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Item PU.1 expression in T follicular helper cells limits CD40L-dependent germinal center B cell development.(American Association of Immunologists, 2015-10-15) Awe, Olufolakemi; Hufford, Matthew M.; Wu, Hao; Pham, Duy; Chang, Hua-Chen; Jabeen, Rukhsana; Dent, Alexander L.; Kaplan, Mark H.; Department of Microbiology and Immunology, IU School of MedicinePU.1 is an ETS family transcription factor important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development, and in limiting Th2 cytokine production. Whether PU.1 has functions in other T helper lineages is not clear. In this report we examined the effects of ectopic expression of PU.1 in CD4+T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1lck−/−) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1lck−/− mice had increased numbers of Tfh cells, increased germinal center B cells, and increased antibody production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1lck−/− mice, compared to control mice. Finally, although blockade of IL-21 did not affect germinal center B cells in Sfpi1lck−/− mice, anti-CD40L treatment of immunized Sfpi1lck−/− mice decreased germinal center B cell numbers and antigen-specific immunoglobulin concentrations. Together, these data indicate an inhibitory role of PU.1 in the function of T follicular helper cells, germinal centers, and Tfh-dependent humoral immunity.Item EFFECTS OF TYPE VI COLLAGEN ON MACROPHAGES(Office of the Vice Chancellor for Research, 2011-04-08) Voiles, Larry; Han, Ling; Lupov, Ivan P.; Anderson, Bailey; Melnikov, Lonya; Pottratz, Sarah Marie; Chang, Hua-ChenEmphysema is an abnormal inflammatory response of the alveoli that lose their elasticity due to destruction of alveolar septi. Collagen, an extracellular matrix protein (ECM), is expressed in the lung, which is important in maintaining the integrity of the tissue. Destruction of the ECM components in the alveolar structure contributes to the development of emphysema. We have found that the gene expression of type VI collagen (COL6A1) is higher in the lungs of emphysema patients as compared to that from normal controls. Type VI collagen (COL6) is found in the pulmonary interstitial compartment where massive macrophages are infiltrated in the inflammatory environment. The hypothesis is that excessive COL6 activates macrophages to mediate inflammatory responses, which may contribute to the pathogenesis of emphysema. The goal is to define the effects of type VI collagen on macrophages. Results from murine bone marrow derived macrophages showed a marked increase in the numbers of CD86-positive cells after soluble COL6 stimulation. To further support the stimulatory function of COL6, human THP-1 cells as well as primary monocytes produced inflammatory cytokines IL-12 and IFNγ following COL6 stimulation. Taken together, our data has demonstrated the stimulatory effects on macrophages by COL6 stimulation, which may mediate the inflammatory responses in the pathogenesis of emphysema.Item Lunasin alleviates allergic airway inflammation while increases antigen-specific Tregs(PLoS, 2015-02-03) Yang, Xiaowei; Zhu, Jingjing; Tung, Chun-Yu; Gardiner, Gail; Wang, Qun; Chang, Hua-Chen; Zhou, Baohua; Department of Pediatrics, IU School of MedicineLunasin is a naturally occurring peptide isolated from soybeans and has been explored in cancer treatment. Lunasin inhibits NF-κB activation and thus pro-inflammatory cytokine and mediator production in macrophages. In this study we demonstrate that lunasin can effectively suppress allergic airway inflammation in two murine models of asthma. In an OVA+Alum sensitization model, intranasal lunasin treatment at the time of OVA challenges significantly reduced total cells counts in bronchoalveolar lavage (BAL) fluid and eosinophilia, peribronchiolar inflammatory infiltration, goblet cell metaplasia and airway IL-4 production. In an OVA+LPS intranasal sensitization model, lunasin treatment either at the time of sensitization or challenge has similar effects in suppress allergic airway inflammation including significantly reduced total cell and eosinophil counts in BAL fluid, inflammatory gene Fizz1 expression in the lung, and IL-4 production by OVA re-stimulated cells from mediastinal lymph nodes. We further show that intranasal instillation of OVA+lunasin significantly increases OVA-specific regulatory T cell (Treg) accumulation in the lung comparing to OVA only treatment. Taken together, our results suggest lunasin as an anti-inflammatory agent can be potentially used in asthma therapy or as an adjuvant to enhance the induction of antigen-specific Tregs and thus boost the efficacy of allergy immunotherapy.Item Activation of Dendritic Cell Function by Soypeptide Lunasin as a Novel Vaccine Adjuvant(Office of the Vice Chancellor for Research, 2015-04-17) Simmons, Sharena J.; Tung, Chun-Yu; Chang, Hua-ChenThe innate immune system is the first line of defense against intruding pathogens. Dendritic cells (DCs) act as messengers between the adaptive and innate immune system, which process and present antigens to mature T helper cells (CD4+T), and Cytotoxic T cells (CD8+T). The addition of an appropriate adjuvant that activates the innate immunity is essential to subsequent development of the adaptive immunity specific to the vaccine antigens. Thus, any innovation capable of improving the immune responses may lead to a more efficacious vaccine. We recently identified a novel immune modulator using a naturally occurring seed peptide called lunasin. Lunasin was originally isolated from soybeans, and it is a small peptide containing 43 amino acids. Our studies had revealed stimulatory effects of lunasin on innate immune cells by regulating expression of a number of genes that are important for immune responses. The objective was to define the effectiveness of lunasin as an adjuvant that enhances immune responses. Immunization of mice with ovalbumin (OVA) and lunasin inhibited the growth of OVA-expressing A20 B-lymphomas, which was correlated with OVA-specific CD8+ T cells. Increased levels of anti-OVA IgG were also observed in mice immunized with OVA and lunasin. These results suggest that lunasin may function as a vaccine adjuvant by promoting DC maturation, which in turn enhances the development of protective immune responses to the vaccine antigens.Item Mechanisms of Gene Regulation by Soy Peptide Lunasin in Innate Immune Cells(Office of the Vice Chancellor for Research, 2015-04-17) Casiano-Rivera, Félix M.; Tung, Chun-Yu; Chang, Hua-ChenLunasin is a seed peptide containing 43 amino acids, originally isolated from soybeans. Recently, a novel function of lunasin was discovered, as it acts as an immune modulating agent regulating gene expression of various innate immune cells. Lunasin strongly activated the expression of genes encoding for type I interferon and inflammatory cytokines. Nonetheless, the molecular mechanisms of lunasin’s gene regulation are relatively unknown. Our current hypothesis states that lunasin is able to induce activation of various transcription factors, resulting in gene expression in immune cells. Human dendritic cells (DCs) or monocytes were purified from peripheral blood mononuclear cells (PBMCs) in order to determine the activation of transcription factors. Phosphorylation of STAT1 and NF-ĸB (p65) were evident in cells treated with lunasin using flow cytometry and Western blotting. The results will ultimately lead to the signaling pathways involved in gene expression regulated by lunasin in innate immune cells. By defining the signaling pathway of lunasin, we can have a better understanding of its application in immune modulation.Item TYPE VI COLLAGEN STIMULATES MACROPHAGES TO PROMOTE INFLAMMATION(Office of the Vice Chancellor for Research, 2012-04-13) Voiles, Larry; Han, Ling; Lupov, Ivan; Chang, Hua-ChenCollagen VI (COL6), an extracellular matrix protein (ECM), is important in maintaining the integrity of the tissue. Our recent findings have demonstrat-ed that excess COL6 is present in the lungs of comorbid patients with em-physema and adenocarcinoma. COL6 localized in the pulmonary interstitium is likely to interact with endothelial, epithelial and infiltrated pulmonary mac-rophages. The hypothesis is that excessive COL6 activates macrophages to promote inflammation, which may exacerbate pulmonary diseases. To test our hypothesis, bone marrow derived macrophages or macrophage cell lines were stimulated with soluble COL6 followed by analysis of activation markers and pro inflammatory cytokines. And results showed an increase in the number of CD86 positive cells and the levels of IL-12 and IFNγ production following stimulation. Taken together, our data have provided a link between increased amounts of COL6 and subsequent immune responses, which may play a role in the pathogenesis of pulmonary inflammatory diseases.Item ENHANCING THE TUMOR FIGHTING CAPACITY OF NK CELLS THROUGH THE USE OF SOYPEPTIDE(Office of the Vice Chancellor for Research, 2012-04-13) Lewis, David; Chang, Hua-Chen; Han, Ling; Voiles, Larry; Henriquez, Sarah M.P.Natural killer or (NK) cells are important components of the innate immune system, which play a major role in the rejection of tumors, and virally in-fected cells. By producing pro-inflammatory cytokines such as IFN-gamma, NK cells are able to exert immunoregulatory functions that influence the adaptive immunity of other immune cells. Due to its critical role in tumor inhibition, researchers, utilizing various cytokines, including IL-12 and IL-2, have fervently pursued the manipulation of NK activity. NK cells respond to cytokines in a dose-dependent manner; however, the toxicity of certain cy-tokines (like IL-2) in high doses prohibits their widespread clinical use. Therefore, efforts to activate NK cells without requiring high doses of cyto-kines is warranted. We recently exploited a soy derived dietary peptide called lunasin to improve the immune functions. The hypothesis was that the lunasin peptide has stimulatory effects on immune cells. To test this hy-pothesis, human peripheral blood mononuclear cells (PBMCs) of healthy do-nors were stimulated with and without lunasin in combination with cytokines IL-12 or IL-2. Our results showed that the lunasin peptide exerts a robust synergistic effect when combined with the selected cytokines. This effect ap-pears to regulate the expression of a number of genes that are important for NK activity. Our findings support the potential clinical use of lunasin in com-bination with cytokine to enhance the tumor fighting capacity of NK cells.Item Enhancement of Cancer Immunotherapy Using Immune Modulating Peptides(Office of the Vice Chancellor for Research, 2013-04-05) Chang, Hua-Chen; Han, Ling; Lewis, David; Tung, Chun-Yu; Srinivasan, Mythily; Robertson, Michael J.; Yeh, Wu-KuangImmune Peptide Therapeutics (IPT) LLC, an Indiana-based small business and its research partner Indiana University previously identified a novel property of lunasin as a distinct class of immune modulating agent that enhances anti-tumor immunity, which may promote disease-free survival by limiting tumor progression, and thus prolong lives of cancer patients. Lunasin, a synthetic 43-amino acid peptide, was originally isolated from soybeans. Our studies have demonstrated that lunasin exerts robust synergistic effects with cytokines on augmenting IFNγ and granzyme B expression by Natural Killer (NK) cells, which is associated with increased tumoricidal activity of NK cells. In addition, this combination regimen is capable of rescuing IFNγ production ex vivo by NK cells from chemotherapy-treated Non-Hodgkin’s Lymphoma (NHL) patients who are immunocompromised with acquired immune deficiency. The long-term goal is to develop an efficacious immunotherapy which will impact the treatment and improve the clinical outcomes for NHL patients. The dose-response study indicates the optimum concentration of lunasin is at the range of μM, which would undermine its use in clinical studies. To enhance the medicinal value lunasin must be optimized for in vitro and in vivo efficacy. The objective is to develop a second generation of lunasin, which will increase its potency to improve the performance. In this study we have implemented several strategies to design and modify the prototype. The newly developed peptide called IPT.103 has 15 amino acids that are in the D-isoform configuration. Activity of IPT.103 has been tested in vitro with EC50 of 0.78 μM as compared to 4.54 μM for lunasin. IPT.103 also has in vivo activity on enhancing the serum levels of IFNγ production using a mouse model. Taken together, we have developed a peptide derivative (IPT.103) that deviates from its parental type lunasin to increase intellectual merit for commercialization as well as support clinical application.Item Overexpression of type VI collagen in neoplastic lung tissues(Spandidos, 2014-08) Voiles, Larry; Lewis, David E.; Han, Ling; Lupov, Ivan P.; Lin, Tsang-Long; Robertson, Michael J.; Petrache, Irina; Chang, Hua-Chen; Department of Biology, IU School of ScienceType VI collagen (COL6), an extracellular matrix protein, is important in maintaining the integrity of lung tissue. An increase in COL6 mRNA and protein deposition was found in the lungs of patients with pulmonary fibrosis, a chronic inflammatory condition with a strong association with lung cancer. In the present study, we demonstrated overexpression of COL6 in the lungs of non-small cell lung cancers. We hypothesized that excessive COL6 in the lung interstitium may exert stimulatory effects on the adjacent cells. In vitro stimulation of monocytes with COL6 resulted in the production of IL-23, which may promote tumor development in an environment of IL-23-mediated lung inflammation, where tissue modeling occurs concurrently with excessive COL6 production. In addition, COL6 was capable of stimulating signaling pathways that activate focal adhesion kinase and extracellular signal‑regulated kinase 1/2 in lung epithelial cells, which may also facilitate the development of lung neoplasms. Taken together, our data suggest the potential role of COL6 in promoting lung neoplasia in diseased lungs where COL6 is overexpressed.Item Effects of Soy Peptide on Dendritic Cells(Office of the Vice Chancellor for Research, 2013-04-05) Shipman, Kaylee; Tung, Chun-Yu; Han, Ling; Patel, Amy; Corn, Caleb; Chang, Hua-ChenInnate immunity is mediated by effector cells, including NK cells, dendritic cells (DCs), macrophages, and polymorphonuclear phagocytes, which can respond immediately after activation through receptors encoded by germ-line genes. Innate immune responses represent the first line of defense in immunosurveillance. Interventions that enhance the functions of innate immunity will be an important armamentarium to human health. We recently exploited a natural dietary soy peptide called lunasin to improve the immune functions. The hypothesis was that lunasin peptide has stimulatory effects on immune cells. To test this hypothesis, human peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated with or without lunasin. We found that lunasin is capable of stimulating DCs to up-regulate chemokines (CCL2, CCL3, and CCL4), cytokines (TNFα and IFNα), and co-stimulatory molecules (CD80, CD86). In addition, lunasin-treated DCs can provide NK with required signals for activation. Taken together, our results support the immunomodulatory activity of soy peptide on DCs, which leads to enhancement of innate immunity.