Intraperitoneal immune cell responses to Eubacterium saphenum in mice

Oral asaccharolytic Eubacterium saphenum, which are newly isolated gram-positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases. Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/gammadelta+) production of cytokines IL-4 and IFN-gamma which are related to cellular and humoral immunity, respectively, was determined. Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E. saphenum or Porphyromonas gingivalis. Intracellular IL-4+ and IFN-gamma+ gammadelta T cells were detected in the exudates after the injection of E. saphenum (4.6 +/- 0.8% and 10.1 +/- 1.4%, respectively) and P. gingivalis (5.3 +/- 1.6% and 10.1 +/- 2.1%, respectively). The intracellular production of IL-4/IFN-gamma in CD4+/CD8+ T cells was rather low indicating that the main response was from gammadelta T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E. saphenum or P. gingivalis. Serum IgG and IgM levels were elevated in animals injected with E. saphenum and similarly with P. gingivalis. The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E. saphenum and P. gingivalis, indicating that E. saphenum may play just as important a role in periodontal diseases as P. gingivalis.     

Changes in immune responses to oxidized LDL epitopes during aging in hypercholesterolemic apoE(-/-) mice

Atherosclerosis is a disease associated with aging and is subject to modulation by both the innate and adaptive immune system. The time course of age-dependent changes in immune regulation in the context of atherosclerosis has not been characterized. This study aims to describe alteration of the immune responses to oxidized LDL (oxLDL) during aging that is associated with changes in plaque size and phenotype in apoE(-/-) mice. Mice fed a Western diet were euthanized at 15-17, 36, or >52 wk of age. The descending aortas were stained for assessment of extent of atherosclerosis. Plaque lipid, macrophage, and collagen content were evaluated in aortic sinus lesions. The adaptive immune response to oxLDL was assessed using anti-malondialdehyde-oxidized LDL (MDA-LDL) and copper-oxidized LDL (Cu-oxLDL) IgG, and the innate immune response was assessed using anti-Cu-oxLDL and phosphorylcholine (PC) IgM. Aging was associated with a significant increase in plaque area and collagen content and a decrease in plaque macrophage and lipid content. MDA-LDL IgG significantly increased at 36 wk but was reduced in mice >52 wk. Cu-oxLDL IgG increased with age and IgG-apoB immune complexes were increased in the >52 wk group. Cu-oxLDL and PC IgM significantly increased with age. The expression of splenic cytokines such as IFN-gamma, IL-4, and IL-10 increased with age. Our study shows a generalized increase in innate immune responses associated with progression of atherosclerosis and a less inflammatory and less lipid-containing plaque phenotype during aging. The adaptive immune response appeared to be less generalized, with a specific reduction in MDA-LDL IgG.     

Abnormal polyclonal B cell activation in NZB/NZW F1 mice

Spleen cells from autoimmune (10-mont-old) NZB/NZW (B/W) mice failed to generate appreciable numbers of antibody-forming cells (AFC) in vitro to TNP-substituted sheep erythrocytes in response to the polyclonal B cell activators (PBA), LPS and PPD, despite normal DNA synthetic responses to these agents and normal AFC responses to TNP-Ficoll. The failure to respond to PBA in old B/W mice was not due to suppressor T cells since anti-brain-associated-theta-treated spleen cells still failed to generate AFC in response to PBA. The defect was age-related since cells from young B/W mice generated vigorous AFC responses to PBA. It is suggested that the failure of the spleen cells of old B/W mice to generate AFC is a result of in vitro polyclonal B cell activation in the course of autoantibody formation.     

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.     

Primary immune responses to H-Y in BALB/c-H-2 k mice

Female mice of the H-2k haplotype have been thought not to reject primary grafts of male skin. However, most BALB/c-H-2k female mice bred in our colony rejected syngeneic male grafts. Graft rejection was sufficient to prime the mice for the subsequent generation of H-Y-specific cytotoxic T cells and delayed-type hypersensitivity responses.     

Abnormal sympathoadrenal development and systemic hypotension in PHD3-/- mice

Cell culture studies have implicated the oxygen-sensitive hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 in the regulation of neuronal apoptosis. To better understand this function in vivo, we have created PHD3(-/-) mice and analyzed the neuronal phenotype. Reduced apoptosis in superior cervical ganglion (SCG) neurons cultured from PHD3(-/-) mice is associated with an increase in the number of cells in the SCG, as well as in the adrenal medulla and carotid body. Genetic analysis by intercrossing PHD3(-/-) mice with HIF-1a(+/-) and HIF-2a(+/-) mice demonstrated an interaction with HIF-2alpha but not HIF-1alpha, supporting the nonredundant involvement of a PHD3-HIF-2alpha pathway in the regulation of sympathoadrenal development. Despite the increased number of cells, the sympathoadrenal system appeared hypofunctional in PHD3(-/-) mice, with reduced target tissue innervation, adrenal medullary secretory capacity, sympathoadrenal responses, and systemic blood pressure. These observations suggest that the role of PHD3 in sympathoadrenal development extends beyond simple control of cell survival and organ mass, with functional PHD3 being required for proper anatomical and physiological integrity of the system. Perturbation of this interface between developmental and adaptive signaling by hypoxic, metabolic, or other stresses could have important effects on key sympathoadrenal functions, such as blood pressure regulation.     

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: Immunological responses to fumonisin B1

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B₁ (FB₁) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 µg of FB₁ reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 µg of FB₁ caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB₁ is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB₁ as an antigen, FB₁ was injected into mice and the number of splenic cells forming rosettes on FB₁-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB₁-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB₁ has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB₁.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - FB₁ fumonisin B₁ - FCS fetal calf serum - PBS phosphate buffered saline - PFC plaque forming cells - RFC rosette forming cells - SRBC sheep red blood cells     

UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.     

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.     

Enhanced pulmonary allergic responses to Aspergillus in CCR2-/- mice

Allergic responses to Aspergillus species exacerbate asthma and cystic fibrosis. The natural defense against live Aspergillus fumigatus spores or conidia depends on the recruitment and activation of mononuclear and polymorphonuclear leukocytes, events that are dependent on chemotactic cytokines. In this study, we explored the relative contribution of the monocyte chemoattractant protein-1 receptor, CCR2, in the pulmonary response to A. fumigatus conidia. Following sensitization to soluble A. fumigatus Ags, mice lacking CCR2 due to targeted deletion were markedly more susceptible to the injurious effects of an intrapulmonary challenge with live conidia compared with mice that expressed CCR2 or CCR2+/+. CCR2-/- mice exhibited a major defect in the recruitment of polymorphonuclear cells, but these mice also had significantly more eosinophils and lymphocytes in bronchoalveolar lavage samples. CCR2-/- mice also had significant increases in serum levels of total IgE and whole lung levels of IL-5, IL-13, eotaxin, and RANTES compared with CCR2+/+ mice. Airway inflammation, hyper-responsiveness to spasmogens, and subepithelial fibrosis were significantly enhanced in CCR2-/- mice compared with CCR2+/+ mice after the conidia challenge. Thus, these findings demonstrate that CCR2 plays an important role in the immune response against A. fumigatus, thereby limiting the allergic airway inflammatory and remodeling responses to this fungus.     

Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1

Here we show that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with human fetal thymic and liver tissues results in long-term, systemic human T-cell homeostasis. In addition, these mice show systemic repopulation with human B cells, monocytes and macrophages, and dendritic cells (DCs). T cells in these mice generate human major histocompatibility complex class I- and class II-restricted adaptive immune responses to Epstein-Barr virus (EBV) infection and are activated by human DCs to mount a potent T-cell immune response to superantigens. Administration of the superantigen toxic shock syndrome toxin 1 (TSST-1) results in the specific systemic expansion of human Vbeta2+ T cells, release of human proinflammatory cytokines and localized, specific activation and maturation of human CD11c+ dendritic cells. This represents the first demonstration of long-term systemic human T-cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.     

ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis

Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share approximately 84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1(-/-) mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1(-/-) mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1(-/-) mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1(-/-) mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1(-/-) mice, with higher IFN-gamma production and lower IL-5 production in MOG35-55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1(-/-) mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE.     

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.     

Impaired immune responses and prolonged allograft survival in Sly1 mutant mice

Adaptive immunity is crucial for protective host defense and the development of immunological disorders. SLY1 was recently identified as an X-chromosomal SH3 protein that is serine phosphorylated (Ser27) upon B-and T-cell receptor engagement. Here, we demonstrate that SLY1 is localized in the cytoplasm and the nucleus of immunocytes. We generated mice expressing a mutant version of SLY1 lacking Ser27 and a functional nuclear localization signal. The defective SLY1 (SLY1(d)) protein is localized exclusively in the cytoplasm. B- and T-cell proliferation is attenuated and T-cell cytokine production is severely reduced. Sly1(d/d) mice exhibit reduced lymphoid organ sizes, diminished marginal zone B-cell numbers, and severely impaired antibody responses against T-dependent and -independent antigens. Importantly, survival of semi-identical cardiac allografts was substantially prolonged in Sly1(d/d) mice. These results define SLY1 as an essential molecular component for the full activation of adaptive immunity.     

Antiviral immune responses in the absence of organized lymphoid T cell zones in plt/plt mice

The paucity of lymph node (LN) T cells (plt) mutation in mice results in strongly reduced T cell numbers in LNs and homing defects of both dendritic cells (DCs) and naive T cells. In this study, we investigated the functional significance of the plt phenotype for the generation of antiviral immune responses against cytopathic and noncytopathic viruses. We found that DC-CD8(+) T cell contacts and the initial priming of virus-specific T cells in plt/plt mice occurred mainly in the marginal zone of the spleen and in the superficial cortex of LNs. The magnitude of the initial response and the maintenance of protective memory responses in plt/plt mice was only slightly reduced compared with plt/+ controls. Furthermore, plt/plt mice mounted rapid neutralizing antiviral B cell responses and displayed normal Ig class switch. Our data indicate that the defective homing of DCs and naive T cells resulting from the plt/plt mutation results in a small, but not significant, effect on the induction of protective antiviral T and B cell immunity. Overall, we conclude that the spatial organization of secondary lymphoid T cell zones via the CCR7-CC chemokine ligand 19/CC chemokine ligand 21 pathway is not an absolute requirement for the initial priming and the maintenance of protective antiviral T and B cell responses.     

Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1

Fever is a common, centrally elicited sign of inflammatory and infectious processes and is known to be induced by the action of PGE2 on its specific receptors in the thermogenic region of the hypothalamus. In the present work, using genetically modified mice, we examined the role of the inducible terminal PGE2-synthesizing enzyme microsomal prostaglandin E synthase-1 (mPGES-1) for the generation of immune-elicited fever. Animals with a deletion of the Ptges gene, which encodes mPGES-1, or their wild-type littermates were given either a subcutaneous injection of turpentine--a model for aseptic cytokine-induced pyresis--or an intraperitoneal injection of interleukin-1beta. While both procedures resulted in typical febrile responses in wild-type animals, these responses were strongly impaired in the mPGES-1 mutant mice. In contrast, both genotypes showed psychogenic stress-induced hyperthermia and displayed normal diurnal temperature variations. Both wild-type and mPGES-1 mutant mice also showed strongly reduced motor activity following turpentine injection. Taken together with previous observations on mPGES-1 induction in the brain vasculature during various inflammatory conditions and its role in endotoxin-induced pyresis, the present findings indicate that central PGE2 synthesis by mPGES-1 is a general and critical mechanism for fever during infectious and inflammatory conditions that is distinct from the mechanism(s) underlying the circadian temperature regulation and stress-induced hyperthermia, as well as the inflammation-induced activity depression.     

Normal tissue depresses while tumor tissue enhances human T cell responses in vivo to a novel self/tumor melanoma antigen,OA1

Antitumor T cells often recognize targets that are nonmutated "self" tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a "knockout." In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-"knockout") individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.     

The role of CXCL12 and CCL7 chemokines in immune regulation,embryonic development,and tissue regeneration

Chemotactic factors direct the migration of immune cells, multipotent stem cells, and progenitor cells under physiologic and pathologic conditions. Chemokine ligand 12 and chemokine ligand 7 have been identified and investigated in multiple studies for their role in cellular trafficking in the setting of tissue regeneration. Recent early phase clinical trials have suggested that these molecules may lead to clinical benefit in patients with chronic disease. Importantly, these two proteins may play additional significant roles in directing the migration of multipotent cells, such as mesenchymal stem cells and hematopoietic progenitor cells. This article reviews the functions of these two chemokines, focusing on recruitment to sites of injury, immune function modulation, and contributions to embryonic development. Additional research would provide valuable insight into the potential clinical application of these two proteins in stem cell therapy.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.