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The coronavirus disease 2019 (COVID-19) is presently the most pressing public health concern worldwide. Cytokine storm is an important factor leading to death of patients with COVID-19. This study aims to characterize serum cytokines of patients with severe or critical COVID-19. Clinical records were obtained from 149 patients who were tested at the Sino-French New City Branch of Tongji Hospital from 30 January to 30 March 2020. Data regarding the clinical features of the patients was collected and analyzed. Among the 149, 45 (30.2%) of them had severe conditions and 104 (69.8%) of that presented critical symptoms. In the meantime, 80 (53.7%) of that 149 died during hospitalization. Of all, male patients accounted for 94 (69.1%). Compared with patients in severe COVID-19, those who in critical COVID-19 had significantly higher levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, and IL-10. Moreover, the passed-away patients had considerably higher levels of TNF-α, IL-6, IL-8, and IL-10 than those survived from it. Regression analysis revealed that serum TNF-α level was an independent risk factor for the death of patient with severe conditions. Among the proinflammatory cytokines (IL-1β, TNF-α, IL-8, and IL-6) analyzed herein, TNF-α was seen as a risk factor for the death of patients with severe or critical COVID-19. This study suggests that anti-TNF-α treatment allows patients with severe or critical COVID-19 pneumonia to recover.

Purpose: The purpose of this study is to identify novel urine protein biomarkers of bladder cancer using a Luminex based screening platform. Materials and Methods: The current study examines urine samples from 66 subjects, comprised of 31 Urology clinic controls and 35 bladder cancer patients, using a Luminex based screening platform. ELISA validation was carried out for the top 4 prospective urine biomarkers using an independent cohort of 20 Urology clinic controls and 60 bladder cancer (BC) subjects. Results: Of the 16 proteins screened by Luminex, 10 showed significant elevation in BC compared to the controls. Eight of these urine proteins were able to differentiate BC from control urine with ROC AUC values exceeding 0.70 at p < 0.0001, with specificity values exceeding 0.9. Upon ELISA validation, urine IL-1α, IL-1ra, and IL-8 were able to distinguish control urine from urine drawn from various bladder cancer stages, with IL-8 being the best discriminator. Compared to members of the IL-1 cytokine family, urine IL-8 was also best at discriminating T1 and/or T2–T4 from Ta BC (ROC AUC ≥ 0.83), as well as high grade from low grade BC (ROC AUC ≥ 0.82). Conclusions: These findings suggest that urine IL-1α, IL-1ra and IL-8 are useful indicators of bladder cancer. Urine IL-8 not only distinguishes bladder cancer from controls, it also discriminates high grade from low grade disease, and the successive clinical stages of bladder cancer. While supportive of previous reports, these findings warrant further analysis in prospective cohorts.

ObjectivesThis study aimed to determine whether plasma and synovial fluid interleukin-34 (IL-34), an inflammatory cytokine reportedly implicated in synovial inflammation-induced joint degeneration, were associated with radiographic severity of knee osteoarthritis (OA) patients and could emerge as knee OA biomarkers.DesignNinety-six knee OA patients and 72 healthy controls were recruited. Plasma and synovial fluid IL-34 levels were quantified using ELISA. IL-34 mRNA and protein expressions in inflamed ( n = 15) and noninflamed synovial tissues ( n = 15) of knee OA patients were determined using real-time polymerase chain reaction and immunohistochemistry, respectively.ResultsSignificant increases in plasma and synovial fluid IL-34 levels were found in knee OA patients—especially those with advanced stage ( P < 0.001, P < 0.001, respectively). Both plasma and synovial fluid IL-34 levels were positively associated with radiographic severity ( r = 0.64, P < 0.001; r = 0.50, P < 0.001, respectively). There was a direct link between plasma and synovial fluid IL-34 ( r = 0.64, P < 0.001). Receiver operating characteristic curve analysis uncovered that the optimal cutoff value of plasma IL-34 as a novel biomarker reflecting knee OA severity was defined at 3750.0 pg/mL (AUC = 0.85), with a sensitivity of 83.1% and a specificity of 74.2%. Further analysis revealed that IL-34 mRNA expression was significantly upregulated in inflamed synovium compared with noninflamed synovium obtained from knee OA patients ( P < 0.001), consistent with protein expression analysis demonstrating IL-34 overexpression localized in the lining and sublining layers of inflamed synovium.ConclusionsAll findings suggest that elevated plasma and synovial fluid IL-34 would reflect knee OA severity and might have potential utility as biomarkers for the disease progression.

Purpose: Exploring and studying the novel target of hepatocellular carcinoma (HCC) has been extremely important for its treatment. The principal objective of this project is to investigate whether myeloid derived growth factor (MYDGF) could accelerate the progression of HCC, and how it works. Methods: Cell proliferation, clonal formation, sphere formation and xenograft tumor experiments were used to prove the critical role of MYDGF in HCC progression. Tumor angiogenesis, immune cell infiltration, macrophage chemotaxis and inflammatory cytokines detection were utilized to clarify how MYDGF remodeled the tumor microenvironment (TME) to accelerate the progress of HCC. Results: Here, we reported a secretory protein MYDGF, which could be induced by hypoxia, was significantly upregulated in HCC and associated with poor clinical outcomes. Using bioinformatics and experimental approaches, we found that MYDGF promotes cell proliferation in vitro and in vivo through a mechanism that might involve enhanced self-renewal of liver CSCs. Furthermore, MYDGF can also promote tumor angiogenesis, induce macrophages to chemotaxis into tumor tissue, and then release various inflammatory cytokines, including IL-6 and TNF-α, which ultimately aggravate inflammation of tumor microenvironment and accelerate HCC progression. Conclusions: We provided evidence that MYDGF could directly affect the self-renewal of liver CSCs, and indirectly aggravate the inflammatory microenvironment to accelerate the progression of HCC.

Introduction Psoriasis is a chronic inflammatory disease affecting the skin with an unclear etiological significance. Aim In this study, we determined the mRNA expression and circulating levels of T helper (Th)/T regulatory (Treg) cytokines in psoriasis and analyzed their association with disease severity and treatment response. Material and methods 189 psoriasis patients and 189 controls were recruited. Circulating Th/Treg cytokines (IL-12, IFN-γ, IL-17, IL-23, TGF-β and IL-4) were measured at baseline and at follow-up after 12 weeks of methotrexate treatment by ELISA and their relative mRNA expression at baseline was estimated by quantitative PCR. Results We observed increased levels of Th1/Th17 cytokines (IFN-γ, IL-17, IL-12 and IL-23) and a decrease in levels of Th2/Treg cytokines (IL-4 and TGF-β) in psoriasis patients at baseline, as compared to controls. Further, we observed that there was a significant decrease in Th1/Th17 cytokines, whilst Th2/Treg cytokine levels were significantly increased on follow-up after treatment with systemic metho trexate, as compared to pre-treatment levels. Our results were further confirmed by the significantly higher mRNA expression of Th1/Th17 cytokine genes and significantly lower mRNA expression of Th2/Treg cytokine genes in patients with psoriasis, as compared to controls. A significant positive correlation of Th1/Th17 cytokines was observed with disease severity in cases, whilst Th2/Treg cytokines correlated negatively with disease severity. Conclusions Our results show that increased Th1/Th17 cytokines and decreased Th2/Treg cytokines, both at the circulatory and gene expression level, play an important role in the immunopathogenesis and severity of psoriasis.

Dendritic cells (DCs) can process the antigens of cancer vaccine and thus stimulate the CD8+ T cells to recognize and kill the tumor cells that express these antigens. However, lack of promising carriers for presenting the antigens to DCs is one of the main barriers to the development of clinically effective cancer vaccines. Another limitation is the risk of inflammatory side effects induced by the adjuvants. It is still unclear how we can develop ideal adjuvant-free DC vaccine carriers without adjuvants. Methods: A 12-mer peptide carrier (CBP-12) with high affinity for Clec9a expressed on DCs was developed using an in silico rational optimization method. The therapeutic effects of the adjuvant-free vaccine comprising CBP-12 and exogenous or endogenous antigenic peptides were investigated in terms of antigen cross-presentation efficacy, specific cytotoxic T lymphocyte response, and antitumor activity. We also explored the mechanism involved in the antitumor effects of the adjuvant-free CBP-12 vaccine. Finally, we assessed the effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Results: Here, we developed CBP-12 as a vaccine carrier that enhanced the uptake and cross-presentation of the antigens, thus inducing strong CD8+ T cell responses and antitumor effects in both anti-PD-1-responsive (B16-OVA) and -resistant (B16) models, even in adjuvant-free conditions. CBP-12 bound to and activated Clec9a, thereby stimulating Clec9a+ DC to product IL-21, but not IL-12 by activating of Syk. The antitumor effects of the CBP-12 conjugated peptide vaccines could be blocked by an IL-21 neutralizing antibody. We also observed the synergistic antitumor effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Conclusions: CBP-12 could serve as an adjuvant-free peptide vaccine carrier for cancer immunotherapy.

Rationale: Interleukin 22 (IL-22) is an epithelial survival cytokine that is at present being explored as therapeutic agents for acute and chronic liver injury. However, its molecular basis of protective activities remains poorly understood. Methods: Here we demonstrate that IL-22 inhibits the deteriorating metabolic states induced by stimuli in hepatocytes. Utilizing cell biological, molecular, and biochemical approaches, we provide evidence that IL-22 promotes oxidative phosphorylation (OXPHOS) and glycolysis and regulates the metabolic reprogramming related transcriptional responses. Results: IL-22 controls metabolic regulators and enzymes activity through the induction of AMP-activated protein kinase (AMPK), AKT and mammalian target of rapamycin (mTOR), thereby ameliorating mitochondrial dysfunction. The upstream effector lncRNA H19 also participates in the controlling of these metabolic processes in hepatocytes. Importantly, amelioration of liver injury by IL-22 through activation of metabolism relevant signaling and regulation of mitochondrial function are further demonstrated in cisplatin-induced liver injury and steatohepatitis. Conclusions: Collectively, our results reveal a novel mechanism underscoring the regulation of metabolic profiles of hepatocytes by IL-22 during liver injury, which might provide useful insights from the bench to the clinic in treating and preventing liver diseases.

Myeloid-derived growth factor (Mydgf), a paracrine protein secreted by bone marrow-derived monocytes and macrophages, was found to protect against cardiac injury following myocardial infarction (MI) in adult mice. We speculated that Mydgf might improve heart function via myocardial regeneration, which is essential for discovering the target to reverse heart failure. Methods: Two genetic mouse lines were used: global Mydgf knockout (Mydgf-KO) and Mydgf-EGFP mice. Two models of cardiac injury, apical resection was performed in neonatal and MI was performed in adult mice. Quantitative reverse transcription-polymerase chain reaction, western blot and flow cytometry were performed to study the protein expression. Immunofluorescence was performed to detect the proliferation of cardiomyocytes. Heart regeneration and cardiac function were evaluated by Masson's staining and echocardiography, respectively. RNA sequencing was employed to identify the key involved in Mydgf-induced cardiomyocyte proliferation. Mydgf recombinant protein injection was performed as a therapy for cardiac repair post MI in adult mice. Results: Mydgf expression could be significantly induced in neonatal mouse hearts after cardiac injury. Unexpectedly, we found that Mydgf was predominantly expressed by endothelial cells rather than macrophages in injured neonatal hearts. Mydgf deficiency impeded neonatal heart regeneration and injury-induced cardiomyocyte proliferation. Mydgf recombinant protein promoted primary mouse cardiomyocyte proliferation. Employing RNA sequencing and functional verification, we demonstrated that c-Myc/FoxM1 pathway mediated Mydgf-induced cardiomyocyte expansion. Mydgf recombinant protein improved cardiac function in adult mice after MI injury with inducing cardiomyocyte proliferation. Conclusion: Mydgf promotes cardiomyocyte proliferation by activating c-Myc/FoxM1 pathway and improves heart regeneration both in neonatal and adult mice after cardiac injury, providing a potential target to reverse cardiac remodeling and heart failure.

Interleukin (IL)-19 is a cytokine clustered in the IL-20 cytokine superfamily with both anti-inflammatory and pro-inflammatory aspects depending on the etiology of inflammatory disease. The function of IL-19 has been evaluated in cutaneous and inflammatory bowel diseases, but has not been studied in liver diseases. Here, we examined the effect of IL-19 on acute liver failure (ALF) using two mouse models of ALF: lipopolysaccharide and D-galactosamine (LPS/GalN)-induced model and concanavalin A (ConA)-induced model. In the LPS/GalN-induced ALF model, which is mainly caused by the innate immune response of liver macrophages, IL-19 knockout (KO) mice showed increased plasma level of liver deviation enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared with wild-type (WT) mice. In histopathology of liver sections, IL-19 KO mice exacerbated liver injury with marked hemorrhagic lesions and hepatocellular death in the liver compared with WT mice. In this model, mRNA expressions of pro-inflammatory chemokines, CCL2 and CCL5 were increased in liver tissue from IL-19 KO mice compared with WT mice. Moreover, the mRNA expressions of IL-19 and its receptor subunit were induced in liver tissue by LPS/GalN administration. However, there is no difference in liver injury between WT and IL-19KO in the ConA-induced ALF model induced by CD4+ T cell activation. These data suggest that IL-19 has a protective effect against inflammation-mediated liver injury, which is dependent on the etiology.

Diabetic retinopathy (DR) is a vision-threatening complication of diabetes mellitus characterized by chronic retinal microvascular inflammation. The involvement of CD4+ T cells in retinal vascular inflammation has been considered, but the specific subset and mechanism of T cell-mediated response during the process remains unclear. Here, we aim to investigate the potential role of follicular helper T (Tfh) cells, a newly identified subset of CD4+ T cells in retinal vascular inflammation in DR. Methods: Patients with DR were enrolled and the PD-1+CXCR5+CD4+ Tfh cells were detected in the peripheral blood by flow cytometry. The streptozotocin (STZ)-induced DR model and oxygen-induced retinopathy (OIR) model were established, and 79-6, an inhibitor of Bcl-6, was injected intraperitoneally to suppress Tfh cells. The Tfh cells-related genes were investigated in the spleen, lymph nodes, and retina of mice by flow cytometry, immunofluorescence, and qPCR. Results: The Tfh cells expanded in the circulation of patients with DR and also increased in circulation, lymph nodes and retinal tissues from the STZ-induced DR mice and OIR mice. Notably, inhibition of Bcl-6, a critical transcription factor for Tfh cells development, prevented upregulation of Tfh cells and its typical IL-21 cytokine, and ameliorated vascular leakage in DR mice or retinal angiogenesis in OIR mice, indicating that Bcl-6-directed Tfh cells could promote vascular inflammation and angiogenesis. Conclusions: Our results suggested that excessive Bcl-6-directed Tfh cells represent an unrecognized feature of DR and be responsible for the retinal vascular inflammation and angiogenesis, providing opportunities for new therapeutic approaches to DR.

MicroRNA-19 (miR-19) is identified as the key oncogenic component of the miR-17-92 cluster. When we explored the functions of the dysregulated miR-19 in lung cancer, microarray-based data unexpectedly demonstrated that some immune and inflammatory response genes (i.e., IL32, IFI6 and IFIT1) were generally down-regulated by miR-19 overexpression in A549 cells, which prompted us to fully investigate whether the miR-19 family (i.e., miR-19a and miR-19b-1) was implicated in regulating the expression of immune and inflammatory response genes in cancer cells. In the present study, we observed that miR-19a or miR-19b-1 overexpression by miRNA mimics in the A549, HCC827 and CNE2 cells significantly downregulated the expression of interferon (IFN)-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L). Furthermore, the ectopic miR-19a or miR-19b-1 expression in the A549, HCC827, CNE2 and HONE1 cells led to a general downward trend in the expression profile of major histocompatibility complex (MHC) class I genes (such as HLA-B, HLA-E, HLA-F or HLA-G); conversely, miR-19a or miR-19b-1 inhibition by the miRNA inhibitor upregulated the aforementioned MHC Class I gene expression, suggesting that miR-19a or miR-19b-1 negatively modulates MHC Class I gene expression. The miR-19a or miR-19b-1 mimics reduced the expression of interleukin (IL)-related genes (i.e., IL1B, IL11RA and IL6) in the A549, HCC827, CNE2 or HONE1 cells. The ectopic expression of miR-19a or miR-19b-1 downregulated IL32 expression in the A549 and HCC827 cells and upregulated IL32 expression in CNE2 and HONE1 cells. In addition, enforced miR-19a or miR-19b-1 expression suppressed IL-6 production by lung cancer and nasopharyngeal carcinoma (NPC) cells. Taken together, these findings demonstrate, for the first time, that miR-19 can modulate the expression of IFN-induced genes and MHC class I genes in human cancer cells, suggesting a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.

CD8+ T cells are thought to be the primary cytotoxic lymphocytes exerting antitumor effects. However, few studies have focused on the antitumor effects of CD8+ T cell-mediated humoral immunity or on interactions between CD8+ T cells and B cells in hepatocellular carcinoma (HCC). We found that the frequency of IL-21-producing CD8+CXCR5+ T cells was higher in HCC tumor tissue than in peritumoral tissue or peripheral blood from the same patients or in blood from healthy donors. Moreover, CD8+CXCR5+ T cells migrated in response to supernatants from primary HCC (HCC-SN) cells, and HCC-SN cells also powerfully induced CXCR5 expression in CD8+ T cells and IL-21 expression in CD8+CXCR5+ T cells. CD8+CXCR5+ T cells from HCC patients, but not those from healthy individuals, stimulated CD19+ B cells to differentiate into IgG-producing plasmablasts. These findings reveal that CD8+CXCR5+ T cells strongly infiltrate HCC tumors, and their infiltration is predictive of a better prognosis. Surprisingly, moreover, CD8+CXCR5+ T cells produced IL-21, which induced B cells to differentiate into IgG-producing plasmablasts and to play a key role in humoral immunity in HCC.

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.

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

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).

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio‐orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence‐based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse‐chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis., ChemBioChem, 21 (13), ISSN:1439-4227, ISSN:1439-7633

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.

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.

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.

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.

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.

// 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.

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.

Atherosclerosis is a chronic inflammatory disease of the arteries associated with various risk factors that promote lipid abnormalities, development and progression of atherosclerotic lesions, plaque rupture, and vascular thrombosis. Atherosclerosis is accelerated in autoimmune diseases. Non-invasive investigations showed increased intima-media thickness (IMT), carotid plaque, and coronary artery calcifications in patients with antiphospholipid syndrome, systemic lupus erythematosus and mixed connective tissue disease compared to controls. The balance between the proinflammatory and anti-inflammatory cytokines allows the immune equilibrium to be maintained. In autoimmune diseases the prevalence of proinflammatory factors leads to premature atherosclerosis. This review presents complementary knowledge on innate and adaptive immunity, cytokines and the role of inflammasomes in progression of early atherosclerosis.

// Ting Lu 1, * , Yan Liu 1, * , Shuang Yu 1 , Congcong Yin 1 , Peng Li 1 , Jingjing Ye 1 , Daoxin Ma 1 , Chunyan Ji 1 1 Department of Hematology, Qilu Hospital, Shandong University, Jinan, PR China * These authors contributed equally to this work Correspondence to: Chunyan Ji, email: jichunyan@sdu.edu.cn Keywords: B-cell non-Hodgkin’s lymphoma, Th22, IL-22, prognosis Received: November 23, 2015 Accepted: July 18, 2016 Published: July 30, 2016 ABSTRACT T helper (Th) 22 cells play important roles in the pathogenesis of autoimmune and inflammatory diseases, and their function in tumors remains uncertain. In the current study, we investigated the alternations and clinical significance of circulating Th22 cells in patients with B-cell non-Hodgkin’s lymphoma (B-NHL). We found that the frequency of Th22 cells was significantly elevated in peripheral blood of newly-diagnosed B-NHL patients, and returned to normal level after chemotherapy. In consistent with increased Th22 frequency, plasma IL-22 and IL-6 levels in B-NHL patients were remarkably increased. Moreover, the increased Th22 frequency was associated with the older age (> 60 yr) and a poorer response to therapy in B-NHL patients. In addition, there existed a statistically positive correlation between circulating Th22 and Th17 frequencies in B-NHL patients. Our data demonstrated that circulating Th22 frequency was associated with the clinical outcome and prognosis of B-NHL patients, indicating that Th22 immune response might play an important role in the development and progression of B-NHL.

Oxidative stress has a critical role in the pathogenesis of Age-related Macular Degeneration (AMD), a multifactorial disease that includes age, gene variants of complement regulatory proteins and smoking as the main risk factors. Stress-induced premature cellular senescence (SIPS) is postulated to contribute to this condition. In this study, we hypothesized that oxidative damage, promoted by endogenous or exogenous sources, could elicit a senescence response in RPE cells, which would in turn dysregulate the expression of major players in AMD pathogenic mechanisms. We showed that exposure of a human RPE cell line (ARPE-19) to a cigarette smoke concentrate (CSC), not only enhanced Reactive Oxygen Species (ROS) levels, but also induced 8-Hydroxydeoxyguanosine-immunoreactive (8-OHdG) DNA lesions and phosphorylated-Histone 2AX-immunoreactive (p-H2AX) nuclear foci. CSC-nuclear damage was followed by premature senescence as shown by positive senescence associated-β-galactosidase (SA-β-Gal) staining, and p16INK4a and p21Waf-Cip1 protein upregulation. N-acetylcysteine (NAC) treatment, a ROS scavenger, decreased senescence markers, thus supporting the role of oxidative damage in CSC-induced senescence activation. ARPE-19 senescent cultures were also established by exposure to hydrogen peroxide (H2O2), which is an endogenous stress source produced in the retina under photo-oxidation conditions. Senescent cells upregulated the proinflammatory cytokines IL-6 and IL-8, the main markers of the senescence-associated secretory phenotype (SASP). Most important, we show for the first time that senescent ARPE-19 cells upregulated vascular endothelial growth factor (VEGF) and simultaneously downregulated complement factor H (CFH) expression. Since both phenomena are involved in AMD pathogenesis, our results support the hypothesis that SIPS could be a principal player in the induction and progression of AMD. Moreover, they would also explain the striking association of this disease with cigarette smoking., Graphical abstract Experiments using a cell line derived from retinal pigment epithelial (RPE) cells support two related hypotheses. (a) An oxidant environment can induce premature senescence in RPE cells. (b) Phenotypic changes of senescent RPE cells could induce and maintain the characteristic lesions of AMD. Notice that, in this model, initial oxidant damage does not induce significant morphological changes. However, structural changes appearing in senescent cells may explain changes in the shape and size of RPE cells overlying AMD lesions. fx1, Highlights • Cigarette smoke, a main risk factor for AMD, induces senescence in RPE cells. • Cigarette smoke-induced senescence is mediated by ROS. • Senescent RPE cells upregulate the inflammatory cytokines IL-6 and IL-8. • Senescent RPE cells increase VEGF expression, but decrease CFH expression. • Senescence of RPE cells could explain AMD development and progression.

Interleukin (IL)-32 is a well-known cytokine associated with inflammation, virus infections and cancer. IL-32θ is a newly identified isoform of IL-32, whose function has yet to be elucidated. In this study, we investigated IL-32θ function in colon cancer stem cells. Using samples from colon cancer patients, we found that the expression of IL-32θ mRNAs was significantly suppressed in tumor regions. We investigated the effects of IL-32θ on colon cancer. Ectopic expression of IL-32θ attenuated invasion, migration in vitro and in vivo tumorigenicity of colon cancer cells. IL-32θ inhibited epithelial-mesenchymal transition (EMT), resulting in the suppression of their migratory and invasive capabilities of HT29 colon cancer cells. In addition, IL-32θ altered various properties of CSCs, including sphere formation and expression of stemness related genes. IL-32θ directly bound to STAT3 and inhibited its nuclear translocation, leading to inhibited transcription of downstream factors, including Bmi1 and ZEB1. We showed that IL-32θ inhibited the STAT3-ZEB1 pathway and consequently inhibited key factors of stemness and EMT. Taken together, our findings reveal that IL-32θ can be a tumor suppressor, indicating that IL-32θ could possibly be used in therapies for colon cancer.

The influence of transfusion of lymphokine-activated T killer cells (T-LAK) on inflammatory responses was examined in dogs after laparotomy. Plasma C-reactive protein (CRP) level, cell numbers of peripheral blood lymphocytes (PBLs) and T lymphocyte subsets (CD3(+), CD4(+) and CD8(+)) and mRNA expression levels of cytokines including interleukin (IL)-2, IL-12, IL-4, IL-10 and transforming growth factor (TGF)-β in peripheral blood mononuclear cells (PBMCs) were measured in dogs with (T-LAK group) or without (control group) a single T-LAK administration immediately after laparotomy. The plasma CRP level initially increased and then decreased to the normal range at 7 days after laparotomy in the T-LAK group, which was earlier than in the control group. The expression level of IL-10 mRNA showed a marked postoperative increase and was significantly higher than the preoperative level on day 7 (P

Multiple sclerosis (MS), also known as disseminated sclerosis or encephalomyelitis disseminate, is an inflammatory disease in which myelin in the spinal cord and brain are damaged. IL-32α is known as a critical molecule in the pathophysiology of immune-mediated chronic inflammatory disease such as rheumatoid arthritis, chronic pulmonary disease, and cancers. However, the role of IL-32α on spinal cord injuries and demyelination is poorly understood. Recently, we reported that the release of proinflammatory cytokines were reduced in IL-32α-overexpressing transgenic mice. In this study, we investigated whether IL-32α plays a role on MS using experimental autoimmune encephalomyelitis (EAE), an experimental mouse model of MS, in human IL-32α Tg mice. The Tg mice were immunized with MOG35-55 suspended in CFA emulsion followed by pertussis toxin, and then EAE paralysis of mice was scored. We observed that the paralytic severity and neuropathology of EAE in IL-32α Tg mice were significantly decreased compared with that of non-Tg mice. The immune cells infiltration, astrocytes/microglials activation, and pro-inflammatory cytokines (IL-1β and IL-6) levels in spinal cord were suppressed in IL-32α Tg mice. Furthermore, NG2 and O4 were decreased in IL-32α Tg mice, indicating that spinal cord damaging was suppressed. In addition, in vitro assay also revealed that IL-32α has a preventive role against Con A stimulation which is evidenced by decrease in T cell proliferation and inflammatory cytokine levels in IL-32α overexpressed Jurkat cell. Taken together, our findings suggested that IL-32α may play a protective role in EAE by suppressing neuroinflammation in spinal cord.

Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ leukemia cells though carrying a sCAR-4N1 expression cassette in the viral genome, forming Ad.4N1, in which 4N1 represents the C-terminal CD47-binding domain of thrombospondin-1. The infection and cytotoxicity of Ad.4N1 in leukemia cells were determined to be mediated by the 4N1-CD47 interaction. Ad.4N1 was further engineered to harbor a gene encoding melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), forming Ad.4N1-IL24, which replicated dramatically faster than Ad.4N1, and elicited significantly enhanced antileukemia effect in vitro and in a HL60/Luc xenograft mouse model. Our data suggest that Ad.4N1 could act as a novel oncolytic adenovirus vector for CD47+ leukemia targeting gene transfer, and Ad.4N1 harboring anticancer genes may provide novel antileukemia agents.

Background: Human immunology research is often limited to peripheral blood. However, there are important differences between blood immune cells and their counterparts residing in secondary lymphoid organs, such as in the case of germinal center (GC) T follicular helper (Tfh) cells and GC B cells. Methods: We developed a versatile ex vivo lymphoid organ culture platform that is based on human pharyngeal tonsils (adenoids) and allows for drug testing. We systematically phenotyped Tfh and GC B cell subsets in explant- and suspension cultures using multicolor flow cytometry and cytokine multiplex analysis. Findings: Phenotypic changes of certain ex vivo cultured immune cell subsets could be modulated by cytokine addition. Furthermore, we optimized an activation-induced marker assay to evaluate the response to T cell stimulation. We provide proof-of-concept that Tfh and GC B cells could be modulated in these cultures by different anti-inflammatory drugs in unstimulated states and upon activation with vaccine-derived antigens. For example, GC B cells were lost upon CD40L blockade, and clinically approved JAK inhibitors impacted Tfh and GC B cells, including down-regulation of their key transcription factor BCL6. BCL6 regulation was affected by IL-6 signaling in T cells and IL-4 in B cells, respectively. Furthermore, we demonstrated that JAK signaling and TNF signaling contributed to the stimulation-induced activation of tonsil-derived T cells. Interpretation: Our optimized methods, assays, and mechanistic findings can contribute to a better understanding of human GC responses. These insights may be relevant for improving autoimmune disease therapy and vaccination efficacy. Funding: This work was supported by a project grant under the joint research cooperation agreement of LMU Munich, LMU University Hospital, and Sanofi-Aventis Deutschland GmbH, as well as by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Emmy Noether Programme BA 5132/1-1 and BA 5132/1-2 (252623821), SFB 1054 Project B12 (210592381), and SFB 914 Project B03 (165054336). Keywords: T follicular helper cells (Tfh), Human ex vivo lymphoid tissue culture, Immunotherapy, CXCR5, BCL6, Germinal center (GC), Activation-induced marker assay (AIM), JAK inhibitor

It has been shown that anti-inflammatory cytokines interleukin-35 (IL-35) and IL-10 could inhibit acute endothelial cell (EC) activation, however, it remains unknown if and by what pathways IL-35 and IL-10 could block atherogenic lipid lysophosphatidylcholine (LPC)-induced sustained EC activation; and if mitochondrial reactive oxygen species (mtROS) can differentiate mediation of EC activation from trained immunity (innate immune memory). Using RNA sequencing analyses, biochemical assays, as well as database mining approaches, we compared the effects of IL-35 and IL-10 in LPC-treated human aortic ECs (HAECs). Principal component analysis revealed that both IL-35 and IL-10 could similarly and partially reverse global transcriptome changes induced by LPC. Gene set enrichment analyses showed that while IL-35 and IL-10 could both block acute EC activation, characterized by upregulation of cytokines/chemokines and adhesion molecules, IL-35 is more potent than IL-10 in suppressing innate immune signatures upregulated by LPC. Surprisingly, LPC did not induce the expression of trained tolerance itaconate pathway enzymes but induced trained immunity enzyme expressions; and neither IL-35 nor IL-10 was found to affect LPC-induced trained immunity gene signatures. Mechanistically, IL-35 and IL-10 could suppress mtROS, which partially mediate LPC-induced EC activation and innate immune response. Therefore, anti-inflammatory cytokines could reverse mtROS-mediated acute and innate immune trans-differentiation responses in HAECs, but it could spare metabolic reprogramming and trained immunity signatures, which may not fully depend on mtROS. Our characterizations of anti-inflammatory cytokines in blocking mtROS-mediated acute and prolonged EC activation, and sparing trained immunity are significant for designing novel strategies for treating cardiovascular diseases, other inflammatory diseases, and cancers. Keywords: Interleukin-35, Interleukin-10, Endothelial cell activation, Mitochondrial ROS, Trained immunity, Atherosclerosis

Background Bariatric surgery leads to early and long-lasting remission of type 2 diabetes (T2D). However, the mechanisms behind this phenomenon remain unclear. Among several factors, gut hormones are thought to be crucial mediators of this effect. Unlike GLP-1, the role of the hormone peptide tyrosine tyrosine (PYY) in bariatric surgery in humans has been limited to appetite regulation and its impact on pancreatic islet secretory function and glucose metabolism remains under-studied. Methods Changes in PYY concentrations were examined in obese patients after bariatric surgery and compared to healthy controls. Human pancreatic islet function was tested upon treatment with sera from patients before and after the surgery, in presence or absence of PYY. Alterations in intra-islet PYY release and insulin secretion were analysed after stimulation with short chain fatty acids (SCFAs), bile acids and the cytokine IL-22. Findings We demonstrate that PYY is a key effector of the early recovery of impaired glucose-mediated insulin and glucagon secretion in bariatric surgery. We establish that the short chain fatty acid propionate and bile acids, which are elevated after surgery, can trigger PYY release not only from enteroendocrine cells but also from human pancreatic islets. In addition, we identify IL-22 as a new factor which is modulated by bariatric surgery in humans and which directly regulates PYY expression and release. Interpretation This study shows that some major metabolic benefits of bariatric surgery can be emulated ex vivo. Our findings are expected to have a direct impact on the development of new non-surgical therapy for T2D correction.

Tuberculosis is becoming a global issue with raising occurrences; particularly in developing countries, the situation is alarming. Besides environmental factors, host genetic factors are vital in disease development. A demographical and genotypic analysis in relation to tuberculosis commencement is conducted in a Pakistani population, and genotypic frequency of EBI3 (rs4740) was analyzed. Allelic frequencies of EBI3 (rs4740) were significantly associated with disease susceptibility in the reviewed population. Analysis for EBI3 (rs4740) genotyping showed a significant association of “GG” with reduced risk for disease. Moreover, females and older age found to be more perilous to develop TB while smoking and a family history of TB are additional risk factors for disease development. Further work with a larger population is necessary to identify the true causative variants of tuberculosis.

Interleukin (IL)-22 regulates tissue inflammation and repair. Here we report participation of the liver in IL-22-mediated cardiac repair after acute myocardial infarction (MI). Methods: We induced experimental MI in mice by ligation of the left ascending artery and evaluated the effect of IL-22 on post-MI cardiac function and ventricular remodeling. Results: Daily subcutaneous injection of 100 µg/kg mouse recombinant IL-22 for seven days attenuated adverse ventricular remodeling and improved cardiac function in mice at 28 days after left anterior descending coronary artery ligation-induced MI. Pharmacological inhibition of signal transducer and activator of transcription (STAT3) muted these IL-22 activities. While cardiomyocyte-selective depletion of STAT3 did not affect IL-22 activities in protecting post-MI cardiac injury, hepatocyte-specific depletion of STAT3 fully muted these IL-22 cardioprotective activities. Hepatocyte-derived fibroblast growth factor (FGF21) was markedly increased in a STAT3-dependent manner following IL-22 administration and accounted for the cardioprotective benefit of IL-22. Microarray analyses revealed that FGF21 controlled the expression of cardiomyocyte genes that are involved in cholesterol homeostasis, DNA repair, peroxisome, oxidative phosphorylation, glycolysis, apoptosis, and steroid responses, all of which are responsible for cardiomyocyte survival. Conclusions: Supplementation of IL-22 in the first week after acute MI effectively prevented left ventricular dysfunction and heart failure. This activity of IL-22 involved crosstalk between the liver and heart after demonstrating a role of the hepatic STAT3-FGF21 axis in IL-22-induced post-MI cardiac protection.