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External beam radiotherapy (EBRT) with carbon ions and endoradiotherapy using radiolabeled tumor targeting agents are emerging concepts in precision cancer therapy. We report on combination effects of these two promising strategies. Tumor targeting 131I-labelled anti-EGFR-antibody (Cetuximab) was used in the prototypic EGFR-expressing A431 human squamous cell carcinoma xenograft model. A 131I-labelled melanin-binding benzamide derivative was utilized targeting B16F10 melanoma in an orthotopic syngeneic C57bl6 model. Fractionated EBRT was performed using carbon ions in direct comparison with conventional photon irradiation. Tumor uptake of 131I-Cetuximab and 131I-Benzamide was enhanced by fractionated EBRT as determined by biodistribution studies. This effect was independent of radiation quality and significant for the small molecule 131I-Benzamide, i.e., >30% more uptake in irradiated vs. non-irradiated melanoma was found (p

Anti-microbial peptides are produced at outer and inner surfaces by epithelia and innate immune cells in response to bacterial infection. Staphylococcus aureus is an enterotoxin producing, Gram-positive pathogen, which is a major cause of soft tissue infections and life-threatening bacteremia and sepsis. Here we show that (i) skin T cells in chronic wounds infected with S. aureus express interleukin-26 (IL-26) in situ, (ii) staphylococcal enterotoxins (SE) trigger IL-26 expression in T cell lines and primary skin T cells, and (iii) IL-26 triggers death and inhibits biofilm formation and growth of S. aureus. Thus, we provide novel evidence that IL-26 is an anti-microbial peptide produced by T cells in response to SE. Accordingly, we propose that IL-26 producing T cells take part in the innate immune response to SE producing S. aureus and thus play a novel role in the primary innate immune defense in addition to their classical role in adaptive immunity. Published version

// Consuelo Amantini 1,* , Valerio Farfariello 2,* , Claudio Cardinali 3,4 , Maria Beatrice Morelli 3,4 , Oliviero Marinelli 1 , Massimo Nabissi 3 , Matteo Santoni 3 , Laura Bonfili 1 , Valentina Cecarini 1 , Anna Maria Eleuteri 1 and Giorgio Santoni 3 1 School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy 2 University of Lille, INSERM U1003 - PHYCEL - Physiologie Cellulaire, Lille, France 3 School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy 4 Department of Molecular Medicine, Sapienza University, Rome, Italy * These authors have contributed equally to this work Correspondence to: Consuelo Amantini, email: // Keywords : ER stress, capsaicin, TRPV1, TRPV1 KO mice, autophagy, Immunology and Microbiology Section, Immune response, Immunity Received : July 28, 2017 Accepted : September 20, 2017 Published : October 11, 2017 Abstract Autophagy and the ubiquitin-proteasome system (UPS) control thymus cell homeostasis under resting and endoplasmic reticulum (ER) stress conditions. Several evidence support a cross-talk between UPS and autophagy; abrogation of UPS responses stimulates autophagy, and vice versa the inhibition of autophagy alters the UPS functions. Herein, we found that TRPV1 activation induces ER stress, proteasome dysfunction and autophagy in thymocytes by modulating the expression of UPR-related genes. The TRPV1-mediated autophagy prevents the UPR activation by inhibiting BiP, Grp94 and ERp57 chaperone protein expression. Thymocytes from TRPV1 KO mice display both autophagy and proteasome dysfunctions, resulting in increased apoptotic cells and reduced total DP thymocyte number. In addition, positive selection of thymocytes triggered by anti-TCRβ/CD2 Ab-mediated costimulation induces apoptosis in thymocytes from TRPV1 KO as compared with WT mice. Stimulation of TRPV1 KO thymocytes with anti-TCRβ/CD2 mAbs modulates the expression of CD4 antigen on purified DP thymocytes, with reduced number of mature, single positive (SP) CD4 and increased number of immature SP CD4 low and DP CD4 low CD8 + thymocytes, further supporting the intrinsic role of TRPV1 in T cell maturation. Finally, a reduction in CD8 + and CD4 + T cells is evidenced in the peripheral blood and spleen of TRPV1 KO, as compared with WT mice. Therapeutic strategy by restraining or stimulating the TRPV1 expression and functions in thymocytes might represent a new pharmacological tool in the regulation of different inflammatory T cell responses.

Anti-tumor necrosis factor (TNF) therapies are successful in the treatment of inflammatory disorders. However, some patients with rheumatoid arthritis (RA) fail to response anti-TNF drugs due to the compensation of other inflammatory signals. In this study, to reduce compensatory responses of interleukin-17A (IL-17A) during TNF-α inhibition, we generated an IgG-like bispecific antibodiy (bsAb) against TNF-α and IL-17A through a combination method of electrostatic Fc pairing and light chain crossover. This bsAb exhibited relatively high stability comparable to natural IgG antibodies, and retained the unaltered affinities to both of two targets. BsAb significantly decreased not only the expression level of neutrophil or Th17 chemokines, but also the secretion of IL-6/IL-8 on fibroblast-like synoviocytes (FLS) from a patient with RA. Meanwhile, TNF-α-mediated cellular cytotoxicity of fibroblasts was neutralized by bsAb. Importantly, we demonstrate that the combined blockade of TNF-α and IL-17A is more efficient than inhibition of either factor alone. Our results suggest the IgG-like anti-TNF-α/IL-17A bispecific molecule overcome the limited therapeutic responses using anti-TNF drugs. It may be a promising therapeutic agent for the treatment of autoimmune diseases.

// Masahiko Ito 1 , Suofeng Sun 1 , Takasuke Fukuhara 2 , Ryosuke Suzuki 3 , Miho Tamai 4 , Toyohiko Yamauchi 5 , Kenji Nakashima 1 , Yoh-ichi Tagawa 4 , Shigetoshi Okazaki 6 , Yoshiharu Matsuura 2 , Takaji Wakita 3 and Tetsuro Suzuki 1 1 Department of Virology and Parasitology, Hamamatsu University School of Medicine, Shizuoka, Japan 2 Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 3 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan 4 School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan 5 Central Research Laboratory, Hamamatsu Photonics K.K., Shizuoka, Japan 6 Department of Medical Spectroscopy, Hamamatsu University School of Medicine, Shizuoka, Japan Correspondence to: Tetsuro Suzuki, email: // Keywords : hepatitis C virus, epigenetic reprogramming, hepatoma cell, oval cell, miR200a, Immunology and Microbiology Section, Immune response, Immunity Received : May 16, 2017 Accepted : June 28, 2017 Published : July 08, 2017 Abstract Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

Excretory/secretory antigens (ESAs) produced by Toxoplasma gondii enable this parasite to invade and survive within the host cells through immunomodulation. In this study, the modulating effects of T. gondii excretory/secretory antigens (TgESAs) on the Ana-1 murine macrophage cell line were evaluated. Ana-1 cells were incubated with several concentrations of TgESAs, and the resulting effects on cellular viability, phagocytotic ability, and apoptosis induction were determined. Pro-inflammatory and anti-inflammatory cytokine secretion, nitric oxide production, toll-like receptor expression, and nuclear translocation of NF-κB were all measured after incubation with TgESAs. After TgESAs treatment, the proliferation and phagocytosis ability of Ana-1 cells decreased, and apoptosis was induced in a dose dependent manner. Analysis of Ana-1 cell culture supernatants showed that TgESAs not only upregulated secretion of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-β1), they also inhibited secretion of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β). Additionally, TgESAs inhibited nitric oxide production, toll-like receptor (TLR) 2 and 4 activation, and the nuclear translocation of NF-κB in lipopolysaccharide-stimulated Ana-1 macrophages. These results suggest TgESAs inhibit the functional activity of Ana-1 murine macrophages by inhibiting TLR-induced NF-κB activation, which suppresses pro-inflammatory cytokine secretion.

// Ying Lu 1 , Xianyu Zhang 1 , Nicolas Bouladoux 2 , Saransh Neel Kaul 3 , Kangxin Jin 4 , Derek Sant’Angelo 5 , Yasmine Belkaid 2 and Damian Kovalovsky 1,6 1 Experimental Immunology Branch, NCI, NIH, Bethesda, MD, USA 2 Mucosal Immunology Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD, USA 3 University of Maryland, College Park, MD, USA 4 Zhongshan Ophthalmic Center, State Key Laboratory for Ophthalmic Researches, Sun Yat-Sen University, Guangzhou, Guangdong, China 5 Cancer Metabolism and Growth Program, Rutgers, Child Health Institute of New Jersey, New Brunswick, NJ, USA 6 Experimental Transplantation and Immunology Branch, NCI, NIH, Bethesda, MD, USA Correspondence to: Damian Kovalovsky, email: // Keywords : innate lymphoid cells, ILC, ILC3, Zbtb1, IFN-γ, Immunology and Microbiology Section, Immune response, Immunity Received : May 16, 2017 Accepted : July 14, 2017 Published : July 27, 2017 Abstract Innate lymphoid cells (ILCs) play a central role conferring protection at the mucosal frontier. In this study, we have identified a requirement of the transcription factor Zbtb1 for the development of RORγt + ILCs (ILC3s). Zbtb1-deficient mice lacked NKp46 + ILC3 cells in the lamina propria of the small and large intestine. This requirement of Zbtb1 was cell intrinsic, as NKp46 + ILC3s were not generated from Zbtb1-deficient progenitors in bone marrow chimeras and Zbtb1-deficient RORγt + CCR6 - NKp46 - ILC3s didn’t generate NKp46 + ILC3s in co-cultures with OP9-DL1 stroma. In correlation with this impairment, Zbtb1-deficient ILC3 cells failed to upregulate T-bet expression, and to acquire IFN-γ production characteristic of NKp46 + cells. Finally, absence of NKp46 + ILC3 cells combined with the absence of T-cells in Zbtb1-deficient mice, led to a transient susceptibility to C. rodentium infections. Altogether, these results establish that Zbtb1 is essential for the development of NKp46 + ILC3 cells.

// Ho-Chang Kuo 1,2 , Sung-Chou Li 3 , Lien-Hung Huang 3 and Ying-Hsien Huang 1,2,4 1 Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan 2 Kawasaki Disease Center, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan 3 Department of Medical Research, Genomics and Proteomics Core Laboratory, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan 4 Department of Pediatrics, Chiayi Chang Gung Memorial Hospital, Kaohsiung, Taiwan Correspondence to: Ying-Hsien Huang, email: // Keywords : matrix metalloproteinase, genetic methylation, Kawasaki disease, Immunology and Microbiology Section, Immune response, Immunity Received : April 05, 2017 Accepted : June 28, 2017 Published : July 28, 2017 Abstract Background: Kawasaki disease (KD) is a type of febrile coronary vasculitis occurring in children. Some researchers have suggested that changes in genetic signatures, such as matrix metalloproteinases (MMPs), are critical markers for cardiovascular diseases. This study aims to provide a comprehensive survey of global DNA methylation levels and MMP transcripts of KD patients compared to control subjects. Materials and Methods: For chips studies, we recruited a total of 18 KD patients, prior to receiving intravenous immunoglobulin (IVIG) and at least 3 weeks after IVIG treatment, as well as 18 healthy and 18 febrile control subjects. We applied Illumina HumanMethylation450 BeadChip and Affymetrix GeneChip® Human Transcriptome Array 2.0 to evaluate their CpG markers and expression levels, respectively. Then we used a separate cohort to carry out real-time quantitative PCR validations of mRNA levels. Results: The expressions of mRNA levels of MMP-8, -9, and -25 were significantly upregulated in KD patients compared to the healthy and febrile controls. Once KD patients underwent IVIG treatment, these MMPs considerably decreased. In particular, the methylation status of CpG sites of MMP-9 indicated a significant opposite tendency between both stages of not only the KD samples but also the controls. We also observed the mRNA level of MMP-9 to be higher in KD patients with coronary arterial lesion formation. Conclusion: This study is the first to report epigenetic hypomethylation, an increased MMP-9 transcript, and the upregulation of MMP-9 in KD patients who had formed coronary arterial lesions.

// Jun Lou 1,2,* , Xiawei Li 1,2,* , Wei Huang 1,2 , Jingjing Liang 1,2 , Mingzhu Zheng 1,2 , Ting Xu 1,2 , Jun Lyu 1,2 , Dan Li 1,2 , Qin Xu 1,2 , Xuexiao Jin 1,2 , Guotong Fu 1,2 , Di Wang 1,2 and Linrong Lu 1,2 1 Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China 2 Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, Hangzhou, China * These authors have equally contributed to this work Correspondence to: Linrong Lu, email: // Keywords : SNX10, macrophage, Listeria monocytogenes, phagosome, innate immunity, Immunology and Microbiology Section, Immune response, Immunity Received : May 16, 2017 Accepted : July 13, 2017 Published : July 27, 2017 Abstract Listeria monocytogenes ( L. monocytogenes ),which is a facultative intracellular bacterial pathogen that causes listeriosis, is widely used to study the mammalian immune response to infection. After phagocytosis by professional phagocytes, L. monocytogenes is initially contained within phagosomes, which mature into phagolysosomes, where the bacteria are degraded. Although phagocytosis and subsequent phagosome maturation is essential for the clearance of infectious microbial pathogens, the underlying regulatory mechanisms are still unclear. SNX10 (Sorting nexin 10) has the simplest structure of the SNX family and has been reported to regulate endosomal morphology, which might be crucial for macrophage function, including phagocytosis and digestion of pathogens, inflammatory response, and antigen presentation. Our results showed that SNX10 expression was upregulated following L. monocytogenes infection in macrophages. It was also revealed that SNX10 promoted phagosome maturation by recruiting the Mon1-Ccz1 complex to endosomes and phagosomes. As a result, SNX10 deficiency decreased the bacterial killing ability of macrophages, and SNX10-deficient mice showed increased susceptibility to L. monocytogenes infection in vivo . Thus, this study revealed an essential role of SNX10 in controlling bacterial infection.

// Jia Yi Guo 1,* , Feng Li 1,* , Yong Bing Wen 1 , Hong Xun Cui 1 , Ma Long Guo 1 , Lin Zhang 2 , Yun Fei Zhang 1 , Yan Jin Guo 1 and Yan Xing Guo 1 1 Luoyang Orthopedic Hospital of Henan Province, Luoyang, Henan, China 2 Department of Surgery, Advanced Clinical Skills Centre, University of Auckland, Auckland, New Zealand * These authors have contributed equally to this work Correspondence to: Hong-Xun Cui, email: // Yan Xing Guo, email: // Keywords : melatonin, redox, Sirt1, NAMPT, NFAT5, Immunology and Microbiology Section, Immune response, Immunity Received : December 19, 2016 Accepted : May 12, 2017 Published : June 03, 2017 Abstract Osteoarthritis (OA) is a degenerative joint disease mainly characterized by cartilage degradation. Interleukin-1β (IL-1β) contributes to OA pathogenesis by enhancing oxidative stress and inflammation. Melatonin reportedly elicits potent protection against OA. However, the role of melatonin and underlying mechanism in IL-1β-stimulated chondrocytes remain largely unclear. In this study, we found that melatonin inhibited IL-1β-induced toxicity and sirtuin 1 (Sirt1) enhancement in human chondrocytes. Melatonin reduced the IL-1β-increased nicotinamide phosphoribosyltransferase (NAMPT) expression and the NAD + level in chondrocytes in a Sirt1-dependent manner. In turn, the inhibitory effect of melatonin on Sirt1 was mediated by NAMPT. Moreover, melatonin suppressed IL-1β-induced Sirt1-mediated matrix metalloproteinase (MMP)-3 and MMP-13 production. Melatonin also decreased the Sirt1-steered nuclear factor of activated T cells 5 (NFAT5) expression in IL-1β-challenged chondrocytes. NFAT5 depletion mimicked the suppressive effects of melatonin on IL-1β-elevated production of inflammatory mediators, including tumor necrosis factor-α (TNF-α), IL-1β, prostaglandin E2 (PGE 2 ), and nitric oxide (NO) in chondrocytes. TNF-α, IL-1β, PGE 2 , or NO decrease caused the similar reduction of MMP-3 and MMP-13 by melatonin in IL-1β-insulted chondrocytes. Highly consistent with in vitro findings, in vivo results demonstrated that melatonin repressed the expression of relevant genes in rat OA pathogenesis in anterior cruciate ligament transection model. Overall, these results indicate that melatonin effectively reduced IL-1β-induced MMP production by inhibiting Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes, suggesting melatonin as a potential therapeutic alternative for chondroprotection of OA patients.

// Arcangelo Liso 1 , Stefano Castellani 1 , Francesca Massenzio 1 , Rosa Trotta 1 , Alessandra Pucciarini 2 , Barbara Bigerna 2 , Pasquale De Luca 3 , Pietro Zoppoli 4 , Filippo Castiglione 5 , Maria Concetta Palumbo 5 , Fabrizio Stracci 6 , Matteo Landriscina 1,7 , Giorgina Specchia 8 , Leon A. Bach 9,10 , Massimo Conese 1 and Brunangelo Falini 2 1 Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy 2 Institute of Haematology, University of Perugia, Perugia, Italy 3 Stazione Zoologica A. Dohrn, Naples, Italy 4 Dipartimento di Medicina Sperimentale e Clinica, Universita degli Studi Magna Graecia, Catanzaro, Italy 5 Institute for Applied Computing, National Research Council of Italy, Rome, Italy 6 Department of Experimental Medicine, Section of Public Health, University of Perugia, Perugia, Italy 7 Laboratory of Preclinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 8 Institute of Hematology, University of Bari, Bari, Italy 9 Department of Medicine, Alfred Hospital, Monash University, Melbourne, Australia 10 Department of Endocrinology and Diabetes, Alfred Hospital, Melbourne, Australia Correspondence to: Arcangelo Liso, email: // Massimo Conese, email: // Keywords : apoptosis, B cells, chemotaxis, dendritic cells, hyperthermia, Immunology and Microbiology Section, Immune response, Immunity Received : April 16, 2017 Accepted : May 12, 2017 Published : June 01, 2017 Abstract Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.

// Marcell Costa de Medeiros 1 , Rajat Banerjee 2 , Min Liu 2 , Giovana Anovazzi 1 , Nisha J. D’Silva 2,3 and Carlos Rossa Junior 1 1 Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Sao Paulo State University, Araraquara, SP, Brazil 2 Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA 3 Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA Correspondence to: Carlos Rossa Junior, email: // Nisha J. D’Silva, email: // Keywords : head and neck squamous cell carcinoma, immune response, T cell, immunosuppression and tumor scape, Immunology and Microbiology Section, Immune response, Immunity Received : January 12, 2017 Accepted : June 01, 2017 Published : June 15, 2017 Abstract The immune system detects shifts from homeostasis and eliminates altered cells. However, neoplastic cells can modulate the host response to escape immunosurveillance thereby allowing tumor progression. Head and neck squamous cell carcinoma (HNSCC) is one of the most immunosuppressive cancers but its role in co-opting the immune system to actively promote tumor growth has not been investigated. In this study, we investigated the influence of soluble factors secreted by HNSCC and non-neoplastic epithelial cells on proliferation, apoptosis, activation, cytokine gene expression and phenotypic polarization of immune cells of healthy donors. Then, we determined if the immunomodulation caused by HNSCC-derived soluble products leads to immunosubversion by assessing proliferation, migration and survival of tumor cells exposed to soluble products secreted by modulated immune cells or co-cultured with immune cells. Soluble products from HNSCC inhibited proliferation and cytokine expression in PBMCs, activation of T cells, and polarization of CD4+ towards the Th17 phenotype. These changes co-opted the immune cells to favor cell proliferation, survival and migration of HNSCC. This immunosubversion was observed both indirectly with secreted products and with direct cell-to-cell contact. We conclude that HNSCC-derived secreted products create an immunosuppressive environment that facilitates evasion of tumor cells and subverts the immune cells into a pro-tumoral phenotype.

// Qin Luo 1 , Hengmin Cui 1,2 , Huidan Deng 1 , Ping Kuang 1 , Huan Liu 1 , Yujiao Lu 1 , Jing Fang 1,2 , Zhicai Zuo 1,2 , Junliang Deng 1,2 , Yinglun Li 1,2 , Xun Wang 1,2 and Ling Zhao 1,2 1 College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Sichuan Agriculture University, Wenjiang, Chengdu, China Correspondence to: Hengmin Cui, email: // Keywords : sodium fluoride, oxidative damage, lesion, dysfunction, kidney, Immunology and Microbiology Section, Immune response, Immunity Received : March 13, 2017 Accepted : April 11, 2017 Published : April 21, 2017 Abstract It has been reported that excessive intake of fluoride can induce renal lesions. However, its pathogenesis is still less understood. Therefore, this study was conducted to investigate oxidative damage and the relationships between the oxidative damage and renal lesions in fluoride-treated mice by using the methods of histopathology, biochemistry, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). A total of 240 ICR mice were randomly divided into four equal groups (sodium fluoride was given orally at the dose of 0, 12, 24 and 48 mg/kg body weight for 42 days, respectively). We found that fluoride in excess of 12 mg/kg induced renal oxidative damage, which was characterized by increasing the levels of reactive oxygen species (ROS) production and contents of malondialdehyde (MDA) and protein carbonyls (PC), and decreasing the abilities of anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR), glutathione (GSH) content, as well as activities and mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px). Concurrently, fluoride caused degeneration and necrosis of the tubular cells, renal tubular hyaline casts and glomeruli swelling, which were consistent with the alteration of renal function parameters including elevated contents of serum creatinine (Cr), serum uric acid (UA), blood urea nitrogen (BUN), and the activities of urinary N-acetyl-b-D-glucosaminidase (NAG), renal lactate dehydrogenase (LDH), and reduced activities of sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and acid phosphatase (ACP) in the kidney. The above-mentioned results showed that fluoride in excess of 12 mg/kg induced renal oxidative damage, which then caused renal lesions and dysfunctions. These findings also clearly demonstrated that oxidative damage is one of the mechanisms of fluoride-induced renal lesions and dysfunctions.

// Lijie Sun 1,2,* , Yan Li 1,2,* , Xiuzhi Jia 1,2,* , Qi Wang 1,2 , Yue Li 3 , Minghui Hu 4 , Linlu Tian 1,2 , Jinfeng Yang 1,2 , Wenjing Xing 1,2 , Weihua Zhang 5 , Jingtao Wang 6 , Hongwei Xu 1,2 , Lihua Wang 7 , Dekai Zhang 3 and Huan Ren 1,2 1 Department of Immunology, Harbin Medical University, Harbin, China 2 Key Laboratory of Infection & Immunity, Heilongjiang Province, Harbin, China 3 Center for Infectious and Inflammatory Disease, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX, USA 4 Department of Clinical Laboratory, The Affiliated Hospital to Qingdao University, Qingdao, China 5 Department of Pathophysiology, Harbin Medical University, Harbin, China 6 Department of Epidemiology and Biostatistics, The Public Health Institute, Harbin Medical University, Harbin, China 7 Department of Neuroscience, The Second Hospital Affiliated to Harbin Medical University, Harbin, China Correspondence to: Huan Ren, email: // Dekai Zhang, email: // Lihua Wang, email: // Keywords : interferon-gamma, astrocyte, interleukin-6, apoptosis, neuroinflammation, Immunology and Microbiology Section, Immune response, Immunity Received : November 21, 2016 Accepted : March 22, 2017 Published : April 09, 2017 Abstract Inflammation eliminates pathogenic infections while also threatening the integrity of the central nervous system. In this study, using in vivo and in vitro models of acute neuroinflammation, we investigated the mechanisms by which inflammation and astrocytes affect neuronal apoptosis. The in vitro model mimicked acute neuroinflammation by incubation in IFN-γ-containing media with primary cultured cerebellar granule neurons, with or without cultured astrocytes. This quickly induced neuronal apoptosis characterized by cleaved caspase-3 expression, Hoechst 33342 staining, and intercellular Ca 2+ influx, whereas the presence of astrocytes significantly protected neurons from these effects. IFN-γ in the inflammation media also promoted astrocyte secretion of IL-6, essential for protection. The supernatants of rat peripheral blood mononuclear cells stimulated by lymphocyte mitogen lipopolysaccharide or concanavalin A were used as inflammation media to verify the results. The in vivo model involved a peripheral challenge with lipopolysaccharide, with or without recombinant IFN-γ, in C57BL/6 mice. This confirmed the in vitro results: anti-IFN-γ antibodies exacerbated the acute course of neuroinflammation and led to neurocyte apoptosis in vivo . The pro-inflammatory cytokine IFN-γ provided neuroprotection during acute neuroinflammation via induction of astrocyte-secreted IL-6. The findings provide novel insights into the mechanisms of neuroprotection by IFN-γ during acute neuroinflammation, and may impact therapies for inflammation-related central nervous system injury and disease.

// Yu-Lin Xu 1,* , Hai-Lin Tang 1,* , Hao-Ran Peng 1 , Ping Zhao 1 , Zhong-Tian Qi 1 and Wen Wang 1 1 Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai, China * These authors have contributed equally to this study Correspondence to: Zhong-Tian Qi, email: // Wen Wang, email: // Keywords : influenza A virus; H7N9 virus; RIP3; necroptosis; proinflammatory cytokines; Immunology and Microbiology Section; Immune response; Immunity Received : December 08, 2016 Accepted : February 23, 2017 Published : March 08, 2017 Abstract Influenza H7N9 virus infection causes an acute, highly contagious respiratory illness that triggers cell death of infected cells and airway epithelial destruction. RIP3 is a key regulator of cell death responses to a growing number of viral and microbial agents. This study aimed to investigate the role of RIP3 in inflammation of influenza H7N9 virus infection. Here, RIP3 knock out (RIP3-/-) mice and littermate wild type mice were infected intranasally with influenza H7N9 virus (A/Fujian/S03/2015) to determine the contribution of RIP3 to the inflammatory response of influenza H7N9 virus infection. It was found that RIP3-/- mice infected with H7N9 virus showed higher survival and less weight loss, compared with wild type littermate mice. In addition, RIP3-/- mice had fewer regions of edema, infiltration with inflammatory cells, and alveolar collapses, and the secretions of IL-1β, IL-6, RANTES and MIP-1 in BALF were significantly decreased on days 3 and 7 p.i. when compared with WT mice. Moreover, caspase 1/IL1β signaling was found to be involved in RIP3 associated inflammation of influenza H7N9 virus, but not RIP3/MLKL dependent necrosis. In the conclusion, our results indicated that RIP3 deficiency can protect mice from the infection of influenza H7N9 virus by downregulating caspase 1/IL1β signaling, which provided evidence of the RIP3 involved necroptosis independent manner.

// Yang Li 1,* , Jin Wang 1,* , Jie Yin 1 , Xinhua Liu 2 , Minghang Yu 1 , Ting Li 1 , Han Yan 1 and Xi Wang 1 1 Department of Cell Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Laboratory of Epigenetics in Development and Tumorigenesis, Tianjin Research Center of Basic Medical Sciences, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin, China 2 Department of Biochemistry and Molecular Biology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China * Yang Li and Jin Wang have contributed equally to this work Correspondence to: Xi Wang, email: // Keywords : NK cell, activation, histone modification, poised state, small molecule inhibitor, Immunology and Microbiology Section, Immune response, Immunity Received : December 08, 2016 Accepted : March 16, 2017 Published : March 29, 2017 Abstract Studies of Natural Killer (NK) cell cytotoxicity have mainly focused on the balance of activating and inhibitory receptors, signaling transduction, calcium influx, formation of immune synapse, and cytolytic degranulation. However, little is known about the chromatin state of NK cells and the impact of its changes during target recognition. In this study, we investigate the contribution of chromatin state dynamics during NK cell activation by comprehensively analyzing a set of microarray data and two sets of Chromatin Immunoprecipitation-Sequencing (ChIP-seq) data. We find that the expression of several histone demethylases and methyltransferases was influenced upon stimulation. Furthermore, we notice that a series of genes, including PI3KCA, NFATC1and TNFSF9, which play important roles during NK cell activation, were at ‘poised’ state prior to activation, and that modifications of H3K4me3 and H3K27me3 on these promotors were sensitive to stimulation with Phorbol Myristate Acetate (PMA) and Ionomycin (Iono) in the NK92MI cell line. Finally, we demonstrate that a series of small molecule inhibitors, which are specific to H3K4 and H3K27 modification, enhance degranulation or the expression levels of IFN-γ and TNF-α. Our results suggest that the histone modification state has a profound impact on NK cell activation, and provide novel insights into the regulation of NK cellular cytotoxicity and immunoregulatory function by chromatin state dynamics.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of LAL resulted in reduced release of 18:2- and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages. We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.

// Hai Chen 1 , Xiaodong Qi 1 , Xue Bai 1 , Ping Qiu 1 , Bin Chen 1 1 Department of Galactophore, The General Hospital of Beijing Military Command, Beijing 100000, China Correspondence to: Hai Chen, email: yuijicshygu@163.com Keywords: CD152, CTLA-4, polymorphisms, breast cancer, immune response Received: July 02, 2016 Accepted: October 13, 2016 Published: March 01, 2017 ABSTRACT Background: The polymorphisms in cluster of differentiation 152 ( CD152 ) gene have been reported to be associated with breast cancer (BC), but relevant findings were far from conclusive. Therefore, we carried out this meta-analysis to combine those results for a clearer perspective on this issue. Results: In our meta-analysis, a total of 8 eligible publications of 19 case-control studies were selected, which totally contained 7,442 BC cases and 7,376 normal controls. Among the five polymorphisms of CD152 gene, +49 G/A, −1661 A/G and −318 C/T significantly increased the risk of BC under corresponding genetic comparisons; while CT60 G/A polymorphism was negatively related to the cancer susceptibility. In addition, −1772 T/C polymorphism of CD152 gene was not associated with the development of BC. Materials and methods: Online databases and other sources were searched for published studies on the relationship between BC susceptibility and CD152 polymorphisms (+49 G/A, −1661 A/G, −1722 T/C, −318 C/T and CT60 G/A). The strength of association was evaluated with pooled odds ratios (ORs) and their corresponding 95% confidence intervals (95% CIs). Heterogeneity evaluation was conducted via Q test. Sensitivity analysis was used to detect the stability of our results. Begg’s funnel plot and Egger’s test were applied to investigate publication bias among selected studies. Conclusions: The polymorphisms +49 G/A, −1661 A/G and −318 C/T may elevate the susceptibility to BC, but the polymorphism CT60 G/A may offer protection against the cancer.

// Si Chen 1,2,* , Bingni Chen 1,2,3,* , Zhongyang Wen 1,2,3,* , Zhong Huang 1,2,3 and Liang Ye 1,2,3 1 Institute of Biological Therapy, Shenzhen University, Shenzhen, China 2 Department of Pathogen Biology and Immunology, Shenzhen University School of Medicine, Shenzhen, China 3 Shenzhen City Shenzhen University Immunodiagnostic Technology Platform, Shenzhen, China * These authors have contributed equally to this work Correspondence to: Liang Ye, email: // Zhong Huang, email: // Keywords : IL-33, IL-33 receptor, IL-10, macrophage, rheumatoid arthritis, Immunology and Microbiology Section, Immune response, Immunity Received : August 11, 2016 Accepted : March 01, 2017 Published : March 16, 2017 Abstract IL-10 is an immunosuppressive cytokine produced and sensed by many immune cells and exerts a protective role in autoimmune diseases. However, the underlying mechanism by which IL-10 contributes to prevent the arthritic inflammation in macrophages is poorly understood. Herein we report on a novel anti-arthritic property of IL-10 through the inhibition of IL-33 signaling by macrophages during collagen-induced arthritis (CIA) development. We show that IL-33 expression rather than its receptor (ST2) is positively correlated with IL-10 level in active RA. IL-10 deficiency in mice leads to significant upregulation of IL-33 expression and aggravates the progression of CIA, while exogenous IL-10 treatment effectively diminishes IL-33 production in IL-10 knockout (IL-10 -/- ) CIA mice. We demonstrate further that the inhibitory effect of IL-10 in suppressing IL-33 production requires STAT3 activation in macrophages. Furthermore, IL-33 stimulated proinflammatory genes are notably increased in IL-10 -/- CIA mice, whereas macrophages treated with recombinant IL-10 exhibit decreased IL-33 amplified inflammation and inhibited IL-33 activated NF-κB signaling pathway. Our findings indicate that IL-10 act as a negative regulator of IL-33/ST2 signaling pathways in vivo , suggesting a potential therapeutic role of IL-10 in autoimmune diseases.

// Daisuke Muraoka 1,2 , Naohiro Seo 1 , Tae Hayashi 1 , Chisaki Hyuga-Amaike 1 , Kana Okamori 1 , Isao Tawara 3 , Naozumi Harada 1 and Hiroshi Shiku 1 1 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan 2 Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan 3 Department of Hematology and Oncology, Mie University Graduate School of Medicine, Mie, Japan Correspondence to: Daisuke Muraoka, email: // Hiroshi Shiku, email: // Keywords : memory T cell, CTL function, STAP2, STAT signaling, SOCS3, Immunology and Microbiology Section, Immune response, Immunity Received : April 18, 2016 Accepted : February 01, 2017 Published : February 16, 2017 Abstract Long-surviving memory CD8 + T cells generated by stimulation with appropriate tumor-associated antigens are the most aggressive and persistent tumoricidal effectors. In this event of memory CD8 + T cell development, the signal transducer and activator of transcription (STAT) proteins function as the crucial intracellular signaling molecules, but the regulatory mechanism of STATs in CD8 + T cells is not fully understood. In this study, we report for the first time, by using murine vaccination models, that signal-transducing adaptor protein-2 (STAP2) maintains the cytotoxicity of long-lived memory CD8 + T cells by controlling a STAT3/suppressor of cytokine signaling 3 (SOCS3) cascade. Following T cell activation, STAP2 expression was transiently reduced but was subsequently recovered and augmented. Analysis using small-interfering RNA (siRNA) demonstrated that restored STAP2 expression was associated with the activation of STAT3/SOCS3 signals and maintenance of cytotoxic T lymphocytes (CTLs) secondary responses by preventing their differentiation into terminal effector cells. Notably, this STAP2-dependent memory differentiation was observed in the spleen, but not in the lymph nodes (LNs). These findings indicate an essential role for STAP2 in the generation of a high-quality memory CD8 + CTLs periphery, and suggest the therapeutic potential of STAP2 in cancer patients.

// Yuan Liu 1,* , Xining Liao 2,* , Ying Wang 3,* , Shiju Chen 1 , Yuechi Sun 1 , Qingyan Lin 1 and Guixiu Shi 1 1 Department of Rheumatology and Clinical Immunology, The First Affiliated Hospital of Xiamen University, Xiamen, China 2 Medical College, Xiamen University, Xiamen, Fujian, China 3 Department of Endocrinology, The ChengGong Hospital Affiliated to Xiamen University, Xiamen, Fujian, China * Yuan Liu, Xining Liao and Ying Wang contribute equally to this study Correspondence to: Guixiu Shi, email: // Keywords : primary Sjogren’s syndrome; anti-MDM2 autoantibody; biomarker;Immunology and Microbiology Section; Immune response; Immunity Received : September 27, 2016 Accepted : January 17, 2017 Published : January 28, 2017 Abstract Introduction: Primary Sjogren’s Syndrome (pSS) is one of the autoimmune diseases characterized by polyclonal autoantibody production. The human homologue of the mouse double minute 2 (MDM2) is an important negative regulator of p53. Our previous study indicated that autoantibody to MDM2 can be detected in systemic lupus erythematosus patients. The purpose of this study is to study anti-MDM2 autoantibody in pSS patients. Methods: Anti-MDM2 autoantibody in sera from 100 pSS patients and 74 normal controls was investigated by ELISA. Positive samples were further confirmed by western blotting. Expression of MDM2 in labial gland tissue from pSS patients and normal controls was checked by immunohistochemistry. The difference in clinical characteristics and laboratory findings between anti-MDM2 positive and anti-MDM2 negative pSS patients was analyzed. Results: The presence of anti-MDM2 autoantibody in pSS patients was 21.0%, significantly higher than normal controls (5.40%). MDM2 was overexpressed in labial gland from pSS patients. pSS patients with positive anti-MDM2 were characterized by longer disease duration and more lymphocytes focal gathering in labial gland. Prevalence of anemia, thrombocytopenia and anti-SSB was significantly higher in pSS patients with anti-MDM2 autoantibody. Titer of anit-MDM2 was negatively associated with hemoglobin level, platelet count, complement 3 level and complement 4 level, positively associated with European Sjogren’s syndrome disease activity index (ESSDAI) and level of IgG. Conclusions: Anti-MDM2 autoantibody may be used as a potential serological biomarker in pSS disease activity evaluation. Study on the role of anti-MDM2 or MDM2 in pSS may help us know the pathogenesis mechanism of pSS better.

Cancer gene therapy has attracted increasing attention for its advantages over conventional therapy in specific killing of tumor cells. Here, we attempt to prove a novel therapeutic approach that targets tumors by harnessing the blood antigen immune response system, which is inherently present in patients with breast cancers. Breast cancer MDA-MB-231 cells expressed blood group H antigen precursor. After ectopic expression of blood group A glycosyltransferase, we found that the H precursor was converted into the group A antigen, appearing on the surface of tumor cells. Incubation with group B plasma from breast cancer patients activated the antigen-antibody-complement cascade and triggered tumor cell killing. Interestingly, expression of blood A antigen also reduced tumorigenesis in breast cancer cells by inhibiting cell proliferation, migration, and tumor sphere formation. Cell cycle analysis revealed that cancer cells were paused at S phase due to the activation of cell cycle regulatory genes. Furthermore, pro-apoptotic genes were unregulated by the A antigen, including BAX, P21, and P53, while the anti-apoptotic BCL2 was down regulated. Importantly, we showed that extracellular HMGB1 and ATP, two critical components of the immunogenic cell death pathway, were significantly increased in the blood A antigen-expressing tumor cells. Collectively, these data suggest that blood antigen therapy induces specific cancer cell killing by activating the apoptosis and immunogenic cell death pathways. Further translational studies are thereby warranted to apply this approach in cancer immuno-gene therapy.

// Fengping Liu 1,4,* , Zongxin Ling 2,3,* , Yonghong Xiao 2,3,* , Longxian Lv 2,3 , Qing Yang 3 , Baohong Wang 2,3 , Haifeng Lu 2,3 , Li Zheng 1 , Ping Jiang 1 , Wei Wang 1,** and Lanjuan Li 2,3,** 1 Department of Urology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 2 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, China 3 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, China 4 Yancheng Medical College, Yancheng, Jiangsu, China * These authors have contributed equally to this work ** These authors also have contributed equally to this work Correspondence to: Wei Wang, email: // Lanjuan Li, email: // Keywords : Akkermansia muciniphila; Escherichia coli; lactobacillus; type 2 diabetes mellitus; urinary microbiota; Immunology and Microbiology Section, Immune response, Immunity Received : November 02, 2016 Accepted : December 13, 2016 Published : December 19, 2016 Abstract Type 2 diabetes mellitus (T2DM) may be associated with altered urinary microbiota in female patients. We investigated alterations of urinary microbiota in Chinese female T2DM patients, and explored the associations between urinary microbiota and a patient’s fasting blood glucose (FBG), urine glucose (UGLU), age, menstrual status, and body mass index (BMI). Midstream urine was collected from 70 female T2DM patients and 70 healthy females. Microbial diversity and composition were analyzed using the Illumina MiSeq sequencing platform by targeting the hypervariable V3-V4 regions of the 16S rRNA gene. We found that bacterial diversity was decreased in T2DM patients. Increased Actinobacteria phylum was positively correlated with FBG, UGLU, and BMI; Lactobacillus abundance decreased with age and menopause; and increased Lactobacillus correlated positively with FBG and UGLU. Decreased Akkermansia muciniphila was associated with FBG and UGLU. Escherichia coli abundance did not differ between the two cohorts. Carbohydrate and amino acid metabolism was reduced in T2DM patients, which were associated with bacterial richness indices such as Chao1 and ACE. Detailed microbiota analysis of well-characterized T2DM patients and healthy controls indicate that Chinese T2DM female patients exhibit dysbiosis of urinary microbiota.

// Juan Fu 1 , Rupashree Sen 1 , David L. Masica 2 , Rachel Karchin 2,3 , Drew Pardoll 3 , Vonn Walter 4 , D. Neil Hayes 5 , Christine H. Chung 6 and Young J. Kim 3,7 1 Department of Otolaryngology - Head & Neck Surgery, SKCCC, Johns Hopkins Hospital, Baltimore, MD, USA 2 Department of Biomedical Engineering and The Institute for Computational Medicine, SKCCC, Johns Hopkins Hospital, Baltimore, MD, USA 3 Bloomberg-Kimmel Institute for Cancer Immunotherapy, SKCCC, Johns Hopkins Hospital, Baltimore, MD, USA 4 Department of Biochemistry and Molecular Biology, Penn State Milton S. Hershey Medical Center, Hershey, PA, USA 5 UNC Chapel Hill School of Medicine, Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA 6 Department of Head & Neck - Endocrine Oncology, Moffitt Cancer Center, Tampa, FL, USA 7 Department of Otolaryngology - Head & Neck Surgery, VICC, Vanderbilt University Medical Center, Nashville, TN, USA Correspondence to: Young J. Kim, email: // Keywords : tumor microenvironment, autologous reconstitution, STAT3, humanized mice, head neck carcinoma, Immunology and Microbiology Section, Immune response, Immunity Received : August 19, 2016 Accepted : December 13, 2016 Published : December 19, 2016 Abstract Correlative studies from checkpoint inhibitor trials have indicated that better understanding of human leukocytic trafficking into the human tumor microenvironment can expedite the translation of future immune-oncologic agents. In order to directly characterize signaling pathways that can regulate human leukocytic trafficking into the tumor, we have developed a completely autologous xenotransplantation method to reconstitute the human tumor immune microenvironment in vivo. We were able to genetically mark the engrafted CD34+ bone marrow cells as well as the tumor cells, and follow the endogenous leukocytic infiltration into the autologous tumor. To investigate human tumor intrinsic factors that can potentially regulate the immune cells in our system, we silenced STAT3 signaling in the tumor compartment. As expected, STAT3 signaling suppression in the tumor compartment in these autologously reconstituted humanized mice showed increased tumor infiltrating lymphocytes and reduction of arginase-1 in the stroma, which were associated with slower tumor growth rate. We also used this novel system to characterize human myeloid suppressor cells as well as to screen novel agents that can alter endogenous leukocytic infiltration into the tumor. Taken together, we present a valuable method to study individualized human tumor microenvironments in vivo without confounding allogeneic responses.

// David J. Paulucci 1 , John P. Sfakianos 1 , Anders J. Skanderup 2 , Kathleen Kan 1 , Che-Kai Tsao 3 , Matthew D. Galsky 3 , A. Ari Hakimi 4 , Ketan K. Badani 1 1 Department of Urology, Icahn School of Medicine at Mount Sinai Hospital, New York, NY, USA 2 Computational Biology Program, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA 3 Department of Medicine, Division of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 4 Department of Surgery—Urology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA Correspondence to: Ketan K. Badani, email: Ketan.Badani@mountsinai.org Keywords: papillary renal cell carcinoma, racial disparities, immune system signaling, targeted therapy, immune response Received: May 03, 2016 Accepted: November 22, 2016 Published: December 23, 2016 ABSTRACT Significant disparities in survival, incidence and possibly response to current therapies exist between black and white patients with renal cell carcinoma (RCC). Recent genomic evidence to account for these disparities has been reported for clear cell RCC. However, racial disparities at the genomic level for papillary RCC (pRCC) which is a genetically distinct and less responsive histologic subtype of RCC have not been reported. Using The Cancer Genome Atlas (TCGA) data, the present study assessed gene-level expression, somatic mutation and pathway differences between 58 black and 58 white patients with pRCC propensity matched on age, gender and pathologic T stage. Distinct tumor biology with differential expression patterns were observed in black vs. white patients with pRCC. Specifically, significance analysis of microarrays was applied to TCGA gene expression data and identified 163 genes and 120 genes overexpressed in black and white patients, respectively (FDR q

// Yingfeng Wei 1 , Chong Lu 1 , Jianing Chen 1 , Guangying Cui 1 , Lin Wang 1 , Tianming Yu 2 , Yue Yang 3 , Wei Wu 1 , Yulong Ding 1 , Lanjuan Li 1 , Toshimitsu Uede 4 , Zhi Chen 1 and Hongyan Diao 1 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 2 Department of Orthopaedics, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 3 Department of Biochemistry, University of Washington, Seattle, Washington, USA 4 Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Correspondence to: Hongyan Diao, email: // Zhi Chen, email: // Keywords : high salt diet, Th17 cells, regulatory T cells, inflammatory bowel diseases, intestinal immunity, Immunology and Microbiology Section, Immune response, Immunity Received : August 01, 2016 Accepted : November 24, 2016 Published : December 01, 2016 Abstract This study focuses on characterizing the effect of a high salt diet (HSD) on intestinal immunity and the risk of inflammatory bowel diseases (IBD). We found that mice on a HSD had an increased frequency of IL-17A producing cells in the intestinal lamina propria (LP) compared to mice on a normal diet (ND). Furthermore, most intestinal IL-17A producing cells were CD4 + TCRβ + cells. A HSD increased the LP T helper 17 (Th17) responses in both the small and large intestines but did not increase the Th17 response of other gut-associated lymphoid organ. Although, HSD did not change the percentage of regulatory T (Treg) cells, HSD significantly inhibit secretion of IL-10 and the suppressive function of Treg cells. Moreover, we found that HSD exacerbates trinitrobenzenesulfonic acid (TNBS) induced colitis, and Th17 response was significantly increased in the colonic LP of HSD TNBS-treated mice compared with the ND TNBS-treated mice. This study demonstrates that HSD stimulates the intestinal Th17 response but inhibits the function of Treg cells. Moreover, HSD exacerbates TNBS induced mice colitis, suggesting that HSD disrupts the intestinal immunity and increases the risk of IBD.

// Weiping Hu 1,* , Guodong Niu 1,* , Hongbo Li 1 , Hanyuan Gao 1 , Rudian Kang 1 , Xiaoqing Chen 1 and Ling Lin 1 1 Rheumatism Department, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China * These authors have contributed equally to this study Correspondence to: Xiaoqing Chen, email: // Ling Lin, email: // Keywords : IFIT1, podocytes, animal study, renal pathological changes, lupus nephritis, Immunology and Microbiology Section, Immune response, Immunity Received : July 28, 2016 Accepted : October 28, 2016 Published : November 03, 2016 Abstract Renal damage is the major cause of SLE associated mortality, and IFIT1 expression was elevated in SLE cases in accordance of previous studies. Therefore, we conducted an animal study to identify the role of IFIT1 expression in renal pathological changes.18 female MRL/lpr mice and same number of female BALB/c mice were enrolled in present study. Quantitative analysis of urine protein, Complement C3 and C4, and anti-ds DNA antibody were conducted. HE and PAS staining and TEM analysis were employed to observe the pathological changes in renal tissue. Significant elevation on urine protein and anti-dsDNA and reduction on Complement C3 and C4 were observed in MRL/lpr mice when comparing the controls in same age. Staining and TEM analysis observed several pathological changes in glomerulus among MRL/lpr mice, including cellular enlargement, basement membrane thickening, and increased cellularcasts. The linear regression analysis found the optical density of IFIT1 was inversely associated with F-actin, Nephrin, and Podocin, but not Synatopodin. In summary, IFIT1 expression is associated with podocytes damage, and capable of suppressing some proteins essential to glomerular filtration.

Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of Epizootic Haemorrhagic Disease Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.

// Hyunjung Baek 1 , Minsook Ye 1 , Geun-Hyung Kang 1 , Chanju Lee 1 , Gihyun Lee 1 , Da Bin Choi 1 , Jaehoon Jung 1 , Hyunseong Kim 2 , Seonhwa Lee 3 , Jin Su Kim 3 , Hyun-ju Lee 4 , Insop Shim 4 , Jun-Ho Lee 5 and Hyunsu Bae 1 1 Department of Physiology, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea 2 K-herb Research Center, Korea Institute of Oriental Medicine, Yuseongdae-ro, Yuseong-gu, Daejeon, Republic of Korea 3 Division of RI Research, Korea Institute of Radiological & Medical Sciences University of Science & Technology, Gongneug-dong, Nowon-ku, Seoul, Republic of Korea 4 Acupuncture and Meridian Science Research Center, College of Korean Medical Science Graduate School, Kyung Hee University, Seoul, Republic of Korea 5 Department of Biotechnology, Chonnam National University, Gwangju, Republic of Korea Correspondence to: Hyunsu Bae, email: // Keywords : Alzheimer’s disease, regulatory T cells (Treg), amyloid-beta (Aβ) pathology, Immunology and Microbiology Section, Immune response, Immunity Received : July 23, 2016 Accepted : September 29, 2016 Published : October 04, 2016 Abstract Alzheimer’s disease patients display neuropathological lesions, including the accumulation of amyloid-beta (Aβ) peptide and neurofibrillary tangles. Although the mechanisms causing the neurodegenerative process are largely unknown, increasing evidence highlights a critical role of immunity in the pathogenesis of Alzheimer’s disease. In the present study, we investigated the role of regulatory T cells (Tregs) on Alzheimer’s disease progression. First, we explored the effect of Tregs (CD4 + CD25 + T cells) and Teffs (CD4 + CD25 - T cells) in an adoptive transfer model. Systemic transplantation of purified Tregs into 3xTg-AD mice improved cognitive function and reduced deposition of Aβ plaques. In contrast, adoptive transfer of Teffs diminished behavioral function and cytokine production. Next, we transiently depleted Treg population using an anti-CD25 antibody (PC61). Depletion of Tregs for four months resulted in a marked aggravation of the spatial learning deficits of six-month-old 3xTg-AD mice. Additionally, it resulted in decreasing glucose metabolism, as assessed by positron emission tomography (PET) with 18 F-2 fluoro-2-deoxy-D-glucose ([F- 18 ] FDG) neuroimaging. Importantly, the deposition of Aβ plaques and microglia/macrophage was increased in the hippocampal CA1 and CA3 regions of the Treg depleted 3xTg-AD compared to the vehicle-treated 3xTg-AD group. Our finding suggested that systemic Treg administration ameliorates disease progression and could be an effective Alzheimer’s disease treatment.

// Ruya Zhang 1,* , Fang Ni 1,2,* , Binqing Fu 1 , Yang Wu 1 , Rui Sun 1 , Zhigang Tian 1 and Haiming Wei 1 1 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Science and Medical Center, University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China 2 Department of Pathophysiology, Anhui Medical University, Hefei, Anhui, China * These authors have contributed equally to this work Correspondence to: Haiming Wei, email: // Zhigang Tian, email: // Keywords : long noncoding RNAs, natural killer cells, CD56, primary lymphocytes, lnc-CD56, Immunology and Microbiology Section, Immune response, Immunity Received : May 06, 2016 Accepted : September 28, 2016 Published : October 04, 2016 Abstract Natural killer (NK) cells are innate immune lymphocytes that play critical roles in host defense against viral infection and surveillance against malignant transformation. Long noncoding RNAs (lncRNAs) are important immune system regulators. Here, we analyzed human primary lymphocyte lncRNA expression profiles to identify NK-lncRNA signatures. We detected numerous novel NK-specific lncRNAs with potential roles in regulating human NK cell differentiation and function. Expression of lnc-CD56, an NK-specific lncRNA, was positively correlated with that of CD56, a classical human NK cell surface marker. We showed that lnc-CD56 may function as a positive regulator of CD56 in primary human NK cells and differentiated NK cells from human CD34 + hematopoietic progenitor cells. Our data provide an annotated human NK cell lncRNA expression catalog and demonstrate a key role for lncRNAs in NK cell biology.

Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy of unknown cause. However, about a quarter of GBS patients have suffered a recent bacterial or viral infection, and axonal forms of the disease are especially common in these patients. Proteomics is a good methodological approach for the discovery of disease biomarkers. Until recently, most proteomics studies of GBS and other neurodegenerative diseases have focused on the analysis of the cerebrospinal fluid (CSF). However, serum represents an attractive alternative to CSF because it is easier to sample and has potential for biomarker discovery. The goal of this research was the identification of serum biomarkers associated with recovery from GBS. To address this objective, a quantitative proteomics approach was used to characterize differences in the serum proteome between a GBS patient and her healthy identical twin in order to lessen variations due to differences in genetic background, and with additional serum samples collected from unrelated GBS (N = 3) and Spinal Cord Injury (SCI) (N = 3) patients with similar medications. Proteomics results were then validated by ELISA using sera from additional GBS patients (N = 5) and healthy individuals (N = 3). All GBS and SCI patients were recovering from the acute phase of the disease. The results showed that Piccolo, a protein that is essential in the maintenance of active zone structure, constitutes a potential serological correlate of recovery from GBS. These results provided the first evidence for the Piccolo's putative role in GBS, suggesting a candidate target for developing a serological marker of disease recovery., This work was partially supported by EU FP7 ANTIGONE project number 278976. LMH was supported by a fellowship from the University of Castilla La Mancha (UCLM), Spain. MV was supported by the Research Plan of UCLM, Spain.

// Kelly C. G. Manfrere 1 , Marina P. Torrealba 1 , Denis R. Miyashiro 2 , Luanda M. S. Oliveira 1 , Gabriel C. de Carvalho 1 , Josenilson F. Lima 1 , Anna Claudia C. C. Branco 1 , Natalli Z. Pereira 1 , Juliana Pereira 3 , Jose A. Sanches, Jr 2,* and Maria N. Sato 1,* 1 Department of Dermatology, Laboratory of Medical Investigation, LIM 56, Tropical Medicine Institute of Sao Paulo, University of Sao Paulo Medical School, Brazil 2 Department of Dermatology, Cutaneous Lymphoma Clinic, Hospital das Clinicas, University of Sao Paulo, Medical School, Brazil 3 Department of Hematology, University of Sao Paulo Medical School, Brazil * These authors share the mentorship, critical revision and supervision of this study Correspondence to: Maria N. Sato, email: // Keywords : Se,zary syndrome, innate immunity, Toll-like receptor agonists, cytokines, type I interferon, Immunology and Microbiology Section, Immune response, Immunity Received : July 28, 2016 Accepted : October 14, 2016 Published : October 21, 2016 Abstract Sezary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.

The immunoregulatory function of T regulatory cells (Tregs) is impaired in multiple sclerosis (MS). Recent studies have shown that umbilical cord-derived mesenchymal stem cells (UC-MSCs) exert regulatory effect on the functions of immune cells. Thus, we investigated whether UC-MSCs could improve the impaired function of Tregs from MS patients. Co-cultures of UC-MSCs with PBMCs of MS patients were performed for 3 days. Flow cytometry was used to determine the frequency of Tregs. A cell proliferation assay was used to evaluate the suppressive capacity of Tregs. ELISA was conducted for cytokine analysis in the co-cultures. Our results showed that UC-MSCs significantly increased the frequency of CD4+CD25+CD127low/- Tregs in resting CD4+ T cells (p

// Andrea Petretto 1,* , Grazia Carbotti 2,* , Elvira Inglese 1 , Chiara Lavarello 1 , Maria Pia Pistillo 3 , Valentina Rigo 2 , Michela Croce 2 , Luca Longo 2 , Stefania Martini 2 , Paola Vacca 2,4 , Silvano Ferrini 2,* and Marina Fabbi 2,* 1 Core Facilities-Proteomics Laboratory, Istituto Giannina Gaslini, Genoa, Italy 2 Department of Integrated Oncological Therapies, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 3 Tumor Epigenetics Unit, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 4 Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy * These authors have equally contributed to this work Correspondence to: Silvano Ferrini, email: // Marina Fabbi, email: // Keywords : IL-27, IFN-γ, proteomic analysis, HLA class I, cancer cells, Immunology and Microbiology Section, Immune response, Immunity Received : July 10, 2016 Accepted : September 17, 2016 Published : September 24, 2016 Abstract IL-27, a member of the IL-12-family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-γ, induces the expression of IL-18BP, IDO and PD-L1 immune regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN-γ display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27-regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels. Importantly, we found that IL-27 induced HLA class I molecule expression in human cancer cells of different histotypes, including tumor cells showing very low expression. IL-27 failed only in a cancer cell line bearing a homozygous deletion in the B2M gene. Altogether, these data point out to a broad set of activities shared by IL-27 and IFN-γ, which are dependent on the common activation of the STAT1 pathway. These data add further explanation to the anti-tumor activity of IL-27 and also to its dual role in immune regulation.

// Yun Guan 1 , Weiqun Li 1 , Zhaohua Hou 1 , Qiuju Han 1 , Peixiang Lan 1 , Jian Zhang 1 , Zhigang Tian 2 and Cai Zhang 1 1 Institute of Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, China 2 Institute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China Correspondence to: Cai Zhang, email: // Keywords : hepatitis B virus; MICA; hepatocarcinoma; GATA-2; GATA-3; Immunology and Microbiology Section; Immune response; Immunity Received : March 30, 2016 Accepted : August 01, 2016 Published : August 12, 2016 Abstract Decreased expression of NKG2D ligands on HBV-infected human hepatoma cells impairs NK cells lysis. However, which components of HBV exert this effect and the precise mechanisms need to be further investigated. In the present study, we observed that the HBx and HBc genes significantly down-regulated MICA expression. Through analysis with the chromatin immunoprecipitation assay, we found that HBV infection promotes the expression of transcription factors GATA-2 and GATA-3, which specifically suppressed MICA/B expression by directly binding to the promoter region of MICA/B. HBx protein, acting as a co-regulator, forms a tripolymer with GATA2 and GATA3, thus promotes the GATA-2 or GATA-3-mediated of MICA/B suppression. HBc protein inhibits MICA/B expression via directly binding to the CpG island in the MICA/B promoter. Thus, our study identified the novel role of transcription factors GATA-2 and GATA-3 in suppressing MICA/B expression and clarified the mechanisms of HBx and HBc in downregulation of MICA/B expression. These findings provide novel mechanisms for the contribution of HBV to hepatoma cells escape from NK cell surveillance.

// Rishein Gupta 1,* , Tanvi Arkatkar 1,* , Jonathon Keck 1 , Gopala Krishna Lanka Koundinya 1 , Kevin Castillo 1 , Sabrina Hobel 2 , James P. Chambers 1 , Jieh-Juen Yu 1 , M. Neal Guentzel 1 , Achim Aigner 2 , Lane K. Christenson 3 and Bernard P. Arulanandam 1 1 South Texas Center for Emerging Infectious Diseases and Center of Excellence in Infection Genomics, University of Texas at San Antonio, San Antonio, TX, USA 2 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Hartelstrase, Leipzig, Germany 3 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA * These authors have contributed equally to this work Correspondence to: Bernard P. Arulanandam, email: // Keywords : Chlamydia muridarum , host immunity, microRNA-155, microRNA-182, CD4+ T-cells, Immunology and Microbiology Section, Immune response, Immunity Received : July 22, 2016 Accepted : August 02, 2016 Published : August 20, 2016 Abstract Anti-chlamydial immunity involves efficient presentation of antigens (Ag) to effector cells resulting in Ag-specific immune responses. There is limited information on inherent underlying mechanisms regulating these events. Previous studies from our laboratory have established that select microRNAs (miRs) function as molecular regulators of immunity in Chlamydia muridarum (Cm) genital infection. In this report, we investigated immune cell type-specific miRs, i.e. miR-155 and -182, and the role in Ag-specific immunity. We observed significant up-regulation of miR-155 in C57BL/6 bone marrow derived dendritic cells (BMDC), and miR-182 in splenic Ag-specific CD4 + T-cells. Using mimics and inhibitors, we determined that miR-155 contributed to BMDC activation following Cm infection. Co-cultures of miR-155 over-expressed in BMDC and miR-182 over-expressed in Ag-specific CD4 + T-cells, or miR-155 -/- BMDC with miR-182 inhibitor treated Ag-specific CD4 + T-cells, resulted in IFN-γ production comparable to Ag-specific CD4 + T-cells isolated from Cm infected mice. Additionally, miR-182 was significantly up-regulated in intranasally vaccinated mice protected against Cm infection. In vivo depletion of miR-182 resulted in reduction in Ag-specific IFN-γ and genital pathology in Cm infected mice. To the best of our knowledge, this is the first study to report an interaction of miR-155 (in Cm infected DC) and miR-182 (in CD4 + T-cell) resulting in Ag specific immune responses against genital Cm.

// Xianyu Zhang 1 , Ying Lu 1 , Xin Cao 2 , Tao Zhen 3 and Damian Kovalovsky 1 1 Experimental Immunology Branch, NCI, NIH, Maryland, USA 2 College of Life Science and Engineering, Northwest University for Nationalities, Gansu Engineering Research Center for Animal Cell, Lanzhou, China 3 Oncogenesis and Development Section, National Human Genome Research Institute, NIH, Maryland, USA Correspondence to: Damian Kovalovsky, email: // Keywords : Zbtb1, lymphoid, development, myeloid, differentiation, Immunology and Microbiology Section, Immune response, Immunity Received : June 18, 2016 Accepted : August 02, 2016 Published : August 17, 2016 Abstract Zbtb1 is a transcription factor that prevents DNA damage and p53-mediated apoptosis in replicating immune progenitors, affecting lymphoid as well as myeloid development when hematopoietic progenitors are in competition in mixed bone marrow chimeras. However, Zbtb1-deficient mice do not have an apparent myeloid deficiency. We report here that Zbtb1-deficient lymphoid-primed multipotent progenitors (LMPPs) are biased to develop towards the myeloid fate in detriment of lymphoid development, contributing to the apparent unaffected myeloid development. Zbtb1 expression was maintained during lymphoid development of LMPP cells but downregulated during myeloid development. Deficiency of Zbtb1 in LMPP cells was sufficient to direct a myeloid fate in lymphoid-inducing conditions and in the absence of myeloid cytokines as shown by upregulation of a myeloid gene signature and the generation of myeloid cells in vitro . Finally, biased myeloid differentiation of Zbtb1-deficient LMPP cells was not due to increased p53-dependent apoptosis as it was not reverted by transgenic Bcl2 expression or p53 deficiency. Altogether, our results show that Zbtb1 expression prevents activation of a default myeloid program in LMPP cells, ensuring the generation of lymphoid cells.

// Raj Kurupati 1,* , Andrew Kossenkov 1,* , Larissa Haut 1 , Senthil Kannan 1,2 , Zhiquan Xiang 1 , Yan Li 1 , Susan Doyle 3 , Qin Liu 1 , Kenneth Schmader 3 , Louise Showe 1 and Hildegund Ertl 1 1 The Wistar Institute, Philadelphia, PA, USA 2 Biomedical Graduate Group, University of Pennsylvania, Philadelphia, PA, USA 3 Development and Division of Geriatrics, GRECC, Durham VA Medical Center and Center for the Study of Aging and Human, Department of Medicine, Duke University Medical Center, Durham, NC, USA * These authors have contributed equally to this work Correspondence to: Hildegund Ertl, email: // Keywords : B cell responses, inactivated influenza vaccine, race, Immunology and Microbiology Section, Immune response, Immunity Received : June 17, 2016 Accepted : August 25, 2016 Published :August 30, 2016 Abstract We conducted a 5-year study analyzing antibody and B cell responses to the influenza A virus components of the inactivated influenza vaccine, trivalent (IIV3) or quadrivalent (IIV4) in younger (aged 35-45) and aged (≥65 years of age) Caucasian and African American individuals. Antibody titers to the two influenza A virus strains, distribution of circulating B cell subsets and the blood transcriptome were tested at baseline and after vaccination while expression of immunoregulatory markers on B cells were analyzed at baseline. African Americans mounted higher virus neutralizing and IgG antibody responses to the H1N1 component of IIV3 or 4 compared to Caucasians. African Americans had higher levels of circulating B cell subsets compared to Caucasians. Expression of two co-regulators, i.e., programmed death (PD)-1 and the B and T cell attenuator (BTLA) were differentially expressed in the two cohorts. Race-related differences were caused by samples from younger African Americans, while results obtained with samples of aged African Americans were similar to those of aged Caucasians. Gene expression profiling by Illumina arrays revealed highly significant differences in 1368 probes at baseline between Caucasians and African Americans although samples from both cohorts showed comparable changes in transcriptome following vaccination. Genes differently expressed between samples from African Americans and Caucasians regardless of age were enriched for myeloid genes, while the transcripts that differed in expression between younger African Americans and younger Caucasians were enriched for those specific for B-cells.

// Doureradjou Peroumal 1 , Thiruvaimozhi Abimannan 1 , Ravichandra Tagirasa 1 , Jyothi Ranjan Parida 3 , Santosh Kumar Singh 1 , Prasantha Padhan 2 and Satish Devadas 1 1 Infectious Disease Biology, Institute of Life Sciences, Chandrashekarpur, Bhubaneswar, Odisha, India 2 Kalinga Institute of Medical Sciences, Patia, Bhubaneswar, Odisha, India 3 Institute of Medical Sciences & SUM Hospital, Kalinga Nagar, Bhubaneswar, Odisha, India Correspondence to: Satish Devadas, email: // Keywords : T helper cells, MAPK, AICD, FasL, rheumatoid arthritis, Immunology and Microbiology Section, Immune response, Immunity Received : October 29, 2015 Accepted : June 20, 2016 Published : July 28, 2016 Abstract Activation Induced Cell Death of T helper cells is central to maintaining immune homeostasis and a perturbation often manifests in aberrant T helper cells that is associated with immunopathologies. Significant presence of T cells positive for IL-17A (Th17) and dual positive for IFN-γ/IL-17A (Th1/Th17) in both effector (CD45RA + RO + ) and memory (CD45RA - RO + ) compartments with differential FasL protein in RA peripheral blood suggested their differential TCR AICD sensitivity. Lowered active caspase-3 in Th17 and Th1/Th17 over Th1 cells confirmed their capability to resist AICD and pointed to early upstream events. Differential MAPK activities, FasL protein and downstream caspase-3 activities in murine Th1 and Th17 cells established distinct TCR mediated signaling pathways and suggested low Erk and p38 activity as pivotal for AICD sensitivity. We extrapolated our mouse and human data and report that Fas-FasL is the preferred death pathway for both Th1 and Th17 and that inherently low Erk2 activity protected Th17 cells from TCR AICD. The presence of significantly higher numbers of aberrant T helper cells in RA also suggest an inflammatory cytokine milieu and AICD insensitive T cell link to sustained inflammation. Re sensitization to apoptosis by targeting MAPK activity especially Erk2 in RA might be of therapeutic value.

The therapeutic efficacy of allergen specific immunotherapy (SIT) on allergic diseases is to be improved. Probiotics can regulate immune response. This study aims to promote the effect of SIT on allergic rhinitis (AR) by co-administration with Clostridium butyricum (Cb). In this study, patients with AR sensitized to mite allergens were enrolled to this study, and treated with SIT or/and Cb. The therapeutic efficacy was evaluated by the total nasal symptom scores (NSS), medication scores, serum specific IgE levels and T helper (Th)2 cytokine levels. The improvement of immune regulation in the AR patients was assessed by immunologic approaches. The results showed that treating AR patients with SIT alone markedly reduced NSS and medication scores; but did not alter the serum specific IgE, Th2 cytokines and skin prick test (SPT) index. The clinical symptoms on AR in SIT group relapsed one month after stopping SIT. Co-administration of Cb significantly enhanced the efficacy of SIT on AR as shown by suppression of NSS, medication scores, serum specific IgE, Th2 cytokines and SPT index; the regulatory B cell frequency was also markedly increased. Such an effect on AR was maintained throughout the observation period even after stopping the treatment. Butyrate blocked the activation of histone deacetylase-1, the downstream activities of epsilon chain promoter activation, and the IgE production in the antigen specific B cells. On the other hand, butyrate induced the IL-10 expression in B cells with a premise of the B cell receptor activation by specific antigens. In conclusion, administration with Cb can markedly enhance the efficacy of SIT on AR.

// Xiao-Fei Song 1,* , Ting-Ting Hu 1,* , Yu Lei 1,* , Hu Li 1 , Li Zhang 1 , Miao Zhang 1 , Bin Liu 1 , Min Chen 1 , Huai-Dong Hu 1 , Hong Ren 1 and Peng Hu 1 1 Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China * These authors have contributed equally to this study Correspondence to: Peng Hu, email: // Keywords : acute hepatitis B; hydrodynamic injection; intrahepatic lymphocytes; viral clearance; Immunology and Microbiology Section; Immune response; Immunity Received : January 08, 2016 Accepted : July 06, 2016 Published : July 18, 2016 Abstract The role of immunity in the pathogenesis of acute hepatitis B virus (HBV) infection is poorly understood. The purpose of this research was to define the intrahepatic immune factors responsible for viral clearance during acute HBV infection. The model of acute HBV infection was established by hydrodynamically transfecting mice with pCDNA3.1-HBV1.3 plasmids which contained a supergenomic HBV1.3-length transgene. The frequency of CD4 + CXCR5 + T cells, CD19 + B cells and their surface molecules in livers, spleens and peripheral blood were detected using flow cytometry. The lymphomononuclear cells isolated from the livers of transfected mice were further stimulated by HBc-derived peptides and then the frequency and cytokine secretion of HBV-specific CD4 + CXCR5 + T cells were detected. We found that the frequency of CXCR5 + in CD4 + T cells was specifically increased; the expression of PD-1 was decreased while the expression of ICOS was increased on intrahepatic CD4 + CXCR5 + T cells. Although the frequency of CD19 + B cells was not affected, the expression of PDL-1, ICOSL and IL-21R on B cells was increased in the livers of mice. The frequency of HBV-specific CD4 + CXCR5 + T cells and the production of IL-21 by intrahepatic CD4 + CXCR5 + T cells of mice with acute HBV infection were increased after stimulation. Furthermore, the expression of function-related molecules of intrahepatic CD4 + CXCR5 + T, including Bcl-6, CXCR5, IL-6, IL-6R, IL-21 and IL-4 in the liver was increased during acute HBV infection. In conclusion, the activation of intrahepatic CD4 + CXCR5 + T cells and B cells was associated with the clearance of HBV during acute infection.

Tumor-associated macrophages (TAMs) polarized to the M2 phenotype play key roles in tumor progression in different cancer types, including lung cancer. MUC1 expression in various types of cancer is an indicator of poorer prognosis. Elevated MUC1 expression has been reported in inflammatory lung macrophages and is associated with lung cancer development. Here, we investigated the role of M2-polarized TAMs (M2-TAMs) in the generation of lung cancer stem cells (LCSCs) and tested pterostilbene, a small-molecule agent that modulates MUC1 expression in lung cancer cells, with the goal of subverting the microenvironment toward a favorable anti-tumor impact. We found that MUC1 was overexpressed in lung cancer patients, which was associated with poor survival rates. M2-TAMs and cancer cell lines were co-cultured in an experimental tumor microenvironment model. The expression levels of MUC1 and cancer stemness genes significantly increased in lung cancer cells in the presence of the M2-TAM cells. Intriguingly, pterostilbene dose-dependently suppressed self-renewal ability in M2-TAMs-co-cultured lung cancer cells, and this suppression was accompanied by downregulation of MUC1, NF-κB, CD133, β-catenin, and Sox2 expression. Moreover, MUC1-silenced M2-TAMs exhibited a significantly lower ability to promote LCSC generation and decreased levels of NF-κB, CD133, and Sox2. The results suggest that MUC1 plays an important role in TAM-induced LCSC progression. Pterostilbene may have therapeutic potential for modulating the unfavorable effects of TAMs in lung cancer progression.

Vascular endothelial growth factor-C (VEGF-C)-induced lymphangiogenesis and increased tissue drainage have been reported to inhibit acute and chronic inflammation, and an activated lymphatic endothelium might mediate peripheral tolerance. Using transgenic mice overexpressing VEGF-C in the skin, we found that under inflammatory conditions, VEGF-C-mediated expansion of the cutaneous lymphatic network establishes an immune-inhibitory microenvironment characterised by increased regulatory T (Treg) cells, immature CD11c+CD11b+ dendritic cells (DCs) and CD8+ cells exhibiting decreased effector function. Strikingly, lymphatic endothelial cell (LEC)-conditioned media (CM) potently suppress DC maturation with reduced expression of MHCII, CD40, and IL-6, and increased IL-10 and CCL2 expression. We identify an imbalance in prostaglandin synthase expression after LEC activation, favoring anti-inflammatory prostacyclin synthesis. Importantly, blockade of LEC prostaglandin synthesis partially restores DC maturity. LECs also produce TGF-ß1, contributing to the immune-inhibitory microenvironment. This study identifies novel mechanisms by which the lymphatic endothelium modulates cellular immune responses to limit inflammation., Oncotarget, 7 (26), ISSN:1949-2553

// Luise Borufka 1 , Erik Volmer 1 , Sarah Muller 1 , Robby Engelmann 2 , Horst Nizze 3 , Saleh Ibrahim 4 and Robert Jaster 1 1 Department of Medicine II, Division of Gastroenterology, Rostock University Medical Center, Rostock, Germany 2 Institute of Immunology and Core Facility for Cell Sorting & Cell Analysis, Rostock University Medical Center, Rostock, Germany 3 Institute of Pathology, Rostock University Medical Center, Rostock, Germany 4 Institute of Experimental Dermatology, University of Lubeck, Lubeck, Germany Correspondence to: Robert Jaster, email: // Keywords : autoimmune pancreatitis, mouse model, dendritic cells, cell culture, gene expression, Immunology and Microbiology Section, Immune response, Immunity Received : April 14, 2016 Accepted : June 09, 2016 Published : June 23, 2016 Abstract Objectives: MRL/MpJ mice spontaneously develop an autoimmune pancreatitis (AIP) and are widely used as a model to study the genetic, molecular and immunological basis of the disease. Here, we have addressed the question whether distinctive features of their dendritic cells (DCs) may predispose MRL/MpJ mice to the chronic inflammation. Methods: Pancreatic lesions were analyzed employing histological methods. Cohorts of young (healthy) MRL/MpJ mice, adult (sick) individuals, and AIP-resistant CAST/EiJ mice were used to establish cultures of bone marrow (BM)-derived conventional DCs (cDCs). The cells were subsequently characterized regarding the expression profile of CD markers and selected genes, proliferative activity as well as cytokine secretion. Results: In pancreatic lesions, large numbers of cells expressing the murine DC marker CD11c were detected in close spatial proximity to CD3 + cells. A high percentage of BM-derived cDCs from adult MRL/MpJ mice expressed typical markers of DC maturation (such as CD83) already prior to a treatment with lipopolysaccharide (LPS). After LPS-stimulation, cDC cultures of both MRL/MpJ mouse cohorts contained more mature cells, proliferated at a higher rate and secreted less interleukin-10 (but also less pro-inflammatory cytokines) than cultures of CAST/EiJ mice. Compared with corresponding cultures of the control strain, LPS-free cultured cDCs from MRL/MpJ mice expressed less mRNA of the inhibitory receptor triggering receptor expressed on myeloid cells 2 (trem2). Conclusions: BM-derived cDCs from AIP-prone MRL/MpJ mice display functional features that are compatible with the hypothesis of an imbalanced DC activation in the context of murine AIP.

// Zhan Zhang 1 , Xinyue Wu 1 , Shuyuan Cao 1 , Li Wang 1 , Di Wang 1 , Hui Yang 1 , Yiming Feng 1 , Shoulin Wang 2 and Lei Li 1 1 Department of Hygiene Analysis and Detection, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, P. R. China 2 Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, P. R. China Correspondence to: Lei Li, email: // Keywords : ulcerative colitis, caffeic acid, dextran sulfate sodium, NF-κB pathway, fecal microbiota, Immunology and Microbiology Section, Immune response, Immunity Received : March 25, 2016 Accepted : May 05, 2016 Published : May 11, 2016 Abstract Emerging evidence shows that dietary agents and phytochemicals contribute to the prevention and treatment of ulcerative colitis (UC). We first reported the effects of dietary caffeic acid (CaA) on murine experimental colitis and on fecal microbiota. Colitis was induced in C57BL/6 mice by administration of 2.5% dextran sulfate sodium (DSS). Mice were fed a control diet or diet with CaA (1 mM). Our results showed that dietary CaA exerted anti-inflammatory effects in DSS colitis mice. Moreover, CaA could significantly suppress the secretion of IL-6, TNFα, and IFNγ and the colonic infiltration of CD3 + T cells, CD177 + neutrophils and F4/80 + macrophages via inhibition of the activation of NF-κB signaling pathway. Analysis of fecal microbiota showed that CaA could restore the reduction of richness and inhibit the increase of the ratio of Firmicute to Bacteroidetes in DSS colitis mice. And CaA could dramatically increase the proportion of the mucin-degrading bacterium Akkermansia in DSS colitis mice . Thus, CaA could ameliorate colonic pathology and inflammation in DSS colitis mice, and it might be associated with a proportional increase in Akkermansia .

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naive allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.

// Jun-Tang Li 1,2,3,* , Wei-Qi Wang 4,5,6,* , Ling Wang 7,* , Ning-Ning Liu 1,* , Ya-Li Zhao 1,* , Xiao-Shan Zhu 1 , Qin-Qin Liu 1 , Chun-Fang Gao 1 , An-Gang Yang 2 and Lin-Tao Jia 3 1 Centre of Inflammation and Cancer Research, 150th Central Hospital of PLA, Luoyang, Henan, China 2 State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi’an, Shaanxi, China 3 State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an, Shaanxi, China 4 State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 5 National Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 6 Shaanxi Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, China 7 Department of Anesthesiology, 150th Central Hospital of PLA, Luoyang, Henan, China * These authors have contributed equally to this work Correspondence to: Jun-Tang Li, email: // An-Gang Yang, email: // Lin-Tao Jia, email: // Keywords : isoflurane, zymosan, neutrophil, NMDA receptor, TLR2, Immunology and Microbiology Section, Immune response, Immunity Received : September 07, 2015 Accepted : April 18, 2016 Published : April 28, 2016 Abstract Neutrophil release of NO/ONOO – induces endothelial cell barrier dysfunction in inflammatory acute lung injury (ALI). Previous studies using zymosan-triggered inflammation and ALI model revealed that zymosan promotes inducible NO synthase (iNOS) expression in neutrophils, and that isoflurane inhibits zymosan-induced oxidative stress and iNOS biosynthesis. However, the underlying mechanisms remain largely unknown. We found here that in zymosan-primed neutrophils, iNOS is transcriptionally activated by NF-κB, whose nuclear translocation is triggered by excessive reactive oxygen species (ROS) and consequently activated p38 MAPK. ROS production is attributed to zymosan-initiated Toll-like receptor 2 (TLR2) signaling, in which the adaptor MyD88 recruits and activates c-Src, and c-Src activates NADPH oxidase to generate ROS. Subanesthetic isoflurane counteracts the aforementioned zymosan-induced signaling by targeting N-methyl-D-aspartic acid (NMDA) glutamate receptor and thereby suppressing calcium influx and c-Src activation. Whereas iNOS accelerates NO/ONOO – production in neutrophils which eventually promote protein leak from pulmonary microvascular endothelial cells (PMVEC), isoflurane reduced NO/ONOO – release from zymosan-treated neutrophils, and thus relieves trans-PMVEC protein leak. This study provides novel insights into the roles of neutrophils and the underlying mechanisms in zymosan-induced ALI, and has implications for the therapeutic potential of subanesthetic isoflurane in attenuating inflammatory responses causing lung endothelial cell damage.

// Hong Zhou 1,2 , Jie Zhang 1 , Fiona Eyers 2 , Yang Xiang 3 , Cristan Herbert 4 , Hock L. Tay 2 , Paul S. Foster 2,* and Ming Yang 2,* 1 Department of Respiratory Medicine, The Second Hospital, Jilin University, ChangChun, Jilin, People’s Republic of China 2 Priority Research Centre for Asthma and Respiratory Diseases, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW, Australia 3 Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China 4 Inflammation and Infection Research Centre, School of Medical Sciences, UNSW Australia, Sydney, Australia * These authors have contributed equally to this wok Correspondence to: Ming Yang, email: // Paul S. Foster, email: // Keywords : microRNA, macrophage, differentiation, transcriptional regulation, Immunology and Microbiology Section, Immune response, Immunity Received : December 23, 2015 Accepted : April 13, 2016 Published : April 22, 2016 Abstract Limited evidence is available about the specific miRNA networks that regulate differentiation of specific immune cells. In this study, we characterized miRNA expression and associated alterations in expression with putative mRNA targets that are critical during differentiation of macrophages. In an effort to map the dynamic changes in the bone marrow (BM), we profiled whole BM cultures during differentiation into macrophages. We identified 112 miRNAs with expression patterns that were differentially regulated 5-fold or more during BMDM development. With TargetScan and MeSH databases, we identified 1267 transcripts involved in 30 canonical pathways linked to macrophage biology as potentially regulated by these specific 112 miRNAs. Furthermore, by employing miRanda and Ingenuity Pathways Analysis (IPA) analysis systems, we identified 18 miRNAs that are temporally linked to the expression of CSF1R, CD36, MSR1 and SCARB1; 7 miRNAs linked to the regulation of the transcription factors RUNX1 and PU.1, and 14 miRNAs target the nuclear receptor PPARα and PPARγ. This novel information provides an important reference resource for further study of the functional links between miRNAs and their target mRNAs for the regulation of differentiation and function of macrophages.

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ T cells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.

// Anandi Narayana Moorthy 1 , Prashant Rai 1,2 , Huipeng Jiao 1 , Shi Wang 3 , Kong Bing Tan 3 , Liang Qin 4 , Hiroshi Watanabe 4 , Yongliang Zhang 1 , Narasaraju Teluguakula 5 and Vincent Tak Kwong Chow 1,2 1 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 2 Infectious Diseases Interdisciplinary Research Group, Singapore-Massachusetts Institute of Technology Alliance in Research and Technology, Singapore 3 Department of Pathology, National University Hospital, Singapore 4 Department of Infection Control and Prevention, Kurume University School of Medicine, Fukuoka, Japan 5 Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA Correspondence to: Vincent Tak Kwong Chow, email: // Keywords : neutrophil extracellular traps, Streptococcus pneumoniae, capsule, serotype, secondary pneumonia, Immunology and Microbiology Section, Immune response, Immunity Received : December 30, 2015 Accepted : March 18, 2016 Published : March 28, 2016 Abstract Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.