Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Serendipitous Discovery of a Novel Murine Astrovirus Contaminating a Murine Helper T-cell Line and Incapable of Infecting Highly Immunodeficient Mice.

The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying <30% capsid identity, yet were closely related to murine astrovirus 2 (85% capsid identity), which had recently been isolated from feral mice in New York City. A MuAstV-2 specific PCR assay was developed and used to eradicate MuAstV-2 from the infected colony using a test and cull strategy. The newly identified MuAstV2 readily transmits to immunocompetent mouse strains by fecal-oral exposure, but fails to infect NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.

Effects of Colonization of Gnotobiotic Swiss Webster Mice with Helicobacter bilis.

Helicobacter bilis (Hb) causes hepatitis in some strains of inbred mice. The current study confirmed that Hb directly causes portal hepatitis in outbred gnotobiotic Swiss Webster (SW) mice, as we previously reported for conventional SW mice. Hbmonoassociated SW mice also developed mild enterocolitis, expanded gut-associated lymphoid tissue (GALT), and tertiary lymphoid tissue in the lower bowel. At 1 and 10 mo after infection, Hb-induced GALT hyperplasia exhibited well-organized, ectopic germinal centers with increased mononuclear cell apoptosis, MHC class II antigen presentation, and pronounced endothelial venule formation, consistent with features of tertiary lymphoid tissue. In the lower bowel, Hb induced mainly B220+ cells as well as CD4+ IL17+, CD4+ IFNγ+, and CD4+ FoxP3+ regulatory T cells and significantly increased IL10 mRNA expression. This gnotobiotic model confirmed that Hb causes portal hepatitis in outbred SW mice but stimulated GALT with an antiinflammatory bias. Because Hb had both anti- and proinflammatory effects on GALT, it should be considered a 'pathosymbiont provocateur' and merits further evaluation in mouse models of human disease.

Helicobacter pylori-infected C57BL/6 mice with different gastrointestinal microbiota have contrasting gastric pathology, microbial and host immune responses.

C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1ß, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.

BATF is required for normal expression of gut-homing receptors by T helper cells in response to retinoic acid.

CCR9 and α4ß7 are the major trafficking receptors for lymphocyte migration to the gut, and their expression is induced during lymphocyte activation under the influence of retinoic acid (RA). We report here that BATF (basic leucine zipper transcription factor, ATF-like), an AP-1 protein family factor, is required for optimal expression of CCR9 and α4ß7 by T helper cells. BATF-deficient (knockout [KO]) mice had reduced numbers of effector T and regulatory T cells in the intestine. The intestinal T cells in BATF KO mice expressed CCR9 and α4ß7 at abnormally low levels compared with their wild-type (WT) counterparts, and BATF KO CD4(+) T cells failed to up-regulate the expression of CCR9 and α4ß7 to WT levels in response to RA. Defective binding of RARα and histone acetylation at the regulatory regions of the CCR9 and Itg-α4 genes were observed in BATF KO T cells. As a result, BATF KO effector and FoxP3(+) T cells failed to populate the intestine, and neither population functioned normally in the induction and regulation of colitis. Our results establish BATF as a cellular factor required for normal expression of CCR9 and α4ß7 and for the homeostasis and effector functions of T cell populations in the intestine.

Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch.

FoxP3(+) T cells populate tumors and regulate anti-tumor immunity. The requirement for optimal population of FoxP3(+) regulatory T cells in tumors remains unclear. We investigated the migration requirement and stability of tumor-associated FoxP3(+) T cells. We found that only memory, but not naïve, FoxP3(+) T cells are highly enriched in tumors. Almost all of the tumor-infiltrating FoxP3(+) T cells express Helios, an antigen associated either with thymus-generated FoxP3(+) T cells or activated T cells in the periphery. The tumor-infiltrating FoxP3(+) T cells largely lack CD62L and CCR7, two trafficking receptors required for T cell migration into secondary lymphoid tissues. Instead, the tumor infiltrating FoxP3(+) T cells highly express memory/tumor-associated CCR8 and CXCR4. Antigen priming is required for induction of this trafficking receptor phenotype in FoxP3(+) T cells and only antigen primed, but not antigen-inexperienced naive, FoxP3(+) T cells can efficiently migrate into tumors. While the migration of FoxP3(+) T cells into tumors was a readily detectable event, generation of induced FoxP3(+) T cells within tumors was unexpectedly inefficient. Genetic marking of current and ex-FoxP3(+) T cells revealed that tumor-infiltrating FoxP3(+) T cells are highly stable and do not readily convert back to FoxP3(-) T cells. Taken together, our results indicate that population of tumors with thymus-generated FoxP3(+) T cells requires an antigen priming-dependent trafficking receptor switch in lymphoid tissues.

Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets.

BACKGROUND: It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells. RESULTS: We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1(low)+, PD-1(medium)++, and PD-1(high)+++ cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells. CONCLUSION: Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response.

Retinoic acid determines the precise tissue tropism of inflammatory Th17 cells in the intestine.

Th17 cells are major effector T cells in the intestine, but the regulation of their tissue tropism within the gut is poorly understood. We investigated the roles of vitamin A and retinoic acid in generation of inflammatory Th17 cells with distinct tissue tropisms within the intestine. We found that Th17 cells with distinct tissue tropisms and pathogenic activities are generated depending on the available concentration of retinoic acid (RA). In contrast to the widespread perception that RA would suppress the generation of Th17 cells, we provide evidence that RA is actually required for generation of Th17 cells with specific tissue tropisms within the gut. Th17 cells induced at suboptimal serum concentrations of RA migrated and induced moderate inflammation mainly in the large intestine, whereas the Th17 cells induced with optimal levels of exogenous RA (approximately 10 nM) migrated to the small intestine and induced more severe inflammation. The Th17 cells, induced in the presence or absence of RA, differentially expressed the trafficking receptors CCR9 and alpha4beta7. CCR9 is required for Th17 cell migration to the small intestine, whereas alpha4beta7 is required for the migration of Th17 cells throughout the whole intestine. Our results identified RA as a major signal that regulates the generation of gut Th17 cells with distinct capacities in migration and inflammatory activities. The results indicate also that specific gut tropism of Th17 cells is determined by the combination of trafficking receptors regulated by the RA signal.

Batf coordinates multiple aspects of B and T cell function required for normal antibody responses.

Batf belongs to the activator protein 1 superfamily of basic leucine zipper transcription factors that includes Fos, Jun, and Atf proteins. Batf is expressed in mouse T and B lymphocytes, although the importance of Batf to the function of these lineages has not been fully investigated. We generated mice (Batf(DeltaZ/DeltaZ)) in which Batf protein is not produced. Batf(DeltaZ/DeltaZ) mice contain normal numbers of B cells but show reduced numbers of peripheral CD4(+) T cells. Analysis of CD4(+) T helper (Th) cell subsets in Batf(DeltaZ/DeltaZ) mice demonstrated that Batf is required for the development of functional Th type 17 (Th17), Th2, and follicular Th (Tfh) cells. In response to antigen immunization, germinal centers were absent in Batf(DeltaZ/DeltaZ) mice and the maturation of Ig-secreting B cells was impaired. Although adoptive transfer experiments confirmed that this B cell phenotype can be driven by defects in the Batf(DeltaZ/DeltaZ) CD4(+) T cell compartment, stimulation of Batf(DeltaZ/DeltaZ) B cells in vitro, or by a T cell-independent antigen in vivo, resulted in proliferation but not class-switch recombination. We conclude that loss of Batf disrupts multiple components of the lymphocyte communication network that are required for a robust immune response.

FoxP3+ regulatory T cells restrain splenic extramedullary myelopoiesis via suppression of hemopoietic cytokine-producing T cells.

Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3(+) regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3(+) regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3(+) regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4(+) T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4(+) T cells are induced from naive CD4(+) T cells in response to antigenic stimulation. FoxP3(+) regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFbeta. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3(+) regulatory T cells. We conclude that FoxP3(+) T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4(+) T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.

High and low vitamin A therapies induce distinct FoxP3+ T-cell subsets and effectively control intestinal inflammation.

BACKGROUND & AIMS: Retinoic acid plays a positive role in induction of FoxP3(+) regulatory T cells. Because retinoic acid is produced as a metabolite of vitamin A in the intestine and FoxP3(+) T cells regulate intestinal inflammation, we investigated the impact of vitamin A status on the regulatory T cells and inflammation in the intestine. METHODS: The SAMP1/YP model is a mouse model of Crohn's disease. We made vitamin A-deficient, vitamin A-excessive, and normal SAMP1/YP mice and assessed the intestinal inflammation. We also investigated the phenotype and function of FoxP3(+) T cells induced in different levels of vitamin A availability in regulation of intestinal inflammation in a T-cell-induced inflammation model in SCID mice. RESULTS: The limited and excessive vitamin A conditions induced distinct FoxP3(+) T-cell subsets in vivo, and both ameliorated the intestinal inflammation in SAMP1/YP mice. The limited vitamin A condition greatly induced unusual CD103(+)CCR7(+) FoxP3(+) cells, while the high vitamin A condition induced CCR9(+)alpha4beta7(+) FoxP3(+) T cells in the intestine. Both FoxP3(+) T-cell populations, when transferred into mice with ongoing intestinal inflammation, were highly effective in reversing the inflammation. Blockade or lack of occupancy of RARalpha is a mechanism to induce highly suppressive CD103(+)CCR7(+) FoxP3(+) cells in both the thymus and periphery in limited vitamin A availability. CONCLUSIONS: Our results identify novel pathways of inducing highly suppressive FoxP3(+) regulatory T cells that can effectively control intestinal inflammation. The results have significant ramifications in treating inflammatory bowel diseases.

[A study on pathological changes of brain after high +Gx exposure in rhesus monkey].

OBJECTIVE: To study the pathological morphological changes of cerebral cortex and hippocampi in rhesus monkey caused by +Gx exposure, and to explore the relation between +Gx level and pathological changes. METHOD: Healthy rhesus monkeys were randomly divided into one control group and three experimental groups (+15 Gx, +18 Gx, +21 Gx). Monkeys in each group were exposed to the corresponding level of +Gx, after that, the required tissue was qualitatively studied on the basis of pathological morphology. RESULT: 1) Different morphology changes were observed in pyramidal neurons and astrocytes in the cerebral cortex and hippocampi by light microscopy. The higher the +Gx level, the more the changes were observed. In addition, the trauma was more serious in the hippocampi. 2) Chromatin marginating, karyotheca fold, apoptosis body and swollen mitochondria with blurred cristae were observed in pyramidal cell under electron microscope after +Gx exposure, and degenerative changes were also observed in some cases after higher +Gx. CONCLUSION: High level of +Gx causes acute pathological trauma of brain tissues in rhesus monkey, and consanguineous relationship exists between pathological changes and level of +Gx.