Expression Of Th2-type Cytokines Research Articles

256-LB: An OGT–TFF2 Axis in the Pathogenesis of Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is a global metabolic disease that comprises a broad spectrum of liver dysfunction ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) with fibrosis. It is largely unknown how the communication between hepatocytes and non-parenchymal cells dictates the disease progression from NAFL to NASH and fibrosis. Dysregulation of protein O-GlcNAcylation in hepatocytes has been implicated in a wide range of liver diseases. In this study, we demonstrate the role of O‑GlcNAc transferase (OGT) in mediating intercellular crosstalk between hepatocytes and immune cells in NASH-fibrosis development. RNA-sequencing analysis of wildtype and hepatocyte-specific OGT knockout mice was performed and trefoil factor 2 (TFF2) was identified as one of the highly induced genes encoding secreted proteins in OGT-deficient hepatocytes. Immunohistochemistry and western blot were performed to reveal decreased OGT and increased TFF2 expression in the liver of NAFLD patients. Mechanically, OGT was found to repress TFF2 transcription in hepatocytes by modifying forkhead box protein A2 (FOXA2). Recombinant TFF2 along with NASH stimuli was applied to a 3D spheroid culture system comprised of hepatocytes and non-parenchymal cells and TFF2 was shown to promote NASH stimuli-induced triglyceride secretion and elevate the expression of pro-inflammatory and fibrogenic genes. ELISA and RT-qPCR were performed to further show that TFF2 treatment induced the expression of Th1-type cytokines (IFN-γ and TNF-α) and Th17-type cytokine (IL17) in CD4 T cells, promoting Th1 and Th17 differentiation. Collectively, these results identify an OGT-TFF2 axis that mediates the crosstalk between hepatocytes and CD4 T cells during NASH pathogenesis, revealing a potential therapeutic target for the treatment of chronic liver disease. Disclosure L. Zhang: None. Q. Wang: None. R. Desrouleaux: None. X. Yang: None. Funding American Diabetes Association (1-19-IBS-119 to X.Y.); National Institutes of Health (R01DK089098, P30DK34989)

Cover story: Eur. J. Immunol. 7'22

European Journal of ImmunologyVolume 52, Issue 7 Cover PictureFree Access Cover story: Eur. J. Immunol. 7'22 First published: 11 July 2022 https://doi.org/10.1002/eji.202270070AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Our cover features H&E staining of colon sections from RAG-1−/− mice after adoptive transfer of regulatory T cell-depleted CD4 T cells purified from Shn-3 deficient mice. Shn3 is a transcription factor expressed by activated T cells and supports the expression of Th2-type cytokines. In this autoimmune colitis model, Shn3-deficient T cells caused enhanced Th1 responses and exacerbated inflammation, and accelerated weight loss in the recipient mice. The image is taken from Cunha et al. (pp. 1077–1094), where the authors present the Th1-Th2 differentiation is controlled by Shn3 which is induced during a late phase of antigen stimulation and promotes GATA3 expression. Volume52, Issue7July 2022 RelatedInformation

Sarcodia suiae Water Extract Promotes the Expression of Proinflammatory and Th1-Type Cytokines and Delay the Onset of Mortality in Cobia (Rachycentron canadum) During Photobacterium damselae subsp. damselae Infection.

Cobia (Rachycentron canadum) is a marine fish of high economic value that grows at a fast rate. However, intensive fish farming has led to disease outbreaks in cobia cultures, which is highly costly to the industry. The impact of infectious diseases on cobia production has led to the inappropriate and increased use of chemicals and antibiotics, which negatively affects the environment and human health and promotes the spread of drug-resistant pathogens. Hence, prophylactic measurements, such as the use of immunomodulators, are required to improve the health of cultured animals against pathogens. In this study, we examined the effects of Sarcodia suiae water extract (SSWE) in cobia in vitro and in vivo. We found that treatment with SSWE could significantly increase the expression of cytokines (e.g., IL-1β, IL-6, IL-10, IL-12, and TNF-α) and chemokines (e.g., IL-8) in primary cultured head kidney leukocytes. Intraperitoneal injection of SSWE (20 μg/g body weight) promoted higher expression of IL-6, IL-8, IL-10, IL-12, chemokines (e.g., CC1), and antibodies (e.g., IgT) in head kidney and spleen tissues of the fish compared with other dose levels. Additionally, we describe for the second time (only after India) of the isolation of Photobacterium damselae subsp. damselae (Phdd) from a deadly epizootic in cage-farmed cobia. An intraperitoneal inoculation of SSWE before Phdd challenge showed that SSWE treatment could delay the onset of mortality of cobia. Finally, fish that received SSWE intraperitoneally before infection with Phdd exhibited elevated expression of Th1-type cytokines, namely, IL-8, IL-12, TNF-α, and IFN-γ. At the same time, the expression of Th2-related factors (such as IL-10 in the head kidney, and IgM and IgT in the spleen) were lower for the fish that received SSWE instead of PBS before the Phdd challenge. The results indicate that SSWE treatment facilitates the induction of Th1-type cytokines in cobia to fight against Phdd infection and has the potential to be used as an immunostimulant and vaccine adjuvant for fish.

Oleanolic Acid Alleviates Atopic Dermatitis-like Responses In Vivo and In Vitro.

Oleanolic acid (OA) is a pentacyclic triterpenoid, abundantly found in plants of the Oleaceae family, and is well known for its beneficial pharmacological activities. Previously, we reported the inhibitory effect of OA on mast cell-mediated allergic inflammation. In this study, we investigated the effects of OA on atopic dermatitis (AD)-like skin lesions and its underlying mechanism of action. We evaluated the inhibitory effect of OA on AD-like responses and the possible mechanisms using a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD animal model and tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated HaCaT keratinocytes. We found that OA has anti-atopic effects, including histological alterations, on DNCB-induced AD-like lesions in mice. Moreover, it suppressed the expression of Th2 type cytokines and chemokines in the AD mouse model and TNF-α/IFN-γ-induced HaCaT keratinocytes by blocking the activation of serine-threonine kinase Akt, nuclear factor-κB, and the signal transducer and activator of transcription 1. The results demonstrate that OA inhibits AD-like symptoms and regulates the inflammatory mediators; therefore, it may be used as an effective and attractive therapeutic agent for allergic disorders, such as AD. Moreover, the findings of this study provide novel insights into the potential pharmacological targets of OA for treating AD.

Small intestinal immune-environmental changes induced by oral tolerance inhibit experimental atopic dermatitis

Atopic dermatitis is a chronic skin inflammatory disease mediated by Th2-type immune responses. Although intestinal immune responses have been shown to play a critical role in the development or prevention of atopic dermatitis, the precise influence of intestinal immunity on atopic dermatitis is incompletely understood. We show here that orally tolerized mice are protected from experimental atopic dermatitis induced by sensitization and epicutaneous (EC) challenge to ovalbumin. Although the expression of Th2-type cytokines in the small intestine of orally tolerized and EC-challenged mice did not change significantly, these mice showed decreased inflammatory responses in the small intestine with restoration of microbial change elicited by the EC challenge. Interestingly, an increase in small intestinal eosinophils was observed with the EC challenge, which was also inhibited by oral tolerance. The role of small intestinal eosinophils and microbiota in the pathogenesis of experimental atopic dermatitis was further substantiated by decreased inflammatory mediators in the small intestine and attenuated Th2-type inflammation in the skin of eosinophil-deficient and microbiota-ablated mice with EC challenges. Based on these data, we propose that the bidirectional interaction between the skin and the intestine has a role in the pathogenesis of atopic dermatitis and that modulation of the intestinal microenvironments could be a therapeutic approach to atopic dermatitis.

Probiotic Lactobacillus rhamnosus GG Promotes Mouse Gut Microbiota Diversity and T Cell Differentiation.

Lactic acid bacteria (LAB) are the primary genera of the intestinal flora and have many probiotic functions. In the present study, Lactobacillus rhamnosus GG (LGG) ATCC 53103 was used to treat BALB/c mice. After LGG intervention, both low and high LGG doses were shown to improve the observed OTU, Chao1, ACE, and Shannon indices, while the Simpson index decreased, demonstrating that LGG can promote intestinal microbiota abundance and diversity. Furthermore, LGG treatment increased the abundances of intestinal Firmicutes, Bacteroides and Actinomycetes while reducing that of Proteobacteria. In addition to its effect on gut the microbiota, LGG could also regulate the host immune system. In the present study, we showed that LGG could affect the percentage of CD3+ T lymphocytes in the spleens (SPLs), mesenteric lymph nodes (MLNs), Peyer’s patches (PPs) and lamina propria lymphocytes (LPLs) of mice, including total CD3+ T, CD3+CD4+ T, and CD3+CD8+ T lymphocytes. Furthermore, LGG could effectively increase the expression of Th1-type cytokines (IFN-γ) and Th2 cytokines (IL-4) in CD4+ T cells, indicating that the proportion of Th1 and Th2 cells in mice with LGG treatment was in a high equilibrium state compared to the control group. In addition, the IFN-γ/IL-4 ratio was greater than 1 in mice with LGG intervention, suggesting that LGG tends to mediate the Th1 immune response. The results of the present study also showed that LGG upregulated the expression of IL-17 in CD4+ T cells and regulated the percentage of CD4+CD25+Foxp3+ Treg cells in various secondary immunological organs, indicating that LGG may promote the balance of Th-17 and Treg cells.

Lactobacillus johnsonii-activated chicken bone marrow-derived dendritic cells exhibit maturation and increased expression of cytokines and chemokines in vitro

Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1β and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.

Blocking the Notch signal transduction pathway promotes tumor growth in osteosarcoma by affecting polarization of TAM to M2 phenotype.

BackgroundOsteosarcoma is a primary malignant tumor that seriously affects the health and life of patients. It is of great clinical significance to explore the molecular mechanism of osteosarcoma development and develop the corresponding therapeutic targets. Th1/Th2 cytokines in the normal human body are in a state of dynamic balance. When this balance is destroyed, it is related to many diseases such as a tumor, autoimmune disease, microbial infection, transplant rejection, among many others.MethodThe model of mouse tumor-associated macrophage (TAM) was induced by being co-cultured with inducer granulocyte-macrophage colony stimulating factor (GM-CSF) and osteosarcoma S180 cells. The Notch1 knockout mice were obtained by gene targeting technology. The distribution of M1- and M2-type TAMs in the tumor was visualized by immunofluorescence staining. And the western-blot testing was used to detect and quantified the protein level of Notch1 and Th1/Th2-type cytokines.ResultsIn this study, the polarization of TAMs to the M2 phenotype occurred after coculture with osteosarcoma S180 cells and secretion level Th1/Th2-type cytokines changed. Also, the expression level of Notch1 reduced significantly. Further, the critical transcription factor Notch1 of the Notch signaling pathway was knocked out in mice. The tumor volume of Notch1 knockout mice was significantly more extensive than of the control mice. The results of microstructural observation on tumor showed that M2-type TAMs infiltrated into tumor increased with increased expression of Th2-type cytokines, but M1-type TAMs reduced with reduced expression of Th1-type cytokines.ConclusionsAccording to our results, the Notch signal transduction pathway participates in tumor occurrence and growth with a negative role by maintaining Th1/Th2 balance.

Eosinophil Activation by Toll-Like Receptor 4 Ligands Regulates Macrophage Polarization.

Eosinophils are terminally differentiated granulocytes that have long been considered as destructive cells associated with Th2 type immune responses such as allergic inflammation and helminth infections. Recently, eosinophils have been actively studied as multifunctional leukocytes regulating an array of physiological responses through interaction with other immune cells. In this study, we examined the expression and function of Toll-like receptors (TLRs) in eosinophilic EoL-1 cells and demonstrated the expression of a number of immune mediators in activated EoL-1 cells and their interaction with the macrophage cell line THP-1 upon TLR4 ligand stimulation. EoL-1 cells differentiated with butyrate increased expression of TLR3, TLR4, and TLR7 at mRNA and protein level with flow cytometry analysis. Mature eosinophils derived from human cord blood CD34+ cells were subjected to RNA-sequencing, and showed the expression of a panel of TLR transcripts and TLR4 was the most highly expressed TLR. Among the cognate ligands of TLR3, TLR4, and TLR7, lipopolysaccharide (LPS) or palmitic acid significantly increased mRNA expression of immune mediators in differentiated EoL-1 cells. Notably, Western blot analysis of palmitic acid-treated differentiated EoL-1 cells showed significantly up-regulated expression of Th2 type cytokines and transcription factors driving eosinophil differentiation. To evaluate functional significance of TLR4 ligand-stimulated eosinophils, we added conditioned media (CM) from EoL-1 cells to differentiated THP-1 cells and assessed the expression of M1 macrophage or M2 macrophage-related markers. M1 and M2 macrophage markers were significantly upregulated by CM from LPS and palmitic acid stimulated EoL-1 cells, respectively. In addition, the adipose tissue of obese mice, where eosinophils are decreased due to obesity-induced inflammation, showed significantly decreased frequency of M2 macrophages, despite an increase in the total macrophage numbers. Based on these collective data, we proposed that eosinophils regulate both inflammatory and anti-inflammatory polarization of macrophages through functional changes induced by different TLR4 ligands.

Combined blockade of IL-25, IL-33 and TSLP mediates amplified inhibition of airway inflammation and remodelling in a murine model of asthma.

Isolated blockade of IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) has been shown to reduce airways inflammation and hyperresponsiveness in murine asthma model. The hypothesis that combined blockade of all three cytokines can accomplish this more effectively has never been addressed. We studied a murine asthma model employing sensitization and challenge with ovalbumin (OVA) or saline control. To discern the effects of IL-33 blockade, we compared outcomes in strain identical, wild-type and IL-33 receptor (St2 -/- ) gene-deleted mice. We then examined, in the St2 -/- animals, the effects of additional, single or combined blockade of IL-25 and TSLP with blocking antibodies. Outcomes included airways reactivity, inflammatory cellular infiltration, epithelial cell metaplasia, deposition of fibrosis-related proteins, local Th2-type cytokine expression and total and specific serum IgE concentrations measured by ELISA and quantitative immunohistochemistry. St2 -/- gene deletion significantly reduced airways reactivity, inflammatory cellular infiltration, lung tissue expression of Th2 cytokines and fibrosis related proteins and serum total IgE in response to OVA sensitization and challenge. Additional administration of anti-IL-25 and anti-TSLP blocking antibodies to the St2 -/- mice further significantly reduced inflammation, Th2 cytokine expression, airways fibrosis and IgE production, while anti-TSLP alone reduced eosinophil infiltration and local IL-4 expression. The airways inflammatory cellular infiltrate and lung tissue expression of Th2 cytokine, but not fibrosis-related proteins were also reduced in the presence of isotype identical, control antibodies. Combined blockade of these three cytokines may better ameliorate airways pathological changes in this murine asthma model, with implications for human asthma.

Bronchial Epithelial Cells Promote the Differentiation of Th2 Lymphocytes in Airway Microenvironment through Jagged/Notch-1 Signaling after RSV Infection

Background: The aim of this study was to investigate the role of Notch-1 signaling through Notch-1 ligands on bronchial epithelial cells (BECs) in regulating the development of T helper 2 (Th2) lymphocytes after RSV infection. Methods: Firstly, we analyzed the expression of cytokines and Notch-1 ligands in BECs by using real-time PCR. Then, RSV-infected BECs were co-cultured with CD4⁺ T cells in a transwell chamber for 48 h, and differentiation of T cells in the lower chamber was determined using flow cytometry and real-time PCR. JAG1 siRNA was then used to determine the effects of Jagged/Notch-1 signaling on the differentiation of Th2. An RSV-infected mouse model was also used to analyze the secretion of Th differentiation-associated cytokines in serum and lung tissues using ELISA, the histopathological changes using HE staining, and the expression of JAG1 and JAG2 in BECs. Results: The results showed that RSV promoted the expression of Th2-type cytokines and Jagged-1 and inhibited the expression of Jagged-2 in normal BECs. RSV-infected BECs induced Th2 differentiation. In addition, JAG1 downregulation inhibited the differentiation of Th2 and promoted differentiation of Th1. In the RSV-infected mouse model, the RSV titer, inflammation decreased with time. IL-4 and IL-17 increased on day 28 and 60, while IFNγ increased on day 7 and 28. Moreover, the expression of Jagged-1 increased and that of Jagged-2 decreased in BECs, which was consistent with IL-4 production in lung tissues. Conclusion: Our data showed that BECs had the potential to promote the differentiation of Th2 lymphocytes through Jagged-1/Notch-1 signaling.

Heat stress, Eimeria spp. and C. perfringens infections alone or in combination modify gut Th1/Th2 cytokine balance and avian necrotic enteritis pathogenesis

Information on the dynamics of the chicken immune system during bacterial or parasite challenge in the presence or absence of stressful situations may provide a better understanding of the complex mechanisms behind these diseases. Necrotic enteritis (NE) had been controlled previously by the proper use of antimicrobial agents; however, more recently, NE has reemerged in many countries. The imposed restrictions on antimicrobial use and/or the intensive productive programs implemented by producers are challenges the birds, leading to large host adaptive responses that in many instances are like those elicited by stressors. This study analyses the effects of heat stress on Th1/Th2 cytokine balance, pathological features, and Toll-like receptor expression in the small intestine of broiler chickens infected with Clostridium perfringens type A in the presence or absence of Eimeria spp. co-infection. This co-infection model was experimentally used because it reproduces the findings commonly observed in the field during avian NE. For this purpose, broiler chickens infected with C. perfringens and/or Eimeria spp. were reared in isolator chambers subjected or not to heat stress intermittently. It was observed that heat stress directs the expression of Th2-type cytokines, increases Toll-like receptor 4 expression in the intestine and reduces the disease severity induced by Eimeria spp. and C. perfringens infections alone or in combination, most likely as a consequence of stress-induced changes in brain-gut axis activity.

IL-1\u03b2 mediates lung neutrophilia and IL-33 expression in a mouse model of viral-induced asthma exacerbation

BackgroundViral-induced asthma exacerbations, which exhibit both Th1-type neutrophilia and Th2-type inflammation, associate with secretion of Interleukin (IL)-1β. IL-1β induces neutrophilic inflammation. It may also increase Th2-type cytokine expression. We hypothesised that IL-1β is causally involved in both Th1 and Th2 features of asthma exacerbations. This hypothesis is tested in our mouse model of viral stimulus-induced asthma exacerbation.MethodWild-type (WT) and IL-1β deficient (IL-1β−/−) mice received house dust mite (HDM) or saline intranasally during three weeks followed by intranasal dsRNA (PolyI:C molecule known for its rhinovirus infection mimic) for three consecutive days to provoke exacerbation. Bronchoalveolar lavage fluid was analysed for inflammatory cells and total protein. Lung tissues were stained for neutrophilic inflammation and IL-33. Tissue homogenates were analysed for mRNA expression of Muc5ac, CXCL1/KC, TNF-α, CCL5, IL-25, TSLP, IL-33, IL-1β, CCL11 and CCL2 using RT-qPCR.ResultsExpression of IL-1β, neutrophil chemoattractants, CXCL1 and CCL5, the Th2-upstream cytokine IL-33, and Muc5ac were induced at exacerbation in WT mice and were significantly inhibited in IL-1β−/− mice at exacerbation. Effects of HDM alone were not reduced in IL-1β-deficient mice.ConclusionWithout being involved in the baseline HDM-induced allergic asthma, IL-1β signalling was required to induce neutrophil chemotactic factors, IL-33, and Muc5ac expression at viral stimulus-induced exacerbation. We suggest that IL-1β has a role both in neutrophilic and Th2 inflammation at viral-induced asthma exacerbations.

Study on the Inhibitory Effects of Ephedra Aconite Asarum Decoction on LPS-Induced Dendritic Cells.

Dendritic cells (DCs) can secrete cytokines stimulated by lipopolysaccharide (LPS), which leads to not just acute inflammatory responses but also Th1 polarization. Furtherly, chronic inflammation or autoimmune diseases could be triggered. As a classic Traditional Chinese Medicine formula, Ephedra Aconite Asarum Decoction with the main ingredients of ephedrine and hypaconitine can show effect on anti-inflammation and immunoregulation. But it remains unclear whether Ephedra Aconite Asarum Decoction controls DCs. In this study, we investigated the effects of Ephedra Aconite Asarum Decoction on LPS-induced bone marrow-derived DCs (BMDCs) in vitro. We found that Ephedra Aconite Asarum Decoction lowered surface costimulators on DCs and reduced the expression of Th1 type cytokines. Yet it is slightly beneficial for shifting to Th2. Our work reveals that the Ephedra Aconite Asarum Decoction can regulate Th1 inflammation through intervening DCs.

In vitro Stimulation of NK Cells and Lymphocytes Using an Extract Prepared from Mycelial Culture of Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and used in traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild Ophiocordyceps is becoming increasingly rare in its natural habitat, and its price limits its use in clinical practice. Therefore, the development of a standardized alternative is a great focus of research to allow the use of Ophiocordyceps as a medicine. To develop an alternative for wild Ophiocordyceps, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vitro immune-modulating effect of CBG-CS-2 on natural killer cells and B and T lymphocytes. CBG-CS-2 stimulated splenocyte proliferation and enhanced Th1-type cytokine expression in the mouse splenocytes. Importantly, in vitro CBG-CS-2 treatment enhanced the killing activity of the NK-92MI natural killer cell line. These results indicate that the mycelial culture extract prepared from Ophiocordyceps exhibits immune-modulating activity, as was observed in vivo and this suggests its possible use in the treatment of diseases caused by abnormal immune function.

Susceptibility and resistance to Echinococcus granulosus infection: Associations between mouse strains and early peritoneal immune responses

In helminth infections, there are no easy associations between host susceptibility and immune responses. Interestingly, immunity to cestodes – unlike most helminths – seems to require Th1-type effectors. In this sense, we reported recently that Balb/c and C57Bl/6 mice are high and low susceptible strains, respectively, to experimental infection by Echinococcus granulosus. However, the role of the early cellular peritoneal response in such differential susceptibility is unknown. Here, we analyzed the kinetics of cytokines expression and cellular phenotypes in peritoneal cells from infected Balb/c and C57Bl/6 mice. Additionally, Principal Components Analysis (PCA) were conducted to highlight the most relevant differences between strains. Finally, the anti-parasite activities of peritoneal cells were assessed through in vitro systems. PCAs clustered C57Bl/6 mice by their early mixed IL-5/TNF-α responses and less intense expression of Th2-type cytokines. Moreover, they exhibited lower counts of eosinophils and higher numbers of macrophages and B cells. Functional studies showed that peritoneal cells from infected C57Bl/6 mice displayed greater anti-parasite activities, in accordance with higher rates of NO production and more efficient ADCC responses. In conclusion, mild Th2-responses and active cellular mechanisms are key determinants in murine resistance to E. granulosus infection, supporting the cestode immune exception among helminth parasites.

Immune-Modulating Activity of Extract Prepared from Mycelial Culture of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild O. sinensis is becoming increasingly rare in its natural habitats, and its price is out of reach for clinical practice. For these reasons, development of a standardized alternative is a great focus of research to allow the use of O. sinensis as a medicine. To develop an alternative for wild O. sinensis, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vivo immune-modulating effect of CBG-CS-2 in mice. Oral administration of CBG-CS-2 supported splenocyte stimulation and enhanced Th1-type cytokine expression from the splenocytes. Importantly, the same treatment significantly enhanced the natural killer cell activity of the splenocytes. Finally, oral administration of CBG-CS-2 enhanced the potential for inflammatory responses. Together, these findings indicate that the mycelial culture extract prepared from O. sinensis exhibited immune-modulating activity and suggest its possible use in the treatment of diseases caused by abnormal immune function.

C-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.

Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

BackgroundCtip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice.Methodology/Principal FindingsTo study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes.Conclusions/SignificanceOur data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2.

Abstract 527: The effects of pemetrexed on innate and adaptive immune cells in subjects with adenocarcinoma of the pancreas

Abstract Fifteen patients with previously surgically resected, or newly diagnosed, non-resectable metastatic adenocarcinoma of the pancreas were consented to an IRB approved study of immune cell functions in subjects treated with pemetrexed followed by gemcitabine. All subjects had a health performance status of 0-1 and no subject had received previous chemotherapy or radiotherapy. Dosages were pemetrexed at 500 mg/m2 followed two weeks later with a second dose of pemetrexed and gemcitabine at 1250 mg/m2. Blood draws were at 5 time-points to investigate baseline levels of innate and adaptive immune cell functions and the effects of pemetrexed on those responses as measured 7 and 14 days after initial therapy as well as the effects of a combination therapy of pemetrexed with gemcitabine. Our study revealed a strong positive correlation between pre-therapy absolute numbers of NK cells and overall survival (p=0.039). Cytolytic units of NK activity positively correlated with numbers of NK cells (p=0.008). Therapy with pemetrexed decreased killing activity with cytolytic units decreasing with each blood draw and reaching significance with combination therapy with gemcitabine (p= 0.02). Expression of Th1-type cytokines were induced and intracellular accumulation measured by flow cytometry. Pemetrexed induced a substantial increase in NK (CD56+) cells producing IFNγ. The increased IFNγ production detected 7 days after initial pemetrexed therapy however had a significant negative correlation with survival (p=0.038) suggesting that NK cell production of IFNγ had a deleterious effect on patient survival. A similar significant negative correlation was obtained when either the combination of anti-CD3, anti-CD28, or Concanavalin A was used to induce cytokine production. Addition of gemcitabine to the therapeutic regimen decreased numbers of cell producing IFNγ to pre-therapy baseline levels. Memory (CD45RO+) T-cells enumerated at baseline also revealed a significant negative correlation with survival (p=0.011) but were decreased by pemetrexed therapy alone or in combination with gemcitabine. FoxP3+, CD8+ T-cells were detected and demonstrated a strong positive correlation with both progression-free interval (p=0.005) and with survival (p=0.026) suggesting that FoxP3+, CD8+ T-cells are beneficial to pancreatic cancer patients. While pemetrexed therapy resulted in a negative correlation with survival by increasing activation of a subset of NK cells to produce IFNγ, addition of gemcitabine abated those responses, decreased IFNγ production thereby resulting in a significant positive correlation between NK cells producing IL-2 and survival (p=0.041). Innate immunity thus is an important defense against pancreatic cancer. In order to eliminate a negative effect of NK cell induction of IFNγ however, pemetrexed should follow rather than precede gemcitabine in initial therapy for pancreatic cancer Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 527. doi:1538-7445.AM2012-527