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.

Comparison of immune responses and intestinal flora in epicutaneously sensitized BALB/c or C57BL/6 mouse models of food allergy

Cutaneous exposure to food allergens through a disrupted skin barrier is recognized as an important cause of food allergy,and the cutaneous sensitized mouse model has been established to investigate relevant allergic disorders.However,the role of different genetic backgrounds of mice on immune responses to food allergens upon epicutaneous sensitization is largely unknown.In this study,two strains of mice,i.e.,the BALB/c and C57BL/6 mice,were epicutaneously sensitized with ovalbumin on atopic dermatitis(AD)-like skin lesions,followed by intragastric challenge to induce IgE-mediated food allergy.Allergic outcomes were measured as clinical signs,specific antibodies and cytokines,and immune cell subpopulations,as well as changes in intestinal barrier function and gut microbiota.Results showed that both strains of mice exhibited typical food-allergic symptoms with a Th2-skewed response.The C57BL/6 mice,rather than the BALB/c mice,were fitter for establishing an epicutaneously sensitized model of food allergy since a stronger Th2-biased response and severer disruptions in the intestinal barrier and gut homeostasis were observed.This study provides knowledge for selecting an appropriate mouse model to study food-allergic responses associated with AD-like skin lesions and highlights the role of genetic variations in the immune mechanism underlying pathogenesis of food allergy.

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.

Helicobacter pylori infection:How does age influence the inflammatory pattern?

The inflammatory pattern during Helicobacter pylori(H.pylori)infection is changeable and complex.During childhood,it is possible to observe a predominantly regulatory response,evidenced by high concentrations of key cytokines for the maintenance of Treg responses such as TGF-β1 and IL-10,in addition to high expression of the transcription factor FOXP3.On the other hand,there is a predominance of cytokines associated with the Th1 and Th17 responses among H.pylori-positive adults.In the last few years,the participation of the Th17 response in the gastric inflammation against H.pylori infection has been highlighted due to the high levels of TGF-β1 and IL-17 found in this infectious scenario,and growing evidence has supported a close relationship between this immune response profile and unfavorable outcomes related to the infection.Moreover,this cytokine profile might play a pivotal role in the effectiveness of anti-H.pylori vaccines.It is evident that age is one of the main factors influencing the gastric inflammatory pattern during the infection with H.pylori,and understanding the immune response against the bacterium can assist in the development of alternative prophylactic and therapeutic strategies against the infection as well as in the comprehension of the pathogenesis of the outcomes related to that microorganism.

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.

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.

Antigen presentation by dendritic cells and their instruction of CD4+ T helper cell responses

Dendritic cells are powerful antigen-presenting cells that are essential for the priming of T cell responses.In addition to providing T-cell-receptor ligands and co-stimulatory molecules for naive T cell activation and expansion,dendritic cells are thought to also provide signals for the differentiation of CD4+T cells into effector T cell populations.The mechanisms by which dendritic cells are able to adapt and respond to the great variety of infectious stimuli they are confronted with,and prime an appropriate CD4+T cell response,are only partly understood.It is known that in the steady-state dendritic cells are highly heterogenous both in phenotype and transcriptional profile,and that this variability is dependent on developmental lineage,maturation stage,and the tissue environment in which dendritic cells are located.Exposure to infectious agents interfaces with this pre-existing heterogeneity by providing ligands for pattern-recognition and toll-like receptors that are variably expressed on different dendritic cell subsets,and elicit production of cytokines and chemokines to support innate cell activation and drive T cell differentiation.Here we review current information on dendritic cell biology,their heterogeneity,and the properties of different dendritic cell subsets.We then consider the signals required for the development of different types of Th immune responses,and the cellular and molecular evidence implicating different subsets of dendritic cells in providing such signals.We outline how dendritic cell subsets tailor their response according to the infectious agent,and how such transcriptional plasticity enables them to drive different types of immune responses.

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.

Mesenchymal stromal cells as potential immunomodulatory players in severe acute respiratory distress syndrome induced by SARSCoV- 2 infection

Severe acute respiratory syndrome coronavirus-2 and the related coronavirus disease-19(COVID-19)is a worldwide emerging situation,which was initially reported in December 2019 in Wuhan,China.Currently,more than 7258842 new cases,and more than 411879 deaths have been reported globally.This new highly transmitted coronavirus is responsible for the development of severe acute respiratory distress syndrome.Due to this disorder,a great number of patients are hospitalized in the intensive care unit followed by connection to extracorporeal membrane oxygenation for breath supporting and survival.Severe acute respiratory distress syndrome is mostly accompanied by the secretion of proinflammatory cytokines,including interleukin(IL)-2,IL-6,IL-7,granulocyte colony-stimulating factor(GSCF),interferon-inducible protein 10(IP10),monocyte chemotactic protein-1(MCP1),macrophage inflammatory protein 1A(MIP1A),and tumor necrosis factor alpha(TNF-α),an event which is known as“cytokine storm”.Further disease pathology involves a generalized modulation of immune responses,leading to fatal multiorgan failure.Currently,no specific treatment or vaccination against severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)has been developed.Mesenchymal stromal cells(MSCs),which are known for their immunosuppressive actions,could be applied as an alternative co-therapy in critically-ill COVID-19 patients.Specifically,MSCs can regulate the immune responses through the conversion of Th1 to Th2,activation of M2 macrophages,and modulation of dendritic cells maturation.These key immunoregulatory properties of MSCs may be exerted either by produced soluble factors or by cell-cell contact interactions.To date,several clinical trials have been registered to assess the safety,efficacy,and therapeutic potential of MSCs in COVID-19.Moreover,MSC treatment may be effective for the reversion of ground-glass opacity of damaged lungs and reduce the tissue fibrosis.Taking into account the multifunctional properties of MSCs,the proposed stem-cell-based therapy may be proven significantly effective in critically-ill COVID-19 patients.The current therapeutic strategy may improve the patient’s overall condition and in parallel may decrease the mortality rate of the current disease.

Type 1 T-cell responses in chlamydial lung infections are associated with local MIP-1a response

Chemokines and their receptors are important mediators of leukocyte trafficking and recruitment and sometimes work as modulators of T-cell responses during infections and inflammation.Modulating the biological activity of chemokines has been found to influence the course of diseases.However,little is known about the role of chemokine responses during chlamydial lung infections.We therefore analyzed the dynamics of multiple chemokines,which are frequently associated with type 1(Th1)T cell immune responses,and their receptors for their expression in the lungs during Chlamydia muridarum(Cm)infections.We also examined the relationship between chemokine responses and the development of Th1 responses as well as the clearance of infection.Our results showed that in parallel with the high levels of gamma interferon(IFN-c)and IL-12 production in the lungs and draining lymph nodes,and the expansion of IFN-c-producing CD4 and CD81 T cells,the production of the cell-related chemokines RANTES,IFN-c-inducible protein-10(IP-10)and macrophage inflammatory protein-1a(MIP-1a)and their receptor CCR1 was elevated in the lung tissues after infection.Interestingly,in a later phase of infection,the expression of RANTES and IP-10 remained elevated but the expression of MIP-1a and CCR1 decreased to a low level,which suggests a closer association with the pattern of Th1 cytokine responses in the process of infection.These results suggest a close association between the MIP-1a response and the Th1-type T-cell responses in chlamydial lung infections .

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.

Platelets promote allergic asthma through the expression of CD154

Platelet activation is associated with multiple immune responses and the pathogenesis of various immune-related diseases. However, the exact role and the underlying mechanism of platelets in the progression of allergic asthma remain largely unclear. In this study, we demonstrate that during antigen sensitization, platelets can be activated by ovalbumin （OVA） aerosol viathe upregulation of CD154 （CD4OL） expression. Platelet transfer promoted allergic asthma progression by inducing more severe leukocyte infiltration and lung inflammation, elevated IgE production and strengthened T helper 2 （Th2） responses in asthma-induced mice. Accordingly, platelet depletion compromised allergic asthma progression. CdI54-deficient platelets failed to promote asthma development, indicating the requirement of CD154 for platelets to promote asthma progression. The mechanistic study showed that platelets inhibited the induction of Foxp3 ＋ regulatory T cells both in vivoand in vitroat least partially through CD154, providing an explanation for the increase of Th2 responses by platelet transfer. Our study reveals the previously unknown role of platelet CD154 in the promotion of asthma progression by polarizing Th2 responses and inhibiting regulatory T-cell generation and thus provides a potential clue for allergic disease interventions.