouse model of experimental colitis. We found distinct expression patterns of IL34 and CSF1 in ileum and colon, with higher IL34 in ileum and, in contrast, higher CSF1 in colon. Furthermore, IL34 and CSF1 expression was increased with inflammation in IBD patients and in experimental colitis. In humans, infiltrating cells of the lamina propria and intestinal epithelial cells expressed IL-34, and TNF-α (tumour necrosis factor α) regulated IL-34 expression in intestinal epithelial cells through the NF-κB (nuclear factor κB) pathway. These data demonstrate the expression pattern of IL-34 in ileum and colon and suggest IL-34 as a new modulator of inflammation in IBD., The macrophage differentiation factor interleukin-34, produced by intestinal epithelial cells, is up-regulated in patients with inflammatory bowel disease, and may be a novel modulator of intestinal inflammation.

IL-35 is a novel heterodimeric and inhibitory cytokine, composed of interleukin-12 subunit alpha (P35) and Epstein-Barr virus -induced gene 3 (EBI3). IL-35 has been reported to be produced by a range of cell types, especially regulatory T cells, and to exert immunosuppressive effects via the STATx signaling pathway. In this study, we demonstrated that IL-35 expression was elevated in both serum and tumors in patients with colorectal cancer. IL-35 mainly expressed in CD4+ T cells in human colorectal cancer tumors and adjacent tissues. Increased IL-35 expression in tumor-adjacent tissues was significantly associated with tumor metastasis. IL-35 inhibited the proliferation of CD4+CD25− T effector cells in vitro in a dose-dependent manner, and its suppression was partially reversed by applying IL-35-neutralizing antibodies. IL-35 treatment activated the phosphorylation of both STAT1 and STAT3 in human CD4+ T cells. Meanwhile, IL-35 induced a positive feedback loop to promote its own production. We observed that Tregs obtained from colorectal cancer patients were capable of inducing more IL-35 production. In addition, EBI3 promoter-driven luciferase activity was higher than that of the mock plasmid after IL-35stimulation. Thus, our study indicates that the high level of IL-35 in colorectal cancer promotes the production of IL-35 via STAT1 and STAT3, which suppresses T cell proliferation and may participate in tumor immunotolerance.

// Janani Panneerselvam 1, 4 , Akhil Srivastava 1, 4 , Ranganayaki Muralidharan 1, 4 , Qi Wang 1, 4 , Wei Zheng 1 , Lichao Zhao 1, 4 , Alshine Chen 3, 4 , Yan D. Zhao 3, 4 , Anupama Munshi 2, 4 , Rajagopal Ramesh 1, 4, 5 1 Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 2 Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 3 Department of Biostatistics and Epidemiology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 4 Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 5 Graduate Program in Biomedical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA Correspondence to: Rajagopal Ramesh, email: rajagopal-ramesh@ouhsc.edu Keywords: IL-24, lung cancer, HMGA1, miR222-3p, metastasis Received: April 21, 2016 Accepted: August 24, 2016 Published: September 02, 2016 ABSTRACT Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive. The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis. To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKT T308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression. Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.