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1.  TLR-2 AND IL-17A IN CHITIN-INDUCED MACROPHAGE ACTIVATION AND ACUTE INFLAMMATION 
Chitin, is a ubiquitous polysaccharide in fungi, insects and parasites. To test the hypothesis that chitin is an important immune modulator, we characterized the ability of chitin fragments to regulate murine macrophage cytokine production in vitro and induce acute inflammation in vivo. Here we show that chitin is a size-dependent stimulator of macrophage interleukin (IL)-17A production and IL-17A receptor (R) expression and demonstrate that these responses are Toll-like Receptor (TLR)-2 and MyD88-dependent. We further demonstrate that IL-17A pathway activation is an essential event in the stimulation of some but not all chitin-stimulated cytokines and that chitin utilizes a TLR-2, MyD88- and IL-17A-dependent mechanism(s) to induce acute inflammation. These studies demonstrate that chitin is a size-dependent pathogen-associated molecular pattern (PAMP) that activates TLR-2 and MyD88 in a novel IL-17A / IL-17AR-based innate immunity pathway.
PMCID: PMC2577310  PMID: 18768886
Monocytes/Macrophages; Cytokines; Inflammation; Lung; Rodent
2.  Chitin Regulation of Immune Responses: An Old Molecule With New Roles 
Current opinion in immunology  2008;20(6):684-689.
Chitin, the second most abundant polysaccharide in nature, is commonly found in lower organisms such as fungi, crustaceans and insects, but not in mammals. Although the non-specific anti-viral and anti-tumor activities of chitin/chitin derivatives were described two decades ago, the immunological effects of chitin have been only recently been addressed. Recent studies demonstrated that chitin has complex and size-dependent effects on innate and adaptive immune responses including the ability to recruit and activate innate immune cells and induce cytokine and chemokine production via a variety of cell surface receptors including macrophage mannose receptor, toll-like receptor 2 (TLR-2), and Dectin-1. They also demonstrated adjuvant effects of chitin in allergen-induced Type 1 or Type 2 inflammation and provided insights into the important roles of chitinases and chitinase-like proteins (C/CLP) in pulmonary inflammation. The status of the field and areas of controversy are highlighted.
doi:10.1016/j.coi.2008.10.002
PMCID: PMC2605627  PMID: 18938241
chitin; chitinases; chitinase-like protein; innate and adaptive immunity
3.  Chitin, Chitinases and Chitinase-like Proteins in Allergic Inflammation and Tissue Remodeling 
Yonsei Medical Journal  2009;50(1):22-30.
Chitin, the second most abundant polysaccharide in nature after cellulose, consist exoskeleton of lower organisms such as fungi, crustaceans and insects except mammals. Recently, several studies evaluated immunologic effects of chitin in vivo and in vitro and revealed new aspects of chitin regulation of innate and adaptive immune responses. It has been shown that exogenous chitin activates macrophages and other innate immune cells and also modulates adaptive type 2 allergic inflammation. These studies further demonstrate that chitin stimulate macrophages by interacting with different cell surface receptors such as macrophage mannose receptor, toll-like receptor 2 (TLR-2), C-type lectin receptor Dectin-1, and leukotriene B4 recepptor (BLT1). On the other hand, a number of chitinase or chitinase-like proteins (C/CLP) are ubiquitously expressed in the airways and intestinal tracts from insects to mammals. In general, these chitinase family proteins confer protective functions to the host against exogenous chitin-containing pathogens. However, substantial body of recent studies also set light on new roles of C/CLP in the development and progression of allergic inflammation and tissue remodeling. In this review, recent findings on the role of chitin and C/CLP in allergic inflammation and tissue remodeling will be highlighted and controversial and unsolved issues in this field of studies will be discussed.
doi:10.3349/ymj.2009.50.1.22
PMCID: PMC2649864  PMID: 19259344
Chitin; chitinases; chitinase-like proteins; immunity; remodeling
4.  Ginsenoside Rg1 and Aluminum Hydroxide Synergistically Promote Immune Responses to Ovalbumin in BALB/c Mice▿  
The combined adjuvant effect of ginsenoside Rg1 and aluminum hydroxide (alum) on immune responses to ovalbumin (OVA) in mice was investigated. BALB/c mice were subcutaneously (s.c.) inoculated twice with OVA alone or in combination with Rg1, alum, or Rg1 plus alum. Samples were collected 2 weeks after the boosting for the measurement of anti-OVA immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-5 (IL-5) produced in singular splenocyte cultures. Delayed-type hypersensitivity (DTH) responses were measured in mice immunized as described above. After 10 days, the mice were injected s.c. with OVA at the footpads. Thereafter, the thickness of the footpads was measured once daily for 5 days. The results indicated that alum enhanced mainly Th2 (IgG1 and IL-5) responses (P < 0.05), while Rg1 enhanced both Th1 (IgG1 and IL-5) and Th2 (IgG2a, IFN-γ, and DTH) responses (P < 0.05). The highest immune responses were found in the mice injected with OVA solution containing both alum and Rg1. In addition, the hemolytic activity of Rg1 was much lower than that of Quil A. Therefore, Rg1 deserves further studies in order to tailor desired immune responses when a mixed Th1/Th2 immune response is needed.
doi:10.1128/CVI.00448-07
PMCID: PMC2238049  PMID: 18094107
5.  Curcumin suppresses ovalbumin-induced allergic conjunctivitis 
Molecular Vision  2012;18:1966-1972.
Purpose
Allergic conjunctivitis (AC) from an allergen-driven T helper 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Because curcumin has shown anti-allergic activity in an asthma and contact dermatitis laboratory models, we examined whether administration of curcumin could affect the severity of AC and modify the immune response to ovalbumin (OVA) allergen in an experimental AC model.
Methods
Mice were challenged with two doses of topical OVA via the conjunctival sac after systemic sensitization with OVA in aluminum hydroxide (ALUM). Curcumin was administered 1 h before OVA challenge. Several indicators for allergy such as serum immunoglubulin E (IgE) antibodies production, eosinophil infiltration into the conjunctiva and Th2 cytokine production were evaluated in mice with or without curcumin treatment.
Results
Mice challenged with OVA via the conjunctival sac following systemic sensitization with OVA in ALUM had severe AC. Curcumin administration markedly suppressed IgE-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice administered curcumin had less interleukin-4 (IL-4) and interleukin-5 (IL-5) (Th2 type cytokine) production in conjunctiva, spleen, and cervical lymph nodes than mice in the non-curcumin-administered group. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS), and curcumin treatment inhibited iNOS production in the conjunctiva.
Conclusions
We believe our findings are the first to demonstrate that curcumin treatment suppresses allergic conjunctival inflammation in an experimental AC model.
PMCID: PMC3413438  PMID: 22876123
6.  Chitins and Chitosans as Immunoadjuvants and Non-Allergenic Drug Carriers 
Marine Drugs  2010;8(2):292-312.
Due to the fact that some individuals are allergic to crustaceans, the presumed relationship between allergy and the presence of chitin in crustaceans has been investigated. In vivo, chitin is part of complex structures with other organic and inorganic compounds: in arthropods chitin is covalently linked to proteins and tanned by quinones, in fungi it is covalently linked to glucans, while in bacteria chitin is diversely combined according to Gram(+/−) classification. On the other hand, isolated, purified chitin is a plain polysaccharide that, at the nano level, presents itself as a highly associated structure, recently refined in terms of regularity, nature of bonds, crystallinity degree and unusual colloidal behavior. Chitins and modified chitins exert a number of beneficial actions, i.e., (i) they stimulate macrophages by interacting with receptors on the macrophage surface that mediate the internalization of chitin particles to be degraded by lysozyme and N-acetyl-β-glucosaminidase (such as Nod-like, Toll-like, lectin, Dectin-1, leukotriene 134 and mannose receptors); (ii) the macrophages produce cytokines and other compounds that confer non-specific host resistance against bacterial and viral infections, and anti-tumor activity; (iii) chitin is a strong Th1 adjuvant that up-regulates Th1 immunity induced by heat-killed Mycobacterium bovis, while down- regulating Th2 immunity induced by mycobacterial protein; (iv) direct intranasal application of chitin microparticles into the lung was also able to significantly down-regulate allergic response to Dermatophagoids pteronyssinus and Aspergillus fumigatus in a murine model of allergy; (v) chitin microparticles had a beneficial effect in preventing and treating histopathologic changes in the airways of asthmatic mice; (vi) authors support the fact that chitin depresses the development of adaptive type 2 allergic responses. Since the expression of chitinases, chitrotriosidase and chitinase-like proteins is greatly amplified during many infections and diseases, the common feature of chitinase-like proteins and chitinase activity in all organisms appears to be the biochemical defense of the host. Unfortunately, conceptual and methodological errors are present in certain recent articles dealing with chitin and allergy, i.e., (1) omitted consideration of mammalian chitinase and/or chitotriosidase secretion, accompanied by inactive chitinase-like proteins, as an ancestral defensive means against invasion, capable to prevent the insurgence of allergy; (2) omitted consideration of the fact that the mammalian organism recognizes more promptly the secreted water soluble chitinase produced by a pathogen, rather than the insoluble and well protected chitin within the pathogen itself; (3) superficial and incomplete reports and investigations on chitin as an allergen, without mentioning the potent allergen from crustacean flesh, tropomyosine; (4) limited perception of the importance of the chemical/biochemical characteristics of the isolated chitin or chitosan for the replication of experiments and optimization of results; and (5) lack of interdisciplinarity. There is quite a large body of knowledge today on the use of chitosans as biomaterials, and more specifically as drug carriers for a variety of applications: the delivery routes being the same as those adopted for the immunological studies. Said articles, that devote attention to the safety and biocompatibility aspects, never reported intolerance or allergy in individuals and animals, even when the quantities of chitosan used in single experiments were quite large. Therefore, it is concluded that crab, shrimp, prawn and lobster chitins, as well as chitosans of all grades, once purified, should not be considered as “crustacean derivatives”, because the isolation procedures have removed proteins, fats and other contaminants to such an extent as to allow them to be classified as chemicals regardless of their origin.
doi:10.3390/md8020292
PMCID: PMC2852840  PMID: 20390107
chitin; chitosan; chitinase; chitinase-like proteins; immunology
7.  Chitin Induces Tissue Accumulation of Innate Immune Cells Associated with Allergy 
Nature  2007;447(7140):92-96.
Allergic and parasitic helminth immunity is characterized by infiltration of tissues with IL-4- and IL-13-expressing cells, including Th2 cells, eosinophils and basophils1. Tissue macrophages assume a distinct phenotype, designated alternatively activated macrophages2. Relatively little is known regarding factors that trigger these host responses. Chitin, a widespread environmental biopolymer of N-acetyl-β-D-glucosamine, confers structural rigidity to fungi, crustaceans, helminths and insects3. Here, we show that chitin induces the tissue accumulation of IL-4-expressing innate immune cells, including eosinophils and basophils, when given to mice. Tissue infiltration was unaffected by the absence of Toll-like receptor-mediated LPS recognition and was abolished by treatment of chitin with the IL-4- and IL-13-inducible mammalian chitinase, AMCase4, or by injection into mice that over-expressed AMCase. Chitin mediated alternative macrophage activation in vivo and production of leukotriene B4, which was required for optimal immune cell recruitment. Chitin is a recognition element for tissue infiltration by innate cells implicated in allergic and helminth immunity and this process can be negatively regulated by a vertebrate chitinase.
doi:10.1038/nature05746
PMCID: PMC2527589  PMID: 17450126
8.  Chitin Modulates Innate Immune Responses of Keratinocytes 
PLoS ONE  2011;6(2):e16594.
Background
Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood.
Methodology/Principal Findings
We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes.
Conclusions/Significance
We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined.
doi:10.1371/journal.pone.0016594
PMCID: PMC3044707  PMID: 21383982
9.  Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein by Use of CpG Oligodeoxynucleotides as an Adjuvant Induces a Protective Immune Response against an Intranasal Chlamydial Challenge  
Infection and Immunity  2002;70(9):4812-4817.
Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 104 inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.
doi:10.1128/IAI.70.9.4812-4817.2002
PMCID: PMC128273  PMID: 12183524
10.  Fungal chitin from asthma-associated home environments induces eosinophilic lung infiltration 
Development of asthma and allergic inflammation involves innate immunity but the environmental contributions remain incompletely defined. Analysis of dust collected from the homes of asthmatic individuals revealed that the polysaccharide chitin is environmentally widespread, and associated with β-glucans, possibly from ubiquitous fungi. Cell wall preparations of Aspergillus isolated from house dust induced robust recruitment of eosinophils into mouse lung, an effect that was attenuated by enzymatic degradation of cell wall chitinand β-glucans. Mice expressing constitutively active acidic mammalian chitinase (AMCase) in the lungs demonstrated a significant reduction in eosinophil infiltration after fungal challenge. Conversely, chitinase inhibition prolonged the duration of tissue eosinophilia. Thus, fungal chitin derived from home environments associated with asthma induces eosinophilic allergic inflammation in the lung, and mammalian chitinases, including AMCase, limit this process.
doi:10.4049/jimmunol.1100972
PMCID: PMC3159725  PMID: 21824866
11.  Vaccination with Novel Immunostimulatory Adjuvants against Blood-Stage Malaria in Mice  
Infection and Immunity  2003;71(9):5178-5187.
An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stage Plasmodium chabaudi AS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-γ) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection with P. chabaudi AS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4+ T cells, IFN-γ, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection with P. chabaudi AS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.
doi:10.1128/IAI.71.9.5178-5187.2003
PMCID: PMC187300  PMID: 12933862
12.  Acrolein exposure suppresses antigen-induced pulmonary inflammation 
Respiratory Research  2013;14(1):107.
Background
Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma.
Methods
C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14–16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14–17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity.
Results
Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.
Conclusion
Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.
doi:10.1186/1465-9921-14-107
PMCID: PMC3852782  PMID: 24131734
Cigarette smoke; Electrophile; Inflammation; Asthma; COPD; Nrf2; NF-κB; JNK
13.  Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase▿  
Infection and Immunity  2007;75(8):4088-4096.
Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.
doi:10.1128/IAI.00623-07
PMCID: PMC1952018  PMID: 17517867
14.  Differential Regulation of Inflammation and Immunity in Mild and Severe Experimental Asthma 
Mediators of Inflammation  2013;2013:808470.
This study aimed at exploring innate and adaptive immunity in allergic asthma by investigation of mRNA expression of pattern recognition receptors, T-cell-specific cytokines, and transcription factors. Mouse models for mild and severe asthma, with similar pathological characteristics observed in humans, were used to study the involved inflammatory markers as a first step in the development of phenotype-directed treatment approaches. In the mild model, mice were sensitized to ovalbumin-Imject Alum and challenged with ovalbumin. In the severe model, mice were sensitized to trinitrophenyl-conjugated ovalbumin and challenged with trinitrophenyl-ovalbumin/IgE immune complex. Pulmonary airway inflammation and mRNA expression of Toll-like receptors (TLRs), NOD-like receptors (NLRs), T cell cytokines, and transcription factors in lung tissue were examined. Different mRNA expression profiles of TLRs, NLRs, T cell cytokines, and transcription factors were observed. In the mild model, Il10 showed the largest increase in expression, whereas in the severe model, it was Infγ with the largest increase. Expression of Tbet was also significantly increased in the severe model. Inflammation and immunity are differentially regulated in mild and severe experimental asthma. This preclinical data may help in directing clinical research towards a better understanding and therapy in mild and severe asthmatic patients.
doi:10.1155/2013/808470
PMCID: PMC3679512  PMID: 23781124
15.  Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells 
Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)–resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c+ monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting “nature's adjuvant,” the inflammatory DC through induction of the endogenous danger signal uric acid.
doi:10.1084/jem.20071087
PMCID: PMC2292225  PMID: 18362170
16.  Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ 
Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.
doi:10.1128/AEM.07506-11
PMCID: PMC3346462  PMID: 22367086
17.  Chitin-microparticles for the control of intestinal inflammation 
Inflammatory bowel diseases  2012;18(9):1698-1710.
Chitin is a polymer of N-acetylglucosamine with the ability to regulate innate and adaptive immune responses. However, the detailed mechanisms of chitin-mediated regulation of intestinal inflammation are only partially known. In this study, Chitin-microparticles (CMPs) or PBS were orally administered to acute and chronic colitis models every three days for six consecutive weeks beginning at weaning age. The effects of this treatment were evaluated by histology, cytokine production, co-culture study and enteric bacterial analysis in DSS-induced colitis or TCRα knockout chronic colitis models. Histologically, chitin-treated mice showed significantly suppressed colitis as compared to PBS-treated mice in both animal models. The production of IFNγ was upregulated in the mucosa of chitin-treated mice compared to control mice. The major source of IFNγ-producing cells was CD4+ T cells. In mouse dendritic cells (DCs), we found that CMPs were efficiently internalized and processed within 48 hours. Mesenteric lymph nodes (MLNs) CD4+ T cells isolated from chitin-treated mice produced 7-fold higher amount of IFNγ in the culture supernatant after being co-cultured with DCs and chitin as compared to the control. Proliferation of CFSElow CD4+ T cells in MLNs and enteric bacterial translocation rates were significantly reduced in chitin-treated mice when compared to the control. In addition, CMPs improved the imbalance of enteric bacterial compositions and significantly increased IL-10-producing cells in non-inflamed colon, indicating the immunoregulatory effects of CMPs in intestinal mucosa. In conclusion, CMPs significantly suppress the development of inflammation by modulating cytokine balance and microbial environment in colon.
doi:10.1002/ibd.22874
PMCID: PMC3586600  PMID: 22241684
chitin; chitinase; colitis; cytokine; animal model
18.  Pivotal Advance: Eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production 
Journal of leukocyte biology  2008;83(4):817-821.
Alum, aluminum-hydroxide-containing compounds, long used as adjuvants in human vaccinations, functions by ill-defined, immunostimulatory mechanisms. Antigen-free alum has been shown to act via a previously unidentified, splenic Gr1+, IL-4-expressing myeloid cell population to stimulate early B cell priming. We demonstrate that the alum-elicited and -activated splenic myeloid cells are IL-4-expressing eosinophils that function to prime B cell responses. Eosinophils are the principal Gr1+, IL-4+ cells in the spleens 6 days following i.p. alum administration. Alum-elicited splenic B cell priming, as evidenced by MHC II cross-linking-mediated calcium mobilization developed in wild-type BALB/c mice, was absent in ΔdblGATA BALB/c eosinophil-deficient mice and could be reconstituted by adoptive eosinophil infusions into the eosinophil-deficient mice. Moreover, early antigen-specific IgM antibody responses in alum-antigen-immunized mice were impaired in eosinophil-deficient mice and were restored with adoptive transfers of eosinophils. Thus, eosinophils, leukocytes of the innate immune system that contain preformed cytokines, including IL-4, have novel, immunomodulatory roles in the initial priming of B cells elicited by the adjuvant alum and in the optimal early B cell generation of antigen-specific IgM.
doi:10.1189/jlb.0607392
PMCID: PMC2735468  PMID: 18192490
BALB/c; MHC II; innate immunity
19.  Importance of Myeloid Dendritic Cells in Persistent Airway Disease after Repeated Allergen Exposure 
Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.
Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.
Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.
Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.
doi:10.1164/rccm.200505-783OC
PMCID: PMC2662981  PMID: 16192450
airway hyperresponsiveness; chronic asthma; cytokine; dendritic cells; eosinophil
20.  Role of SHIP-1 in the Adaptive Immune Responses to Aeroallergen in the Airway 
PLoS ONE  2010;5(11):e14174.
Background
Th2-dominated inflammatory response in the airway is an integral component in the pathogenesis of allergic asthma. Accumulating evidence supports the notion that the phosphoinositide 3-kinase (PI3K) pathway is involved in the process. We previously reported that SHIP-1, a negative regulator of the PI3K pathway, is essential in maintaining lung immunohomeostasis, potentially through regulation of innate immune cells. However, the function of SHIP-1 in adaptive immune response in the lung has not been defined. We sought to determine the role of SHIP-1 in adaptive immunity in response to aeroallergen stimulation in the airway.
Methodology/Principal Findings
SHIP-1 knockout (SHIP-1−/−) mice on BALB/c background were immunized with ovalbumin (OVA) plus aluminum hydroxide, a strong Th2-inducing immunization, and challenged with OVA. Airway and lung inflammation, immunoglobulin response, Th2 cytokine production and lymphocyte response were analyzed and compared with wild type mice. Even though there was mild spontaneous inflammation in the lung at baseline, SHIP-1−/− mice showed altered responses, including less cell infiltration around the airways but more in the parenchyma, less mucus production, decreased Th2 cytokine production, and diminished serum OVA-specific IgE, IgG1, but not IgG2a. Naïve and OVA sensitized SHIP-1−/− T cells produced a lower amount of IL-4. In vitro differentiated SHIP-1−/− Th2 cells produced less IL-4 compared to wild type Th2 cells upon T cell receptor stimulation.
Conclusions/Significance
These findings indicate that, in contrast to its role as a negative regulator in the innate immune cells, SHIP-1 acts as a positive regulator in Th2 cells in the adaptive immune response to aeroallergen. Thus any potential manipulation of SHIP-1 activity should be adjusted according to the specific immune response.
doi:10.1371/journal.pone.0014174
PMCID: PMC2994819  PMID: 21151496
21.  A polysaccharide isolated from Agaricus blazei Murill (ABP-AW1) as a potential Th1 immunity-stimulating adjuvant 
Oncology Letters  2013;6(4):1039-1044.
In the present study, a low molecular weight polysaccharide, ABP-AW1, isolated from Agaricus blazei Murill was assessed for its potential adjuvant activity. ABP-AW1 is considered to create a ‘depot’ of antigen at a subcutaneous injection site. ICR mice were immunized with 100 μg ovalbumin (OVA) alone or with 100 μg OVA formulated in 0.9% saline containing 200 μg aluminum (alum) or ABP-AW1 (50, 100 and 200 μg) on days 1 and 15. Two weeks after the secondary immunization, splenocyte proliferation, the expression of surface markers, cytokine production and the OVA-specific antibody levels in the serum were determined. The OVA/ABP-AW1 vaccine, in comparison with OVA alone, markedly increased the proliferation of splenic lymphocytes and elicited greater antigen-specific CD4+ T cell activation, as determined by splenic CD4+CD69+ T cells and Th1 cytokine interferon (IFN)-γ release. The combination of ABP-AW1 and OVA also enhanced IgG2b antibody responses to OVA. In conclusion, these data indicated that ABP-AW1 significantly enhanced the humoral and cellular immune responses against OVA in the mice, suggesting that ABP-AW1 stimulated Th1-type immunity. We suggest that ABP-AW1 may serve as a new adjuvant.
doi:10.3892/ol.2013.1484
PMCID: PMC3796401  PMID: 24137460
polysaccharide; Agaricus blazei; Th1; vaccine; immune
22.  Chitin elicits CCL2 from airway epithelial cells and induces CCR2-dependent innate allergic inflammation in the lung 
Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages, and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. Here, we show that chitin induces alternative activation of macrophages in vivo, but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas antibody neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17 and CCL22 expression. Furthermore, CCR2KO mice exposed to chitin had diminished ROS products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL13 and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo.
doi:10.4049/jimmunol.1200689
PMCID: PMC3424300  PMID: 22851704
CCL2; CCR2; chitin; macrophage; eosinophil; epithelial; airway; lung; asthma; allergy
23.  Syk Activation in Dendritic Cells Is Essential for Airway Hyperresponsiveness and Inflammation 
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
doi:10.1165/rcmb.2005-0298OC
PMCID: PMC2644204  PMID: 16339999
AHR; dendritic cells; eosinophils; mice; Syk
24.  Mast Cells Can Amplify Airway Reactivity and Features of Chronic Inflammation in an Asthma Model in Mice 
The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell–deficient WBB6F1-W/Wv mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F1-+/+ normal mice. However, W/Wv mice that had undergone selective reconstitution of tissue mast cells with in vitro–derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/Wv mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/Wv mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the “asthma model” investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice.
PMCID: PMC2193222  PMID: 10934234
eosinophil; epithelium; hyperresponsiveness; tissue remodeling; T lymphocyte
25.  Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice 
BMC Immunology  2010;11:8.
Background
Allergy has been an increasing problem in several parts of the world. Prenatal exposure to allergen and microbial components may affect the development of allergies in childhood, as indicated by epidemiological and experimental studies. We investigated the capacity for allergic sensitisation in offspring after induction of a Th1- or a Th2-polarised immune response to the same allergen in mothers during pregnancy.
Results
During pregnancy, mice were immunised with ovalbumin (OVA) given with either one of the Th2-adjuvants pertussis toxin (PT) or Al(OH)3 (aluminium hydroxide), or with the Th1 adjuvant CpG. Offspring were immunised with OVA in Al(OH)3 as young adults. Serum and supernatants from ex vivo stimulated or non-stimulated spleen cells from mothers and offspring were analysed for OVA-specific antibodies and cytokines, respectively. Mothers immunised with OVA together with either Al(OH)3 or PT had increased levels of OVA-specific IgE and IgG1 compared to naive mothers, whereas mothers immunised with OVA together with CpG had increased levels of OVA-specific IgG2a compared to naive mothers. In general the highest levels of IL-5, IL-10, and IFNγ were observed in spleen cells from mothers immunised with PT and OVA. Upon immunisation, offspring from mothers immunised with OVA and either PT or Al(OH)3 showed reduced levels of OVA-specific IgE and IgG1 and increased levels of OVA-specific IgG2a antibodies compared to offspring from naive mothers. Maternal immunisation with CpG and OVA did not affect antibody responses in offspring.
Conclusion
Allergic sensitisation in the offspring was affected by the type of adjuvant used for immunisation of the mothers with the same allergen. Th2 polarisation of the immune response in the mothers was found to give reduced IgE levels upon sensitisation of the offspring, whereas no reduction was achieved with Th1 polarisation in the mothers.
doi:10.1186/1471-2172-11-8
PMCID: PMC2846879  PMID: 20193059

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