Interactions of thymic stromal lymphopoietin with interleukin-4 in adaptive immunity during Aspergillus fumigatus keratitis

AIM:To investigate the potential interactions of thymic stromal lymphopoietin(TSLP)with interleukin-4(IL-4)in adaptive immunity during fungal keratitis(FK).METHODS:An FK mouse model was induced with Aspergillus fumigatus(AF)hyphal infection.Mice were divided into several groups:untreated,phosphate buffer saline(PBS),infected with AF,and pretreated with a scrambled siRNA,a TSLP-specific siRNA(TSLP siRNA),murine recombinant TSLP(rTSLP),immunoglobulin G(IgG),murine recombinant IFN(rIFN-γ),murine recombinant IL-4(rI L-4),rIL-13,murine recombinant IL-17A(rIL-17A),and murine recombinant IL-17F(rIL-17F)groups.Quantitative realtime reverse transcription-polymerase chain reaction(qRTPCR)and enzyme-linked immunosorbent assay(ELISA)or Western blot were performed to determine mRNA and protein levels in the inflamed cornea.Cytokine locations were observed by immunofluoresence staining after AF hyphal infection.RESULTS:Compared to those in the untreated group,TSLP and T helper type 1(Th1)cytokine levels in the AF group were upregulated at 24 h post infection(hpi),and those of T helper type 2(Th2)and T helper type 17(Th17)cytokines were increased at 5 d post infection(dpi).Th2 cytokine levels were decreased in the TSLP siRNA-pretreated group and increased in the rTSLP-pretreated group compared with the AF group.The TSLP level was increased in the rIL-4-pretreated group,but there were no significant changes among the other groups.Immunofluorescence staining showed cytokine locations after AF hyphal infection.CONCLUSION:TSLP induces a Th2 immune response and promots Th2 T cell differentiation in vivo.IL-4 promotes TSLP secretion.Therefore,TSLP with IL-4 regulates adaptive immunity in FK.

Acupoint catgut-embedding therapy ameliorates DNCB-induced atopic dermatitis in BALB/c mice by regulating Th2 type immune response and reducing infiltration of CD4^(+)and CD8^(+)cells

Background:This study aimed to assess how acupoint catgut-embedding therapy influences Th2-type immune response and the infiltration of CD4^(+)and CD8^(+)cells in DNCB-induced atopic dermatitis in BALB/c mice.It also conducted an initial examination of the underlying molecular mechanisms.Methods:Seventy-two mice were randomly divided into four groups:normal control,DNCB-induced atopic dermatitis model(AD),AD with acupoint catgut-embedding treatment(ADA),and AD with sham-acupoint catgut-embedding treatment.After DNCB challenge to induce AD,the ADA group received acupoint catgut-embedding therapy treatment at Zusanli(ST 36)and Quchi(LI 11)acupoints every other week from day 8.Mice in the AD with sham-acupoint catgut-embedding treatment group underwent the same procedure as the ADA group but without catgut implantation.Severity was assessed using SCORAD on treatment days 1,10,and 20.On day 18,nine mice per group were euthanized,and the remaining on day 28.Histopathological changes were observed using hematoxylin-eosin and immunohistochemistry staining.TNF-α,IL-4,IL-6,and IL-13 levels were analyzed by ELISA,and GATA3 and STAT6 protein levels by western blot.Results:After 20 days of acupoint catgut-embedding therapy treatment,mice showed reduced dermatitis scores compared to DNCB-induced AD-like mice.Significant decreases occurred in serum IL-4,IL-6,IL-13,and TNF-αlevels.Skin analysis revealed marked reductions in CD4^(+)and CD8^(+)cell infiltration,as well as GATA3 and STAT6 protein levels.Conclusion:Acupoint catgut-embedding therapy may effectively alleviate atopic dermatitis by suppressing Th2 immune responses via the STAT6-GATA3 pathway and reducing CD4^(+)and CD8^(+)T cell infiltration in skin lesions.

Th immune response induced by H pylori vaccine with chitosan as adjuvant and its relation to immune protection

AIM： To study the immunological protective effect of H pylori vaccine with chitosan as an adjuvant and its mechanism. METHODS： Female BALB/c mice were randomly divided into seven groups and orally immunized respectively with PBS, chitosan solution, chitosan particles, H pylori antigen, H pylori antigen plus cholera toxin （CT）, H pylori antigen plus chitosan solution, Hpylori antigen plus chitosan particles once a week for four weeks. Four weeks after the last immunization, the mice were challenged twice by alive Hpylori （1 × 10^9 CFU/mL） and sacrificed. Part of the gastric mucosa was embedded in paraffin, cut into sections and assayed with Giemsa staining. Part of the gastric mucosa was used to quantitatively culture Hpylori. EUSA was used to detect cytokine level in gastric mucosa and anti- Hpylori IgG1, IgG2a levels in serum. RESULTS： In the groups with chitosan as an adjuvant, immunological protection was achieved in 60% mice, which was significantly higher than in groups with H pylori antigen alone and without H pylori antigen （P 〈 0.05 or 0.001）. Before challenge, the level of IFN and IL-12 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in the control group and the group without adjuvant （P 〈 0.05 or 0.005）. After challenge, the level of IFN and IL-12 was significantly higher in the groups with adjuvant than in the groups without adjuvant and antigen （P 〈 0.05 or 0.001）. Before challenge, the level of IL-2 in gastric mucosa was not different among different groups. After challenge the level of IL-2 was significantly higher in the groups with adjuvant than in the control group （P 〈 0.05 or 0.001）. Before challenge, the level of IL-10 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in other groups without adjuvant （P 〈 0.05 or 0.01）. After challenge, the level of IL-10 was not different among different groups. Before challenge, the level of IL-4 in gastric mucosa was significantly higher in the groups with chitosan as an adjuvant than in other groups without adjuvant （P 〈 0.05）. After challenge, the level of IL-4 was significantly higher in the groups with chitosan particles as an adjuvant than in the group with CT as an adjuvant （P 〈 0.05）, and in the group with chitosan solution as an adjuvant, the level of IL-4 was significantly higher than that in control group, non-adjuvant group and the groups with CT （P 〈 0.05 or 0.001）. The ratio of anti- Hpylori IgG2a/ IgG1 in serum was significantly lower in the groups with chitosan as an adjuvant than in the groups with CT as an adjuvant or without adjuvant （P 〈 0.01）. CONCLUSION： H pylori vaccine with chitosan as an adjuvant can protect against H pylori infection and induce both Thl and Th2 type immune response.

Analysis of Specific Th1/Th2 Helper Cell Responses and IgG Subtype Antibodies in Anti-CD4 Monoclonal Antibody Treated Mice with Autoimmune Cardiomyopathy

The cytokine repertoire of ADP/ATP carrier-specific humoral immune responses and the cytokine-dependent anti-ADP/ATP carrier antibody IgG subclasses were examined in a cohort of ADP/ATP carrier-immunized BALB/c mice treated with anti-CD4 monoclonal antibody. Eighteen male BALB/c mice （6–8 weeks old） were randomized into 3 groups： dilated cardiomyopathy （DCM） group, DCM-tolerance （Tol） group and control group. The mice in DCM group were immunized with the peptides derived from human ADP/ATP carrier protein for 6 months and mice in the control group were sham-immunized, while the mice in DCM-Tol group were immunized with ADP/ATP carrier protein and anti-CD4 McAb simultaneously. Serum autoantibody against ADP/ATP carrier and IgG subclasses were measured by ELISA, intracellular cytokines IFN-γ and IL-4 of Th cells were moni- tored with flow cytometry, and splenic T cell cytokines IFN-γ, IL-2, IL-4 and IL-6 were detected by using real-time fluorescent quantitative PCR. The results showed that the autoantibody against ADP/ATP carrier was found in all mice in DCM group, and the antibody level, serum IgG1 and IgG2a subclasses, cytokines in T cells and Th cells were all elevated in DCM group, as compared with those in control group （P〈0.01）. On the other hand, in DCM-Tol group, the autoantibody level and contents of all the cytokines were significantly different from those in DCM group （P〈0.01）, and were close to those in control group. And the levels of IgG1, IgG2a, IgG2b and IgG3 were influenced, to varying degrees, by anti-CD4 McAb as compared with those in DCM group. All these four types of IgG subclasses were substantially decreased in DCM-Tol group as compared with DCM group. It is concluded that the treatment with anti-CD4 McAb could prevent the activation of T cells, reverse the abnormal secretion of cytokines and the imbalance between Th1/Th2 cell subsets and abnormal production of autoantibody against ADP/ATP carrier, and eventually avoid myocardial injuries.

A new DNA vaccine fused with the C3d-p28 induces a Th2 immune response against amyloid-beta

To enhance anti-amyloid-beta （Aβ） antibody generation and induce a Th2 immune response, we constructed a new DNA vaccine p（Aβ3-10 ）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant. In this study, we administered this adjuvant intramus-cularly to female C57BL/6J mice at 8-10 weeks of age. Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody, isotypes, and cytokines in splenic T cel s. A 3-（4,5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to detect the prolifera-tion rate of splenic T cel s. Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques. The p（Aβ3-10）10-C3d-p28.3 vaccine induced high titers of anti-amyloid-βantibodies, which bound to Aβplaques in APP/PS1 transgenic mouse brain tissue, demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease. Moreover, the vaccine elicited a pre-dominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes, indicating that the vaccine could shift the cel ular immune response towards a Th2 phenotype. This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile. Our results indicate that the p（Aβ3-10）10-C3d-p28.3 vaccine is a promising immunothera-peutic option for Aβvaccination in Alzheimer’s disease.

Inactivated Pseudomonas PE(△Ⅲ)exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

The Dual Effects of Interleukin-18 in Tumor Progression

Interleukin-18 （IL-18） was discovered as an interferon-y-inducing factor and had a critical role in inflammatory and immune respouse. It stimulates natural killer （NK） and T cells and enhances Thl immune response. These activated immune cells eliminate cancer cells and virus-infected cells effectively. However, IL-18 has also been found to promote tumor progression. Higher expression or secretion level of IL-18 is detected in various cancer cells in comparison with normal control, and IL-18 is able to induce angiogenesis, migration/metastasis, proliferation and immune escape. These dual effects and the mechanism of IL-18 need to be investigated further as it relates to cancer.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors （TLRs） recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 （rBCSP31） to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases （MAPKs） by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-KB in macrophages. In addition, continuous exposure （〉24 h） of RAW264.7 cells to rBCSP31 significantly enhanced I FN-y-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4＋ T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2-/- and TLR4-/- mice was lower than that from C57BL/6 macrophages, and the activation of NF-KB and MAPKs was attenuated in macrophages from TLR2-/- and TLR4-/- mice. In addition, CD4＋ T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-y and IL-2 compared with CD4＋ T cells from TLR2-/- and TLR4-/- mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2-/- and TLR4-/- mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2-/- and TLR4-/- mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Thl immune response.

Maternal helminth infection protects offspring from high-fat-diet-induced obesity through altered microbiota and SCFAs

Helminth-induced Th2 immunity and gut microbiota have been recently shown to be highly effective in modulating metabolic syndromes in animal models.This study aimed to determine whether maternal immunity and microbial factors affect the induction and development of obesity in offspring.Here,Heligomosomoides polygyrus(Hp)-infected or control female C57BL/6J mice mated with normal males and their offspring were fed a high-fat diet(HFD)for 9 weeks after weaning.Our results showed that Hp-induced maternal outcomes during gestation and lactation significantly impacted offspring metabolic phenotypes.This was evidenced by results showing that offspring from helminth-infected mothers on an HFD(Hp-offspring+HFD)gained significantly less body weight than those from uninfected mothers(Cont-offspring+HFD).Hp-offspring+HFD exhibited no Th2 phenotype but displayed a pattern of gut microbiota composition similar to that of Hp-infected mothers.Cross-fostering experiments confirmed that the helminth-induced maternal attenuation of offspring obesity was mediated through both prenatal and postnatal effects.Our results further showed that helminth-infected dams and their offspring had a markedly altered gut microbiome composition,with increased production of short-chain fatty acids(SCFAs).Intriguingly,Hp-infected mothers and Hp-offspring+HFD showed increased SCFA receptor(GPR)expression in adipose and colonic tissues compared to noninfected mothers and Cont-offspring+HFD,respectively.Moreover,SCFA supplementation to the pups of uninfected control mothers during lactation protected against HFD-induced weight gain,which corresponded with changes in gut bacterial colonization.Collectively,our findings provide new insights into the complex interaction of maternal immune status and gut microbiome,Hp infection,and the immunity and gut microbiome in obese-prone offspring in infant life.