Monitoring of immune activation using biochemical changes in a porcine model of cardiac arrest

In animal models, immune activation is often difficult to assess because of the limited availability of specific assays to detect cytokine activities. In human monocytes/macrophages, interferon-gamma induces increased production of neopterin and an enhanced activity of indoleamine 2,3-dioxygenase, which degrades tryptophan via the kynurenine pathway. Therefore, monitoring of neopterin concentrations and of tryptophan degradation can serve to detect the extent of T helper cell 1-type immune activation during cellular immune response in humans. In a porcine model of cardiac arrest, we examined the potential use of neopterin measurements and determination of the tryptophan degradation rate as a means of estimating the extent of immune activation. Urinary neopterin concentrations were measured with high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) (BRAHMS Diagnostica, Berlin, Germany). Serum and plasma tryptophan and kynurenine concentrations were also determined using HPLC. Serum and urine neopterin concentrations were not detectable with HPLC in these specimens, whereas RIA gave weakly (presumably false) positive results. The mean serum tryptophan concentration was 39.0 +/- 6.2 micromol/l, and the mean kynurenine concentration was 0.85 +/- 0.33 micromol/l. The average kynurenine-per-tryptophan quotient in serum was 21.7 +/- 8.4 nmol/micromol, and that in plasma was 20.7 +/- 9.5 nmol/micromol (n = 7), which corresponds well to normal values in humans. This study provides preliminary data to support the monitoring of tryptophan degradation but not neopterin concentrations as a potential means of detecting immune activation in a porcine model. The kynurenine-per-tryptophan quotient may serve as a short-term measurement of immune activation and hence permit an estimate of the extent of immune activation.     

LPS-induced NF-κB expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase

Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-γ (IFN-γ). In parallel, IFN-γ induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-κB (NF-κB) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-κB expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-κB activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-κB inducible reporter system.In cells stimulated with LPS, a significant induction of NF-κB was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-κB activation, neopterin release and trp degradation (all p < 0.001). We conclude that there is a parallel induction of NF-κB, neopterin formation and trp degradation in monocytic THP-1 cells, which is elicited by pro-inflammatory triggers like LPS during innate immune responses.     

Memory and effector T cells modulate subsequently primed immune responses to unrelated antigens

Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated antigen, depending on whether the second antigen was administered in the same or opposite type of adjuvant. The regulatory effects of the first wave of T cell immunity on the subsequent T cell responses to an unrelated antigen attenuated over time. Notably, following challenge with the second antigen, there was a mutual cross-regulation between the first and second wave of humoral responses to unrelated antigens. Thus, immunization with one antigen not only primes immune responses to that antigen, but also influences subsequently primed immune responses to unrelated antigens.     

Leishmania donovani Infection of a Susceptible Host Results in Apoptosis of Th1-Like Cells: Rescue of Anti-Leishmanial CMI by Providing Th1-Specific Bystander Costimulation

A protective immune response against Leishmania donovani infection is mediated by T-helper type 1 (Th1) cells. Th1 induced cell-mediated immunity (CMI), as assessed by anti-leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down-regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite downregulates the expression of a Th1-specific costimulatory molecule, M150, on the surface of infected BALB/c mice-derived macrophages. Th cells are rendered unresponsive to anti-CD3 Ab-mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL-2, IL-4 and IFN-γ compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti-CD3 Ab induced unresponsiveness and IFN-γ but not IL-4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.     

Toll-like receptor (TLR)2 and TLR3 sensing is required for dendritic cell activation, but dispensable to control Schistosoma mansoni infection and pathology

Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. However, relatively little is known about their functions in innate/acquired responses to complex eukaryotic microorganisms, including helminth parasites. That Schistosoma mansoni eggs activate myeloid DCs through TLR2 and TLR3 has been shown by us and others, but the consequences of this combined activation are still unknown. We show that the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs. Strikingly, DCs sensitized with ovalbumin in the presence of parasite eggs dramatically reduce the release of Th2-type cytokines by ovalbumin-specific T lymphocytes, an effect that fully depends on TLR3. Finally, although TLR2 and TLR3 have no role in host resistance and in egg-induced granuloma formation in S. mansoni-infected mice, they individually and additionally increase the Th1/Th2 balance of the immune response. Thus, TLR2 and TLR3 sensing is required to shape the immune response during murine schistosomiasis, but is dispensable to control infection and pathology.     

Roles of cytokines in the pathogenesis and therapy of type 1 diabetes

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans by autoreactive T helper 1 (Th1) cells characterized by their cytokine secretory products, interleukin-2 (IL-2) and interferon γ (IFNγ). Th1-type cytokines (IL-2 and IFNγ) correlate with T1D, whereas Th2 (IL-4 and IL-10), Th3 (transforming growth factor beta [TGFβ]), and T regulatory cell-type cytokines (IL-10 and TGFβ) correlate with protection from T1D. Paradoxically, however, administrations of Th1-type cytokines (IL-2 and IFNγ) and immunotherapies that induce Th1-type cytokine responses actually prevent T1D, at least in animal models. Therefore, immunotherapies that inhibit IL-2 production/action will block Th1 cell/cytokine-driven effector mechanisms of pancreatic islet β-cell destruction; however, anti-IL-2 therapy will not allow immune tolerance to be established. In contrast, immunotherapies that increase IL-2 production/action may correct an immunodeficiency in IL-2 production that appears to underlie the autoimmunity of T1D, thereby restoring immune tolerance to islet β-cells and prevention of T1D.     

TLR4-dependent effects of ISAg treatment on conventional T cell polarization in vivo

We recently demonstrated that the polysaccharide component of the Korean medicinal herb Angelica gigas (immuno-stimulatory fraction of A. gigas; ISAg) induces anticancer effects in mice by activating natural killer (NK) and natural killer T (NKT) cells. However, it is unclear whether the use of ISAg in vivo can affect the differentiation of conventional T cells. Here, we investigated the effects of ISAg on the activation of conventional CD4⁺ and CD8⁺ T cells. We found that the administration of ISAg induced the polarization of CD4⁺ T cells toward the acquisition of the Th1 phenotype in vivo. Additionally, in mice treated with ISAg, CD8⁺ T cells produced more IFNγ than in control mice treated with PBS. Moreover, treatment with ISAg activated CD4⁺ and CD8⁺ T cells as well as NK and NKT cells, resulting in the secretion of Th1-type cytokines in a toll-like receptor 4 (TLR4)-dependent manner, implying that TLR4 is critical for an optimal Th1 response. Interestingly, ISAg treatment increased the number of Foxp3⁺ Treg cells, but not of Th2 cells, compared to control mice treated with PBS, indicating that ISAg possesses an immunomodulatory capacity that can control adaptive immune responses. Taken together, our results indicate that ISAg possesses a Th1-enhancing activity that could be used to treat Th2-mediated allergic immune diseases such as atopic dermatitis.     

Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.     

T cell receptor stimulation, reactive oxygen species, and cell signaling

In the immune system, much of the focus on reactive oxygen species (ROS) has been regarding their role in antimicrobial defense as part of the innate immune system. In addition to this role, it is now becoming clear that ROS are used by cells of the adaptive immune system as regulators of signal transduction by cell surface receptors. The activation of T lymphocytes through their specific antigen receptor [T cell receptor (TCR)] is vital in regulating the immune response. Much experimental evidence has suggested that activation of T cells is redox dependent and recent studies have shown that engagement of the TCR induces rapid production of ROS. This review examines the evidence for TCR-stimulated generation of ROS and discusses the role(s) of receptor-stimulated ROS production in T cell signal transduction and gene expression.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Helicobacter pylori-pulsed dendritic cells induce H. pylori-specific immunity in mice

Background: The growing concern over the emergence of antibiotic‐resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)‐based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow‐derived DCs pulsed with phosphate‐buffered saline (PBS‐DC) or live H. pylori SS1 (HP‐DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP‐DC or PBS‐DC immunized mice. The effect of regulatory T‐cell (Treg) depletion by anti‐CD25 antibody on HP‐DC vaccine efficacy was also evaluated. Results: HP‐DC induced a Th1‐dominant response in vitro. In vivo, HP‐DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP‐DC immunized mice expressed a higher level of IFN‐γ compared to PBS‐DC immunized mice; no difference was found for interleukin‐5 expressions in the stomach. A lower bacterial colonization post‐H. pylori challenge was observed in HP‐DC immunized mice compared to PBS‐DC immunized mice with no significant difference in gastritis severity. H. pylori‐specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti‐CD25 antibody. Conclusion: DC‐based anti‐H. pylori vaccine induced H. pylori‐specific helper T‐cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

Orally Administered Bisphenol A Disturbed Antigen Specific Immunoresponses in the Naïve Condition

Bisphenol A [2,2-bis(4-hydoxyphenyl)propane; BPA] is an endocrine disrupter widely used in polycarbonate plastics and epoxy resins. We investigated the effects of orally administered BPA on antigen-specific responses of the naïve immune system.

BPA was orally administered to T cell receptor transgenic mice, and the antigen-specific responses of immune cells were investigated. Administered BPA moderately reduced interleukin (IL)-2, 4, and interferon (IFN)-γ secretion and increases in IgA and IgG2a production.

Additionally, it was found that orally administered BPA increased antigen-specific IFN-γ production of T cells and modified whole antigen presenting cells (APCs) to suppress antigen-specific cytokine production from T cells.

These findings suggest that BPA can augment the Th1-type responses of naïve immune systems, though the bioavailability of orally administered BPA was low in our experiments.     

Regulatory role of miR‐2909 in cell‐mediated immune response

Keeping in view the micromanagement of immune response by micro RNAs, the present study was directed to explore the role of miR‐2909 in the differentiation and maturation of T‐lymphocytes within the population of normal human peripheral blood mononuclear cells maintained in in vitro culture. The results of such a study revealed that miR‐2909 had the inherent capacity to significantly increase Treg (CD4+CD25+Foxp3+) cell population and dominant Th1‐type cytokine (especially with decrease in IL‐4 level and higher levels of INF‐β and INF‐γ) profile. Based upon these results, we propose that miR‐2909 may modulate native immunity in general and help in providing protective immunity against viral infections in particular. Copyright © 2012 John Wiley & Sons, Ltd.     

Intrapulmonary administration of leukotriene B(4) augments neutrophil accumulation and responses in the lung to Klebsiella infection in CXCL1 knockout mice

In prior studies, we demonstrated that 1) CXCL1/KC is essential for NF-κB and MAPK activation and expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine in Klebsiella-infected lungs, and 2) CXCL1 derived from hematopoietic and resident cells contributes to host immunity against Klebsiella. However, the role of CXCL1 in mediating neutrophil leukotriene B(4) (LTB(4)), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. In this study, we investigated 1) the role of CXCL1 in LTB(4), NADPH oxidase, and inducible NO synthase (iNOS) expression in lungs and neutrophils, and 2) whether LTB(4) postinfection reverses innate immune defects in CXCL1(-/-) mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB(4) levels, and decreased ROS and iNOS production in the lungs of CXCL1(-/-) mice after Klebsiella pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB(4) after infection. To treat immune defects in CXCL1(-/-) mice, we intrapulmonarily administered LTB(4). Postinfection, LTB(4) treatment reversed immune defects in CXCL1(-/-) mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB(4) also enhanced myeloperoxidase, H(2)O(2,) RNS production, and bacterial killing in K. pneumoniae-infected CXCL1(-/-) neutrophils. These novel results uncover important roles for CXCL1 in generating ROS and RNS in neutrophils and in regulating host immunity against K. pneumoniae infection. Our findings suggest that LTB(4) could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.     

轮状病毒致乳鼠肠道外感染Th1/Th2细胞因子变化的研究

目的通过复制轮状病毒（RV）肠道外感染乳鼠的动物模型,检测接种后乳鼠体内Th1/Th2平衡改变,对RV肠道外感染后机体免疫状态进行初步研究。方法48只乳鼠随机均分为3组：肠道外组、肠道内组和正常对照组。肠道外组通过腹腔注射猴RVSA11株,肠道内组灌胃等量RV悬液,对照组无特殊处理。分别在接种后第4天、第8天处死乳鼠,收集标本,观察心、肝、肾、肺等脏器病理变化,用ELISA法检测血清中IL-10和IFN-γ的表达。结果光镜下肠道外组乳鼠肾、肝、肺和脾脏出现病理改变。感染后第4天,肠道内、外组乳鼠血清IFN-γ水平均高于正常组,到第8天明显下降,基本达到基线水平;IL-10在肠道外组第4天增高,到第8天小幅下降,但仍然高于正常组;而肠道内组IL-10无明显改变。结论RV肠道外感染早期呈现Th1-Th2混合反应,而后期则以IL-10的表达为主,T细胞向Th2型免疫应答方向偏离,Th1/Th2细胞因子失衡机制可能是RV肠道外感染的重要因素。