Plasmodium yoelii Infection Enhances the Expansion of Myeloid-Derived Suppressor Cells via JAK/STAT3 Pathway.

Myeloid-derived suppressor cells (MDSCs), the negative immune regulators, have been demonstrated to be involved in immune responses to a variety of pathological conditions, such as tumors, chronic inflammation, and infectious diseases. However, the roles and mechanisms underlying the expansion of MDSCs in malaria remain unclear. In this study, the phenotypic and functional characteristics of splenic MDSCs during Plasmodium yoelii NSM infection are described. Furthermore, we provide compelling evidence that the sera from P. yoelii-infected C57BL/6 mice containing excess IL-6 and granulocyte-macrophage colony-stimulating factor promote the accumulation of MDSCs by inducing Bcl2 expression. Serum-induced MDSCs exert more potent suppressive effects on T cell responses than control MDSCs within both in vivo P. yoelii infection and in vitro serum-treated bone marrow cells experiments. Serum treatment increases the MDSC inhibitory effect, which is dependent on Arg1 expression. Moreover, mechanistic studies reveal that the serum effects are mediated by JAK/STAT3 signaling. By inhibiting STAT3 phosphorylation with the JAK inhibitor JSI-124, effects of serum on MDSCs are almost eliminated. In vivo depletion of MDSCs with anti-Gr-1 or 5-fluorouracil significantly reduces the parasitemia and promotes Th1 immune response in P. yoelii-infected C57BL/6 mice by upregulating IFN-γ expression. In summary, this study indicates that P. yoelii infection facilitates the accumulation and function of MDSCs by upregulating the expression of Bcl2 and Arg1 via JAK/STAT3 signaling pathway in vivo and in vitro. Manipulating the JAK/STAT3 signaling pathway or depleting MDSCs could be promising therapeutic interventions to treat malaria.

Characterization of γδT cells in lung of Plasmodium yoelii-infected C57BL/6 mice.

BACKGROUND: Malaria has high morbidity and mortality rates in some parts of tropical and subtropical countries. Besides respiratory and metabolic function, lung plays a role in immune system. γδT cells have multiple functions in producing cytokines and chemokines, regulating the immune response by interacting with other cells. It remains unclear about the role of γδT cells in the lung of mice infected by malaria parasites. METHODS: Flow cytometry (FCM) was used to evaluate the frequency of γδT cells and the effects of γδT cells on the phenotype and function of B and T cells in Plasmodium yoelii-infected wild-type (WT) or γδTCR knockout (γδT KO) mice. Haematoxylin-eosin (HE) staining was used to observe the pathological changes in the lungs. RESULTS: The percentage and absolute number of γδT cells in the lung increased after Plasmodium infection (p < 0.01). More γδT cells were expressing CD80, CD11b, or PD-1 post-infection (p < 0.05), while less γδT cells were expressing CD34, CD62L, and CD127 post-infection (p < 0.05). The percentages of IL-4+, IL-5+, IL-6+, IL-21+, IL-1α+, and IL-17+ γδT cells were increased (p < 0.05), but the percentage of IFN-γ-expressing γδT cells decreased (p < 0.05) post-infection. The pathological changes in the lungs of the infected γδT KO mice were not obvious compared with the infected WT mice. The proportion of CD3+ cells and absolute numbers of CD3+ cells, CD3+ CD4+ cells, CD3+ CD8+ cells decreased in γδT KO infected mice (p < 0.05). γδT KO infected mice exhibited no significant difference in the surface molecular expression of T cells compared with the WT infected mice (p > 0.05). While, the percentage of IFN-γ-expressing CD3+ and CD3+ CD8+ cells increased in γδT KO infected mice (p < 0.05). There was no significant difference in the absolute numbers of the total, CD69+, ICOS+, and CD80+ B cells between the WT infected and γδT KO infected mice (p > 0.05). CONCLUSIONS: The content, phenotype, and function of γδT cells in the lung of C57BL/6 mice were changed after Plasmodium infection. γδT cells contribute to T cell immune response in the progress of Plasmodium infection.

Changes of CD103-expressing pulmonary CD4+ and CD8+ T cells in S. japonicum infected C57BL/6 mice.

BACKGROUND: Recent studies have shown that CD103 is an important marker for tissue-resident memory T cells (TRM) which plays an important role in anti-infection. However, the role of CD103+ TRM was not elucidated in the progress of S. japonicum infection induced disease. METHODS: 6-8 weeks old C57BL/6 mice were infected by S. japonicum. Mice were sacrificed and the lungs were removed 5-6 weeks after infection. Immunofluorescent staining and Q-PCR were performed to identify the expression of CD103 molecule. Single cellular populations were made, percentages of CD103 on both CD4+ and CD8+ T lymphocytes were dynamical observed by flow cytometry (FCM). Moreover, the expression of memory T cells related molecules CD69 and CD62L, T cell function associated molecules CD107a, IFN-γ, IL-4, IL-9, and IL-10 were compared between CD103+ CD4+ and CD8+ T cells by FCM. RESULTS: CD103+ cells were emerged in the lung of both naive and S. japonicum infected mice. Both the percentage and the absolute numbers of pulmonary CD4+ and CD8+ cells were increased after S. japonicum infection (P < 0.05). The percentage of CD103+ cells in CD8+ T cells decreased significantly at the early stage of S. japonicum infection (P < 0.05). Increased CD69, decreased CD62L and CD107a expressions were detected on both CD4+ and CD8+ CD103+ T cells in the lungs of infected mice (P < 0.05). Compared to CD8+ CD103+ T cells, CD4+ CD103+ T cells from infected mice expressed higher level of CD69 and lower level CD62L molecules (P < 0.05). Moreover, higher percentage of IL-4+, IL-9+ and IL-10+ cells on CD4+ CD103+ pulmonary T cells was found in infected mice (P < 0.05). Significantly increased IL-4 and IL-9, and decreased IFN-γ expressing cells were detected in CD8+CD103+ cells of infected mice (P < 0.05). CONCLUSIONS: CD103-expressing pulmonary CD4+ and CD8+ T cells play important roles in mediating S. japonicum infection induced granulomatous inflammation in the lung.

A Schistosoma japonicum Infection Promotes the Expansion of Myeloid-Derived Suppressor Cells by Activating the JAK/STAT3 Pathway.

Myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immune cells from the myeloid lineage, play an important part in suppression of host immune responses during many pathologic conditions, including cancer and infectious diseases. Thus, understanding the functional diversity of these cells as well as the underlying mechanisms is crucial for the development of disease control strategies. The role of MDSCs during Schistosoma japonicum infection, however, is not clear, and there is a lack of systematic study so far. In this study, we provide strong evidence that the soluble egg Ag (SEA) and schistosome worm Ag (SWA) of S. japonicum enhance the accumulation of MDSCs. Ag-induced MDSCs have more potent suppressive effects on T cell responses than do control MDSCs in both in vivo S. japonicum infection and in vitro SEA- and SWA-treated mouse bone marrow cells experiments. Interestingly, the enhanced suppressive activity of MDSCs by Ag administration was coupled with a dramatic induction of the NADPH oxidase subunits gp91phox and p47phox and was dependent on the production of reactive oxygen species. Moreover, mechanistic studies revealed that the Ag effects are mediated by JAK/STAT3 signaling. Inhibition of STAT3 phosphorylation by the JAK inhibitor JSI-124 almost completely abolished the Ag effects on the MDSCs. In summary, this study sheds new light on the immune modulatory role of SEA and SWA and demonstrates that the expansion of MDSCs may be an important element of a cellular network regulating immune responses during S. japonicum infection.

Interleukin-27 enhances TNF-α-mediated activation of human coronary artery endothelial cells.

Immune cells and inflammatory mediators play important roles in the development of atherosclerotic vascular inflammation. IL-27 is a member of the IL-6/IL-12 family that can promote Th1 responses and augment the release of inflammatory mediators from human mast cells and monocytes. However, the direct effect of IL-27 on human coronary artery endothelial cells was unclear. In this study, the effects of IL-27 and TNF-α on the cell surface expression of adhesion molecules, inflammatory cytokines, and chemokines were investigated. Results showed that IL-27 alone could significantly promote the release of chemokine CXCL10. However, IL-27 could further significantly enhance the TNF-α-mediated upregulation of adhesion molecules ICAM-1 and VCAM-1, inflammatory cytokine IL-6, as well as chemokines CCL5 and CXCL10 from human coronary artery endothelial cells. The release of IL-6, CCL5, and CXCL10 were significantly suppressed by specific signaling molecule inhibitors, implying that the induction of these mediators from the human coronary artery endothelial cells could be differentially regulated by the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and nuclear factor-κB pathways. These results provided new insights into the effect of IL-27 on the TNF-α mediated activation of human coronary artery endothelial cells in atherosclerotic vascular inflammation.

Characteristics of Th9 cells in Schistosoma japonicum-infected C57BL/6 mouse mesenteric lymph node.

Interleukin 9 (IL-9) is an effective cytokine secreted by newly defined Th9 cells, which is involved in allergic and infectious diseases. In this study, lymphocytes were isolated from mesenteric lymph node (MLN), spleen, liver, lung, and Peyer's patches (PP) of C57BL/6 mice 5-6 weeks after S. japonicum infection, intracellular cytokine staining was done to detect the percentage of IL-9-producing CD4+ T cells. The qPCR and ELISA were used to verify the content of IL-9 in MLN. The population of IL-9-producing lymphocyte subset was identified by FACS. In addition, the dynamic changes and cytokine profiles of Th9 cells in the MLN of infected mice were detected by FACS. ELISA was used to detect IL-9 induced by soluble egg antigen (SEA) from isolated lymphocytes in mouse MLN. The results showed that the percentage of IL-9-secreting Th9 cells in the MLN of the infected mouse was higher than that in the spleen, liver, lung, or PP. Though CD8+ Tc cells, NKT cells, and γδT cells could secrete IL-9, CD4+ Th cells were the main source of IL-9 in S. japonicum-infected C57BL/6 mice (P < 0.05). The percentage of Th9 cells in MLN of infected mouse increased from week 3-4, and reached a peak at week 5-6, then began to decrease from week 7-8 (P < 0.05). Moreover, Th9 cells could also secrete a small amount of IL-4, IFN-γ, IL-5, and IL-10. Our results suggested a higher percentage of Th9 cells was induced in the MLN of S. japonicum-infected mice, which might play an important role in the early stage of S. japonicum-induced disease.

TLR7 controls myeloid-derived suppressor cells expansion and function in the lung of C57BL6 mice infected with Schistosoma japonicum.

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune responses against Schistosoma japonicum (S. japonicum) infection. However, the role of Toll-like receptor 7 (TLR7) in the mouse lung during S. japonicum infection and the myeloid-derived suppressor cells (MDSCs) affected by the absence of TLR7 are not clearly understood. In this study, the results indicated that the MDSCs were accumulated and the proportion and activation of CD4+ and CD8+ T cells were decreased in the lung of mice at 6-7 weeks after S. japonicum infection. Then, the expression of TLR7 was detected in isolated pulmonary MDSCs and the results showed that the expression of TLR7 in MDSCs was increased after infection. Furthermore, TLR7 agonist R848 could down-regulate the induction effect of the soluble egg antigen (SEA) on pulmonary MDSCs in vitro. Meanwhile, TLR7 deficiency could promote the pulmonary MDSCs expansion and function by up-regulating the expression of PD-L1/2 and secreting of IL-10 in the mice infected with S. japonicum. Mechanistic studies revealed that S. japonicum infection and the antigen effects are mediated by NF-κB signaling. Moreover, TLR7 deficiency aggravates S. japonicum infection-induced damage in the lung, with more inflammatory cells infiltration, interstitial dilatation and granuloma in the tissue. In summary, this study indicated that TLR7 signaling inhibits the accumulation and function of MDSCs in S. japonicum infected mouse lung by down-regulating the expression of PD-L1/2 and secreting of IL-10, via NF-κB signaling.

TLR7 modulating B-cell immune responses in the spleen of C57BL/6 mice infected with Schistosoma japonicum.

B cells played an important role in Schistosoma infection-induced diseases. TLR7 is an intracellular member of the innate immune receptor. The role of TLR7 on B cells mediated immune response is still unclear. Here, C57BL/6 mice were percutaneously infected by S. japonicum for 5-6 weeks. The percentages and numbers of B cells increased in the infected mice (p < 0.05), and many activation and function associated molecules were also changed on B cells. More splenic cells of the infected mice expressed TLR7, and B cells were served as the main cell population. Moreover, a lower level of soluble egg antigen (SEA) specific antibody and less activation associated molecules were found on the surface of splenic B cells from S. japonicum infected TLR7 gene knockout (TLR7 KO) mice compared to infected wild type (WT) mice (p < 0.05). Additionally, SEA showed a little higher ability in inducing the activation of B cells from naive WT mice than TLR7 KO mice (p < 0.05). Finally, the effects of TLR7 on B cells are dependent on the activation of NF-κB p65. Altogether, TLR7 was found modulating the splenic B cell responses in S. japonicum infected C57BL/6 mice.

Interferon Regulatory Factor 4 Regulates the Development of Polymorphonuclear Myeloid-Derived Suppressor Cells Through the Transcription of c-Myc in Cancer.

The accumulation of myeloid-derived suppressor cells (MDSCs) is one of the major obstacles to achieve an appropriate anti-tumor immune response and successful tumor immunotherapy. MDSCs in tumor-bearing hosts are primarily polymorphonuclear (PMN-MDSCs). However, the mechanisms regulating the development of MDSCs remain poorly understood. In this report, we showed that interferon regulatory factor 4 (IRF4) plays a key role in the development of PMN-MDSCs, but not monocytic MDSCs. IRF4 deficiency caused a significant elevation of PMN-MDSCs and enhanced the suppressive activity of PMN-MDSCs, increasing tumor growth and metastasis in mice. Mechanistic studies showed that c-Myc was up-regulated by the IRF4 protein. Over-expression of c-Myc almost abrogated the effects of IRF4 deletion on PMN-MDSCs development. Importantly, the IRF4 expression level was negatively correlated with the PMN-MDSCs frequency and tumor development but positively correlated with c-Myc expression in clinical cancer patients. In summary, this study demonstrated that IRF4 represents a novel regulator of PMN-MDSCs development in cancer, which may have predictive value for tumor progression.

Adjustments of γδ T Cells in the Lung of Schistosoma japonicum-Infected C56BL/6 Mice.

Many kinds of lymphocytes are involved in Schistosoma japonicum (S. japonicum) infection-induced disease. γδ T cells comprise a small number of innate lymphocytes that quickly respond to foreign materials. In this study, the role of γδ T cells in the lung of S. japonicum-infected C56BL/6 mice was investigated. The results demonstrated that S. japonicum infection induces γδ T cell accumulation in the lung, expressing higher levels of CD25, MHCII, CD80, and PDL1, and lower levels of CD127 and CD62L (P < 0.05). The intracellular cytokines staining results illustrated higher percentages of IL-4-, IL-10-, IL-21-, and IL-6-producing γδ T cells and lower percentages of IFN-γ-expressing γδ T cells in the lung of infected mice (P < 0.05). Moreover, the granuloma size in lung tissue was significantly increased in Vδ-/- mice (P < 0.05). In the lung of S. japonicum-infected Vδ-/- mice, both type 1 and type 2 immune responses were decreased significantly (P < 0.05). In addition, the expression of CD80 and CD69 on B cells was decreased significantly (P < 0.05), and the SEA-specific antibody was markedly decreased (P < 0.05) in the blood of infected Vδ-/- mice. In conclusion, this study indicates that γδ T cells could adjust the Th2 dominant immune response in the lung of S. japonicum-infected mice.

VP19 is important for the envelope coating of white spot syndrome virus.

VP19 is a major envelope protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. However, the exact function of VP19 in WSSV assembly and infection is unknown. To understand the function of VP19, the gene was knocked down by RNA interference. We found that the dsRNA specific for vp19 gene dramatically reduced the replication of WSSV genomic DNA in infected animals. Further investigation by transmission electron microscopy showed that inhibition of VP19 prevented envelope coating of progeny virions, resulting in a high amount of immature virus particles without outer layer (envelope) in the host cells. This finding was further confirmed by SDS-PAGE analysis, which showed the loss of VP19 and other envelope proteins from the improperly assembled virions. These results suggest that VP19 is essential for WSSV envelope coating.

Expression of TLR2, TLR3, TLR4, and TLR7 on pulmonary lymphocytes of Schistosoma japonicum-infected C57BL/6 mice.

Despite the paramount role of TLRs in the induction of innate immune and inflammatory responses, there is a paucity of studies on the role of TLRs in Schistosoma japonicum infection. Here, we observed obvious infiltration of inflammatory cells in S. japonicum-infected C57BL/6 mouse lungs. Expression and release of IFN-γ, IL-4, and IL-17 were significantly higher in pulmonary lymphocytes from infected mice compared with control mice in response to anti-CD3 plus anti-CD28 mAbs. Higher percentages of TLR2, TLR3, TLR4, and TLR7 were expressed on such lymphocytes, and the TLR agonists PGN, Poly I:C, LPS, and R848 induced a higher level of IFN-γ. However, a higher level of IL-4 was found in the supernatant of pulmonary lymphocytes from infected mice stimulated by these TLR agonists plus CD3 Ab. Only R848 plus anti-CD3 mAb could induce a higher level of IFN-γ in such lymphocytes. TLR expressions were then compared on different pulmonary lymphocytes after infection, including T cells, B cells, NK cells, NKT cells, and γδT cells. The expression levels of TLR3 on T cells, B cells, NK cells, and γδT cells were increased in the lungs after infection. NK cells also expressed higher levels of TLR4 after infection of control mice. Collectively, these findings highlight the potential role of TLR expression in the context of S. japonicum infection.

Detection of T lymphocyte subsets and related functional molecules in follicular fluid of patients with polycystic ovary syndrome.

Immune responses play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the characteristics of T lymphocyte subsets in PCOS remain insufficiently understood. In this study, lymphocytes of follicular fluid (FF) were obtained from oocyte retrieval before in-vitro fertilization (IVF) in infertile women with or without PCOS. The levels of cluster of differentiation 25 (CD25), CD69, programmed death 1 (PD-1), interferon-γ (IFN-γ), interleukin 17A (IL-17A) and IL-10 in T lymphocytes were determined by flow cytometry. Our results showed that the percentage of FF CD8+ T cells was significantly decreased in infertile patients with PCOS (P < 0.05). Furthermore, the levels of CD69 and IFN-γ were significantly decreased and the level of PD-1 was increased in both CD4+ and CD8+ T cells from infertile patients with PCOS (P < 0.05). Moreover, the expression of PD-1 on CD4+ or CD8+ T cells was positively correlated with the estradiol (E2) levels in the serum and reversely correlated with the expression of IFN-γ in CD4+ or CD8+ T cells in infertile patients with PCOS. These results suggested that T cell dysfunction may be involved in the pathogenesis of PCOS.

Schistosoma japonicum Infection Promotes the Response of Tfh Cells Through Down-Regulation of Caspase-3-Mediating Apoptosis.

CD4+ T follicular helper (Tfh) cells, a new subset of immune cells, have been demonstrated to be involved in granulomatous responses to Schistosoma japonicum (S. japonicum) infection. However, the role and underlying mechanisms of Tfh cell aggregation in S. japonicum infection remain incompletely understood. In this study, we provide evidence that S. japonicum infection enhances the accumulation of Tfh cells in the spleen, lymph nodes, and peripheral blood of C57BL/6 mice. Infection-induced Tfh cells exhibited more potent effects directly on B cell responses than the control Tfh cells (P < 0.05). Furthermore, reduced apoptosis of Tfh cells was found both in S. japonicum infected mice and in soluble egg antigen (SEA) treated Tfh cells (P < 0.05). Mechanistic studies reveal that caspase-3 is the primary drivers of down-regulated apoptotic Tfh cell death in S. japonicum infection. In summary, this study demonstrates that Tfh cell accumulation might have an impact on the generation of immune responses in S. japonicum infection, and caspase-3 signaling mediated apoptosis down-regulation might responsible for the accumulation of Tfh cell in this course.

TLR7 Modulated T Cell Response in the Mesenteric Lymph Node of Schistosoma japonicum-Infected C57BL/6 Mice.

Toll-like receptors (TLRs) play an important role in regulating immune responses during pathogen infection. However, roles of TLRs on T cells reside in the mesenteric lymph node (MLN) were not be fully elucidated in the course of S. japonicum infection. In this study, T lymphocytes from the mesenteric lymph node (MLN) of S. japonicum-infected mice were isolated and the expression and roles of TLR2, TLR3, TLR4, and TLR7 on both CD4+ and CD8+ T cells were compared. We found that the expression of TLR7 was increased in the MLN cells of S. japonicum-infected mice, particularly in CD4+ and CD8+ T cells (P < 0.05). R848, a TLR7 agonist, could enhance the production of IFN-γ from MLN T cells of infected mice (P < 0.05), especially in CD8+ T cells (P < 0.01). In TLR7 gene knockedout (KO) mice, the S. japonicum infection caused a significant decrease (P < 0.05) of the expression of CD25 and CD69, as well as the production of IFN-γ and IL-4 inducted by PMA plus ionomycin on both CD4+ and CD8+ T cells. Furthermore, the decreased level of IFN-γ and IL-4 in the supernatants of SEA- or SWA-stimulated mesenteric lymphocytes was detected (P < 0.05). Our results indicated that S. japonicum infection could induce the TLR7 expression on T cells in the MLN of C57BL/6 mice, and TLR7 mediates T cell response in the early phase of infection.

TLR3 Modulates the Response of NK Cells against Schistosoma japonicum.

Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious diseases. NK cells possess many kinds of TLRs that allow them to sense and respond to invading pathogens. Our previous study found that NK cells could modulate the immune response induced by Schistosoma japonicum (S. japonicum) in C57BL/6 mice. In the present study, the role of TLRs in the progress of S. japonicum infection was investigated. Results showed that the expression of TLR3 on NK cells increased significantly after S. japonicum infection by using RT-PCR and FACS (P < 0.05). TLR3 agonist (Poly I:C) increased IFN-γ and IL-4 levels in the supernatant of cultured splenocytes and induced a higher percentage of IFN-γ- and IL-4-secreting NK cells from infected mouse splenocytes (P < 0.05). Not only the percentages of MHC II-, CD69-, and NKG2A/C/E-expressing cells but also the percentages of IL-4-, IL-5-, and IL-17-producing cells in TLR3+ NK cells increased significantly after infection (P < 0.05). Moreover, the expression of NKG2A/C/E, NKG2D, MHC II, and CD69 on the surface of splenic NK cells was changed in S. japonicum-infected TLR3-/- (TLR3 KO mice, P < 0.05); the abilities of NK cells in IL-4, IL-5, and IL-17 secretion were decreased too (P < 0.05). These results indicate that TLR3 is the primary molecule which modulates the activation and function of NK cells during the course of S. japonicum infection in C57BL/6 mice.

Characteristics of IL-9 induced by Schistosoma japonicum infection in C57BL/6 mouse liver.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9+ Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8+ Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

NOD2 and TLR2 ligands trigger the activation of basophils and eosinophils by interacting with dermal fibroblasts in atopic dermatitis-like skin inflammation.

The skin of patients with atopic dermatitis (AD) has a unique predisposition for colonization by Staphylococcus aureus (S. aureus), which contributes to the inflammation and grim prognosis of AD. Although the mechanism underlying the S. aureus-induced exacerbation of AD remains unclear, recent studies have found a pivotal role for pattern recognition receptors in regulating the inflammatory responses in S. aureus infection. In the present study, we used a typical mouse model of AD-like skin inflammation and found that S. aureus-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and toll-like receptor 2 (TLR2) ligands exacerbated AD-like symptoms, which were further deteriorated by the in vivo expansion of basophils and eosinophils. Subsequent histological analyses revealed that dermal fibroblasts were pervasive in the AD-like skin lesions. Co-culture of human dermal fibroblasts with basophils and eosinophils resulted in a vigorous cytokine/chemokine response to the NOD2/TLR2 ligands and the enhanced expression of intercellular adhesion molecule-1 on the dermal fibroblasts. Basophils and eosinophils were primarily responsible for the AD-related cytokine/chemokine expression in the co-cultures. Direct intercellular contact was necessary for the crosstalk between basophils and dermal fibroblasts, while soluble mediators were sufficient to mediate the eosinophil-fibroblast interactions. Moreover, the intracellular p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and nuclear factor-kappa B signaling pathways were essential for NOD2/TLR2 ligand-mediated activation of basophils, eosinophils, and dermal fibroblasts in AD-related inflammation. This study provides the evidence of NOD2/TLR2-mediated exacerbation of AD through activation of innate immune cells and therefore sheds light on a novel mechanistic pathway by which S. aureus contributes to the pathophysiology of AD.

NOD-like receptors mediated activation of eosinophils interacting with bronchial epithelial cells: a link between innate immunity and allergic asthma.

Key intracytosolic pattern recognition receptors of innate immunity against bacterial infections are nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). We elucidated the NOD1 and NOD2-mediated activation of human eosinophils, the principal effector cells for allergic inflammation, upon interacting with human bronchial epithelial BEAS-2B cells in allergic asthma. Eosinophils constitutively expressed NOD1,2 but exhibited nonsignificant responses to release chemokines upon the stimulation by NOD1 ligand γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 ligand muramyl dipeptide (MDP). However, iE-DAP and MDP could significantly upregulate cell surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and ICAM-1 on BEAS-2B cells, as well as induce chemokines CCL2 and CXCL8 release in the coculture system (all P<0.05). Both eosinophils and BEAS-2B cells were the main source for CXCL8 and CCL2 release in the coculture system upon iE-DAP or MDP stimulation. Direct interaction between eosinophils and BEAS-2B cells is responsible for CCL2 release, and soluble mediators are implicated in CXCL8 release. ERK and NF-κB play regulatory roles for the expression of adhesion molecules and chemokines in coculture. Treatment with NOD1,2 ligand could induce the subepithelial fibrosis and significantly enhance the serum concentration of total IgE, chemokine CCL5 for eosinophils and T helper type 2 (Th2) cells and asthma Th2 cytokine IL-13 in bronchoalveolar lavage fluid of ovalbumin-sensitized allergic asthmatic mice (all P<0.05). This study provides further evidence of bacterial infection-mediated activation of NOD1,2 in triggering allergic asthma via the activation of eosinophils interacting with bronchial epithelial cells at inflammatory airway.

Activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine IL-31 and alarmin IL-33: implications in atopic dermatitis.

BACKGROUND: IL-31 is a pruritogenic cytokine, and IL-33 is an alarmin for damaging inflammation. They together relate to the pathogenesis of atopic dermatitis (AD). Eosinophil infiltration into the inner dermal compartment is a predominant pathological feature of AD. We herein investigated the in vitro inflammatory effects of IL-31 and IL-33 on the activation of human eosinophils and dermal fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: Receptors, adhesion molecules and signaling molecules were assessed by Western blot or flow cytometry. Chemokines and cytokine were quantitated by multiplex assay. Functional IL-31 receptor component IL-31RA, OSMR-ß and IL-33 receptor component ST2 were constitutively expressed on the surface of eosinophils. Co-culture of eosinophils and fibroblasts significantly induced pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with IL-31 and IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was up-regulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, nuclear factor-κB and phosphatidylinositol 3-kinase-Akt pathways. Using specific signaling molecule inhibitors, the differential induction of IL-31 and IL-33-mediated release of cytokines and chemokines such as IL-6 and CXCL8 from co-culture should be related to their distinct activation profile of intracellular signaling pathways. CONCLUSIONS/SIGNIFICANCE: The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms.