Biodegradable Chitosan Decreases the Immune Response to Trichinella spiralis in Mice

The purpose of this study was to evaluate the potential of chitosan units released during natural degradation of the polymer to activate the immune system against T. spiralis infection. High molecular weight chitosan was injected intraperitoneally into C57BL/6 mice. Flow cytometry and cytokine concentration, measured by ELISA, were used to characterize peritoneal cell populations during T. spiralis infection. The strong chemo-attractive properties of chitosan caused considerable infiltration into the peritoneal cavity of CD11b+ cells, with reduced expression of MHC class II, CD80, CD86, Dectin-1 or CD23 receptors in comparison to T. spiralis-infected mice. After prolonged chitosan biodegradation, cell populations expressing IL-4R, MR and Dectin-1 receptors were found to coexist with elevated IL-6, IL-10, TGF-β and IgA production. IgA cross-reacted with T. spiralis antigen and chitosan. It was found that chitosan treatment attracted immune cells with low activity, which resulted in the number of nematodes increasing. The glucosamine and N-acetyl-D-glucosamine residues were recognized by wheat germ agglutinin (WGA) lectin and therefore any biodegradable chitosan units may actively downregulate the immune response to the parasite. The findings are relevant for both people and animals treated with chitosan preparations.


Introduction
There are many proposals for the use of polysaccharide biomaterials in medicine. Due to their physical and chemical properties, many of these substances are suitable for therapeutic purposes [1,2]. However, before these compounds can find full use as drug carriers, implants or regenerative materials in dressings, their immunogenic properties need to be clearly identified and characterized [3]. Polysaccharides are recognized by distinct cell receptors, and may induce or modulate the immune response, depending on their size and structure [4][5][6].
Chitosan and its derivatives are believed to be non-toxic, bioactive, biocompatible and biodegradable substances with low immunogenicity [7,8]. The polymer acts as a temporary extracellular matrix; it is endocytosed by cells and eventually broken down by lysozyme, after which chitosan chains with low degrees of acetylation may remain active in the body for several months [9]. Chitosan is soluble in dilute acids, and the number fraction of N-acetylglucosamine (GlcNAc) residues in the polymer chain, designated by the degree of acetylation, influences cell response by modulating the solubility, reactivity, biodegradability and activity of the polymer [10,11]. During biological degradation, the length of the released chains will influence the degree to which chitosan may differentially affect the outcome of the immune response. The polymer is gradually degraded, and its distinct surface activity or shared moieties may affect receptor cell signaling during immune activation. The peritoneal cell suspension in the glass smear showed that macrophages and monocytes were present but neutrophils dominated ( Figure 1B). Thirty days post infection, the number of these cells significantly increased in mice infected with T. spiralis but dropped in mice treated with chitosan.
An increase in the percentage of CD11b + cells in the peritoneal cavity was observed in the enteral phase of infection ( Figure 1C). The pattern of response changed on day 30 of the experiment, and the highest percentage of CD11b + cells appeared in mice infected with T. spiralis, with the number being slightly lower in the mice injected with chitosan. The peritoneal cell suspension in the glass smear showed that macrophages and monocytes were present but neutrophils dominated ( Figure 1B). Thirty days post infection, the number of these cells significantly increased in mice infected with T. spiralis but dropped in mice treated with chitosan.
An increase in the percentage of CD11b + cells in the peritoneal cavity was observed in the enteral phase of infection ( Figure 1C). The pattern of response changed on day 30 of the experiment, and the highest percentage of CD11b + cells appeared in mice infected with T. spiralis, with the number being slightly lower in the mice injected with chitosan.
Resident macrophages and migrating monocytes were recognized based on the level of CD11b + and F4/80 receptor expression as CD11b hi F4/80 hi and CD11b + F4/80 lo , respectively ( Figure 1C,E). The number of resident macrophages fell significantly only in groups injected with chitosan in the enteral phase of infection. The percentage of monocytes, CD11b + F4/80 lo cells, fell in mice infected with T. spiralis, but still constituted over 85% of the CD11b + cell population in mice given chitosan ( Figure 1C). In all groups of infected and/or treated mice, the percentage of monocytes dropped during the muscle phase of T. spiralis infection in comparison to untreated and uninfected mice. Chitosan significantly affected the percentage of myeloid cells in the peritoneal cavity and strongly attracted CD11b + monocytes into the peritoneum. Chitosan induced chronic peritonitis, accompanied by the precipitation and adhesion of chitosan onto the mesentery epithelium with granuloma formation on the surface of the liver ( Figure 1F).
Five days after infection, the number of cells in the peritoneal cavity was lower in mice infected with the nematode than uninfected mice. The response of peritoneal cells was enhanced 30 days after infection with domination of resident macrophages.

Changes in the Percentage of Cells Expressing Immune Receptors
The percentage of CD11b + monocytes in the peritoneal cavity expressing surface immune markers was modified differently by T. spiralis and by chitosan (Figure 2A-C). Resident macrophages and migrating monocytes were recognized based on the level of CD11b + and F4/80 receptor expression as CD11b hi F4/80 hi and CD11b + F4/80 lo , respectively ( Figure 1C,E). The number of resident macrophages fell significantly only in groups injected with chitosan in the enteral phase of infection. The percentage of monocytes, CD11b + F4/80 lo cells, fell in mice infected with T. spiralis, but still constituted over 85% of the CD11b + cell population in mice given chitosan ( Figure  1C). In all groups of infected and/or treated mice, the percentage of monocytes dropped during the muscle phase of T. spiralis infection in comparison to untreated and uninfected mice. Chitosan significantly affected the percentage of myeloid cells in the peritoneal cavity and strongly attracted CD11b + monocytes into the peritoneum. Chitosan induced chronic peritonitis, accompanied by the precipitation and adhesion of chitosan onto the mesentery epithelium with granuloma formation on the surface of the liver ( Figure 1F).
Five days after infection, the number of cells in the peritoneal cavity was lower in mice infected with the nematode than uninfected mice. The response of peritoneal cells was enhanced 30 days after infection with domination of resident macrophages.

Changes in the Percentage of Cells Expressing Immune Receptors
The percentage of CD11b + monocytes in the peritoneal cavity expressing surface immune markers was modified differently by T. spiralis and by chitosan (Figure 2A   Five days post T. spiralis infection, the percentage of cells expressing MHC class II, MR, Dectin r CD23 increased in untreated mice, while the percentage of cells with MHC class II, CD80, CD8 ctin-1 or CD23 expression dramatically fell in mice treated with chitosan. Thirty days po fection, the cell populations expressing MHC class II, IL-4R and Dectin-1 were increased, but tho pressing the costimulatory receptor CD86, and MR or CD23 were reduced. After a prolonge riod of biodegradation, chitosan increased the proportions of IL-4R + , MR + and Dectin-1 + cells at 3 I. After chitosan treatment, the percentage of cells demonstrating enhanced receptor expressio ll during the intestinal phase of T. spiralis infection; however, chitosan supported IL-4R and Decti elated cell activation in the muscle phase.

. The Level of Cytokines in the Peritoneal Fluid
Five days post infection with T. spiralis, chitosan appeared to influence the levels yeloperoxidase (MPO) and cytokines in mice: higher levels of MPO, MCP-1, TNF-α, IL-12p70, IL IL-10 and TGF-β were found in infected mice treated with chitosan compared to those who we t. Thirty days post infection, chitosan treatment resulted in MPO, MCP-1, TNF-α, IL-12p70 and IL evels falling to control values, but IL-6, IL-10 and TGF-β were elevated ( Figure 3). After chitosan treatment, the percentage of cells demonstrating enhanced receptor expression fell during the intestinal phase of T. spiralis infection; however, chitosan supported IL-4R and Dectin-1 related cell activation in the muscle phase.

The Level of Cytokines in the Peritoneal Fluid
Five days post infection with T. spiralis, chitosan appeared to influence the levels of myeloperoxidase (MPO) and cytokines in mice: higher levels of MPO, MCP-1, TNF-α, IL-12p70, IL-6, IL-10 and TGF-β were found in infected mice treated with chitosan compared to those who were not. Thirty days post infection, chitosan treatment resulted in MPO, MCP-1, TNF-α, IL-12p70 and IL-4 levels falling to control values, but IL-6, IL-10 and TGF-β were elevated ( Figure 3).

IgA Response Specific to Muscle Larvae Somatic Antigen
In untreated infected mice, the level of peritoneal IgA specific to T. spiralis muscle larvae antigens was around 2.5 times greater at 30 days after infection (DAI) than at 5 DAI. However, the IgA levels at both 5 DAI and 30 DAI were higher in the mice injected with chitosan ( Figure 4A).
Western blot analysis revealed IgA cross-reactivity to muscle larvae somatic proteins of noninfected mice injected with high molecular weight (HMW) chitosan (Ch) only, and of mice infected

IgA Response Specific to Muscle Larvae Somatic Antigen
In untreated infected mice, the level of peritoneal IgA specific to T. spiralis muscle larvae antigens was around 2.5 times greater at 30 days after infection (DAI) than at 5 DAI. However, the IgA levels at both 5 DAI and 30 DAI were higher in the mice injected with chitosan ( Figure 4A).
Western blot analysis revealed IgA cross-reactivity to muscle larvae somatic proteins of non-infected mice injected with high molecular weight (HMW) chitosan (Ch) only, and of mice infected with T. spiralis at 30 DAI. The IgA of mice injected with chitosan and the IgA of T. spiralis

The Level of T. spiralis Infection in Mice Injected with Chitosan
The numbers of adult forms and muscle larvae were greater in mice injected with chitosan than in control infection; however, the number of newborn larvae was unchanged ( Figure 5).

The Level of T. spiralis Infection in Mice Injected with Chitosan
The numbers of adult forms and muscle larvae were greater in mice injected with chitosan than in control infection; however, the number of newborn larvae was unchanged ( Figure 5).

The Level of T. spiralis Infection in Mice Injected with Chitosan
The numbers of adult forms and muscle larvae were greater in mice injected with chitosan than in control infection; however, the number of newborn larvae was unchanged ( Figure 5).  cyclic, CH 2 OH groups) as shown in Figure 6, spectrum A. The analysis of peritoneal fluids confirmed the presence of chitosan; however, the spectra were slightly changed. The single peak initially present at 1700 cm −1 had been spread to the range 1690-1550 cm −1 , and a new band at 1742 cm −1 was observed, suggesting the presence of new free carboxyl groups ( Figure 6, spectrum B). The FTIR spectrum of the sample isolated from precipitated material in the granuloma 35 days after chitosan injection exhibited characteristic bands only for adipic acid ( Figure 6, spectrum C) [26]. The chitosan injected into the peritoneal cavity had biodegraded.

Biodegradation of Chitosan Injected into the Peritoneal Cavity
Attenuated total reflectance (ATR) analysis was performed. The FTIR spectra of the chitosan contained bands typical of chitosan as a salt: 3365 cm −1 (amide), 3292 cm −1 (N-H stretching), 2878 cm −1 (C-H stretching, present also in adipic acid), 1640 cm −1 (amide I), 1563 cm −1 (amide II), 1550 cm −1 (NH3 + ), 1155 cm −1 (O-C-O stretching), 1100-1020 cm −1 (C-O stretching of: C-OH, C-O-C cyclic, CH2OH groups) as shown in Figure 6, spectrum A. The analysis of peritoneal fluids confirmed the presence of chitosan; however, the spectra were slightly changed. The single peak initially present at 1700 cm −1 had been spread to the range 1690-1550 cm −1 , and a new band at 1742 cm −1 was observed, suggesting the presence of new free carboxyl groups ( Figure 6, spectrum B).
The FTIR spectrum of the sample isolated from precipitated material in the granuloma 35 days after chitosan injection exhibited characteristic bands only for adipic acid ( Figure 6, spectrum C) [26]. The chitosan injected into the peritoneal cavity had biodegraded.

Discussion
The present study examines the role played by biodegradable chitosan particles in the modification of the immune response to T. spiralis infection. As noted previously in a different model [27], chitosan is believed to induce chronic peritonitis, accompanied by the precipitation and adhesion of polysaccharide onto the mesentery epithelium with granuloma formation at the surface of the liver. The strong chemo-attractive properties of chitosan resulted in a huge number of cells infiltrating the peritoneal cavity.
In mice injected with chitosan, the number of cells in the peritoneum dramatically increased and the proportion of CD11b hi F4/80 hi cells of the total CD11b + population was depleted. The large resident macrophage population was mostly replaced by another myeloid cell population with the CD11b + F4/80 lo phenotype of a nonresponsive state and a non-resident cell phenotype [28,29]. In mice infected with T. spiralis, the percentage of CD11b + cells expressing MHC class II molecules was found to be greater, and these cells might be derived from blood monocytes which had entered the peritoneal cavity after stimulation with parasitic antigens [30,31].

Discussion
The present study examines the role played by biodegradable chitosan particles in the modification of the immune response to T. spiralis infection. As noted previously in a different model [27], chitosan is believed to induce chronic peritonitis, accompanied by the precipitation and adhesion of polysaccharide onto the mesentery epithelium with granuloma formation at the surface of the liver. The strong chemo-attractive properties of chitosan resulted in a huge number of cells infiltrating the peritoneal cavity.
In mice injected with chitosan, the number of cells in the peritoneum dramatically increased and the proportion of CD11b hi F4/80 hi cells of the total CD11b + population was depleted. The large resident macrophage population was mostly replaced by another myeloid cell population with the CD11b + F4/80 lo phenotype of a nonresponsive state and a non-resident cell phenotype [28,29]. In mice infected with T. spiralis, the percentage of CD11b + cells expressing MHC class II molecules was found to be greater, and these cells might be derived from blood monocytes which had entered the peritoneal cavity after stimulation with parasitic antigens [30,31].
In the muscle phase of T. spiralis infection, the regulatory cell phenotype was favored, and the percentage of the CD11b + cell population co-expressing IL-4R and MR indicated the presence of alternatively activated macrophages [32]. The macrophage marker F4/80 is also expressed at low levels on monocytes [33]. The cells were characterized as not fully activated, and the percentage expressing MHC class II, CD80, CD86, IL-4R, MR, Dectin-1 and CD23 receptors was lower. The number of myeloid cells and the level of myeloperoxidase remained temporarily elevated, but then fell as the chitosan precipitated [34].
During T. spiralis infection, a mixed Th1/Th2 immune response develops [35]. A skewed immune response profile can be seen following chitosan treatment, reflected in an increase in MCP-1 and IL-12p70 production, typical for Th1-induced responses; a rather weak IL-4 response was also observed, together with greatly elevated IL-6 and IL-10 levels. The clear increase in TGF-β and IL-10 observed after chitosan treatment is typical of a regulatory response [36][37][38]. TGF-β is known to be essential for parasite survival [39], T. spiralis stages induce the production of mediators that increase Treg cell numbers in the host [40]. In addition, native T. spiralis excretory-secretory products have been found to stimulate increased IL-10 and TGF-β production by dendritic cells (DCs) [41]. Chitosan treatment also greatly increased TGF-β and IL-10 production, and a greater number of the nematodes survived.
Chitosan administered into the peritoneal cavity was biodegraded, which acted as a source of polymer chains with a distinct molecular weight; this could skew the Th2 response into a Treg response following recognition by DCs [42]. The structural analysis of the chitosan in the peritoneal cavity, confirmed by the examination of nuclear magnetic resonance difference spectra, indicated the presence of chemical functional groups; the findings may be considered as the spectrum of the degradation products and adipic acid residue [26]. In addition, the presence of new COOH groups confirmed the degradation of chitosan into shorter chains [43][44][45].
Chitosan strongly attracted neutrophils [46]. The glass smears revealed the presence of numerous neutrophils in the peritoneal fluid until day 10 following chitosan treatment. These cells are able to recognize chitosan even without injury being present, and in vivo chemotaxis of neutrophils to biodegradable chitosan continues until all particles are completely internalized inside phagocytes [47]. In inflammatory disorders, neutrophils are the main source of chitinases [48]. During endocytosis, chitosan would be biodegraded into its primary structural units such carbohydrate residues [49], and this is important for the dynamics of the immune response. Chitosan clearance is also accompanied by mesenchymal stem cell recruitment and by granuloma tissue formation [47], which was also observed in our studies.
The exact mechanisms that mediate the degradation of chitosan in vivo remain poorly defined. The polymer prepared by enzymatic hydrolysis and analyzed by HPLC revealed that the chitosan oligosaccharide mixture contained oligosaccharides with different degrees of polymerization [49]. The stimulatory effects of chitosan oligosaccharide on macrophages are mediated in a size-dependent and pathway-specific manner, and although the present study provides no direct evidence that the enzymes released in vivo directly degraded the polymer before endocytosis, our findings nevertheless suggest that HMW chitosan was degraded in the peritoneal cavity, as demonstrated by the changes observed in the FTRI profile spectra. Particles of the chitosan mixture would be phagocytosed by cells. This aspect of our study requires further evaluation, especially the kinetics regarding the degree of depolymerization of the high molecular weight chitosan particles deposited into the peritoneal cavity. In addition, the incoming macrophages may persist around chitosan materials until the compound is completely cleared [50]. The chitosan attracted macrophages, and induced cell migration and proliferation at the site of the destroyed tissue, as also noted in previous studies [51,52].
The binding specificities of GlcN and/or GlcNAc residues of chains released by chitosan biodegradation were identified by WGA lectin bind assays. These may play a crucial role in the outcome of this response, probably by elevating the levels of the Dectin-1 receptor [53]. The inflammatory response of the cells could be abrogated by the overloading of GlcN or GlcNAc [54,55]. The peritoneal cells of the studied mice demonstrated low expression of the immune receptors essential for antigen recognition and presentation, and the immature myelocytes newly attracted into the peritoneal cavity were characterized by weak immune activation [56]. The activation of T cells without co-stimulation may lead to T cell anergy, deletion or the development of immune tolerance [57]. The low level of the cellular response contributed to a low level of protection during colonization of the intestinal enterocytes or muscles by larvae.
Neutrophil infiltration of the small intestinal epithelium has already been shown to contribute to the stimulation of epithelial cell cytokine production in rats infected with T. spiralis: The intestinal phase of the parasite infection influences the subsequent muscle invasion with increasing numbers of eosinophils and neutrophils [58]. Chitosan attracted neutrophils into the peritoneal cavity and reduced the local activation of the intestinal epithelium, thus possibly allowing more adult nematodes to survive [59]. Larvae in the muscle not exposed to the neutrophils attracted into the peritoneum, thus avoiding inflammation during nurse cell formation, would also be protected. This neutrophil accumulation may be caused by peritoneal cell damage provoked by highly adherent chitosan [46,60].
T. spiralis larvae express distinct carbohydrate moieties on antigens; these may possess immunogenic properties or help the parasite to evade the host immune response by various mechanisms [41]. The percentage of monocytes, B lymphocytes and other peritoneal cells expressing the CD23 receptor, a marker of allergy and helminth infection with a weak affinity for IgE, increased after infection but was slightly reduced by chitosan. The reduced CD23 + cell population would be associated with reduced immune activation [61] and weaker protection against intestinal stages of the nematode.
Chitosan treatment induced the production of significantly higher levels of IgA. The immunoglobulin recognized T. spiralis muscle larvae antigenic determinants rich in GlcNAc as WGA [62]. The IgA of mice injected with chitosan or infected with T. spiralis were seen to cross-react with the nematode antigens as a consequence of immunoglobulin affinity [63]: an increase in IgA antibody production may grow during T. spiralis infection [64,65]. It is important to stress that chitosan administration was also associated with an increased IgA response in the peritoneal cavity, together with an elevation in TGF-β, IL-10 and IL-6 production. TGF-β and IL-6 supported plasma cell survival and induced IgA secretion [66,67]. This increase in IgA production in response to chitosan units with GlcN residues is an interesting observation, as it illustrates the induction of a form of immune tolerance mechanism in response to a common residue in food, commensal bacterial components or parasites [68]. Recent studies have shown that GlcN possesses immunosuppressive activity, and being abundant in the molecules of the parasite, it may have immunomodulatory properties [53]. More detailed studies are needed to explain the immunoregulatory properties of separate residues released from biodegraded chitosan units.
In conclusion, high molecular weight chitosan administered into the peritoneal cavity of mice before nematode infection provoked an accumulation of myeloid cells in situ. After prolonged deposition and biodegradation of chitosan, immune activation was redirected into a tolerant milieu, resulting in a greater number of nematodes in the host.

Schedule of the Experiment
All experimental procedures were performed according to the Polish Law on Animal Experimentation and EU Directive 2010/63/UE. The study was approved by the First Warsaw Local Ethics Committee for Animal Experimentation (approval ID 151/2011).
Male seven-week-old C57BL6 mice were kept in standard light:dark conditions (12 h:12 h) with ad libidum access to water and pellet food. Experimental groups consisted of five mice.
High molecular weight chitosan with >75% degree of deacetylation was obtained from the chitin of crustacean shells (HMW; 310 to >375 kDa based on the viscosity range of 800-2000 mPaS) (Sigma, Steinheim, Germany). This was dissolved as 1% stock solution in 0.75% adipic acid (StanLab, Lublin, Poland) and autoclaved. The endotoxin level in stock solution was found to be <0.2 EU/mL in LAL assay (Pierce LAL chromogenic endotoxin quantitation kit; Thermo Fisher Scientific, Rockford, IL, USA). The animals were injected intraperitoneally with 200 µL amounts of 500 µg chitosan (final adipic acid concentration: 0.35%) daily for 10 days, starting five days before infection.
Attenuated Total Reflectance (ATR) spectra of the initial chitosan and peritoneal samples were recorded as described below.
T. spiralis larvae were isolated from BALB/c mice by artificial digestion of carcasses with pepsin/HCl solution. On day 5 after chitosan administration, the mice were alimentary infected with 400 larvae L1 T. spiralis. Four groups of mice were examined on days 5 and 30 after T. spiralis infection: mice infected with the nematode (5 DAI) and (30 DAI) but not treated with chitosan, infected mice previously treated with chitosan (Ch/5 DAI) and (Ch/30 DAI), mice which were uninfected and untreated (Ctr), mice only administered chitosan (Cht/Ctr). Adult forms were isolated from the small intestine using the Baermann technique. Muscle larvae were inspected after digestion of muscle tissue with pepsin/HCl solution. Ten females were cultured separately in RPMI 1640 Medium (Gibco, Paisley, UK) with 10% Glutamax (Gibco) for 48 h, and the number of newborn larvae (NBL) per female was estimated.

Cell Isolation, Cytokine and Myeloperoxidase Measurement
Cells were isolated from the peritoneal cavity; 5 mL of RPMI 1640 medium supplemented with penicillin/streptomycin (100 µg/mL) and 2 mM L-glutamine (all from Gibco), was injected into the peritoneal cavity and lavages were collected separately on ice and centrifuged (1200 rpm, 4 • C); the volume of collected fluid was measured. The supernatant was frozen at −80 • C for cytokine analysis. The cells were washed, counted with Trypan blue exclusion and a concentration of 2.5 × 10 6 cells per mL was used for receptor phenotyping.
Peritoneal fluid was assayed for myeloperoxidase (MPO) and cytokines using commercially-available enzyme-linked immunosorbent assay (ELISA) reagents for MCP-1, TGF-β, IL-10 (e-Bioscences, San Diego, CA, USA) and TNF-α, IL-12p40, IL-4, IL-6 (BD Biosciences, Pharmingen, San Diego, CA, USA), MPO ELISA Kit (MyeloPeroxidase; R & D Systems, Minneapolis, MN, USA) according to the suppliers' guidelines. For the TGF-β measurement, the samples were acidified and latent; samples were taken to measure the amount of active cytokine excreted into the culture medium. The plates were read at 490 nm or 450 nm (MPO) using a u-Quant spectrophotometer (Bio-Tek, Acton, MA, USA). The mean optical densities (OD) of the cultures, read in triplicate, were compared with the standard curves prepared using recombinant proteins.  Samples were centrifuged at 18,000× g for five minutes at 4 • C and the supernatant was stored at −80 • C until use. IgA in the peritoneal fluid was measured by ELISA; 96 well polysorp plates (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) were coated with 100 µL of 10 µg/mL T. spiralis L1 somatic extract or 10 µg/mL chitosan in a buffer pH 6.5 overnight in 4 • C. The wells were then blocked with 200 µL of 5% non-fat milk in PBS (pH 7.2) for one hour and incubated with 100 µL of undiluted peritoneal fluid for two hours at room temperature. RPMI medium was used as a negative control. After extensive washing with PBS Tween (0.05%) specific IgA was detected with anti-mouse IgA-HRP complex diluted 1:5000 (Novus Biologicals, Abingdon, UK); after a one hour incubation period, 100 µL TMB substrate (Sigma-Aldrich Inc., St. Louis, MO, USA) was added. The reaction was stopped after 20 min with 50 µL 2 N H 2 SO 4 and absorbance was measured at λ = 450 nm with a µQuant spectrophometer (Bio-Tek, Acton, MA, USA).

IgA Specific Response Detected by Electrophoresis and Immunoblotting
Protein samples of T. spiralis L1 somatic extracts were boiled in Laemelli buffer and centrifuged for 10 min at 15,000× g; 10 µg of parasite antigen was separated on 4% and 12% SDS PAGE gel for 50 min at a constant 150 V using a Bio-Rad TetraCell system (Bio-Rad Laboratories, Richmond, BC, Canada). The proteins were transferred onto a nitrocellulose membrane (Sigma, 0.45 µm pore size) for one hour at 18 V with SemiDry Transfer apparatus (BioRad Laboratories, Hercules, CA, USA). After transfer, the membrane was blocked with 5% non-fat milk in PBS (pH 7.2) for two hours. Subsequently, membranes were incubated overnight in 4 • C with undiluted peritoneal fluid. After extensive washing with PBS Tween (0.05%), the membrane was incubated with anti-mouse IgA-HRP complex diluted 1:5000 (Novus Biologicals) for one hour. Protein bands were detected with DAB/H 2 O 2 solution for 20 min. The membrane was dried and analyzed in a Molecular Imager Gel Doc TM XR + visualization system.

Identification of N-Acetyl-glucosamine Residues in T. spiralis Somatic Antigen
After electrophoresis and transfer of proteins onto nitrocellulose, the membranes were blocked with 1% BSA in PBS. N-acetyl-glucosamine epitopes on T. spiralis antigens were developed using a Biotinylated Lectin Kit I (Vector Laboratories, Burlingame, CA, USA) using Wheat Germ Agglutinin (WGA). The blots were washed with 10 mM TRIS-buffered saline (TBS, pH 7.4), blocked overnight with 3% (w/v) bovine serum albumin (Sigma) in TBS (TBS-BSA), washed again with TBS and incubated for two hours with lectin diluted in TBS-BSA 1:1000. After extensive washing with TBS, the blots were incubated for two hours at room temperature in TBS-BSA containing avidin-alkaline phosphatase (Sigma-Aldrich Inc., St. Louis, MO, USA) diluted 1:100,000. The blots were washed with TBS and subsequently developed using BCIP/NBT (SIGMA FAST™ 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. Sigma-Aldrich Inc.). The reaction was stopped by bathing the blots in distilled water.

Fourier Transform Infrared Spectroscopy (FTIR) Analysis
Samples of 200 µL of peritoneal fluid were taken from mice injected with 5 mL of RPMI at 37 • C after a 1.5 min massage of the abdomen. Samples were evaluated after being cooled to 4 • C, before being frozen at −20 • C until use. Attenuated Total Reflectance (ATR) analyses of the initial chitosan and peritoneal samples were recorded on a Genesis II FTIR spectrophotometer (Mattson, Madison, WI, USA) equipped with an ATR device (MIRacleTM PIKE Technologies, Madison, WI, USA) and a zinc selenide (ZnSe) crystal. For FTIR analysis, the initial chitosan and peritoneal samples were poured on the spectrophotometric windows from CaBr 2 (Sigma-Aldrich) and evaporated at room temperature (25 • C) for 24 h. FTIR spectra were collected in the wavenumber range between 4000 cm −1 and 600 cm −1 , at a resolution of 4 cm −1 using 64-times scanning [69].

Histopathological Examination of Granuloma on the Surface of the Liver
The organs were isolated and immediately immersed in Neg-50™ Frozen Section Medium (Thermo Scientific, Dreieich, Germany) and frozen in liquid nitrogen. The samples were stored at −80 • C and then cut into 10 µm sections in cryomicrotome at −20 • C, and fixed at 90% methanol for five minutes. Standard staining with Cole's hematoxylin and acid eosin Y was performed. The preparations were observed under a light microscope.

Statistical Analysis
The results are presented as mean and standard error. Data was analyzed with ANOVA and the Student's t-test. Differences between groups were considered significant when the p value < 0.05.

Conclusions
Chitosan appears to augment cellular infiltration, with an immunoregulative phenotype supporting T. spiralis infection, and its biodegraded particles, rich in N-acetyl-D-glucosamine and D-glucosamine, may be involved in the process. These findings may be relevant for both people and animals treated with chitosan preparations.